Common Gamma Chain Research Articles

Effect of cytokines (IL-2, IL-7, and IL-15) having common γ-chain of receptors on differentiation and maturation of CD4+/CD8+ T-cells in a CD45RA T-lymphocyte population in vitro

The effect of cytokines (IL-2, IL-7, and IL-15) having a common γ-chain of receptors on the maturation and differentiation of CD3+CD45RA+CD4+/CD8+ lymphocytes in homeostatic cultivation model in vitro was analyzed. It was found that the maximum IL-2 concentration in the helper CD45RA+-T-cell population mediates an increase in the number of CD45RA+CD4+ T lymphocytes with the phenotype of mature and immature terminally differentiated TEMRA T cells. IL-15 leads to the production of lymphocytes with CD27–CD62L+ phenotype (presumably, TEMRA, in which the CD62L expression persists). In the CD45RA+CD8+ T lymphocyte populations, the studied cytokines (IL-2, IL-7, and IL-15) initiate the production of mature TEMRA (E) T lymphocytes and memory T cells with the CD45RA−CD27+CD62L+ central phenotype (TCM).

Innate IFN-γ-Producing Cells Developing in the Absence of IL-2 Receptor Common γ-Chain.

IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.

Developmental and Functional Control of Natural Killer Cells by Cytokines.

Natural killer (NK) cells are effective in combating infections and tumors and as such are tempting for adoptive transfer therapy. However, they are not homogeneous but can be divided into three main subsets, including cytotoxic, tolerant, and regulatory NK cells, with disparate phenotypes and functions in diverse tissues. The development and functions of such NK cells are controlled by various cytokines, such as fms-like tyrosine kinase 3 ligand (FL), kit ligand (KL), interleukin (IL)-3, IL-10, IL-12, IL-18, transforming growth factor-β, and common-γ chain family cytokines, which operate at different stages by regulating distinct signaling pathways. Nevertheless, the specific roles of each cytokine that regulates NK cell development or that shapes different NK cell functions remain unclear. In this review, we attempt to describe the characteristics of each cytokine and the existing protocols to expand NK cells using different combinations of cytokines and feeder cells. A comprehensive understanding of the role of cytokines in NK cell development and function will aid the generation of better efficacy for adoptive NK cell treatment.

Maternal T and B cell engraftment in two cases of X-linked severe combined immunodeficiency with IgG1 gammopathy

X-linked severe combined immunodeficiency (X-SCID), caused by defects in the common gamma chain, is typically characterized by T and NK cell defects with the presence of B cells. T cell dysfunction and impaired class-switch recombination of B cells mean that patients typically have defects in class-switched immunoglobulins (IgG, IgA, and IgE) with detectable IgM. Here, we describe two patients with X-SCID with IgG1 gammopathy, in whom we identified maternal T and B cell engraftment. Exclusively, maternal B cells were found among the IgD−CD27+ class-switched memory B cells, whereas the patients' B cells remained naïve. In vitro stimulation with CD40L+IL-21 revealed that peripheral blood cells from both patients produced only IgG1. Class-switched maternal B cells had restricted receptor repertoires with various constant regions and few somatic hypermutations. In conclusion, engrafted maternal B cells underwent class-switch recombination and produced immunoglobulin, causing hypergammaglobulinemia in patients with X-SCID.

IL2RG, identified as overexpressed by RNA-seq profiling of pancreatic intraepithelial neoplasia, mediates pancreatic cancer growth.

Pancreatic ductal adenocarcinoma evolves from precursor lesions, the most common of which is pancreatic intraepithelial neoplasia (PanIN). We performed RNA-sequencing analysis of laser capture microdissected PanINs and normal pancreatic duct cells to identify differentially expressed genes between PanINs and normal pancreatic duct, and between low-grade and high-grade PanINs. One of the most highly overexpressed transcripts identified in PanIN is interleukin-2 receptor subunit gamma (IL2RG) encoding the common gamma chain, IL2Rγ. CRISPR-mediated knockout of IL2RG in orthotopically implanted pancreatic cancer cells resulted in attenuated tumor growth in mice and reduced JAK3 expression in orthotopic tumors. These results indicate that IL2Rγ/JAK3 signaling contributes to pancreatic cancer cell growth in vivo.

B Cell-Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B Cell Immune Responses.

