Annual Review of Immunology Volume 27 2009

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Annual Review of Immunology

Annu. Rev. Immunol. 2009.27:621-668. Downloaded from arjournals.annualreviews.org by Karolinska Institutet on 03/29/09. For personal use only.

Contents

Volume 27, 2009

Frontispiece Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p x Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1 Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. LaPorte, and K. Christopher Garcia p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29 Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 61 Immune Therapy for Cancer Michael Dougan and Glenn Dranoff p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 83 Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff and V. Hugh Perry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p119 The Liver as a Lymphoid Organ Ian Nicholas Crispe p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147 Immune and Inﬂammatory Mechanisms of Atherosclerosis Elena Galkina and Klaus Ley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p165 Primary B Cell Immunodeﬁciencies: Comparisons and Contrasts Mary Ellen Conley, A. Kerry Dobbs, Dana M. Farmer, Sebnem Kilic, Kenneth Paris, Soﬁa Grigoriadou, Elaine Coustan-Smith, Vanessa Howard, and Dario Campana p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199 The Inﬂammasomes: Guardians of the Body Fabio Martinon, Annick Mayor, and Jürg Tschopp p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p229 Human Marginal Zone B Cells Jean-Claude Weill, Sandra Weller, and Claude-Agn`es Reynaud p p p p p p p p p p p p p p p p p p p p p p267

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Aire Diane Mathis and Christophe Benoist p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p287 Regulatory Lymphocytes and Intestinal Inﬂammation Ana Izcue, Janine L. Coombes, and Fiona Powrie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p313 The Ins and Outs of Leukocyte Integrin Signaling Clare L. Abram and Clifford A. Lowell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p339

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Recent Advances in the Genetics of Autoimmune Disease Peter K. Gregersen and Lina M. Olsson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p363 Cell-Mediated Immune Responses in Tuberculosis Andrea M. Cooper p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p393 Enhancing Immunity Through Autophagy Christian Munz ¨ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423 Alternative Activation of Macrophages: An Immunologic Functional Perspective Fernando O. Martinez, Laura Helming, and Siamon Gordon p p p p p p p p p p p p p p p p p p p p p p p p451 IL-17 and Th17 Cells Thomas Korn, Estelle Bettelli, Mohamed Oukka, and Vijay K. Kuchroo p p p p p p p p p p p p p p485 Immunological and Inﬂammatory Functions of the Interleukin-1 Family Charles A. Dinarello p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p519 Regulatory T Cells in the Control of Host-Microorganism Interactions Yasmine Belkaid and Kristin Tarbell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p551 T Cell Activation Jennifer E. Smith-Garvin, Gary A. Koretzky, and Martha S. Jordan p p p p p p p p p p p p p p p591 Horror Autoinﬂammaticus: The Molecular Pathophysiology of Autoinﬂammatory Disease Seth L. Masters, Anna Simon, Ivona Aksentijevich, and Daniel L. Kastner p p p p p p p p p621 Blood Monocytes: Development, Heterogeneity, and Relationship with Dendritic Cells Cedric Auffray, Michael H. Sieweke, and Frederic Geissmann p p p p p p p p p p p p p p p p p p p p p p p p669 Regulation and Function of NF-κB Transcription Factors in the Immune System Sivakumar Vallabhapurapu and Michael Karin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p693

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Contents

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Review in Advance first posted online on November 13, 2008. (Minor changes may still occur before final publication online and in print.)

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Translating Molecular Insights in Autoimmunity into Effective Therapy Marc Feldmann Kennedy Institute of Rheumatology Division, Imperial College London, London W6 8LH, UK; email: [email protected]

Annu. Rev. Immunol. 2009. 27:1–27

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

cytokines, rheumatoid arthritis, TNF, anti-TNF

This article’s doi: 10.1146/annurev-immunol-082708-100732

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

Autoimmunity and the pathogenesis of autoimmune diseases was a major focus of the Walter and Eliza Hall Institute, where I started my research career. After my initial studies on immune cell culture and immune regulation, I returned to an analysis of the pathogenesis of human autoimmunity in London. Linking upregulated antigen presentation to autoimmunity led to an investigation of the role of cytokines in rheumatoid arthritis (RA), in collaboration with Ravinder Maini. These experiments led to the concept of a TNF-dependent cytokine cascade driving the manifestations of RA, which led to successful clinical trials of anti-TNF monoclonal antibody in RA patients, heralding a major change in medical practice. This success was made possible by enthusiastic support from many laboratory and clinical colleagues and taught us that cytokines are important rate-limiting steps and hence good therapeutic targets. My current scientiﬁc challenge is exploring the hypothesis of whether all major medical needs can be approached via cytokine blockade.

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GROWING UP IN AUSTRALIA

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Postwar France was poor, and despite my Polish father’s French accountancy degree, life was difﬁcult. My Jewish parents thus sought greater opportunities elsewhere. My father had two cousins, both doctors, one in New Haven, Connecticut, in the United States, and the other in Melbourne, Australia, and they both ﬁled immigration papers for my family. The Australian papers came a month before the U.S. papers. Who knows what might have happened had U.S. bureaucracy been speedier. As an eight-year-old, a month’s journey by ship to Australia was an adventure going into the unknown, to a land of kangaroos and much promise, visiting the pyramids en route. Learning English in a land welcoming immigrants was not a major hurdle. Immigrants have a very strong motivation to work hard and succeed. As a child, the image of my father coming home tired from his work as an accountant in a factory, to study anew for his accountancy degree, as his French qualiﬁcation was not recognized, had a profound impact on me. Once qualiﬁed, he built up a prospering sole-proprietorship serving other immigrants. Having helped balance books of accounts on weekends and holidays, I found the thought of following in his footsteps far too boring! My father’s cousin, more ﬂamboyant, was a doctor, and that seemed to my older brother and me a more challenging and possibly more satisfying profession, so we both became medical students at the University of Melbourne. Medical studies opened up new vistas. Some courses were painstaking and meticulous—ﬁve terms of learning and regurgitating anatomy is a chore that is mercifully no longer imposed— but others were exciting and challenging. Biochemistry teachers encouraged successful students to read more widely. My ﬁrst exposure to the Annual Reviews of Biochemistry was an eye-opener, illustrating the questioning and uncertainties of emerging ideas and knowledge, rather than the deﬁnite facts and platitudes that we students usually received in lectures.

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Microbiology provided my ﬁrst glimpse of immunology, with eight lectures on serology taught by a microbiologist on the use of antisera to diagnose infections. A few years later, I learned that this course bore no resemblance to the major discoveries about cellular immunity being made contemporaneously on the other side of the Sydney Road from the university campus, at the Walter and Eliza Hall Institute of Medical Research, by luminaries Jacques Miller, Gus Nossal, and others, who eventually became my mentors. Clinical studies were stressful for a young, immature student like me, who at 17 started medicine straight from high school. We had already experienced death early in our studies, by working with cadavers in anatomy. The University of Melbourne clinical studies were performed in two hospitals: at the Royal Melbourne Hospital, over the road from the university, which took a large group of students, and at St. Vincent’s Hospital in Fitzroy, which took one-ﬁfth the number of students that Royal Melbourne did. I suspect that I am impatient. I got married while still a medical student, and my son was born while I was working in hospital and my daughter while I was completing my PhD. I made what in retrospect was a pivotal decision: to go to St. Vincent’s, the hospital with fewer students, because I was impatient to learn clinical medicine more rapidly by seeing the most interesting patients, which I hoped being part of the smaller group would allow. It was indeed easier to learn clinical medicine, but I paid the price later when, after qualiﬁcation, I and the other young doctors had to see all comers and not just the interesting cases. St. Vincent’s was in a poor, hard-drinking suburb of Melbourne, so almost 50% of patients arriving in casualty (emergency room) were chronic alcoholics, who have neither the most pleasant demeanors nor the most intellectually challenging problems. I therefore began exploring research opportunities earlier than I might have if I had chosen the more academic hospital.

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SCIENCE IS DIFFERENT FROM MEDICINE: GOLDEN YEARS AT THE WALTER AND ELIZA HALL INSTITUTE The Walter and Eliza Hall Institute of Medical Research (WEHI), across the road from the university, was made famous by the Nobel Prize winner Sir Frank MacFarlane Burnet, who had been a pioneer both in virology and subsequently in immunology. The institute had recently appointed a young and dynamic new director, Gustav Nossal. I went to meet him and to see the institute. The contrast with the rest of the university was stark: WEHI was clearly in a different league. Gus accepted me as a PhD student for the following year, to work on new techniques of tissue culture for generating immune responses in vitro. Little did I know that I had applied far too late, but Gus’s intuition was to make an exception and seek another PhD studentship for me. When I arrived in February, after a summer break recovering from the stresses of endless on-call rotations in the hospital, an arduous apprenticeship compared with today’s European Union work regulation–restricted hours, I was greeted by Erwin Diener, the Swiss scientist

Gus had chosen to supervise my ﬁrst tentative steps into science (see Figure 1). The project was wonderful, optimizing in vitro lymphoid cell cultures that were independently being developed in the United States by Bob Mishell and Dick Dutton (1) and at WEHI by John Marbrook (2) and Erwin Diener (3). The project was wonderful because of its potential inﬂuence on virtually all aspects of immunology. In vitro experiments are truly reductionist, and if that is to your taste, all the elements involved can be controlled: cells puriﬁed and quantitated, antigen concentration maintained precisely, other stimuli controlled. But this control comes at the same price as all the reductionist science still popular today (e.g., gene knockouts): Concepts generated in one precise circumstance often do not extrapolate to complex and nonreductionist reality. With Erwin, I started to improve the current culture methods. It was already possible to generate antibody production from mouse spleen cells. The antigen used, salmonella ﬂagellin, was popular at WEHI, having been used by Gus Nossal and Gordon Ada to help validate Burnet’s clonal selection theory (reviewed in 4). This antigen had been used to demonstrate that one cell produced only one antibody,

Mentors

Jacques Miller

Gustav Nossal

Erwin Diener

Figure 1 My mentors at the Walter and Eliza Hall Institute of Medical Research, Gustav Nossal, Jacques Miller, and Erwin Diener. www.annualreviews.org • Effective Therapy for Autoimmunity

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even from lymph nodes of multiply immunized mice. Assaying ﬂagellin immunity was laborious: Cell suspensions were incubated with bacteria, which adhered to the antibody-forming cells. The suspension was plated on agar, and colonies were grown for a few hours to enable discrimination of cells that had bound multiple bacteria from single bacteria (5). The competing single-cell assay was the hemolytic plaque assay, developed by Niels Jerne (6) and his collaborators. It used complement-mediated lysis, either on agar or between glass slides. An improvement I engineered was to convert the cumbersome bacterial assay to a plaque assay, coating the red cells with protein. For multipurpose use, I developed the technique of using anti-sheep red cell Fab fragments (7), which could be derivatized with haptens, e.g., DNP or proteins, such as, for example, myelin basic protein (MBP), work I did in collaboration with fellow PhD student Vanda Lennon (8). Initially, we performed these cultures on a small scale, 10–20 ﬂasks permitting 3–6 groups, producing 3–6 sets of data to compare. But as techniques improved, many more questions arose, and so the glassware proliferated, as did the need for more washing up, more media, more sera incubators, etc. I needed more resources. Thus, I learned early at WEHI the virtue of collaborations, pooling intellectual and material resources to enhance scientiﬁc productivity. Effective collaboration has been a key part of WEHI’s success as an international scientiﬁc powerhouse over its long history. Erwin had two laboratory technicians, and as he was a reﬂective scientist, not prone to an excessive number of experiments, his technicians were encouraged to assist me. As a beginning PhD student, I found this to be a wonderful situation, as was having Erwin’s patient help in developing my scientiﬁc writing skills. Jacques Miller was at his magniﬁcent prime when I started at WEHI. With Graeme Mitchell, he had just published a series of three landmark papers (9–11) documenting that thymus-derived lymphocytes did not in themselves make antibody or develop into antibodyforming cells, but rather interacted with

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and activated bone marrow–derived antibodyforming cell precursors. He had also recently been elected a fellow of the Royal Society, ahead of the other two scientiﬁc giants at WEHI, Gus Nossal and Don Metcalf. Jacques Miller’s unit studied the function of thymus-derived cells, later renamed T cells by Ivan Roitt et al. (12, 13), whereas Gus Nossal’s unit, where I worked, studied antibody formation from B cells, bursa-equivalent or bone marrow–derived lymphocytes. Growing up in Australia inevitably engendered the love of playing sport. There were no tennis courts in the vicinity of WEHI, but there were squash courts buried in the bowels of the Royal Melbourne Hospital. I played regularly with Tony Basten, a postdoc in Jacques’s unit, and so over sweat and drinks we evolved a collaboration to try to recreate in tissue culture the T-B interactions that Miller and Mitchell had reported in irradiated mice. Tony provided a series of irradiated mice repopulated with thymus cells only, a source of relatively pure T cells (9), and I put them in culture with a variety of other populations, usually adult thymectomized bone marrow– grafted mice (14) that Tony had also provided, where B (but not T) lymphocyte repopulation takes place. To study the process in more detail, I developed a variant of the MarbrookDiener culture system that I had been using to study a variety of immunological processes in vitro, including immunological tolerance. This is illustrated in Figure 2 (15). It permitted separating the two cell populations to assess whether direct cell contact or cell-free mediators were sufﬁcient. The results we obtained (16) were published back-to-back with Anneliese Schimpl/Eberhard Wecker’s (17) results generated in the alternative Mishell-Dutton culture system. Other scientiﬁc interests that I have pursued subsequently were nurtured at WEHI. Ken Shortman was the head of the Biochemistry Unit, but his main focus was cell separation: how to purify cell subsets. Using these techniques enabled me to study lymphocyte-macrophage interactions, the

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process now known as antigen presentation, an acknowledged vital key step in the generation of immune responses. Macrophages are very adherent, so they can be enriched by adherence, and lymphocytes, relatively nonadherent, can also be enriched through this technique. But a more physiological approach was to use recirculating cell populations in vivo enriched in lymphocytes, and access to these cell populations is possible by thoracic duct drainage. John Sprent, a fellow PhD student in Jacques’s unit, was working extremely long hours collecting such cells after the tricky surgery to cannulate the duct and prevent the tiny tube from blocking up. He generously provided these cells, and we showed that thoracic duct cells alone were not able to respond to a particulate antigen, sheep red cells in vitro, unless supplemented by adherent macrophages (18). Vanda Lennon was also my contemporary at WEHI, a PhD student in the Clinical Research Unit headed by Ian Mackay. She studied experimental allergic encephalomyelitis, assisted by Patrick Carnegie. In these animals, we generated autoantibodies to MBP and devised a project to use MBP-coated red sheep cells to detect where the antibody-forming cells to MBP were present in animals. We duly found them in the brain (19). In hindsight, these research interests nurtured at WEHI were recombined in 1983 to help me conceive of a new hypothesis linking upregulated antigen presentation and autoimmunity, triggered by the immunohistological data of Franco Bottazzo (20) and others [e.g., Klareskog & Wigzell (21)] that there was augmented HLA class II expression in autoimmune disease tissues, such as the thyroid in Graves’ disease, or rheumatoid joints. By the time Gus Nossal returned from his sabbatical in Paris, where he had gone to examine reports by Alain Bussard (22) that peritoneal cells could make multifunctional antibody against red cells, I was generating a lot of in vitro culture data. Some months later, when Erwin Diener left to head a new immunology department in Edmonton, Canada, Gus de-

T cells Upper compartment Lower compartment

Nuclepore membrane

B cells Dialysis membrane

M E DI U M

Figure 2 Double-chamber cultures, formed by concentric glass tubes, suspended in a reservoir of medium. T cells were placed in the upper compartment and B cell–containing populations in the lower compartment. Adapted from Reference 15.

cided that I could keep the two technicians in Erwin’s charge, which enabled me to carry on with a wider range of projects and not confront the classic PhD student’s dilemma of too many ideas for the time and limited resources. Gus’s decision had some interesting implications, and it was not popular with other staff. Still a PhD student, I was heading a little group working in immune tissue culture. In the methodological aspects, Alan Harris, who had trained with Renato Dulbecco in culturing tumor cells at the Salk Institute, was very helpful, querying the methods we used with spleen cells, compared with his own work with cancer cells. Gus offered me new students, and I had the opportunity to initiate John Schrader into the intricacies of immune cell culture. It was a challenge, from which both John and I escaped unscathed (23). Hermann Wagner was the ﬁrst of a group of talented young German medical scientists who came to WEHI to train, followed by Martin Rollinghof and Harold von Boehmer. Hermann had worked in complement and was intrigued www.annualreviews.org • Effective Therapy for Autoimmunity

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by immune cell killing; he wanted to develop an in vitro system for generating cytotoxic T cells (CTL). We set about doing this using the Marbrook-Diener culture system; the experiments succeeded and resulted in an in vitro generation of CTL. Hermann developed this research path enthusiastically over the ensuing years (24). Wunderlich and Canty at NIH had previously generated similar results using the Mishell-Dutton system (25). Taking part in the collaborative atmosphere at WEHI was an incredible learning experience. Gus had a saying that I paraphrase: “Not publishing your data is a luxury few can afford.” I took that to heart and suspect few PhD students have published more from their thesis time than I did, owing to the multiple collaborations evaluating immune responses in vitro. Many of these papers were written late at night, fuelled by coffee and the music of the Rolling Stones.

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MOVING TO LONDON Gus was a friend and contemporary of most senior immunologists, so contemplating a postdoctoral position in the laboratory of another famous scientist was a realistic possibility for me. High on my list were Gerald Edelman’s lab in New York and Avrion Mitchison’s lab in London. Because of funding problems in Australia (regular occurrences in all laboratories), Gus decided to delegate to me attendance at a small, intimate immunology conference at Brook Lodge, a retreat in Kalamazoo, Michigan, owned by the Upjohn Company and used for conferences. I was very privileged to be able to take part; so many luminaries whose papers I had read, such as Baruj Benacerraf, James Gowans, Fritz Bach, and Mel Cohn, were present. On the way, I visited Avrion (Av) Mitchison and Gerald Edelman and compared which lab might be more suitable for me. Av was a most charming host, inviting me to stay in his house and give a talk to his colleagues at the National Institute of Medical Research (NIMR) at Mill Hill, where there was a wonderful intellectual and friendly 6

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atmosphere (e.g., 26, 27). So I decided to join Av’s group, but not at NIMR, but rather at the new Imperial Cancer Research Fund (ICRF) Immunology Unit he was starting at University College. Talented young colleagues there from the beginning, Marty Raff, Mel Greaves, and Nancy Hogg, were soon joined by Peter Beverley, Reg Gorczynski, Robert Tigelar, and Geoff Shellam, with Liz Simpson a frequent visitor. Subsequently, Mike Owen, Benny Chain, and Mary Collins joined us. All have gone on to major scientiﬁc careers and contributions. It was an exciting place to be, rich in intellectual resources and modern scientiﬁc equipment, with Av’s friendship with the Herzenbergs securing him one of the very ﬁrst ﬂuorescence-activated cell sorters (FACS) (28). Scientiﬁc visitors abounded, to give seminars in the crowded, small seminar room in the Department of Zoology, with Av, lying back in the front row with his feet up, eyes almost closed, as they gave their seminar, but very much awake, as question time revealed. Memorable was a young Peter Doherty coming to tell the world of his very surprising ﬁndings with Rolf Zinkernagel (29) of the genetic restrictions in CTL activation and of the various interpretations they were exploring. Moving to London had amazing advantages. No longer was there the tyranny of distance and isolation that have so preoccupied legions of Australian scientists to this day, and the United States, where almost half of science was being performed, was now only between 7 and 10 hours away and could be visited for a few days. The exhausting 24-hour trips from Melbourne to the scientiﬁc centers of Europe or the United States were no longer necessary. For example, there was no need to choose which of two major conferences to attend: Travel time and exhaustion level were no longer deciding factors. I was invited to many conferences, and I went to a lot. There was more money for research than in Australia, and more scientists were available for the skilled, laborintensive immunology research. Added to these career beneﬁts were the numerous cultural attractions of London. Av readily organized an

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appointment for me to the research staff of ICRF, and my planned return to Australia after a two-year fellowship was postponed, indeﬁnitely as it turned out. I did make trips back with my family so that my children could visit their grandparents. On one of these trips, I bought my ﬁrst souvenir of tribal art from Australia, an aboriginal bark painting, from a Melbourne dealer. A love of tribal art, encountered ﬁrst in Fiji on the way to London, has remained with me. My ofﬁce is now crowded with African and New Guinea masks and sculptures. I believe (an untested hypothesis) that being surrounded by original creations fosters creativity!

THE EARLY MAGICAL DAYS OF CYTOKINES In the early 1970s, I developed a multichambered culture ﬂask to study whether cell contact was necessary, and this generated a keen interest in intercellular mediators (15). The technology back then for identifying these mediators was far inferior to today’s. And so although important biological activities were present in supernatants and were given names to reﬂect that [e.g., osteoclast-activating factor, macrophageactivating factor, T cell growth factor, B cell– stimulating factor (30–33)], their molecular identity remained a mystery. An approximate molecular weight was as far as the effort got, as the potency of cytokines meant that there was very little protein. The number of potential mediators described was growing fast, and to try to make sense of this, Joe Oppenheim and others initiated the ﬁrst of a series of conferences that grew into the Cytokine Conferences. The ﬁrst was near NIH in 1977, and the second was at Ermatigen, Switzerland, in 1979. The ﬁrst conference focused on clarifying the problems of the ﬁeld; by the second, attendees suggested that some bioactivities might coexist within the same or related molecular species. We agreed upon the nomenclature interleukin, with IL-1 potentially encompassing lymphocyte-activating factor, osteoclastactivating factor, and endogenous pyrogen, all

based chieﬂy on similar molecular weight and origin, and IL-2 being T cell growth factor. A consensus paper was published from this conference chieﬂy reﬂecting input from Kendall Smith and Joe Oppenheim (34–37). But the real turning point in this ﬁeld came with the use of new technology, driven by perceived clinical need. Molecular biology techniques had invaded immunology in the mid- to late 1970s and had instigated real progress, such as cloning of antibody genes (38) and clarifying the generation of antibody diversity. Interferons (anti-viral mediators) were considered to be potential cancer cures (39), and so a lot of work was emerging in the late 1970s to scale up their production. By 1979 (40), Tada Taniguchi had cloned the ﬁrst type I interferon (IFN) cDNA, closely followed by David Goeddel and Sidney Pestka (41) and Shigekazu Nagata and Charles Weissman (42). Cloning of interferon was closely followed by the cloning of IFN-γ and other important interleukins, IL1 (43), IL-2 (44), IL-4 (45), IL-6, etc., in the 1980s. By 1984, the cloning of tumor necrosis factor (TNF) and lymphotoxin were reported, ﬁrst presented at a Cytokine Conference at Schloss Elmau by David Goeddel. By this time, I had started collaborating with Ravinder (known usually as Tiny) Maini (see Figure 3) on the role of cytokines in the pathogenesis of rheumatoid arthritis (RA). The properties of pure TNF described by Goeddel (46) were highly suggestive of those relevant to RA. The molecular biologists were providing new tools for elucidating the properties and function of cytokines, and, with that, many aspects of pathology and medicine were to change dramatically.

CYTOKINES AND UNCOVERING MOLECULAR CLUES TO AUTOIMMUNITY Science progresses by testing new ideas or hypotheses. In the early 1980s, there was increasing realization that in various autoimmune disease sites, there was upregulation of major histocompatibility complex (MHC) www.annualreviews.org • Effective Therapy for Autoimmunity

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Key collaborators

Ravinder (Tiny) Maini

Fionula Brennan

Mike Shepard

Jim Woody

Figure 3 Important collaborators throughout my career: Ravinder Maini, Fionula Brennan, Mike Shepard, Jim Woody.

expression, especially of MHC class II. This was found in rheumatoid synovium by Klareskog and Wigzell (21) in Sweden and Janossy (47) in London, and in endocrine autoimmune tissue, thyroid, and pancreas by Franco Bottazzo and Ricardo Pujol-Borrell (20). Franco came to see me to discuss whether this upregulated class II expression had any immunological meaning. To someone like me, having worked for many years on mechanisms of T cell activation, including antigen presentation, the answer was obvious. 8

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But it seemed a bit too obvious. Could upregulation of antigen presentation, induced presumably by environmental events, be sufﬁcient to trigger autoimmunity in genetically susceptible individuals? The latter point is critical because of the importance of genetics, especially MHC, in regulation of the immune response, as Hugh McDevitt (48) ﬁrst showed. Experiments in both mice and humans had demonstrated that autoantigen-reactive T cells were present in nondiseased individuals. How might upregulated MHC be connected to autoimmunity? There seemed to be a clear scenario, based on Steeg and Oppenheim’s (49) ﬁnding that IFN-γ upregulated MHC class II expression. The pathway might run as follows: Local tissue infection, perhaps viral, or other local damage would release cytokines and autoantigens, activating local cells to augment their MHC class II and antigenpresenting function. The cytokines and autoantigens would then be able to activate nontolerant autoantigen-reactive T cells, which in turn would activate effector cells, B cells to generate autoantibody, and macrophages to produce cytokines and other mediators, together causing more tissue damage, cytokine release, and so the vicious cycle of an ongoing disease. With the possibility of abnormal suppressor or regulatory T cells, I could thus envision the pathogenesis of a chronic disease. I deﬁned this scenario in early 1983, while staying with my family in a holiday home we had just bought in Begur, on the Costa Brava. While there, I had the time and freedom to think critically and write this hypothesis. With coauthors who had generated the relevant data, it was published in the Lancet as an untested hypothesis, a format that now seems very antiquated (50). When was the last major concept published without any supporting data? Of course, that would raise the possibility that a rival group could scoop yours by generating the experimental evidence. Nevertheless, 25 years later, this hypothesis is still a reasonable approximation, and rereading it is not at all embarrassing.

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Testing this new concept experimentally was exciting. The necessary techniques were already available: Cell culture methods had progressed rapidly, and the understanding based on Kendall Smith’s work on T cell growth factor (35) and on antigen presentation permitted rapid evaluation of the new hypothesis. Very important in testing this hypothesis were two young colleagues, Jonathan Lamb (now a professor in Edinburgh, then a postdoc who had greatly improved human T cell cloning techniques while in Jim Woody’s lab) (51) and Marco Londei (an enthusiastic and bright medical graduate new to the lab but keen to make his mark in medical research). We passed the ﬁrst test swiftly: adherent thyroid cells, a population including many epithelial cells and some antigen-presenting cells, were able to restimulate, after inﬂuenza peptide incubation, MHCcompatible inﬂuenza-speciﬁc T cell clones (52). Other tests took a little longer. Cloning T cells from Graves’ disease samples was a challenge that Marco relished; he cultured the lymphocytic inﬁltrate ﬁrst with IL-2 to select for in vivo–activated T cells and then cloned them (53). Seeking cells that were restimulated by autologous adherent thyroid cells but not allogeneic thyroid epithelial cells was accomplished, and he obtained wonderful pictures of T cells stretched and adherent to epithelium, as well as more quantitative proliferative data. Subsequent work in collaboration with thyroid experts Basil Rapoport and Sandy McLachlan (54) and postdocs Sonia Quaratino and Colin Dayan identiﬁed the diversity of autoantigens recognized, thyroid-stimulating hormone receptor, thyroglobulin, and, most often, thyroid peroxidase (55). We analyzed the cytokines able to upregulate epithelial cell MHC expression and showed that IFN-γ and TNF were both important, varying with cell type. This work was driven by Ricardo Pujol-Borrell and my PhD student Ian Todd (56). So the cellular basis, the outline of the hypothesis, had been rapidly tested and substantiated between 1983 to 1986. Scientiﬁc interest in this concept was high; transgenesis was becoming an effective research tool, and so trans-

genic mice overproducing IFN-γ in the islet cells of the pancreas, driven by the insulin promoter, were generated by Nora Sarvetnick at Genentech, and these mice duly developed autoimmune diabetes (57). But of course many questions remained unsolved. Were the epithelial cells really the antigen-presenting cells initiating disease? This went against the dogma. Did the epithelial cells have a role in disease maintenance or in recruiting immune cells? To evaluate the medical signiﬁcance properly, we needed to identify the intercellular mediators involved, cytokines or others. But this was not possible with the operative samples of thyroid that could be obtained after the disease was quiescent enough to permit safe surgery. Furthermore, thyroid diseases have never been seen as major unmet needs because their treatments, while imperfect, have been good enough for a long time.

WONDERFUL COLLABORATIONS AND FRIENDS The ethos of effective collaboration—the pooling of diverse skills and resources to permit a more effective attack on a major challenge— had pervaded WEHI. Having learned the power of effective collaboration, I joined forces with Franco Bottazzo on my ﬁrst serious venture into autoimmunity, which made considerable progress. But personality differences limited this joint venture. I sought a more important autoimmune disease, like thyroid with a local site of disease that could be immunologically studied, and RA was an obvious choice. Nathan Zvaiﬂer, a leading U.S. rheumatologist, had come in the late 1970s to do a sabbatical with Av Mitchison, and I had the good fortune to be charged with looking after him. I suspect we both learned a lot from each other: Nat gained some insights into the ever increasing complexity of immunology, and I discovered that RA was a major immunological disease with many aspects not yet understood, which made it an important unmet need. When I subsequently rang to ask him who in London was www.annualreviews.org • Effective Therapy for Autoimmunity

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the best person to work with in this ﬁeld, he unhesitatingly said Ravinder (Tiny) Maini at the Kennedy Institute of Rheumatology (KIR), whom I duly rang. His enthusiasm for a potential new collaboration was evident, and he was in my ofﬁce accompanied by Lindsay, his laboratory technician, within two days. That was the start of a truly wonderful collaboration and friendship, which has transformed our careers and enabled the very difﬁcult task of translating laboratory science into effective therapy. It began with a detour, however. Systemic lupus erythematosus (SLE) was the major disease being studied by Maini’s group at the time, and so I explored whether the techniques used in Graves’ disease might be useful in studying SLE. With Tiny’s encouragement, I rapidly got involved in arthritis research, successfully applied for an Arthritis Research Campaign grant, and then was invited to see the new research center being built on the site of the Charing Cross Hospital, which had plenty of lab space compared with my base at the ICRF Tumor Immunology Unit at University College. Professors from Charing Cross and Westminster Medical School had gotten together to raise support for this new center, and Mary Glen-Haig, then chief administrator of the hospital, had found donors. Her friend Sir William Shapland, chief executive of the building ﬁrm Bernard Sunley & Sons, was the chairman of the Sunley Trust and of the group planning this research development, the Charing Cross Sunley Research Center. Because my work on autoimmunity was progressing well and because, being an optimist, I could envisage that it would eventually be tested in patients, I no longer felt it was appropriate to be personally supported by a cancer research organization, even if it was very rich and broad-minded, as the ICRF led by Walter Bodmer and Mike Crumpton was. Moving to an empty building (even if only four to ﬁve miles away and with only a few key staff ) to build up a team is traumatic and difﬁcult. One always underestimates the ﬁnancial and equipment needs. We had ambitious plans to discover the key molecular mediators in active RA synovium and develop new therapies

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aimed at interfering with them. I do not know the percentage of teams with such ambitious therapeutic goals that actually succeed, but it is certainly not high. We had certain key assets, including two leaders, one at the laboratory end and the other at the clinical end (Tiny), but we also had an appreciable overlap of understanding. Another asset was excellent team spirit, facilitated by the involvement of several fellows and students who had previously worked with one or the other of us. Working in the rapidly developing cytokine ﬁeld was enthralling, but its clinical importance had yet to be established or understood. It was a wonderful challenge, which was chieﬂy supported ﬁnancially by a variety of research charities. The Arthritis Research Campaign was the major one; it has had a large, long-term investment in KIR since its beginning in the 1960s and in my work at the Sunley Research Center from 1985, before I joined KIR (the Sunley Research Center became incorporated into the Kennedy Institute in 1992). This long-term funding made such risky research much more possible than funding on threeyear grants that The Wellcome Trust, Nufﬁeld Foundation, and Sunley Trust all contributed. Most importantly for the long-term challenge of this work was that Tiny and I were good friends. It is not clear that we would have worked so closely and effectively for over 20 years, overcoming various problems, had we not had the trust in each other that friendship brings. Joining me initially at the Sunley from University College were several key postdocs, including Marco Londei, whose work on Graves’ disease I mentioned above, and the late Glenn Buchan, from Otago, New Zealand. Glenn had begun successfully to use molecular biological techniques to study cytokine and cytokine receptor expression in synovium and was involved in reﬁning them to permit use with small human diseased tissue samples (58, 59). Regrettably, he died early in 2008 from cancer. Very important for the extensive grant writing that was needed was that my secretarial assistant Philippa Wells also decided to move with me.

