Chain Gammac Research Articles

ID: 64: Targeting the IL-7R pathway in leukemia

Acute lymphoblastic leukemia, derived from immature T or B cells, is the most common cancer in children. Current chemotherapeutic regimen are effective in over 80% of T-ALL, but the failure rate and the harshness of current treatments makes new approaches desirable. We and others have recently identified gain-of-function mutations in IL-7R alpha in T cell acute lymphoblastic leukemia (T-ALL) which serve as driver oncogenes. Most of these mutations consist of insertions containing cysteine into the juxtamembrane region, creating homodimers. These signal independently of IL-7 or the gamma-c chain of the IL-7 receptor, and constitutively activate Jak1. Two approaches have been undertaken to target the IL-7R pathway in T-ALL: inhibition of Jak1, and development of monoclonal antibodies (MAbs) against IL-7R alpha. Ruxolitinib is a recently FDA approved inhibitor of Jak1, and we show it is an effective inhibitor of cells driven by mutant IL-7R alpha in vitro and in immunodeficient mice. Tofacitinib, which is more selective for Jak3, was effective in vitro but there was no therapeutic window in vivo in immunodeficient mice. Two novel mouse MABs were developed that are directed against two different epitopes on human IL-7R alpha, and they recognize both mutant and WT proteins. MAbs were chimerized with human IgG1 to optimize antibody-dependent cell mediated cytotoxicity (ADCC). These MAbs were highly effective in ADCC assays, mediating NK cell killing of T-ALL cells harboring mutant IL-7R alpha, and they also target normal T cells which express IL-7R alpha. These approaches are being developed as new therapeutics for acute lymphoblastic leukemia.

Limited engraftment of low-risk myelodysplastic syndrome cells in NOD/SCID gamma-C chain knockout mice

Limited engraftment of low-risk myelodysplastic syndrome cells in NOD/SCID gamma-C chain knockout mice

Human IL-Rbeta chains form IL-2 binding homodimers.

Two types of functional interleukin-2 receptor (IL-2Ralpha/IL-2Rbeta/gammac and IL-2Rbeta/gammac) have already been characterized in humans. Here we describe a new form consisting of IL-2Rbeta/beta homodimers that assemble spontaneously in the absence of gammac. Co-transfection of COS-7 cells with constructs expressing IL-2Rbeta chains tagged with either HA or MYC sequences results in the formation of IL-2Rbeta:HA/IL-2Rbeta:MYC complexes detectable by coimmunoprecipitation. The formation of these IL-2Rbeta:HA/IL-2Rbeta:MYC dimers is also observed in the absence of IL-2. Moreover, in COS cells expressing chimeras of IL-2Rbeta fused to fluorescence reporters such as IL-2Rbeta:ECFP and IL-2Rbeta:EYFP, we also observed specific FRET at the surface of living cells, as expected for dimer formation. Transiently transfected COS-7 cells expressing IL-2Rbeta bind 125I-labeled IL-2 (homodimers, Kd = 1nM) as cells expressing both IL-2Rbeta and gammac chains (heterodimers, Kd = 1 nM). IL-2Rbeta/IL-2Rbeta could represent either a decoy receptor or a new form of IL-2R involved in signaling when gammac expression is low.

Gamma Chain Receptor Interleukins: Evidence for Positive Selection Driving the Evolution of Cell-to-Cell Communicators in the Mammalian Immune System

The interleukin-2 receptor (IL-2R) gamma chain, or common gamma chain (gammac), is the hub of a protein interaction network in the mammalia that is central to defense against disease. It is the indispensable subunit of the functional receptor complexes for a group of interleukins known as the gamma-chain-dependent interleukins (IL-2, IL-4, -7, -9, -15, and -21). The gammac links these proteins through their interaction with it and their competition for its recruitment. The gammac-dependent interleukins also interact with each other to either enhance or suppress expression through manipulation of expression of receptor subunits. Given the influence of protein-protein interactions on evolution, such as those documented for many genes including the reproductive proteins of the sperm and egg coat, here we have asked whether there is a common thread in the evolution of these interleukins. Our findings indicate that positive selection has acted by fixing a large number of amino acid replacement mutations in every single one of these interleukins, this adaptive evolution is also observed in a lineage-specific manner. Crucially, however, there does not appear to have ever been an instance of adaptive evolution in the gammac chain itself, thereby providing an insight into the evolution of this hub protein. These findings highlight the importance of adaptive evolutionary events in the evolution of this central network in the immune system and suggest underlying causes for differences in defense responses in the mammalia.

