CD30 Ligand

CD30 Ligand Paola Romagnani1, Carmelo Mavilia2 and Sergio Romagnani2,* 1

Department of Physiopathology, Endocrinology Unit, University of Florence, Viale Morgagni 85, Florence, 50134, Italy 2 Department of Internal Medicine, Section of Immunoallergology and Respiratory Diseases, University of Florence, Viale Morgagni 85, Florence, 50134, Italy * corresponding author tel: 39-055-413663, fax: 39-055-412867, e-mail: [email protected] DOI: 10.1006/rcwy.2000.05007.

SUMMARY CD30 ligand (CD30L) is a member of the TNF family which is present on a variety of cells, including normal resting B cells, granulocytes, eosinophils, a fraction of bone marrow precursors, subsets of activated macrophages, and T cells (both CD4‡ and CD8‡), and is highly expressed by thymic medullary epithelial cells. In vitro, the interaction of CD30L with its receptor (CD30) has been proved to induce either cell proliferation or cell death depending on the cell type receiving the signal and/or the microenvironmental conditions in which the signal is provided. This suggests an important role for CD30L in the regulation of different immune functions, including thymic negative selection, and in the pathogenesis of some immune-mediated disorders.

BACKGROUND

Discovery CD30 ligand (CD30L), a member of the TNF superfamily (Armitage, 1994), was demonstrated by screening cell lines with a soluble fusion protein consisting of the extracellular portion of human CD30 linked to the hinge, CH2, and CH3 domains of human immunoglobulin G1 (IgG1) heavy chain (CD30-Fc). The chimeric receptor was expressed in mammalian cells, purified to homogeneity as a 190 kDa disulfide-linked homodimer, and used to identify by flow cytometry a murine T cell clone and

CD3-stimulated human peripheral blood T cells as sources of surface ligand. To isolate cDNA clones encoding the cognate ligand, a cDNA library was constructed with mRNA isolated from the murine T cell clone expressing the ligand. Transfected COS cells were then screened for the surface expression of a CD30L by autoradiography using a radio-iodinated CD3-Fc probe, and the clone obtained was sequenced. A human CD30L cDNA clone was then cloned, characterized and sequenced (Smith et al., 1993).

Structure CD30L is expressed as type II membrane protein on the surface of different cell types, particularly hematopoietic cells, from different species and has a molecular weight of approximately 40 kDa (26 kDA in humans) as well as multiple N-linked glycosylation sites in the extracellular domain of the molecule.

Main activities and physiological roles Although the physiologic role of CD30L is still unclear, its interaction with the putative receptor (CD30) may result in either cell proliferation or cell death depending on the cell type involved and the microenvironmental conditions in which the signal is delivered, thus suggesting an important regulatory role for this molecule in the function of the immune system.

466 Paola Romagnani, Carmelo Mavilia and Sergio Romagnani

GENE AND GENE REGULATION

Accession numbers EMBL/GenBank: U97594, U97595, U97596, U97597, U97598, U97599, U97600

regulated by a promoter lacking the TATA box region. The genomic organization of CD30L is similar to that of other TNF family members, in which the first exon encodes the intracellular domain, whereas exons 2±4 encode the intracellular domain (Croager and Abraham, 1997).

Chromosome location

Sequence See Figure 1. The CD30L gene shows clear sequence homology to the other members of the TNF family. Among these members, CD30L exhibits the lowest sequence similarity: 12±18% identity with the various other family members. The structure for the human CD30L gene is composed of four exons and three intervening introns spaced over 17.1 kb of genomic DNA. The exons varied in length from 43 bp (exon 2) to 392 bp (exon 4), whereas introns varied in length from approximately 12 kb for the most 50 intron to 0.8 kb for the most 30 intron. The initiation site for the transcript consists of 114 bp before methionine. The promoter region lacks a TATA motif, and the presence of poly-dT element directly upstream from the transcription start site suggests that CD30L expression is

The human CD30L gene is localized to the long arm of chromosome 9 (9q33), the murine CD30L gene to the proximal region of chromosome 4.