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In retrospect, certain experimental choices were vital for understanding the role of cytokines in RA. First, we needed to focus on which mediators were actively synthesized at the site of disease, using mRNA analysis through the cloning of cytokine genes. Second, for studying regulation of cytokine production in rheumatoid synovium, we did not use the classical techniques of culturing and passaging rheumatoid synovial cells, a complex mixture of cells, until only adherent synovial ﬁbroblastlike cells are left. This technique made no sense to me, as 80–90% of the initial synovium, the hemopoietic-derived immune cells, were thus discarded, and, of course, after several passages, the environment of the remaining cells was very distinct from the initial environment (60). Hence, we used short-term cultures of the entire cell mixture in synovium (3–7 days maximum) to study synovial cytokine regulation. These experiments were carried out chieﬂy by postdoc Fionula Brennan (58, 61), now a professor at KIR. From her work emerged the ﬁrst evidence that TNF might be a therapeutic target. She demonstrated that in rheumatoid but not osteoarthritic mixed synovial cell cultures, anti-TNF antibodies dramatically reduced the production of IL-1 (61). Subsequently, this was extended to anti-TNF downregulating a range of other proinﬂammatory cytokines, GM-CSF, IL-6, and IL-8, which was all encouraging news (62–64). But it was worrying that anti-TNF also reduced the anti-inﬂammatory mediators, such as IL-10, IL-1 receptor antagonist, and soluble TNF receptors. This work is summarized by the TNF-dependent cytokine network concept, illustrated in Figure 4, which has proved a useful approximation to the truth. Richard Williams tested in animal models the hypothesis that TNF was a therapeutic target, using the collagen-induced arthritis model that he had already established in Tiny’s group (65). All animal models are imperfect; this one was less imperfect than others and was very useful in demonstrating the need for high concentrations of antibody for maximal efﬁcacy. It also showed clearly, by immunohistology, that leukocyte inﬁltration was markedly

Anti-inflammatory IL-10, IL-1ra, sTNF-R Immune system

TNF-α

IL-1 IL-6, IL-8, GM-CSF, etc. Pro-inflammatory

Figure 4 TNF-dependent cytokine cascade in rheumatoid arthritis (RA). This was an important component of the scientiﬁc rationale for anti-TNF therapy in RA.

reduced, and it demonstrated joint protection of both cartilage and bone. This joint protection was known in 1991 in the mouse but was not veriﬁed in humans with RA until 1999. In this work, we were greatly assisted by Robert Schreiber, who donated his anti-mouse TNF monoclonal antibody, unique at the time. He generously provided this hamster antibody in large amounts, without which we could not have done the work. It is of interest that coming from other vantage points, two other groups, of Thorbecke (66) and Piguet (67), concurrently demonstrated the beneﬁt of TNF blockade in mouse models of RA. Tiny Maini had started his research career at KIR with Dudley Dumonde. He had been involved in studying lymphocyte mitogenic factors in the late 1960s and had coined the term lymphokines (68) well before there was technology for identifying such rare molecules. So he was very aware of the importance and potency of such mediators, and exploring the role of cytokines in RA was thus for him also a natural progression. While I encouraged my colleagues to improve the sensitivity of techniques needed to quantitate cytokines, Tiny had the unenviable task of masterminding the collection of abundant samples of rheumatoid synovium needed and characterizing their clinical phenotype and biological marker proﬁle. To this day, I am puzzled that it is so difﬁcult to get the majority of surgeons to take the extra 1–2 minutes to ensure that the correct tissue www.annualreviews.org • Effective Therapy for Autoimmunity

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is collected in a sterile manner into the appropriate bottle of medium. The rare exceptions appear to be surgeons who understand the research process and the dependence of medical progress, indeed of all progress, on research. The cytokine ﬁeld was revolutionized by the molecular biology skills of the biotech industry. Scientists at a number of companies, e.g., David Goeddel, Pat Gray, and Axel Ullrich at Genentech and Craig Smith at Immunex, cloned cytokine genes, expressed the proteins, and generated antibodies. I made contacts with these companies, and they donated antibodies and cytokine reagents without the awful material transfer agreements that plague collaborative research today. Particularly important were antibodies to TNF, produced at Genentech, which had ﬁrst cloned it. At Genentech the extramural program manager then was Michael Shepard, who took a great interest in our work and helped us considerably to unravel the role of TNF in RA. He supplied all the cytokines, cDNAs, and antibodies to TNF and to LTα/TNF-β. After a few years, he returned to his own cancer research career, which was very productive, and he has found fame as the initiator, scientiﬁc champion, program manager, and developer of the anti-HER-2 antibody, trastuzumab, better known as Herceptin®, which has saved many thousands of lives of patients with breast cancer (69, 70). A personal reminiscence: When David Goeddel ﬁrst presented his group’s work on cloned TNF in 1984 at Schloss Elmau, I went to talk to him and was told that as the TNF project was a collaboration with a European company only they could supply European labs with TNF reagents. However, the European company was worried that my hypothesis that cytokines such as TNF might be involved in pathogenesis of disease would negatively inﬂuence the development of what they had wanted, which was a cytokine cancer cure. So they did not want to help me, but fortunately the Genentech research-driven culture did. James Woody, a U.S. Navy medically trained researcher, was a prot´eg´ee of Ken Sell who had beneﬁted from getting his PhD in the UK in

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three years, much quicker than was possible in the United States. Jim was rising rapidly in the U.S. Navy Medical Research Command, and Ken Sell, his chief, sent him to Av Mitchison in London to obtain his PhD. Somehow he ended up under my supervision. And being a very bright, diligent, well-organized scientist, he duly ﬁnished in the minimum time, often bringing his children to the lab at weekends in order to do so. He returned to Bethesda, jumping from being a PhD student to running a big laboratory for the U.S. Navy. From there, his career progressed in leaps and bounds, emulating his mentor Ken Sell to reach the top of the U.S. Navy Medical Research. While he was at the Navy, we kept in close touch, and he knew of our burgeoning work on TNF. The U.S. Navy funded some of this research. By the time Jim had ﬁnished his 20 years in the Navy, in 1991, and was considering pharmaceutical and biotech opportunities, he was aware that we were close to deﬁning TNF-α as a therapeutic target in RA. So it was very pleasing that he opted to join an emerging biotech company, Centocor, a pioneer in developing the monoclonal antibody ﬁeld. John Ghrayeb and his team at Centocor had grafted the human constant region to antibody variable genes from a murine anti-TNF hybridoma generated in Jan Vilcek’s laboratory (71), in response to Tony Cerami’s powerful arguments that blocking TNF-α might save thousands from death in sepsis (72). So in early 1991, before Jim had ofﬁcially started, I visited Centocor and presented our work leading up to the deﬁnition of TNF-α as a therapeutic target in RA. It received a warm reception, especially from Hubert Schoemaker, the chairman/CEO. Some of the company scientists were more skeptical, especially their only rheumatologist who somehow knew that anti-CD4 antibody would be much more effective for RA therapy than blocking TNF. Centocor was focused on sepsis, and in Europe, their IgM monoclonal antibody to LPS had been approved, on scant data. In the United States, it had not yet been approved, and so an interesting deal was set up, basically that I (and my colleagues) would help them to deﬁne

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the mechanism of action of their LPS antibody and how it protected despite its low afﬁnity, and they would help us with testing our TNF therapeutic target. Our academic-led project was not a normal clinical program. Jim Woody as chief scientist ran it instead of the clinical group, with the help of a number of Centocor staff, including Hanny Bijl, Dick McCluskey, Carrie Wagner, and Tom Schaible. The chimeric monoclonal antibody cA2 had already been administered in high dose to several dozen patients with sepsis. It failed to correct the septic shock, but importantly they did not get worse. This reassured us that its use in RA trials would not lead to overwhelming infections.

THE EXCITEMENT OF CLINICAL TRIALS The ﬁrst trial was an open study with no placebo controls, nonblinded owing to the unknown risks of blocking TNF in rheumatoid patients. Ten patients were initially planned to be treated with the high dose found necessary in mice. When the striking results of the ﬁrst patients were disclosed to the company, Centocor did not know exactly what to do, so it asked us to treat 10 more. Of course with 20 patients responding, it was easier to draw conclusions and publish than with only 10 (73). The results had matched or even exceeded our expectations. Over the slow (3 h) infusion of the antibody, many of the patients commented that they were already feeling better, less tired. Over the next day or two, reductions in stiffness and pain were noted. Large effusions in knee joints rapidly diminished. There had been concerns that blocking TNF, a host defense molecule, might promote infection, and so we had taken the precaution of starting the infusions slowly, with just one patient ﬁrst, treating them as inpatients, and we had our own nurse spend the night in their room in order to treat possible problems as rapidly and effectively as possible. It was a very thrilling time. All the patients we treated improved dramatically, despite having had long-standing active RA refractory

to current treatment. The ﬁrst two to three months were especially interesting because we did not know how long the beneﬁt would last. Patients returned to their normal activities, holidayed, played golf, etc., and were really happy. They thought the improvement might be long lasting. But it was not to be. There were 12 to 18 weeks of marked beneﬁt before relapse. There were no cures, but nevertheless there had been major improvement and a clear pointer for the future. I helped coorganize a conference in Arad, Israel, near the Dead Sea, with my friend David Naor. It was there, in midSeptember 1992, that Maini ﬁrst presented the dramatic results of the ﬁrst clinical trial. There were scientists from other companies, from Immunex, Genentech, Roche, etc., and the disclosure, probably premature owing to our naivet´e, started the race toward the clinic, as these companies had already produced TNF inhibitors for use in sepsis, based on Tony Cerami’s work (74). To establish what might happen with longerterm anti-TNF treatment, we sought ethical permission to retreat some of the patients from this ﬁrst trial after they had relapsed. Eight of 20 were retreated, up to three times. In each case, there was reintroduction of signiﬁcant clinical and biochemical beneﬁt (e.g., reduction in Creactive protein), suggesting that if TNF was blocked, other cytokines did not rapidly take over to drive the cytokine network (75). But this initial experiment was not a formal proof. There had been no controls or randomization, much needed in clinical trials with potentially high placebo responses. To do that, a formal double-blind (patients and clinicians), randomized, placebo-controlled trial was performed. Three European rheumatology friends, Joachim Kalden of Erlangen, Ferdinand Breedveld of Leiden, and Josef Smolen of Vienna, joined in with Tiny and me. There were issues to resolve, such as what to use as placebo. We chose human albumin to avoid immunizing patients to mouse antibodies, and the primary end point was limited to four weeks for ethical considerations and to reduce drop-outs in the placebo-controlled trial. Again, the results were www.annualreviews.org • Effective Therapy for Autoimmunity

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very clear and convincing. Both the high dose used previously and a tenth of the dose worked well, but the placebo infusion did not (76). We collected large samples—400 ml of blood from these patients—to do a very detailed analysis of the post-treatment events. The mechanism-ofaction studies were very informative, most importantly because they conﬁrmed that there was a very rapid diminution in other proinﬂammatory cytokines, for example IL-6. The reduction to baseline of a downstream cytokine in a few hours is evidence of a direct effect of anti-TNF (76, 77). The mechanism-of-action studies were performed in considerable detail, possible because it was an academic-led study, with the blood samples under our control. Few other clinical programs to date have been analyzed in such detail. We also looked at cellular changes in the blood: They were less informative than we had hoped but showed a rapid reduction in circulating neutrophils and monocytes. More interesting was a rapid increase in lymphocyte counts, which tend to be low in active RA, with more activated cells in the blood. The rapidity of the change suggested that there was a change of trafﬁcking, probably an exit of T lymphocytes from the joints. We were able to explore the hematology in more detail, and important pathogenic clues emerged. There was an increase in the hemoglobin concentration (usually low) in the patients within the four weeks of the trial. The high platelet counts in RA and high ﬁbrinogen, both potentially linked to accelerated atherosclerosis, tended to normalize (78). This was a clue that the abnormal cardiovascular outcomes in RA might be improved, but it took a long time for other groups to establish this, in large post-registration registries in the UK that Alan Silman, David Isenberg, and their colleagues have ably run (79, 80), as well as those in Sweden (81, 82) and other countries. Cell inﬁltration in the joints is a hallmark of chronic RA. The reduction in joint swelling suggested that fewer cells persisted after therapy. Biopsy studies clearly showed that to be the case, with reductions in lymphocyte and

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macrophage numbers and a thinner lining layer. How did this occur? Attempts to show increased apoptosis were not successful, but we did ﬁnd reductions in markers of cell recruitment. Thus, endothelial-speciﬁc E-selectin was reduced, both as detected in synovium by immunohistology and in serial serum samples as soluble form. Also reduced were intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, both involved in cell recruitment to tissues. The more quantitative serum soluble adhesion molecule assays were consistent with the less quantiﬁable histologic reductions. In a similar way, we also found that numerous chemokines are reduced in the synovium as well as in the blood (83, 84). Markers of tissue destruction were also diminished. Serum matrix metalloproteinase precursors are elevated in active RA and were reduced after anti-TNF therapy (85). Of course, serum assays fail to demonstrate what is active in the joint, but they do reﬂect the biosynthesis during active disease. Rheumatoid joints are very cellular and have often been described as resembling tumors. To sustain this augmented mass, new vessels are needed, and so angiogenesis is readily apparent. Ewa Paleolog and colleagues observed that angiogenic factors are also augmented in RA, and it was of great interest that the most potent of these, vascular endothelial growth factor (VEGF) (86), was rapidly but partly diminished after anti-TNF therapy. However, it took a lot of subsequent immunohistological analysis by Peter Taylor and colleagues to demonstrate a reduction in blood vessels (87). It was remarkable how much molecular work could be performed from one small clinical trial: We obtained many of longitudinal samples but still had many unanswered questions. The results all pointed toward normalization of the pathological processes, and while they did not show that TNF causes arthritis, they showed that TNF is a very important driver of active disease. In the mouse, the elegant work of Kollias and his colleagues has shown that transgenic mice overexpressing TNF does cause an erosive polyarthritis, even in mice lacking T and

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B cells (88, 89). We had learned much about disease mechanisms from the detailed work on the ﬁrst placebo-controlled trial. But there was a subsequent disaster. The freezer holding most of the samples from the next trial, a longer phase II, over six months, defrosted. The consternation, angst, frustration was awful; it was physically painful to think of the major scientiﬁc opportunities lost. In the phase III trial, Centocor controlled the samples to speed up the trial process and the hoped-for drug approval; hence, far fewer samples were collected, and the opportunity of investigating biological markers in greater detail was lost. By 1993, there was clear evidence of clinical beneﬁt, even in the most disease-active patients, but no cures. Retreatment was successful, but we were concerned about immunogenicity of this antibody, which, while chimerized (2 faces, 3/4 human constant region), still had the mouse variable regions that were likely to be immunogenic (71). Whether it was a drug that could be used for long-term therapy was unclear, so research was planned to learn how to augment the beneﬁt and reduce immunogenicity. As is the case for all major diseases (e.g., cancer, hypertension), combination treatment is necessary to optimize clinical beneﬁt. So we used the mouse model of collagen-induced arthritis to pilot potential approaches to augment beneﬁt. It was not difﬁcult to produce anti-TNF nonresponder mice. We needed to reduce the treatment dose to 50 μg/twice per week instead of the efﬁcacious 300 μg (65). In this model, using suboptimal doses of anti-TNF antibody, a range of additional T cell–directed therapies were tested, and cyclosporine, antiCD4, and CTLA4-Ig (90–92) were all effective, suggesting that there was enhancement of the clinical beneﬁt if T cell function was also reduced. In these experiments, there was synergy, as the effects of anti-CD4, cyclosporine, and CTLA4-Ig as monotherapy after disease onset were rather modest, if present at all (91). So from these animal model studies, especially the anti-CD4 experiment, the clinical trial design evolved in which patients with an inade-

quate response to methotrexate (MTX) were treated with various concentrations of antiTNF (by that time known as cA2, later inﬂiximab, later Remicade®), in order to augment their response. MTX is effective in a signiﬁcant proportion of rheumatoid patients, as demonstrated and championed by Michael Weinblatt in Boston (93, 94). In the 1990s, its impact was growing, and it was becoming recognized as the most effective disease-modifying antirheumatic drug. As it would not be possible to use two unlicensed drugs together (anti-CD4 is unlicensed), Tiny Maini and I chose to use MTX, which had been reported, among its legion of effects, to inhibit T cell function, promote apoptosis, and reduce IFN-γ production (95, 96), effects that resembled those of anti-CD4. Patients who had an inadequate response to MTX were abundant, and so our trial was designed to ﬁll an important clinical need. But an issue was the risk, especially of infection, in the combination. So a very low dose of MTX was chosen, 7.5 mg/week. However, at this time Centocor was struggling ﬁnancially, and the long-term clinical trial we had envisaged was shortened to 24 weeks, 12 weeks on therapy and 12 weeks further follow-up. Nevertheless, the results were very interesting and have been inﬂuential. Lowerdose cA2, 1 mg/kg at weeks 0, 2, 4, 8, and 12, was effective, with about 25–30% of patients showing 50% beneﬁt [using the American College of Rheumatology (ACR) 50 criteria], only up to week 4 if used alone. But with low-dose MTX, there was clear synergy, with 60–70% ACR 50 up to week 24 (97, 98). The results using higher doses of cA2, 3 mg/kg and 10 mg/kg, also showed the added beneﬁt of the addition of cA2 to MTX. It is now the combination most extensively used in routine practice, with about 70% of patients given the existing three anti-TNF drugs also being given MTX because of the increased efﬁcacy (99, 100). After Tiny had ﬁrst presented the exciting initial clinical results to Centocor management, Jim Woody made cA2 available to Sander Van Deventer, an enterprising gastroenterologist in Amsterdam, who successfully treated a Crohn’s disease www.annualreviews.org • Effective Therapy for Autoimmunity

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patient with ﬁstulas (101). Centocor then prioritized resources to Crohn’s clinical trials, to our dismay. This became the ﬁrst approved indication for cA2, now known as inﬂiximab or Remicade®. Despite its clinical priorities and cash limitations, Centocor agreed to fund an important imaging mechanism-of-action trial to investigate leukocyte trafﬁcking to joints before and after anti-TNF therapy. This was performed by Peter Taylor, who now ably leads the Kennedy clinical trials group, together with A.M. Peters, a leukocyte imaging expert at Hammersmith Hospital. This trial demonstrated that antiTNF reduces leukocyte trafﬁc to joints (84). This was an important clinical trial because reduced recruitment of inﬂammatory cells to disease sites probably accounts for the ability of anti-TNF to ameliorate so many diseases. With the success of the phase II trial in which MTX was supplemented with cA2, the multinational phase III was planned and eventually successfully executed with Peter Lipsky (a friend from the 1970s from his time as a postdoc at NIH with Alan Rosenthal) as the U.S. trial leader and Tiny as the European leader. The complexities and grind of phase III trials made this a nonexciting and stressful, though necessary, experience compared with the earlier trials. But through the whole process, working with Tiny was an enjoyable, educational experience, as we blended his rheumatological and other clinical skills with my immunology and cytokinology and entered ﬁelds new to us, where success rates had been dauntingly low.

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PROS AND CONS OF WORKING WITH INDUSTRY Academia and industry often do not mix well. Having studied medicine, I was interested in the practical application of immunological research. My industrial interactions started while I was still a postdoc. James Howard worked on immunity to polysaccharides (102), a ﬁeld analogous to one of my research topics, immunity to polymerized ﬂagellin (103). Both were repeated polymers that induced thymus-independent an16

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tibody responses. He invited me to consult with his group at Wellcome Research Labs, a small pharmaceutical company, known locally at the time as University of Beckenham for its academic bent. It had recruited Nobel Prize winners John Vane and James Black to run its research. This was a wonderful start to the pros. There was much to learn, and one was paid extra, which is very appreciated early in one’s career, with growing children and increasing bills to pay! My second exposure was with ICI Pharmaceuticals, the precursor of Zeneca, now AstraZeneca, when my PhD student Eric Culbert joined them: I have had a number of longterm consulting relationships, helping friends and colleagues. For example, I consulted with ed David Webb, ﬁrst at Syntex, then OSI, Syrrx, and now Celgene; Michael Moore; Jim Woody at Centocor, then Roche. I advised Michael Shepard, ﬁrst at Genentech, on the Herceptin project, then adenoviral gene therapy while he was at Canji, then targeted cancer therapy at Newbiotics, and now at Receptor Biologix. These long-term relationships were in many ways very educational. Thus, our work on adenoviral inhibitors for studying cytokine and other intracellular signaling pathways developed from an awareness of the utility of adenoviruses developed while helping Canji. But the work with Centocor was on quite a different scale, and despite its many frustrations at times, it was very beneﬁcial and helped drive anti-TNF therapy forward. Like all the best interactions, mutual beneﬁt is essential. In 1992, Centocor was a rapidly growing biotech company that thought it was going to be the one to capitalize on treating sepsis with monoclonal antibodies. It had an IgM (Centoxin) anti-LPS monoclonal antibody approved on limited data in Europe and was looking forward to new data and approval in the United States. When Jim Woody joined them as chief scientist, he was keen that we help them to ﬁll a gap, to understand how Centoxin mediated its beneﬁt (104). As this research would involve our ﬁeld of expertise, cytokines, it was logical. In return, it would be easier for him to encourage his

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colleagues to invest in and become interested in our untested and not yet accepted concept of TNF as the therapeutic target for RA. My colleague Peter Katsikis (105) duly found a novel mechanism by which complement may assist in endotoxin clearance, and Jim succeeded in getting Centocor to provide ﬁnancial and antibody resources to test anti-TNF therapy. The success of this interaction led to a promising novel therapy for a major unmet need and, in my opinion, eventually saved Centocor from extinction, as the endotoxin project failed and its share price crashed in 1992. Centocor had charismatic leaders Hubert Schoemaker and Michael Wall, and it tenaciously survived its problems with sepsis therapy and went on to be acquired for almost $5 billion by Johnson & Johnson in 1999. The early trials with Jim Woody at the helm and Harlan Weissman, a key player, were run harmoniously, as were the ﬁrst placebo-controlled trial and the six-month phase II trial. But then as the likelihood increased that clinical success would translate into commercial success, the management of the trials became focused on commercial speed of completion, and thus were run very differently. By this time, Jim Woody had left Centocor to become president of Roche in Palo Alto, California, and Hubert Schoemaker had been weakened by a brain tumor and its very aggressive therapy. We were left to manage the Centocor-KIR relationship with no internal champions. It was not congenial: Agreements and promises were reneged; stress ensued. Matters important to academics, such as publication and presentation rights, were challenged and arbitrarily overturned; issues of the nationality of presenters of key data were raised. It is almost unbelievable that it was felt in the late 1990s that an American was a more credible presenter of data than a European. Agreements about authorship were subsequently ignored by the company. Even worse is what happened after the drug was approved and started to sell and the other two TNF inhibitors also were sold. The respect scientists have for each other’s discoveries is often not shared by industry and its

lawyers. In Bob Dylan’s words, “money doesn’t talk, it swears.” I regret that anti-TNF is yet another of the British inventions that was not commercialized in Britain, but rather in the United States. This happened despite very extensive discussion in the late 1980s/early 1990s with the UK’s leading monoclonal antibody company. But this company missed the golden opportunity taken up by Centocor and its U.S. rivals, Immunex and Abbott, that has resulted in approximately $11 billion in sales of anti-TNFs in 2007. But overall, I am an enthusiastic supporter of working with the biotech and pharmaceutical industry. Many of the skills needed to get new treatments into the patient population are possessed by industry: medicinal chemistry, pharmacology, and especially the ﬁnancial resources for major clinical trials. These are complimentary with academia, and if these complimentary skills were harnessed more appropriately, society would undoubtedly beneﬁt. My actions with many ongoing industrial relationships probably speak louder than any words on this topic, and the practical outcome of the interaction between academia and industry is that there are now no more RA patients in wheelchairs.

PROMOTING TRANSLATIONAL RESEARCH What is translational research? There is no agreed deﬁnition, and that is part of the problem, but by conventional usage it is research designed to further human health: to bridge new discoveries in basic research and the applied research in clinical trials of therapeutic products. A major challenge in research has always been which experiment to perform, among the countless possibilities. Sir Peter Medawar has elegantly expounded on this, and “The Art of the Soluble” is not an exact science (106). How high to aim at any one time can result in major disappointment if the effort fails, but if it succeeds, wonderful gains can be achieved. So every scientist expresses their unique personality in their choice of projects and approach. I was acutely aware that despite the major www.annualreviews.org • Effective Therapy for Autoimmunity

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advances in cellular and molecular understanding of immunology, its key practical applications, vaccines, date back 200 years to the time before immunology was a major science. Why had there been so little progress, despite stellar advances such as the discovery of monoclonal antibodies by Kohler & Milstein (107)?

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Communication remains a problem in all complex organizations and societies, and scientiﬁc meetings have an important role in science. I ﬁrst became interested in organizing scientiﬁc meetings in 1983, wanting to promote the relatively new techniques of T cell cloning and their potential for helping unravel disease pathogenesis. This meeting took place in 1984 (108), after I had worked out how to raise funds for these small, intense, focused meetings. Critical to the venture was James Woody, when he was in the U.S. Navy, who convinced the Navy to support meetings. As he has progressed in his career, via Centocor and Roche, he has for many years been the major funder of many scientiﬁc meetings and made the meeting organization much less stressful for me. A key beneﬁt of organizing these meetings has been that my colleagues and I learned, made contacts and friends, and initiated important collaborations, with the help of discussions on the lawns and at the bar during these conferences, chieﬂy held at Trinity College, Oxford (109, 110). Many experiments were hatched at these meetings, which have been variously named T Cell Activation in Health and Disease; T Cells and Cytokines in Health and Disease; From Laboratory to the Clinic. I have held 20 of these meetings, coorganized with Andrew McMichael, a fellow at Trinity, again with the skilled enthusiasm of my long-term personal assistant, Philippa Wells, who has now capably helped me for 27 years, all ﬁnanced through friends and acquaintances in the biotech and pharmaceutical industries.

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But while translational research is now a hugely popular theme, from NIH with Dr. Zerhouni’s roadmap to the newly revamped Medical Research Council under Sir Leszek Borysiewicz, it is still very ill deﬁned, and many fail to understand its basic principles. Translation is the apex of the pyramid, an activity suitable for only a minority of projects. It depends ﬁrst on excellent quality of science, but second on science that reveals an important rate-limiting step in the complex biology that occurs in vivo. Not only that, the complex biology must be relevant to humans and their diseases, and not just to mice. I paraphrase the late Judah Folkman, who said in the late 1990s, when there was hype about blocking angiogenesis curing cancer: If you are a mouse with cancer, we can help you, but if you are human, it may take another 20 years. Humans differ from mice, most obviously in their longevity. With longevity there is a need for more robustly regulated biological systems for the many years before reproduction. So while mouse systems need to function for 10–12 weeks and are subject to Darwinian evolutionary selection pressure only for this period, for humans the selection process is more than 100 times longer, for over 20 years. We expect differences in complexity to emerge, so mice are not likely always to be an accurate model for human pathophysiology. It is thus puzzling and a continual challenge that many medical journals still fail to appropriately prioritize and encourage research performed with rare human disease material, often because all the controls cannot be performed as well as in mice. The latter is the current band wagon, as much progress is based on elegant genetically engineered experiments. But many failures in successful translation of laboratory research into disease relevance are likely due to technical issues such as the overuse of reductionist systems, interspecies differences, or the use of transformed cell lines, with their many mutations, as surrogates for normal human cells.

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Directing the Kennedy Institute of Rheumatology Directing KIR has been a major focus of my activity for the past six years since Tiny Maini retired as director. The KIR is the largest research institute dedicated to rheumatology and has been supported effectively by renewable long-term (ﬁve-year) major grants from the Arthritis Research Campaign in the UK covering 40–50% of the total budget and increasingly by the Kennedy Institute of Rheumatology Trust. The director’s role is to evolve strategy, recruit the best talent, provide them with the best resources, and let them get on with their research. Leading by example works better for most scientists than direction. KIR’s focus is translational research, from laboratory to clinic and back again, and it has a wide range of expertise from molecular science, proteomics, molecular modeling, etc., through cytokine biology, immunology, inﬂammation, matrix biology, and signaling to clinical research and trials. KIR has long been a global resource for research and training in rheumatology and related disciplines. My challenge is to leave it in an even better state than I found it when my friend Tiny transferred it to my care. It has been a privilege to work there long term with many talented colleagues, almost 25 years with Sir Ravinder Maini, 20 years with Fionula Brennan, almost that long with Brian Foxwell, and 10 years with the burgeoning osteoarthritis team leaders, Jeremy Saklatvala and Hideaki Nagase. The team spirit, mutual support, enthusiasm, and intellectual challenges make it a pleasure. Interacting with the Faculty of Medicine, Imperial College, which the KIR joined in 2000, opened up new avenues and access to many multidisciplinary colleagues in other branches of medicine, engineering, chemistry, etc. Taking part in the creation of the UK’s ﬁrst Academic Health Sciences Centre led by Stephen Smith, from the merger of Hammersmith, Charing Cross, and St. Mary’s hospitals, has been educational. The administrative issues at KIR, in a constantly changing scientiﬁc environment, are a challenge and are less

entertaining than the science, but the prospect of helping to deliver the fruits of research more effectively for our patients makes it worthwhile.

CONCLUSIONS Maintaining Life/Work Balance Science is fun, and should be fun. It is the ultimate experience in solving puzzles, puzzles that no one has previously solved, and you are even paid to solve them. If you are not able to enjoy the excitement and thrill of science, to enjoy the roller-coaster ride, and to shrug off the inevitable frustrations of failed experiments, malfunctioning equipment and colleagues, and rejected papers and grants, then a career in science will be more pain than pleasure and perhaps is not a wise choice for you. But with the fun and excitement comes the inevitable huge work load, and maintaining a life/work balance is a challenge that few can successfully manage. For those working in Europe, at least there is the hallowed tradition that long holidays are beneﬁcial, but my U.S. colleagues seem to take far fewer holidays. They spend more time in the lab, but does that add up to greater productivity? Having enjoyed outdoor activities and sports while growing up in Australia, I know long holidays provide not only an opportunity to enjoy family, friends, and the splendor of our planet, but also time to think creatively and strategically. Some of my best ideas emerged thousands of miles from the laboratory. A challenge for all scientists is to optimize their productivity; my warning is that more time in the lab might not be the best way. Eventually, we all learn that time is life’s most precious commodity.

Do We Value Practical Research Contributions? All of life is inﬂuenced by fad and fashion, and science is no exception. The term “blue sky research” (for pure basic research) clearly implies basic research’s desirability, whereas in contrast applied research implies sweat rather than

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inspiration. But is this really true? I could make an argument for the reverse. Thus, much basic research is not inspired, and, in the past quarter of a century, it has evolved from many projects following a preset pattern chieﬂy purifying a protein, to cloning the mRNA for a gene, to making transgenics and knockouts. Clearly, as techniques become better deﬁned, much diligence is necessary, but how much really new, creative, inspirational research takes place per project? I suspect that what really matters in research is its quality and imagination, and both are always needed for the best, pure, basic, blue sky, applied, or translational research to succeed at the highest level. My own experience is that society does indeed value research contributions of a practical or applied nature. If it is practical or applied, the effects of the research are easier to measure than they are with basic research. However, there appears to be greater delay before success in practical research is recognized, which is inevitably frustrating. The frustration stems in part from the delay of being recognized or rewarded only by objective concrete delivery, rather than by subjective promise or potential. In due course my work with my many collaborators has resulted in much personal recognition, including election to the National Academies of Science in the UK and Australia, honors such as the European Inventor of the Year award in the Lifetime achievement category, the Curtin Medal of Australian National University, and the award, together with Ravinder Maini, of a series of prestigious international prizes for medical research such as the Crafoord Prize of the Royal Swedish Academy of Science and the Albert Lasker Award for Clinical Medical Research. But the greatest reward of practical contributions, added to the respect of one’s peers, is the heartwarming acknowledgment by patients of the positive impact on their lives. It is an unexpected pleasure.

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what might happen next. What I have helped to achieve, ﬁrst, is that a cytokine, TNF, is now recognized as a very good therapeutic target for a cluster of chronic inﬂammatory diseases, including RA, juvenile RA, Crohn’s disease, ankylosing spondylitis, psoriatic arthritis, psoriasis, and ulcerative colitis (111). This raises the question of how important TNF is as a ﬁre alarm for noxious signals bringing in the ﬁre ﬁghters, leukocytes, and whether most conditions currently treated by corticosteroids might be treatable by anti-TNF. Second, my colleagues and I have helped to demonstrate that biological therapeutics, that is, monoclonal antibodies and antibody-like fusion proteins, can be used for chronic diseases in the long term, now very long term (up to 10 years and running). This has inevitably inﬂuenced the pharmaceutical industry, and now a very signiﬁcant percentage of new therapeutics entering trials are of this type. There are, of course, major beneﬁts. An important one is that biologics, with a large surface of interaction with their target, are more speciﬁc and selective than the small molecular, organic chemicals traditionally favored by the pharmaceutical industry. Hence, their side effects are more predictable because they are mechanism related. The unfortunate TeGenero disaster, in which an activating anti-CD28 monoclonal antibody was used to try to stimulate regulatory T cells, is worth noting. Some believe it was unexpected or unpredictable. However, most human immunologists like myself, who are aware of the variable toxicity of OKT3, an anti-CD3 antibody, which polyclonally activates T cells, believe it was extremely predictable (112). This disaster and the ensuing publicity have markedly inﬂuenced clinical trial capacity in the UK. There have been a number of subsequent successes for monoclonal antibodies and cytokine blockade. Anti-CD20 antibody, developed for lymphoma based on Ron Levy’s work, has been very successful (113) and was introduced to rheumatology by Jo Edwards (114). IL-1 blockade with IL-1 receptor antagonist has been approved but, because it

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has been less effective than TNF blockade, has not been widely used in RA (115). It has been very useful in, for example, MuckleWells syndrome. There is clear effectiveness of blocking the IL-6 receptor with an antibody developed by Tadamitsu Kishimoto (116, 117), and there is great anticipation for the utility of blocking RANK ligand for bone disorders (118). So an interesting possibility emerges. Are all diseases and unmet medical needs treatable by cytokine blockade? As an optimist I believe that will be close to the truth. The 100 or so cytokines (a term that I use to encompass interleukins, growth factors, IFNs, chemokines, members of the TNF family, etc.) are involved in all key biological processes, for example cell proliferation, cell motility, inﬂammation, immunology, angiogenesis, ﬁbrosis, etc. Hence, all diseases involve alterations in cytokine expression, and many are upregulated. These are potential therapeutic targets. My colleagues and I are pursuing important new therapeutic endeavors that might be treatable by cytokine blockade. For example, with Claudia Monaco, we are studying treatment of atherosclerosis; with Mervyn Maze, we are studying post-operative cognitive dysfunction; and with Tracy Hussell, Brian Foxwell, and Kendall Smith, we are studying acute respira-

tory distress induced by avian ﬂu. Only time will tell if these endeavors will succeed, but inevitably the ﬁeld of cytokine blockade or anticytokine medicine will ﬂourish in many more directions. Why should one bother to read semihistorical personal reviews? I am not sure, but there may be lessons for the less experienced. If so, one is that we now have wonderful technologies for permitting scientiﬁc progress in the ﬁeld of disease pathogenesis and therapy. These can unravel molecular mechanisms of diseases and permit the discovery, design, and development of new treatments that impact millions of lives. But this will not work for most projects, as most projects and hypotheses fail. But it will succeed for some. So there are enormous opportunities remaining to use science for the beneﬁt of human health and welfare. But the hurdles are tough and the risks high. I encourage as many clinicians as possible to spend the time and training, as I did, to merge both science and medicine, as there is a critical shortage of individuals, for example physician-scientists, who can synthesize these components to bring the translational discoveries to patients. Perhaps a summary of an exciting adventure that has beneﬁted many patients might encourage and challenge you to venture into that arena and see what you might achieve.

DISCLOSURE STATEMENT Over the years, I have interacted extensively with companies and so have been a paid consultant or scientiﬁc advisory board member to many companies involved in arthritis and cytokine work: Amgen, Astra-Zeneca, Abbott, Centocor, Glaxo, Celgene, Immunex, Merck, Roche, Wyeth, Novo Nordisk, Schering Plough, Boehringer-Ingelheim, Synovis Ltd., Xenova plc, Hydra Biosciences, Receptor BioLogix, Inc., Nuon Therapeutics, Canji, Inc., Trillium Therapeutics, Inc., Sandoz (now Novartis), Alza, Inc., Almirall Prodesfarma, Ferring AS, and Calyx Therapeutics. I or close colleagues have received grants in the past three years from Roche, Wyeth, Novo Nordisk, Celgene, Nuon Therapeutics, and Receptor Biologix, Inc. I have patents in the anti-TNF therapy ﬁeld and many others (I was European Inventor of the Year in 2007). I have signiﬁcant ﬁnancial holdings in Johnson & Johnson and Schering Plough.