Unusual interleukin-4 and -13 signaling in human normal and tumor lung fibroblasts.

IL-4 and IL-13 act on human lung fibroblasts through specific receptors differing in their composition. Indeed, the gammac chain is constitutively expressed in tumor lung myofibroblast but not in normal cells. Here, we have analysed the signal transduction induced by IL-4 and IL-13 in both cell types, in order to better understand the molecular mechanisms underlying tumor stromal development. The IL-4Ralpha chain is constitutively phosphorylated and pre-associated with the JAK1 protein in both cell types. In normal cells, we detected the activation of the classic IRS-2 or JAK1/STAT6 pathways, the phosphorylation of JAK2, while Tyk2 was constitutively phosphorylated and not modified by both cytokines. In addition to these pathways, in lung tumor myofibroblasts, IL-4 and IL-13 induced the phosphorylation of JAK3 and increased the phosphorylation of Tyk2. Interestingly, in both cell types IL-4 and IL-13 triggered an unusual pattern of STAT1 and STAT3 activation. These events probably correspond to a tissue-specific signaling important for the immunoregulatory functions of airways fibroblasts. Indeed, the inflammatory-like pattern of STATs signaling triggered by IL-4 and IL-13 in these cells may favor the homing of inflammatory and/or metastatic cells. In lung myofibroblasts, these properties could be modified through the different pattern of JAK activation.

Characterization of the gammac chain among 27 unrelated Japanese patients with X-linked severe combined immunodeficiency (X-SCID).

X-linked severe combined immunodeficiency (X-SCID) is a rare fatal disease that is caused by mutations in the gene encoding the gammac chain. In this study, 27 unrelated Japanese patients with X-SCID were examined in terms of their genetic mutations and surface expression of the gammac chain. Among 25 patients examined, excluding two patients with large deletions, 23 different mutations were identified in the IL2RG gene, including 10 novel mutations. One patient bearing an extracellular mutation and all three of the patients bearing intracellular mutations after exon 7 expressed the gammac chain on the cell surface. Overall, 84% of patients lacked surface expression of the gammac chain leading to a diagnosis of X-SCID.

Altered natural killer cell differentiation in CD34+ progenitors from chronic myeloid leukemia patients.

IL-15 and SCF fail to induce NK differentiation and proliferation of CD34+ hematopoietic progenitors from chronic myeloid leukemia patients in contrast to normal stem cells although, both normal and leukemic CD34+ cells display comparable expression of c-kit or IL-15 receptor subunits. Interestingly, confocal microscopy analysis revealed that leukemic and most normal CD34+ cells produce and secrete IL-15, as shown by its trafficking through the Golgi apparatus and early endosomes. However, only leukemic progenitors express the membrane bound IL-15. Colocalization and internalization of IL-15Rbeta/gammac and IL-15Ralpha/gammac complexes indicated that IL-15 was specifically uptaken by leukemic progenitors. We also demonstrated that in both normal and leukemic progenitors, the signaling kinase Jak3 is constitutively pre-associated with the gammac chain. Anti-IL-15 neutralizing mAb treatment resulted in down-regulation of gammac chain and disruption of gammac/Jak3 interaction in normal but had no effect in leukemic progenitors. Our results suggest the existence in both normal and leukemic CD34+ cells of a constitutive production of a bioactive IL-15 that does not lead to NK differentiation and further indicate that membrane bound IL-15 and constitutive activation of gammac are hallmarks of leukemic progenitors. Oncogene (2000).

Critical role of the membrane-proximal, proline-rich motif of the interleukin-2 receptor gammac chain in the Jak3-independent signal transduction.

