Sheela Ramanathan

SOCS1 links cytokine signaling to p53 and senescence.

SOCS1 is lost in many human tumors, but its tumor suppression activities are not well understood. We report that SOCS1 is required for transcriptional activity, DNA binding, and serine 15 phosphorylation of p53 in the context of STAT5 signaling. In agreement, inactivation of SOCS1 disabled p53-dependent senescence in response to oncogenic STAT5A and radiation-induced apoptosis in T cells. In addition, SOCS1 was sufficient to induce p53-dependent senescence in fibroblasts. The mechanism of activation of p53 by SOCS1 involved a direct interaction between the SH2 domain of SOCS1 and the N-terminal transactivation domain of p53, while the C-terminal domain of SOCS1 containing the SOCS Box mediated interaction with the DNA damage-regulated kinases ATM/ATR. Also, SOCS1 colocalized with ATM at DNA damage foci induced by oncogenic STAT5A. Collectively, these results add another component to the p53 and DNA damage networks and reveal a mechanism by which SOCS1 functions as a tumor suppressor.

Impaired Post-Thymic Development of Regulatory CD4+25+ T Cells Contributes to Diabetes Pathogenesis in BB Rats

One of the BB rat diabetes (diabetes mellitus (DM)) susceptibility genes is an Ian5 mutation resulting in premature apoptosis of naive T cells. Impaired differentiation of regulatory T cells has been suggested as one possible mechanism through which this mutation contributes to antipancreatic autoimmunity. Using Ian5 congenic inbred rats (wild-type (non-lyp BB) and mutated (BB)), we assessed the development of BB regulatory CD8−4+25+T cells and their role in the pathogenesis of DM. BB rats have normal numbers of functional CD8−4+25+Foxp3+ thymocytes. The proportion of CD25+ cells among CD8−4+ recent thymic emigrants is also normal while it is increased among more mature CD8−4+ T cells. However, BB CD8−4+25+Foxp3+ thymocytes fail to undergo homeostatic expansion and survive upon transfer to nude BB rats while Foxp3 expression is reduced in mature CD8−4+25+ T cells suggesting that these cells are mostly activated cells. Consistent with this interpretation, peripheral BB CD8−4+25+ T cells do not suppress anti-TCR-mediated activation of non-lyp BB CD8−4+25− T cells but rather stimulate it. Furthermore, adoptive transfer of unfractionated T cells from diabetic BB donors induces DM in 71% of the recipients while no DM occurred when donor T cells are depleted of CD8−4+25+ cells. Adoptive transfer of 106 regulatory non-lyp BB CD8−4+25+ T cells to young BB rats protects the recipients from DM. Taken together, these results demonstrate that the BB rat Ian5 mutation alters the survival and function of regulatory CD8−4+25+ T cells at the post-thymic level, resulting in clonal expansion of diabetogenic T cells among peripheral CD8−4+25+ cells.

IL-6, in Synergy with IL-7 or IL-15, Stimulates TCR-Independent Proliferation and Functional Differentiation of CD8+ T Lymphocytes

Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8+ T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8+ T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8+ T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8+ T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8+ T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8+ T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.

Suppressor of Cytokine Signaling 1 Regulates IL-15 Receptor Signaling in CD8+CD44high Memory T Lymphocytes

T lymphocyte survival, proliferation, and death in the periphery are dependent on several cytokines. Many of these cytokines induce the expression of suppressor of cytokine signaling-1 (SOCS1), a feedback inhibitor of JAK kinases. However, it is unclear whether the cytokines that regulate T lymphocyte homeostasis are critically regulated by SOCS1 in vivo. Using SOCS1−/−IFN-γ−/− mice we show that SOCS1 deficiency causes a lymphoproliferative disorder characterized by decreased CD4/CD8 ratio due to chronic accumulation of CD8+CD44high memory phenotype T cells. SOCS1-deficient CD8+ T cells express elevated levels of IL-2Rβ, show increased proliferative response to IL-15 and IL-2 in vitro, and undergo increased bystander proliferation and vigorous homeostatic expansion in vivo. Sorted CD8+CD44high T cells from SOCS1−/−IFN-γ−/− mice respond 5 times more strongly than control cells, indicating that SOCS1 is a critical regulator of IL-15R signaling. Consistent with this idea, IL-15 stimulates sustained STAT5 phosphorylation in SOCS1-deficient CD8+ T cells. IL-15 strongly induces TNF-α production in SOCS1-deficient CD8+ T cells, indicating that SOCS1 is also a critical regulator of CD8+ T cell activation by IL-15. However, IL-15 and IL-2 induce comparable levels of Bcl-2 and Bcl-xL in SOCS1-deficient and SOCS1-sufficient CD8+ T cells, suggesting that cytokine receptor signals required for inducing proliferation and cell survival signals are not identical. These results show that SOCS1 differentially regulates common γ-chain cytokine signaling in CD8+ T cells and suggest that CD8+ T cell homeostasis is maintained by distinct mechanisms that control cytokine-mediated survival and proliferation signals.

