IL15

IL-15 Thomas A. Waldmann* and Yutaka Tagaya Metabolism Branch, National Cancer Institute, NIH Building 10, Room 4N115, 10 Center Drive, MSC 1374, Bethesda, MD 20892-1374, USA * corresponding author tel: 301-496-6653, fax: 301-496-0056, E-mail: [email protected] DOI: 10.1006/rwcy.2000.03008.

SUMMARY IL-15 is a 14±15 kDa member of the four  helix bundle family of cytokines. IL-15 expression is controlled at the levels of transcription, translation, and intracellular trafficking. In particular, IL-15 is posttranslationally regulated by multiple controlling elements that impede translation, including 13 upstream AUGs of the 50 UTR, two unusual signal peptides, and the C-terminus of the mature protein. IL-15 uses two distinct receptor and signaling pathways. In T and NK cells the type 1 IL-15 receptor includes IL-2/ 15R and c subunits which are shared with IL-2 as well as an IL-15-specific receptor subunit, IL-15R. Mast cells respond to IL-15 with a receptor system that does not share elements with the IL-2R but uses a novel 60±65 kDa IL-15RX subunit. This type 2 receptor signaling involves JAK2/STAT5 activation rather than the JAK1/JAK3 and STAT5/STAT3 system used by the type 1 system in activated T cells. In addition to its other functional activities in immune and nonimmune cells, IL-15 plays a pivotal role in the development, survival and function of NK cells. Abnormalities of IL-15 expression have been described in patients with rheumatoid arthritis, inflammatory bowel disease, and in diseases associated with the retroviruses HIV and HTLV-I. New approaches inhibiting the action of IL-15, its receptor or its signaling pathway may be of value in the therapy of these diseases.

BACKGROUND

Discovery In May 1994, two groups simultaneously reported identifying the cytokine now termed IL-15 (Bamford

et al., 1994; Burton et al., 1994; Grabstein et al., 1994). As a part of an analysis of the cytokines produced by the HTLV-I-associated adult T cell leukemia (ATL) cell line, HuT-102, Burton, Bamford, Waldmann, and coworkers (1994) reported purifying a T cell stimulatory cytokine provisionally termed IL-T. In the same month the group of Grabstein, Giri and coworkers (1994) by analysis of the cell line CV-1 EBNA identified a novel cytokine, molecularly cloned the gene encoding it, and termed the cytokine IL-15. Using an appropriate anti-IL-15 antibody, IL-T and IL-15 were shown to be identical (Bamford et al., 1996a).

Alternative names Initially the alternative names IL-T and IL-15 were utilized. Now all groups use the term IL-15.

Structure IL-15 is a 14±15 kDa glycoprotein whose mature form consists of 114 amino acids (Grabstein et al., 1994). It is a member of the four  helix bundle family of cytokines.

Main activities and pathophysiological roles IL-15 stimulates the proliferation of activated CD4± CD8±, CD4‡CD8‡, CD4‡, and CD8‡ cells and dendritic epidermal T cells (Burton et al., 1994; Grabstein et al., 1994; Edelbaum et al., 1995; Zhang et al., 1998; Garcia et al., 1998). Although IL-15 does not have an effect on resting B cells it induces

214 Thomas A. Waldmann and Yutaka Tagaya proliferation and immunoglobulin synthesis by B cells costimulated by PMA or by an immobilized antibody to immunoglobulin M (Armitage et al., 1995). One of the most critical functions of IL-15 is a pivotal role in the development, survival, and activation of natural killer (NK) cells (Carson et al., 1994; MroÂzek et al., 1996; Suzuki et al., 1997; Mingari et al., 1997; Ogasawara et al., 1998). IL-15 also has unique functions on nonlymphoid cells, including actions on muscle, brain, microglia, and mast cells (Lee et al., 1996; Tagaya et al., 1996a, 1996b; Hanisch et al., 1997; Quinn et al., 1997).

GENE AND GENE REGULATION

Accession numbers See Table 1.

Chromosome location The IL-15 gene was mapped to chromosome 4q31 (human) and to the central region of chromosome 8 (mouse) by fluorescence in situ hybridization (Anderson et al., 1995a). The IL-15 gene consists of nine exons (exon 1±8 and a newly discovered exon 4a). The nine introns span at least 35 kb. This exon± intron organization contrasts with the four exon± three intron architectural pattern observed with IL-2, IL-4, IL-5, and IL-13 (Seigel et al., 1984).