The interleukin-2 receptor (IL-2R) consists of three subunits, the IL-2Ralpha, IL-2Rbetac, and IL-2Rgammac chains. The essential role of the IL-2Rgammac cytoplasmic domain, consisting of 86 amino acids, in signal transmission has been well documented. Particularly, the carboxyl ter-minal region containing 48 amino acids. is essential for the association with and activation of the Jak3 protein tyrosine kinase. On the other hand, little is known about the role of the rest of the IL-2Rgammac cytoplasmic region consisting of the membrane-proximal 38 amino acids. We show that a truncated mutant form of IL-2Rgammac which lacks the membrane-distal 48 amino acids is still capable of inducing the activation of Jak1 and Stat3/Stat5 in the absence of Jak3 activation. This membrane-proximal region can also mediate the IL-2-induced tyrosine phosphorylation of the p85 subunit of phosphatidylinositol-3-kinase (PI3K). Furthermore, these signalling events are completely abrogated when mutations are introduced into the proline-rich motif in this region. In this study, we identified a Jak3-independent signalling pathway(s) from the membrane-proximal region of IL-2Rgammac. Our results indicate that the proline-rich motif in this region plays a critical role in this signalling pathway(s). The present study may provide further insight into the mechanism of cellular responses mediated by IL-2 and other cytokines which utilize the IL-2Rgammac for their signal transmission.

Oligomerization and Scaffolding Functions of the Erythropoietin Receptor Cytoplasmic Tail

Signal transduction by the erythropoietin receptor (EPOR) is activated by ligand-mediated receptor homodimerization. However, the relationship between extracellular and intracellular domain oligomerization remains poorly understood. To assess the requirements for dimerization of receptor cytoplasmic sequences for signaling, we overexpressed mutant EPORs in combination with wild-type (WT) EPOR to drive formation of heterodimeric (i.e. WT-mutant) receptor complexes. Dimerization of the membrane-proximal portion of the EPOR cytoplasmic region was found to be critical for the initiation of mitogenic signaling. However, dimerization of the entire EPOR cytoplasmic region was not required. To examine this process more closely, we generated chimeras between the intracellular and transmembrane portions of the EPOR and the extracellular domains of the interleukin-2 receptor beta and gammac chains. These chimeras allowed us to assess more precisely the signaling role of each receptor chain because only heterodimers of WT and mutant receptor chimeras form in the presence of interleukin-2. Coexpression studies demonstrated that a functional receptor complex requires the membrane-proximal region of each receptor subunit in the oligomer to permit activation of JAK2 but only one membrane-distal tail to activate STAT5 and to support cell proliferation. Thus, this study defines key relationships involved in the assembly and activation of the EPOR signal transduction complex which may be applicable to other homodimeric cytokine receptors.

Interleukin-15 differentially enhances the expression of interferon-gamma and interleukin-4 in activated human (CD4+) T lymphocytes.

In this study interleukin (IL)-15 was examined for its ability to modulate the expression of interferon-gamma (IFN-gamma) and IL-4 in activated human T lymphocytes. The effect of IL-15 was compared with IL-2 and IL-7, cytokines all known to use the IL-2 receptor gammaC chain. The results demonstrate that the extent of upregulation of IFN-gamma and IL-4 mRNA was dependent on the applied cytokine (IL-2>IL-15>IL-7) and on the stimulatory signal. IFN-gamma and IL-4 mRNAs were upregulated by IL-15 in concanavalin A- (twofold) and anti-CD3 plus anti-CD28- (fivefold) stimulated T lymphocytes. IFN-gamma mRNA accumulation, but not IL-4 mRNA, was additively upregulated by IL-15 plus IL-7 (ninefold) in anti-CD3 stimulated T lymphocytes, and bypassed the requirement of CD28 signalling. Fluorescence-activated cell sorting (FACS) experiments demonstrated that IFN-gamma mRNA was upregulated by IL-15 in both CD4+ and CD8+ T lymphocytes, whereas IL-4 mRNA accumulation predominantly occurred in CD4+ cells. Preincubation of highly purified CD4+ T lymphocytes during 7 days with IL-15 and/or IL-7, followed by activation, also showed enhanced IL-4 protein secretion, but predominantly upregulated IFN-gamma protein. The net effect was a dramatically increased IFN-gamma/IL-4 ratio. Taken together, IL-15 and IL-7 can act as costimulatory signals, which may favour a T helper 1 (Th1) immune response, particularly in the absence of sufficient CD28 costimulation.