Regulatory sites and corresponding transcription factors Several transcription factor-binding sites have been identified on the human CD30L gene. These include: AP4, localized from ÿ429 to ÿ420; ELK1/cETS1 from ÿ285 to ÿ273; MZF1 from ÿ80 to ÿ73; Ik2 from ÿ456 to ÿ445, from ÿ185 to ÿ173 and from ÿ109 to ÿ98; E47 from ÿ561 to ÿ547 and from ÿ20 to ÿ6; PBX1 from ÿ541 to ÿ528; and C/EPBP from ÿ439 to ÿ480. ELK1/cETS1, E47 and Ik2 represent

Figure 1 Nucleotide sequence for CD30L. Sequence 1 ccaagtcaca 61 aactgaatca 121 ccagggctgc 181 ccggcgggct 241 acagccactc 301 cagaggacgg 361 tcagaagacc 421 caagtggcaa 481 ggagtcagat 541 tgccaactgc 601 ctcatcaaca 661 acgaaacacg 721 accatatcag 781 aatgtgttgt 841 gaagaaagcg 901 aactttagac 961 tggaaagata 1021 aggcagggaa 1081 ggatgtccca 1141 gagcctccag 1201 aaatgaaggg 1261 tctgcagatg 1321 aacttcccta 1381 actgtcactc 1441 ccccagggaa 1501 tacaccacca 1561 ggcataatga 1621 gtcagacaga 1681 tcaattcact 1741 tccagtctgc 1801 tagaaccaga 1861 ttgtatacaa

tgattcagga gatgaagaga agcaagcact ccgtggccag tggctctgtg actccattcc tcttatgtat agcatctaaa atcaggatgg agtttcttgt agcatatcaa tataccagaa tcaatgtgga ccatcttctt cctctctacc caagacaaac cagctccagg gcaatgtagt gcccaaccac ttaaaatggg gagctgggtg gcctaatgct tactggcaga tggcactagg aaccaggctc caatggaaaa gtctgaaggg gacagcaaga ggcaaatgcc taggcaggaa aacccatatc aatggttgcc

ttcaggggga gataaggtgt caacggaatg ccacctgggg ccttgtcttc caactcacct cctgaaaaga caaaaccaag gaatctggtg acaatgccca aaaacaggcc tctctctcaa tacattccag atacagtaat atacagtatt tacacagggt gttaaaaaga gtggtgggca taattcactg gcttcagtac tgagtgttta tagttgtcca tacacttgtg aggctgatct ggacagcccc caagttcaaa cagtcctcct cagttaacaa tgaatggtct agatgcctct aagggtgact aaggcaacat

gaatccttct tggaacagag atgggcccag gatgtgggga agactatata aagaatggac gcccctcctg gagacacagc catgcatgtg accacgagcc gcagctattt ctatttgacc acggtggcca ctattatggt gttggtcgtt gacaacgtcc ccctcaaagg aggaaattgc gctccattca agaagtcatg ggcctacctc ttgtcttgga acaaagatgg cattctccat atccaattcc ctggtttgta cttcatcatt aataattctg tcgatctgaa gttggagctt ctggtgacag tgtgtgagtc tggaatgcaa ttcttgctgg attacctgca ggtcaacacc tacatagata caagcacctt tcctcttgag tcagactgaa cagtttctct tggccttcag tcatccctccaaacacttgg gcaaaaagaa attaaatagtatacttctcc ttctgtctct gagtttttag tgaagtatct ttcagatagc gagccccaca cagaatcaga agggatgaat tatggtcttg atctatttct tctgttttga cagagcagct agcaactctg ccctaatggg cactgtgccc ttcacgggat acttctttta agtcgcgatc aaggactctc tcacacagga actgaaccat gcccagttta tgcctgtctg tgtactccat atgaccccac ccctaggaac ctgttcctga gatggaaagc acaaatttaa gacttttact tacagatcct ggacagaaag tctccaggtt acatgaggca ggaataagaa cgtaggtaaa gaaatagggt gtggtcactc gtctgaagga agcaacagag aagtggggaa aagttcttgt ctctggccag aggtgtggta aagcccggct tccggtatga gaaattaaac aaaattataa gaattc