Suppressor of Cytokine Signaling 1 Stringently Regulates Distinct Functions of IL-7 and IL-15 In Vivo during T Lymphocyte Development and Homeostasis

SOCS1−/− mice accumulate within the thymus and periphery CD8+ lymphocytes that express memory cell markers and display heightened in vitro responses to common γ-chain cytokines. To investigate whether dysregulated homeostasis of T lymphocytes and acquisition of memory phenotype by CD8+ cells in SOCS1−/− mice were mediated by IL-7 and/or IL-15 in vivo, we have generated SOCS1−/−IL-7−/−, SOCS1−/−IL-15−/− and SOCS1−/−IL-7−/−IL-15−/− mice. We observed that in mice lacking SOCS1, either IL-7 or IL-15 skewed thymocyte development toward CD8 lineage, whereas IL-15 is the principal mediator of dysregulated homeostasis in the periphery. Homeostatic proliferation of SOCS1−/− CD8+ lymphocytes in Rag1−/−, Rag1−/−IL-7−/−, Rag1−/−IL-15−/−, and Rag1−/−IL-7−/−IL-15−/− mice showed that SOCS1 deficiency did not overcome the requirement for IL-7 and IL-15 to sustain homeostatic expansion. Differential expression of memory phenotype markers CD44, CD122, and Ly6C by SOCS1−/−IL-15−/− CD8+ lymphocytes suggest that multiple signals contributed to the memory cell differentiation program. To address whether increased IL-15 responsiveness of SOCS1−/− CD8+ lymphocytes required prior TCR sensitization, we generated SOCS1−/− H-Y TCR transgenic (Tg) mice. Using female SOCS1−/− H-Y TCRtg mice in Rag1+/+ and Rag1−/− backgrounds, we show that acquisition of the memory phenotype by SOCS1-deficient CD8+ lymphocytes did not require prior antigenic stimulation, but required the presence of activated T cells. SOCS1 deficiency accelerated the maturation of CD8 single-positive thymocytes expressing Tg TCR, but did not compromise negative selection in HY-TCRtg males. Our findings illustrate distinct functions for IL-7 and IL-15 in T lymphocyte development and homeostasis, and stringent regulation of these processes by SOCS1.

SOCS1 controls liver regeneration by regulating HGF signaling in hepatocytes

BACKGROUND & AIMS Frequent repression of the Socs1 (suppressor of cytokine signaling 1) gene in hepatocellular carcinoma (HCC) and increased susceptibility of SOCS1-deficient mice to hepatocarcinogens suggest a tumor suppressor role for SOCS1 in the liver, but the underlying mechanisms remain unclear. Here we investigated the role of SOCS1 in regulating hepatocyte proliferation following partial hepatectomy and HGF stimulation. METHODS Because Socs1(-/-) mice die prematurely due to deregulated IFNγ signaling, we used Socs1(-/-)Ifng(-/-) mice to study the role of SOCS1 in liver regeneration following partial hepatectomy. We examined the activation of signaling molecules downstream of IL-6 and hepatocyte growth factor (HGF) receptors in the regenerating liver, primary hepatocytes, and in human hepatoma cells. We examined the interaction between SOCS1 and the HGF receptor c-Met by reciprocal immunoprecipitation. RESULTS Socs1(-/-)Ifng(-/-) mice displayed accelerated liver regeneration with increased DNA synthesis compared to Ifng(-/-) and wild type mice. The regenerating liver of Socs1(-/-)Ifng(-/-) mice did not show increased IL-6 signaling, but displayed earlier phosphorylation of Gab1, a signaling adaptor downstream of c-Met. Following HGF stimulation, hepatocytes from Socs1(-/-)Ifng(-/-) mice displayed increased phosphorylation of c-Met and Gab1, cell migration and proliferation. Accordingly, SOCS1 overexpression attenuated HGF-induced phosphorylation of c-Met, Gab1, and ERK1/2 in hepatoma cells, and decreased their proliferation and migration. SOCS1 interacted with the Tpr-Met, an oncogenic form of the Met receptor. CONCLUSIONS SOCS1 attenuates c-Met signaling and thus negative regulation of HGF signaling could be an important mechanism underlying the anti-tumor role of SOCS1 in the liver.

Increased antigen responsiveness of naive CD8 T cells exposed to IL-7 and IL-21 is associated with decreased CD5 expression.

