Chantal Leblanc

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.

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.

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.

Erratum to: Interleukin-15 plays an essential role in the pathogenesis of autoimmune diabetes in the NOD mouse

Fig. 6 Inhibition of IL-15 signalling in NOD mice decreases type 1 diabetes. (a) Female NOD mice were administered, by i.p. injection, 200 μg TM-β1 mAb (white circles, n012) or control mAb (black circles, n08) three times a week for 4 weeks (indicated by the grey bar). Injected mice were followed for diabetes development up to 7 months of age. Data from mice from two different experiments were pooled. p<0.01 (logrank test) The online version of the original article can be found at http://dx.doi.org/ 10.1007/s00125-012-2675-1.