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Division of Immunology, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, CanadaCentre de Recherche Clinique Etienne-Le Bel, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Québec, Canada
Division of Immunology, Department of Pediatrics, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 North 12th Avenue, Sherbrooke Quebec J1H 5N4, Canada.
Division of Immunology, Department of Pediatrics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, CanadaCentre de Recherche Clinique Etienne-Le Bel, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Québec, Canada
Suppressor of cytokine signaling 1 (SOCS1) is a critical regulator of T lymphocyte homeostasis. SOCS1-deficient mice accumulate CD8+ T cells, which display a memory-like phenotype and proliferate strongly to IL-15. Socs1−/- mice develop inflammatory skin lesions, however, the underlying mechanisms are not well understood. In order to investigate the role of SOCS1 in regulating CD8+ T cells potentially reactive to tissue antigens (Ags) of the skin, we generated Socs1−/- mice expressing MHC-I–restricted Pmel-1 transgenic TCR specific to the melanoma-derived gp100 Ag, which is also expressed by normal melanocytes. Socs1−/- Pmel-1 cells express increased levels of memory markers CD44, Ly6C, CD122, and CD62L, and show downregulation of TCR and upregulation of CD5, suggesting in vivo TCR stimulation. However, stimulation of Socs1−/-Pmel-1 cells with gp100-derived peptide induced only marginal proliferation in vitro despite eliciting strong effector functions, which was associated with elevated Blimp-1 induction. Following adoptive transfer to Rag1−/- mice, Socs1−/-Pmel-1 cells underwent lymphopenia-induced proliferation and caused severe skin pathology characterized by inflammatory lesions in ears, muzzle, extremities, and eyes. These findings underscore the importance of SOCS1 in regulating potentially skin-reactive cytotoxic T lymphocytes, which could get activated under conditions that promote Ag-nonspecific, cytokine-driven proliferation.
). This study showed that SOCS1 deficiency downmodulated the chemokine receptor CCR7 in CD4+ T lymphocytes, allowing them to migrate to peripheral tissues and cause inflammation. However, mechanisms underlying the initiation of inflammatory skin lesions in Socs1−/- mice have not yet been fully elucidated.
SOCS1 is essential to maintain T lymphocyte homeostasis, which is critically dependent on IL-7 and IL-15 (
). For survival, naive CD8+ T cells requires IL-7 as well as self-peptide–MHC-I complexes that provide basal TCR signaling, whereas memory CD8+ T cells require IL-15 but not TCR stimulation (
Self-class I MHC molecules support survival of naive CD8 T cells, but depress their functional sensitivity through regulation of CD8 expression levels.
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.
). Socs1−/- CD8+ T cells bearing the male Ag-specific H-Y transgenic TCR, which shows minimal reactivity toward environmental Ag, also display a memory-like phenotype (
). These observations allowed us to postulate that SOCS1 deficiency may enable naive CD8+ T cells to respond to minimally cross-reactive environmental Ag or self-peptide–MHC-I complexes. Similar activation of potentially self-reactive CD8+ T lymphocyte clones in Socs1−/- mice bearing a polyclonal TCR repertoire could initiate and perpetuate the inflammatory processes, leading to chronic skin lesions.
To test the above hypothesis, we generated Socs1−/- mice expressing transgenic Pmel-1 TCR. The Pmel-1 TCR is reactive to the human melanoma Ag gp100 (hgp100), which is homologous to mouse pmel-17 (mgp100) expressed by normal melanocytes (
). The Pmel-1 TCR does not cause any pathology in mice, indicating that the antigenic reactivity of Pmel-1 TCR transgenic CD8+ T cells (Pmel-1 cells) is efficiently controlled by immune tolerance mechanisms (
We have recently shown that combinations of inflammatory and homeostatic cytokines, for instance IL-15 and IL-21, can synergistically induce Ag-nonspecific proliferation of naive CD8+ T cells that is accompanied by increased sensitivity toward cognate Ag and weak TCR ligands (
) would allow Ag-nonspecific activation of Pmel-1 cells in vivo by endogenous cytokines and enhance their reactivity toward mgp100 expressed by normal melanocytes. Our findings indeed support this notion and demonstrate a critical role for SOCS1 in preventing CD8+ T cells that possess minimal reactivity toward skin tissue Ag from gaining autoaggressive potential.
