Blocking common γ chain cytokine signaling ameliorates T cell-mediated pathogenesis in disease models.

The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.

IL2RG-deficient minipigs generated via CRISPR/Cas9 technology support the growth of human melanoma-derived tumours.

OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development. MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice. CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.

The anti-inflammatory potential of cefazolin as common gamma chain cytokine inhibitor.

A continuing quest for specific inhibitors of proinflammatory cytokines brings promise for effective therapies designed for inflammatory and autoimmune disorders. Cefazolin, a safe, first-generation cephalosporin antibiotic, has been recently shown to specifically interact with interleukin 15 (IL-15) receptor subunit α (IL-15Rα) and to inhibit IL-15-dependent TNF-α and IL-17 synthesis. The aim of this study was to elucidate cefazolin activity against IL-2, IL-4, IL-15 and IL-21, i.e. four cytokines sharing the common cytokine receptor γ chain (γc). In silico, molecular docking unveiled two potential cefazolin binding sites within the IL-2/IL-15Rß subunit and two within the γc subunit. In vitro, cefazolin decreased proliferation of PBMC (peripheral blood mononuclear cells) following IL-2, IL-4 and IL-15 stimulation, reduced production of IFN-γ, IL-17 and TNF-α in IL-2- and IL-15-treated PBMC and in IL-15 stimulated natural killer (NK) cells, attenuated IL-4-dependent expression of CD11c in monocyte-derived dendritic cells and suppressed phosphorylation of JAK3 in response to IL-2 and IL-15 in PBMC, to IL-4 in TF-1 (erythroleukemic cell line) and to IL-21 in NK-92 (NK cell line). The results of the study suggest that cefazolin may exert inhibitory activity against all of the γc receptor-dependent cytokines, i.e. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.

Identification of a γc Receptor Antagonist That Prevents Reprogramming of Human Tissue-resident Cytotoxic T Cells by IL15 and IL21.

BACKGROUND & AIMS: Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells. METHODS: We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8+ T-cell lines, or CD8+ T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B. RESULTS: Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines. CONCLUSIONS: We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell-mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21.

Specific Inhibition of Soluble γc Receptor Attenuates Collagen-Induced Arthritis by Modulating the Inflammatory T Cell Responses.

IL-17 produced by Th17 cells has been implicated in the pathogenesis of rheumatoid arthritis (RA). It is important to prevent the differentiation of Th17 cells in RA. Homodimeric soluble γc (sγc) impairs IL-2 signaling and enhances Th17 differentiation. Thus, we aimed to block the functions of sγc by inhibiting the formation of homodimeric sγc. The homodimeric form of sγc was strikingly disturbed by sγc-binding DNA aptamer. Moreover, the aptamer effectively inhibited Th17 cell differentiation and restored IL-2 and IL-15 signaling impaired by sγc with evidences of increased survival of T cells. sγc was highly expressed in SF of RA patients and increased in established CIA mice. The therapeutic effect of PEG-aptamer was tested in CIA model and its treatment alleviated arthritis pathogenesis with impaired differentiation of pathogenic Th17, NKT1, and NKT17 cells in inflamed joint. Homodimeric sγc has pathogenic roles to exacerbate RA progression with differentiation of local Th17, NKT1, and NKT17 cells. Therefore, sγc is suggested as target of a therapeutic strategy for RA.

Regulation of T cell proliferation with drug-responsive microRNA switches.

As molecular and cellular therapies advance in the clinic, the role of genetic regulation is becoming increasingly important for controlling therapeutic potency and safety. The emerging field of mammalian synthetic biology provides promising tools for the construction of regulatory platforms that can intervene with endogenous pathways and control cell behavior. Recent work has highlighted the development of synthetic biological systems that integrate sensing of molecular signals to regulated therapeutic function in various disease settings. However, the toxicity and limited dosing of currently available molecular inducers have largely inhibited translation to clinical settings. In this work, we developed synthetic microRNA-based genetic systems that are controlled by the pharmaceutical drug leucovorin, which is readily available and safe for prolonged administration in clinical settings. We designed microRNA switches to target endogenous cytokine receptor subunits (IL-2Rß and γc) that mediate various signaling pathways in T cells. We demonstrate the function of these control systems by effectively regulating T cell proliferation with the drug input. Each control system produced unique functional responses, and combinatorial targeting of multiple receptor subunits exhibited greater repression of cell growth. This work highlights the potential use of drug-responsive genetic control systems to improve the management and safety of cellular therapeutics.

