Julien Gagnon

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.

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 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.

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.

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.

Regulation of Cytokine-Driven Functional Differentiation of CD8 T Cells by Suppressor of Cytokine Signaling 1 Controls Autoimmunity and Preserves Their Proliferative Capacity toward Foreign Antigens

We have previously shown that naive CD8 T cells exposed to IL-7 or IL-15 in the presence of IL-21 undergo Ag-independent proliferation with concomitant increase in TCR sensitivity. In this study, we examined whether CD8 T cells that accumulate in suppressor of cytokine signaling 1 (SOCS1)-deficient mice because of increased IL-15 signaling in vivo would respond to an autoantigen expressed at a very low level using a mouse model of autoimmune diabetes. In this model, P14 TCR transgenic CD8 T cells (P14 cells) adoptively transferred to rat insulin promoter-glycoprotein (RIP-GP) mice, which express the cognate Ag in the islets, do not induce diabetes unless the donor cells are stimulated by exogenous Ag. Surprisingly, SOCS1-deficient P14 cells, which expanded robustly following IL-15 stimulation, proliferated poorly in response to Ag and failed to cause diabetes in RIP-GP mice. SOCS1-deficient CD8 T cells expressing a polyclonal TCR repertoire also showed defective expansion following in vivo Ag stimulation. Notwithstanding the Ag-specific proliferation defect, SOCS1-null P14 cells produced IFN-γ and displayed potent cytolytic activity upon Ag stimulation, suggesting that SOCS1-null CD8 T cells underwent cytokine-driven functional differentiation that selectively compromised their proliferative response to Ag but not to cytokines. Cytokine-driven homeostatic expansion in lymphopenic RIP-GP mice allowed SOCS1-null, but not wild-type, P14 cells to exert their pathogenic potential even without Ag stimulation. These findings suggest that by attenuating cytokine-driven proliferation and functional differentiation, SOCS1 not only controls the pathogenicity of autoreactive cells but also preserves the ability of CD8 T cells to proliferate in response to Ags.

Increased generation of CD8 single positive cells in SOCS1-deficient thymus does not proportionately increase their export.

Mice lacking suppressor of cytokine signaling 1 (SOCS1) accumulate CD8(+) T lymphocytes in the thymus and in the periphery. Whereas IL-7 and IL-15 promote the generation of CD8 single positive (SP) thymocytes, IL-15 drives the expansion of CD8 T cells in the periphery. Here, we investigated whether increased production of CD8 SP thymocytes is accompanied by their increased export in SOCS1-deficient mice. In vivo labeling with bromodeoxyuridine showed increased cycling of CD8 SP thymocytes in SOCS1-deficient mice. However, SOCS1-deficient thymi contained increased proportion of CD24(lo)CD69(lo) SP thymocytes as well as increased expression of Qa-2 in both CD4 and CD8 SP compartments. Analysis of recent thymic emigrants (RTE) following intrathymic labeling with fluorescein isothiocyanate revealed less efficient export of CD8 RTEs from SOCS1-deficient thymi and comparable CD4:CD8 ratio among RTEs in SOCS1-null and control mice. These findings show that the rate of export of CD8 SP thymocytes is not proportional to their generation in SOCS1-deficient thymi and suggest the existence of homeostatic mechanisms controlling the egress of CD8 T cells.