Results
SOCS1-deficient Pmel-1 cells display signs of in vivo Ag stimulation
), Socs1−/-Pmel-1 mice became sick early and died by 6 weeks of age. These mice showed retarded growth and often developed white spots around eyes, ears, and in extremities (Figure 1a). In both control and Socs1−/-Pmel-1 mice, most CD8+ T cells expressed the Vβ13-containing transgenic TCR (Figure 1b). Socs1−/-Pmel-1 cells showed increased expression of CD44, CD122, and Ly6C (Figure 1c), a phenotype similar to memory-like cells arising from lymphopenia-induced proliferation (LIP;
). Interestingly, these cells showed elevated levels of CD5 (Figure 1d), a negative regulator of TCR signaling that is downmodulated by cytokine stimulation (
). However, upregulation of CD69 or downmodulation of CD62L, the hallmarks of acute TCR stimulation, was not evident in Socs1−/-Pmel-1 cells (Figure 1e). Accordingly, stimulation of purified Pmel-1 cells via TCR cross-linking showed marginal or negligible decrease in protein tyrosine phosphorylation, activation of the key signaling protein LAT, or induction of calcium flux response in SOCS1-deficient cells compared with control cells (Supplementary Figure S1 online). These results indicate that Socs1−/- Pmel-1 cells not only display cytokine-induced upregulation of memory cell markers, but also show altered expression of cell surface molecules that are modulated by TCR activation without displaying acute changes of TCR stimulation.
Figure 1Socs1−/-Pmel-1 cells display signs ofin vivoantigen stimulation. (a) Appearance of 4-week-old Socs1−/-Pmel-1 and Socs1+/+ Pmel-1 control mice. (b–e) Pooled brachial, inguinal, cervical, and mesenteric lymph nodes from 3-week-old mice of the indicated genotype from the same litter were stained for CD4, CD8, TCR Vβ13 (b) and the indicated memory cell markers (c) or molecules that are modulated following antigen stimulation (d and e), and evaluated by flow cytometry. Numbers within quadrants of dot plots indicate the proportion of cells. Numbers within histograms denote mean channel values. Dotted lines were placed in histograms for visual comparison. Data shown are representative of similar results from more than three mice from different litters. SOCS1, suppressor of cytokine signaling 1.
). To determine whether the pathological lesions and premature death of Socs1−/-Pmel-1 mice resulted from increased Ag responsiveness of Pmel-1 cells to endogenous melanocyte gp100, we stimulated lymph node cells from control and Socs1−/-Pmel-1 mice with mgp10025–33 peptide. We observed that SOCS1-deficient cells proliferated poorly (Figure 2a, left panel), which was not due to impaired Ag presentation as purified Socs1−/-Pmel-1 cells stimulated with the same peptide presented by irradiated wild-type splenocytes also showed reduced proliferation (Figure 2a, right panel). In both instances, exogenous IL-2 reversed the defective Ag-induced proliferation of Socs1−/-Pmel-1 cells (Figure 2a). Previous reports have shown that Pmel-1 cells respond more strongly to hgp10025–33 (KVPRNQDWL) than to mgp10025–33 (EGSRNQDWL), as the former binds H-2Db with greater affinity (
). Proliferation induced by hgp10025–33 was also impaired in Socs1−/-Pmel-1 cells, which was reversed by IL-2 (Figure 2b). Furthermore, Socs1−/-Pmel-1 cells responded poorly to immobilized anti-CD3/anti-CD28 antibodies (Figure 2c, left panel). Nonetheless, Socs1−/-Pmel-1 cells proliferated robustly to IL-15 or IL-15 plus IL-21 (Figure 2c, right panel).