Table 1 Accession numbers for IL-15 DNA sequences Species

Type

Accession numbers

Human

cDNA

U14407

cDNA (short signal peptide isoform)

AF031167, X94222

Genomic

X91233

0

5 upstream

AF038163

Simian

cDNA

U03099

Murine

cDNA

U14332

cDNA (short signal peptide isoform)

AB022307

50 upstream

AF038164, AB006745

Rat

cDNA

AF015719

Bovine

cDNA

U42433

Chicken

cDNA (provisional)

AF000631

Relevant linkages There are no relevant linkages to other genes.

Regulatory sites and corresponding transcription factors Human IL-15 mRNA contains a 50 untranslated region (50 UTR) of at least 352 nucleotides, a coding sequence of 486 nucleotides, and a 30 UTR of at least 400 nucleotides (Grabstein et al., 1994). There are two alternative leader peptides, one with 48 amino acids and one with 21 amino acids (Grabstein et al., 1994; Meazza et al., 1996; Onu et al., 1997; Tagaya et al., 1997). The classical long (48 amino acid) signal peptide associated with all secreted IL-15 is encoded by a 1.6 kb cDNA. It is encoded by exons 3, 4, and 5 of the human IL-15 gene (Anderson et al., 1995a) (Figure 1). The short 21 amino acid signal peptide is encoded by a 1.2 kb cDNA that lacks the elements encoded by exon 1. This signal peptide is encoded by exon 5 and by an additional 119 nucleotide sequence inserted between exons 4 and 5 (new exon 4a) (Meazza et al., 1996; Onu et al., 1997; Tagaya et al., 1997). The introduction of the 119 nucleotides of exon 4a disrupts the 48 amino acid signal sequence by inserting a premature termination codon and then provides an alternative initiation codon with a poor Kozak context (TTCATGG). As noted below, IL-15 associated with the short 21 amino acid signal peptide is not secreted but rather is stored intracellularly, appearing in nuclear and cytoplasmic components. There is widespread constitutive expression of IL15 mRNA in a variety of tissues. The regulation of IL-15 expression is multifaceted. Modest control occurs at the level of transcription and a dominant control occurs posttranscriptionally at the levels of translation and intracellular trafficking. The cloning of the human and murine 50 flanking regions of the IL-15 gene has permitted the study of the mechanisms underlying the constitutive and induced transcriptional regulation of IL-15 mRNA. A series of conserved motifs between mouse and human IL-15 50 regulatory regions has been identified; these putative transcriptional motifs include GCF, NFB, IRF-E, myb, IRE, NF-IL-6, and INF-2 (Azimi et al., 1998; Washizu et al., 1998). As demonstrated through the use of a series of reporter assays using IL-15 promoter deletion mutants, the IRF-E motif is critical for the activation of the IL-15 promoter (Azimi et al., 1998; Ogasawara et al., 1998). The IL-15 generated acts on NK cell precursors stimulating their development into mature NK cells. Another transcription factor that

IL-15 215 Figure 1

Generation of two IL-15 isoforms from the human IL-15 locus. Intron 4

2

3

LSP-IL-15

4

4a

1–3

5

3 4 5

6

7

8

5– 8

48aa SP 21aa SP SSP-IL-15

3, 4

I4 4a 5

5– 8

ATG (alternative) TAG (termination)

appears to play an important role in IL-15 transcription is NFB acting on an NFB motif adjacent to the IRF-E motif (Azimi et al., 1998; Washizu et al., 1998). Posttranscriptional Regulation of IL-15 Protein Expression IL-15 is predominantly regulated posttranscriptionally at the levels of translation and translocation. In particular, although IL-15 mRNA is widely expressed constitutively it has been difficult to demonstrate IL-15 in supernatants of many cells that express such mRNA (Grabstein et al., 1994; Bamford et al., 1996a, 1996b). There are multiple controlling elements that impede translation, including 13 upstream AUGs of the 50 UTR, two unusual signal peptides, and the C-terminus of the mature protein. Initial studies focused on the 50 UTR of IL-15 mRNA (Bamford et al., 1996a). In general, the 50 UTR of effectively translated messages are short, simple, and unencumbered by AUGs upstream of the initiation AUG (Kozak, 1987). In contrast to this pattern, the 50 UTR of the message encoding IL-15 is long, quite complex, and includes multiple upstream AUGs (5 in mice, 13 in humans). Kozak (1991) has emphasized that the presence of such AUGs in the 50 UTR of mRNAs may dramatically reduce the efficiency of their translation. An IL-15 construct with all upstream AUGs deleted produced approximately 12-fold more IL-15 than cells transfected with the wild-type construct (Bamford et al., 1996a; Waldmann and Tagaya, 1999). Using expression constructs that exchange the signal peptide, coding sequences of IL-2 and IL-15 linked to the alternative mature protein coding sequence, it was shown that the IL-15 long signal peptide and the initiation ATG codon were important factors in the negative regulation of

