The IL-15 receptor {alpha} chain cytoplasmic domain is critical for normal IL-15Ralpha function but is not required for trans-presentation

IL-15 is critical for natural killer (NK)-cell development and function and for memory CD8(+) T-cell homeostasis. The IL-15 receptor consists of IL-15Ralpha, IL-2Rbeta, and the common cytokine receptor gamma chain (gamma(c)). IL-15Ralpha is known to "trans-present" IL-15 to an IL-2Rbeta/gamma(c) heterodimeric receptor on responding cells to initiate signaling. To investigate the importance of the IL-15Ralpha cytoplasmic domain, we generated a chimeric receptor consisting of the extracellular domain of IL-15Ralpha and intracellular domain of IL-2Ralpha (IL-15Ralpha(ext)/IL-2Ralpha(int)) and examined its function in 32D cells, in knock-in (KI) mice, and in adoptive-transfer experiments. The chimeric protein exhibited decreased cell-surface expression, and KI mice exhibited diminished NK, NKT, and CD8(+) T-cell development and defects in T-cell functional responses. However, 32D cells expressing the chimeric receptor had less IL-15-induced proliferation than wild-type (WT) transfectants with similar levels of IL-15Ralpha expression, indicating a signaling role for the IL-15Ralpha cytoplasmic domain beyond its effect on expression, and demonstrating that the IL-2Ralpha and IL-15Ralpha cytoplasmic domains are functionally distinct. Interestingly, adoptive-transfer experiments indicated that the chimeric IL-15Ralpha(ext)/IL-2Ralpha(int) receptor still supports trans-presentation. These experiments collectively indicate that IL-15Ralpha can act in cis in addition to acting in trans to present IL-15 to responding cells.     

Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain

We have identified a type I cytokine receptor, which we have termed novel interleukin receptor (NILR), that is most related to the IL-2 receptor beta chain (IL-2Rbeta) and physically adjacent to the IL-4 receptor alpha chain gene on chromosome 16. NILR mRNA is most highly expressed in thymus and spleen, and is induced by phytohemagglutinin in human peripheral blood mononuclear cells. NILR protein was detected on human T cell lymphotropic virus type I-transformed T cell lines, Raji B cells, and YT natural killer-like cells. Artificial homodimerization of the NILR cytoplasmic domain confers proliferation to Ba/F3 murine pro-B cells but not to 32D myeloid progenitor cells or CTLL-2 murine helper T cells. In these latter cells, heterodimerization of IL-2Rbeta and the common cytokine receptor gamma chain (gamma(c)) cytoplasmic domains allows potent proliferation, whereas such heterodimerization of NILR with gamma(c) does not. This finding suggests that NILR has signaling potential but that a full understanding of its signaling partner(s) is not yet clear. Like IL-2Rbeta, NILR associates with Jak1 and mediates Stat5 activation.     

Role of common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of murine mast cells

The regulatory roles of the common cytokine receptor gamma chain (gamma(c))- and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using gamma(c)-deficient (gamma(c)(-)) and Jak3-deficient (Jak3(-)) mice. Although the mast cells in gamma(c)(-) and Jak3(-) mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in gamma(c)(-) and Jak3(-) mice as compared with that in wild-type mice. Among gamma(c)-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow-derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from gamma(c)(-) and Jak3(-) mice. In addition, IL-4Ralpha, gamma(c), and Jak3, but not IL-2Rbeta or IL-7Ralpha, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Ralpha1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from gamma(c)(-) and Jak3(-) mice. These results indicate that gamma(c)- and Jak3-dependent signaling is essential for IL-4- and IL-9-induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a gamma(c)- and Jak3-dependent manner.     