Figure 2Socs1−/-CD8+ T cells show impaired antigen (Ag)-induced proliferation. (a) Lymph node cells (left panel) from 2- to 3-week-old Socs1−/- and control Pmel-1 mice were stimulated with mgp10025–33 with or without IL-2. Purified CD8+ T cells (right panel) were stimulated with mgp10025–33 presented by irradiated C57BL/6 splenocytes. (b, c) Lymph node cells were stimulated with hgp10025–33 with or without IL-2, anti-CD3ε, and anti-CD28 mAb-coated Dynabeads or cytokines. (d) Lymph node cells from female Socs1−/- or control Rag1−/-H-Y TCR transgenic mice were stimulated with H-Y peptide or cytokines. Cell proliferation was evaluated by [3H]-thymidine incorporation after 2 days (for Ag) or 3 days (cytokines) stimulation. Representative data from at least three independent experiments are shown. c.p.m., counts per minute; SOCS1, suppressor of cytokine signaling 1.
TCR reactivity toward MHC–self-peptide complexes may underlie the defective Ag-induced proliferation of Socs1−/- CD8+ T cells
We have previously shown that Socs1−/- CD8+ T cells expressing the P14 transgenic TCR, specific to a viral Ag, also displayed impaired Ag-induced proliferation (
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.
), it seems paradoxical that Socs1−/-Pmel-1 and Socs1−/-P14 cells, which are likely to have been exposed to abundant inflammatory cytokines in vivo, show impaired Ag-induced proliferation. Ag-stimulated CD8+ T cells can become transiently refractory to TCR stimulation due to “Ag-induced non-responsiveness” (
). Hence, it is possible that the decreased Ag responsiveness of Socs1−/- CD8+ T cells may be a consequence of increased Ag responsiveness resulting from persistent exposure to cognate self (mgp100) or cross-reactive environmental Ag. Alternately, Socs1−/- CD8+ T cells may respond strongly to self-peptide–MHC-I complexes needed for naive T cell survival (
), leading to refractoriness toward subsequent TCR stimulation. To investigate these possibilities, we examined Ag responsiveness of Socs1−/- H-Y TCR transgenic CD8+ T cells reactive to the male-specific H-Y Ag (
). The H-Y TCR displays low affinity toward its cognate peptide and shows negligible reactivity to environmental Ag. As shown in Figure 2d, Socs1−/-H-Y cells proliferated poorly to the cognate peptide despite robust proliferation to cytokines. These results indicate that increased cytokine responsiveness of Socs1−/- CD8+ T cells may augment their functional avidity toward MHC-I–self-peptide complexes that may attenuate their proliferation to subsequent Ag stimulation, irrespective of their TCR affinity toward cognate peptides.
Socs1−/-Pmel-1 cells show increased Ag-specific cytolytic activity
CD8+ T cells that develop Ag-induced non-responsiveness lose their ability to proliferate to Ag because of impaired IL-2 production but retain their effector functions (
). Therefore, we examined Ag-induced cytotoxic T lymphocyte (CTL) activity of Socs1−/-Pmel-1 cells. As shown in Figure 3a (upper panel), Socs1−/-Pmel-1 cells stimulated with mgp10025-33, but not unstimulated cells, efficiently lysed mgp10025–33-loaded EL-4 targets. These cells did not lyse target cells loaded with a null peptide (SGPSNTPPEI), which binds to H-2Db (
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.
). Control Pmel-1 cells showed minimal lytic activity toward mgp10025–33-loaded targets, whereas the same cells efficiently lysed hgp10025–33-loaded targets (Figure 3a, upper and lower panels). Socs1−/-Pmel-1 cells also showed increased lytic activity toward hgp10025–33-loaded targets. These results indicate that SOCS1 deficiency drives Pmel-1 cells into a state of Ag-specific proliferative unresponsiveness without compromising their effector functions.
Figure 3Socs1−/-Pmel-1 cells display increased antigen (Ag)-specific cytolytic activity after Ag or cytokine stimulation. (a) Lymph node cells from control and Socs1−/- Pmel-1 mice were stimulated with 1μgml−1 of mouse (upper panel) or human (lower panel) gp100-derived peptide. After 2 days, stimulated cells were equalized for CD8+ T cell numbers, and incubated with 51Cr-loaded EL4 target cells that were pulsed with hgp10025–33 peptide to measure cytotoxic T lymphocyte (CTL) activity. Freshly isolated cells were used as controls. (b) Lymph node cells from Socs1−/- Pmel-1 mice were stimulated with IL-15 and IL-21, either alone or together for 36hours, and equivalent numbers of CD8 cells were tested for CTL activity against hgp100 peptide–loaded EL4 targets. Representative data from two independent experiments with similar results are shown. SOCS1, suppressor of cytokine signaling 1.