LITERATURE CITED 1. Mishell RI, Dutton RW. 1966. Immunization of normal mouse spleen cell suspensions in vitro. Science 153:1004–6 www.annualreviews.org • Effective Therapy for Autoimmunity

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2. Marbrook J. 1967. Primary immune response in cultures of spleen cells. Lancet 2:1279–81 3. Diener E, Armstrong WD. 1967. Induction of antibody formation and tolerance in vitro to a puriﬁed protein antigen. Lancet 2:1281–85 4. Ada GL, Nossal GJ. 1987. The clonal-selection theory. Sci. Am. 257:62–69 5. Diener E. 1968. A new method for the enumeration of single antibody-producing cells. J. Immunol. 100:1062–70 6. Jerne NK, Nordin AA. 1963. Plaque formation in agar by single antibody-producing cells. Science 140:405 7. Feldmann M. 1971. Induction of immunity and tolerance to the dinitrophenyl determinant in vitro. Nat. New Biol. 231:21–23 8. Lennon V, Feldmann M. 1972. The detection of autoantibody-forming cells. I. An assay for plaqueforming cells to the basic protein of myelin in guinea-pigs. Int. Arch. Allergy Appl. Immunol. 42:627–40 9. Miller JF, Mitchell GF. 1968. Cell to cell interaction in the immune response. I. Hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes. J. Exp. Med. 128:801–20 10. Mitchell GF, Miller JF. 1968. Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes. J. Exp. Med. 128:821–37 11. Nossal GJ, Cunningham A, Mitchell GF, Miller JF. 1968. Cell to cell interaction in the immune response. 3. Chromosomal marker analysis of single antibody-forming cells in reconstituted, irradiated, or thymectomized mice. J. Exp. Med. 128:839–53 12. Roitt IM, Greaves MF, Torrigiani G, Brostoff J, Playfair JH. 1969. The cellular basis of immunological responses. A synthesis of some current views. Lancet 2:367–71 13. Greaves MF, Roitt IM, Rose ME. 1968. Effect of bursectomy and thymectomy on the responses of chicken peripheral blood lymphocytes to phytohaemagglutinin. Nature 220:293–95 14. Feldmann M, Basten A. 1971. The relationship between antigenic structure and the requirement for thymus-derived cells in the immune response. J. Exp. Med. 134:103–19 15. Feldmann M, Basten A. 1972. Cell interactions in the immune response in vitro. 3. Speciﬁc collaboration across a cell impermeable membrane. J. Exp. Med. 136:49–67 16. Feldmann M, Basten A. 1972. Speciﬁc collaboration between T and B lymphocytes across a cell impermeable membrane in vitro. Nat. New Biol. 237:13–15 17. Schimpl A, Wecker E. 1972. Replacement of T-cell function by a T-cell product. Nat. New Biol. 237:15– 17 18. Feldmann M. 1972. Cell interaction in the immune response in vitro. II. The requirement for macrophages in lymphoid cell collaboration. J. Exp. Med. 135:1049–58 19. Lennon V, Feldmann M, Crawford M. 1972. The detection of autoantibody-forming cells. II. Cells in lymph nodes and central nervous system containing antibody to myelin basic protein. Int. Arch. Allergy Appl. Immunol. 43:749–58 20. Hanafusa T, Pujol-Borrell R, Chiovato L, Russell RC, Doniach D, Bottazzo GF. 1983. Aberrant expression of HLA-DR antigen on thyrocytes in Graves’ disease: relevance for autoimmunity. Lancet 2:1111– 15 21. Klareskog L, Forsum U, Scheynius A, Kabelitz D, Wigzell H. 1982. Evidence in support of a selfperpetuating HLA-DR-dependent delayed-type cell reaction in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 79:3632–36 22. Bussard AE, Lurie M. 1967. Primary antibody response in vitro in peritoneal cells. J. Exp. Med. 125:873– 92 23. Schrader JW, Feldmann M. 1973. The mechanism of antigenic competition. I. The macrophage as a site of a reversible block of T-B lymphocyte collaboration. Eur. J. Immunol. 3:711–17 24. Wagner H, Feldmann M. 1972. Cell-mediated immune response in vitro. I. A new in vitro system for the generation of cell-mediated cytotoxic activity. Cell. Immunol. 3:405–20 25. Wunderlich JR, Canty TG. 1970. Cell mediated immunity induced in vitro. Nature 228:62–63 26. Mitchison NA. 1971. The carrier effect in the secondary response to hapten-protein conjugates. I. Measurement of the effect with transferred cells and objections to the local environment hypothesis. Eur. J. Immunol. 1:10–17

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116. Nishimoto N, Kishimoto T. 2006. Interleukin 6: from bench to bedside. Nat. Clin. Pract. Rheumatol. 2:619–26 117. Maini RN, Taylor PC, Szechinski J, Pavelka K, Broll J, et al. 2006. Double-blind randomized controlled clinical trial of the interleukin-6 receptor antagonist, tocilizumab, in European patients with rheumatoid arthritis who had an incomplete response to methotrexate. Arthritis Rheum. 54:2817–29 118. Miller PD, Bolognese MA, Lewiecki EM, McClung MR, Ding B, et al. 2008. Effect of denosumab on bone density and turnover in postmenopausal women with low bone mass after long-term continued, discontinued, and restarting of therapy: a randomized blinded phase 2 clinical trial. Bone 43:222–29

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Structural Biology of Shared Cytokine Receptors Xinquan Wang, Patrick Lupardus, Sherry L. La Porte, and K. Christopher Garcia Howard Hughes Medical Institute, Stanford University School of Medicine, Departments of Molecular and Cellular Physiology, and Structural Biology, Stanford, California 94305; email: [email protected], [email protected], [email protected], [email protected]

Annu. Rev. Immunol. 2009. 27:2.1–2.32

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

interleukin, signaling, structure

This article’s doi: 10.1146/annurev.immunol.24.021605.090616

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

Recent structural information for complexes of cytokine receptor ectodomains bound to their ligands has signiﬁcantly expanded our understanding of the macromolecular topology and ligand recognition mechanisms used by our three principal shared cytokine signaling receptors—gp130, γc , and βc . The gp130 family receptors intricately coordinate three structurally unique cytokine-binding sites on their four-helix bundle cytokine ligands to assemble multimeric signaling complexes. These organizing principles serve as topological blueprints for the entire gp130 family of cytokines. Novel structures of γc and βc complexes show us new twists, such as the use of a nonstandard sushitype α receptors for IL-2 and IL-15 in assembling quaternary γc signaling complexes and an antiparallel interlocked dimer in the GM-CSF signaling complex with βc . Unlike gp130, which appears to recognize vastly different cytokine surfaces in chemically unique fashions for each ligand, the γc -dependent cytokines appear to seek out some semblance of a knobs-in-holes shape recognition code in order to engage γc in related fashions. We discuss the structural similarities and differences between these three shared cytokine receptors, as well as the implications for transmembrane signaling.

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INTRODUCTION

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The interaction of cell surface receptor extracellular domains with secreted ligands is essential to most types of cell signaling and cellcell communication. This initial step and the subsequent activation of membrane-proximal and -distal intracellular signaling cascades lead to speciﬁc, although often redundant, cellular responses that control cell proliferation, differentiation, maturation, and survival. Cell surface receptors usually bind their ligands through highly speciﬁc molecular interactions to provide the tight regulation necessary for control of physiological responses. However, researchers increasingly appreciate that many receptor systems exhibit, to a greater or lesser extent, cross-reactivity with a spectrum of different ligands. There are many examples of degenerate, shared receptors with central roles in signaling (1). In neurobiology, the p75 neurotrophin receptor can recognize a family of neurotrophic factors, including nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 (2). The glial cell line–derived neurotrophic factor family ligands, which include glial cell line–derived neurotrophic factor, neurturin, artemin, and persephin, share the RET receptor as the signaling subunit in their receptor complexes (3). In the immune system, shared receptors exist in both adaptive (T cell receptors, costimulatory molecules B7/CD28) (4) and innate immunity (NKG2D natural killer receptor, scavenger, and pattern-recognition receptors such as RAGE and Toll) (5). However, the most widespread roles for shared receptors are found for cytokines (6–9), which are secreted growth factors that control cell growth and proliferation primarily in the immune and hematopoietic systems. There are three major shared receptors in the class I cytokine receptor family: the common gamma chain (γc ), gp130, and the common beta chain (βc ), which participate in the formation of receptor complexes for nearly 20 different cytokines (Figure 1). Important insights into the mechanisms for cross-reactivity of shared cytokine receptors

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have emerged from structural studies of complexes between cytokines and extracellular domains of their receptors (10). During the past several years, crystal structures have been determined for gp130, γc , and βc complexes with cytokines, including gp130 bound to human herpes virus (HHV)-8 IL-6 (11), IL-6 (12), and leukemia inhibitory factor (LIF) (13); LIF complexed with LIF receptor (14); γc bound to IL-2 (15, 16) and IL-4 (17); and more recently βc bound to GM-CSF (18). Gp130, γc , and βc share a rudimentary core structural blueprint for the assembly of the extracellular cytokinereceptor signaling complexes; however, there are many important deviations between these three systems that result in substantially different signaling complex topologies. Collectively, these structures allow us to delineate common and unique structural features for both ligand recognition and assembly of signaling complexes by these three major shared cytokine receptors in the class I family, which is the focus of this review. Owing to space constraints, we could not cite all contributors to this ﬁeld. We refer the reader to excellent treatises on various aspects of cytokine structure, receptor interaction, and signaling; see References 10, 19–35.

THE CLASS I CYTOKINE RECEPTORS Cytokines represent a diverse group of small soluble proteins that when secreted by one cell can act on the same cell, in an autocrine fashion, or on another cell, in a paracrine fashion (36). Through binding to speciﬁc cell surface receptors, they initiate signals that are critical to a diverse spectrum of functions, including induction of immune responses, cell proliferation, differentiation, and apoptosis. Structural analysis has allowed the grouping of cytokines into different structural classes, including the helical cytokines (37), the trimeric tumor necrosis factor (TNF) family (38), the cysteine knot growth factors (39), and the β-trefoil growth factors (40). Cytokines can also be classiﬁed according to the type of receptor that they engage.

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IL-2

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Figure 1 Diversity of shared cytokine-receptor interactions. Shared cytokine receptors γc (a), gp130 (b), and βc (c) are represented schematically on a cell membrane. The respective interacting cytokines with known three-dimensional structures are shown with cylinder representations of the four-helix bundles. (Abbreviations: LIF, leukemia inhibitory factor; OSM, oncostatin-M; CNTF, cillary neurotrophic factor; CLC, cardiotrophin-like cytokine; CHR, cytokine-binding homology region; HHV-8, human herpes virus; GM-CSF, granulocyte-macrophage colony-stimulating factor.)

On the basis of common structural features, the cytokine receptors are grouped into six major families: class I cytokine receptors, class II cytokine receptors, TNF receptors, IL-1 receptors, tyrosine kinase receptors, and chemokine receptors (36, 41, 42). The class I cytokine receptors, also known as the hematopoietin receptors, constitute the largest group among the cytokine receptor family (41, 43, 44). These are type I membrane proteins with an N-terminal extracellular and C-terminal intracellular orientation. The extracellular segments of the class I cytokine receptors show a modular architecture, which is characterized by a ∼200 residue–long cytokinebinding homology region (CHR) (45) possessing the classical binding motif for cytokines, as structurally delineated in the human growth hormone (hGH) receptor complex (46). The CHR module consists of two ﬁbronectin typeIII (FNIII) domains connected by a linker, and it represents the signature recognition module for helical cytokines that is present on every type I cytokine receptor (Figure 1). The upper, N-terminal domain contains four (47) con-

served cysteine residues that form interstrand disulﬁde bonds. The lower, C-terminal domain has a conserved Trp-Ser-X-Trp-Ser motif (45, 46). Mutagenesis studies have shown an essential structural role for these amino acids in maintaining the tertiary structure of the protein, but they are not involved in cytokine interaction (48). These signature sequence and structural features have been used to identify novel cytokine receptors in several genomes (49–51). The cytokine-binding site for most CHR modules is at the apex of the elbow region, consisting mainly of the interstrand loops connecting the β-strands from both N- and Cterminal domains (46). The basic CHR module is present in every class I cytokine receptor, and for some, such as the receptors for hGH (46, 52) and erythropoietin (EPO), a single CHR is sufﬁcient to mediate ligand binding and receptor homodimerization. However, many other class I cytokine receptors require additional domains, such as the Ig-like domain and additional membrane-proximal ﬁbronectin domains, found in the gp130 family, to function and respond to cytokines (11, 12). The α

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receptors of IL-2 and IL-15 of the γc family are atypical cytokine receptors in that they do not contain CHR, but rather sushi domains (discussed below) (53, 54). The deﬁning structural feature of the cytokines recognized by the class I cytokine receptors is a four-helix bundle motif (43, 55), which is composed of four amphipathic helices having solvent-facing hydrophilic sides and hydrophobic sides that form the core of the helical bundle. These four helices are oriented into a unique up-up-down-down topology that is only found in the helical cytokines (43, 55). Structural predictions, later conﬁrmed by several crystal and NMR structures, indicated that these cytokines could be further subclassiﬁed on the basis of the length of the helices (19, 37). Short-chain cytokines, represented by IL-2 and IL-4, have helices of 8–10 residues. The longchain cytokines, such as gp130 family cytokines, hGH, and EPO, have helices of 10–20 residues. Finally, some cytokines, such as IL-5 and interferon (IFN)-γ, have two four-helix bundles forming an eight-helix architecture (55–57). Cytokine binding induces receptor oligomerization that leads to the juxtaposition of the intracellular domains of the signaling subunits. Unlike receptors for many growth factors (e.g., insulin, epidermal growth factor) that have intracellular domains possessing tyrosine kinase activity on the same polypeptide chain, the class I cytokine receptors have no intrinsic enzymatic activity. Rather, the intracellular domains of the class I cytokine receptors are constitutively associated with tyrosine kinases of the Janus kinase ( JAK) family, and to a more restricted degree the TYK kinases (26, 31, 32, 34). After the JAK/TYK kinases are activated by ligand-induced receptor oligomerization, they phosphorylate themselves and the intracellular domains of the receptors. The phosphorylated tyrosine residues in the receptors then serve as the docking sites for a second family of proteins, the signal transducer and activator of transcription (STATs). Binding of STATs to the intracellular domains of the receptors leads to their tyrosine phosphorylation and subsequent dissociation

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from the receptors. The phosphorylated STATs form dimers and translocate into the nucleus, where they bind to DNA recognition sequences and act as transcription factors (33, 34, 58). Besides the major JAK-STAT signaling pathway, the class I cytokine receptors also use other signaling mechanisms such as the RAS-RAF-MAP kinase pathway (59), PI3 kinase (60), and insulin receptor substrate (61). The structural aspects of JAK-STAT communication during receptor signaling are poorly understood and represent a major future frontier in cytokine receptor structural biology.

SHARED RECEPTORS IN THE CLASS I CYTOKINE RECEPTOR FAMILY Although the original structural paradigm for cytokine receptor complexes was derived from the homodimeric hGH system (21, 46), most of the class I cytokine receptors do not signal through homodimerization (Figure 2). In fact, most form heterodimers (e.g., IL-4, IL-7, etc.) (17), and some even form heterotrimers (IL-2 and IL-15 receptors) (15, 16), tetramers (viral IL-6/gp130, G-CSF/G-CSFR) (11, 62), hexamers (human IL-6/IL-6Rα/gp130) (12), and even dodecamers (GM-CSF/GM-CSFRα/βc ) (18). A signiﬁcant feature of these heterooligomeric receptor complexes is the use of a common, shared receptor subunit as a signaltransducing chain together with a cytokinespeciﬁc chain. When subgrouped by shared receptors, there are three major classes of heteroreceptor complexes in the class I cytokine receptor family: those that use βc , those that use gp130, and those that use γc (Figure 2). In most cases, the shared receptors do not show appreciable afﬁnity for cytokines, but in the presence of cytokine-speciﬁc α receptors they can form high-afﬁnity cytokine receptor complexes that are capable of initiating intracellular signaling cascades. Such a characteristic afﬁnity-conversion effect is used by shared receptors as a means of imposing tissue speciﬁcity (8). Shared receptors also must recognize different cytokines that have relatively low

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sequence identities, requiring that they be polyspeciﬁc for different ligand surface structures and chemistries, yet speciﬁc enough not to cross-react with inappropriate cytokines. In this respect, the shared receptors may teach us much about the basic structural and chemical mechanisms of protein-protein cross-reactivity.

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Gp130 is the founding member of the tall cytokine receptors and is the common signaltransducing receptor component for the gp130, or IL-6/IL-12, family of cytokines (Figure 1b and Figure 2b) that exhibit highly pleiotropic biological activities (23, 63). There are currently ten members in the gp130 family of cytokines: IL-6, IL-11, LIF, cillary neurotrophic factor (CNTF), oncostatin M (OSM), cardiotrophin 1 (CT-1), NNT-1/BSF3 [also known as cardiotrophin-like cytokine (CLC)], and IL-27. There are two viral homologs of IL6, one from HHV-8 IL-6 and another from the Rhesus macaque rhadinovirus (Rm IL-6) (Figure 1b). Except for the viral IL-6 homologs that bind directly to gp130 alone (11, 64), the signaling functions of gp130 cytokines are mediated through a set of receptor complexes that are formed by combining gp130 with other receptors (Figure 2b) (63). The association of gp130 with cytokine-speciﬁc, nonsignaling receptor IL-6Rα (65) or IL-11Rα executes the activities of IL-6 or IL-11, respectively. Other signaling receptors such as LIF receptor (LIFR) and OSM receptor (OSMR) can also participate in signaling complexes with gp130. The nonsignaling CNTF α receptor (CNTFRα) can recognize the cytokines CNTF, CT-1, and CLC as part of a quaternary signaling complex with gp130 and LIFR (66–68). Thus, CNTFRα exhibits the ability to bind three different cytokines, which is a rare example of degeneracy by an α receptor. Gp130 can also engage the recently identiﬁed heterodimeric cytokine IL-27 (p28/EBI3) in conjunction with the signaling receptor TCCR (T cell cytokine receptor, also known as WSX-1) (69). A major distinguishing feature of gp130 cytokines is that they possess

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Figure 2 Diversity of shared receptor-receptor interactions. Shared cytokine receptors βc (a), gp130 (b), and γc (c) and their various receptor partners are depicted. These complexes are formed by the combination of ligand-speciﬁc α and/or β receptors with shared cytokine receptors. (Abbreviations: TCCR, T cell cytokine receptor; TSLPR, thymic stromal-derived lymphopoietin receptor.)

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the unique site III receptor-binding site at the tip of the cytokine that is necessary for gp130 activation (12, 70, 71). The extracellular part of gp130 is composed of six contiguous β-sandwich domains with a single Ig domain at the top (D1), followed by one CHR module (D2 and D3), and three ﬁbronectin III-like domains (D4–D6) leading to the cell membrane (Figure 1b). Both the CHR and Ig domains are necessary for full activation. There are three known crystal structures of complexes involving gp130: the IL6/IL-6Rα/gp130-D1D2D3 hexamer (12), the HHV-8 IL-6/gp130-D1D2D3 tetramer (11), and LIF/gp130-D2D3 (13). In addition, there are two low-resolution, electron microscopic (EM) three-dimensional reconstructions of the entire extracellular complexes of IL-6 (72) and IL-11 (73). The crystal structures established that gp130 cytokines use the canonical sites I and II to engage the elbow regions of the α receptor and gp130 CHR, respectively. Site III in the cytokine engages the Ig domain of gp130 so that each gp130 contacts two different cytokines in an antiparallel fashion (Figure 3a). This basic assembly template is used by all gp130 cytokine receptor family members, including the nonshared members of the tall receptor family such as G-CSF, leptin, and OSMR. Originally, G-CSF was crystallized with only the CHR of its receptor (74). Subsequently, a mutational study of G-CSF based on the viral IL-6/gp130 complex structure (which revealed the ﬁrst site III) determined that GCSF contains a site III (75). Recently, the full complex of the G-CSFR Ig domain plus CHR has been solved with G-CSF (62), and this complex is almost identical in structure to that of the viral IL-6/gp130 complex and other gp130 complexes in the use of site III (11, 12). Several recent advances have established the site III paradigm for heterodimeric gp130/LIFR signaling complexes. The 4.0 A˚

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structure of the D1–D5 domains of LIFR in complex with LIF conﬁrmed that LIFR uses a high-afﬁnity site III interaction in which the interhelical loops at one tip of LIF engage the D3 Ig domain of LIFR in an almost orthogo2.6

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nal orientation (Figure 3b) (14). Binding measurements have also conﬁrmed that LIFR and CNTF alone interact via a high-afﬁnity site III that is presumably analogous to that of the LIF/LIFR interaction. A recent single-particle EM analysis of full-length gp130 and LIFR in complex with CNTFRα and CNTF (76) has conﬁrmed the architecture of the quaternary gp130/LIFR/CNTFRα/CNTF complex proposed in Boulanger et al. (13) and for the trimeric LIF/LIFR/gp130 complex modeled in Huyton et al. (14) (Figure 3c). The structure of an intact gp130/LIFR heterodimeric complex is an important milestone for research on this class of receptors, as there has been some controversy about the functionally active domains of LIFR involved in cytokine-mediated complex formation with gp130. With the basic site II/III architecture of the heterodimeric gp130/LIFR signaling complex predicted from a variety of structural and biochemical data, it is now clear that other gp130related heterodimeric cytokines such as IL-12 (p35/p40) (77), IL-23 (p19/p40) (78), and IL27 (p28/EBI3) (79) also engage their cognate receptors in some variation of this basic organizing principle (69, 80, 81). Interestingly, in the case of these heterodimeric cytokines (which consist of a four-helix bundle cytokine in complex with a soluble α receptor), the gp130like receptors IL-12Rβ1 (IL-12 and -23) and TCCR/WSX-1 (IL-27) lack an N-terminal Iglike domain and hence engage site II via their CHR domains. Site III is then free to interact with the second signaling receptor IL-12Rβ2, IL-23R, or gp130, respectively, all of which contain the top-mounted Ig domain required for site III interaction. In this fashion, the presence of the Ig domain serves as a structural beacon for the receptor that engages in site III interaction. IL-12 and IL-23 also represent unique examples of two cytokines sharing both an α receptor (p40) and a signaling receptor (IL-12Rβ1), while gaining speciﬁcity by using different site III receptors (IL-12Rβ2 and IL-23R). An additional characteristic feature of the gp130 family receptors is that that they are

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IL-6Rα gp130 Figure 3 Structures of human IL-6/IL-6Rα/gp130 hexameric complex (a), mouse LIFR in complex with human LIF (b), and human CNTF/ CNTFRα/LIFR/gp130 (c) assembled from known crystallographic, biochemical, and electron microscopic data. In (a) the model shown was derived from the crystal structure of the IL-6 hexamer headpiece (12) together with the single-particle reconstruction of the entire extracellular complex (72). IL-6 signaling is mediated through homodimerization of gp130 in a symmetric hexameric arrangement with a nonsignaling IL-6Rα receptor. In (b) the model shown derives from the 4 A˚ crystal structure of the LIF/LIFR complex (14) missing the membrane-proximal domains that are depicted as cartoons. In (c), the quaternary LIFR/gp130/CNTF/ CNTFRα complex is derived from a combination of the crystal structures of LIF/gp130 (13), CNTF (163), and a single-particle reconstruction of the entire quaternary complex (76). CNTF signals through the asymmetric heterodimerization of gp130 and LIFR and the nonsignaling CNTFα receptor. In panel (d ), the assembly pathway for IL-6 signaling is depicted as elucidated from References 12, 164. IL-6 ﬁrst engages IL-6Rα through a site I interaction to form a composite interface (site II) that recruits gp130. This trimeric structure can then engage a second trimer through two site III interfaces to form a productive signaling complex.

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taller than other cytokine receptors, by virtue of three additional membrane-proximal domains. A three-dimensional reconstruction of negatively stained six-domain gp130 complexed with IL-6 and the IL-6Rα receptor indicated that the gp130 membrane-proximal legs are bent back toward one another through the ﬂexing of a hinge between the D3 and D4 domains (73). This leg closure has also been observed in a cryoelectron microscopic analysis of the hexameric gp130/IL-11/IL-11Rα complex (74). Thus, although the FNIII leg domains have retained conformational ﬂexibility to allow for the close apposition of the intracellular domains required for intracellular signaling, it remains unclear whether the unliganded gp130 exists in this bent conformation or if the engagement of the shorter cytokine/Rα binary complex forces gp130 to bend in order to accommodate the height differences.

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THE COMMON BETA CHAIN: βc βc is a type I transmembrane protein that serves as a shared signaling subunit for the receptors of IL-3, IL-5, and GM-CSF (Figure 1c and Figure 2a), which are related cytokines involved in the regulation of hematopoiesis and inﬂammation (82–84). Although βc does not measurably bind any of the ligands alone, its coexpression with cytokine-speciﬁc α receptors enhances the afﬁnity of cytokine binding. The activated receptor complex, consisting of the cytokine ligand plus the α and βc receptors, initiates the intracellular signaling pathway mainly through JAK2 associated with the cytoplasmic domain of βc receptors (85). The extracellular part of βc has four ﬁbronectin domains, forming two contiguous CHR modules (Figure 1c), with features conserved among the class I cytokine receptors. The crystal structure of the unliganded extracellular domain of βc shows it to exist as an unusual, intertwined, strand-swapped, antiparallel homodimer (86). This structure, together with mutagenesis studies (87), led to the proposition that the possible cytokine-binding site is composed of D1 of one chain and D4 of another chain in the βc homod2.8

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imer that join in an antiparallel fashion. Excitingly, the recent crystal structure of the βc in complex with the GM-CSFα receptor (GMRα) and GM-CSF conﬁrms that βc engages the cytokine via a composite D1/D4 interface similar to site II in gp130 and γc , while the accessory receptor GMRα engages the cytokine via a site I–like interface (Figure 4) (18). It has been reported that GMRα does not engage a JAK kinase (85), leaving the JAK2-bound βc as the sole carrier of the signal-transducing kinase. The asymmetric unit of the βc /GMRα/GMCSF complex consists of a 2:2:2 hexamer with the C termini of βc ∼ 140 A˚ apart, therefore making it hard to reconcile how JAK kinases bound to βc subunits of a single βc dimer could be activated. Importantly, crystallographic contacts between βc D4 domains of two separate βc /GMRα/GM-CSF hexamers suggested βc signaling may be mediated by two hexamers dimerized into a dodecameric structure (18). Site-directed mutagenesis of this interface abrogated GM-CSF-induced signaling; thus, it appears that a second βc dimer in complex with GMRα and GM-CSF is necessary to complete the active signaling unit. These studies clarify what turns out to be a highly interesting deviation from the typical cytokine receptor signaling paradigm and can likely be extrapolated to explain the activation mechanisms of the other βc -family cytokines IL-3 and IL-5.

THE COMMON GAMMA CHAIN: γc γc serves as a shared signaling receptor for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (Figure 1a and Figure 2c) (8). The biological importance of γc is illustrated by the fact that mutations in either γc or the associated JAK3 kinase can abolish the function of all γc -dependent cytokines and cause X-linked severe combined immunodeﬁciency diseases (X-SCID) (88, 89). One interesting twist in the γc family is that the IL-2 receptor (IL-2R) and IL-15R signaling complexes are heterotrimers composed of structurally unique α subunits and shared IL2Rβ and γc subunits (Figure 2c). This is in

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GM-CSF GMRα βc Figure 4 The 2:2:2 GM-CSF/GMRα/βc complex viewed from the side (a) and from the top (b) (18). GMRα engages the cytokine GM-CSF via a canonical site I interaction, whereas the βc receptor engages site II on GM-CSF by using a composite cytokine-binding homology region (CHR) interface generated by domain 1 (D1) of one βc subunit and domain 4 (D4) of the second βc .

contrast to type I IL-4R, IL-7R, IL-9R, and IL21R, which heterodimerize cytokine-speciﬁc α subunits and γc (Figure 2c). Another interesting twist in the γc family is limited sharing of several α receptors including IL-2Rβ, IL-4Rα, and IL-7Rα to recognize different cytokines as well as different receptors (Figure 2c). IL-2Rβ serves as a receptor for both IL-2 and IL-15. IL-4Rα heterodimerizes with γc to form type I IL-4R, and with IL-13Rα1 to form type II IL4R. Type II IL-4R is also the functional recep-

tor of IL-13 (discussed below) (25, 90, 91). IL7Rα can also form a receptor heterodimer with TSLPR (thymic stromal-derived lymphopoietin receptor) to recognize TSLP (92, 93).

STRUCTURAL STUDIES OF CYTOKINE-RECEPTOR COMPLEXES IN IL-2 AND IL-4/IL-13 SYSTEMS The cytokine IL-2 is a prototype member of cytokines and has pleiotropic actions in

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the immune system (28, 94, 95). Produced mainly by activated T cells, IL-2 promotes the proliferation, differentiation, and survival of mature T and B cells and the cytolytic activity of natural killer (NK) cells (95). There are three receptor chains for IL-2: IL-2Rα, IL-2Rβ, and γc , which form three different receptor complexes on different target cells. Isolated IL-2Rα has been termed the low-afﬁnity IL-2 receptor (Kd ≈ 10 nM) and is not currently believed to be involved in signal transduction (96). IL-2Rβ and γc form the intermediateafﬁnity complex (Kd ≈ 1 nM) expressed on NK cells, macrophage, and resting T cells (95), although IL-2Rβ alone has very low afﬁnity (Kd ≈ 100 nM) and γc alone has no detectable binding afﬁnity for IL-2 (97, 98). The heterodimerization of IL-2Rβ and γc in the presence of IL-2 is necessary and sufﬁcient for effective signaling through the activation of JAK1 and JAK3 kinases associated with the intracellular domains of the IL-2Rβ and γc , respectively (99, 100). A complex with three subunits, IL-2Rα, IL-2β, and γc , is the highafﬁnity complex (Kd ≈ 10 pM) for IL-2 and is the receptor form on activated T cells (101). The high-afﬁnity receptor complex mediates most biological effects of IL-2 in vivo (102). Prior to structural analysis, the Ciardelli group published a series of elegant papers measuring the various rate constants and afﬁnities for soluble forms of the different compositions of complexes (103–107). A similar analysis of the soluble complex assembly has also been conducted using thermodynamic measurements instead of kinetic studies (97). Therefore, the IL-2 receptor system has been one of the most rigorously characterized receptor systems using both cellular and biochemical approaches. IL-4 is another principal regulatory cytokine during the immune response and is crucially important in allergy and asthma (90). Once resting T cells are antigen activated and expand in response to IL-2, the fate decision of Th1 versus Th2 is inﬂuenced by IL-4. Th2 cells secrete IL4, which both stimulates Th2 in an autocrine fashion and acts as a potent B cell growth factor to promote humoral immunity (90). There

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are two types of receptor complexes for IL-4 (Figure 2c) (61, 91). Type I IL-4R is predominantly expressed on the surface of hematopoietic cells and consists of IL-4Rα and γc . Type II IL-4R consists of IL-4Rα and IL-13Rα1 and is predominantly expressed on the surface of nonhematopoietic cells, and this receptor complex is also the functional receptor of IL-13 (108– 110). The crystal structures of the high-afﬁnity IL-2/IL-2Rα/IL-2Rβ/γc quaternary complex and IL-4/IL-4Rα/γc , IL-4/IL-4Rα/IL13Rα1, and IL-13/IL-4Rα/IL-13Rα1 ternary complexes have all been determined (Figure 5) (15, 17). The structural comparison between IL-2/IL-2Rα/IL-2Rβ/γc and IL-4/IL-4Rα/γc complexes allows us to probe the basis by which γc can recognize six distinct cytokines. For the convenience of comparison between these two structures, and to eliminate redundancy in the chapter, we describe them in parallel throughout the following sections.

OVERALL STRUCTURE The ﬁrst complex structure of γc to be solved was the IL-2/IL-2Rα/IL-2Rβ/γc quaternary complex (15, 16) (Figure 5a), which is composed of one copy each of IL-2, IL-2Rα, IL2Rβ, and γc . Viewed from the perspective of the cell membrane, IL-2Rα sits on top of IL-2, and receptors IL-2Rβ and γc form a Y shape in which IL-2 sits in the fork (Figure 5a). The overall structural organization of the IL4/IL-4Rα/γc ternary complex is very similar to that of the IL-2 quaternary complex with a 1:1:1 stoichiometry (Figure 5b) (17), except for the absence of a top-mounted IL-2Rα. In the IL-4/IL-4Rα/γc ternary complex, receptor IL-4Rα and γc form a Y-shape heterodimer that binds to IL-4 in the classical site I/site II paradigm (Figure 5b).

BINARY COMPLEX OF IL-2Rα/IL-2 After resting T cells are activated by antigen, expression of IL-2Rα is upregulated in order to sensitize the T cells to low concentrations

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www.annualreviews.org • Structural Biology of Shared Cytokine Receptors

Extracellular complex structures of IL-2/IL2Rα/IL-2Rβ/γc (a) (15), IL-4/IL4Rα/γc (b), IL-4/IL4Rα/IL-13Rα1 (c), and IL-13/IL-4Rα/IL13Rα1 (d ) (17) extracellular signaling complexes depicted on a cell membrane.

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of IL-2, which is required for clonal expansion. The human IL-2Rα receptor chain, also known as Tac antigen or CD25, is a ∼55-kDa polypeptide consisting of an extracellular domain of 219 residues, a transmembrane domain of 19 residues, and a short cytoplasmic tail containing 19 residues (111–113). The short cytoplasmic tail does not participate in the signal transduction, although it is highly conserved between mice and humans, suggesting some important functional roles that remain unknown (100). From sequence analysis, IL2Rα also clearly lacks the signature features of class I cytokine receptors such as IL-2Rβ and γc . With the goal of targeting IL-2Rα with therapeutics for immunosuppression, the crystallization of the IL-2/IL-2Rα was an important benchmark in the ﬁeld (114). In fact, monoclonal antibody anti-Tac identiﬁes IL-2Rα and blocks the interaction of IL-2 with IL-2Rα (115, 116). The humanized form of anti-Tac (daclizumab, or Zenapax®) has been approved by the FDA for use in preventing renal transplant rejection (117). In several other clinical trials, daclizumab also provided a reduction of rejection in patients receiving liver, cardiac, and pancreatic islet transplants (118–120). In 2005, the structure of a recombinant soluble form of IL-2Rα complexed with IL-2 was solved to 2.8 A˚ (53) and revealed a very unusual structure for both the IL-2Rα as well as its mode of interaction with IL-2. The globular part of the IL-2Rα extracellular region is composed of two domain-swapped sushi modules (D1 and D2). Strands A and B from one sushi domain (A-B on C-D-E) are exchanged with strands F and G from another sushi domain (F-G on H-I-J), so the two domain-swapped sushi modules in IL-2Rα now become F on G-C-D-E (D1 domain) and A on B-H-I-J (D2 domain) (Figure 6a). As a result of this domain swap, IL-2Rα uses a composite surface to dock into a groove on IL-2 between the A and B helices, the same surface that serves as a binding site for antagonist drugs (121). This can be considered the dorsal surface of the cytokine with respect to the membrane, poised to present IL-2 to the side-oncoming IL-2Rβ and

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γc (Figures 5a, 6c). The IL-2/IL-2Rα binding interface (site a) has a total buried surface of 1670 A˚ 2 and is dominated by two hydrophobic clusters with a surrounding polar periphery, which may contribute to IL-2Rα’s high-afﬁnity binding and exquisite speciﬁcity. A recent mutational study identiﬁed IL-2 hot-spot residues Glu-62, Tyr-45, and Phe-42 as the most energetically critical residues in the receptor/IL-2 interface (122). Interestingly, these residues also serve as the hot-spots for several small molecule drugs, suggesting that the small molecule and receptor use the same energetic mechanism for binding. The implications are that residue contact footprints in a protein/protein interface may, in some cases, serve as a useful surrogate scaffold for design of small molecules. The IL-2/IL-2Rα complex represents the initiating step for formation of the quaternary signaling complex, so many anticipated that IL2Rα’s role was simply to capture free IL-2 and present it to IL-2Rβ and γc (Figure 7a and Table 1). Moreover, the expression level of IL-2Rα is 10- to 20-fold higher than that of IL-2Rβ on activated T cells (123). Thus, the excess of IL-2Rα molecules and relatively highafﬁnity binding to IL-2 would facilitate efﬁcient capture of free IL-2 and its delivery to IL-2Rβ through a restricted two-dimensional handoff on the same cell membrane. That IL2Rα did not appear to make any contact with either IL-2Rβ or γc in the quaternary signaling complex was a surprise (Figure 5a, 6c) (15). The linker connecting the globular domains of IL-2Rα to the cell membrane, which is disordered in the structure, does not appear capable of forming receptor-receptor contact with IL2Rβ even if fully extended (Figure 6c). This is a rather surprising ﬁnding given the longstanding observation that the coexpression of IL-2Rα and IL-2Rβ forms the pseudo-highafﬁnity complex that can bind to IL-2 with a Kd of ∼30 pM, much higher than IL-2 binding to IL-2Rβ alone (Kd ≈ 100 nM) (124). Isothermal titration calorimetry (ITC) experiments with the soluble receptor ectodomains also showed a twofold afﬁnity increase between IL-2 and IL-2Rβ in the presence of IL-2Rα

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a

b D1

C-ter

C-ter

H

G E D C

B

F

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I J

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H I

J IL-15

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trans

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IL-2Rβ γc

IL-2Rβ cis

cis

γc

Figure 6 IL-2/IL-2Rα and IL-15/IL-15Rα binary complexes. (a) IL-2Rα is composed of two sushi modules (D1 and D2) that swap the β-strands, forming a noncanonical sushi fold topology (53). (b) IL-2Rα and IL-15Rα contact the dorsal surface of IL-2 and IL-15, respectively (130, 131). (c) IL-2 quaternary complex and modeled IL-15 quaternary complexes. IL-2Rα presents IL-2 in cis, whereas IL-15Rα presents IL-15 in cis or trans. www.annualreviews.org • Structural Biology of Shared Cytokine Receptors

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IL-2Rβ IL-2

IL-2Rα

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IL-2

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IL-13 IL-13

IL-13

Site I

Site II + III IL-4Rα

IL-13Rα1

IL-13Rα1

IL-13Rα1

Figure 7 The sequential assembly pathways of the IL-2/IL-2Rα/IL-2Rβ/γc quaternary (a), IL-4/IL-4Rα/γc (b), IL-4/IL-4Rα/IL-13Rα1 (c), and IL-13/IL-4Rα/IL-13Rα1 (d ) ternary complexes. See Table 1 for the interaction afﬁnity and thermodynamic parameters of each binding site.

