IL-7 Receptor

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IL-7 Receptor Hergen Spits* Division of Immunology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands * corresponding author tel: 31-20-5122063, fax: 31-20-5122057, e-mail: [email protected] DOI: 10.1006/rwcy.2000.14003.

SUMMARY The presently accepted configuration of the functional interleukin 7 (IL-7) receptor is one of a twochain receptor consisting of an  chain (IL-7R) and a chain ( common, c). The latter polypeptide is denoted common because it is also a component of the receptors for IL-2, IL-4, IL-9, and IL-15. The IL7R chain is a member of the cytokine receptor superfamily. The IL-7R chain plays an essential, nonredundant role in T and B cell development in the mouse. In addition, the IL-7R is involved in survival and proliferation of functional peripheral T cells. The IL-7R chain is also essential for the formation of Peyer's patches in the mouse. In human the IL-7R chain appears to be critical for T but not for B cell development, as two IL-7R-deficient severe combined immunodeficiency patients presented with B and NK cells but lacked T cells. The receptor has at least two ligands: IL-7 and TSLP-1. The latter cytokine interacts with a complex of the IL-7R chain and an as yet to be defined receptor distinct from c.

of IL-R and c. The discovery of this latter receptor component is described in the chapter on the IL-2 receptor.

Main activities and pathophysiological roles The IL-7R chain plays an essential, nonredundant role in T and B cell development in the mouse (Peschon et al., 1994) (see also for reviews, Akashi et al., 1998; DiSanto and Rodewald, 1998; Maeurer et al., 1998). In addition, the IL-7R plays a role in survival and proliferation of functional peripheral T cells (Maraskovsky et al., 1996). The IL-7R chain is also essential for the formation of Peyer's patches in the mouse (Adachi et al., 1998). In humans, the IL-7R chain appears to be critical for T but not for B cell development as two IL-7R-deficient severe combined immunodeficiency patients presented with B and NK cells but lacked T cells (Puel et al., 1998).

GENE

BACKGROUND

Accession numbers

Discovery

Human gene: AF043123-9, AF043124-9, AF0431259, AF043126-9, AF043127-9, AF043128-9 (these accession numbers denote the different exons) Human cDNA: NPI_002185 Mouse cDNA: M29697

The cloning of the IL-7R chain was reported in 1990. cDNA clones encoding the murine (IL-7) receptor were isolated from an IL-7-dependent pre-B cell line (Park et al., 1990) and that for human IL-7R from an SV40-transformed lung cell line (Goodwin et al., 1990). IL-7R cDNAs expressed in COS-7 cells bound radiolabeled IL-7, producing curvilinear Scatchard plots containing high- and low-affinity classes. The functional receptor for IL-7 is a complex

Chromosome location and linkages Human IL-7R maps to chromosome 5p13 (Lynch et al., 1992). The chromosomal localization of murine IL-7R is unknown. Human and mouse genes both

1482 Hergen Spits contain eight exons and seven introns with a total size of 19 kb in the human and 24 kb in the mouse. The first exon contains the 50 UTR, signal peptide, and the N-terminus of the mature protein. The remainder of the extracellular region is encoded by exons 2 to 6. In addition, exon 6 also encodes the transmembrane part of the cytoplasmic region and exons 7 and 8 encode the rest of the cytoplasmic region. The entire 30 UTR is encoded by exon 8. Differential splicing results in mRNA encoding a secreted form of the human IL7R chain. This form lacks the sequences in exon 6 that encode the transmembrane region. Inspection of the entire 50 region up to the start of translation of the murine IL-7R revealed a TATA box and a CAAT sequence. Furthermore an AP-1 and an AP-2 sequence were present. Interestingly an interferon response element (IRE)-like sequence was found. The 50 region contains several consensus binding sites for the glucocorticoid receptor. The region ÿ2495 to +5 contains promotor sequences that are active in a murine pre-B cell line (Pleiman et al., 1991).