MyD88 and FcR common γ-chain (Fcer1g, FcRγ) elicit proinflammatory responses to exogenous Ags. Deletion of these receptors in autoimmune models has generally led to reduced overall disease. In B cells, Myd88 is required for anti-DNA and anti-RNA autoantibody responses, whereas Fcer1g is not expressed in these cells. The roles of these receptors in myeloid cells during B cell autoimmune activation remain less clear. To investigate the roles of Myd88 and Fcer1g in non-B cells, we transferred anti-self-IgG (rheumatoid factor) B cells and their physiologic target Ag, anti-chromatin Ab, into mice lacking Fcer1g, Myd88, or both and studied the extrafollicular plasmablast response. Surprisingly, we found a markedly higher and more prolonged response in the absence of either molecule; this effect was accentuated in doubly deficient recipients, with a 40-fold increase compared with wild-type recipients at day 10. This enhancement was dependent on CD40L, indicating that Myd88 and FcRγ, presumably on myeloid APCs, were required to downregulate T cell help for the extrafollicular response. To extend the generality, we then investigated a classic T cell-dependent response to (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken γ globulin and found a similar effect. Thus, these results reveal novel regulatory roles in the B cell response for receptors that are typically proinflammatory.

Selective Inhibition of Janus Kinase 3 Has No Impact on Infarct Size or Neurobehavioral Outcomes in Permanent Ischemic Stroke in Mice.

Janus kinase 3 (JAK3) is associated with the common gamma chain of several interleukin (IL) receptors essential to inflammatory signaling. To study the potential role of JAK3 in stroke-induced neuroinflammation, we subjected mice to permanent middle cerebral artery occlusion and investigated the effects of JAK3 inhibition with decernotinib (VX-509) on infarct size, behavior, and levels of several inflammatory mediators. Results from our double immunofluorescence staining showed JAK3 expression on neurons, endothelial cells, and microglia/macrophages in the ischemic mouse brain (n = 3). We found for the first time that total and phosphorylated/activated JAK3 are dramatically increased after stroke in the ipsilateral hemisphere (**P < 0.01; n = 5–13/group) in addition to increased IL-21 expression after stroke (**P < 0.01; n = 5–7/group). However, inhibition of JAK3 confirmed by reduced phosphorylation of its activation loop at tyrosine residues 980/981 does not reduce infarct volume measured at 48 h after stroke (n = 6–10/group) nor does it alter behavioral outcomes sensitive to neurological deficits or stroke-induced neuroinflammatory response (n = 9–10/group). These results do not support a detrimental role for JAK3 in acute neuroinflammation following permanent focal cerebral ischemia. The functional role of increased JAK3 activation after stroke remains to be further investigated.

Abstract 1595: IL15/IL15Rα heterodimeric Fc-fusions with extended half-lives

Abstract IL15 and IL2 are similar cytokines that stimulate the proliferation and differentiation of B cells, T cells, and NK cells. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. IL2Rα is highly expressed on Tregs, and the therapeutic benefit of IL2 for cancer treatment has been limited for this reason. IL15 is produced by monocytes and dendritic cells and functions as a stabilized heterodimeric complex with membrane-bound IL15Rα present on the same cells. This IL15/IL15Rα complex is presented in trans to NK cells and CD8+ T cells expressing IL2Rβ and the common gamma chain. It has been shown that recombinant IL15/IL15Rα heterodimer is highly active. Currently there are no approved versions of recombinant IL15 although several clinical trials are ongoing. As potential drugs, cytokines suffer from a very fast clearance that hinders favorable dosing. Consequently, a more druggable version of IL15 would consist of the IL15/IL15Rα complex coupled with slower clearance. We created various IL15/IL15Rα heterodimeric Fc-fusions in an effort to facilitate production, promote FcRn-mediated recycling of the complex, and thus prolong half-life. We engineered IL15/IL15Rα heterodimeric Fc-fusions by either fusing IL15 to one side of a heterodimeric Fc-region, and the sushi domain of IL15Rα to the other side, or by creating a single-chain IL15/IL15Rα that was attached to one side of a heterodimeric Fc-region. These Fc-fusions were tuned for optimal activity by engineering the linker regions between IL15/IL15Rα and the Fc and/or by engineering substitutions on IL15 at the IL15/IL2Rβ or IL15/gamma chain interface. In vitro proliferation of T and NK cells in healthy PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days. In vivo activity was evaluated using a mouse model in which human PBMCs are engrafted into NSG mice (huPBMC-NSG) and measuring the extent of T cell engraftment by flow cytometry as well as IFNγ. PK was evaluated in C57BL/6J mice. IL15/IL15Rα heterodimeric Fc-fusions were successfully produced with favorable yields. The Fc-fusions enhanced proliferation of T and NK cells in vitro. Treatment of huPBMC-NSG mice with weekly doses of IL15/IL15Rα heterodimeric Fc-fusions promoted enhanced T cell engraftment and elevated levels of IFNγ in a dose dependent manner. Severe graft versus host disease was observed in treated mice. Little activity was seen with a comparable weekly dose of recombinant IL15. PK in C57BL/6J mice indicated half-lives of several days for the IL15/IL15Rα heterodimeric Fc-fusions, which are significantly longer than the &amp;lt;1 hr half-life reported for IL15. Together, these data indicate that IL15/IL15Rα heterodimeric Fc-fusions demonstrate the high activity of IL15 with a more favorable PK profile. Citation Format: Matthew J. Bernett, Christine Bonzon, Rumana Rashid, Rajat Varma, Kendra N. Avery, Irene W. Leung, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. IL15/IL15Rα heterodimeric Fc-fusions with extended half-lives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1595. doi:10.1158/1538-7445.AM2017-1595