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Table 1 Interaction affinity and thermodynamic parameters of binding sites in IL-2/IL-2Rα/IL-2Rβ/γc quaternary complex, IL-4/IL-4Rα/γc , IL-4/IL-4Rα/IL-13Rα1, and IL-4/IL-4Rα/IL-13Rα1 ternary complexesa Complex IL-2/IL-2Rα/Rβ/γ

IL-4/IL-4Rα/γc

Kd (nM)

ΔH (Kcal/mol)

Site a

Binding site

10

−5.2

18.4

−10.5

Site I

63

−6.9

8.9

−9.5

Site II

12

−10.3

0.72

−10.4

Site I

1

−11.2

3.5

−12.2

559

−11.7

−10.5

−8.6

1

−11.2

3.5

−12.2

Site II IL-4/IL-4Rα/IL-13Rα1

Site I

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a

ΔG (Kcal/mol)

487

−4.8

13.0

−8.7

Site I

30

−15.0

−15.6

−10.3

Site II+III

20

−5.8

16.0

−10.6

Site II+III IL-13/IL-4Rα/IL-13Rα1

ΔS (cal/molK)

Data compiled from References 17 and 97.

(97). Kinetic studies of both membrane-bound and soluble ectodomains have shown that the on rate of IL-2 for IL-2Rβ is 3 to 20 times faster in the presence of IL-2Rα (96, 98). A simple mechanistic explanation for these afﬁnity data would be the presence of a composite binding surface for IL-2 contributed by both IL-2Rα and IL-2Rβ, which is not seen in the structure (Figure 5a). So what is the basis of the cooperativity? One explanation is that the afﬁnity-enhancing role of IL-2Rα is independent of structural effects and is achieved by simply capturing and concentrating free IL-2 at the cell surface, as mentioned above. The other possibility is an IL-2Rα-induced conformational change in IL-2 that favors the binding to IL-2Rβ. After comparison of the IL-2 structures in the quaternary complex, binary complex, and unbound states, one local conformational adjustment on IL-2 upon IL-2Rα binding was found at the beginning of the helix C, where several turns of the helix are slightly unwound and translated forward by approximately 1.0 A˚ toward IL-2Rβ. This local conformational change enables the movement of IL-2 residue Asn-88 into hydrogen-bonding proximity to IL-2Rβ residue Arg-42 (Figure 8a). Consistent with this, mutation of Asn-88 in IL2 ablates binding to IL-2Rβ (125). We take this to suggest that IL-2Rα may induce and stabilize a favorable IL-2Rβ-binding conformation of the IL-2 C helix in IL-2, in addition to its roles in capture and delivery.

IL-15 is the only other cytokine that uses a speciﬁc sushi-domain α receptor (IL-15Rα) (126, 127). In addition, IL-15 uses IL-2Rβ and γc as its signaling components in the receptor heterotrimer (128, 129). Because IL-15Rα has only one sushi domain in the extracellular part, there is no possibility for a strand exchange as observed in IL-2Rα, which has been conﬁrmed by a NMR structure of the IL-15Rα sushi domain and two complex crystal structures of IL15Rα sushi domain with IL-15 from human and mouse (54, 130, 131). In these structures, the IL-15Rα sushi domain shows a canonical sushi fold topology (Figure 6b). The IL-15/IL-15Rα and IL-2/IL-2Rα binary complexes have similar cytokine-receptor docking modes: The α receptor sushi domain binds to the dorsal surface of the cytokine (Figure 6b), and the cytokinereceptor interaction footprints of IL-15Rα on IL-15 and IL-2Rα on IL-2 also have substantial overlap (130–132), but IL-15Rα has an approximately 1000-fold higher binding afﬁnity to IL15 than that of IL-2Rα to IL-2 (126). Compared to the dominant hydrophobic patches, with a surrounding polar periphery seen in the IL-2/IL-2Rα binding interface, the interaction area between IL-15 and IL-15Rα is dominated by salt bridges and hydrogen bonds with superior shape complementarity (130, 131). The charge-charge interactions may cause the low Koff value between IL-15 and IL-15Rα that is responsible for the high afﬁnity. Similar to IL-2/IL-2Rα, the presence of the IL-15Rα

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Binding site I in IL-2Rβ quaternary complex

a

Binding site I in IL-4Rα ternary complex

Helix C

CC'1

Helix B Helix A

Helix C R42

Helix A N88 D20 R88

CC1'

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EF1

H133

Y134

E9

D72 Y13

EF1 BC2

Y182 AB1

Binding site IIa in IL-2 quaternary complex

b

BC2

FG2

Binding site IIa in the IL-4 ternary complex Helix C

CC1'

Helix D CC1'

Helix A EF1

E122 R121

Q126 Y103

Helix A

Y124

C209 FG2

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Y182

C160

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BC2

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C160 BC2

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γc

E122

S127

Y103 Q126 C209 C160

T118

S125

S130 I129

EF1

T123

Y124

Y103 C209

R121

C160

Figure 8 Speciﬁc versus degenerate binding sites I and IIa in the IL-2/IL-2Rα/IL-2Rβ/γc and IL-4/IL-4Rα/γc complexes. (a) Left panel shows the binding site I between IL-2 helices A and C and IL-2Rβ loops in the elbow region. The corresponding binding site I in the IL-4 ternary complex is shown in right panel. (b) Binding site IIa in the IL-2 quaternary complex (γc /IL-2) and in the IL-4 ternary complex (γc /IL-4) are shown in left and right panels, respectively. 2.16

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endows IL-15 with a much higher afﬁnity for IL-2Rβ and γc (30, 133). One possible explanation for this is the fact that IL-15Rα is known to present IL-15 in trans, from cell to cell, to the IL-2Rβ/γc complex (134) (Figure 6c). The disconnection of IL-15Rα from lying in the same membrane as the IL-2Rβ and γc components may require a more profound allostery for IL-15Rα to serve effectively as an afﬁnity converter for IL-15. In other words, the trans-signaling role of IL-15Rα necessitates that it enhance IL-15 binding to IL-2Rβ through space rather than simply through surface capture on the same cell membrane. Although trans-presentation is currently believed to be the major mechanism by which IL-15 exerts its biological effects in vivo, the assembly of a cis IL-15/IL-15Rα/IL-2Rβ/γc quaternary complex on the surface of the same cell is also used (131, 135) (Figure 6c).

IL-2Rβ/IL-2 AND IL-4Rα/IL-4 INTERFACES IL-2Rβ and IL-4Rα are functional and structural counterparts in their respective signaling complexes, and both form one of the two major signaling subunits in their γc receptor complexes. The ∼75-kDa human IL-2Rβ chain is composed of an extracellular domain of 214 residues, a transmembrane domain of 25 residues, and an intracellular domain of 286 residues (136). The ∼140-kDa human IL-4Rα chain has 207 residues in the extracellular domain, 24 in the transmembrane region, and 569 in the intracellular domain (137). Although the nomenclature is confusing, IL-2Rβ is analogous to the α receptors for other γc cytokines, but because IL-2 has the additional nonstandard initiating receptor, IL-2Rα, IL-2Rβ is then referred to as the β receptor on the basis of the sequence of interactions with IL-2. The intracellular domains of IL-2Rβ and IL4Rα possess the box 1 and box 2 motifs at the membrane-proximal region that constitute the binding sites for JAK1 (34). The cytokine binding–induced association of IL-2Rβ or IL4Rα with γc will bring their intracellular do-

mains into close proximity, inducing the activation of the JAK kinases (Figure 5a,b). After the capture of IL-2 by IL-2Rα, the delivery of IL-2 to IL-2Rβ in cis represents the second step in the formation of the quaternary IL-2 receptor complex (Figure 7a and Table 1). The binding interface between IL-2 and IL-2Rβ (site I) buries ∼1350 A˚ 2 and is formed by residues from helices A and C in IL-2 and loops CC 1, EF1, BC2, and FG2 in IL-2Rβ (Figure 8a). The interface is highly polar, with eight hydrogen bonds directly between IL-2 and IL-2Rβ residues and seven buried water molecules mediating the interactions between IL-2 and IL-2Rβ by forming hydrogen bonds with protein atoms (Figure 8a). Solvent exchange with the layer of water molecules between IL-2Rβ and IL-2 could explain the fast on and off rates and the weak afﬁnity of the IL-2/IL-2Rβ binary complex. Two residues of IL-2 that have been shown by mutagenesis to be critical for IL-2Rβ binding, Asp-20 (138) and Asn-88 (125), are involved in hydrogen bonding networks to both water molecules and side chains on IL-2Rβ. There is excellent knob-inhole shape complementarity between IL-2Rβ and IL-2 (Figure 8a). As mentioned, IL-2Rβ is also used by IL-15 to form a quaternary complex along with the IL-15Rα and γc (30, 133) (Figure 6c). IL-15 has limited sequence homology (19%) with IL-2, so its contacts with IL-2Rβ are almost certainly through a unique set of interactions. The apparently central role that water molecules play in bridging hydrogen bonds between IL-2Rβ and IL-2 would contribute to the ability of IL-2Rβ to cross-react through remodeling of this hydration layer to accommodate the IL-15 residues. The ﬁrst step in the formation of the IL-4/IL-4Rα/γc complex is the binding of IL-4 with IL-4Rα receptor (139, 140) (Figure 7b and Table 1). These interactions were ﬁrst elucidated in the IL-4/IL-4Rα binary complex (141). The comparison of IL-4/IL-4Rα binary and IL-4/IL-4Rα/γc ternary complex structures reveals that the engagement of γc does not cause substantial conformational changes

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in the mode of interaction with IL-4Rα. Minor differences in peripheral interface contacts between the IL-4 type I binary and ternary complexes are likely due to the differing resolutions of the structures and crystal packing forces. The interacting residues from helices A and C in IL-4 and loops CC 1, EF1, AB1, BC2, and FG2 in IL-4Rα, bury a total surface area of ∼1520 A˚ 2 (site I) (Figure 8a). The chemical nature of the IL-4/IL-4Rα interface is also highly polar, similar to that of the IL-2/IL-2Rβ interface. The interacting residues in the IL-4/IL-4Rα interface can be grouped into two major clusters centered at Glu-9 (IL-4) to Tyr-134 (IL-4Rα) and Arg-88 (IL-4) to Asp-72 (IL-4Rα), respectively, each containing an inner polar core surrounded by outer hydrophobic residues (Figure 8a). Considering there are only two bridging water molecules in the IL-4/IL-4Rα interface, the buried polar and charged interactions contribute to the rapid on rate (Kon ≈ 1.3 × 106 M−1 s−1 ) and slow off rate (Koff ≈ 2.1 × 10−3 s−1 ) that result in high-afﬁnity binding between IL-4 and IL-4Rα (142). An exhaustive double-mutant analysis of the IL-4-IL-4Rα interaction has been carried out by the Sebald group (143), and we refer the reader to this paper for a rigorous, energetic deconvolution of the cytokine-receptor interface. In summary, both the IL-2 and IL-4 interactions with their respective α chains are characterized by polar and charged contacts. This is consistent with the high degree of speciﬁcity these receptors show for their cytokine, in contrast to γc ’s degeneracy. Although not completely generalizable, polar and charged contacts primarily mediate speciﬁcity in proteinprotein interactions because their energetic content is greatly affected by the precise structural context within an interface (144). Van der Waals and hydrophobic interactions are more suitable to a promiscuous binding surface because these are less structure-selective interactions that are a result more of water exclusion than of speciﬁc structural context and pairwise atomic contacts (144).

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RECRUITMENT OF γc BY IL-2/IL-2Rαβ AND IL-4/IL-4Rα The interaction of the IL-2/IL-2Rα/IL-2Rβ ternary complex with γc is the last step in formation of the IL-2 signaling complex on activated T cells (Figure 7a and Table 1). IL2 has very low afﬁnity for γc alone (Kd ≈ 700 uM) (97, 98), requiring precomplexation with IL-2Rα/IL-2Rβ (in the high-afﬁnity complex) or IL-2Rβ alone (in the intermediateafﬁnity complex) to bind γc with Kd in the low nM range. IL-2Rβ and γc do not have a measurable afﬁnity for one another (97). The two weak interactions, IL-2 with γc and IL-2Rβ with γc , combine to produce a high afﬁnity for γc . In the complex structure, two contact surfaces, a small one between IL-2 and γc and a larger one between IL-2Rβ and γc , form the interaction surface between IL-2/IL-2Rαβ and γc (Figure 5a). The IL-2/γc binding surface (site IIa) reﬂects its degenerate recognition capabilities, showing a remarkable ﬂatness and almost tangential contact with IL-2 (Figure 8b). The IL-2/γc interface is the smallest of the four protein/protein interfaces in the complex, burying ∼970 A˚ 2 of surface area. In contrast to the IL-2Rβ interface, the γc binding surface is rather devoid of extended side chain–speciﬁc interactions with IL-2 and exhibits primarily main chain contact (Figure 8b). Unlike the IL-2/IL-2Rβ interface that has a broad array of speciﬁc polar interactions, the IL-2/γc interface is composed of small contact patches (Figure 8b). The ﬁrst one is composed of residue Tyr-103 from γc and residues Ser127 and Ser-130 from IL-2. In γc , the Tyr-103 side chain does not protrude outward toward IL-2 but is instead pinned back via a hydrogen bond with the Cys-209 main chain and is therefore positioned so that its aromatic ring packs ﬂat against the side chains of Ser-127 and Ser-130 in IL-2 (Figure 8b). The second contact patch is around residue Gln-126 in IL-2, whose side chain is ﬂattened and almost parallel to the surface formed by main chain atoms

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of residues Pro-207 to Ser-211 in the FG2 loop of γc (Figure 8b). The second part of the composite interface between IL-2/IL-2Rαβ and γc is formed by extensive interactions between the D2 domains of IL-2Rβ and γc (site IIb) (Figure 5a). The residues in the IL-2Rβ/γc interface bury over 1750 A˚ 2 of surface area, which is the most extensive seen so far between receptor domains in cytokine-receptor complexes. The IL-2Rβ/γc interface is predominantly composed of polar interactions, with a total of 17 hydrogen bonds surrounding a small hydrophobic stripe. After the initial binding of IL-4 with the IL-4Rα receptor, the association of γc is also mediated through a composite interface: IL4/γc and IL-4Rα/γc (Figure 7b and Table 1). The IL-4/γc interface buries a total surface of ∼1020 A˚ 2 , which is slightly larger than that of the IL-2/γc interface but still the smallest in the IL-4/IL-4Rα/γc complex (site IIa) (Figure 8b). The Tyr-103 residue and the FG2 loop ﬁxed by the Cys-160 to Cys-209 disulﬁde bond in γc interact with IL-4 in a binding mode similar to that of γc within the IL-2 receptor complex (Figure 8b). There is low sequence identity between IL-2 and IL-4, and so the contacting residues in IL-4 with Tyr-103 are Glu-122 and Ser-125, which still contact the aromatic ring of Tyr-103 through van der Waals contacts instead of forming speciﬁc polar interactions (Figure 8b). In the IL-4/γc binding interface, the Arg-121 residue from IL4, which replaces the critical Asn-126 position in IL-2, contacts with main chain atoms of residues Pro-207 to Ser-211 in γc through its long methylene side chain (Figure 8b). While maintaining these two binding epitopes that are also observed in the IL-2/γc interface, the IL-4/γc interface has an additional hydrophobic patch consisting of Tyr-124 in IL-4 and the interacting Leu-208 and Tyr-182 in γc (Figure 8b). Also contributing to the IL-4Rα/γc interface are extensive receptor-receptor contacts between the D2 domains of the respective CHR (site IIb) (Figure 5b). However, the packing between IL-4Rα/γc is less intimate than that

of the IL-2Rβ/γc interface, as evidenced by its much smaller buried surface (∼1200 A˚ 2 versus ∼1750 A˚ 2 ). The IL-4Rα/γc interface also has extensive polar interactions surrounding a small hydrophobic stripe in the center, dominated by Tyr-154 from IL-4Rα and Phe-186 from γc . The IL-4Rα-γc interaction is much weaker (Kd ∼ uM) (17) in the IL-4 ternary complex than that between IL-2Rβ and γc in the IL-2 quaternary complex (Table 1). The fact that receptor-receptor contacts seen in the respective complexes are less extensive explains much of this afﬁnity difference.

DEGENERATE CYTOKINE RECOGNITION BY γc Protein-protein interactions and, in fact, most receptor-ligand interactions are usually characterized by speciﬁcity for one ligand. Therefore, the molecular basis for γc recognition of six different cytokines has been not only a fundamental question in understanding cytokine recognition, but also a basic problem in physical chemistry. As a shared signaling subunit, the engagement of γc is the last step in the formation of functional signaling complexes (Figure 7a,b). On the basis of the structural information from the IL-2 quaternary complex and the IL-4 ternary complex, we can conclude that the preformed complexes of cytokines with the α receptors provide a composite binding site for γc that is composed of two interaction interfaces: cytokine/γc and α receptor/γc . The small buried surface area (∼970 A˚ 2 between IL-2 and γc and ∼1020 A˚ 2 between IL-4 and γc ) and the formation of relatively nonspeciﬁc atomic interactions characterize the cytokine/γc binding interface. In contrast, the α receptor/γc binding interface has a large buried surface area (∼1750 A˚ 2 between IL-2Rβ and γc and ∼1200 A˚ 2 between IL-4Rα and γc ) composed predominantly of speciﬁc polar interactions. Extensive mutagenesis work has identiﬁed Tyr-103, Cys-160, and Cys-209 in γc as critical residues for the engagement of all γc dependent cytokines (145–147). Because certain residues around these hot-spot positions

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were exclusively implicated in the binding of different cytokines, such as Ile-100 and Leu102 for IL-4 binding and Asn-44, Leu-161, and Glu-162 for IL-21 binding, the γc -binding sites for different cytokines overlap but are not identical (146, 147). The side chain of Tyr-103 does not form speciﬁc side chain interactions with residues in IL-2 and IL-4, but it presents its aromatic ring for contact with residues Ser127 and Ser-130 in IL-2 and Glu-122 and Ser125 in IL-4 (Figure 8b). Residues Cys-160 and Cys-209 form a unique disulﬁde bond that connects loops FG2 and BC2 in the D2 domain of γc (Figure 8b). This disulﬁde bond ﬁxes the bent conformation of loop FG2, where the main chain atoms from residues Ser-207 to Pro211 directly contact the methylene side chain of residue Gln-126 in IL-2 or Arg-121 in IL4 (Figure 8b). The critical role of these hotspot residues in γc is also illustrated by the fact that some of their mutations have been found in the human γc gene of X-SCID patients (88). Although the cytokines recognized by γc have an average 19% sequence identity among them, helix D is the most conserved region, which is also the major contact area used by γc dependent cytokines to bind γc . Phe-114 and Ile-115 are two strictly conserved positions at the N terminus of helix D in IL-2, but they are not involved in γc binding, and their hydrophobic side chains point into the helical core. Residue 126 in IL-2 helix D is a conserved Asn in the γc -dependent cytokines IL-9, IL-15, and IL-21, while IL-4 and IL-7 contain Arg121 and Lys-139 at this position. The Gln-126 in IL-2 and Arg-121 at this position in IL-4 both contribute to binding with γc in their respective complexes (Figure 8b). Although this position serves as a common contact point for γc , its importance in binding with γc can vary with different cytokines. In IL-2, Gln-126 is the major γc -binding determinant, and mutation of this site greatly reduces binding afﬁnity (148). In IL-4, Arg-121 is only one of the minor γc binding determinants, and the nearby residues Ile-11, Asn-15, and Tyr-124 serve as the major determinants (146).

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In the absence of obvious conserved hotspot residues in γc -dependent cytokines, shape complementarity appears to play a dominant role. This assertion is supported by the observation that both IL-2 and IL-4 provide a shallow groove that accommodates the protruding hot-spot binding residues in γc . In IL-2, the bulky side chains from residues Thr-123, Qln126, Ser-127, Ile-129, and Ser-130 form the walls of a canyon, which receives a protruding ridge on γc composed of Tyr-103 and the Cys-160 to Cys-209 disulﬁde bond (Figure 8b). The groove in IL-4 is surrounded by residues Thr-118, Arg-121, Glu-122, Tyr-124, and Ser125, which are in the same positions with those of IL-2 (Figure 8b). The formation of the groove for the placement of hot-spot residues in γc would only require that residues have side chains with similar volume and shape, which can be achieved without the need for strictly conserved residues. We expect that other γc dependent cytokines, IL-7, IL-9, IL-15, and IL-21, will also form a similar shallow groove on helix D that serves as the docking site for the protruding binding site on γc . The functional role of this groove appears to facilitate complex formation with γc by guiding a perfect geometrical alignment of the D2 domains of the cytokine-speciﬁc α receptor and γc , resulting in the numerous interatomic contacts (H-bonds, van der Waals, etc.) in the D2/D2 interface between α receptor and γc . In other words, the knob-in-hole shape complementarity between the groove on the cytokine and the protruding γc -binding site acts as a guide to align the receptor D2 domains for the intimate interaction that they exhibit.

RECRUITMENT OF IL-13Rα1 IN TYPE II IL-4 AND IL-13 RECEPTOR COMPLEXES As previously mentioned, IL-4Rα represents an important subfamily of γc -cytokine receptors in that it serves as a shared signaling receptor within three different cell surface complexes. IL-4Rα and IL-13Rα1 form receptor heterodimers on cells of nonhematopoietic

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stem cell origin, functioning as the type II receptor complex for both IL-4 and IL-13. IL-13Rα1 is derived from the same ancestral subgroup as γc (149), and its divergence derives from its extra N-terminal Ig-like domain that has contact with the dorsal surfaces of both IL-4 and IL-13 (site III) in IL-4/IL4Rα/IL-13Rα1 and IL-13/IL-4Rα/IL-13Rα1 complexes (Figure 5c,d ). The IL-13Rα1 D1 domain–binding sites on IL-4 and IL-13 have extensive overlap with the respective α receptor–binding sites on IL-2 and IL-15. In site III of both complexes, the C strand of IL13Rα1 interacts with the C-D strand of IL-4 and IL-13, forming an antiparallel beta sheet. The residues Trp-65 and Ile-78 on the C strand of IL-13Rα1 form a hydrophobic patch that opposes complementary hydrophobic residues in IL-13—these interactions are missing between IL-4 and IL-13Rα1 (Figure 5c,d ). Consistent with this structural data, mutational studies have shown that after deleting the D1 domain, the IL-13Rα1 D2D3 CHR module does not detectably bind to IL-13, but it can still form a ternary complex with IL-4 and IL-4Rα (17). This difference in the energetics of site III interactions between the respective cytokines likely explains the requirement of IL-13Rα1 D1 domain for signaling in the IL-13 type II complex, in contrast to the IL-4 type II complex (150). Although IL-4 and IL-13 use the same IL4Rα/IL-13Rα1 receptor heterodimer for signaling, the type II IL-4 and IL-13 complexes assemble in the reverse cooperative sequences (Figure 7c,d ). Similar to the type I IL-4 complex, type II IL-4 complex is formed by the initial high-afﬁnity binding of IL-4 with IL4Rα (site I) with a subnanomolar Kd , followed by the recruitment of IL-13Rα1 (site II and site III) with a much lower afﬁnity of 487 nM (Figure 7c and Table 1). In contrast, for the type II IL-13 complex, IL-13 ﬁrst binds to IL-13Rα1 with an afﬁnity of 30 nM, and the IL-13/IL-13Rα1 binary complex then recruits IL-4Rα with an afﬁnity of 20 nM (Figure 7d and Table 1). The driver (receptor for initial cytokine interaction) and trigger (receptor recruited for

signaling) terminology was previously proposed for the assembly of γc heterodimeric complexes (151). The type II IL-4 and IL-13 complexes have switched the driver and trigger in their respective assembly pathways. The recruitment of the trigger IL-4Rα in the type II IL-13 complex is more energetically favorable (Kd ≈ 20 nM) than the recruitment of the trigger IL-13Rα1 by the type II IL-4 complex (Kd ≈ 487 nM). Measurement of STAT6 phosphorylation induced by IL-4 and IL-13 in the human epithelial carcinoma cell line A549 (expressing IL-4Rα and IL-13Rα1, but not γc ) revealed that IL-4 is more potent (17). In this A549 cell line, the expression level of IL-13Rα1 is higher than that of IL-4Rα. In other cell lines where IL-13Rα1 expression level is limiting, IL-13 can become more potent than IL-4 in stimulating signaling (152). It was therefore proposed that when IL-13Rα1 is abundant, the highafﬁnity binding of IL-4 with its driver IL-4Rα would determine the signaling potency. When IL-13Rα1 is limiting, the IL-13 and IL-13Rα1 binding afﬁnity (Kd ≈ 30 nM) would still allow the efﬁcient formation of IL-13/IL-13Rα1 binary complex, and the subsequent relatively high-afﬁnity binding (Kd ≈ 20 nM) with trigger IL-4Rα could favor the formation of the type II IL-13 complex, resulting in more potent IL-13-induced signaling. These studies showed that membrane-proximal signaling events induced by a cytokine could be collectively inﬂuenced by many factors: the structural aspects of extracellular cytokine receptor interactions (e.g., receptor orientation and conformation), the concentration of cytokine, receptor expression level, the sequence of receptor assembly, and cytokine receptor binding afﬁnity. Each of these factors could potentially be manipulated to effect a therapeutic endpoint in this system that has obvious clinical importance for asthma.

COMPARISON OF γc WITH gp130 With a raft of structures of both γc and gp130 complexes with cytokines, we can now assess the similarities and differences between the structural mechanisms by which these

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a IL-2/γC

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Figure 9 Cross-reactivity of the cytokine-binding homology region (CHR) of γc and gp130. (a) Contact surface of γc and gp130 when bound to IL-2, IL-4, HHV-8 IL-6, IL-6, and LIF. Hydrophobic residues are colored as blue surface area and hydrophilic residues as red surface area. The helices and the contacting residues for each of the cytokines are shown docked onto the surface of the receptor. (b) Surface representations showing the contact surface of each cytokine. Note that IL-2 and IL-4 have a similar distribution of hydrophobic and hydrophilic residues in the contact area. For gp130 family cytokines, HHV-8 IL-6 has primarily hydrophobic contact surface area. The contact area on IL-6 is more polar, and LIF has the most signiﬁcant hydrophilic contact surface. (c) The packing environment of the common binding epitope residues Tyr-103 (γc ) and Phe-169 (gp130) against the cytokines.

shared receptors cross-react (Figure 9). The ectodomain of γc is composed of one CHR domain, whose elbow region at the interdomain boundary is the contact area for six different short-chain cytokines. While the ectodomain of gp130 is taller than that of γc and consists of one top-mounted Ig domain, one CHR module, and three extra membrane-proximal ﬁbronectin domains (Figure 3), the main gateway entry point for all long-chain gp130 family cytokines is also the elbow of the CHR module, analogous to γc (12). The structural analogies between the CHR modules on γc and gp130 are, then, very clear. Their cytokinebinding surfaces are similar in the distribu2.22

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tion of hydrophobic and hydrophilic residues, with a largely hydrophobic core shared region and discontinuous peripheral polar patches, but gp130 has more buried surface area within the cytokine-receptor interface compared with γc , consistent with gp130 engaging the larger longchain cytokines (Figure 9a). The buried surface area on γc contributing to IL-2 and IL-4 binding is ∼500 A˚ 2 , whereas the buried surface area on gp130 upon binding human IL6, human LIF, and viral IL-6 is 710, 700, and 610 A˚ 2 , respectively. Gp130 also has a larger core hydrophobic region of its interfaces. This can possibly be understood from the standpoint that gp130 is a shared receptor capable

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of binding to some cytokines, such as LIF and OSM, in the absence of an α receptor, therefore not requiring the receptor-receptor contact necessary in the γc complexes. γc , in contrast, requires an α receptor for all cytokine interactions and therefore has a smaller binding interface with cytokines, as the energetics are distributed across both α receptor and cytokine. In the hydrophobic core of the interfaces of both γc complexes, Tyr-103 is the hot-spot residue that contributes about 20% of the buried surface area upon binding with IL-2 and IL-4 (Figure 9c). Gp130 has Phe-169, an analog of Tyr-103 in γc , in the center of the hydrophobic core of its binding site, that contributes the largest fraction of buried surface area and is critical for ligand engagement of all cytokines (71, 153–155) (Figure 9c). Another noteworthy observation is the rigidity of the binding surfaces on γc and gp130. A comparison between the unliganded (153) and liganded forms of gp130 shows almost no rotameric ﬂexibility in the side chains of interacting residues. Although we do not have the structure of unliganded γc , the superimposition of γc structures onto the complex of IL-2 and IL-4 shows a similar rigidity of the side chains of interacting residues. Thus, conformational plasticity is most likely not used by either shared receptor as a means of cross-reactivity. More generally, the idea that conformational plasticity will be a mechanism to enable cross-reactivity in protein-ligand interactions has largely been supplanted by the observation that degeneracy can be provided simply through enthalpyentropy compensation of rigid interacting surfaces (13, 156). The binding epitopes on γc -dependent cytokines for γc are on helices A and D, whereas the binding epitopes for gp130 on the gp130 family cytokines are on helices A and C. The binding surfaces on IL-2 and IL-4 for γc are quite similar in the distribution of hydrophobic and hydrophilic residues (Figure 8b). Structurally nonidentical but positionally analogous residues on the respective γc cytokines form the grooves similar in topology and chemical nature for the docking of γc residue Tyr-103

and the loop formed by the Cys-160 to Cys209 disulﬁde bond (Figure 8b). In contrast, the surfaces contributing to the binding to gp130 in HHV-8 IL-6, LIF, and IL-6 are very different in the distribution of hydrophilic and hydrophobic residues (Figure 8b). The gp130-binding surface on HHV-8 IL-6 has the largest hydrophobic area, and the binding surface on IL-6 is signiﬁcantly more polar. The contact surface on LIF is the most polar, consistent with four well-deﬁned water molecules that participate in an intermolecular hydrogen bond network observed in the crystal structure of LIF in complex with gp130 (13). The topology and chemical nature of the observed grooves in gp130 family cytokines for the docking of Phe-169 from gp130 vary greatly between different cytokines (Figure 8c). The groove on HHV-8 IL-6 represents one extreme with a deep pocket. The surface grooves on human IL-6 and LIF that accommodate Phe-169 are more similar in overall topology, but are not as deep as observed in HHV-8 IL-6. In human IL-6, the Phe-169 does not sit deeply in the pocket, but instead packs directly against the side chains from IL-6. The gp130-binding groove on LIF is the most polar of all three cytokines. The polar head groups of hydrophilic residues from LIF form the walls of the pocket and direct the aromatic ring of Phe169 to pack against LIF in a similar fashion to the packing of Phe-169 against human IL-6. Analysis of the binding surfaces on γc and gp130 indicates that they use chemically inert complementary surfaces to bind to different cytokines, as opposed to adjusting their main chain and/or side chain conformations to interact speciﬁcally with divergent cytokine residues. This observation contrasts with notions of receptor promiscuity through binding site ﬂexibility (157). Because structural adaptation is not used by γc or gp130 as a means of cross-reactivity, the basis for degenerate recognition lies in the unique chemistry of the CHR epitope. Extensive thermodynamic studies of the binding between gp130 and different cytokines by ITC have revealed that their interactions are all primarily entropy driven, presumably because of desolvation of the

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interacting surfaces (LIF: 5 cal/molK; human IL-6: 45 cal/molK; OSM: 30 cal/molK; and CNTF: 62 cal/molK), albeit to varying extents commensurate with the surface polarity of the cytokine (10). We interpret these data to mean that gp130 uses desolvation as a structurally insensitive means of cross-reacting with structurally unique surfaces. The multiple solventexposed aromatic residues are likely covered with immobilized water clathrates in the unbound state, so that expulsion of the water into bulk solvent would be extremely entropically favorable. ITC analysis of γc interactions with the preformed IL-2/IL-2Rβ and IL2/IL-2Rα/IL-2Rβ complexes shows that the entropic contribution is smaller than that of gp130 (IL-2/IL-2Rβ: 4.85 cal/molK; IL-2/IL2Rα/IL-2Rβ: 0.72 cal/molK) (97). We still lack comparative data for the binding of γc with other γc -dependent cytokines, but we expect that γc uses similar thermodynamic solutions for the recognition of different cytokines. The entropy contribution may be smaller than that of gp130 because γc has a smaller contact area. Also, compared with gp130, the entropy contribution by the elbow region (site II) of γc to binding likely plays less of a role in cytokine recognition because of the extensive D2-D2 interactions between γc and the α receptors. The D2-D2 contacts are more polar in nature than are the cytokine-elbow region contacts and would presumably be more enthalpically driven, which would offset the entropy-driven recognition of the cytokine. Thus, as for gp130, γc uses enthalpy-entropy compensation to modulate its binding afﬁnity for diverse surface chemistries.