PROTEIN

Accession numbers Human: NP_002176 Mouse: AAA39304

Description of protein Murine IL-7R gene encodes a protein of 439 amino acids with a calculated molecular weight of 49.5 kDa. On T cells the protein has a molecular weight of 90 kDa. The associated c chain has a molecular weight of 74 kDa. IL-7R is a type I membrane protein with a single transmembrane domain. The extracellular domain contains features of the cytokine receptor superfamily. The cytoplasmic domain with 195 amino acids does not contain consensus sequences of protein kinases. The crystal structure of the IL-7R has not been determined. Based on the model of the prolactin receptor, intrachain disulfide bridges are most probably formed between the first and second and between the third and fourth cysteine residues. The receptor contains a WS motif (WS  WS) at the C-terminus of the extracellular domain.

Relevant homologies and species differences The IL-7R protein is a member of the cytokine receptor superfamily which is characterized by

conserved extracellular domains fit to bind helical cytokines. IL-7R is similar to other receptors of the

c family including c, IL-2R , IL-4R, and IL-9R. These are characterized by two fibronectin type IIIlike domains containing four cysteine residues in the N-terminus domain and a WS motif at the Cterminus. Human and mouse IL-7R are not speciesspecific as both murine and human IL-7 interact with human T cells and vice versa.

Affinity for ligand(s) Reconstitution experiments with human IL-7R and

c revealed three classes of affinity: an uncharacterized low-affinity complex (Kd=145 nM) and complexes of intermediate (Kd=250 pM) and high (Kd=40 pM) affinity. The latter two consist of the IL-7R alone and complexes of IL-7R and c, respectively. High-affinity (79 pM) and low-affinity (16 nM) IL-7R were identified on the murine stromal cell line IxN/2b. Treatment of these cells with anti- c antibody reduced the affinity to 255 pM without affecting the low-affinity receptor and the combination of anti- c plus anti IL-7R eliminated the highaffinity receptor altogether while the low-affinity binding was not affected. These findings suggest the existence of a c-independent IL-7R-containing IL7 receptor complex (Sugamura et al., 1996). The nature of the low-affinity complex is unknown but may involve a yet to be identified receptor component. The IL-7R chain is also part of the receptor for murine thymus-derived lymphopoietin 1 (TSLP-1). In the functional TSLP-1R complex IL7R does not seem to pair with c but with another yet to be described receptor (Levin et al., 1999). The affinity of this complex for TSLP-1 is unknown.

Cell types and tissues expressing the receptor See Table 1 (for references in reviews, see Akashi et al., 1998; DiSanto and Rodewald, 1998; Maeurer et al., 1998).

Release of soluble receptors cDNA clones have been isolated that potentially encode a soluble receptor. These clones lack the sequences in exon 6 encoding the transmembrane domain (Pleiman et al., 1991). It has not been

IL-7 Receptor 1483 Table 1 Cell types and tissues that express the IL-7 receptor Fetal NK cell/dendritic cell precursors in fetal lymph nodes Common T/NK/B lymphoid cell precursors Cryptopatch-associated lymphoid precursors Developing T cells All subsets of CD3ÿ CD4ÿ CD8ÿ thymocytes A fraction of CD4+CD8+ DP cells SP CD4+ or CD8+ thymocytes TCR  cells Thymic NK-1.1+ T cells Developing B cells All pre-pro, pro-, and pre-B cell stages Mature T cells Bone marrow-derived macrophages Malignant cell types Colorectal cancer cells Renal cancer cells Cutaneous T cell lymphomas Human intestinal epithelial cells

reported whether a soluble receptor protein is indeed secreted by cells expressing it. The function of the soluble receptor is unknown.

SIGNAL TRANSDUCTION

Associated or intrinsic kinases The IL-7R complex requires both the  chain and c for relaying signals. These two chains do not have intrinsic catalytic activity, but associate with tyrosine phosphokinases that are instrumental in transducing signals triggered by binding of IL-7 to the receptor complex. The src kinase p59fyn has been shown to associate with the receptor complex following activation with IL-7 in a human pre-B cell line (Seckinger and Fougereau, 1994). Association of p59fyn with the receptor was also found in T cells (Venkitaraman and Cowling, 1992). In both T cells and thymocytes p56lck has been shown to associate with IL-7R. The role of p56lck and p59fyn in IL-7 signaling is unknown. Fyn is certainly not essential in IL-7R signaling since Fynÿ/ÿ mice have no reduction in the size of the thymus and numbers of T and B cells in the periphery are normal (Appleby