CD30 Ligand 467 the sites for transcription factors specific to lymphoid cells. The transcription factors involved are still unknown.

Cells and tissues that express the gene Several cell types express the CD30L gene, including normal hematopoietic cells, cells from hematopoietic tumors of both myeloid and lymphoid origin, and cells from some nonhematopoietic tumors (yolk sac and embryonal carcinomas). In some cells (neutrophils, eosinophils, B lymphocytes, hematopoietic precursors, and medullary thymic epithelial cells), CD30L gene expression is constitutive; in others (CD4‡ and CD8‡ T cells and macrophages) it is induced after activation.

PROTEIN

Accession numbers

domain). The dominant form of ligand in different species exists as a molecule of approximately 40 kDa, but the molecular weight of the human protein is 26 kDa.

Posttranslational modifications There is extensive use of multiple N-linked glycosylation sites present in the extracellular domain of the molecule. These sites, as well as six out of seven cysteines (five in the extracellular and one in the transmembrane portion) are conserved. Human CD30L possesses five, rather than six, potential Nlinked glycosylation sites in the extracellular domain; these five sites are conserved, as are six of the seven cysteines (five extracellular, one transmembrane). The single cytoplasmic cysteine in mouse CD30L is not conserved in the human ligand. CD30L possesses clear homology with TNF, TNF , and CD40L, which is limited to the terminal portion involved in binding to their respective receptors.

SwissProt: P32971

CELLULAR SOURCES AND TISSUE EXPRESSION

Sequence

Cellular sources that produce

See Figure 2.

Although CD30L was initially isolated from activated T cells, its expression was then described on several cell types. These include subsets of both CD4‡ and CD8‡ activated T cells and activated macrophages (Smith et al., 1993), normal resting B cells (Younes et al., 1996), granulocytes (Gruss et al., 1994), including eosinophils (Pinto et al., 1996), a fraction of bone marrow myeloid precursors, erythroblasts, and megakaryocytes (Gattei et al., 1997). More recently, a high expression of CD30L has been observed on medullary thymic epithelial cells (TECs), including those present in the outer part of Hassal's corpuscles (Romagnani et al., 1998a). A variety of tumors derived from hematopoietic cells of myeloid and lymphoid origin (Gattei et al.,

Important homologies Human and murine CD30L are expressed as 236 and 239 amino acid membrane proteins showing a 72% amino acid sequence identity (Smith et al., 1993). Both proteins have the classic characteristics of a type II membrane protein: first, the lack of an obvious signal peptide; second, an internal 21 residue hydrophobic domain (transmembrane domain) 46 residues from the N-terminus (cytoplasmic domain); and third, an 172 residue C-terminal domain containing six potential N-linked glycosylation sites (extracellular

Figure 2 Amino acid sequence for CD30L. Sequence MDPGLQQALNGMAPPGDTAMHVPAGSVASHLGTTSRSYFYLTTATLALCLVFTVATI MVLVVQRTDSIPNSPDNVPLKGGNCSEDLLCILKRAPFKKSWAYLQVAKHLNKTKLS WNKDGILHGVRYQDGNLVIQFPGLYFIICQLQFLVQCPNNSVDLKLELLINKHIKKQA LVTVCESGMQTKHVYQNLSQFLLDYLQVNTTISVNVDTFQYIDTSTFPLENVLSIFLY SNSD.