Abstract 2663: Characterization of the pharmacodynamic immune response to a novel immunotherapeutic agent, ALKS 4230, in mice and non-human primates

Abstract ALKS 4230 is a selective agonist of the intermediate-affinity IL-2 receptor (IL-2R). A phase 1 study is ongoing to evaluate the safety and tolerability of ALKS 4230 in the treatment of patients with refractory solid tumors. The selectivity of ALKS 4230 is achieved through the stable fusion of circularly permuted IL-2 to the extracellular portion of the IL-2Rα chain, CD25. The resulting fusion protein is sterically prevented from binding to the high-affinity IL-2R complex, comprised of IL-2Rα, IL-2Rβ, and common gamma chain, expressed preferentially on CD4+ FOXP3+ regulatory T cells (CD4+ Tregs) yet retains full ability to signal through the intermediate-affinity IL-2R complex, comprised of IL-2Rβ and common gamma chain, expressed on memory CD8+ T cells and NK cells. Repeated dosing of ALKS 4230 drives the significant expansion of various CD8+ T cell and NK cell populations without activation and minimal expansion of CD4+ Tregs in mice and non-human primates (NHP). The kinetics of the immunological responses in mice and NHP demonstrate that the pharmacodynamic effects persist beyond systemic exposure of ALKS 4230. Data demonstrating the effects of ALKS 4230 on tumor-infiltrating lymphocyte populations in syngeneic tumor models will also be presented. Citation Format: Jared E. Lopes, Heather C. Losey, Reginald L. Dean, Heather L. Flick, Michael R. Huff, Rosemarie A. Moroso, Lei Sun, Juan C. Alvarez. Characterization of the pharmacodynamic immune response to a novel immunotherapeutic agent, ALKS 4230, in mice and non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2663. doi:10.1158/1538-7445.AM2017-2663

Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment

Loss-of-function mutations in the common gamma (γc) chain cytokine receptor subunit give rise to severe combined immunodeficiency characterized by lack of T and natural killer cells and infant death from infection. Hematopoietic stem cell transplantation or gene therapy offer a cure, but despite successful replacement of lymphoid immune lineages, a long-term risk of severe cutaneous human papilloma virus infections persists, possibly related to persistent γc-deficiency in other cell types. Here we show that keratinocytes, the only cell type directly infected by human papilloma virus, express functional γc and its co-receptors. After stimulation with the γc-ligand IL-15, γc-deficient keratinocytes show significantly impaired secretion of specific chemokines including CXCL1, CXCL8, and CCL20, resulting in reduced chemotaxis of dendritic cells and CD4+ T cells. Furthermore, γc-deficient keratinocytes also exhibit defective induction of T-cell chemotaxis in a model of stable human papilloma virus-18 infection. These findings suggest that persistent γc-deficiency in keratinocytes alters immune cell recruitment to the skin, which may contribute to the development and persistence of warts in this condition and would require different treatment approaches.