et al., 1992), in contrast to IL-7Rÿ/ÿmice. In addition, the phenotype of p56lckÿ/ÿ mice is distinct from that of IL-7Rÿ/ÿ mice (Molina et al., 1992). Thus, these src kinases do not seem to be important in IL-7R signaling. However, an attenuating role of these enzymes in certain aspects of IL-7R signaling cannot be excluded. Tyrosine kinases belonging to the family of Janus kinases are essential for IL-7R signaling. Two Janus kinases, JAK1 and JAK3, associate with the complex. JAK1 binds to IL-7R and JAK3 to c (Leonard and O'Shea, 1998). Both kinases become phosphorylated when IL-7 binds to its receptor, but the extent of phosphorylation of JAK3 is much higher than that of JAK1. JAK3ÿ/ÿ mice have a phenotype similar to that of IL-7Rÿ/ÿ mice (Nosaka et al., 1995; Thomis et al., 1995), strongly suggesting that JAK3 activity is required for IL-7R-mediated signaling during T and B cell development. In contrast to JAK3, which is specifically expressed in lymphoid cells, JAK1 is ubiquitously expressed. Nonetheless, analysis of JAK1ÿ/ÿ mice revealed a dedicated role of JAK1 in T and B cell development (Rodig et al., 1998). These mice were born but all pups died within 24 hours after birth. The thymus of newborn JAK1ÿ/ÿ mice was 260-fold reduced in size as compared with heterozygous controls. CD4+CD8+ double positive (DP) cells were present, but the CD4ÿ CD8ÿ double negative (DN) compartment was increased. No further analysis was performed and it is therefore unknown whether the DN cells in the thymus of these mice were stromal cells or T cell precursors. B cells were strongly reduced in numbers. Importantly, whereas wild-type fetal liver cells form colonies in IL-3 and IL-7, JAK1ÿ/ÿ fetal liver cells formed colonies in IL-3 but not in IL-7 (Rodig et al., 1998). These findings demonstrate that JAK1 is an essential constituent of IL-7R signaling. Another kinase that associates with the IL-7R complex is phosphatidyl 3-kinase (PI-3 kinase). JAK3 appears also to control PI-3 kinase activity (Sharfe et al., 1995). PI-3 kinase phosphorylates the D3 position of the inositol group of phosphoinositide lipids to generate phosphatidylinositol-3 phosphate (PtdIns(3)P), PtdIns(3,4)P2 and PtdIns(3,4)P3 (Kapeller and Cantley, 1994). The latter two products are important for cellular proliferation. The enzyme consists of a 85 kDa adapter and a 110 kDa catalytic unit. Upon ligand binding the p85 unit is tyrosine phosphorylated and associates with many receptors, including the IL-7R chain (Venkitaraman and Cowling, 1994). Stimulation of human thymocytes with IL-7 was shown to activate both isoforms of p85,  and , in human thymocytes (Dadi et al., 1994). Interestingly, phosphorylation of p85 required JAK3,

1484 Hergen Spits which was shown to associate with p85 (Sharfe et al., 1995). In a later study these authors presented evidence that two pools of PI-3 kinase are activated by IL-7: one is associated with the IL-7R chain and another with insulin receptor substrate 1 (IRS-1) and IRS-2 (Sharfe and Roifman, 1997). PI-3 kinase appears to be important for survival/proliferation of both T and B cell precursors. Using human IL-7R mutants transfected into murine pro-B cell lines, Venkitaraman and Cowling demonstrated association of PI-3 kinase with the IL-7R chain through an SH2 domain recognition motif (YXXM) spanning residue Tyr449 in its cytoplasmic domain (Venkitaraman and Cowling, 1994). Interestingly, this residue was found to be critical for IL-7-mediated proliferation of murine pre-B cells. Induction of IgH VDJ gene recombination, however, did not require Tyr449 residue (Corcoran et al., 1996). These findings indicate that IL-7R-transduced signals can control both survival and/or proliferation and differentiation through distinct pathways. More recently it was demonstrated that PI-3 kinase also mediates survival and proliferation of human T cell precursors. Pallard et al. (1999) observed that the Tyr449 in the cytoplasmic domain of the IL-7R is important for survival and expansion of human thymocytes. A dominant negative mutant of p85 that binds to the receptor but fails to interact with p100, was introduced into human T cell precursors by retrovirus-mediated gene transfer. This mutant was found to inhibit survival and expansion of the T cell precursors in a fetal thymic organ culture but did not inhibit differentiation of these cells. The study of Pallard coworkers left unresolved which isoform, p85 or , is recruited by IL-7 binding. These two isoforms are encoded by different genes. Two recent studies failed to provide evidence for a role of p85 in T cell development as the size of the thymus and the distribution of thymocyte subsets was unaffected in p85ÿ/ÿ mice (Fruman et al., 1999; Suzuki et al., 1999). The observation that the level of p85 in these mice is elevated compared with wild-type mice strongly suggests a compensatory role of p85 in T cell development of p85ÿ/ÿ mice.