468 Paola Romagnani, Carmelo Mavilia and Sergio Romagnani 1997) also express CD30L. These include acute myeloid leukemias (M3, M4, and M5 phenotypes), Hodgkin's disease (HD), B lineage acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia, B cell non-Hodgkin's lymphomas, hairy cell leukemia, T cell prolymphocytic leukemia, a fraction of multiple myelomas, peripheral T non-Hodgkin's lymphoma, and adult T cell leukemia/lymphoma (Gruss et al., 1994, 1996; Trentin, 1997; Younes et al., 1997a). CD30L is also expressed by cells of some nonhematopoietic tumors, such as yolk sac and nullipotent embryonal carcinoma (Pera et al., 1997). The cells expressing CD30L in mice and humans are listed in Table 1.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators CD30L is constitutively expressed by some cell types (B cells, neutrophils, eosinophils, and others; see above), whereas in other cells it can be induced by different stimuli. CD30L expression was induced on T cells by ionomycin A, TPA, PMA, ConA, PHA, and anti-CD3 antibody, the strongest expression resulting from the combination of ionomycin with PHA or PMA (Smith et al., 1993; Gruss et al., 1994; Wiley et al., 1996; Gattei et al., 1997). CD30L was induced on monocytes by lipopolysaccharide (LPS) and IL-1 Table 1 Cell and tissue sources of CD30L Cell types

Cell state

References

Resting Activated Human Monocytes

ÿ

+

Smith et al., 1993

T lymphocytes

ÿ

+

Smith et al., 1993

B lymphocytes

+

Younes et al., 1996

Granulocytes

+

Gruss et al., 1994

Eosinophils

+

Pinto et al., 1996

Myeloblasts

+

Gattei et al., 1997

Erythroblasts

+

Gattei et al., 1997

Megakaryocytes +

Gattei et al., 1997

Medullary TEC

Romagnani et al., 1998a, b

++

Murine Macrophages

ÿ

+

Smith et al., 1993

T lymphocytes

ÿ

+

Smith et al., 1993

(Smith et al., 1993). Cytokines regulating eosinophil proliferation (IL-3, IL-5, and GM-CSF) were able to enhance CD30L expression on eosinophils (Pinto et al., 1996). So far, no inhibitory stimuli for CD30L expression have been reported, although the possibility that the production of sCD30 may downregulate CD30L has been suggested (Younes et al., 1997b).

RECEPTOR UTILIZATION CD30L binds to CD30.

IN VITRO ACTIVITIES

In vitro findings The in vitro activities of CD30L/CD30 interactions have been investigated by mimicking the activity of CD30L with agonistic anti-CD30 antibodies (Gruss et al., 1994, 1995, 1996; Del Prete et al., 1995; McDonald et al., 1995) or by using a recombinant form of CD30L (Powell et al., 1998). CD30L/CD30 interactions in vitro may indeed result in either cell proliferation or cell death, depending on the cell type receiving the signal and perhaps also on the microenvironmental conditions in which the signal is delivered (Gruss et al., 1994). Opposite effects of the CD30L are not caused by CD30 mutations and do not correlate with differences in calcium mobilization after CD30 crosslinking (Jung et al., 1994). The in vitro activities of CD30L/CD30 interactions observed in different cell types are listed in Table 2.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS None are known.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Role in experiments of nature and disease states The pathophysiologic roles of CD30L in normal humans and disease states are unclear because (i) its