IL-15 protein expression (Bamford et al., 1998). In particular, the linkage of the IL-2 signal peptide to the IL-15 mature coding sequence increased IL-15 production 20-fold compared with the construct with the IL-15 signal peptide. Reciprocally, linkage of the IL-15 signal peptide to the IL-2 mature coding sequence reduced IL-2 production 40-fold compared with that obtained with the wild-type IL-2 signal peptide linked to the IL-2 mature coding sequence. Finally, a third negative element may exist in the C-terminus of the IL-15 mature protein coding sequence or protein (Bamford et al., 1998). The variety of negative regulatory features controlling IL-15 expression may be required in light of the potency of IL-15 as an inflammatory cytokine that if indiscriminately expressed could lead to serious disorders including autoimmune diseases. In terms of a more positive role for IL-15, by maintaining a pool of translationally inactive IL-15 mRNA, diverse cells may respond rapidly to an intracellular infection or to other stimuli by transforming IL-15 mRNA into a form of mRNA that can be translated effectively. Two isoforms of human IL-15 exist: one isoform has a short 21 amino acid putative signal peptide whereas the other isoform has an unusually long 48 amino acid signal peptide. In addition to their role in the regulation of IL-15 translation, the signal peptides influence intracellular trafficking of IL-15 (Meazza et al., 1996; Onu et al., 1997; Tagaya et al., 1997; Waldmann and Tagaya, 1999). The 21 amino acid IL-15 isoform is translated but IL-15 is not secreted; instead it is distributed into the cytoplasm and the nucleus (Tagaya et al., 1997). The IL-15 associated with the long 48 amino acid signal peptide presents a more complex trafficking pattern. Some is demonstrable in the cytoplasm. The other forms enter the endoplasmic reticulum where they are glycosylated. An alternatively processed form with partial

216 Thomas A. Waldmann and Yutaka Tagaya processing of the signal peptide is retained within the cell, whereas the fully processed form is secreted (Waldmann and Tagaya, 1999).

Cells and tissues that express the gene On northern blot analysis there is widespread expression of IL-15 mRNA in the placenta, skeletal muscle, kidneys, lung, heart, fibroblasts, epithelial cells, dendritic cells, and monocytes (Grabstein et al., 1994; Bamford et al., 1996a). IL-15 mRNA could not be demonstrated by northern blot analysis in normal resting or phytohemagglutinin-activated T cells, although through the use of the more sensitive RNase protection assay (RPA) IL-15 mRNA was demonstrated in normal T cells (Azimi et al., 1999). Although IL-15 mRNA is widely distributed, the modest IL-15 protein that has been identified was produced by monocytes and dendritic cells. Table 2 Accession numbers for IL-15 protein sequences Species

Type

Accession numbers

Human

Protein

P40933

Protein (short signal peptide isoform)

CAA63913, AAB97518

Murine

Protein

P48346

Rat

Protein

P97604

Bovine

Protein

Q28028

PROTEIN

Accession numbers See Table 2.

Sequence Sequences of the human and murine short and long signal isoforms of IL-15 are shown in Figure 2.

Description of protein IL-15 is a 14±15 kDa glycoprotein whose mature form consists of 114 amino acids (Grabstein et al., 1994). It has two cystine disulfide crosslinkages at positions Cys42±Cys88 (homologous to IL-2) and Cys35± Cys85. IL-15 is a member of the four  helix bundle cytokine family, which includes cytokines IL-2, IL-3, IL-5, IL-6, IL-7, and IL-9 (Bazan, 1990).

Discussion of crystal structure The predicted folding topology of IL-15 suggests three loops connecting the four helixes in an up up down down configuration (Grabstein et al., 1994).