a downstream effector of PI-3 kinase as well (Pallard et al., 1999). The substrates of IL-7-activated PKB are not yet known. Two sets of observations suggest a role for the p38 MAP kinase in IL-7-mediated proliferation of mature T cells. In the first place activation of a murine T cell line by IL-7 resulted in phosphorylation of p38 MAP kinase and secondly the proliferative response of human T cells was inhibited by the highly selective p38 MAP kinase inhibitor SB203580 (Crawley et al., 1997). It is unknown which signaling pathway activates p38 MAP kinase; p21ras is not activated by IL-7 and therefore does not act in this pathway. The same report also documented activation of the stress-activated protein kinase (SAP kinase)/Jun-Nterminal kinase (JUN kinase) by IL-7, but there is no evidence that activation of this enzyme is important for the proliferative response induced by IL-7 (Crawley et al., 1997). The p38 inhibitor SB203580 influenced negative selection through the TCR in a fetal thymic organ culture (FTOC) but did not affect thymic cellularity (Sugawara et al., 1998). Since blocking the IL-7/IL-7R interaction resulted in a strong reduction of thymic cellularity and SB203580 did not, it is unlikely that p38 MAP kinase is involved in IL-7-mediated control of early T cell development in the thymus. A potential constituent of the IL-7R signaling is the serine/threonine kinase pim-1 as pro/pre B cells of pim-1ÿ/ÿ mice do not proliferate in response to IL-7 (Domen et al., 1993). Evidence for a direct link between pim-1 and the IL-7R complex is lacking, however, since the B and T cell phenotype of pim-1deficient mice is comparable to that of wild-type mice. It is possible that the related kinases pim-2 and pim-3 compensate the pim-1 deficiency, but this has yet to be verified.

Cytoplasmic signaling cascades

In general JAKs phosphorylate the receptor chains, providing docking sites for SH2 domains of STATs (Leonard and O'Shea, 1998). STATs are recruited to the phosphorylated sites of the receptor and become phosphorylated. The phosphorylated STATs can dimerize, translocate to the nucleus and stimulate expression of cytokine-inducible genes. Two studies documented activation of STAT1 by IL-7 (Zeng et al., 1994; van der Plas et al., 1996). The phenotype of

Recently it was demonstrated that protein kinase B (PKB) can be activated by IL-7 (Pallard et al., 1999). PKB seems to play a central role in PI-3 kinasemediated protection against apoptosis in a wide range of cell types (Coffer et al., 1998). As activation of PKB as well as of PI-3 kinase requires residue Tyr449, it is very likely that in human T cell precursors PKB is