The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor.

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.

Differential expression of T-cell growth factors in rejecting murine islet and human renal allografts: conspicuous absence of interleukin (IL)-9 despite expression of IL-2, IL-4, IL-7, and IL-15.

Interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15, all T-cell growth factors (TCGFs), utilize the common IL-2 receptor gammac chain as a critical signaling component in their receptor complexes. We have bred IL-2-/- and IL-4-/- double knockout (DKO) mice and showed vigorous islet allograft rejection by DKO hosts. The identity of TCGFs that support the IL-2- and IL-4-independent allograft rejection is unclear. We analyzed IL-9 gene expression in rejecting islet allografts in wild-type and in DKO mice, as well as in human renal transplant biopsy specimens, by reverse transcriptase polymerase chain reaction and compared the expression of IL-9 with that of other TCGFs. IL-9 gene expression was not detected in rejecting murine islet allografts in either wild-type or DKO recipient mice despite robust expression of other TCGFs, including IL-7 and IL-15. IL-9 transcripts were also not expressed in any of the human renal transplant biopsies obtained 4 to 251 days after transplantation, regardless of the presence or absence of histological evidence of rejection. Despite expression of IL-9 by DKO splenic cells upon in vitro mitogenic stimulation, IL-9 alone was unable to stimulate the proliferation of concanavalin A-activated splenic leukocytes harvested from DKO mice. IL-9 is conspicuously absent despite vigorous expression of IL-2, IL-4, IL-7, and IL-15 genes during acute allograft rejection.

Delineation of the Regions of Interleukin-2 (IL-2) Receptor β Chain Important for Association of Jak1 and Jak3: Jak1-INDEPENDENT FUNCTIONAL RECRUITMENT OF Jak3 TO IL-2Rβ

Interleukin-2 (IL-2) induces heterodimerization of the IL-2 receptor beta (IL-2Rbeta) and gammac chains of its receptor and activates the Janus family tyrosine kinases, Jak1 and Jak3. Whereas Jak1 associates with IL-2Rbeta, Jak3 associates primarily with gammac but also with IL-2Rbeta. We analyzed four IL-2Rbeta mutations that diminish IL-2-induced proliferation and found that each also decreased IL-2-induced signal transducer and activator of transcription (STAT) activation. For this reason, and because the mutations were in the IL-2Rbeta membrane-proximal region, we investigated and found that each mutation diminished IL-2Rbeta association with both Jak1 and Jak3. This suggested that these Jaks might interact with the same region of IL-2Rbeta; however, certain IL-2Rbeta internal deletions and C-terminal truncations differentially affected the association of Jak1 and Jak3. Interestingly, just as Jak1-IL-2Rbeta association is Jak3-independent and functionally important, we show that Jak3-IL-2Rbeta association is Jak1-independent and implicate this association as being important for IL-2-induced Stat5 activation. Moreover, Jak1 and Jak3 could associate only in the presence of IL-2Rbeta, suggesting that these kinases can simultaneously bind to IL-2Rbeta. Thus, our data not only demonstrate that somewhat more distal as well as membrane-proximal cytoplasmic regions of a type I cytokine receptor are important for Jak kinase association but also suggest that two IL-2Rbeta-Jak kinase interactions are important for IL-2 signaling.

TGF-beta down-regulates stromal IL-7 secretion and inhibits proliferation of human B cell precursors.