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CONCLUSIONS AND FUTURE PERSPECTIVES Gp130, βc , and γc —our principal shared cytokine receptors—appear to use a variety of both distinct and similar mechanisms to recognize diverse cytokines and ultimately to assemble into productive signaling complexes. The complexes exhibit the basic core template in which the CHR of the cytokine-speciﬁc α re2.24

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ceptors and the shared receptors engage the sides of the four-helix bundle in the typical site I/site II manner seen in homodimeric receptor complexes. The presence of an additional site III interacting Ig domain distinguishes gp130 from γc , and the antiparallel βc dimer demarks the most obvious signature of the βc complexes. In gp130, the heterodimers between gp130 and α receptors are nonproductive because in most cases the α receptors do not contain intracellular signaling motifs. Thus, site III is necessary to dimerize gp130, each of which is bound to a JAK, and signal. This stands in contrast to the γc family, in which the α receptors do contain intracellular signaling domains, and so the γc /α receptor heterodimers are productive: γc does not need to be dimerized. Another major difference is the extensive receptor-receptor contact seen between γc and the α receptors. The relatively energetic role of this contact versus the cytokine/γc contact remains to be determined, but it may be that the receptor-receptor contact between the D2 domains of the CHRs is the primary driving force for heterodimerization and that the role of the cytokines is to tip the energetic balance toward heterodimer formation. Finally, the chemical basis of degeneracy is also nicely paralleled in both receptors. Gp130 and γc are structurally rigid and present relatively ﬂat, hydrophobic binding sites for cytokine engagement that are rather devoid of highly charged and speciﬁc polar contacts. There are several important future questions that remain regarding the extracellular structures of these shared receptors. For gp130, several cytokines such as CNTF, OSM, and LIF heterodimerize gp130 with LIFR. Now that the basic template for dimerization of gp130 and LIFR has been elucidated by EM and structural studies, a high-resolution structure of the intact heterodimer remains an exciting puzzle to solve. In the γc family, with only two complex structures so far, there is some indication of the presence of a possible recognition code between cytokines and γc . This would be very exciting and different from gp130 cytokines, which do not appear to share any sequence or structural motifs necessary for gp130 engagement. The

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mechanism by which βc cross-reacts with IL-3 and IL-5 awaits additional complex structures for comparison with GM-CSF. However, the major structural frontier for cytokine receptors remains to obtain a better picture of the intracellular machinery. So far, we have no idea about the tertiary structure of any cytokine receptor intracellular domain. These regions may be unstructured unless bound to adaptors JAK and STAT. For STAT molecules, there are several structures now bound to DNA (158, 159) and also recently bound to phosphorylated peptides from

cytokine receptors (160). However, we do not have any full-length JAK structures. The crystal structures of kinase domains of JAKs are a step forward (161, 162), but ultimately we need to know how the N- and C-terminal ends of JAK communicate, as well as how the cytokine receptors box1 and box2 bind to JAK proteins. To fulﬁll these goals, higher-order imaging techniques such as EM and tomography will likely be used to obtain snapshots of entire cytokine receptors in lipid environments to preserve the integrity of the transmembrane regions.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS The authors acknowledge the contribution of Figure 4 from Michael Parker and Jack KingScott prior to publication. We also acknowledge helpful discussions with Warren Leonard, Nicole Hanick, Mathis Rickert, and Marty Boulanger. This work was supported by NIH (RO1-AI51321), Keck Foundation, Sandler Foundation, Damon Runyon Cancer Research Foundation, and the Howard Hughes Medical Institute. LITERATURE CITED 1. Garcia KC, ed. 2004. Advances in Protein Chemistry, Vol. 68. London/San Diego: Academic. 506 pp. 2. Dechant G, Barde YA. 2002. The neurotrophin receptor p75(NTR): novel functions and implications for diseases of the nervous system. Nat. Neurosci. 5:1131–36 3. Airaksinen MS, Saarma M. 2002. The GDNF family: signalling, biological functions and therapeutic value. Nat. Rev. Neurosci. 3:383–94 4. Rudolph MG, Stanﬁeld RL, Wilson IA. 2006. How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 24:419–66 5. Strong RK. 2002. Asymmetric ligand recognition by the activating natural killer cell receptor NKG2D, a symmetric homodimer. Mol. Immunol. 38:1029–37 6. Nicola NA. 1994. Cytokine pleiotropy and redundancy: a view from the receptor. Stem Cells 12(Suppl. 1):3–14 7. Taga T, Kishimoto T. 1995. Signaling mechanisms through cytokine receptors that share signal transducing receptor components. Curr. Opin. Immunol. 7:17–23 8. Ozaki K, Leonard WJ. 2002. Cytokine and cytokine receptor pleiotropy and redundancy. J. Biol. Chem. 277:29355–58 9. Donnelly RP, Sheikh F, Kotenko SV, Dickensheets H. 2004. The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain. J. Leukoc. Biol. 76:314–21 10. Boulanger MJ, Garcia KC. 2004. Shared cytokine signaling receptors: structural insights from the gp130 system. Adv. Protein Chem. 68:107–46 11. Chow D, He X, Snow AL, Rose-John S, Garcia KC. 2001. Structure of an extracellular gp130 cytokine receptor signaling complex. Science 291:2150–55 www.annualreviews.org • Structural Biology of Shared Cytokine Receptors

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Immunity to Respiratory Viruses Jacob E. Kohlmeier and David L. Woodland Trudeau Institute, Saranac Lake, New York 12983; email: [email protected]

Annu. Rev. Immunol. 2009. 27:3.1–3.22

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

lung, T cell, memory, inﬂuenza

This article’s doi: 10.1146/annurev.immunol.021908.132625

Abstract

c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

The respiratory tract is characterized by an extensive surface area that is in direct contact with the environment, posing a signiﬁcant problem for effective immune surveillance. Yet most respiratory pathogens are quickly recognized and controlled by a coordinated response involving the innate and adaptive arms of the immune system. The investigation of pulmonary immunity to respiratory viruses during a primary infection has demonstrated that multiple innate and adaptive immune mechanisms are necessary for efﬁcient antiviral responses, and the inhibition of any single mechanism can have disastrous consequences for the host. Furthermore, the investigation of recall responses in the lung has shown that protection from a secondary challenge infection is a complex and elegant process that occurs in distinct stages. In this review, we discuss recent advances that describe the roles of individual components during primary and secondary responses to respiratory virus infections and how these discoveries have added to our understanding of antiviral immunity in the lung.

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INTRODUCTION RSV: respiratory syncytial virus

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PRR: patternrecognition receptor Chemokines: a family of chemoattractant cytokines important for inﬂammation and immune system homeostasis

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The mucosal surfaces of the respiratory, intestinal, and genital tracts are the primary portals of entry for a wide range of pathogens. The lung in particular is in direct and continuous contact with the surrounding environment, sampling nearly 10 liters of air each minute. Despite this continual exposure to potential antigens, the lung is generally maintained in a quiescent, noninﬂamed state. However, once a pathogen establishes a productive infection in the lung, an orchestrated process of pathogen recognition, inﬂammatory cytokine production, and cell migration leads to the generation of robust adaptive immune responses that eradicate the invading organism while limiting collateral damage to the lung tissue. Respiratory viruses, such as inﬂuenza virus, parainﬂuenza virus, respiratory syncytial virus (RSV), severe acute respiratory syndrome coronavirus (SARS-CoV), rhinovirus, and adenovirus, are important human pathogens that establish acute infections usually localized to the upper respiratory tract. Despite the presence of these viruses in the population at endemic levels, it has taken the emerging threat of potential pandemics to thrust this class of pathogens into the public spotlight. For instance, the emergence of the highly pathogenic H5N1 inﬂuenza virus in 1997 has generated substantial public health concern owing to its extreme virulence and its potential to spread rapidly through the human population (1). Currently, there is considerable effort to develop improved vaccines capable of providing broad protection against these different types of viruses. However, achieving the goal of developing safe and effective vaccines to these pathogens has been complicated by our incomplete knowledge of how the immune system recognizes, contains, and eradicates respiratory viruses. An increasing number of reports have taken advantage of respiratory virus infection models in an attempt to develop a better understanding of mucosal immune responses in general and to provide new insight into speciﬁc pathogens that are major causes of morbidity

Kohlmeier

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and mortality worldwide. The use of small animal models to investigate immunity to these pathogens has generated a wealth of information regarding the dynamics of immune responses in the lung and has identiﬁed many individual components of the response necessary for the successful resolution of infection. Perhaps the best-characterized models are mouseadapted strains of inﬂuenza and murine parainﬂuenza viruses, which elicit robust T cell and B cell responses similar to infections in humans. The power of these models for understanding antiviral immunity in the lung has been enhanced by the advent of technologies for identifying antigen-speciﬁc responses and by the availability of a wide array of genetic tools. Here, we discuss recent advances in our understanding of antiviral immunity in the lung, from the initiation of innate and adaptive responses following primary virus infection to the recall of antigen-speciﬁc T cells during a secondary response, with a focus on lessons learned from murine models of inﬂuenza and parainﬂuenza virus infections.

INITIATION OF IMMUNE RESPONSES IN THE LUNG Innate Recognition of Infection A common feature of respiratory virus infections is that the initial infection is established in epithelial cells lining the respiratory tract. Epithelial cells, as well as alveolar macrophages and dendritic cells (DCs), continually sample the constituents of the airway lumen and detect the presence of an invading virus through pattern-recognition receptors (PRRs) (Figure 1) (2). The recognition of pathogen-associated molecular patterns by these receptors initiates a cascade of signals that results in the production of cytokines and chemokines. The release of these inﬂammatory mediators into the surrounding environment alerts the innate immune system to the presence of infection and establishes a localized antiviral state. In addition, chemokines provide the necessary signals for the recruitment

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Lung airways

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Lung parenchyma

Lung-draining lymph node

Respiratory dendritic cell

Naive T cell

Alveolar macrophage

Naive B cell

Red blood cell

Memory T cell

Lymph node dendritic cell

Virus-speciﬁc B cell

Figure 1 The resting pulmonary immune system. The lung airways, lung parenchyma, and lung-draining lymph nodes are three key sites of the antiviral immune response to respiratory viruses. In the absence of infection, alveolar macrophages and dendritic cells sample the constituents of the airway lumen for the presence of invading pathogens. Memory T cells from prior respiratory virus infections are localized to each of these sites, with large numbers of cells present in the airways and parenchyma for several months postinfection. Virus-speciﬁc B cells are also localized to each of these sites, with resting memory B cells widely distributed while long-lived plasma cells are localized primarily to lymphoid tissue associated with the respiratory tract and the bone marrow.

of circulating leukocytes to the site of infection. Finally, the combination of inﬂammatory cytokines and PRRs initiates the process of DC maturation and trafﬁcking that is re-

quired for the induction of adaptive immune responses. The best described of the PRRs are those of the Toll-like receptor (TLR) family, which in www.annualreviews.org • Immunity to Respiratory Viruses

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Type I interferons: a family of inﬂammatory cytokines, including IFN-α and IFN-β, produced in response to PRR signaling

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NK cell: natural killer cell

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mammals is composed of up to 15 unique receptors that are expressed by numerous cell types and recognize a wide range of microbial proteins, lipids, and nucleic acids (3). With respect to respiratory viruses, TLR3, 7, and 9 recognize various products of viral replication (dsRNA, ssRNA, and unmethylated CpG DNA, respectively) (4–7), whereas TLR4 recognizes the fusion (F) protein of RSV (8). TLRs that recognize nucleic acids are located in late endosomes. This location optimizes the TLRs’ ability to interact with viral nucleic acids while limiting their access to host-derived nucleic acids (9, 10). Although TLRs expressed on the cell surface (TLR4) or within the cell (TLR3, 7, 8, and 9) utilize different signaling pathways, each of these receptors can activate the transcription of interferon (IFN)-inducible genes (11). In addition, several recent reports have demonstrated that viral RNA is also recognized by several RNA helicases. Retinoic acid-inducible gene I (RIG-I) interacts with 5 -triphosphate RNA and is important for early cytokine production in response to numerous RNA viruses (12–15). Melanoma differentiation-associated gene 5 (MDA5) is a related helicase that recognizes polyinosinic polycytidylic acid and is crucial for innate recognition of picornaviruses (16). Similar to signaling through TLRs, the pathways utilized by RNA helicases ultimately trigger IFN regulatory factor (IRF) and nuclear factor-κB (NF-κB) activation (17). The key difference between these molecules and TLRs is that the RNA helicases are localized throughout the cytosol, rather than being regulated to intracellular compartments. Thus, viruses that infect cells by direct membrane fusion and do not enter endosomes can nevertheless trigger innate immune responses via RNA helicases.

The Early Inflammatory Response The innate recognition of viral components through PRRs described above leads to a program of gene expression that promotes a localized antiviral state and elicits the recruitment of inﬂammatory cells to the site of infection. 3.4

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Foremost among the early cytokines produced following infection are the pleiotropic antiviral cytokines of the type I interferon family, of which IFN-α and IFN-β are most commonly associated with early antiviral responses in the lung. Although nearly all cell types are capable of producing type I IFNs, numerous studies in mice and humans have shown that plasmacytoid DCs (pDCs) are the primary source of IFN-α and IFN-β following infection with a systemic virus (18). With respect to respiratory viruses, a recent study has provided in vivo evidence that alveolar macrophages are the primary producers of IFN-α during a parainﬂuenza virus infection and necessary for efﬁcient virus clearance (19). Notably, this study demonstrated that although IFN-α production by pDCs was largely TLR-dependent, alveolar macrophages required RIG-I signaling for optimal IFN-α production. However, the importance of alveolar macrophage-derived IFNα remains uncertain, as subsequent work has demonstrated that alveolar macrophage depletion had no effect on virus clearance during RSV infection (20). Therefore, type I IFN production in the lung appears to employ a level of redundancy, with alveolar macrophages or pDCs predominating depending on the type of virus infection. Type I IFNs produced following respiratory virus infections form a feedback loop by signaling through the IFN-α/β receptor and act in concert with PRR signaling to promote sustained production of proinﬂammatory cytokines such as TNF-α, IL-1, and IL-6 from lung-resident innate immune cells (21, 22). These proinﬂammatory cytokines and PRR-mediated signals also prompt alveolar macrophages, DCs, and epithelial cells to initiate a coordinated program of chemokine production following virus infection. DCs secrete successive waves of chemokines following inﬂuenza virus infection, beginning with those capable of recruiting inﬂammatory cells such as neutrophils and NK cells, and followed by chemokines associated with the recruitment of monocytes and memory T cells (23). Epithelial cells and alveolar macrophages also contribute

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to early chemokine production following infection and/or inﬂammation, particularly those chemokines capable of recruiting monocytes and memory T cells (24, 25). The chemokines produced during the innate immune response in the lung are capable of recruiting a wide range of innate and adaptive immune cell types. However, during a primary respiratory virus infection, the innate immune cells recruited to the lung are predominantly neutrophils and NK cells (Figure 2). The role that neutrophils play in respiratory virus clearance has not been well deﬁned, despite the large number of these cells present in the lung following infection. In fact, it has recently been shown that inhibition of neutrophil recruitment to the lung following inﬂuenza virus infection had no effect on the course of infection, suggesting that these cells do not play an essential role in virus clearance (26). In contrast, NK cells directly recognize inﬂuenza virus– and parainﬂuenza virus–infected cells through the interaction of the activating receptor NCR1 (NKp46 in humans) with hemagglutinin glycoprotein (27). NK cell recognition of virus-infected cells in coordination with proinﬂammatory cytokines resulted in enhanced cytolysis and IFN-γ production by NK cells (28–30). A recent deﬁnitive study investigating the importance of NK cells for protection against inﬂuenza virus showed a signiﬁcant increase in the number of NK cells in the lung beginning around day 3 postinfection. Importantly, this study also demonstrated that inﬂuenza virus infection was lethal in mice lacking the NK cell–activating receptor NCR1 (31). In addition to establishing a localized inﬂammatory environment and recruiting innate immune cells, PRR- and cytokine-mediated signals are important for the maturation and trafﬁcking of DCs to the draining lymph nodes preceding the initiation of the adaptive immune response. Under steady-state (i.e., noninﬂammatory) conditions, the trafﬁcking of DCs from the lung compartment to the draining lymph nodes is a continuous process that is dependent on the chemokine receptor CCR7 (32). The DCs that enter the lymph nodes under these

conditions are not fully mature, and it is believed that this process plays a role in the establishment of immune tolerance in the lung. Following inﬂuenza virus infection, lung-resident DCs increase expression of molecules involved in antigen presentation such as MHC class II, CD80, CD86, and CD40. Beginning as early as 6 h postinfection, there is an increase in the trafﬁcking of DCs from the lung to the draining lymph nodes that is maintained for several days (33, 34). In addition, the trafﬁcking of respiratory DCs to the lymphoid tissues is essential for the generation of adaptive immunity, as blocking DC migration abrogates antigen-speciﬁc T cell responses. Curiously, the trafﬁcking of DCs to the draining lymph nodes during both steady-state and inﬂammatory conditions is dependent on CCR7, suggesting that the accelerated recruitment following infection is not mediated by different chemokine receptors. However, investigators (35, 36) recently demonstrated that the interaction of the chemokine CCL5 with its receptor CCR5 is indirectly responsible for this enhanced trafﬁcking following infection by increasing the expression of CCR7 expression on DCs and enhancing migration across high endothelial venules. Thus, the innate recognition of infection leading to inﬂammatory cytokine and chemokine expression results in both the maturation and accelerated trafﬁcking of DCs, enabling more efﬁcient antigen presentation to T cells in the draining lymph nodes. A unique aspect of antiviral immunity in the lung is the potential for adaptive responses to be generated in local lymphoid structures such as nasal-associated lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) (37). These structures exhibit similar organization to encapsulated lymph nodes with distinct T and B cell zones, high endothelial venules, and the expression of homeostatic chemokines important for DC and naive T cell migration (38). Importantly, these structures can signiﬁcantly contribute to the antiviral response, as mice devoid of secondary lymphoid tissues are able to mount effective, albeit delayed, virusspeciﬁc T cell and B cell responses that are www.annualreviews.org • Immunity to Respiratory Viruses

BALT: bronchusassociated lymphoid tissue

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Innate response (3-6 days p.i.)

Adaptive response (7-10 days p.i.)

Lung airways

Lung airways

Lung parenchyma

Lung parenchyma

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Lung-draining lymph node

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Virus-speciﬁc B cell

Alveolar macrophage

Neutrophil

Red blood cell

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Lymph node dendritic cell

Eﬀector T cell

Naive T cell

Virus

Naive B cell Figure 2 Innate and adaptive immune responses during a primary respiratory virus infection. During the innate response, virus detection in the lung by PRRs initiates a program of cytokine and chemokine production that leads to the recruitment of neutrophils and NK cells from the circulation to the lung airways and lung parenchyma. The inﬂux of innate immune cells and the production of cytokines limits early virus replication prior to the adaptive response. Concurrently, activated, antigen-bearing DCs migrate to the lung-draining lymph node, where they interact with antigen-speciﬁc naive T cells and generate a population of differentiated effector T cells. During the adaptive response, effector CD4+ T cells provide help to virus-speciﬁc B cells within the lymph node, and effector CD4+ and CD8+ T cells exit the lymph node and migrate to the lung. Large numbers of effector T cells accumulate in the lung airways and lung parenchyma, and through the production of cytokines and the lysis of infected epithelial cells, virus is cleared around 10 days postinfection. It is important to note that virus-speciﬁc antibody, which is not illustrated, also plays a critical role in virus clearance. 3.6

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originated and propagated within the BALT (39). Although the contribution of these localized lymphoid structures to the overall immune response in mice with normal secondary lymphoid tissues is difﬁcult to dissect, the proximity of these structures to the site of virus replication, and their ability to support a robust and diverse adaptive response suggests that they may play an important role in antiviral immunity.

ADAPTIVE IMMUNITY DURING PRIMARY RESPONSES T Cell Responses Naive CD4+ and CD8+ T cells within lymphoid tissues continually scan the surface of DCs for the presence of cognate antigen/MHC complexes (40, 41). This dynamic process, combined with the protrusion of dendrites that increases the DC surface area and the circulation of naive T cells through the lymph node, enhances the probability that extremely small numbers of antigen-speciﬁc naive T cell precursors, which range from 20–1200 cells of a given speciﬁcity in mice, will come into contact with their cognate antigen and enter into a program of proliferation and differentiation (42–44). As antigen-bearing mature DCs enter the lung-draining lymph nodes following respiratory virus infection, naive T cells speciﬁc for that antigen form stable interactions with the DCs, and the signals delivered by antigen recognition through the T cell receptor in addition to accessory signals delivered through costimulatory molecules result in T cell priming (45, 46). The initial priming of naive antigenspeciﬁc CD4+ and CD8+ T cells occurs within 72 h following inﬂuenza virus infection and initiates a program of sustained proliferation resulting in the accumulation of large numbers of virus-speciﬁc effector T cells (47–49). The instructions delivered by DCs during this initial expansion phase can have a dramatic impact on the survival and function of the responding T cells. For example, expression of FasL on DCs following inﬂuenza infection has been shown to regulate the magnitude of the CD8+ T cell

response (50). In addition, factors such as TCR avidity, costimulation, and the local inﬂammatory milieu all contribute to the generation of differentiated effector T cells prior to their exit from the lymph node and subsequent trafﬁcking to the lung (51–54). The appearance of antigen-speciﬁc effector T cells at the site of virus infection (i.e., the lung airways and lung parenchyma) is ﬁrst observed around days 6–7 postinfection with inﬂuenza and parainﬂuenza viruses (Figure 2). Chemokines expressed in the lung are recognized by blood-borne effector T cells, leading to changes in integrin afﬁnity that allow for tight binding to the blood vessel wall and extravisation into the surrounding tissue (55, 56). Endothelial selectins are also important for this process, as mice lacking expression of the molecules or their receptors showed a dramatic decrease in the trafﬁcking of CD4+ and CD8+ T cells to the lung (57, 58). Having migrated from the circulation into the lung tissue, effector T cell–expressed adhesion molecules are important for movement and survival within the interstitial spaces and airways of the lung. In addition, the expression of β1 integrins has been shown to control the localization of effector T cells to distinct compartments of the lungs. For example, cells expressing the integrin α1β1 (VLA-1) are predominantly associated with collagen Type IV–rich areas surrounding the airways and blood vessels, whereas cells expressing α2β1 (VLA-2) are predominantly associated with the collagen Type I–rich areas of the interstitial spaces (59). Analyses of chemokine and chemokine receptor expression in the lung during the adaptive phase of the immune response have shown elevated expression of numerous molecules associated with effector T cell trafﬁcking (60, 61). Surprisingly, very few published reports have directly investigated the role of speciﬁc chemokine receptors in the trafﬁcking of effector T cells during acute respiratory virus infections. One reason for this gap may be that the presence of multiple chemotactic signals in the lung at this stage of infection and the redundant nature of the chemokine system have made www.annualreviews.org • Immunity to Respiratory Viruses

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dissecting the importance of individual receptors in effector T cell recruitment a difﬁcult task (62). Nevertheless, several studies have identiﬁed roles for speciﬁc chemokine receptors in effector T cell trafﬁcking to the lung under resting or inﬂammatory conditions using different (i.e., nonviral) models that may provide some insight for future studies employing respiratory virus infections. For example, effector CD8+ T cells require CCR5 for migration from the pulmonary vasculature into the lung parenchyma in naive, uninfected mice (63). Also, an analysis of CD4+ T cells to the lung airways in asthmatic humans has shown that CCR6 and CXCR3 may be important for their trafﬁcking to this site (64). With regard to respiratory viruses, the trafﬁcking of effector T cells during RSV infection is at least partially dependent on CX3CR1, suggesting a potential role for this chemokine receptor in other paramyxovirus infections (65). To elucidate a role for speciﬁc chemokine receptors during acute respiratory virus infections requires future studies focused on receptors that are known to contribute to recruitment during inﬂammation, combined with studies already conducted that have characterized the expression of chemokine receptors on respiratory virus–speciﬁc T cells (66, 67). The continual migration of effector T cells from lymphoid tissues during an acute infection results in a massive increase in the numbers of antigen-speciﬁc cells in the lung airways and lung parenchyma from days 7–10 postinfection (68). The arrival of effector T cells has an immediate and dramatic impact on the viral load through the expression of cytokines and the direct lysis of infected cells. Inﬂuenzaspeciﬁc CD4+ and CD8+ effector T cells in the lung predominantly produce IFN-γ and TNF-α, and CD4+ effector T cells also produce IL-2 and IL-10 (69–72). CD8+ effector T cells localize to the respiratory epithelium and induce apoptosis of infected epithelial cells through Fas-FasL interactions or the exocytosis of cytolytic granules containing perforin and granzymes (73, 74). Together, these effector mechanisms contribute to the rapid decline in viral load beginning around day 7 postinfec-

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tion and result in virus clearance around day 10 postinfection (75, 76).

B Cell Responses B cell responses and virus-speciﬁc antibody play an important role in the clearance of inﬂuenza virus during primary infection, especially during infection with a highly pathogenic virus (77). Studies with B cell–deﬁcient mice have shown that, although early virus control (days 3–6 postinfection) is not impaired, these mice fail to clear the virus and ultimately succumb to infection (78, 79). The protective effect of B cells in these studies appears to be mediated at least in part through the production of virus-speciﬁc IgM because mice lacking only this isotype had delayed virus clearance and increased mortality (80, 81). Also, virusspeciﬁc IgM has been shown to provide protection from inﬂuenza-induced pathology in the presence of T cells (82). Together, these studies clearly deﬁne a role for the early production of virus-speciﬁc IgM in B cell–mediated protection during inﬂuenza virus infection. However, it is important to note that a direct comparison of B cell–deﬁcient and IgM-deﬁcient mice found that B cell–deﬁcient mice were more susceptible to inﬂuenza virus infection (81). Therefore, the production of neutralizing isotype-switched, virus-speciﬁc antibody during the later stages of the primary response is required for optimal virus clearance and antibody-mediated protection (83). The conventional model of isotypeswitching involves direct contact between antigen-speciﬁc CD4+ T cells and antigenpresenting B cells in lymphoid tissues. This antigen-dependent interaction, in addition to CD40-CD40L interactions and cytokine signaling, drives B cell proliferation and antibody isotype switching (84). Although this model of B cell activation and antibody production is the primary mechanism for virus-speciﬁc antibody production during inﬂuenza virus infection, several studies have demonstrated that these interactions are not absolutely required for virus-speciﬁc IgA and IgG antibodies. For

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example, inﬂuenza virus–speciﬁc IgA production during the early stages of infection is CD4+ T cell dependent but does not require cognate T-B cell interactions (85). In addition, CD4 T cell–deﬁcient mice can generate virusspeciﬁc IgG, although the inﬂuenza-speciﬁc antibody titers are considerably decreased in these mice (79). Taken together, these studies demonstrate that both T cell–dependent and –independent mechanisms contribute to inﬂuenza-speciﬁc antibody production.

T CELL AND B CELL MEMORY TO RESPIRATORY VIRUSES T Cell Memory The peak of effector T cell numbers in the lung following inﬂuenza and parainﬂuenza virus infections generally occurs around 10 days postinfection and coincides with virus clearance. The resolution of infection and waning inﬂammation has a dramatic impact on the virus-speciﬁc T cell population, initiating a program of contraction in which 90–95% of effector T cells are deleted by apoptosis (86). The outcome of this process is the establishment of a stable pool of memory T cells that persists in both peripheral and lymphoid tissues (87–89). Considerable progress has been made in recent years identifying the cues that instruct antigenspeciﬁc effector T cells to develop into longlived memory T cells, the factors that maintain the memory T cell population over time, the anatomical location and trafﬁcking patterns of different memory T cell subsets, and the relationship between different memory T cell subsets and the efﬁcacy of the recall response. In the initial months following a respiratory virus infection, antigen-speciﬁc CD4+ and CD8+ T cells can be found throughout lymphoid and nonlymphoid tissues in mice and humans, with the highest frequency of these cells located in the lung airways and lung parenchyma (90–93). However, the number of antigen-speciﬁc T cells in the lung wanes over time, and by one year postinfection the frequency and number of antigen-speciﬁc T cells

is similar between the lung and other peripheral or lymphoid tissues (90, 94). Importantly, the decline in memory T cell numbers in the lung over time correlates with a decline in the ability of antigen-speciﬁc T cells to control viral load during a secondary challenge (95). The large number of antigen-speciﬁc T cells in the lung following respiratory virus infection is believed to be maintained by persistent depots of inﬂuenza antigens that are presented within the draining lymph nodes for several months following virus clearance (96, 97). This antigen depot is capable of inducing T cell activation (as measured by CD69 expression) and low levels of proliferation (as measured by CFSE dilution) for up to 60 days postinfection. However, the low level of proliferation supported by this antigen depot may be sufﬁcient to account for the higher number of antigen-speciﬁc T cells found in the lung for several months postinfection. In support of this hypothesis, the waning antigenspeciﬁc T cell numbers observed beginning several months postinfection coincides with the disappearance of prolonged antigen presentation. However, although antigen-speciﬁc T cell numbers at sites such as the lung airways decline for several months postinfection, this population of cells stabilizes at a low level and is maintained indeﬁnitely (94, 98, 99). Thus, once the depot of persistent antigen has been cleared, the low number of antigen-speciﬁc memory T cells found in the lung airways is maintained by a background level of recruitment from the circulation (100). Memory T cells generated by respiratory viruses are heterogeneous in terms of their phenotype and function. This heterogeneity has led to the classiﬁcation of memory T cells into two subsets based on their preferential migration of peripheral (effector memory T cells, TEM ) or lymphoid (central memory T cells, TCM ) tissues (101). These subsets can be delineated on the basis of expression of CD62L and CCR7, which direct entry into lymphoid tissues. The majority of virus-speciﬁc memory T cells present throughout the body 1– 6 months postinfection express a TEM phenotype (CD62Llo and CCR7− ) and preferentially www.annualreviews.org • Immunity to Respiratory Viruses

TEM : effector memory T cell TCM : central memory T cell

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migrate to nonlymphoid sites (67, 102). Over time, the systemic memory T cell pool undergoes a gradual conversion to a TCM phenotype (CD62Lhi and CCR7+ ) that results in their localization to the lymph nodes and also to the bone marrow (103–105). Although there is conﬂicting evidence regarding the relationship between the TEM and TCM lineages, current evidence suggests that these two subsets are distinct populations generated during the initial infection, and the outgrowth of the TCM population over time is due to increased homeostatic turnover (106, 107). Regardless of the relationship between TEM and TCM , the passage of time leads to a shift in the preferential localization of virus-speciﬁc memory T cells, and in turn alters the dynamics and efﬁcacy of the recall response. For several months following virus clearance, large numbers of TEM are present in the lung and are able to provide immediate antiviral effector functions upon secondary infection. Over time, however, the number of virus-speciﬁc TEM able to provide this immediate response in the lung dramatically declines. Instead, the majority of virus-speciﬁc cells exist as TCM present in lymphoid tissues capable of rapid proliferation and the generation of new effector T cells following a secondary challenge. Therefore, the ability of antigen-speciﬁc memory T cells to immediately recognize and respond to a secondary virus challenge at the site of infection is lost over time.

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B Cell Memory Similar to the kinetics of T cell memory, inﬂuenza-speciﬁc memory B cells are rapidly established in multiple tissues following virus clearance. However, it is evident that there are profound differences between T cell and B cell memory with regard to their generation, trafﬁcking, and maintenance. B cell memory is characterized by two distinct populations of cells: long-lived plasma cells that continually secrete antibody and memory B cells that persist in a quiescent state (108, 109). The generation of B cell memory, particularly the generation of long-lived plasma cells, is dependent on cog3.10

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nate T-B cell interactions and CD40 signaling that occurs in the germinal center (110, 111). In support of this ﬁnding, investigators demonstrated that the inﬂuenza virus–speciﬁc IgG response generated in CD40-deﬁcient mice rapidly wanes and is undetectable by 60 days postinfection (79). In addition, it has recently been demonstrated that invariant natural killer T cells can also provide B cell help and enhance IgG responses (112). Therefore, although the data demonstrate that contact-dependent interactions are required for the generation of longlived B cell memory, these interactions can involve different cell types. Following inﬂuenza virus clearance, plasma cells leave the germinal centers and migrate to the bone marrow, where they continue to secrete virus-speciﬁc antibody. In addition, longlived plasma cells secreting inﬂuenza virus– speciﬁc IgA are localized and maintained in lymphoid tissues lining the respiratory tract (113). In contrast, resting memory B cells are widely dispersed to many tissues, where they can remain for many months. Interestingly, these cells localize at a higher frequency in the lung tissue following inﬂuenza virus infection, suggesting that the nature of the infection may alter the migratory capacity of these cells (114). Although it is unclear whether speciﬁc adhesion molecules or chemokine receptors play a role in the tissue-speciﬁc migration of memory B cells, the localization of these cells to the lung would allow them to rapidly recognize and respond to a secondary inﬂuenza virus challenge.

RECALL RESPONSES TO SECONDARY CHALLENGE It is well accepted that neutralizing, virusspeciﬁc antibodies (humoral immunity) provide optimal protection against most respiratory viruses by blocking the ability of the virus to establish infection. However, many viruses have evolved mechanisms to circumvent antibody-mediated immunity, allowing for secondary infections of closely related virus strains. For example, variability in the coat proteins of inﬂuenza virus enables the virus to evade

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neutralizing antibody and allows yearly inﬂuenza epidemics (115). Therefore, antibodymediated protection alone, while effective against secondary infection with a homologous virus, does not protect against variant viruses that arise through mutation. In contrast, the internal proteins of inﬂuenza virus, where many T cell epitopes are located, are highly conserved across many strains. Therefore, memory T cells speciﬁc for internal virus antigens are able to mount recall responses against heterologous virus strains (cellular immunity) and can provide broader protection against secondary challenge. In addition, non-neutralizing antibody to conserved internal proteins can enhance the memory T cell response to an inﬂuenza challenge (116). This section focuses on the experimental evidence that has been generated describing the cellular immune response to secondary virus infection in the absence of neutralizing antibody.

Lung Conditioning Much has been learned regarding the factors that inﬂuence memory T cell recall responses in animal models. However, a key consideration when interpreting the data from animal models is that these studies are often performed on speciﬁc pathogen-free mice. Although this limits the number of variables that could impact the results of an experiment, the immune response in a completely naive lung may not reﬂect the normal situation (117, 118). For example, the lungs of naive mice are devoid of lymphoid structures, such as BALT, that are present after respiratory virus clearance (119). Another issue is that prior inﬂuenza virus infections alter the responses to unrelated pathogens (120). Infection history can dramatically alter the clinical outcome of new infections depending on both the type of pathogen and order in which different pathogens are encountered (121). These studies have suggested a role for prior infections or inﬂammation in modifying the lung environment, thereby altering the manner in which the innate immune system reacts to subsequent inﬂammatory cues (122).

T Cell Recall Responses in the Lung As stated previously, the resolution of a primary respiratory virus infection generates a substantial number of antigen-speciﬁc memory T cells that are localized to both lymphoid and peripheral sites, such as the lung airways and the lung parenchyma. Several seminal studies have demonstrated that these respiratory virus– speciﬁc memory T cells mediate accelerated virus clearance and enhance survival following secondary challenge with related viruses (95, 123). More recently, evidence has emerged that memory T cell responses are far more complex than previously appreciated and that distinct populations of memory T cells segregated by their anatomical location and migratory capacity mediate different stages of the recall response. Thus, the recall response can be divided temporally, based on when these populations encounter virus-infected cells in the lung, and functionally, based on the steps required for their accumulation at this site (Figure 3). Importantly, it is the combination of these stages (discussed below) that results in the enhanced speed and magnitude of the recall response. Virus-speciﬁc memory T cells in the lung airways are believed to provide an initial line of defense against a secondary infection because these cells would be the ﬁrst to encounter antigen (124). Virus-speciﬁc memory T cells are present in considerable numbers for at least several months postinfection, despite their proximity to the harsh external environment and presence of mucus and surfactants (94, 125, 126). However, likely owing to the harsh environment, this population of cells is highly dynamic and is maintained by a process of continual recruitment from the circulation (100). Although lung airway memory T cells lack effector functions such as cytolytic activity (127), these cells are able to produce cytokines in response to antigen or inﬂammatory cytokines (98). Importantly, a direct role for these cells in protective immunity was demonstrated by the transfer of antigen-speciﬁc memory T cells to the lung airways of naive mice, which resulted in signiﬁcantly reduced viral titers following infection (124). Therefore, the ﬁrst stage of the www.annualreviews.org • Immunity to Respiratory Viruses

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Early stage of recruitment (2-5 days p.i.)

Late stage of recruitment (4-7 days p.i.)