CD30 Ligand 469 Table 2 In vitro activities of CD30L Target

Effect

Reference

ATL cell lines

Proliferation

Gruss et al., 1994

H-RS cell lines

Proliferation

Gruss et al., 1994

IL-6, TNF production

Gruss et al., 1995

CD54 expression and release

Gruss et al., 1996

NFB activation

McDonald et al., 1995

Memory CD4+

Amplification of TH2 T cell responses

Del Prete et al., 1995

CD4+ T cell clones

Proliferation

Del Prete et al., 1995

IL-4 and IL-5 production

Del Prete et al., 1995

Human

NFB activation

McDonald et al., 1995

T cell hybridomas

TCR-dependent cell death

Tian et al., 1995; Lee et al., 1996

LCAL cell lines

Growth inhibition; cytolysis

Gruss et al., 1994

HIV-infected T cells

Enhanced HIV expression

Biswas et al., 1995; Maggi et al., 1995

EBV-transformed B cell lines

Enhanced Ig production

Gruss et al., 1994

CTL lines

IL-5 production

Bowen et al., 1996

B lymphocytes

Growth and differentiation

Shanebeck et al., 1995

Murine

biological activities in vitro are heterogeneous depending on the cellular target, and (ii) experiments with CD30L knockouts or CD30L transgenic animals are still lacking (see above). Such roles can, therefore, be only suspected on the basis of in vitro studies, as well as on the demonstration of CD30L expression in given tissues and/or certain pathologic conditions. The possibility that CD30L can not only act as a signaling ligand for CD30‡ cells, but also receive and transduce signals to the ligand-bearing cell (`reverse signaling') has also been reported (Wiley et al., 1996). Peripheral blood T cells exposed to suboptimal concentrations of anti-CD3 increased their metabolic activity, proliferated, and produced IL-6 in response to the crosslinking of CD30L, suggesting that, as for other TNF family members and their cognate receptors, bidirectional signaling may occur (Wiley et al., 1996). Possible Role in Thymic Negative Selection One possible role of CD30L has been suggested on the basis of the demonstration that CD30 knockout mice have an impaired negative selection (Amakawa et al., 1996). Accordingly, high CD30L expression in human TECs has been observed, which is associated

with the presence of a remarkable number of CD4‡ CD8‡ CD45RO‡ IL-4R‡ CD30‡ thymocytes selectively localized to the medullary areas (Romagnani et al., 1996a) (Figure 3). Thus, during thymus development, CD30L expressed by medullary TECs may contribute to the negative selection of autoantigenactivated, CD30-expressing thymocytes (Amakawa et al., 1996; Romagnani et al., 1998b). Amplification of Type 2 TH Responses and B Cell Function The reason why, for mature activated T cells, the interaction with CD30L-expressing cells results in a costimulatory rather than an apoptotic signal remains to be clarified. Since CD30 is preferentially expressed by TH0/TH2 cells, it has been suggested that CD30L can, on effector cells, provide a signal for the amplification of TH2 responses (Del Prete et al., 1995). No inhibition of the development of TH2 responses has, however, been observed in CD30 knockout mice (Barner et al., 1997). On the other hand, it is clear that CD30L provides an important signal for the proliferation of, and possibly also immunoglobulin production by, both murine and human B lymphocytes (Gruss et al., 1994; Shanebeck et al., 1995).

470 Paola Romagnani, Carmelo Mavilia and Sergio Romagnani Figure 3 High CD30L expression in medullary epithelial cells of postnatal human thymus. (A) Immunostaining of a thymic section with anti-CD30L antibody (M81; Genzyme Diagnostics, Cambridge, MA) using the avidin± biotin±peroxidase method and the AEC substrate (Romagnani et al., 1998a). CD30L-immunoreactivity (red) in the medulla is visible (40). (B) CD30Limmunoreactivity (red) in the outer part of a Hassal's corpuscle (1000). (C) CD30L immunoreactivity (red) in the outer part of one Hassal's corpuscle (large arrow) and in some cells (small arrows) scattered in the medullary area (250). (D) Double immunostaining for CD30L and cytokeratin in the thymic medulla. CD30L was identified using M81 antibody and the AEC substrate (red) and cytokeratin by using an anti-pan-cytokeratin antibody (C11; Sigma Immunochemicals, Milano, Italy) and the vector SG substrate (bluish grey) (Romagnani et al., 1998a). Hassal's corpuscles (large arrows) and single cells (small arrows) staining for both CD30L and cytokeratin (purple-brown), as well as many cells staining for cytokeratin alone, are visible (250). (Full colour figure can be viewed online.) Figure 3A±B

CD30 Ligand 471 Figure 3C±D (Contd.)