Development of lymphoid progenitors in vivo requires interaction with a bone marrow stromal microenvironment containing multiple cytokines involved in the development of nonlymphoid hemopoietic lineages. We tested the effect of one such cytokine, TGF-beta, on the proliferation of early human clonogenic lymphoid progenitors using a stroma-dependent in vitro culture system. TGF-beta caused a dose-dependent inhibition of lymphoid progenitor colonies that was reversible at low TGF-beta doses by addition of exogenous IL-7 to the cultures. IL-7 was unable to reverse the inhibitory effect of higher TGF-beta concentrations or inhibition caused by IL-1alpha, IL-4, or TNF-alpha. Stromal IL-7 mRNA expression and protein secretion were markedly down-regulated by TGF-beta, suggesting that inhibition of stromal IL-7 secretion partially accounts for the inhibitory effect of TGF-beta on lymphopoiesis in this culture system. It is likely that higher TGF-beta concentrations do not inhibit lymphopoiesis by down-regulating IL-7 receptor expression, since this cytokine did not reduce IL-7R alpha or gamma c mRNA levels in normal B cell precursors. Since direct stromal contact is required for in vitro lymphopoiesis, the potential regulation of the IL-7 pathway by cell adhesion was examined. Adhesion of human B cell precursors to stroma did not alter stromal IL-7 expression or expression of IL-7R alpha or gamma c-chains by B cell precursors. These results indicate that TGF-beta is a significant negative regulator of stroma-dependent proliferation of early human lymphoid progenitors and acts in part by down-regulating stromal IL-7 secretion.

IL2RGbase: a database of gamma c-chain defects causing human X-SCID.

Abstract X-linked severe combined immunodeficiency (X-SCID) is an immune disorder caused by mutations in the X-linked gene IL2RG, which encodes the common γ chain of the lymphocyte receptors for interleukin 2 (IL-2) and many other cytokines. A database of human X-SCID mutations (IL2RGbase) has been assembled, and this article summarizes the first 136 entries from unrelated patients.

Distinct Tyrosine Residues within the Interleukin-2 Receptor β Chain Drive Signal Transduction Specificity, Redundancy, and Diversity

To explore the basis for interleukin (IL)-2 receptor (IL-2R) signaling specificity, the roles of tyrosine-based sequences located within the cytoplasmic tails of the beta and gammac chains were examined in the murine helper T cell line HT-2. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, cellular proliferation, and the induction of various genes were monitored. All four of the cytoplasmic tyrosine residues as well as the distal portion of the gammac proved dispensable for the entire spectrum of IL-2R signaling responses studied. Conversely, select tyrosine residues within the beta chain were essential and differentially required for various signaling events. Specifically, activation of c-fos gene expression was found to occur exclusively through the most membrane proximal tyrosine, Tyr-338, whereas proliferation and the activation of STAT-5 were induced either through Tyr-338 or through the two C-terminal tyrosine residues, Tyr-392 and Tyr-510. These tyrosine residues mediated the induction of two different STAT-5 isoforms, which were found to form heterodimers upon receptor activation. In contrast to the tyrosine dependence of c-fos and STAT-5 induction, bcl-2 gene induction proceeded independently of all IL-2Rbeta tyrosine residues. Thus, the tyrosine-based modules present within the IL-2Rbeta cytoplasmic tail play a critical role in IL-2R signaling, mediating specificity, redundancy, and multifunctionality.

The human T-cell leukemia/lymphotropic virus type 1 p12I proteins bind the interleukin-2 receptor beta and gammac chains and affects their expression on the cell surface

p12I is a small hydrophobic protein encoded by the human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) that interacts with the 16-kDa component of the H+ vacuolar ATPase and cooperates with bovine papillomavirus 1 E5 oncoprotein in cell transformation. Just as an important step in E5 action appears to be its binding to the platelet-derived growth factor receptor, it was found that p12I binds specifically to both the beta and gamma(c) chains of the interleukin-2 receptor (IL-2R). The IL-2R beta and gamma(c) chains associated with p12I are endoglycosidase-H sensitive, suggesting that their interaction occurs in a pre-Golgi compartment. p12I stabilizes the immature forms of the IL-2R beta and gamma(c) chains and decreases their cell surface expression. The interactions of p12I with IL-2R beta and gamma(c) may have important implications in the immunosuppressive effect of HTLV-1 in vivo as well as in the ligand-independent HTLV-1-mediated T-cell proliferation.