Lung airways

Lung airways

Lung parenchyma

Lung parenchyma

Lung-draining lymph node

Lung-draining lymph node

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Memory T cell

Alveolar macrophage

Virus-speciﬁc B cell

Red blood cell

Neutrophil

Lymph node dendritic cell

NK cell

Naive T cell

Eﬀector T cell

Naive B cell

Virus

Figure 3 Dynamics of the T cell recall response to secondary virus challenge. Similar to a primary response, neutrophils and NK cells are recruited to the lung in response to localized inﬂammation. In addition, circulating memory CD8+ T cells are also recruited from the circulation to the lung in a CCR5-dependent manner during the early phase of infection, thereby increasing the number of antigen-speciﬁc cells at the site of infection and limiting virus replication. Memory T cells in lymphoid tissues are activated by antigenbearing DCs from the lung, and, owing to their increased precursor frequency and activation status compared with naive T cells, they are able to rapidly generate large numbers of secondary effector T cells. Secondary effector T cells migrate to the lung airways and lung parenchyma and mediate rapid virus clearance. Although not illustrated, the generation of secondary adaptive immune responses can also occur in lymphoid tissues (such as BALT) within the lung. 3.12

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recall response involves cells present at the site of infection that limit viral replication during the ﬁrst several days of infection while antigenbearing DCs are trafﬁcking to lymphoid tissues. An established paradigm of secondary T cell responses is the more rapid generation of effector T cells compared with a primary T cell response. Despite this more rapid generation, in respiratory virus infections this still allows for approximately four days between initial virus infection and the appearance of secondary effector T cells in the lung. However, several groups have demonstrated a dramatic increase in the number of virus-speciﬁc CD8+ T cells in the lung airways prior to the appearance of secondary effector T cells (128, 129). This increase in virus-speciﬁc CD8+ T cells could not be accounted for by localized proliferation within the airways, as these cells had not recently divided and the increase in cell number did not require cognate antigen stimulation. Rather, these studies showed that increased numbers of virusspeciﬁc CD8+ T cells in the airways was due to the inﬂammation-dependent recruitment of nondividing memory T cells from the circulation (130). Thus, the recruitment of circulating memory CD8+ T cells to the lung airways in response to inﬂammation serves to increase the number of antigen-speciﬁc memory T cells at the site of virus replication prior to the secondary effector T cell response. Although these studies had clearly demonstrated that the recruitment of memory CD8+ T cells occurs in response to localized inﬂammation, the mechanism that directs circulating memory CD8+ T cells to the lung airways and the importance of this process during secondary respiratory virus challenge had not been determined. Recently, we demonstrated that CCR5 expressed on memory CD8+ T cells was required for their recruitment to the airways during the early stages of the recall response (131). The role for CCR5 in memory CD8+ T cell recruitment is unique for the lung airways, as no defect in recruitment was observed in the lung parenchyma. Importantly, inhibiting memory CD8+ T cell recruitment to the airways resulted in signiﬁcantly higher

virus titers during the early phase of infection. Therefore, the second stage of the recall response involves the CCR5-dependent recruitment of circulating memory CD8+ T cells to the lung airways, resulting in increased numbers of antigen-speciﬁc cells at the site of virus replication and a further decrease in viral load. The ﬁnal stage of the recall response involves the activation of memory T cells in the draining lymphoid tissue by antigen-bearing DCs, resulting in the proliferation and expansion of secondary effector T cells that can migrate to the lung and eradicate the infection. Although this process occurs in a similar manner during both primary and secondary infections, the reduced stimulatory requirements, more rapid acquisition of effector functions, and increased precursor frequency of memory T cells compared with naive T cells allows for the accelerated generation of effector T cells (132, 133). Despite the reduced activation requirements of memory T cells, professional antigenpresenting cells (i.e., DCs) are still required for the optimal generation of secondary effector T cells during a recall response (134). The importance of memory T cell priming by DCs during an inﬂuenza virus infection was shown when inﬂuenza-speciﬁc memory CD8+ T cells transferred into mice lacking bone marrow–derived DCs failed to provide protection from virus challenge (135). The heterogeneity of the memory T cell population can also have a substantial impact on the magnitude of the secondary effector T cell response. We have shown that the ability of memory CD8+ T cells to proliferate and generate effector T cells that accumulate in the lung following secondary virus infection improves over time, and this improvement in recall efﬁcacy was observed in both the TEM and TCM populations (67). However, there have been conﬂicting reports regarding the relative contributions of the TEM and TCM populations to recall responses (136–140). The discrepancy in these ﬁndings suggested that the division of memory CD8+ T cell subsets solely between effector and central memory cells was insufﬁcient to describe their recall potential. A more www.annualreviews.org • Immunity to Respiratory Viruses

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thorough examination of the TEM and TCM subsets revealed that both populations could be further delineated based on the expression of activation markers such as CD27, CD43, and killer cell lectin-like receptor G1 (KLRG1) (141). Similar to the expression of CD62L that deﬁnes the TEM and TCM subsets, the distribution of the memory CD8+ T cell pool deﬁned by these activation markers changes over time, so that by one year postinfection most cells display a resting (CD27hi CD43lo ) phenotype. Importantly, the memory T cell subsets deﬁned by activation marker expression showed substantial differences in their ability to mount recall responses to respiratory viruses. Memory CD8+ T cells with the most activated phenotype (CD27lo CD43lo ) were 5- to 20-fold less efﬁcient at generating secondary effector T cells that could accumulate in the lung than were memory CD8+ T cells with the most resting phenotype (CD27hi CD43lo ) (141). Therefore, the third and ﬁnal stage of the recall response depends on the stimulation of memory T cells by DCs in the lymphoid tissues that give rise to a new population of secondary effector T cells. Furthermore, the ability of memory T cells to mount a robust recall response depends on the activation status of these cells when they re-encounter antigen.

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CONCLUDING REMARKS Our understanding of the mechanisms that the immune system employs to identify and eliminate acute respiratory viruses has grown considerably over the past decade. The discovery of different PRRs and the role they play in the initiation of the immune response has illustrated how the innate immune system distinguishes between host and pathogen. The dissection of the inﬂammatory response into individual cytokines and chemokines has allowed us to determine the importance of these molecules for protective responses, and to determine the contribution of different cell types to antiviral immunity. The characterization of memory T cell and B cell subsets has demonstrated the importance of these populations for protection from a secondary challenge and shown how different subsets of these cells contribute to the recall response. However, it is apparent that the various facets of antiviral immunity in the lung are far more complex than originally thought, and subtleties discovered so far hint at additional issues that must be resolved. Future studies investigating the innate and adaptive immune responses in ever greater detail will be required to develop a comprehensive picture of antiviral immunity in the lung.

SUMMARY POINTS 1. Viral nucleic acids recognized by TLR3, 7, and 9 in the endosome, or by RNA helicases in the cytosol, initiate a signaling cascade that activates IRFs and results in the production of type I IFNs. 2. Inﬂammatory signals trigger the production of chemokines by epithelial cells, alveolar macrophages, and DCs that attract innate immune cells prior to the generation of adaptive immunity. The early recruitment of NK cells to the lung and their recognition of virus-infected cells by the activating receptor NCR1 are essential for protection from an inﬂuenza virus infection. 3. The appearance of virus-speciﬁc effector T cells expressing cytokines and cytolytic molecules in the lung and the production of virus-speciﬁc IgM and IgG by B cells in the lymphoid tissue combine to resolve acute respiratory virus infections within approximately 10 days postinfection. 4. Following virus clearance, memory T cells are established in lymphoid and peripheral tissues, including a large population of antigen-speciﬁc cells that is localized to the lung

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airways and lung parenchyma. Virus-speciﬁc memory B cells are localized to the lymphoid tissue associated with the respiratory tract (primarily IgA-secreting cells) and the bone marrow (primarily IgG-secreting cells).

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5. Changes in the systemic memory T cell pool from a predominantly TEM to TCM phenotype over time, coupled with the disappearance of residual antigen in the lymph nodes, results in the redistribution of virus-speciﬁc memory T cells from peripheral to lymphoid tissues. 6. The recall response of memory T cells to a secondary virus infection in the lung airways can be separated into three distinct stages. The ﬁrst phase involves virus-speciﬁc memory T cells located in the lung airways, the second phase involves the CCR5-dependent recruitment of circulating memory T cells to the airways, and the third stage involves the appearance of secondary effector T cells that were generated in lymphoid tissue.

FUTURE ISSUES 1. How do different DC subsets in the lung impact the innate response to virus and inﬂuence the quality of the adaptive immune response? 2. Which chemokine receptors, or combination of chemokine receptors, are required for the migration of effector T cells to the various compartments of the lung? 3. Does tissue-speciﬁc imprinting occur during a respiratory virus infection that directs the preferential migration of memory T cells and memory B cells to the lung? 4. How is T cell memory generated during a respiratory virus infection, and what are the relationships between memory T cells that express different activation markers. 5. How do previous infections alter the lung environment and what are the consequences of these alterations for innate immunity? 6. By what mechanisms do memory T cells in the lung airways limit early virus replication during a secondary infection, and do these cells inﬂuence the development of the secondary immune response by altering the course of infection?

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We thank M. Blackman for a critical reading of the manuscript and A. Bernat for graphic design. This work was supported by funds from the Trudeau Institute and by NIH grants AI067967, AI076499, AG021600, and T32 AI49823 to D.L.W., and F32 AI071478 to J.E.K. LITERATURE CITED 1. Horimoto T, Kawaoka Y. 2001. Pandemic threat posed by avian inﬂuenza A viruses. Clin. Microbiol. Rev. 14:129–49 www.annualreviews.org • Immunity to Respiratory Viruses

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89. Joshi NS, Cui W, Chandele A, Lee HK, Urso DR, et al. 2007. Inﬂammation directs memory precursor and short-lived effector CD8+ T cell fates via the graded expression of T-bet transcription factor. Immunity 27:281–95 90. Marshall DR, Turner SJ, Belz GT, Wingo S, Andreansky S, et al. 2001. Measuring the diaspora for virus-speciﬁc CD8+ T cells. Proc. Nat. Acad. Sci. USA 98:6313–18 91. Cauley LS, Cookenham T, Miller TB, Adams PS, Vignali KM, et al. 2002. Cutting edge: virus-speciﬁc CD4+ memory T cells in nonlymphoid tissues express a highly activated phenotype. J. Immunol. 169:6655–58 92. Masopust D, Vezys V, Usherwood EJ, Cauley LS, Olson S, et al. 2004. Activated primary and memory CD8 T cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin. J. Immunol. 172:4875–82 93. de Bree GJ, van Leeuwen EM, Out TA, Jansen HM, Jonkers RE, van Lier RA. 2005. Selective accumulation of differentiated CD8+ T cells speciﬁc for respiratory viruses in the human lung. J. Exp. Med. 202:1433–42 94. Hogan RJ, Usherwood EJ, Zhong W, Roberts AD, Dutton RW, et al. 2001. Activated antigen-speciﬁc CD8+ T cells persist in the lungs following recovery from respiratory virus infections. J. Immunol. 166:1813–22 95. Liang S, Mozdzanowska K, Palladino G, Gerhard W. 1994. Heterosubtypic immunity to inﬂuenza type A virus in mice. Effector mechanisms and their longevity. J. Immunol. 152:1653–61 96. Jelley-Gibbs DM, Brown DM, Dibble JP, Haynes L, Eaton SM, Swain SL. 2005. Unexpected prolonged presentation of inﬂuenza antigens promotes CD4 T cell memory generation. J. Exp. Med. 202:697–706 97. Zammit DJ, Turner DL, Klonowski KD, Lefrancois L, Cauley LS. 2006. Residual antigen presentation after inﬂuenza virus infection affects CD8 T cell activation and migration. Immunity 24:439–49 98. Marsland BJ, Harris NL, Camberis M, Kopf M, Hook SM, Le Gros G. 2004. Bystander suppression of allergic airway inﬂammation by lung resident memory CD8+ T cells. Proc. Nat. Acad. Sci. USA 101:6116–21 99. Kohlmeier JE, Miller SC, Woodland DL. 2007. Cutting edge: antigen is not required for the activation and maintenance of virus-speciﬁc memory CD8+ T cells in the lung airways. J. Immunol. 178:4721–25 100. Ely KH, Cookenham T, Roberts AD, Woodland DL. 2006. Memory T cell populations in the lung airways are maintained by continual recruitment. J. Immunol. 176:537–43 101. Sallusto F, Lenig D, Forster R, Lipp M, Lanzavecchia A. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708–12 102. Masopust D, Vezys V, Marzo AL, Lefrancois L. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291:2413–17 103. Wherry EJ, Teichgraber V, Becker TC, Masopust D, Kaech SM, et al. 2003. Lineage relationship and protective immunity of memory CD8 T cell subsets. Nat. Immunol. 3:225–34 104. Mazo IB, Honczarenko M, Leung H, Cavanagh LL, Bonasio R, et al. 2005. Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells. Immunity 22:259–70 105. Becker TC, Coley SM, Wherry EJ, Ahmed R. 2005. Bone marrow is a preferred site for homeostatic proliferation of memory CD8 T cells. J. Immunol. 174:1269–73 106. Marzo AL, Klonowski KD, Le Bon A, Borrow P, Tough DF, Lefrancois L. 2005. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6:793–99 107. Moulton VR, Bushar ND, Leeser DB, Patke DS, Farber DL. 2006. Divergent generation of heterogeneous memory CD4 T cells. J. Immunol. 177:869–76 108. Bachmann MF, Kundig TM, Odermatt B, Hengartner H, Zinkernagel RM. 1994. Free recirculation of memory B cells versus antigen-dependent differentiation to antibody-forming cells. J. Immunol. 153:3386–97 109. Slifka MK, Antia R, Whitmire JK, Ahmed R. 1998. Humoral immunity due to long-lived plasma cells. Immunity 8:363–72 110. Noelle RJ, Roy M, Shepherd DM, Stamenkovic I, Ledbetter JA, Aruffo A. 1992. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Nat. Acad. Sci. USA 89:6550–54

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111. Lee BO, Moyron-Quiroz J, Rangel-Moreno J, Kusser KL, Hartson L, et al. 2003. CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses. J. Immunol. 171:5707–17 112. Galli G, Pittoni P, Tonti E, Malzone C, Uematsu Y, et al. 2007. Invariant NKT cells sustain speciﬁc B cell responses and memory. Proc. Nat. Acad. Sci. USA 104:3984–89 113. Liang B, Hyland L, Hou S. 2001. Nasal-associated lymphoid tissue is a site of long-term virus-speciﬁc antibody production following respiratory virus infection of mice. J. Virol. 75:5416–20 114. Joo HM, He Y, Sangster MY. 2008. Broad dispersion and lung localization of virus-speciﬁc memory B cells induced by inﬂuenza pneumonia. Proc. Nat. Acad. Sci. USA 105:3485–90 115. Stamboulian D, Bonvehi PE, Nacinovich FM, Cox N. 2000. Inﬂuenza. Infect. Dis. Clin. N. Am. 14:141–66 116. Rangel-Moreno J, Carragher DM, Misra RS, Kusser K, Hartson L, et al. 2008. B cells promote resistance to heterosubtypic strains of inﬂuenza via multiple mechanisms. J. Immunol. 180:454–63 117. Goulding J, Snelgrove R, Saldana J, Didierlaurent A, Cavanagh M, et al. 2007. Respiratory infections: Do we ever recover? Proc. Am. Thorac. Soc. 4:618–25 118. Kim EY, Battaile JT, Patel AC, You Y, Agapov E, et al. 2008. Persistent activation of an innate immune response translates respiratory viral infection into chronic lung disease. Nat. Med. 14:633–40 119. Moyron-Quiroz JE, Rangel-Moreno J, Hartson L, Kusser K, Tighe MP, et al. 2006. Persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs. Immunity 25:643– 54 120. Walzl G, Tafuro S, Moss P, Openshaw PJ, Hussell T. 2000. Inﬂuenza virus lung infection protects from respiratory syncytial virus-induced immunopathology. J. Exp. Med. 192:1317–26 121. Chen HD, Fraire AE, Joris I, Welsh RM, Selin LK. 2003. Speciﬁc history of heterologous virus infections determines antiviral immunity and immunopathology in the lung. Am. J. Pathol. 163:1341–55 122. Williams AE, Edwards L, Humphreys IR, Snelgrove R, Rae A, et al. 2004. Innate imprinting by the modiﬁed heat-labile toxin of Escherichia coli (LTK63) provides generic protection against lung infectious disease. J. Immunol. 173:7435–43 123. Cerwenka A, Morgan TM, Dutton RW. 1999. Naive, effector, and memory CD8 T cells in protection against pulmonary inﬂuenza virus infection: homing properties rather than initial frequencies are crucial. J. Immunol. 163:5535–43 124. Hogan RJ, Zhong W, Usherwood EJ, Cookenham T, Roberts AD, Woodland DL. 2001. Protection from respiratory virus infections can be mediated by antigen-speciﬁc CD4+ T cells that persist in the lungs. J. Exp. Med. 193:981–86 125. Ostler T, Hussell T, Surh CD, Openshaw P, Ehl S. 2001. Long-term persistence and reactivation of T cell memory in the lung of mice infected with respiratory syncytial virus. Eur. J. Immunol. 31:2574–82 126. Wiley JA, Hogan RJ, Woodland DL, Harmsen AG. 2001. Antigen-speciﬁc CD8+ T cells persist in the upper respiratory tract following inﬂuenza virus infection. J. Immunol. 167:3293–99 127. Vallbracht S, Unsold H, Ehl S. 2006. Functional impairment of cytotoxic T cells in the lung airways following respiratory virus infections. Eur. J. Immunol. 36:1434–42 128. Chen HD, Fraire AE, Joris I, Brehm MA, Welsh RM, Selin LK. 2001. Memory CD8+ T cells in heterologous antiviral immunity and immunopathology in the lung. Nat. Immunol. 2:1067–76 129. Topham DJ, Castrucci MR, Wingo FS, Belz GT, Doherty PC. 2001. The role of antigen in the localization of naive, acutely activated, and memory CD8+ T cells to the lung during inﬂuenza pneumonia. J. Immunol. 167:6983–90 130. Ely KH, Cauley LS, Roberts AD, Brennan JW, Cookenham T, Woodland DL. 2003. Nonspeciﬁc recruitment of memory CD8+ T cells to the lung airways during respiratory virus infections. J. Immunol. 170:1423–29 131. Kohlmeier JE, Miller SC, Smith J, Lu B, Gerard C, et al. 2008. The chemokine receptor CCR5 plays a key role in the early memory CD8+ T cell response to respiratory virus infections. Immunity 29:101–13 132. Seder RA, Ahmed R. 2003. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nat. Immunol. 4:835–42 133. Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, et al. 2003. Duration of antiviral immunity after smallpox vaccination. Nat. Med. 9:1131–37 134. Zammit DJ, Cauley LS, Pham QM, Lefrancois L. 2005. Dendritic cells maximize the memory CD8 T cell response to infection. Immunity 22:561–70 www.annualreviews.org • Immunity to Respiratory Viruses

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135. Castiglioni P, Hall de S, Jacovetty EL, Ingulli E, Zanetti M. 2008. Protection against inﬂuenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells. J. Immunol. 180:4956–64 136. Roberts AD, Woodland DL. 2004. Cutting edge: effector memory CD8+ T cells play a prominent role in recall responses to secondary viral infection in the lung. J. Immunol. 172:6533–37 137. Vaccari M, Trindade CJ, Venzon D, Zanetti M, Franchini G. 2005. Vaccine-induced CD8+ central memory T cells in protection from simian AIDS. J. Immunol. 175:3502–7 138. Bachmann MF, Wolint P, Schwarz K, Oxenius A. 2005. Recall proliferation potential of memory CD8+ T cells and antiviral protection. J. Immunol. 175:4677–85 139. Stock AT, Jones CM, Heath WR, Carbone FR. 2006. Cutting edge: central memory T cells do not show accelerated proliferation or tissue inﬁltration in response to localized herpes simplex virus-1 infection. J. Immunol. 177:1411–15 140. Klonowski KD, Marzo AL, Williams KJ, Lee SJ, Pham QM, Lefrancois L. 2006. CD8 T cell recall responses are regulated by the tissue tropism of the memory cell and pathogen. J. Immunol. 177:6738–46 141. Hikono H, Kohlmeier JE, Takamura S, Wittmer ST, Roberts AD, Woodland DL. 2007. Activation phenotype, rather than central- or effector-memory phenotype, predicts the recall efﬁcacy of memory CD8+ T cells. J. Exp. Med. 204:1625–36

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Review in Advance first posted online on November 13, 2008. (Minor changes may still occur before final publication online and in print.)

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Immune Therapy for Cancer Michael Dougan and Glenn Dranoff

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Department of Medical Oncology and Cancer Vaccine Center, Dana-Farber Cancer Institute and Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; email: glenn [email protected]

Annu. Rev. Immunol. 2009. 27:4.1–4.35

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

monoclonal (antibody), inﬂammation, vaccine, adjuvants, immunotherapy

This article’s doi: 10.1146/annurev.immunol.021908.132544 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0001$20.00

Abstract Over the past decade, immune therapy has become a standard treatment for a variety of cancers. Monoclonal antibodies, immune adjuvants, and vaccines against oncogenic viruses are now well-established cancer therapies. Immune modulation is a principal element of supportive care for many high-dose chemotherapy regimens. In addition, immune activation is now appreciated as central to the therapeutic mechanism of bone marrow transplantation for hematologic malignancies. Advances in our understanding of the molecular interactions between tumors and the immune system have led to many novel investigational therapies and continue to inform efforts for devising more potent therapeutics. Novel approaches to immune-based cancer treatment strive to augment antitumor immune responses by expanding tumor-reactive T cells, providing exogenous immune-activating stimuli, and antagonizing regulatory pathways that induce immune tolerance. The future of immune therapy for cancer is likely to combine many of these approaches to generate more effective treatments.

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INTRODUCTION

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Immune therapy is already established as a central component of many cancer treatment regimens (Table 1). Tumors express a wide variety of proteins that can be recognized by the immune system. In addition to microbial proteins, mutated proteins, and fusion proteins, the immune system can recognize developmentally and tissue-restricted proteins, as well as proteins that are highly overexpressed by cancer cells. Established therapies employ a variety of manipulations to activate antitumor immunity. These include passive immunization Table 1

with monoclonal antibodies, the introduction of adjuvants into the tumor microenvironment, and the systemic delivery of cytokines. Immune therapy can ameliorate the toxic effects of standard chemotherapy and is an essential element in the curative mechanism of bone marrow transplantation for hematologic malignancies. Vaccination against and treatment for microbial infections can effect sterilizing immunity against cancer-promoting microorganisms, acting as tumor prophylaxis. Investigational immune therapies for cancer seek to build upon established treatment regimens to devise more efﬁcacious and less toxic cancer therapy. A wide variety of novel

Approved immune therapies for cancera

Established therapies

Indication

References

Monoclonal antibodies Rituximab Ibritumomab tiuxetan Tositumomab Alemtuzumab Gentuzumab Trastuzumab Cetuximab Panitumumab Bevacizumab

NHL, CLL NHL NHL CLL AML Breast cancer Colorectal cancer Colorectal cancer Colorectal, lung

11, 14 12 13 9 10 18 20 21 24–26

Immune adjuvants BCG Imiquimod

Superﬁcial bladder cancer Basal cell carcinoma, VIN, actinic keratosis

39–42 45, 46

Cytokines IFN-α IL-2 TNF-α

melanoma, RCC melanoma, RCC Soft tissue sarcoma, melanoma

49, 50 47, 48 54, 55

Supportive therapy G(M)-CSF Leucovorin

Myelosuppressive chemotherapy MTX rescue

56–58 59

Prophylactic immune therapy HBV vaccine HPV vaccine Antibiotics (H. pylori ) NSAIDs (FAP, ulcerative colitis)

Hepatocellular carcinoma Cervical cancer Gastric cancer, MALT lymphoma Colorectal cancer

61–62 63 69–71 72–75

Bone marrow transplantation Allogeneic DLI

Hematologic malignancies Hematologic malignancies

77–81 82

a Abbreviations: AML, acute myelogenous leukemia; BCG, bacilli Calmette-Gu´erin; CLL, chronic lymphocytic leukemia; DLI, donor lymphocyte infusion; FAP, familial adenomatous polyposis; HBV, hepatitis B virus; HPV, human papilloma virus; MALT, mucosal-associated lymphoid tissue; MTX, methotrexate; NHL, non-Hodgkin’s lymphoma; NSAID, nonsteroidal anti-inﬂammatory drug; RCC, renal cell carcinoma; VIN, vulvar intraepithelial neoplasia.

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strategies have been developed based on fundamental advances in our understanding of the interactions between tumors and the immune system. Collectively, these strategies attempt to augment protective antitumor immunity and to disrupt the immune-regulatory circuits that are critical for maintaining tumor tolerance.

CURRENT IMMUNE THERAPIES

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Monoclonal Antibodies Antibodies play an essential role in providing protective immunity to microorganisms, and the administration of tumor-targeting monoclonal antibodies has proven to be one of the most successful forms of immune therapy for cancer. The infusion of manufactured monoclonal antibodies can generate an immediate immune response while bypassing many of the limitations that impede endogenous immunity. The target speciﬁcity and optimal afﬁnity of manufactured antibodies can be carefully selected, and the quantity of antibody infused can be set to achieve biologically active antibody titers rapidly. Because therapeutic antibodies are initially produced in animals, even self-proteins to which the immune system is generally tolerant can be targeted, a signiﬁcant advantage given that most tumor proteins are also expressed on nonmalignant tissues. Monoclonal antibody therapies are typically not as toxic as conventional cytotoxic cancer chemotherapy, although binding to nonmalignant cells can, in some cases, precipitate significant adverse reactions (1–4). The animal origin of monoclonal antibodies can also lead to treatment-limiting hypersensitivity reactions; however, these reactions have been minimized by substituting human IgG1 sequences outside of the antibody-binding domain (5–7). Mice that exclusively express human antibody genes have also been used to produce fully humanized antibodies (8). Nine monoclonal antibodies, targeting six tumor-associated proteins, are clinically approved for the treatment of cancer. Five of these antibodies bind surface proteins that

are highly expressed on hematologic tumors: CD52 in chronic lymphocytic leukemia (CLL) (alemtuzumab), CD33 in acute myelogenous leukemia (AML) (gentuzumab), and CD20 in non-Hodgkin’s lymphoma (NHL) and CLL (rituximab, ibritumomab tiuxetan, and tositumomab) (9–13). Of these, rituximab is the most widely used and has now been added to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) as part of the standard treatment for NHL; in combination with CHOP, rituximab induces 76% complete remissions, contrasted with 63% for CHOP therapy alone (11, 14). Four monoclonal antibodies (trastuzumab, cetuximab, panitumumab, and bevacizumab) have been approved for the treatment of solid tumors, although none of these has yet shown efﬁcacy comparable with rituximab in NHL (15–22). Trastuzumab, cetuximab, and panitumumab bind proteins of the epidermal growth factor receptor (EGFR) family, either targeting EGFR itself (cetuximab and panitumumab) or targeting the related protein HER2/neu (transtuzumab). EGFR proteins play an important role in transmitting growth signals to a variety of epithelial tumors and have also been targeted by small molecule signal transduction inhibitors (23). Both EGFR-targeting antibodies have been approved for the treatment of metastatic colorectal cancer in patients who have previously failed standard chemotherapy (17, 19–21). In this patient population, cetuximab and panitumumab increase progressionfree survival and are associated with 10–20% and 10% response rates, respectively (20, 21). Trastuzumab was the second monoclonal antibody approved for cancer therapy and is used, either alone or in combination with paclitaxel, for the treatment of invasive, HER2/neupositive breast cancer, which represents approximately 20–30% of invasive breast cancers. Treatment with trastuzumab is associated with variable response rates, ranging from 11% to 26% when used as monotherapy to 50% when used in combination with chemotherapy. In comparison, chemotherapy alone has a response rate of 32% (18). www.annualreviews.org • Immune Therapy for Cancer

CLL: chronic lymphocytic leukemia AML: acute myelogenous leukemia NHL: non-Hodgkin’s lymphoma

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NSCLC: non-small cell lung cancer

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ADCC: antibodydependent cellular cytotoxicity

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Bevacizumab is the only monoclonal antibody with anticancer activity that does not directly target malignant cells. Instead, bevacizumab binds vascular endothelial growth factor (VEGF), a critical mediator of tumor angiogenesis. Inhibiting angiogenesis slows the delivery of nutrients and oxygen to tumors, inhibiting growth without severely compromising normal tissue function (15, 16). Bevacizumab has shown some efﬁcacy in a range of solid tumors, including nonsquamous nonsmall cell lung cancer (NSCLC), metastatic colon cancer, metastatic HER2/neu-negative breast cancer, renal cancer, and pancreatic cancer (15, 16, 24–26). By binding to their targets, antibodies exercise their functions through several effector mechanisms, including steric inhibition and neutralization, complement activation, and activation of cell-mediated cytotoxicity. Each of these mechanisms may play a role in the antitumor activity of monoclonal antibodies; however, at present the relative importance of these mechanisms is not completely clear. Several monoclonal antibodies are known to inhibit signaling downstream of their targets. Cetuximab and pantitumumab are potent inhibitors of EGFR signaling, acting both to block interactions with epidermal growth factor and to prevent conformational changes in the receptor that are required for dimerization and signaling (27–29); similarly, rituximab and trastuzumab alter signaling downstream of their targets (30, 31). Bevacizumab directly binds the soluble growth factor VEGF, preventing VEGF-dependent effects on the vasculature (32). Two monoclonal antibodies have been used to deliver cytotoxic therapy directly to tumors by conjugating them to radioactive isotypes (ibritumomab tiuxetan and 131 I tositumomab) or to toxic chemicals (gemtuzumab) (10, 12). Antibody binding alone is probably sufﬁcient to provide some antitumor activity; however, therapeutic monoclonal antibodies may also function by recruiting other elements of the immune system to malignant cells. At sufﬁcient densities, IgG1 antibodies can activate compleDougan

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ment, leading to direct cytotoxicity through the formation of complement pores in the membrane of antibody-coated cells. Some evidence indicates that complement-dependent cytotoxicity may contribute to the antitumor effects of rituximab (33). In addition to activating complement, antibodies can activate cells of the innate immune system through binding to fragment c receptors (FcRs). Ligation of activating FcRs (FcγRI, FcγRIIA, FcγRIIC, and FcγRIII) on neutrophils, monocytes, and natural killer (NK) cells can lead to antibody-dependent cellular cytotoxicity (ADCC). Antibody-sensitive tumors grown in mice that lack FcγRs become resistant to both rituximab and trastuzumab, suggesting that recognition of IgG1 by FcγRs on innate immune cells contributes signiﬁcantly to the antitumor activity of these antibodies (34). Although suggestive, the importance of ADCC in monoclonal antibody activity was not supported by clinical data until more recently. FcR polymorphisms that enhance monocyte and NK cell recognition of antibody-coated tumors are highly correlated with the clinical response to rituximab, cetuximab, and trastuzumab (35–37). One year following rituximab treatment, lymphoma patients homozygous for FCGR3A-158V, encoding the highafﬁnity FcγRIIIA, had a 90% objective response rate, compared with a 67% objective response rate for patients carrying the lowafﬁnity FCGR3A-158F polymorphism (35). Similarly, breast cancer patients homozygous for FCGR3A-158V had signiﬁcantly higher objective response rates following trastuzumab treatment compared with patients carrying FCGR3A-158F (82% versus 40%), and this higher response rate was associated with signiﬁcantly longer progression-free survival (36). A smaller study examining cetuximab treatment in colorectal cancer found that patients with either of the high-afﬁnity polymorphisms, FCGR3A-158V or FCGR3A-131R, had a median progression-free survival of 3.7 months compared with 1.1 months for patients who carried neither high-afﬁnity polymorphism (37). In addition to monoclonal antibody therapy,

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vaccination against the speciﬁc B cell receptors carried by lymphoma cells (anti-idiotype vaccines) also show increased efﬁcacy in association with high-afﬁnity FcγR polymorphisms (38). These ﬁndings implicate cells of the innate immune system in the clinical response to monoclonal antibodies. Furthermore, these ﬁndings underscore the ability of innate immune cells, when appropriately targeted, to have powerful antitumor effects. Given the capacity of immune cells to inhibit tumor growth, one of the principal goals of tumor immune therapy is to develop novel strategies for targeting immune cells toward malignant tissues.

Immune Adjuvants Cancer cells often express a variety of abnormal proteins that can serve as targets for an immune response (antigens). Although spontaneous immune responses to these antigens can occur, these reactions are rarely sufﬁcient to cause tumor regression; however, the local administration of immune-activating agents (adjuvants) can induce tumor-associated inﬂammation and protective immunity. In general, immune adjuvant–based therapies have only proven effective against early stage tumors; yet in this context they can be remarkably effective with minimal risk of serious adverse reactions. The standard of care for superﬁcial bladder cancer is surgical removal of the tumor followed by immune therapy with an intravesicular injection of live bacilli Calmette-Gu´erin (BCG). In most patients, BCG provokes a local, selflimiting inﬂammatory reaction in the bladder wall (39, 40). Therapy is associated with increased urinary frequency, hematuria, cystitis, and fever, but it is generally well tolerated and carries a very low risk of disseminated infection (40). Several clinical trials have shown that, when combined with surgery, immune therapy with BCG is more effective than conventional chemotherapy (40–42). In one trial with a tenyear follow-up, surgery and BCG combination therapy was associated with progression-free survival in 61.9% of patients, compared with 37% of patients who received surgery alone

(41). Although the precise mechanism of BCG action is not known, the degree of local immune activation correlates with disease efﬁcacy, implicating immune activation by BCG rather than direct antitumor activity (39). Microbes often elicit immune responses by activating pattern-recognition receptors such as members of the Toll-like receptor (TLR) family. Puriﬁed TLR ligands have been evaluated as immune adjuvants and have shown considerable activity in preclinical models. The TLR7 agonist imiquimod was initially approved for the treatment of external warts caused by human papilloma virus (HPV) infection; however, imiquimod has also demonstrated efﬁcacy against low-grade epithelial tumors and precancerous lesions (43, 44). Imiquimod is approved for the treatment of basal cell carcinoma, as well as actinic keratosis, the precursor lesion of cutaneous squamous cell carcinoma (43, 44). More recently, imiquimod has been evaluated in the nonsurgical treatment of vulvar intraepithelial neoplasia (VIN) (45). VIN, like cervical intraepithelial neoplasia (CIN), is typically associated with HPV 16 and 18 infection and can be managed surgically (45, 46); however, surgical treatment can be disﬁguring, making less invasive treatment strategies worthwhile. Topical treatment of grade 2 and grade 3 VIN with imiquimod led to a 25% reduction in lesion size in 21 of the 26 treated patients, with 9 complete responses. In contrast, none of the lesions regressed in the placebotreated group (45). As a result of these ﬁndings, immune adjuvant therapy with imiquimod is likely to become part of the standard treatment for VIN, and imiquimod is currently under evaluation in other neoplasias associated with HPV infection.

VIN: vulvar intraepithelial neoplasia

Cytokines Cytokines, secreted proteins with immunemodulating properties, can be delivered systemically to activate antitumor immunity. Although response rates are low, both the cytokines IL-2 and interferon (IFN)-α have been used to treat advanced melanoma and renal cell www.annualreviews.org • Immune Therapy for Cancer

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carcinoma (RCC), tumors that are generally refractory to standard chemotherapy (47–50). IFN-α is an important mediator in antiviral immunity, and IL-2 is a potent T cell growth factor, although how these functions contribute to the pharmacologic effect of these cytokines is at present unclear. Work in animal models suggests that IFN-α may play a role in antitumor immunity, and clinical responses to IFN-α are associated with therapy-induced autoimmunity, linking the effectiveness of IFN-α to an induction of an immune response (51–53). The side effects of cytokine administration are severe and often dose limiting. Typically, cytokines induce symptoms that mirror those of systemic infection, including hypotension, vomiting, diarrhea, fever, and malaise (47, 49). Despite the limitations of cytokine therapy, both IL-2 and IFN-α can induce durable responses in a subset of patients with melanoma. IFN-α is most effective prior to distant metastasis (stage III disease); in this setting, IFN-α has a 16% overall response rate and a 5% complete response rate (49). Unlike IFN-α, IL-2 can induce responses in patients with metastatic melanoma, although the overall response rate (16%) and the complete response rate (6%) are similarly low (47). In addition to treatment of melanoma, both IFN-α and IL-2 therapy have been approved for the treatment of advanced RCC (48, 50). The importance of these cytokines in advanced RCC treatment has recently been reduced owing to newer therapeutics such as inhibitors of the mammalian target of rapamycin (mTOR) pathway and anti-VEGF treatments, both of which have fewer side effects and similar efﬁcacy to IFN-α and IL-2 (54). In contrast to systemic cytokine therapy used primarily for immune modulation, local administration of the cytokine tumor necrosis factor (TNF)-α has been used to treat soft tissue sarcomas (STSs) of the limb and melanoma, making use of the toxic effects on both tumor cells and the tumor vasculature that are mediated by this cytokine (55, 56). In addition to its antitumor activity, TNF-α is the primary medi-

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RCC: renal cell carcinoma

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ator in septic shock; as a consequence, systemic release of TNF-α can be life threatening. To prevent life-threatening hypotension, TNF-α must be delivered locally using isolated limb perfusion. Isolated limb perfusion is achieved by surgically ligating the main artery and vein of the affected limb to create a bypass circuit; a cardiac bypass pump and oxygenator can then maintain limb perfusion, enabling isolated release of TNF-α without a substantial risk of escape into the systemic circulation. Although many limb STSs can be removed surgically, severe disﬁguration and amputation are often necessary in recurrent or locally advanced disease (55, 56). Using isolated limb perfusion to deliver TNF-α in combination with the alkylating agent melphalan leads to a high response rate (70–85%) with a relatively high chance of limb preservation in patients who otherwise would require amputation to prevent disease progression (55, 56).