Socs1−/-Pmel-1 cells become pathogenic following LIP
Intriguingly, Socs1−/-Pmel-1 cells stimulated with IL-15 and IL-21 displayed high cytolytic activity as Ag-stimulated cells (Figure 3b). As these cells proliferate robustly to these cytokines (Figure 2c, right panel), we postulated that Socs1−/-Pmel-1 cells might become pathogenic under lymphopenia conditions, when CD8+ T cells undergo cytokine-driven homeostatic proliferation. To test this hypothesis, we labeled splenocytes from control or Socs1−/-Pmel-1 mice with 5-(6)carboxyfluorescein diacetate succinimidyl ester (CFSE), and adoptively transferred these cells into Rag1−/- recipients. More than 98% of CD8+ T cells recovered from the recipients of either control or Socs1−/-Pmel-1 cells expressed the transgenic TCR (Figure 4a). Socs1−/- CD8+ T cells underwent pronounced LIP in Rag1−/- mice within 5 days after cell transfer, whereas Pmel-1 cells from wild-type donors underwent limited expansion (Figure 4a). Socs1−/-Pmel-1 cells showed increased expression of CD44 and Ly6C, whereas CD62L and CD69 levels were comparable between Socs1−/- and control cells (Figure 4b). Furthermore, both control and Socs1−/- Pmel-1 cells underwent limited expansion in Rag1−/-Il15−/- mice, confirming the requirement of IL-15 for homeostatic expansion (Figure 4c). However, Socs1−/-Pmel-1 cells expanded in Rag1−/-Il15−/- recipients contained a pool of rapidly proliferating CFSElo cells, which may arise from TCR stimulation (
Figure 4Socs1−/-Pmel-1 cells undergo massive lymphopenia-induced proliferation. (a) Lymph node cells from Socs1−/- and control Pmel-1 cells were labeled with 5-(6)carboxyfluorescein diacetate succinimidyl ester (CFSE) and 10 × 106 cells were adoptively transferred to Rag1−/- mice. After 5 days, pooled lymph node cells were stained for CD8, and proliferation of CD8+ T cells was evaluated by flow cytometry. (b) In parallel, markers of lymphopenia-induced proliferation were evaluated on CD8+ T cells. (c) Rag1−/- mice lacking IL-15 were used as recipients of CFSE-labeled Socs1−/- and control Pmel-1 cells, and proliferation of donor CD8+ T cells was evaluated after 5 days. Representative data from three independent experiments are shown. SOCS1, suppressor of cytokine signaling 1.
Rag1−/- recipients that received Socs1−/-Pmel-1 cells developed severe inflammatory skin lesions 2 months after cell transfer, whereas wild-type Pmel-1 cells failed to cause disease (Figure 5a). Somewhat unexpectedly, Rag1−/-Il15−/- recipients harboring Socs1−/-Pmel-1 cells also developed lesions, albeit with a delay (Figure 5a). Rag1−/- recipients harboring Socs1−/- Pmel-1 cells developed lesions in areas accessible to scratching, particularly in flanks, ears, muzzle, and eyes (Figure 5b A, B; Supplementary Figure S2 online: i, v). Histology of the affected muzzle skin showed moderate-to-severe lymphocytic infiltration of dermis and hair follicles (Figure 5b, C, D vs. E). Cross-section of ear lobes from Rag1−/- mice harboring Socs1−/-Pmel-1 cells showed acanthosis, infiltration of lymphocytes and melanophages, basal cell vacuolization, and reactive keratinocyte atypia, resembling lichenoid-type interface dermatitis (Supplementary Figure S2 online: ii, iii vs. iv). Some very sick mice developed cataract and showed lymphocytic infiltration of peri-orbital and sub-conjunctival tissues, with melanophages arising from the underlying choroid (Supplementary Figure S2 online: vi, vii). These observations show that reversal of the Ag-specific proliferative defect by lymphopenia-driven expansion allows Socs1−/-Pmel-1 cells to unleash their effector functions and cause autoimmune tissue destruction.