Limiting {gamma}c expression differentially affects signaling via the interleukin (IL)-7 and IL-15 receptors

X-linked severe combined immunodeficiency (SCID-X1) results from mutations in the IL2RG gene, which encodes the common gamma chain (gammac) of the receptors for interleukin (IL)-2, 4, 7, 9, 15, and 21. Affected infants typically lack T and natural killer (NK) cells as a consequence of loss of signaling via the IL-7 receptor (IL-7R) and the IL-15R, respectively. In some infants, however, autologous NK cells are observed despite failure of T-cell ontogeny. The mechanisms by which mutations in gammac differentially impact T- and NK-cell ontogeny remain incompletely understood. We used SCID-X1 patient-derived EBV-transformed B cells to test the hypothesis that the IL-15R-mediated signaling is preferentially retained as gammac expression becomes limiting. Signal transduction via the IL-15R was readily detected in control EBV-transformed B cells, and via the IL-7R when modified to express IL-7Ralpha. Under the same experimental conditions, patient-derived EBV-transformed B cells expressing trace amounts of gammac proved incapable of signal transduction via the IL-7R while retaining the capacity for signal transduction via the IL-15R. An equivalent result was obtained in ED-7R cells modified to express varying levels of gammac. Collectively, these results confirm that signal transduction via the IL-15R, and hence NK ontogeny, is preferentially retained relative to the IL-7R as gammac expression becomes limiting.     

Transformation of hematopoietic cells and activation of JAK2-V617F by IL-27R, a component of a heterodimeric type I cytokine receptor

From a patient with acute myeloid leukemia (AML), we have identified IL-27Ra (also known as TCCR and WSX1) as a gene whose expression can induce the transformation of hematopoietic cells. IL-27Ra (IL-27R) is a type I cytokine receptor that functions as the ligand binding component of the receptor for IL-27 and functions with the glycoprotein 130 (gp130) coreceptor to induce signal transduction in response to IL-27. We show that IL-27R is expressed on the cell surface of the leukemic cells of AML patients. 32D myeloid cells transformed by IL-27R contain elevated levels of activated forms of various signaling proteins, including JAK1, JAK2, STAT1, STAT3, STAT5, and ERK1/2. Inhibition of JAK family proteins induces cell cycle arrest and apoptosis in these cells, suggesting the transforming properties of IL-27R depend on the activity of JAK family members. IL-27R also transforms BaF3 cells to cytokine independence. Because BaF3 cells lack expression of gp130, this finding suggests that IL-27R-mediated transformation of hematopoietic cells is gp130-independent. Finally, we show that IL-27R can functionally replace a homodimeric type I cytokine receptor in the activation of JAK2-V617F, a critical JAK2 mutation in various myeloproliferative disorders (MPDs). Our data demonstrate that IL-27R possesses hematopoietic cell-transforming properties and suggest that, analogous to homodimeric type I cytokine receptors, single-chain components of heterodimeric receptors can also enhance the activation of JAK2-V617F. Therefore, such receptors may play unappreciated roles in MPDs.     

Function of the interleukin-2 (IL-2) receptor gamma-chain in biologic responses of X-linked severe combined immunodeficient B cells to IL-2, IL-4, IL-13, and IL-15

The interleukin-2 (IL-2) receptor gamma-chain is a common component of several members of the cytokine receptor superfamily including those for IL-2, IL-4, IL-7, IL-9, IL-15, and possibly IL-13, and has recently been renamed the common gamma-chain (gamma c-chain). Transfection experiments have shown that the gamma c-chain participates in signal transduction by IL-2, IL-4 and IL-7, but a functional role for the gamma c-chain in biological responses by normal T cells and B cells to these cytokines has not been established. In this study, we have used X- linked severe combined immunodeficiency (X-SCID) as a naturally occurring gamma c-chain gene disruption model to examine the role of the gamma c-chain in human B-cell responses to IL-2, IL-4, IL-13, and IL-15. Our experiments show that B cells from two X-SCID patients with characterized gamma c-chain gene mutations do not respond to IL-2 or IL- 15, but respond as well or better than normal B cells to both IL-4 and IL-13 in assays for B-cell activation, proliferation, and IgE secretion. This finding raises important questions about the function of the gamma c-chain in receptors for IL-4 and IL-13, and the nature of the immune defect in X-SCID.     