Important homologies IL-15 shares no sequence homology with IL-2 or with other members of the cytokine superfamily. However,

Figure 2 Sequences of the human and murine short and long signal isoforms of IL-15. Human IL-15 (short signal peptide isoform):

–21 1 51 101

MVLGTIDLCS NWVNVISDLK SLESGDASIH QSFVHIVQMF

CFSAGLPKTE A KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL INTS

Human IL-15 (long signal peptide isoform):

–48 1 51 101

MKILKPYMRN NWIDVRYDLE LHEYSNMTLN QSFIRIVQMF

TSISCYLCFL LNSHFLTEAG IHVFILGCVS VGLPKTEA KIESLIQSIH IDTTLYTDSD FHPSCKVTAM NCFLLELQVI ETVRNVLYLA NSTLSSNKNV AESGCKECEE LEEKTFTEFL INTS

Murine IL-15 (short signal peptide isoform):

–26 1 51 101

MAPSSKELGF NWIDVRYDLE LHEYSNMTLN QSFIRIVQMF

VSSYSCVSVG LPKTEA KIESLIQSIH IDTTLYTDSD FHPSCKVTAM NCFLLELQVI ETVRNVLYLA NSTLSSNKNV AESGCKECEE LEEKTFTEFL INTS

Murine IL-15 (long signal peptide isoform):

–48 1 51 101

MKILKPYMRN NWIDVRYDLE LHEYSNMTLN QSFIRIVQMF

TSISCYLCFL LNSHFLTEAG IHVFILGCVS VGLPKTEA KIESLIQSIH IDTTLYTDSD FHPSCKVTAM NCFLLELQVI ETVRNVLYLA NSTLSSNKNV AESGCKECEE LEEKTFTEFL INTS

IL-15 217 structural homology among these members is clearly conserved. There is 97% sequence identity between human and simian IL-15 and 82% sequence identity between human and porcine IL-15 (Anderson et al., 1995a).

IL-15 mRNA is upregulated by IFN ‡LPS acting through IRF-1 and an IRF-E-binding site in the promoter region (Ogasawara et al., 1998, Azimi et al., 1998).

Posttranslational modifications

RECEPTOR UTILIZATION

There are three Asn residues (119, 127, and 160) that in two cases are sites for N-glycosylation (Grabstein et al., 1994).

IL-15 uses two receptor and signaling pathways (Bamford et al., 1994; Grabstein et al., 1994; Giri et al., 1994; Tagaya et al., 1996a, 1996b). The type 1 receptor expressed in T and NK cells is made up of three distinct membrane components. Two of these components, IL-2/IL-15R and c, are shared with IL-2 (Bamford et al., 1994; Grabstein et al., 1994; Giri et al., 1994). The two cytokines have their own private  chains: IL-2R for IL-2 and IL-15R for IL-15 (Giri et al., 1995). The c chain is also shared by IL-4, IL-7, and IL-9 (Kondo et al., 1993; Noguchi et al., 1993). The IL-15R, the private chain for IL-15, is a type 1 membrane protein with a predicted signal peptide of 32 amino acids, a 173 amino acid extracellular domain, a single membrane spanning region of 21 amino acid, and a 37 amino acid cytoplasmic domain (murine IL-15R, Giri et al., 1995). It is not a member of the cytokine receptor superfamily. However, a comparison of IL-2R and IL-15R revealed the shared presence of a conserved motif known as a GP-1 motif, or a SUSHI domain (Giri et al., 1995). Another factor linking IL-2R and IL-15R is the demonstration that the IL-2R and IL-15R genes have a similar intron-exon organization. Moreover, they are closely linked in both human (10p15-14) and murine genomes (chromosome 2) (Anderson et al., 1995b). IL-15R binds IL-15 with a Kd (dissociation constant) of 10ÿ11 M, 1000-fold higher affinity than that of IL-2R for IL-2. IL-2/15R in association with c is able to bind IL-15 at a lower affinity (Kd of 10ÿ9 M) and in select cells can transduce an IL-15 signal in the absence of IL-15R. IL-15R has a wide cellular distribution (Anderson et al., 1995b), being expressed in T cells, B cells, monocytes, thymic and bone marrow stromal cells, as well as liver, heart, spleen, lungs, skeletal muscle, and activated endothelial cells. The type 1 receptor/signaling pathway in activated T cells utilizes JAK3 and JAK1 as well as STAT3 and STAT5 (Witthuhn et al., 1994; Johnston et al., 1995; Lin et al., 1995). The signaling pathways in T cells also involves the phosphorylation of the srcrelated cytoplasmic tyrosine kinases p56lck and p72syk, the induction of the expression of the Bcl-2 antiapoptotic protein, and the stimulation of the Ras/ Raf and MAP kinase pathway leading to fos/jun activation (Miyazaki et al., 1995).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-15 mRNA is constitutively expressed in a variety of tissues including placenta, skeletal muscle, kidney, lung, heart, fibroblasts, epithelial cells, dendritic cells, and monocytes (Grabstein et al., 1994; Bamford et al., 1996a,b). Low levels of IL-15 mRNA are also produced by T cells (Azimi et al., 1998). How-ever, IL15 protein has only been demonstrated in normal cells from activated monocytes and dendritic cells. Cell lines that produce IL-15 protein include HuT-102 (HTLV-I‡ T cells), CV-1/EBNA (monkey kidney epithelial cell), 10P12 (murine mast cell), and 9 of 11 squamous cell carcinomas studied.