Exposure of naive CD8 T cells to the synergistic combination of interleukin (IL)‐7 and IL‐21 enables them to respond strongly to subsequent antigen stimulation. Mechanisms underlying the increased antigen responsiveness of such cytokine‐primed CD8 T cells remain unknown. In this study, we showed that a brief exposure of <24 h to IL‐7 and IL‐21 is sufficient enough to sensitize naive P14 T‐cell receptor (TCR) transgenic CD8 T cells to respond to limiting quantities of antigen, resulting in increased proliferation, interferon‐γ secretion and antigen‐specific cytolytic activity. Cytokine‐induced increase in TCR responsiveness occurs even in the absence of costimulatory signals. Cytokine priming upregulates the expression of the γc chain and increases IL‐2 production after antigen stimulation, thus enhancing autocrine stimulation. Notably, cytokine priming induces a rapid and profound downmodulation of CD5, implicated in the negative regulation of TCR signaling, by induction of the transcriptional repressor E47. These findings show that increased antigen responsiveness of cytokine‐primed CD8 T cells results from the modulation of multiple cell‐surface molecules, which influence cytokine receptor and TCR signaling.

Exposure to IL-15 and IL-21 Enables Autoreactive CD8 T Cells To Respond to Weak Antigens and Cause Disease in a Mouse Model of Autoimmune Diabetes

Autoreactive CD8+ T lymphocytes play a key role in the pathogenesis of several autoimmune diseases. It is not yet well understood how autoreactive CD8+ T cells, which express TCRs with low reactivity toward self-Ags, gain the ability to respond to autoantigens to cause disease. Previously, we have shown that prior stimulation of CD8+ T cells with synergistic combinations of cytokines produced by the innate immune response, such as IL-21 and IL-15, induces Ag-independent proliferation. Such “cytokine-primed” CD8 T cells displayed increased responsiveness to limiting quantities of the cognate Ag. In this paper, we report that prior stimulation with IL-15 and IL-21 also enables CD8+ T cells to respond to weakly agonistic TCR ligands, resulting in proliferation, cytokine secretion, and cytolytic activity. Using a transgenic mouse model of autoimmune diabetes, we show that cytokine-primed autoreactive CD8+ T cells induce disease following stimulation by weak TCR ligands, but their diabetogenic potential is dependent on continuous availability of IL-15 in vivo. These findings suggest that inflammatory cytokines could facilitate the triggering of autoreactive CD8+ T cells by weak autoantigens, and this mechanism may have important implications for autoimmune diseases associated with microbial infections and chronic inflammation.

Antigen-nonspecific activation of CD8+ T lymphocytes by cytokines: relevance to immunity, autoimmunity, and cancer

Development of T lymphocytes and their survival in the periphery are dependent on signals emanating from cytokine receptors as well as the T cell antigen receptor (TCR). These two signaling pathways play distinct and complementary roles at various stages of T cell development, maturation, survival, activation and differentiation. During immune response to foreign antigens initiated by TCR signaling, cytokines play a key role in the expansion of activated T cells. Even though the initial activation of T cells occurs via the TCR, this requirement can be overcome under certain circumstances. During lymphopenia, cytokines trigger memory CD8+ T cells to undergo antigen non-specific homeostatic expansion, whereas naïve CD8+ T cells require both cytokines and TCR signaling. Recent reports show certain combinations of cytokines can induce proliferation and effector functions of naïve CD8+ T cells without concomitant stimulation via the TCR. While such antigen non-specific stimulation of naïve T cells might significantly boost the adaptive immune response, it could also have an undesirable effect of triggering potentially autoreactive cells. Understanding the mechanisms and the regulation of cytokine-driven stimulation of naïve CD8+ T cells may lead to novel strategies of intervention for autoimmune diseases. On the other hand, in vitro expansion of naïve CD8+ T cells by certain combinations of cytokines could be used to generate tumor-specific cells with ideal properties for cellular immunotherapy of cancer.

Excessive production of nitric oxide by macrophages from DP-BB rats is secondary to the T-lymphopenic state of these animals.

Activated macrophages are the first mononuclear cells to migrate to the pancreas of DP-BB rats at the initiation of insulitis. These cells produce an excess of NO, which has been implicated as a mediator of both (β-cell damage and inhibition of T-cell proliferation in this rat strain. Genetic studies have shown that the impaired proliferative response of T-cells segregates with one of the diabetes-susceptibility genes of the DP-BB rat, lyp, which is responsible for a peripheral T-lymphopenia. This observation suggests that the dysregulated expression of inducible NO synthase (iNOS) is under the control of lyp itself or a gene in linkage disequilibrium with lyp. Using two models of hemopoietic chimeras—DP-BB rats reconstituted with isocongenic Tcells and irradiated (WF × DP-BB)F1 animals reconstituted with bone marrow of both parental strains—we demonstrated that the production of NO by DP-BB macrophages is normal when these cells originate from a non–T-lymphopenic environment. Consequently, these macrophages no longer inhibit the stimulation of DNA synthesis in activated T-cells. Macrophages of young WF rats were found to produce high levels of NO, which inhibited T-cell proliferation in vitro. This observation strongly suggests that upregulation of NO synthesis in DP-BB macrophages represents the abnormal persistence of a phenomenon restricted to the first few weeks of life in non-diabetes-prone rats. Taken together, these results demonstrate that the elevated production of NO by DP-BB macrophages results from the lyp mutation and represents a crucial mechanism through which T-lymphopenia contributes to the development of diabetes.