The molecular basis of IL-21-mediated proliferation

Interleukin-21 (IL-21) is a type I cytokine that modulates functions of T, B, natural killer (NK), and myeloid cells. The IL-21 receptor (IL-21R) is closely related to the IL-2 receptor beta chain and is capable of transducing signals through its dimerization with the common cytokine receptor gamma chain (gamma(c)), the protein whose expression is defective in humans with X-linked severe combined immunodeficiency. To clarify the molecular basis of IL-21 actions, we investigated the role of tyrosine residues in the IL-21R cytoplasmic domain. Simultaneous mutation of all 6 tyrosines greatly diminished IL-21-mediated proliferation, whereas retention of tyrosine 510 (Y510) allowed full proliferation. Y510 efficiently mediated IL-21-induced phosphorylation of Stat1 and Stat3, but not of Stat5, and CD8(+) T cells from Stat1/Stat3 double knock-out mice exhibited decreased proliferation in response to IL-21 + IL-15. In addition, IL-21 weakly induced phosphorylation of Shc and Akt, and consistent with this, specific inhibitors of the MAPK and PI3K pathways inhibited IL-21-mediated proliferation. Collectively, these data indicate the involvement of the Jak-STAT, MAPK, and PI3K pathways in IL-21 signaling.     

Restoration of lymphoid populations in a murine model of X-linked severe combined immunodeficiency by a gene-therapy approach.

X-linked severe combined immunodeficiency (XSCID) is a life-threatening syndrome in which both cellular and humoral immunity are profoundly compromised. This disease results from mutations in the IL2RG gene, which encodes the common cytokine receptor gamma chain, gamma(c). Previously, we generated gamma(c)-deficient mice as a murine model of XSCID. We have now used lethally irradiated gamma(c)-deficient mice to evaluate a gene therapeutic approach for treatment of this disease. Transfer of the human gamma(c) gene to repopulating hematopoietic stem cells using an ecotropic retrovirus resulted in an increase in T cells, B cells, natural killer (NK) cells, and intestinal intraepithelial lymphocytes, as well as normalization of the CD4:CD8 T-cell ratio and of serum Ig levels. In addition, the restored cells could proliferate in response to interleukin-2 (IL-2). Thus, our results provide added support that gene therapy is a feasible therapeutic strategy for XSCID. Moreover, because we used a vector directing expression of human gamma(c) to correct a defect in gamma(c)-deficient mice, these data also indicate that human gamma(c) can cooperate with the distinctive cytokine receptor chains such as IL-2Rbeta and IL-7Ralpha to mediate responses to murine cytokines in vivo.     

Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors

Activation of human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors, ectopically expressed in FDCP-mix multipotent cells, stimulates self-renewal or myeloid differentiation, respectively. These receptors are composed of unique alpha subunits that interact with common beta(c) subunits. A chimeric receptor (hGM/beta(c)), comprising the extracellular domain of the hGM-CSF receptor alpha subunit (hGM Ralpha) fused to the intracellular domain of hbeta(c), was generated to determine whether hbeta(c) activation is alone sufficient to promote differentiation. hGM-CSF activation of hGM/beta(c), expressed in the presence and absence of the hbeta(c) subunit, promoted maintenance of primitive phenotype. This indicates that the cytosolic domain of the hGM Ralpha chain is required for differentiation mediated by activation of the hGM Ralpha, beta(c) receptor complex. We have previously demonstrated that the alpha cytosolic domain confers signal specificity for IL-3 and GM-CSF receptors. Bioinformatic analysis of the IL-3 Ralpha and GM Ralpha subunits identified a tripeptide sequence, adjacent to the conserved proline-rich domain, which was potentially a key difference between them. Cross-exchange of the equivalent tripeptides between the alpha subunits altered receptor function compared to the wild-type receptors. Both the mutant and the corresponding wild-type receptors promoted survival and proliferation in the short-term but had distinct effects on developmental outcome. The mutated hGM Ralpha promoted long-term proliferation and maintenance of primitive cell morphology, whereas cytokine activation of the corresponding hIL-3 Ralpha mutant promoted myeloid differentiation. We have thus identified a region of the alpha cytosolic domain that is of critical importance for defining receptor specificity.     

Cellular localization of interleukin 13 receptor alpha2 in human primary bronchial epithelial cells and fibroblasts.