Eliciting and inhibitory stimuli including exogenous and endogenous modulators Freshly isolated monocytes express only low levels of IL-15 mRNA that was upregulated when the monocytes were activated with IFN or LPS/IFN (Bamford et al., 1996a). In addition, infection of monocytes with herpesvirus 6, herpesvirus 7, bacillus Calmette-GueÂrin (BCG), Mycobacterium tuberculosis, Toxoplasma gondii, Salmonella choleraesuis, Mycobacterium leprae, Cryptococcus neoformans, or Candida albicans was associated with an upregulation of IL-15 mRNA and in some cases protein expression (Carson et al., 1995; Flamand et al., 1996; Doherty et al., 1996; Atedzoe et al., 1997; Mody et al., 1998; Nishimura et al., 1998). In T cells HTLV-I-encoded Tax protein transactivates IL-15 gene transcription through an NFB site (Azimi et al., 1998).

218 Thomas A. Waldmann and Yutaka Tagaya IL-15 uses a distinct type 2 receptor/signal transduction pathway in mast cells. Mast cells respond to IL-15 with a receptor system that does not share elements with the IL-2R but uses a novel 60±65 kDa IL-15RX subunit (Tagaya et al., 1996a, 1996b). In mast cells, IL-15 signaling through the type 2 receptor involves JAK2/STAT5 activation rather than the JAK1/JAK3 and STAT5/STAT3 system used in activated T cells (Tagaya et al., 1996a, 1996b).

IN VITRO ACTIVITIES

In vitro findings The response to IL-15 through the class 1 receptors requires the expression of IL-15R. This in turn may require activation of T cells, B cells, and NK cells. Thus although IL-15 does not have an effect on resting B cells, it induces proliferation and immunoglobulin synthesis by B cells costimulated by PMA or by an immobilized antibody to immunoglobulin M (Armitage et al., 1995). Furthermore, when used in concert with CD40 ligand (CD40L) it is an inducer of polyclonal IgM, IgG1, and IgA but not IgG4 or IgE (Armitage et al., 1995). The effect of IL-15 on immunoglobulin secretion can be modulated by IL-10 (increased secretion) or by IL-4 (decreased secretion) (Salvucci et al., 1996). The effect of IL-4 may be caused by its diversion of the shared c receptor, a conclusion supported by fluorescence resonance energy transfer (FRET) analysis of receptor subunit association after cytokine addition (Damjanovich et al., 1997). IL-15 stimulates the proliferation of activated CD4±CD8±, CD4‡CD8‡, CD4‡, and CD8‡ cells (Burton et al., 1994; Grabstein et al., 1994; Edelbaum et al., 1995; Zhang et al., 1998; Garcia et al., 1998). One of the most critical functions of IL-15 through the type 1 receptor is a role in the development, survival and activation of NK cells (Carson et al., 1994; MroÂzek et al., 1996; Mingari et al., 1997; Suzuki et al., 1997; Ogasawara et al., 1998). Mice made deficient in IL-2/IL-15R , c, IRF-1, JAK3, or STAT5a/b that are required for IL-15 action are deficient in NK cells (Macchi et al., 1995; Russell et al., 1995; Suzuki et al., 1997; Imada et al., 1998; Ogasawara et al., 1998; Ohteki et al., 1998; Teglund et al., 1998). Furthermore, the addition of IL-15 to bone marrow progenitor cells or to mature postnatal thymocytes or to fetal thymic organ cultures leads to the development of NK cells (Cavazzana-Calvo et al., 1996; Mingari et al., 1997). IL-15 also has unique functions on nonlymphoid cells. Acting through the type 2 receptor it stimulates