A diverse repertoire of memory CD4 T cells is maintained in the absence of the common gamma chain

Abstract After successfully combating a pathogen, a small number of antigen-experienced T cells are retained in the body to offer better protection against future infections by the same agent. In the case of CD8 T cells, the stable maintenance of these memory phenotype T cells critically requires homeostatic turnover driven by cytokines such as IL-7 and IL-15. Intriguingly, CD4 T cells that are deficient in the shared receptor for IL-2, IL-7 and IL-15 – i.e. the cytokine common gamma chain (γc), can still generate memory phenotype (MP) T cells in vivo. Here we show that such naturally generated γc-deficient CD4 MP T cells have a diverse T-cell receptor repertoire usage, despite a profound deficiency in the naïve CD4 T cell compartment in such mice. Furthermore, adoptive transfer experiments show that γc-deficient CD4 MP T cells can be stably maintained even in a competitive environment (of a γc+ host) where other CD4 MP T cells can utilize IL-2, IL-7 and IL-15. CD4 MP T cells from γc-deficient animals also differentiated to IFN-γ producers. Therefore, unlike CD8 memory T cells, the maintenance of a diverse repertoire of CD4 memory T cells is independent of trophic cytokines such as IL-7 and IL-15.

PD-1 signals instruct a critical metabolic switch for maintenance of T cell memory

Abstract PD-1 is highly expressed on CD8 T cells during activation, both in acute and chronic viral infections. Inhibitory signaling in cytotoxic T cells through the PD-1 axis is well characterized during chronic infections and has led to the development of clinical checkpoint blockade therapies that restore function to otherwise exhausted CD8 T cells. However, the functional significance of transient PD-1 expression on T cells early after activation during acute infections remains yet to be characterized. It is paradoxical that expression of PD-1 (an inhibitory receptor) during activation temporally coincides with rapid proliferation and production of copious amounts of signature effector cytokines (IFN-γ and TNF-α). Our studies using PD-1−/−antigen-specific CD8 T cells revealed an unexpected lack of an inhibitory role for PD-1 early during priming - PD-1−/−T cells exhibited similar proliferation, accumulation, cytotoxicity and polyfunctionality compared to WT T cells. Surprisingly, PD-1−/−T cells underwent precipitous contraction, leading to near ablation of the memory pool. Mechanistically, PD-1−/−memory Tcells showed a severe defect in antigen-independent homeostatic proliferation in vivo, despite identical expression of common gamma-chain cytokine receptors as WT T cells. Notably, PD-1−/−memory T cells failed to optimally switch the critical utilization of glucose to fatty acid as an energy source, which impaired their homeostatic maintenance and resulted in attrition. These findings show a previously unrecognized role of PD-1 signaling in programming a metabolic switch that is critical for the maintenance of memory CD8 T cells and could make it a novel target for manipulating vaccine-induced memory T cell longevity.

331 An open label clinical trial of the JAK inhibitor tofacitinib for alopecia areata

Alopecia areata (AA) is a common autoimmune disease, with a lifetime risk of approximately 2%, affecting 5.3 million individuals in the US. AA results in unpredictable nonscarring hair loss. There are no FDA approved treatments. Our previous work used whole genome expression studies to identify active immune circuits in AA, including the common gamma chain IL-15 and IL-2 cytokine pathways and the IFN pathway. Since receptors for both of these pathways signal through JAK kinases, this work invited clinical exploration of a small molecule pan-JAK inhibitor, tofacitinib. Tofacitinib is FDA approved for use in patients with rheumatoid arthritis. In preclinical trials in C3H/HeJ mice, tofacitinib prevented the development of AA and successfully reversed established disease both topically and orally. We therefore initiated an open-label trial of tofacitinib in 12 patients with moderate to severe AA. Eleven of 12 patients completed treatment. There were minimal adverse events. Due to limited response to the initial dose of 5mg BID, the dose was escalated to 10mg. 7 of 12 patients achieved at least 50% regrowth demonstrating a 60% response rate for the primary outcome. ALADIN analysis of RNAseq experiments performed on scalp biopsies taken throughout the trial further revealed three key findings: molecular response to treatment was observable as early as 4 weeks of treatment in low-dose responders; patients with delayed clinical response (dose escalation required, recovery observed at >24 weeks), were nonetheless identifiable as responsive at earlier time points; and that molecular gene expression signatures can be used to predict non-responders prior to initiating treatment. Our study of tofacitinib in AA has shown dramatic clinical responses in moderate-to-severe AA, and provides a strong rationale for larger clinical trials using JAK inhibitors in AA.

Modulation of Tim-3 Expression by Antigen-Dependent and -Independent Factors on T Cells from Patients with Chronic Hepatitis B Virus Infection.