Supportive Therapy Many forms of conventional chemotherapy have dose-limiting toxic effects on the bone marrow, including effects on cells of the immune system. Immune toxicity, in particular neutropenia, can lead to substantial morbidity and mortality. Because neutrophils are central effectors in most antibacterial responses, loss of neutrophils can predispose cancer patients to life-threatening bacterial sepsis. Supportive therapy aimed at rescuing immune cells is thus a critical component of many chemotherapy regimens for both solid and hematopoietic tumors, as well as many bone marrow transplantation protocols (57, 58). To prevent neutropenia, many high-dose chemotherapeutic regimens are followed with an infusion of recombinant granulocyte (G) or granulocyte-macrophage (GM) colony stimulating factor (CSF) (57, 58). Both G-CSF and GM-CSF act on the bone marrow to increase neutrophil production, reducing the risk of febrile neutropenia, infection-related mortality, and early mortality by more than 40% (57).

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Evidence favoring a particular CSF is minimal; however, a single head-to-head trial found that G-CSF led to more rapid neutrophil recovery, although this kinetic difference was not associated with a clinically meaningful risk reduction (59). Bone marrow rescue is also a routine component of high-dose methotrexate (MTX) regimens (60). MTX, which inhibits dihydrofolate reductase (DHFR), a critical enzyme in the folic acid pathway required for thymidine synthesis, is used to treat a variety of tumors, including acute lymphoblastic leukemia (ALL), NHL, and breast cancer (60). High-dose therapy with MTX can block the replication of normal cells, leading to substantial toxicity in both gut epithelial cells and the bone marrow. This toxic effect can be mitigated by following MTX with an activated folic acid derivative (leucovorin), which bypasses DHFR and allows thymidine synthesis to continue. For reasons that are, at present, unclear, leucovorin tends to have a greater effect on nonmalignant cells compared with malignant ones, enabling leucovorin rescue to prevent bone marrow and gut epithelial cell loss without compromising the antitumor activity of MTX (60).

Prophylactic Immune Therapy A number of cancers are caused by microbial infections, either directly or through the induction of chronic inﬂammation (61). As a result, therapies aimed at eradicating or preventing these infections act prophylactically against their associated tumors. Because microbes carry many conserved structures that can be recognized as foreign by the immune system, classic vaccination strategies to induce protective immunity can be used without having to overcome the signiﬁcant barriers inherent in targeting sporadic tumors. The hepatitis B virus (HBV) vaccine was the ﬁrst available vaccine that provided protection against an infection with known oncogenic potential. HBV infections of the liver can lead to chronic hepatitis, which can, in turn, predispose people to the de-

velopment of hepatocellular carcinoma (HCC) (61). Although the HBV vaccine induces protective antibody titers in 95% of vaccinated individuals, the large population studies necessary to demonstrate a vaccine-induced reduction in adult HCC have not been performed (62). In Taiwan, where HBV is endemic, however, vaccination has been associated with a drop in HCC incidence among children, especially boys, with HCC cases falling from 1.08 per 100,000 between 1981 and 1984 to 0.49 per 100,000 between 1990 and 1996, establishing the principle of antitumor efﬁcacy (63). More recently, a vaccine against HPV 16 and 18 has been developed speciﬁcally to prevent cervical cancer. Worldwide, cervical cancers cause more than 250,000 deaths per year, and HPV 16 and 18 are associated with 70% of these tumors, in addition to tumors of the vagina and vulva (64–66). Other strains of HPV cause genital warts, and two of these strains (HPV 6 and 11) are also included in the most widely used vaccine (64). Vaccination of girls against HPV induces potent antiviral immunity that prevents viral acquisition, leading to 98% efﬁcacy against HPV 16– and 18–associated CIN and cervical carcinoma (64). In addition to cervical and other genital epithelial tumors in women, both HPV 16 and 18 are associated with penile, perineal, and perianal cancer, as well as tumors of the head and neck; trials are currently underway to evaluate the HPV 16 and 18 vaccine as prophylactic therapy for these cancers as well (67, 68). Like viruses, bacteria, including the bacteria Helicobacter pylori, have also been associated with tumor development. H. pylori is the primary cause of stomach cancer, the second most common cause of cancer-related death in the world, and is also an important cause of mucosal-associated lymphoid tissue (MALT) lymphomas (66, 69). Although infection of the gastric mucosa with H. pylori is typically asymptomatic, these infections can lead to chronic gastritis and the development of gastric ulcers; in rare individuals, H. pylori–associated chronic inﬂammation can then precipitate tumor

www.annualreviews.org • Immune Therapy for Cancer

MTX: methotrexate ALL: acute lymphoblastic leukemia

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FAP: familial adenomatous polyposis NSAID: nonsteroidal anti-inﬂammatory drug GVL: graft-versusleukemia CML: chronic myelocytic leukemia

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formation (61, 69). H. pylori colonization can be cleared with appropriate antibiotics, and evidence suggests that treatment of H. pylori prior to alterations in the gastric mucosa can decrease the risk of stomach cancer (70). In contrast, after the development of precancerous mucosal changes, the risk of tumor formation no longer depends on the continued presence of H. pylori (70). The situation with MALT lymphomas is somewhat different. Many cases of MALT lymphoma are treatable with antibiotics even after the development of cancer, and resistance to antibiotic therapy is associated with genomic alterations in the tumor cells (71, 72). A strong correlation exists between chronic inﬂammation of the colon and colorectal cancer, even in the absence of a clear infectious cause. This inﬂammation has been directly implicated in the development of colorectal cancer owing to the efﬁcacy of several antiinﬂammatory treatments in reducing cancer risk (73–78). The efﬁcacy of anti-inﬂammatory therapy is most apparent in patients at high risk for the development of colorectal cancer. Treatment of patients with ulcerative colitis, a severe inﬂammatory disease of the colon, with the anti-inﬂammatory drug 5-aminosalicylic acid (5-ASA) is associated with a 50% decrease in the odds of colorectal cancer development (73). Similarly, anti-inﬂammatory COX-2 inhibitors are approved for reducing precancerous polyp formation in patients with the genetic colon cancer syndrome familial adenomatous polyposis (FAP). Although no anti-inﬂammatory agents have been approved for the treatment of sporadic colorectal cancer, the use of nonsteroidal anti-inﬂammatory drugs (NSAIDs) like aspirin is associated with an 18–39% decrease in the risk of sporadic colorectal tumors, although side effects mitigate the overall beneﬁt of this approach in a relatively low-risk population (76, 77).

Bone Marrow Transplantation Many hematologic malignancies are treated with bone marrow ablative therapy, which is then followed by bone marrow transplantation 4.8

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from a healthy donor. Although bone marrow transplantation has a very high response rate, in a large number of hematologic tumors relapses following transplantation are still fairly common (79, 80). Intriguingly, for many tumors the risk of relapse is substantially higher for patients who receive a syngeneic transplant (for example, from an identical twin) than for patients who receive allogeneic transplants, indicating a graft-versus-leukemia (GVL) effect associated with allogeneic transplantation (79). In ALL, the risk of relapse is 36% with a syngeneic transplant, compared with 26% with an allogeneic transplant; in AML the difference in relapse rates is 52% compared with 16%; and in chronic myelocytic leukemia (CML) the difference is 40% compared with 7% (79). Allogeneic transplant recipients also run the risk of developing graft-versus-host disease (GVHD), a potentially lethal syndrome characterized by a transplant-mediated immune assault on the recipient’s tissues. The severity of GVHD is inversely correlated with the risk of tumor relapse, suggesting that similar immune mechanisms are responsible both for GVHD and for the GVL effect (80). The GVL effect is believed to be mediated by a combination of cytotoxic T cells and NK cells, and more recent work has begun to elucidate the speciﬁc targets of the GVL response in tumor cells (81, 82). Fully mismatched major histocompatibility complex (MHC) bone marrow transplantation is now possible using nonmyeloablative treatment regimens. These nonmyeloablative conditions lead to bone marrow chimerism, with hematopoetic cells deriving from both the donor and the recipient (83). As might be expected, given the proposed GVL mechanism, nonmyeloablative transplants may reduce the risk of relapse, especially in AML. In a series of 20 patients transplanted using a nonmyeloablative protocol, only two developed relapsed disease (83). Perhaps the clearest evidence indicating an important role for donor immune cells in preventing post-transplantation relapses comes from the success of donor leukocyte infusion (DLI) (84, 85). In DLI, leukocytes harvested

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from the peripheral blood of transplant donors are infused back into transplant recipients following disease relapse. The rationale behind DLI is to provide potent antitumor immune cells to bring the expanding tumor back under immune control. This immune therapeutic approach has been remarkably successful in CML, leading to complete remission of relapsed disease in 70% of infused patients (84). DLI is also effective in relapsed ALL, AML, and multiple myeloma, although complete remissions are far less common (15–29%) (84). Because of its success, immune therapy with DLI is now considered a standard treatment for relapsed leukemia following bone marrow transplantation.

THE INTERACTION BETWEEN THE IMMUNE SYSTEM AND CANCER The immune system interacts with tumors throughout their development, and the consequences of this interaction have substantial implications for cancer therapy (Figure 1). Although some host responses may inhibit tumor growth and progression, the immune system can also promote cancer by provoking chronic inﬂammation and, in turn, elaborating factors that drive tumor growth, survival, and angiogenesis. Failure of immunity can predispose a person to tumor development, particularly in the context of oncogenic viruses.

NK cells

Microbial products?

MyD88

Endogenous adjuvant receptors

NKG2D ligands

Microbial receptors

Myeloid cells

Inﬂammatory cytokines DNA damage

TNF-α, IL-1β, IL-6

NF-κB STAT3

NF-κB

IL-23?

CD4

IL-17 Angiogenesis

Tumor

Proliferation Survival Growth

IFN-γ Tumor necrosis?

Tumor apoptosis (MFG-E8)

Tregs

CD4

CD8

T cells

Immunosuppressive cytokines (TGF-β, IL-10) Negative costimulation (PD-L1) IDO

Figure 1 Complex interactions with the immune system shape tumor development. Chronic inﬂammatory response can be initiated by microbial products or endogenous adjuvants released from necrotic cells. These signals activate nuclear factor-κB (NF-κB) in myeloid cells, leading to the production of inﬂammatory cytokines (TNF-α, IL-1β, and IL-6), which in turn activate NF-κB in the tumor. Tumor-intrinsic NF-κB activation promotes growth, survival, and proliferation. IL-23 produced by myeloid cells can promote the generation of IL-17-secreting T cells, which can further support tumor growth. Genotoxic stress in tumor cells can activate NK cell ligands, which can synergize with endogenous tumor-speciﬁc CD4+ and CD8+ T cells that produce IFN-γ and restrict tumor development. Tumors can suppress nascent immune responses through a variety of mechanisms, including immunosuppressive cytokines (TGF-β and IL-10) and metabolites [indoleamine 2,3-dioxygenase (IDO)] and the expression of negative costimulatory molecules [programmed death ligand 1 (PD-L1)]. Tumors can also promote Treg recruitment and differentiation, in part through the recognition of apoptotic cells by the MFG-E8 (milk fat globule epidermal growth factor 8) pathway. www.annualreviews.org • Immune Therapy for Cancer

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Established tumors use immune-regulatory circuits to generate an immune-suppressive environment, which can act as a substantial barrier to the induction of therapeutic antitumor immunity. Substantial progress in our understanding of the molecular mechanisms governing the interaction between tumors and the immune system has been the basis for multiple investigational immune therapies for cancer, and these fundamental advances continue to provide insights with potential applications in novel treatments.

Infections and Inflammation Several infectious microorganisms are strongly linked to tumor development yet do not appear to encode oncogenic proteins (61, 86). Both HBV and HCV are highly associated with liver cancer; similarly, schistosomal infections are associated with tumors of the bladder and colon. The bacterial species Helicobacter pylori is linked to most cancers of the stomach, as well as to MALT lymphomas. In each of these cases, the pathogen establishes a chronic infection characterized by continuous inﬂammation and failure to achieve immune clearance; the chronic inﬂammation itself appears to cause the associated cancers. In fact, even in the absence of obvious infection, chronic inﬂammation can provoke tumor formation. Several autoimmune diseases increase the risk of B cell lymphoma, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren’s syndrome (87). Similarly, both ulcerative colitis and Crohn’s disease, inﬂammatory diseases of the gastrointestinal track, increase the risk of colorectal cancer (88). As mentioned above, evidence from NSAID use suggests that the role of inﬂammation in cancer may extend beyond tumors that arise in the context of frank inﬂammatory disease. NSAID use is associated with a decreased incidence of sporadic colon cancer, and COX-2 inhibitors decrease the risk of tumor formation in patients with FAP (74–77). Inﬂammation has also been linked to lung cancer: Both cigarette smoking and asbestos inhalation lead 4.10

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to pulmonary inﬂammation, and NSAIDs can decrease the risk of lung cancer (61, 89). Over the past few years, signiﬁcant progress has been made toward elucidating the molecular mechanisms linking inﬂammation to cancer. The proinﬂammatory cytokine TNF-α plays an essential, early role in several cancer models, as well as in chronic inﬂammatory diseases of both mice and humans (61, 86). Consistent with the importance of TNF-α in human cancer, increased levels of TNF-α have been linked to multiple myeloma, HCC, breast cancer, bladder cancer, and gastric cancer (61, 86). TNF-α, largely produced by cells of the innate immune system such as macrophages and mast cells, can have myriad effects in the tumor microenvironment. TNF-α promotes cell growth and survival as well as angiogenesis and the recruitment of immune effector cells. Although the events downstream of TNF-α that are critical for tumor development are not all known, nuclear factor-κB (NF-κB) family transcription factors appear to play a signiﬁcant role in linking TNF-α to cancer (61, 86). NF-κB controls the transcription of a number of proteins involved in cell survival, division, and growth; in addition, NF-κB is important for the production of many inﬂammatory cytokines and chemokines, including TNF-α itself. NFκB can function both tumor-intrinsically and -extrinsically to promote cancer development. In a mouse model of colon cancer, speciﬁc ablation of NF-κB signaling in immune cells led to reductions in tumor growth, whereas ablation in the colonic epithelium dramatically decreased tumor incidence (90). Depending on the system examined, other acute inﬂammatory cytokines can drive cancer development, including IL-6 and IL-1. IL-6 has been implicated in many of the same processes as TNF-α, acting both as a mitogen and as an angiogenic factor, primarily through activation of the transcription factor signal transducer and activator of transcription (STAT) 3 (91). IL-6 is a central mediator in mouse models of inﬂammatory liver and colon cancer, and IL6 has been implicated in an autocrine growth pathway downstream of EGFR in a subset of

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human lung cancers (61, 86, 92). IL-1 can also activate NF-κB and promote tumor formation in carcinogen-induced skin cancer models; in humans, polymorphisms in IL-1 have been linked to gastric cancer (86, 93, 94). Given their apparent importance, strategies to target acute inﬂammatory cytokines or downstream signaling molecules are under evaluation (95). Many tumor-promoting cytokines are produced largely by innate immune cells; however, cells of the adaptive immune system can also promote tumor formation. Overexpression of the cytokine IL-23 dramatically increases the risk of cancer in mouse models, and IL-23 levels are increased in a variety of human tumors (96). IL-23 functions to support the differentiation of a subset of T cells that preferentially produce IL-17, a cytokine that is overexpressed in several human cancers, including cervical cancer and NSCLC (61). IL-17 production can drive both TNF-α and IL-6 secretion from many different cell types, linking IL-17 to innate immunity and the acute inﬂammatory cytokines and suggesting that therapies targeting IL-17 or IL-23 could limit tumor-promoting inﬂammation (61). Despite substantial progress in identifying relevant inﬂammatory mediators, many of the early steps that initiate tumor-promoting inﬂammation are, at present, unknown. In several mouse models of cancer, inﬂammation is initiated by signals downstream of microbial pattern-recognition receptors. In these models, the signaling adaptor protein myeloid differentiation factor 88 (MyD88) plays a crucial role (97–99). MyD88 is an indispensable mediator of most TLR signaling pathways as well as IL-1 receptor signaling. Loss of MyD88 reduces the number of skin tumors formed in two different carcinogen-induced skin cancer models, as well as in a carcinogen-induced model of liver cancer (97, 98). In APCmin/+ mice, a model of human FAP, MyD88 deﬁciency reduces both the size and the frequency of tumors, as well as tumor-associated cytokine production (99). Even with MyD88 positioned upstream of the initiation of inﬂammation, the factors responsible for engaging MyD88 are still ob-

scure. TLRs can recognize a wide range of microbial products, potentially implicating occult infections or recognition of endogenous ﬂora in the onset of tumor-promoting inﬂammation. Alternatively, MyD88 may function to mediate sterile inﬂammation downstream of the IL-1 receptor, which would be consistent with the importance of IL-1 in the 7,12-dimethylbenz[a]anthracene, 12O-tetradecanoylphorbol-13-acetate (DMBA/ TPA) model of skin cancer (97). Other sterile inﬂammatory signals, such as those mediated by the receptor for advanced glycation endproducts (RAGE), have also been implicated in tumor formation (100). Because acute, self-limiting inﬂammation is generally insufﬁcient to induce tumor formation, defects in the normal mechanisms of immune regulation may be common features of tumor-promoting inﬂammation. In many cases, for example in the context of infection or autoimmunity, the speciﬁc factors preventing the resolution of inﬂammation are unknown; however, in several mouse models, defects in immune-regulatory proteins increase tumor susceptibility. In the dextran sulfate sodium model of colitis, loss of TIR8, a negative regulator of TLR and IL-1 receptor signaling, exacerbates cancer development (101). Similarly, loss of the secreted IL-1 antagonist, IL-1Ra, increases the growth rate of skin tumors in DMBA/TPA-treated mice (93). A more thorough understanding of how immuneregulatory pathways control cancer development may provide novel opportunities for therapeutic intervention.

Spontaneous Immunity Whereas chronic inﬂammation generally promotes tumor development, in some contexts, adaptive immunity can suppress tumor formation. The role of adaptive immunity in controlling tumor growth is most obvious in the case of cancers of viral origin; however, immunedeﬁcient states are also associated with a small increase in the risk of tumors that are not known to have infectious etiologies. www.annualreviews.org • Immune Therapy for Cancer

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HTLV: human T-lymphotropic virus

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HHV8: human herpes virus 8

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Oncogenic viruses. Several human viruses encode proteins that can directly contribute to tumor formation, including HPV, human T-lymphotropic virus (HTLV), EpsteinBarr virus (EBV), and human herpes virus 8 (HHV8/Kaposi sarcoma virus). Control of these viruses represents perhaps the most direct mechanisms by which the immune system can suppress cancer, as well as one of the most attractive targets for tumor immune therapy. As discussed earlier, a vaccine against HPV, the primary cause of cervical cancer and several other anogenital tumors, has become the ﬁrst vaccine against an oncogene-encoding virus, serving as a model for further prophylactic anticancer immune therapy (64, 68). Like human immunodeﬁciency virus (HIV), HTLV is a retrovirus that infects CD4+ T cells; however, in contrast to HIV, HTLV promotes T cell survival and proliferation (102). Infection with HTLV provokes strong antiviral cytotoxic T cell responses, yet these responses are typically unable to clear the infection (102). In a subset of patients, over the course of 20 to 30 years, persistent HTLV leads to the development of adult T cell leukemia/lymphoma (ATLL) (103); strategies to augment antiHTLV responses thus represent an attractive avenue for treating these tumors. The herpes virus EBV primarily infects B cells and is associated with Burkett’s lymphoma and nasopharyngeal carcinoma in a minority of infected individuals (104). EBV is one of the most prevalent infectious diseases: Greater than 90% of the world’s population has been infected, with the vast majority of infected individuals living as asymptomatic carriers. EBV lymphomas are more common in immunesuppressed individuals, implicating the immune system in the control of these tumors; furthermore, EBV lymphoproliferative disease following bone marrow transplant can be controlled through DLIs that contain EBV-reactive T cells (104, 105). Like EBV, HHV8 infections typically do not cause overt disease, and most HHV8-associated skin tumors occur in patients infected with HIV (106). In the context of HIV, HHV8-associated Kaposi sarcomas occur in paDougan

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tients with low CD4+ T cell counts, and effective antiretroviral therapy can inhibit their development, strongly implicating the immune system in the control of these tumors (106). Tumors of nonviral origin. Increasing evidence suggests that beneﬁcial immune responses to tumors can occur spontaneously in humans; however, these immune responses are unlikely to control tumor development in most patients. Spontaneous immune inﬁltrates are common in many cancers, often correlating with a favorable prognosis; this correlation is particularly strong for inﬁltrates composed of activated CD8+ T cells and memory CD45RO+ T cells (107, 108). Several types of cancer are associated with the development of distinct autoimmune syndromes. These syndromes are thought to occur following the induction of spontaneous antitumor responses, and cancer patients who have autoantibodies characteristic of these paraneoplastic syndromes in the absence of frank autoimmune disease have improved prognosis (51, 107). In addition to evidence of immune activation, the microenvironment of most established human cancers is generally immune suppressive; this suggests that overcoming immune rejection may be an important feature of early tumor growth, yet whether the development of such an environment poses a legitimate barrier for early tumors is unclear (109–111). Extensive work performed in experimental systems has elucidated some of the mechanisms underlying spontaneous antitumor immunity, and has formed the basis for the cancer immunoediting hypothesis. This hypothesis divides the immune response to cancer into three phases: a tumor destructive “elimination” phase, a stable “equilibrium phase,” and “escape” phase characterized by tumor progression. Immune responses that eliminate tumors or delay their growth involve the production of IFN-γ, as well as the generation of tumorreactive cytotoxic T cells (51, 107). Mice deﬁcient in IFN-γ are more susceptible to methylcholanthrene (MCA)-induced sarcomas, as are mice lacking T cells and mice unable to produce

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perforin, a key effector protein in T cell cytotoxicity (51, 107). Several strains of immunedeﬁcient mice, including mice lacking T cells and mice deﬁcient in IFN-γ, are also more susceptible to sporadic tumor development, although a role for microbial infections in these tumors cannot yet be excluded (51, 107). In addition to preventing tumor formation, the adaptive immune system may also be able to hold established tumors in check, leading to equilibrium between the host response and tumor growth. Recent studies in animals have identiﬁed a subset of mice that develop stable tumors in response to low-dose MCA treatment; these tumors can be induced to grow by transient immune suppression, establishing a role for protective immunity in restricting tumor growth (112). Clear evidence for a similar phenomenon in human cancer patients is lacking; however, several observations suggest that such immune control may occur. Rarely, patients develop recurrent malignancies decades after the removal of their primary tumor (51, 107). Similarly, tumors have been inadvertently transmitted along with an organ transplant taken from a donor who was apparently tumor free after having undergone cancer surgery years earlier (51, 107). Plasma cells from patients with the precursor lesion of multiple myeloma, monoclonal gammopathy of undetermined signiﬁcance (MGUS), express high levels of MHC class I chain–related gene A (MICA), which can be recognized by NKG2D on NK cells and stimulate the production of anti-MICA antibodies (113). The secretion of soluble MICA, which leads to downregulation of NKG2D, is associated with progression to multiple myeloma, linking tumor development to suppression of endogenous immunity (113). Although these observations are not deﬁnitive, establishing such immune control of tumor growth is one of the principal goals of cancer immune therapy.

Immunogenic Cell Death Cell death is a ubiquitous feature of developing tumors, which are often characterized by

disorganized growth and an inadequate blood supply. The speciﬁc mechanisms underlying tumor cell death can dramatically inﬂuence interactions with the immune system, having implications for the development of tumor immune therapies. Many malignant as well as nonmalignant cells die through apoptosis, a highly regulated cell death pathway that results in loss of membrane lipid polarity and exposure of phosphatidylserine (PS) on the outer leaﬂet of the plasma membrane (114). Exposed PS is recognized by at least three distinct pathways, each of which can lead to rapid phagocytosis of apoptotic cells by macrophages and dendritic cells (DCs). Phagocytosis of apoptotic debris promotes the production of anti-inﬂammatory cytokines and immune tolerance. By antagonizing these pathways, the immune-suppressive effect of apoptosis can be circumvented, potentially enhancing strategies to generate antitumor immunity. This approach has proved successful in an experimental therapeutic vaccine for melanoma, where antagonism of one of these pathways, mediated by the binding of milk fat globule epidermal growth factor 8 (MFG-E8) to PS and the subsequent binding of MFG-E8 to αv integrins, signiﬁcantly augmented vaccine efﬁcacy (115). In many cases, tumor cells do not undergo apoptosis and instead die through necrosis. In contrast to apoptosis, necrotic cell death appears to be immune stimulatory (116). The molecular mechanisms linking necrosis to immune activation are just beginning to be elucidated; however, they appear to rely on the aberrant exposure of speciﬁc cellular components, such as uric acid and the DNA-binding protein high-mobility group box 1 (HMG-B1), which are recognized by activating receptors on innate immune cells (116). Necrotic cell death may act to prime endogenous antitumor immune responses or may augment immune therapies. In addition, some evidence indicates that immunogenic cell death may be promoted by speciﬁc chemotherapeutic agents, such as the DNA-targeting anthracyclines, indicating the potential for more conventional cancer treatments to augment antitumor immunity (117). www.annualreviews.org • Immune Therapy for Cancer

MGUS: monoclonal gammopathy of undetermined signiﬁcance

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MDSC: myeloidderived suppressor cell IDO: indoleamine 2,3-dioxygenase

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Similarly, DNA damage caused by chemotherapy or defective repair machinery may increase the expression of stress ligands such as MICA that can be targeted by NK cells (118). By increasing the expression of NK cell ligands, tumors with genetic damage can be recognized by the innate immune system. Strategies to take advantage of these immunogenic death and damage pathways may synergize with immune therapy to generate more potent antitumor responses.

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Immune Suppression in the Tumor Microenvironment Immune suppression is a common feature of the tumor microenvironment and a substantial barrier to tumor immune therapy. The microenvironment of tumors is established through the activity of both myeloid and lymphoid regulatory cells, as well as through the production of immune-suppressive factors by malignant cells themselves. Many tumor-inﬁltrating macrophages have an immune-suppressive phenotype (109). These macrophages, referred to as myeloid-derived suppressor cells (MDSCs), are characterized by the expression of both CD11b and GR1 (109). MDSCs are abundant in many tumors arising in both humans and mice and can exert powerful anti-inﬂammatory effects. The anti-inﬂammatory activity of MDSCs is mediated at least in part through the production of two enzymes involved in arginine metabolism: arginase 1 and nitrous oxide synthase (NOS) (109). Both arginase 1 and NOS exert powerful suppressive effects on T cells, although whether these effects are mediated by altered T cell metabolism, increased production of hydrogen peroxide, or other as yet undeﬁned mechanisms is not clear (109). In addition to MDSC, regulatory T cells (Tregs) also heavily inﬁltrate many tumors (110). These cells, characterized by the expression of the transcription factor FoxP3 as well as CD4 and CD25, play a key role in the regulation of adaptive immunity. Tregs can suppress immune responses through the secretion 4.14

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of suppressive cytokines like TGF-β and IL35 as well as through a poorly understood, contact-dependent mechanism (110, 119). The presence of large Treg inﬁltrates is correlated with poor prognosis in several types of cancer, including cancers of the ovary, breast, and liver (110). As is discussed below, Tregs are a potential barrier to developing productive immune therapies for cancer, and they represent an attractive target for enhancing antitumor immunity. The elaboration of immune-suppressive factors by tumor cells represents yet another powerful barrier to the generation of antitumor immunity. Tumor cells often secrete immunesuppressive cytokines such as IL-10, TGF-β, and VEGF (110). These factors not only inhibit cytotoxic immune responses but may also promote the formation or recruitment of additional regulatory cells (110). Many tumors express the immune-suppressive enzyme indoleamine 2,3dioxygenase (IDO), an enzyme that is involved in tryptophan metabolism (111). IDO appears to exert its suppressive effect through the depletion of tryptophan and the production of antiinﬂammatory tryptophan metabolites (111). In addition to immune-suppressive soluble mediators, many tumors express surface receptors that can inhibit T cell activation, such as programmed death ligand 1 (PD-L1), which may play an important role in downmodulating antitumor T cell responses (120). The possibility of blocking the PD-L1 pathway as a mechanism for enhancing tumor immunity is under active investigation, as is discussed below.

NOVEL APPROACHES TO IMMUNE THERAPY A variety of novel strategies for eliciting protective antitumor immune responses are under development. Several of these strategies attempt to target novel pathways identiﬁed through basic research. Although tumor-reactive cells are present in many experimental and therapeutic settings, a large number of regulatory pathways appear to prevent these reactive cells from generating productive antitumor responses. As

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a result, many novel immune therapies for cancer attempt to circumvent these regulatory pathways. Immune suppression can be inhibited by antagonizing regulatory molecules or by directly targeting regulatory cells. Alternatively, T cell costimulation can be augmented to reverse the effects of immune suppression. The antigenic targets and frequency of spontaneous, tumor-reactive cells may not be sufﬁcient for inhibiting tumor growth; consequently, a variety of therapeutic strategies seek to increase the number of tumor-reactive cells either through vaccination or ex vivo expansion. In addition to novel therapeutics, immune-modulating agents developed to treat nonmalignant conditions are now being tested for efﬁcacy in cancer. Similarly, immune-modulating cancer therapeutics have the potential to become important treatments for nonmalignant conditions, including autoimmunity. The frequency of such therapeutic crossovers should only increase as we expand our understanding of the molecular interactions between tumors and the immune system.

Second-Generation Monoclonal Antibodies Impressive clinical responses to monoclonal antibody therapy have already been achieved, yet many tumors remain largely refractory to approved antibody therapies. Second-generation monoclonal antibodies, addressing some of the limitations of current antibodies, are under development to enhance antitumor efﬁcacy. The high molecular weight of the four-chain antibody structure can inhibit diffusion, potentially contributing to weak responses in solid tumors. By removing the antibody Fc region to generate F(ab )2 fragments, diffusion of antibodies into tumors can be signiﬁcantly improved (121). Unfortunately, many important antibody functions are mediated by the Fc region. As a result, such F(ab )2 fragments are likely to be most useful as targeting mechanisms for antibody-conjugated cytotoxic therapy. Monoclonal antibodies have been modiﬁed to alter serum half-life, either to extend the

period over which the antibodies exert their biology effects or to accelerate the clearance of toxin-conjugated antibodies (122, 123). The neonatal FcR (FcRn) is largely responsible for determining the serum half-life of antibodies; by introducing mutations that enhance or diminish FcRn binding, antibody half-life can be extended or reduced (122, 123). Recent evidence, both from animal models and from clinical trials, supports an important role for the innate immune system in the antitumor activity of therapeutic monoclonal antibodies. In particular, the mechanism underlying monoclonal antibody–mediated killing appears to involve the activation of complement as well as the ability to direct monocyte and NK cell cytotoxicity. In addition, antibodies can assist DCs in the acquisition of tumorassociated antigen and in the presentation of these antigens to T cells, thus linking monoclonal antibody therapy to the induction of adaptive immunity (124, 125). Many strategies to improve the efﬁcacy of monoclonal antibodies seek to enhance antibody-mediated immune activation. ADCC can be augmented through modiﬁcation of the antibody Fc region to produce a more favorable binding proﬁle for the FcRs expressed on monocytes and NK cells (34, 126– 128). These modiﬁcations include mutations in the amino acid structure of the Fc region as well as alterations in the Fc glycosylation pattern (34, 126–128). A triple alanine substitution mutant trastuzumab (S298A/E333A/K334A) has signiﬁcantly improved binding to FcγRIIIA, the principal activating FcR on monocytes and NK cells; consistent with improved binding, this substituted trastuzumab has a superior ability to activate ADCC in vitro (126). Loss of fucosylation on antibody N-linked glycosyl groups has a similar effect on FcγRIIIA binding, again leading to improved activation of ADCC (127). Interestingly, loss of fucosylation appears to maximize ADCC, and the additional amino acid substitutions may be unable to enhance ADCC further (128). Antibodymediated killing can also be enhanced by decreasing binding to the inhibitory FcγR, www.annualreviews.org • Immune Therapy for Cancer

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FcγRIIB, implicating both positive and negative signals in the response to antibody therapy (34). Like ADCC, complement activation may play an important role in the antitumor activity of monoclonal antibodies. Several antibody isotypes, including IgM, IgG1, and IgG3, are capable of ﬁxing complement through the binding of complement factor C1q and the subsequent activation of the C1r/s proteases. Although IgM pentamers are the most efﬁcient complement-ﬁxing antibodies, these multimeric structures are too bulky to make useful therapeutics; as a result, strategies for enhancing therapeutic complement activation have focused on modifying IgG1 to increase its binding afﬁnity for C1q (129). Several mutations in the IgG1 Fc region enhance C1q binding and complement activation; however, some of these complement-activating mutants also negatively affect ADCC, narrowing the range of potentially useful modiﬁcations. The double alanine substitution K326A/E333A (which overlaps at E333 with the ADCC-activating triple mutant) enhances complement activation by rituximab without negatively affecting ADCC (129). In an experimental system, complement activation has also been achieved through the direct inhibition of complement regulatory proteins using a chimeric, bi-speciﬁc antibody capable of both tumor binding and complement inhibition (130).

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Novel Immune Adjuvants Immune adjuvants have already proven useful in the treatment of a range of early stage tumors. Unfortunately, neither the TLR7 agonist imiquimod nor BCG, the two immune adjuvants currently approved for cancer therapy, are suitable for systemic delivery. As a result, current research has focused on identifying systemically active immune adjuvants which could be used to treat a wider range of tumors. TLR9 agonists. Unlike imiquimod, agonists of TLR9 can activate productive immune responses when delivered into the circulation 4.16

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(131). Similar to TLR7, TLR9 is part of the recognition system for microbial nucleic acids. TLR9 is involved in transducing signals from unmethylated CpG DNA, acting to skew T cell responses toward the production of IFN-γ, a critical cytokine mediator of tumor immunity (132). In humans, TLR9 is expressed on only a subset of cells, including plasmacytoid dendritic cells (pDCs) and B cells (132, 133). A variety of synthetic TLR9 agonists have been developed, several of which are undergoing clinical testing in a wide range of tumors (131, 134–139). Intratumor injection of the TLR9 agonist PF-3512676 is being tested in phase I/II trials as monotherapy for both basal cell carcinoma and metastatic melanoma (134, 135). Systemic PF-3512676 is under evaluation in phase I trials as monotherapy for cutaneous T cell lymphoma (CTCL) and following rituximab therapy for NHL; phase II trials have also begun to examine PF-351267 combination therapy with taxanes for NSCLC (136– 138). In each of these ongoing trials, TLR9 agonist treatment has been associated with the induction of immune reactions and some evidence of antitumor activity, although complete responses have been rare (134–138). α-galactosylceramide. α-galactosylceramide (α-galcer, KRN7000), a lipid derived from marine sponges, was originally isolated more than a decade ago in a screen for biological molecules with anticancer activity (140, 141). α-galcer is a speciﬁc agonist for a subset of rapidly activated T cells known as NKT cells. Unlike most T cells, which recognize peptide bound to MHC molecules, NKT cells respond to lipid antigens displayed by the MHC class I homolog CD1d (141). Most NKT cells can recognize α-galcer bound to CD1d and respond within several hours by producing cytokines; as a result, α-galcer is now often used as a pan-NKT cell agonist (141). NKT cells may play a critical role in skewing immune responses toward the production of speciﬁc effector cytokines, including IFN-γ (141). The administration of α-galcer can have potent antitumor effects in a wide range of murine

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cancer models, suggesting that NKT cells may play a broad role in antitumor immunity (141). Additional α-galcer derivatives are also under investigation in an attempt to generate still more potent antitumor activity (142). Several phase I clinical trials have investigated the potential for using NKT cell activation through α-galcer as a cancer treatment (143–147). The intravenous administration of α-galcer is well tolerated in cancer patients and leads to measurable immune activation; however, total NKT cell numbers vary dramatically across individuals, and responses are only substantial in patients who have high baseline circulating NKT cells (143). DCs loaded with α-galcer have also been used in early clinical trials, where they have been associated with signs of immune activation and NKT cell expansion (144, 145, 147, 148). Because many malignancies are associated with an apparent reduction in NKT cells, the dependence of α-galcer therapy on baseline NKT cell numbers may pose an obstacle to in vivo activation therapies (149). In a recent attempt to circumvent this limitation, autologous NKT cells from patients with NSCLC were cultured and activated ex vivo and then reinfused into cancer patients (146). Although objective responses were not observed, this strategy did boost NKT cell numbers in most patients, despite the relatively low purity of the transfused NKT cell product (146).