mast cell proliferation (Tagaya et al., 1996a, 1996b). Although the type of receptor has not been defined, IL-15 acts on skeletal muscles to induce muscle fiber hypertrophy (Quinn et al., 1997), on microvasculature to promote angiogenesis (Angiolillo et al., 1997), and on microglia and astrocytes (Lee et al., 1996; Hanisch et al., 1997). The action of IL-15 on T cells, B cells, and NK cells can be blocked by select antibodies to the IL-2/IL-15R chain (Bamford et al., 1994; Grabstein et al., 1994).

Bioassays used The cytokine-dependent CTLL-2 murine T cell line can be used in a bioassay for IL-2 or IL-15. In the presence of antibodies to IL-2, it is IL-15 specific. This cell line detects both human and murine IL-15. Similarly, the human cytokine-dependent KIT-225K6 cell line is responsive to human IL-2, IL-7, and IL-15 and can be used in bioassays of IL-15 levels. Human IL-15 can also be measured using a cytokinespecific ELISA assay. There is no commercial assay system available for detecting murine IL-15.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Mice made deficient in IRF-1 by gene targeting do not manifest inducible IL-15 expression (Ogasawara et al., 1998; Ohteki et al., 1998). Such mice can be used to define the in vivo effects of IL-15. These mice lack NK cells but produce NK cells from precursors following IL-15 addition. Furthermore, IL-15 has been shown to be important for the development of NK-T cells and intestinal intraepithelial lymphocytes.

Species differences No species differences have been examined in in vivo IL-15 action.

Knockout mouse phenotypes No published reports are available on IL-15 knockout or transgenic mice. IL-15R knockout mice (Lodolce

IL-15 219 et al., 1998) are markedly lymphopenic despite grossly normal T cell and B cell development. This lymphopenia is due to decreased proliferation and decreased homing of IL-15Rÿ/ÿ lymphocytes to peripheral lymph nodes. These mice are also deficient in NK cells, NK-T cells, CD8‡ lymphocytes, and

TCR  intraepithelial lymphocytes (Lodolce et al., 1998).

Pharmacological effects IL-15 acting through the IL-2/15R and c receptors leads to the induction of the expression of IL-2R by T lymphocytes (Ye et al., 1996; Treiber-Held et al., 1996; Bulfone-Paus et al., 1997).

Endogenous inhibitors and enhancers The effect of IL-15 on immunoglobulin secretion is increased by IL-10 and decreased by IL-4 (Salvucci et al., 1996).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Using available ELISA and bioassay procedures IL-15 cannot be demonstrated in normal serum. Inducible IL-15 appears to be required for the generation, maintenance, and survival of NK cells and for the development of natural killer T cells, select CD8 cells and TCR  intraepithelial lymphocytes (reviewed in Waldmann and Tagaya, 1999).

Role in experiments of nature and disease states Failure to produce IL-15 has not been demonstrated in disease states although it presumably would be associated with the absence of NK cell development. Abnormalities involving increased IL-15 expression have been reported in inflammatory autoimmune diseases. McInnes and coworkers (1996, 1997) reported elevated levels of IL-15 in rheumatoid