T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) was up-regulated on viral specific T cells and contributed to T cells exhaustion during chronic hepatitis B virus (HBV) infection. However, modulation of Tim-3 expression was still not fully elucidated. To evaluate the potential viral and inflammatory factors involved in the inductor of Tim-3 expression on T cells, 76 patients with chronic HBV infection (including 40 chronic hepatitis B [CHB] and 36 asymptomatic HBV carriers [AsC]) and 40 of normal controls (NCs) were enrolled in this study. Tim-3 expressions on CD4+ and CD8+ T cells were assessed in response to HBV-encoding antigens, HBV peptide pools, and common γ-chain (γc) cytokines stimulation by flow cytometry. HBV peptides and anti-CD3/CD28 directly induced Tim-3 expression on T cells. γc cytokines also drive Tim-3 up-regulations on both CD4+ and CD8+ T cells in patients with chronic HBV infection. However, γc cytokines did not enhance the Tim-3 inductions by either anti-CD3/CD28 or HBV peptides stimulation. Furthermore, γc cytokines-mediated Tim-3 induction could not be abrogated by γc cytokine receptor-neutralizing antibodies. The current results suggested that elevation of Tim-3 expression on T cells could be regulated by both antigen-dependent and -independent manner in patients with chronic HBV infection. The role of γc cytokines in modulation of inhibitory pathway might be evaluated as immunotherapies in humans.

Murine Th17 cells utilize IL-2 receptor gamma chain cytokines but are resistant to cytokine withdrawal-induced apoptosis.

Adoptive cellular therapy (ACT) with the Th17 subset of CD4+ T cells can cure established melanoma in preclinical models and holds promise for treating human cancer. However, little is known about the growth factors necessary for optimal engraftment and anti-tumor activity of Th17 cells. Due to the central role of IL-2 receptor gamma chain (IL2Rγ-chain) cytokines (IL-2, IL-7, and IL-15) in the activity and persistence of many T cell subsets after adoptive transfer, we hypothesized that these cytokines are important for Th17 cells. We found that Th17 cells proliferated in response to IL-2, IL-7, and IL-15 in vitro. However, in contrast to many other T cell subsets, including conventionally activated CD8+ T cells, we found that Th17 cells were resistant to apoptosis in the absence of IL2Rγ-chain cytokines. To determine whether Th17 cells utilize IL2Rγ-chain cytokines in vivo, we tracked Th17 cell engraftment after adoptive transfer with or without cytokine depletion. Depletion of IL-7 and/or IL-2 decreased initial engraftment, while depletion of IL-15 did not. Supplementation of IL-2 increased initial Th17 engraftment. To assess the clinical relevance of these findings, we treated melanoma-bearing mice with Th17 cell adoptive transfer and concurrent cytokine depletion or supplementation. We found that simultaneous depletion of IL-2 and IL-7 decreased therapeutic efficacy, depletion of IL-15 had no effect, and IL-2 supplementation increased therapeutic efficacy. Our results show that Th17 cells are responsive to IL2Rγ-chain cytokines, and provide insight into the application of these cytokines for Th17-based therapeutic strategies.

Soluble common gamma chain exacerbates COPD progress through the regulation of inflammatory T cell response in mice.

Cigarette smoking (CS) is a major cause of considerable morbidity and mortality by inducing lung cancer and COPD. COPD, a smoking-related disorder, is closely related to the alteration of immune system and inflammatory processes that are specifically mediated by T cells. Soluble common gamma chain (sγc) has recently been identified as a critical regulator of the development and differentiation of T cells. We examined the effects of sγc in a cigarette smoke extract (CSE) mouse model. The sγc level in CSE mice serum is significantly downregulated, and the cellularity of lymph node (LN) is systemically reduced in the CSE group. Overexpression of sγc enhances the cellularity and IFNγ production of CD8 T cells in LN and also enhances Th1 and Th17 differentiation of CD4 T cells in the respiratory tract. Mechanistically, the downregulation of sγc expression mediated by CSE is required to prevent excessive inflammatory T cell responses. Therefore, our data suggest that sγc may be one of the target molecules for the control of immunopathogenic progresses in COPD.