DOWNSTREAM GENE ACTIVATION

Transcription factors activated

IL-7 Receptor 1485 STAT1-deficient mice, however, makes it unlikely that STAT1 plays an essential role in IL-7R signaling in vivo as the thymus of these mice is normal and T and B cell development proceeds undisturbed (Meraz et al., 1996). STAT3 and STAT5 have been shown to be activated by IL-7 in several cell types (Leonard and O'Shea, 1998). It is unlikely that STAT3 is involved in IL-7R signaling in vivo. STAT3 deficiency results in embryonic lethality preventing analysis of the role of STAT3 in T cell development in conventional STAT3ÿ/ÿ mice. Recently, mice were generated with a STAT3 deficiency specifically in the T cell lineage by conditional gene targeting using the Cre-loxP system (Takeda et al., 1998). Floxed-STAT3 mice were mated with transgenic mice with Cre recombinase under control of the T cell-specific Lck promoter. Although STAT3 was not expressed in the thymus of these mice, the cellularity of the thymus and distribution of thymic subsets was the same as in control mice (Takeda et al., 1998). Thymocytes of these mice responded normally to IL-7 in vitro. Together, these findings indicate that STAT3 is not involved in IL-7-mediated control of T cell development. IL-7 activates STAT5 in human PBMCs and thymocytes (Lin et al., 1995; Pallard et al., 1999). STAT5 was originally identified as a prolactininduced mammary gland transcription factor. Two STAT5 genes, 5a and 5b, encode proteins that are approximately 95% identical in amino acid sequence. STAT5a and STAT5b differ in their C-terminal transactivation domains and exert relatively specific actions. STAT5-deficient mice display immunological defects. STAT5aÿ/ÿ mice have a reduced expansion of peripheral T cells which is associated with a diminished IL-2-mediated induction of the IL-2R chain. STAT5b-deficient mice exhibit a reduced expansion of peripheral NK cells and a slightly reduced cellularity of the thymus (Imada et al., 1998). In contrast, mice deficient for both STAT5a and STAT5b were reported to have no major decrease in thymic cellularity (Moriggl et al., 1999). In addition, the distributions of CD4+, CD8+SP and DP cells were normal, although the number of peripheral T cells reduced progressively when these mice aged (Moriggl et al., 1999). The mechanism of this reduction is not clear but it is likely that an inability of STAT5-deficient T cells to respond to IL-2 is a contributing factor. The reported findings on the phenotype of mice deficient for either STAT5a and STAT5b or both argues against a role for STAT5 isoforms in IL-7-mediated control of T cell development. However, Pallard et al. (1999) reported that overexpression of dominant negative mutant of STAT5b in human T cell precursors disrupted their

development into mature T cells in an in vitro FTOC. The mutant used by Pallard and coworkers lacks the C-terminal transactivating domain and inhibits transactivation of wild-type STAT5 and one would expect that overexpression of dominant negative STAT5b has the same effect as STAT5 deficiency. There is presently no explanation for these discordant effects of the absence of STAT5a and STAT5b and of overexpression of dominant negative STAT5b on T cell development. It is possible that there are species differences in the requirement of STAT5 in murine and human T cell development. Another possibility is that the effect of dominant negative STAT5b on development of human T cell precursors is a consequence of a gain of function of the mutant resulting in activities beyond those of inhibiting transcription mediated by full-length wildtype STAT5a or STAT5b. Mel-18 is a mammalian homolog of Drosophila melanogaster Polycomb group genes. Mice deficient for this gene show a severe combined immunodeficiency phenotype with a strongly reduced thymic size (Akasaka et al., 1997). Thymocytes of these mice fail to respond to IL-7, suggesting an involvement of mel18 in cell cycle progression in response to the interaction of IL-7 and the IL-7R complex.

Genes induced Presently there are no genes known that are proven to be direct targets of IL-7R-signaling.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors The effects of activation of the IL-7R with IL-7 are discussed in the chapter on IL-7. Disrupting IL-7/IL7R interactions also affect human T cell development. Antibodies to the human IL-7R strongly inhibited development of fetal liver and thymic T cell precursors in a hybrid human/mouse fetal thymic organ culture (Plum et al., 1996; Pallard et al., 1999). Development of the human T cell precursors was arrested at the CD3ÿ CD4ÿ CD8ÿ stage.

1486 Hergen Spits

Phenotypes of receptor knockouts and receptor overexpression mice IL-7R-deficient mice were reported by Peschon et al. (1994). These mice had severely reduced numbers of T and B cells. NK cell numbers were not affected by the IL-7R deficiency and also development of myeloid cells was normal. Another IL-7Rÿ/ÿmouse was generated by Ikuta and collaborators (Maki et al., 1996). In these mice exon 2 was targeted rather than the exon 3 in the mice generated by Peschon and coworkers. The phenotype of these latter mice was similar to that of exon 3-targeted mice although some differences were noted. Peschon et al. (1998) reported that the T cell deficiency in IL-7R(exon 3)ÿ/ÿ mice was variable. While all IL-7R-deficient mice demonstrated a reduction in thymic cellularity, the level of the reduction varied. In around 65% of the mice the size of the thymus was only 0.1% of the wild type, while the thymus of the other mice was between 1 and 10% of the wild type. In the mice with