arthritis. They suggested that IL-15 may precede TNF in the cytokine cascade and suggest a role for IL-15 in development of inflammatory arthritis. Abnormalities have also been reported in other inflammatory diseases including active ulcerative colitis, Crohn's disease, type C chronic liver disease, sarcoidosis, T cell-mediated alveolitis, and in multiple sclerosis (Agostini et al., 1996; Kakumu et al., 1997; Kivisakk et al., 1998). Furthermore, the observation that IL-15 stimulates mast cell proliferation suggests a potential role for this cytokine in mastocytosis (Tagaya et al., 1996a, 1996b). Increased IL-15 expression has also been observed in retroviral diseases and neoplasia. HTLV-I-infected T cells of patients with the neurological disorder tropical spastic paraparesis (TSP/HAM) express the HTLV-Iencoded transactivator p40tax. The expression of tax induces IL-15 expression, which in turn participates along with IL-2 in an autocrine cytokinemediated, spontaneous proliferation of T cells when they are studied ex vivo (Azimi et al., 1999). This proliferation can be inhibited in part by antibodies to IL-15 or to IL-2 and virtually completely abrogated by simultaneously administered antibodies to both cytokines or to both cytokine receptors, suggesting that two cytokine-mediated autocrine systems occur as a consequence of HTLV-I infection. Similarly, the production of IL-15 by HTLV-I-associated T cells in adult T-cell leukemia (ATL) associated with HTLV-I tax transactivation has been observed. In the case of the ATL cell line HuT-102 that permitted the identification of IL-15, the overproduction of IL-15 was associated with the generation of a fusion message wherein the HTLV-I virus R region was joined in a chimeric RNA with the IL-15 mature protein-coding region (Bamford et al., 1996a). In particular, the predominant IL-15 mRNA expressed by HuT-102 was a chimeric mRNA with a 118 nucleotide segment of the R region of the long terminal repeat (LTR) joined to the 50 UTR of IL-15. This fusion yielded a marked increase in IL-15 mRNA transcription. Furthermore, the introduction of the R segment eliminated 10 of the 13 upstream AUGs that, as indicated above, behave as impediments to translation. IL-15 serum levels were elevated in HIV-infected individuals (Kacani et al., 1997). Several of the tumor cell lines examined including those from lung, ovarian, melanoma, some leukemia, osteosarcoma, and especially rhabdomyosarcoma expressed elevated levels of IL-15 mRNA (Meazza et al., 1996; Barzegar et al., 1998). This mRNA usually represented the short signal peptide (SSP) IL-15 isoform which is not secreted but trafficks to the cytoplasm and the nucleus.

220 Thomas A. Waldmann and Yutaka Tagaya

IN THERAPY

Preclinical ± How does it affect disease models in animals? IL-15 has not been used in in vivo therapeutic trials.

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. IL-2 is effective in the treatment of renal cell carcinoma and malignant melanoma and in the therapy of patients with AIDS. In parallel, IL-15 was shown to help correct impaired proliferative responses of CD4‡ lymphocytes studied ex vivo from HIVinfected individuals without some of the in vivo toxic effects when it was applied to mice. Thus IL-15 could provide an alternative therapeutic option in the treatment of patients with select tumors or AIDS (Chehimi et al., 1997). The majority of therapeutic efforts involving the IL-15/IL-15R are being directed toward inhibiting IL-15 action. The scientific basis for this approach is the suggestion that IL-15 might contribute to the pathogenesis of rheumatoid arthritis, inflammatory bowel disease, and other autoimmune disorders. The administration of the IL-15 inhibitor, the soluble IL-15R chain prevented the development of murine collageninduced arthritis (Ruchatz et al., 1998). In addition, intragraft IL-15 transcripts were increased in renal allograft rejection patients (Pavlakis et al., 1996). A correlation existed between IL-15 transcripts within grafts being rejected as compared with nonrejected renal allografts, suggesting that IL-15 may play a role in T cell- and NK cell-mediated rejection (Chehimi et al., 1997). Moreover, IL-15 transcripts were present in the allografts in association with pancreatic islet allograft rejection (Kim et al., 1998). The blocking of IL-15R with a receptor antagonist enhanced acceptance of pancreatic islet cell allografts. Furthermore, the use of a humanized antibody to IL-2/IL-15R that blocked IL-15 action enhanced acceptance of renal allografts in cynomolgus monkeys (Tinubu et al., 1994). An attempt was made to use IL-15 cDNA (the intracellular isoform) to suppress fibrosarcoma growth through the activation of NK celland T cell-mediated immunity (Kimura et al., 1999).

Pharmacokinetics IL-15 disappears rapidly from the blood following intravenous administration to mice. The survival

of IL-15 is shorter than that of IL-2 which binds to 2-macroglobulin.

Toxicity It appears that IL-15 is associated with less severe capillary leak syndrome than is the IL-2 molecule.

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LICENSED PRODUCTS Human IL-15 may be obtained from Peprotech (Rocky Hill, NJ, USA), Genzyme Inc. (Cambridge, MA, USA), and R&D Systems (Minneapolis, MN, USA). Antibodies to IL-15 may be obtained from Serotec Inc. (Raleigh, NC, USA), Genzyme, and R&D Systems. A human IL-15 ELISA assay is available from R&D Systems. Monoclonal antibodies to murine IL-15 may be obtained from PharMingen (San Diego, CA, USA).