Targeting the binding interface on a shared receptor subunit of a cytokine family enables the inhibition of multiple member cytokines with selectable target spectrum.

The common γ molecule (γc) is a shared signaling receptor subunit used by six γc-cytokines. These cytokines play crucial roles in the differentiation of the mature immune system and are involved in many human diseases. Moreover, recent studies suggest that multiple γc-cytokines are pathogenically involved in a single disease, thus making the shared γc-molecule a logical target for therapeutic intervention. However, the current therapeutic strategies seem to lack options to treat such cases, partly because of the lack of appropriate neutralizing antibodies recognizing the γc and, more importantly, because of the inherent and practical limitations in the use of monoclonal antibodies. By targeting the binding interface of the γc and cytokines, we successfully designed peptides that not only inhibit multiple γc-cytokines but with a selectable target spectrum. Notably, the lead peptide inhibited three γc-cytokines without affecting the other three or non-γc-cytokines. Biological and mutational analyses of our peptide provide new insights to our current understanding on the structural aspect of the binding of γc-cytokines the γc-molecule. Furthermore, we provide evidence that our peptide, when conjugated to polyethylene glycol to gain stability in vivo, efficiently blocks the action of one of the target cytokines in animal models. Collectively, our technology can be expanded to target various combinations of γc-cytokines and thereby will provide a novel strategy to the current anti-cytokine therapies against immune, inflammatory, and malignant diseases.

Therapeutic activity of multiple common γ-chain cytokine inhibition in acute and chronic GVHD.

The common γ chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-γ, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3(-/-) T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common γ-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation.

Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2Rγ) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2Rγ abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2Rγ in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2Rγ completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2Rγ interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2Rγ contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2Rγ, indicating IL2Rγ and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2Rγ in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth.

Integrated mRNA-microRNA profiling of human NK cell differentiation identifies MiR-583 as a negative regulator of IL2Rγ expression.

Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3' UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.

Combination of donor splenocyte transfusion with blockade of γc signal synergizes to inhibit alloreactive T-cell proliferation and induces apoptosis.

BACKGROUND: The common γ chain (γc) plays a critical role in regulating proliferation, differentiation, and apoptosis of peripheral T-cells. It was previously confirmed that blocking the γc signal can successfully induce transplant tolerance in a murine model. Here we investigated the potential mechanism. METHODS: Splenocytes from C57BL/6 mice were transfused into T-cell deficient Balb/c nude mice that were reconstituted with syngeneic wild-type T-cells labeled with 5-carboxyfluorescein diacetate succinimidyl ester (CFSE). After 24 hours, recipients received i.p. injection of mixture of anti-γc mAbs, or with isotype control IgG2a. The labeled T-cells were harvested from recipient spleens after 12 and 48 hours. T-cell proliferation and apoptosis were detected by flow cytometry. RESULTS: T-cell proliferation was markedly inhibited and apoptotic T cells could be detected at 12 hours after the mAbs injection. Proliferation was inhibited at 48 hours, but the proportion of apoptotic T-cells was not more than at 12 hours. In the control group, however, T-cells actively proliferated and no significant apoptosis was detected at either time point. CONCLUSIONS: The results suggested that blockade of γc signals can synergize with donor splenocyte transfusion and lead to inhibition of antigen-specific T-cell proliferation and induction of apoptotic T-cell death. This protocol may develop a novel approach to induce donor-specific tolerance.

Blockade of gammac signals in combination with donor-specific transfusion induces cardiac allograft acceptance in murine models.