Immune-Modulating Antibodies A large number of novel monoclonal antibodies with immune-modulating activity are under development for the treatment of cancer. Several of these antibodies directly antagonize negative regulatory circuits that are thought to be important in limiting antitumor responses; similarly, agonistic antibodies that activate T cell coreceptors are being developed to drive cytotoxic T cell responses. Negative regulatory receptors. The most clinically advanced immune-modulating antibodies block cytotoxic T lymphocyte antigen

(CTLA)-4, an important negative immuneregulatory receptor expressed on a variety of immune cells, including activated T cells and Tregs (150). In addition to sending its own negative signals, CTLA-4 binds to B7-1 and B72 with substantially higher afﬁnity and avidity than does CD28, enabling CTLA-4 to effectively outcompete CD28 (150). In the absence of CTLA-4, mice develop a lethal multiorgan inﬂammatory disease, underscoring the central importance of CTLA-4 in immune homeostasis (151, 152). A large number of studies in animal models have demonstrated enhanced antitumor activity following CTLA-4 blockade, particularly when used in conjunction with other tumor vaccination strategies (150, 153). Two CTLA-4-blocking antibodies (ipilimumab and tremelimumab) with afﬁnities <1 nM have entered clinical testing, predominantly in advanced melanoma (153–155). A series of phase I and phase II trials have shown that both antibodies have clinical activity, with objective response rates ranging from 5% to 15%, including some complete responses. Based on these early studies, advanced registration trials for these antibodies are currently underway (155). In addition to single-agent trials, some of these early trials have combined anti-CTLA-4 therapy with other immune-modulating therapy, and these combination regimens appear to enhance efﬁcacy (155). CTLA-4 blockade has been associated with grade 3 and grade 4 autoimmune toxicities, establishing the importance of this negative regulatory pathway in maintaining normal immune homeostasis in addition to suppressing antitumor immunity (153–155). Treatmentassociated colitis has been the most signiﬁcant autoimmune reaction following CTLA-4 antibody administration; however, many other autoimmune reactions have been observed, including dermatitis (in the case of melanoma), hypophysitis, and pulmonary alveolitis (153– 155). The precise mechanism of action of CTLA4-blocking therapy is still incompletely understood. In particular, as mentioned above, CTLA-4 is expressed on both effector and www.annualreviews.org • Immune Therapy for Cancer

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Tregs. The relative importance of activity against these two cell types is unclear, with some reports suggesting a role for CTLA-4 on Tregs in immune suppression, and others ﬁnding evidence of increased Treg numbers in lymph nodes following CTLA-4 blockade (154). The negative immune-regulatory receptor PD-1 is also under evaluation as an antibody target for cancer immune therapy. Similar to CTLA-4, PD-1 is an inhibitory receptor expressed on activated T cells, as well as on B cells and monocytes (156). PD-1 has two principal ligands, PD-L1 and PD-L2 (156). PD-L2 is expressed on antigen-presenting cells, whereas PD-L1 is expressed broadly, both within the immune system and on nonimmune tissues (156). PD-L1 expression can be upregulated by both IFN-α/β and IFN-γ, leading to high expression in a variety of inﬂamed tissues (156). Similarly, PD-L1 is often highly expressed on tumors, and expression of PD-1 is correlated with an unfavorable prognosis in cancers of lung, kidney, pancreas, and ovaries (157–161). These ﬁndings suggest a role for PD-L1 in limiting T cell responses within the tumor microenvironment. Consistent with the importance of the PD1/PD-L1 axis in tumor development, in animal models inhibition of PD-1, either through genetic ablation or with blocking antibodies, can improve antitumor immunity (156, 162). A fully human PD-1-blocking monoclonal antibody has recently been developed that is capable of augmenting T cell responses in vitro (161). A phase I trial of the PD-1-blocking antibody CT-011 was recently completed in 17 patients with various hematologic malignancies (163). In this patient population, PD-1 blockade was safe and associated with a complete response in one patient with NHL (163).

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Immune-suppressive cytokines. Tumors and tumor-associated immune cells can produce a range of cytokines with the ability to suppress tumor immunity. The antiinﬂammatory cytokines IL-10 and TGF-β are among the most important immunesuppressive cytokines produced by tumors, and 4.18

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strategies to reverse this immune suppression represent an attractive target for immune therapy (164–168). Targeting TGF-β can substantially augment antitumor immunity in animal models (165, 166), suggesting that analogous strategies in humans may show clinical efﬁcacy. Anti-TGF-β monoclonal antibodies have been safely administered in clinical trials for systemic sclerosis (CAT-192) and for corneal scarring (CAT-152/lerdelimumab) (169, 170), opening up the possibility for clinical investigation in cancer patients. Similarly, a monoclonal antibody against IL-10 has been developed for testing in SLE (171). Blocking IL-10 may be particularly useful in B cell lymphomas, where, in addition to suppressing antitumor immunity, IL-10 can induce expression of B cell growth factors (168). Inﬂammatory cytokines are also under investigation as immune therapy targets. Antibodies against TNF-α have recently been evaluated in clinical trials for RCC, where treatment was associated with partial responses in 16% of patients (95). TNF family costimulatory receptors. Monoclonal antibodies that act as agonists of stimulatory receptors can directly augment antitumor immune responses. Several such antibodies have been developed to target TNF family costimulatory receptors, including glucocorticoid-induced tumor necrosis factor receptor (GITR), CD134 (OX40), CD137 (4-1BB), and CD40. GITR, CD134, and CD137 are each costimulatory receptors expressed on T cells, and ligation of these receptors enhances cytotoxic T cell function (172). In animal models, agonistic antibodies directed against each of these proteins can signiﬁcantly increase the efﬁcacy of antitumor immune therapy (173–177). Clinical trials are planned to evaluate both GITR- and CD134-speciﬁc antibodies in cancer therapy, and an anti-CD137 antibody (BMS-663513) is now in phase I and phase II testing in a variety of solid tumors, including melanoma and NSCLC.

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CD40 plays a central role in the crosstalk between CD4+ T cells, DCs, and B cells. Antibodies against CD40 differ from other immune-stimulating antibodies because, in addition to immune-modulating activity, antiCD40 can also directly bind CD40-expressing B cell tumors, enabling the activation of conventional antibody-dependent killing mechanisms. The balance of these effects is likely dependent on whether the antibody activates or blocks CD40, and both types of antibodies are under evaluation (178, 179). Activating antibodies against CD40 have shown efﬁcacy in animal tumor models, where responses required immune activation (178). Consistent with these ﬁndings, a recent phase I trial of the anti-CD40 antibody CP-870893 showed encouraging preliminary results in patients with advanced melanoma (180). Although not an activating receptor, the receptor for the cytokine tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) is also under investigation as a target for agonistic therapy either with TRAIL itself or with monoclonal antibodies. Work in experimental systems suggests that signals through the TRAIL receptor can have potent effects on tumors cells, directly inducing apoptosis, without inducing substantial systemic toxicities (181).

Targeting Regulatory Cells The immune-suppressive tumor microenvironment is, in large part, maintained through the anti-inﬂammatory activity of both innate and adaptive regulatory cells. In particular, Tregs seem to play an important role in damping antitumor immune responses. Tregs are overrepresented in a wide range of tumors, and increased numbers of Tregs correlates with poor prognosis in a variety of tumors (182–185); in one series of 70 patients with ovarian cancer, high Treg numbers were associated with a 25-fold increase in the risk of death after controlling for disease stage and surgical debulking (182). Tregs may also pose a signiﬁcant barrier to tumor vaccination strategies, which can induce increases in antigen-speciﬁc Tregs (186). Given the poten-

tial importance of Tregs in limiting antitumor immunity, methods for directly targeting them may be of clinical use. The drug denileukin diftitox is a conjugate of Diphtheria toxin and IL-2 and was initially developed to treat T cell malignancies. In more recent work, denileukin diftitox has shown some selective toxicity against Tregs (187, 188). This selective effect may relate to the high expression of the IL-2 receptor α chain (CD25) that characterizes these cells. Inclusion of denileukin diftitox in a cancer vaccine setting has shown early promising results (188). Additional Treg-targeted therapies are under investigation in experimental systems, including interfering with Treg chemotaxis by blocking interactions between CCL22 and CCR4, blocking IL-35, and antagonizing Treg induction following the phagocytosis of apoptotic cells using a dominant negative form of MFG-E8 (115, 119, 189).

Therapeutic Cancer Vaccines Prophylactic cancer vaccines have been effective in a number of spontaneous tumor models in mice, including models of breast, prostate, and colon cancer (190–192). In cancer patients, vaccination strategies have been limited to advanced disease, owing to the investigational nature of current therapies. These therapeutic vaccines present a substantial challenge, given that they must bypass immune-regulatory mechanisms that have already led to tumor tolerance. The enormity of this challenge is underscored by the lack of effective vaccination strategies to treat chronic infectious diseases, despite the large number of foreign antigens present in infectious organisms. Many strategies for generating therapeutic immune responses to cancer have been attempted; antigen-speciﬁc vaccines, DC vaccines, and cytokine-based, whole tumor cell vaccines are among the most promising and best studied of these strategies. Antigen-specific vaccines. A wide range of vaccines based on single-tumor antigens has www.annualreviews.org • Immune Therapy for Cancer

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been tested in experimental systems, and several speciﬁc antigen vaccines are currently in clinical trials. These vaccines, which make use of recombinant proteins or antigenic peptides mixed with immune adjuvants, can elicit coordinated T and B cell immune responses and have some efﬁcacy in a variety of human tumors. The targets selected in these strategies have been diverse, yet many belong to a class of proteins referred to as cancer testes antigens. Cancer testes antigens are expressed by tumor cells as well as endogenously within the immune-privileged environment of the testes, making immune response against them highly selective for tumor tissue. Two cancer testes antigens, MAGE-A3 and NY-ESO-1, have been evaluated as vaccine targets in a series of clinical trials. MAGE-A3 is highly expressed on a wide range of tumors (193, 194), and adjuvant-mixed, recombinant MAGE-A3 protein or peptide vaccines can elicit potent antitumor B and T cell responses that can be associated with objective responses, and the tumor-speciﬁc cells persist for years following vaccination (194). Phase I and II MAGE-A3 vaccine trials have been completed in melanoma and NSCLC, and a phase III trial in NSCLC is currently underway (193, 194). Like MAGE-A3, NY-ESO1 is widely expressed in human cancers (195, 196). Multiple clinical trials have examined NYESO-1-based vaccines in a variety of cancers, including melanoma, ovarian carcinoma, and NSCLC (195, 196). Vaccines using adjuvants along with peptide or recombinant protein have elicited strong immune reactions, including coordinated antibody and cytotoxic T cell responses following vaccination with recombinant protein and CpG (196). In addition to cancer testes antigens, some tumors express proteins with a narrow distribution in nonmalignant tissues; tissue-restricted proteins have been successfully targeted in melanoma, where a variety of melanocyte-speciﬁc proteins often continue to be expressed (197). Many tumors overexpress proteins with relatively low endogenous expression, and these overexpressed proteins can be effective tar-

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gets of single-antigen vaccines. Similarly, the HER2/neu protein is highly expressed on a subset of breast cancers and, as was discussed above, has been effectively targeted by the monoclonal antibody trastuzumab. Vaccination against HER2/neu can induce coordinated antitumor immune responses that can spread beyond the initial antigenic determinants used in the vaccine (198). Targeting HER2/neu using a vaccine may be a particularly valuable approach in combination with trastuzumab, where the two therapies may synergize to augment antitumor immunity. Dendritic cell vaccines. DCs are potent inducers of adaptive immunity, driving the activation of T cells in response to invading microorganisms. The ability to culture DCs from human peripheral blood monocytes has generated signiﬁcant interest in using DCs in novel cancer vaccination strategies (199). Immature monocyte-derived DCs can be readily loaded with antigenic peptides or proteins in vitro; these antigen-loaded DCs can then be used in an autologous transplant to induce antigenspeciﬁc T cell responses (199). DCs must be activated before they are capable of inducing immunity. Activated DCs have improved antigen-presenting abilities, as well as increased expression of T cell costimulatory proteins; activation also confers migratory potential to DCs, a necessary step to bring them into contact with naive T cells in lymph nodes. DCs can be activated by a range of stimuli, including inﬂammatory cytokines and microbial products, although these stimuli do not always lead to equivalent DC activation (200, 201). Most clinical trials use an inﬂammatory cytokine cocktail to mature DCs, although other approaches, including the use of microbial pattern-recognition receptor agonists, have also been considered (202, 203). An alternative approach, which has been tested in experimental models, bypasses in vitro activation and instead injects immature, antigenloaded DCs into an inﬂamed tissue (204, 205). Once exposed to the inﬂammatory environment, antigen-loaded DCs can mature in a

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more physiological fashion and migrate to draining lymph nodes (204, 205). This vaccination approach offers at least two advantages. First, DCs acquire antigen while immature, which should ensure appropriate antigen processing. Second, this approach obviates the need for large quantities of recombinant cytokines, which can signiﬁcantly increase costs. Antigens can also be delivered directly to DCs in vivo using antibodies that bind DC surface receptors such as DEC205; targeting DCs using this strategy has proved successful in experimental models and is under development for use in patients (206–208). The delivery of antigen to cultured DCs has been accomplished through a variety of approaches, including the direct loading of antigenic peptides or long overlapping peptide mixtures, exposure to whole recombinant protein, transfection with antigen-encoding mRNA, and fusion with tumor cells (199, 209–211). Each of these approaches has its advantages, and the most effective mechanisms for generating immunity in cancer patients has not yet been established (199, 209–211). A large number of DC vaccination trials have been conducted in cancer patients using a range of protocols, some with encouraging preliminary results (212). Large-scale phase III trials of DC vaccines, including a trial in metastatic melanoma, have thus far failed to demonstrate a protective beneﬁt (212, 213). DC vaccination protocols are clearly not yet optimized, and generating more effective vaccines continues to be an area of active research. Cytokine-based tumor vaccines. Coadministered cytokines have been used as adjuvants in a variety of cancer vaccination strategies (214). When combined with vaccines, cytokines can boost immune responses through the recruitment and maturation of a wider variety of immune effector cells (214). Several cytokines, including IL-2, IL-12, IFN-α, and GM-CSF, have been evaluated as vaccine adjuvants; among these, GM-CSF has been the most widely studied (214). GM-CSF has been particularly potent when used to prime immune

responses against whole tumor cell vaccines. GM-CSF primarily acts on myeloid cells and functions to recruit and mature DCs, enhancing the presentation of tumor antigens to the immune system. In mouse models, GM-CSFtransfected tumor cell vaccines can induce complete protective immunity when used prophylactically against syngeneic tumors, and when used therapeutically these vaccines can delay tumor growth (215). Similarly, in cancer patients, injection of autologous, irradiated, whole tumor cells engineered to produce GM-CSF (GVAX) can induce coordinated B and T cell responses to a wide range of tumor antigens. In melanoma patients, these immune reactions have been associated with both partial and complete responses (216). Autologous whole-cell vaccines have the advantage of providing multiple potential tumor antigens, yet these patient-speciﬁc vaccines are also difﬁcult to manufacture on a large scale. To facilitate large-scale vaccine production, GMCSF-secreting allogeneic tumor vaccines have also been evaluated in clinical trials. These allogeneic vaccines, which are derived from tumor cell lines, have been tested in both pancreatic cancer and hormone-resistant prostate cancer, where they have shown promising early results (217–219). Based on these early studies, phase III efﬁcacy trials of GVAX in hormone-resistant prostate cancer are currently underway. Although some patients have had encouraging responses to GVAX, this strategy may be most effective when used in combination with other immune-modulating therapies (215, 220, 221). The anti-CTLA-4 monoclonal antibody ipilimumab has been tested in patients who have been previously vaccinated using DCs or GVAX (220, 221). In these patients, ipilimumab is associated with augmented antitumor immunity and objective responses in a subset of patients (220, 221). Inﬂammatory side effects were associated with therapy, yet treatment did not provoke severe autoimmunity (220, 221). Intriguingly, tumor necrosis was highly associated with an increased CD8+ -to-FoxP3+ ratio in tumor-inﬁltrating T cells (Figure 2). This suggests that clinical responses to GVAX and www.annualreviews.org • Immune Therapy for Cancer

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FoxP3

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Figure 2 The ratio of tumor-inﬁltrating CD8+ T cells to FoxP3+ Tregs after GVAX followed by ipilimumab infusion is tightly correlated with the extent of tumor necrosis. (a) Upper row: Minimal necrosis of melanoma metastasis. Lower row: Extensive necrosis of melanoma metastasis. (Magniﬁcation: H&E, × 4; CD8, × 20; FoxP3, × 40.) (b) Numbers of tumor-inﬁltrating FoxP3+ Tregs and CD8+ T cells versus tumor necrosis (reprinted from Reference 200).

CTLA-4 blockade come from a loss of immune suppression coupled with the induction of cytotoxic T cells (220, 221). These preliminary results suggest that combination immune therapies may have signiﬁcant clinical efﬁcacy, although this potential awaits testing in randomized clinical trials. 4.22

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Adoptive T Cell Therapy Adoptive T cell therapy relies on the in vitro expansion of endogenous, cancer-reactive T cells, which are harvested from cancer patients, manipulated, and then reintroduced as a mechanism for generating productive tumor immunity. Adoptive T cell therapy has had some

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promising early clinical results and has been associated with clinical responses in a minority of patients with metastatic melanoma (222–224). CD8+ cytotoxic T lymphocytes are the primary effector cells in adoptive T cell therapy (225, 226); however, CD4+ T cells may also play an important role in maintaining CD8+ cytotoxic function, and transplantation of tumorreactive CD4+ T cells has been associated with some efﬁcacy in metastatic melanoma (227). T cells used in adoptive therapy can be harvested from a variety of sites, including peripheral blood, malignant effusions, resected lymph nodes, and tumor biopsies. Although T cells harvested from the peripheral blood are technically easier to obtain, tumor-inﬁltrating lymphocytes (TILs) obtained from biopsies may contain a higher frequency of tumor-reactive cells (228). Practically, obtaining sufﬁcient cells from tumor biopsies is difﬁcult, although this approach has been used successfully in patients with melanoma (228). Two alternative approaches attempt to circumvent low levels of endogenous antitumor reactivity in the peripheral blood by directly supplying T cells with the ability to recognize tumors. T cells harvested from the peripheral blood can be engineered to express T cell receptors (TCRs) that have been selected for tumor recognition. This approach has been tested in metastatic melanoma; however, because TCR recognition of antigen is MHC restricted, each engineered TCR can only be used in patients with the required MHC allele (229). MHC restriction can be bypassed by engineering T cells to express novel chimeric fusion proteins that link the antigen-binding domain of the B cell receptor with the signaling component of the TCR complex (230–232). These “T-bodies” can directly bind tumor antigens, leading to T cell activation. Transfusion of T-body-expressing T cells have been tested in RCC, ovarian carcinoma, and pediatric neuroblastoma. Although T-body-expressing cells are generally well tolerated, severe autoimmune hepatitis was precipitated in RCC, presumably owing to expression of the target antigen, car-

bonic anhydase IX, in the biliary tract (232). This adverse event clearly demonstrates the reactive potential of T-body-expressing T cells, but it also underscores the importance of antigen selection. Once harvested, T cells can be expanded either through polyclonal stimulation with activating antibodies or through exposure to speciﬁc tumor antigens, although this second approach requires the identiﬁcation of relevant targets. Given the frequency of antigen loss variants in current clinical trials, the selection of appropriate targets may be challenging, potentially making polyclonal stimulation a more attractive approach (222–224). In addition to antigen-loss variants, adoptive T cell therapies have been limited by the replicative potential of cultured T cells. Several strategies, including the enforced expression of costimulatory proteins and telomerase, have been used to attempt to extend the life span of cultured T cells (233, 234). IL-15 has also been considered as a possible additive to cultures to enhance the production of cytotoxic cells (226). Intriguingly, engraftment of adoptively transferred T cells appears to be enhanced in lymphodepleted hosts, and strategies to combine pretreatment with lymphodepleting chemotherapy and adoptive T cell transplantation appear to increase treatment efﬁcacy signiﬁcantly (235, 236). As with other strategies to generate therapeutic antitumor immunity, adoptive T cell therapy alone may be insufﬁcient to induce clinically meaningful responses in most cancer patients. Combining adoptive T cell therapy with an autologous tumor vaccine mixed with BCG has produced promising results in RCC, where a 27% objective response rate was observed in a recent phase II trial (237). Furthermore, adoptively transferred T cells stimulated with pneumococcus can be activated in vivo using a pneumococcal vaccine (238). These results suggest that combination therapies using vaccines to increase the frequency of tumor-reactive T cells prior to, or immediately after, adoptive therapy may be another mechanism for increasing overall treatment efﬁcacy. www.annualreviews.org • Immune Therapy for Cancer

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CONCLUSION

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Single modality immune manipulations have had signiﬁcant success in some therapeutic settings; however, the complexity of the interaction between developing tumors and immune cells may often necessitate combination therapy in order to generate protective antitumor immunity. By disabling key regulatory pathways in conjunction with strategies to expand and activate tumor-reactive cells, the efﬁcacy of tumor immune therapy may improve substantially. Yet disabling regulatory pathways increases the risk of treatment-limiting autoimmunity, as has already been observed. One of the principal challenges of immune therapy for cancer will be to identify those mechanisms of immune suppres-

sion that are most valuable to speciﬁc tumors and least necessary for the maintenance of normal immune tolerance. A further challenge will be to establish meaningful criteria for the evaluation of novel immune therapies. Survival is the most important endpoint for evaluating cancer therapy; however, objective criteria based on changes in the size of tumor masses may underestimate immune therapies. Productive antitumor immune responses often lead to swelling and ﬁbrosis, which do not diminish the size of tumor masses but instead alter their composition, decreasing the overall number of viable tumor cells. As a result, using immune correlates and an assessment of tumor composition may be more a meaningful way to evaluate and optimize investigational immune therapies.

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Microglial Physiology: Unique Stimuli, Specialized Responses Richard M. Ransohoff1 and V. Hugh Perry2 1

Neuroinﬂammation Research Center, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195; email: [email protected]

2

School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK; email: [email protected]

Annu. Rev. Immunol. 2009. 27:119–45

Key Words

The Annual Review of Immunology is online at immunol.annualreviews.org

microglia, macrophage, inﬂammation, neurodegenerative disease, brain cytology, activation, regulation, central nervous system

This article’s doi: 10.1146/annurev.immunol.021908.132528 c 2009 by Annual Reviews. Copyright All rights reserved 0732-0582/09/0423-0119$20.00

Abstract Microglia, the macrophages of the central nervous system parenchyma, have in the normal healthy brain a distinct phenotype induced by molecules expressed on or secreted by adjacent neurons and astrocytes, and this phenotype is maintained in part by virtue of the blood-brain barrier’s exclusion of serum components. Microglia are continually active, their processes palpating and surveying their local microenvironment. The microglia rapidly change their phenotype in response to any disturbance of nervous system homeostasis and are commonly referred to as activated on the basis of the changes in their morphology or expression of cell surface antigens. A wealth of data now demonstrate that the microglia have very diverse effector functions, in line with macrophage populations in other organs. The term activated microglia needs to be qualiﬁed to reﬂect the distinct and very different states of activationassociated effector functions in different disease changes. Manipulating the effector functions of microglia has the potential to modify the outcome of diverse neurological diseases.

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INTRODUCTION TO MICROGLIA

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Resident tissue macrophages are present in all tissues of the body, and the central nervous system (CNS) is no exception. An extensive literature had debated the origin of the most numerous of the brain macrophages, the microglia, speciﬁcally whether they are of myeloid lineage or of neuroectodermal origin. There is little need to rehearse these different points of view; the fact that it took so long to establish the myeloid origin of microglia tells us that these cells have a phenotype that is distinct from other tissue macrophages. Many lines of evidence have converged to establish that the microglia are of myeloid lineage, which was conclusively conﬁrmed by their absence from the CNS of PU.1-null mice (1), although the brains of PU.1-null mice are readily repopulated by microglia following bone marrow transplantation (2). Because microglia and the other populations of macrophages associated with the different structural compartments of the CNS are part of the mononuclear phagocyte system (MPS), we can identify more speciﬁc questions: When do macrophages ﬁrst invade the CNS? Are microglia derived from a speciﬁc pool of progenitors or monocytes? How are their numbers maintained in the steady state? What are the signals that lead to microglial activation? What are the phenotypes and functions of these cells in health and disease? This review relies on studies in humans, mice, and rats. It is worth mention that cells that are functionally equivalent to microglia are observed in the invertebrate CNS. Studies carried out in the leech (Hirudo medicinalis) reveal a population of neuroectodermal cells termed small glia or microglia. Leech microglial cells move rapidly to nerve crush lesions, at least partly via purinergic signaling, as described also for mammalian microglia (see below), and they phagocytose debris. Leeches show axonal sprouting and accurate reestablishment of synaptic contacts after nerve crush. Intriguingly, blockade of microglial accumulation at the lesion site impairs axon sprouting (3, 4).

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Macrophage Populations of the CNS Microglia in the adult mammalian CNS have a small cell soma, little perinuclear cytoplasm, and a number of ﬁne, branched processes that are covered in ﬁne protrusions. The cells are readily revealed in rodent and human CNS by lectin cytochemistry or immunocytochemistry for selected antigens, and they occupy a territory that does not overlap with adjacent microglia. The absence or low levels of expression of many antigens typically found on other macrophage populations indicate that the CNS microenvironment plays a critical role in deﬁning the phenotype (see the discussion below in Regulation of the Microglia Phenotype in the Normal Healthy CNS). Despite the downregulated phenotype of microglia in the normal healthy brain, in vivo imaging studies demonstrate that the ﬁne processes of microglia continually palpate and monitor their local microenvironment (5, 6). Microglia are distributed throughout the CNS and vary in density in both rodents and humans, with subtle variations in morphology in different cytoarchitectural regions (7). It is unclear what local factors determine their numerical and morphological variations or whether these variations reﬂect functional differences, although there are subtle regional diversities in expression of important immune receptors (8). In addition to the microglia, there are macrophage populations associated with the perivascular space [the perivascular macrophages (PVMs)], the circumventricular organs, the choroid plexus, and the meninges. These macrophages have different phenotypes when compared with the microglia and appear differentially constrained by their local microenvironment. PVMs express antigens not expressed on the microglia. For example, the mannose receptor is detected on PVMs of mouse and human (9) and CD163 in rat and human (10). Similarly, a signiﬁcant proportion of meningeal macrophages express these receptors. The PVMs and meningeal populations are more overtly phagocytic than are the microglia, and the expression of major

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histocompatibility complex (MHC) antigens is more widespread in these populations of cells relative to the microglia. Macrophages are also present in regions of the brain where they are exposed to the blood, including the circumventricular organs and the choroid plexus, and the phenotype of these macrophages is distinct from the microglia and PVMs, perhaps reﬂecting the role of serum proteins in the regulation of microglia phenotype (11). An important question regarding the immune privilege of the CNS is whether there exists a population of dendritic cells (DCs), the professional antigen-presenting cells (APCs) of the immune system (12). Since the ﬁrst reports demonstrating a lack of MHC class II expression in the CNS, there has been no convincing demonstration of DCs, either by expression of relevant DC surface markers or by DC function in the healthy brain parenchyma (13), although DCs are present in the meninges and the choroid plexus. This situation is radically changed in the diseased brain, however (see the section below on Multiple Sclerosis).

Origin of Microglia In postnatal rodents, immunocytochemical studies using antibodies to F4/80 (ERM1), CR3, and FcγRII/III suggested that the microglia entered the brain from the circulation and presumably were derived from circulating monocytes (14). At earlier stages of development (prior to the development of the vasculature), cells of myeloid origin can be found within the embryonic mouse CNS (embryonic day 8), where they proliferate (15). In the zebra ﬁsh, yolk sac–derived macrophages enter the developing brain and retina, where they develop into immature microglia (16). The lineage origin of these cells from the progenitor cells in the yolk sac is unclear, but evidence suggests that these primitive macrophages develop along a PU.1-independent pathway (17). Whether any of these primitive macrophages contribute to the microglial populations that appear later in development or persist into adulthood is not known. In one study (18), more

than 20% of microglia isolated from immature brain expressed the hematopoietic stem cell marker CD34, and 90% expressed the B220 antigen, an isoform of CD45 typically associated with B cells and a subset of DCs. These markers are subsequently downregulated during maturation.

Maintaining the Microglial Population Investigators widely believe that the microglia are a long-lived population of tissue macrophages, but how the resident populations of brain macrophages are maintained in homeostasis and during disease is not yet resolved. Recent studies show that resident tissue macrophage populations may arise from speciﬁc subsets of monocytes rather than simply from stochastic recruitment or entry of monocytes into tissues. At least two distinct subsets of monocytes are found in mouse, Gr-1hi /CCR2+ /Ly6Chi /CX3CR1+ and Gr-1lo /CCR2− /Ly6Clo /CX3CR1++ (19). Geissmann et al. (20) reported that Gr1lo /CCR2− cells may give rise to tissue macrophages, including the microglia, in the steady state. Those investigations did not clarify how many cells enter the brain and into which compartment; furthermore, the cells that apparently entered the parenchyma did not persist for more than a few days. It has been suggested that either CX3CR1 or homeostatic tissue macrophage turnover mediates recruitment, but Cardona et al. (21) demonstrated that CX3CR1 is not required for entry into the brain. Thymidine labeling studies in adult mice demonstrate that microglia undergo DNA synthesis, divide, and contribute to the maintenance of this population (22), akin to the situation found for other long-lived resident myeloid cell populations. To address the question of the relative contribution of circulating monocytes or intrinsic division to the steady-state population, investigators have typically used radiation chimeras or parabionts. However, the complications introduced by whole body irradiation, including irradiation of the brain, have led to some www.annualreviews.org • Microglial Physiology

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confusion. The use of radiation chimeras and parabiosis to study microglia turnover is discussed below in the section on Irradiation Bone Marrow Chimerism and Parabiosis. The extent to which other populations of brain macrophages, PVMs, or meningeal cells turn over is also poorly understood. Several bone marrow chimera studies have suggested that these cells turn over more rapidly than microglia, but again there are technical issues to consider (discussed below). By injecting India ink into the brain parenchyma, PVMs were labeled that could be identiﬁed even two years later (23), suggesting that this population may also be long-lived.

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RESEARCH METHODS—THEIR STRENGTHS AND LIMITATIONS In Vitro In vitro microglial culture. In vitro systems provide a critical tool for exploring many aspects of microglia biology. Removal of these cells from their microenvironment will release them from the normal constraints that play such a critical role in their phenotype. Thus, using in vitro systems assumes we recognize either that the cells must be studied rapidly after isolation or that the investigator must attempt to recapitulate features of the CNS microenvironment that will in turn give clues about the factors regulating their phenotype. A signiﬁcant problem is the identiﬁcation of the microglia phenotype in vitro, as there is no single microglia marker and the microglial phenotype is deﬁned in vivo by a combination of morphology and often lack of or low expression of multiple macrophage antigens. This circumstance has lead to a relatively loose use of the term “process bearing” or other terms for the characterization of microglia in vitro, and all too often no attempt is made to address whether they differ from macrophages. Despite these caveats, many studies rely on the isolation and expansion of microglia from the neonatal brain as a model for the study of resident microglia function. Another limitation of studying neonatal 122

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microglia is that they have not experienced the CNS milieu in vivo in the context of an intact, mature blood-brain barrier (BBB). Ponomarev et al. (24) recently described a protocol for isolating adult rodent microglia. The importance of astrocyte-microglia interactions as part of the environmental regulation is well illustrated in experiments by Rosenstiel and colleagues (25), who have shown that microglia and other macrophage populations grown on an astrocyte monolayer develop a highly ramiﬁed morphology that is associated with a downregulation of nuclear factor-κB (NF-κB). Whether expression of a spectrum of cell surface antigens is also downregulated has not been systematically studied. Isolation of adult microglia and neonatal microglia shows these cells to be poor or immature APCs, although treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) will lead to differentiation to a more DC-like phenotype (26). Isolation of adult microglia and subsequent culture in low levels of macrophage colony-stimulating factor-1 (M-CSF) lead to proliferation of the surviving cells that then develop a modestly ramiﬁed morphology that could be maintained for many weeks (24), consistent with the notion that M-CSF is a key component of tissue macrophage survival and phenotype. Mice lacking M-CSF do exhibit normal morphology and apparent function of microglia, however (27). Elucidation of many aspects of macrophages and microglia clearly can be readily accomplished in vitro, but the direct relevance of these observations needs to be established through in vivo studies before accepting them at face value. In most in vitro studies, there has been no attempt to replicate the CNS microenvironment, and the state of the cells grown on glass or tissue culture plastic is likely more relevant to inﬂammatory cells rather than to steady-state microglia. Brain slices. An intermediate approach to in vivo characterization or the isolation of microglia from their natural environment is to investigate their properties in accessible acute

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brain slices or organotypic cultures. Acute brain slices suffer from the obvious consequences of the slicing process, and one might imagine that the microglia would be rapidly activated, but in contrast to isolated in vitro microglia, the ion channel expression in the slice cultures is distinct from that found in cells in vitro and consistent with a downregulated or resident phenotype (28). Organotypic cultures permit the study of microglia with a resident phenotype after several days in vitro, and subsequent injury to these slices leads to somewhat predictable responses given what we know of the in vivo condition and the responses of macrophages (29). Few reported studies have used slice culture, and wider exploitation of this approach might be productive, given the evident limitations of in vitro culture of isolated microglia.

In Vivo Immunohistochemistry and in situ hybridization. Given the marked limitations imposed by studying microglia in vitro (see above), much effort has been devoted to evaluating microglial biology in the intact CNS. Immunohistochemistry (IHC) has been widely used to characterize microglia, both in human tissues and in those from experimental animals. The strengths of this approach are manifold, and no other technique can provide a comparable wealth of information. Different from RNA analysis [such as in situ hybridization (ISH), Northern blotting, or reverse transcriptasecoupled polymerase chain reaction (RT-PCR)], IHC detects protein, which is more likely to be functionally relevant than mRNA. Furthermore, unlike Western blotting or enzymelinked immunosorbent assay (ELISA), IHC enables protein both to be detected and to be localized to speciﬁc cell types with regional differentiation. With proper controls, IHC supports quantitative morphometry. In addition, using high-resolution techniques such as confocal microscopy with immunoﬂuorescence, subcellular localization can be achieved, occasionally with functional implications. For example, stimulus-dependent transcription factors,

such as the signal transducers and activators of transcription (STATs) and the NF-κB components, translocate to the nucleus upon ligand engagement of upstream receptors. Therefore, the detection of STATs in microglial nuclei implies ligation of relevant cytokine receptors (30). IHC can accordingly yield an incomparably vivid depiction, albeit static, of physiological and pathological processes. Against these strengths lies the cardinal weakness that IHC is fraught with artifact, much of which is deceptively subtle and the causes of which are legion. Artifactual immunostaining can appear crisp and speciﬁc with low or absent background. Because of IHC’s importance in clarifying neurobiological processes, guidelines for validating antibody staining in neuroscience have been proposed (31, 32). These guidelines are useful both for those performing experiments and for those evaluating experimental results. At the outset, it is evident that simple technical controls such as omission of primary antibody represent only a ﬁrst step in this crucial process. Pertinent suggestions include use of Western immunoblotting to verify presence of the an