Tissue-specific Activity of the γc Chain Gene Promoter Depends upon an Ets Binding Site and Is Regulated by GA-binding Protein

The gammac chain is a subunit of multiple cytokine receptors (interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15), the expression of which is restricted to hematopoietic lineages. A defect in gammac leads to the X-linked severe combined immunodeficiency characterized by a block in T cell differentiation. In order to better characterize the human gammac promoter and define the minimal tissue-specific promoter region, progressive 5'-deletion constructs of a segment extending 1053 base pairs upstream of the major transcription start site were generated and tested for promoter activity in various hematopoietic and nonhematopoietic cell types. The -1053/+34 construct allowed promoter activity only in cells of hematopoietic origin, and tissue specificity was conserved in all other constructs tested. The region downstream of -90 appeared critical for basal promoter activity. It contains two potential Ets binding sites conserved in the murine gammac promoter gene, one of which was found essential for functional promoter activity as determined by mutational analysis. The functional Ets binding site was found to bind Ets family proteins, principally GA-binding protein and Elf-1 and could be transactivated by GABPalpha and -beta synergistically. These results indicate that, as already reported for the IL2Rbeta promoter, GA-binding protein is an essential component of gammac basal promoter activity. Although GABP expression is not restricted to the hematopoietic lineage, its interaction with other specific factors may contribute to the tissue-specific expression of the gammac gene.

γc Gene Transfer Into SCID X1 Patients’ B-Cell Lines Restores Normal High-Affinity Interleukin-2 Receptor Expression and Function

SCID X1 is characterized by faulty T-cell and natural killer cell differentiation caused by mutation of the gamma-c chain gene encoding a number of multiple cytokine receptors (interleukin-2 [IL-2], IL-4, IL-7, IL-9, and IL-15 receptors). To assess the feasibility of inducing long-term expression and function of the gamma-c chain, Epstein-Barr virus (EBV)-transformed B-cell lines from two patients with SCID X1 were transduced with a Moloney-derived retroviral vector containing the gamma-c chain cDNA. The viral LTR was used as the promoter. Immediately after two cycles of coculture with the psi-crip clone producing the MFG(B2)-gamma-c cDNA vector, gamma-c expression, assessed by detection of the mRNA and membrane protein expression, was found in 15% to 20% of cells. The degree of membrane expression was similar to that in control EBV-B cells. Expression increased steadily over 6 months, becoming detectable in 100% of cells, and remained stable thereafter for a total of 9 months, reflecting positive selection of transduced cells. A study of provirus integration sites showed multiple integration. The expressed gamma-c was functional, because it restored high-affinity IL-2 receptor binding, IL-2 endocytosis, and IL-2-triggered phosphorylation of JAK-3 tyrosine kinase. Similar results were obtained with the two B-cell lines. These results show that efficient gamma-c gene transfer into B-cells lacking functional gamma-c is feasible and results in strong and stable expression of a functional gamma-c chain, apparently conferring a selective growth advantage in culture. Further in vitro studies of gamma-c gene transfer into gamma-c- hematopoietic progenitors are being conducted to assess the feasibility of correcting lymphocyte differentiation defects.

Convergence of signaling transduced by prolactin (PRL)/cytokine chimeric receptors on PRL-responsive gene transcription.

Ligand binding to cytokine receptors rapidly triggers tyrosine phosphorylation of Janus family tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats). Jak2 activation is mediated by PRL receptor homodimers as well as by receptors for the interleukin (IL)-3, IL-5, and granulocyte macrophage-colony stimulating factor, which share the common beta c-subunit. Otherwise, Jak1 and Jak3 are involved in IL-2 signaling through heterodimerization of the IL-2 receptor-beta (IL-2R beta) and gamma c-chains. Stat5, a member of the Stat family, confers the PRL response on milk protein genes. Here we show that chimeric PRL receptors that contain the transmembrane and cytoplasmic domains of the IL-2R beta or beta c-chains transduce in response to PRL tyrosine phosphorylation and activation of Jak1 and Jak2, respectively. Tyrosine phosphorylation of Stat5, activation of its DNA-binding activity assessed in bandshift experiments using a lactogenic hormone responsive region (LHRR) probe, and transcriptional induction of a beta-casein promoter luciferase construct in stably transfected CHO cells are observed with both chimeras upon PRL stimulation. Our results demonstrate that distinct cytoplasmic domains of these cytokine receptors elicit convergent signaling pathways and provide evidence that beta c and IL-2R beta function as a complete signal transducer. Our data strengthen previous observations that Stat5 activation is not dependent on the activation of a specific Jak kinase and also suggest that neither Jak3 nor gamma c have a specific role in this process.