The gammac cytokines play an important role in proliferation and survival of T cells. Blocking the gammac signals can cause the activated donor-reactive T cells losing the ability to proliferate, and getting into apoptosis pathway, which contributes to induction of the peripheral tolerance. In this study, we induced the transplant tolerance through blocking the gammac in combination with donor-specific transfusion (DST) in the cardiac transplantation. Following DST, on the day 2, 4 and 6, C57BL/6 recipients received anti-gammac monoclonal antibodies (mAbs) injection, and those in control group were not given anti-gammac mAbs. On the day 7, Balb/c cardiac allografts were transplanted. All recipients in experimental group accepted cardiac allografts over 30 days, and two of them accepted allografts without rejection until sacrifice on the 120 day. Animals only receiving DST rejected grafts within 5 days, and the mice receiving cardiac transplantation alone rejected grafts within 9 days. Our study showed that blockade of gammac signaling combined with DST significantly prolonged allograft survival, which was probably associated with inhibition of antigen-specific T-cell proliferation and induction of apoptosis.

Defucosylated anti-CCR4 monoclonal antibody exerts potent ADCC against primary ATLL cells mediated by autologous human immune cells in NOD/Shi-scid, IL-2R gamma(null) mice in vivo.

There is a lack of suitable small animal models to evaluate human Ab-dependent cellular cytotoxicity (ADCC) in vivo, because of the species incompatibility between humans and animals or due to nonspecific allogeneic immune reactions. To overcome these problems, we established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice as recipients, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T cell leukemia/lymphoma (ATLL) cells and a therapeutic chimeric anti-CCR4 mAb, the Fc region of which is defucosylated to enhance ADCC. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in Ab-based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future.

The cellular amount of the common gamma-chain influences spontaneous or induced cell proliferation.

Mutations of the IL2RG encoding the common gamma-chain (gamma(c)) lead to the X-linked SCID disease. Gene correction through ex vivo retroviral transduction restored the immunological impairment in the most of treated patients, although lymphoproliferative events occurred in five of them. Even though in two cases it was clearly documented an insertional mutagenesis in LMO2, it is conceivable that gamma(c) could have a role per se in malignant lymphoproliferation. The gamma(c) is a shared cytokine receptor subunit, involved also in growth hormone (GH) receptor signaling. Through short interfering RNA or using X-linked SCID B lymphoblastoid cell lines lacking gamma(c), we demonstrate that self-sufficient growth was strongly dependent on gamma(c) expression. Furthermore, a correlation between gamma(c) amount and the extent of constitutive activation of JAK3 was found. The reduction of gamma(c) protein expression also reduced GH-induced proliferation and STAT5 nuclear translocation in B lymphoblastoid cell lines. Hence, our data demonstrate that gamma(c) plays a remarkable role in either spontaneous or GH-induced cell cycle progression depending on the amount of protein expression, suggesting a potential role as enhancing cofactor in lymphoproliferation.

Disruption of the gamma c cytokine network in T cells during HIV infection.

The common gamma chain (gammac)-sharing cytokines (IL's-2, 4, 7, 9, 15, and 21) play a vital role in the survival, proliferation, differentiation and function of T lymphocytes. As such, disruption of their signaling pathways would be expected to have severe consequences on the integrity of the immune system. Indeed, it appears that the signaling network of these cytokines is both disrupted and exploited by HIV at various stages of infection. IL-2 secretion and signaling downstream of its receptor are impaired in T cells from chronically-infected HIV+ patients. Elevated plasma IL-7 levels and decreased IL-7Ralpha expression in patient T cells results in significantly decreased responsiveness to this critical cytokine. Interestingly, IL-2 and IL-15 are also able to render CD4+ T cells permissive to HIV infection through their influence on the activity of the APOBEC3G deaminase enzyme. Herein, we describe the current state of knowledge on how the gammac cytokine network is affected during HIV infection, with a focus on how this impairs CD4+ and CD8+ T cell function while also benefiting the virus itself. We also address the use of cytokines as adjuncts to highly active antiretroviral therapy to bolster immune reconstitution in infected patients.