Glomerular common gamma chain confers B- and T-cell–independent protection against glomerulonephritis

Crescentic glomerulonephritis is a life-threatening renal disease that has been extensively studied by the experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM-GN) model. Although T cells have a significant role in this model, athymic/nude mice and rats still develop severe renal disease. Here we further explored the contribution of intrinsic renal cells in the development of T-cell-independent GN lesions. Anti-GBM-GN was induced in three strains of immune-deficient mice (Rag2-/-, Rag2-/-Il2rg-/-, and Rag2-/-Il2rb-/-) that are devoid of either T/B cells or T/B/NK cells. The Rag2-/-Il2rg-/- or Rag2-/-Il2rb-/- mice harbor an additional deletion of either the common gamma chain (γC) or the interleukin-2 receptor β subunit (IL-2Rβ), respectively, impairing IL-15 signaling in particular. As expected, all these strains developed severe anti-GBM-GN. Additionally, bone marrow replenishment experiments allowed us to deduce a protective role for the glomerular-expressed γC during anti-GBM-GN. Given that IL-15 has been found highly expressed in nephritic kidneys despite the absence of lymphocytes, we then studied this cytokine invitro on primary cultured podocytes from immune-deficient mice (Rag2-/-Il2rg-/- and Rag2-/-Il2rb-/-) compared to controls. IL-15 induced downstream activation of JAK1/3 and SYK in primary cultured podocytes. IL-15-dependent JAK/SYK induction was impaired in the absence of γC or IL-2Rβ. Wefound γC largely induced on podocytes during humanglomerulonephritis. Thus, renal lesions are indeed modulated by intrinsic glomerular cells through the γC/IL-2Rβ receptor response, to date classically described only in immune cells.

Severe combined immunodeficiency: From its discovery to the perspective.

Severe combined immunodeficiency (SCID) is impaired in lymphocyte development and function. Affected children have extreme susceptibility to infections, which are fatal in the first year of life without treatment. The estimate of incidence is one in approximately 50,000 live birth. The first series of diseases were described in 1950s, and all patients died in infancy. The first transplant for SCID was carried out in 1968, and it has been described that SCID patients could be treated by hematopoietic stem cell transplantation (HSCT) since then. Adenosine deaminase and common gamma chain were identified to be causative genes for SCID in 1972 and 1993, respectively. SCID arises from a variety of genetic defects. The early intervention of healthy SCID infants without infections affords higher survival rate, and newborn screening (NBS) was suggested. T-cell receptor (TCR) exicision circles (TRECs) are circular DNA formed from the leftover fragment generated from the TCR rearrangement. TRECs can be measured from a small aliquot of DNA such as Guthrie card by quantitative PCR. SCID patients lack TRECs, and TRECs quantification is useful for NBS for SCID. NBS for SCID have been already carried out in most of the Unite States, and the early introduction is desired in Japan to save SCID children.

In vitro Homeostatic Proliferation of Human CD8 T Cells.

Long-lived T-cell-mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in part by the common gamma-chain cytokines IL-7 and IL-15. To further explore the mechanisms governing maintenance of effector functions in long-lived memory T cells during antigen-independent proliferation, human naïve and memory CD8 T cells can be sorted from peripheral blood mononuclear cells (PBMCs), labeled with the proliferation-tracking dye carboxyfluorescein succinimidyl ester (CFSE), and then purified based on their levels of cell division. This allows investigators to assess differences in the desired molecular target in cells that have undergone cytokine-driven proliferation. We provide here a protocol for assessing epigenetic programs in divided and undivided human naïve and memory CD8 T cells following 7 days in culture with IL-7 and IL-15 to illustrate how this approach can shed light on the mechanism(s) that governs the preservation of effector functions during homeostasis of long-lived memory CD8 T cells.

Interleukin-12 bypasses common gamma-chain signalling in emergency natural killer cell lymphopoiesis.

Differentiation and homeostasis of natural killer (NK) cells relies on common gamma-chain (γc)-dependent cytokines, in particular IL-15. Consequently, NK cells do not develop in mice with targeted γc deletion. Herein we identify an alternative pathway of NK-cell development driven by the proinflammatory cytokine IL-12, which can occur independently of γc-signalling. In response to viral infection or upon exogenous administration, IL-12 is sufficient to elicit the emergence of a population of CD122+CD49b+ cells by targeting NK-cell precursors (NKPs) in the bone marrow (BM). We confirm the NK-cell identity of these cells by transcriptome-wide analyses and their ability to eliminate tumour cells. Rather than using the conventional pathway of NK-cell development, IL-12-driven CD122+CD49b+ cells remain confined to a NK1.1lowNKp46low stage, but differentiate into NK1.1+NKp46+ cells in the presence of γc-cytokines. Our data reveal an IL-12-driven hard-wired pathway of emergency NK-cell lymphopoiesis bypassing steady-state γc-signalling.