BACKGROUND: Interleukin (IL) 13 is a key cytokine in asthma, regulating fibrosis, airway remodeling, induction of immunoglobulin E synthesis by B cells, bronchial hyperresponsiveness, and mucus production. IL-13 signals through the type II IL-4 receptor (IL-4R), which is composed of the IL-4Ralpha and the IL-13Ralpha1 chains. Another IL-13 binding chain, IL-13Ralpha2, binds IL-13 with high affinity but has no known signaling capability and is thought to serve as a decoy receptor providing tight regulation of IL-13 responses. METHODS: In this study, we investigated the cellular localization of IL-13Ralpha2 in human primary bronchial epithelial cells and fibroblasts using flow cytometry and confocal microscopy, as well as the in vivo expression of IL-13Ralpha2 in the human bronchial mucosa by means of immunohistochemistry. RESULTS: IL-13Ralpha2 is predominantly an intracellular rather than a membrane-bound molecule in both human primary bronchial epithelial cells and fibroblasts and displays a diffuse granular cytoplasmic distribution in both cell types. IL-13Ralpha2 protein is expressed in vivo in the human bronchial mucosa with its expression being higher in bronchial epithelial cells than bronchial fibroblasts both in vivo and in vitro. CONCLUSIONS: IL-13Ralpha2 is expressed by both human primary bronchial epithelial cells and fibroblasts as an intracellular protein with a diffuse cytoplasmic distribution. In vivo, IL-13Ralpha2 is expressed in the human airway mucosa mainly by bronchial epithelial cells.     

Characterization of cDNAs encoding the murine interleukin 2 receptor (IL-2R) gamma chain: chromosomal mapping and tissue specificity of IL-2R gamma chain expression.

The IL-2R gamma chain (IL-2R gamma) is an essential component of high- and intermediate-affinity IL-2Rs, playing critical roles for ligand binding and internalization. Recently, our laboratory has demonstrated that IL-2R gamma mutation results in X chromosome-linked severe combined immunodeficiency in humans, suggesting that IL-2R gamma plays a vital role in thymic maturation of human T cells. We now report the isolation and characterization of cDNAs encoding murine IL-2R gamma. The open reading frame encodes 369 aa, identical in length to that encoded by the human IL-2R gamma cDNA. Murine IL-2R gamma and human IL-2R gamma have 69% and 70% identity at the nucleotide and amino acid levels, respectively. As expected, the murine IL-2R gamma retains the WSXWS motif and four cysteine residues characteristic of cytokine receptor superfamily members. IL-2R gamma mRNA distribution shows significant tissue specificity, with particularly high-level expression in spleen and thymus, and higher expression in single-positive (CD4+8- or CD4-8+)-enriched thymocytes than in double-negative (CD4-8-) thymocytes. Finally, we have localized the murine IL-2R gamma gene, Il2rg, to the X chromosome between Rsvp and Plp and demonstrated that a defect in IL-2R gamma is not responsible for the X chromosome-linked xid mutation, which maps to this same region. The cloning of the murine IL-2R gamma cDNA will facilitate the investigation of the role of this protein in lymphocyte function and thymic development.     

Converting IL-15 to a superagonist by binding to soluble IL-15R{alpha}

IL-15 is normally presented in vivo as a cell-associated cytokine bound to IL-15Ralpha. We show here that the biological activity of soluble IL-15 is much improved after interaction with recombinant soluble IL-15Ralpha; after injection, soluble IL-15/IL-15Ralpha complexes rapidly induce strong and selective expansion of memory-phenotype CD8(+) cells and natural killer cells. These findings imply that binding of IL-15Ralpha to IL-15 may create a conformational change that potentiates IL-15 recognition by the betagamma(c) receptor on T cells. The enhancing effect of IL-15Ralpha binding may explain why IL-15 normally functions as a cell-associated cytokine. Significantly, the results with IL-2, a soluble cytokine, are quite different; thus, IL-2 function is markedly inhibited by binding to soluble IL-2Ralpha.     

IL-2 receptor alpha(-/-) mice and the development of primary biliary cirrhosis

Recently, we identified a child born with a genetic deficiency of IL-2 receptor alpha (IL-2Ralpha, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first-degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL-2Ralpha/CD25 deficient (IL-2Ralpha(-/-)) mice and wild-type littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL-2Ralpha(-/-), but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4(+) and CD8(+) T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN-gamma, TNF-alpha, IL-2 and IL-12p40. Of importance is the finding that the IL-2Ralpha(-/-) mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC-E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL-2Ralpha(-/-) mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC-E2 and subsequent biliary ductular cell damage.