Regulatory Role in Malignant Disorders The importance of CD30L in human diseases is still unclear, but there is strong evidence to suggest that it plays a critical pathophysiologic role in the regulation of the growth of malignant lymphomas, particularly Hodgkin's disease, large cell anaplastic lymphomas (LCALs), Burkitt's lymphomas and CD30‡ cutaneous T cell lymphomas (Gruss et al., 1994, 1995; Tian et al., 1995; Lee et al., 1996). In Hodgkin's disease, CD30L enhances cytokine production, such as that of IL-6 and TNF, as well as surface intercellular adhesion molecule 1 (ICAM-1/ CD54) expression and shedding, by Hodgkin and Reed-Sternberg (H-RS) cells (Gruss et al., 1994).

CD30L transduction in H-RS cells involves the activation of a tyrosine kinase and of a mitogenactivated protein kinase (Wendtner et al., 1995). Of interest is the demonstration of high CD30L expression by eosinophils present in HD-involved lymph nodes, as well as its ability to transduce proliferative signals on CD30‡ target cells, including H-RS cells (Gruss et al., 1995). These data suggest that eosinophils may contribute to the deregulated network of CD30L/CD30-mediated interactive signals between H-RS cells and the surrounding reactive cells in HD-involved tissues (Pinto et al., 1996). In lymphoma patients with CD30‡ tumors, CD30L expression was limited to a few B cells and was

472 Paola Romagnani, Carmelo Mavilia and Sergio Romagnani reduced in those showing a high level of sCD30, suggesting that the enhanced production of sCD30 by these tumors may provide a mechanism to escape the apoptosis-inducing activity of CD30L (Younes et al., 1997b). A direct growth-inhibitory effect, or even cytolytic cell death, was induced by CD30L on CD30‡ LCAL. The CD30L/CD30 interaction seems to play some role in the autocrine regulation of embryonal carcinoma stem cells (Pera et al., 1998). Role in HIV Infection The importance of CD30L/CD30 interaction in the progression of HIV infection has been supported by studies performed both in vivo and in vitro. High levels of sCD30 were found in the sera of HIVinfected individuals, which appeared to act as an independent predictor of unfavorable prognosis for the development of full-blown disease (Pizzolo et al., 1994). Other authors have confirmed the elevated level of sCD30 in the serum of HIV-infected patients (Rizzardi et al., 1997; Sabin et al., 1997), but no association between sCD30 serum elevation and more rapid HIV progression has been found (Sabin et al., 1997). The in vitro studies, however, have clearly shown that CD30 crosslinking strongly enhances HIV replication in chronically HIV-infected T cell lines (Biswas et al., 1995). This effect was found to be mediated by NF (B activation, which in turn activated viral LTR (Biswas et al., 1995). Accordingly, both agonistic anti-CD30 antibodies and CD30L-expressing, glutaraldehyde-fixed CD8‡ human T cell clones significantly enhanced HIV expression in human CD4‡ T cells obtained from HIV-infected individuals (Maggi et al., 1995). Taken together, these data suggest that the activation of CD30 expression in HIV-infected CD4‡ T cells, which occurs mainly during TH2 responses, may allow the interaction of these cells with CD30L-expressing cells and, therefore, favor HIV replication. Role in Inflammatory Disorders A high level of CD30L expression has been observed on alveolar macrophages in patients with sarcoidosis (Nicod and Isler, 1997), but the meaning of this finding remains obscure.

IN THERAPY So far, no therapeutic applications based on the use of CD30L, its possible enhancers or inhibitors have been described.

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