Figure 5Socs1−/-Pmel-1 cells cause severe skin lesions following lymphopenia-induced proliferation. (a) Lymph node cells containing 5 × 106 CD8+ T cells from Socs1−/- or control Pmel-1 mice were adoptively transferred to Rag1−/- or Rag1−/-Il15−/- recipients and monitored for pathological manifestations. (b) (A) Representative Rag1−/- mice that received Socs1−/- (knockout (KO)) or control (wild type (WT)) Pmel-1 cells at 8 weeks after cell transfer. (B–D) Inflammatory lesions of the muzzle in Rag1−/- mice harboring KO cells (B), showing lymphocytic and histiocytic infiltration in the dermis (C, between braces) with unaffected epidermis (C, arrowhead) and vibrissae (C, arrow), and dense lymphocytic and histiocytic infiltration around hair follicles (D). (E) Skin section of Rag1−/- mice harboring control Pmel-1 cells. SOCS1, suppressor of cytokine signaling 1.
Ag stimulation strongly induces Blimp-1 in Socs1−/-Pmel-1 cells
To gain insight into the ability of Socs1−/-Pmel-1 cells to cause skin lesion in Rag1−/- recipients, we phenotyped the adoptively transferred Pmel-1 cells at 6 weeks after transfer. Both wild-type and Socs1−/-Pmel-1 cells undergoing LIP have upregulated CD44, Ly6C, and CD127 to the same extent, whereas the expression of CD25, CD132, and CCR7 showed negligible changes (Figure 6a). These cells have also upregulated CD5 and KLRG-1, which are modulated by cytokine and Ag stimulation (
). A subset of these cells downregulated CD62L (Figure 6a), suggesting TCR stimulation in vivo. Overall, there was no appreciable phenotypic difference between Socs1−/- and control Pmel-1 cells recovered from Rag1−/- mice 2 months after adoptive transfer. Recent studies have shown that Blimp-1, a transcriptional repressor induced by Ag stimulation, favors differentiation of effector CD8+ T cells during viral infections (
). We observed that Ag stimulation markedly upregulated Blimp-1 in Socs1−/- Pmel-1 cells compared with control cells (Figure 6b). Ag-stimulated SOCS1-deficient cells also expressed Bcl-6, which is implicated in promoting cell proliferation (
). IL-15 and IL-21, which induced strong proliferation of Socs1−/-Pmel-1 cells, did not upregulate Blimp-1, whereas IL-21–mediated induction of Bcl-6 was comparable in Socs1−/- and control Pmel-1 cells (Figure 6b). Besides, IL-15 inhibited IL-21–induced Bcl-6 in both Socs1−/- and control Pmel-1 cells. These results suggest that cytokine-driven proliferation, increased Ag sensitivity, and Ag-induced upregulation of Blimp-1 may collectively contribute to the autoaggressive potential of Socs1−/-Pmel-1 cells.
Figure 6Socs1−/-Pmel-1 cells show increased Blimp-1 expression after antigen stimulation. (a) Socs1−/- and control Pmel-1 cells recovered 6 weeks after transfer to Rag1−/- recipients were evaluated for the indicated cell surface markers. Freshly isolated control Pmel-1 cells were included for comparison. (b) Freshly isolated Socs1−/- and control Pmel-1 cells were stimulated with antigen or the indicated cytokines. After 48hours, cell lystaes were analyzed by western blot to evaluate Blimp-1 expression. Actin was used to ensure equivalent protein loading. Data shown are representative of two independent experiments with similar results. SOCS1, suppressor of cytokine signaling 1.
In this study, we have shown that SOCS1 has a crucial role in preventing the activation of potentially skin-reactive CD8+ T cells. Socs1−/-Pmel-1 cells undergo robust cytokine-induced proliferation but proliferate poorly to gp100. This Ag-specific proliferative unresponsiveness of Socs1−/-Pmel-1 cells presumably develops as a consequence of constant TCR stimulation by endogenous gp100 expressed by normal melanocytes. Nonetheless, Socs1−/-Pmel-1 cells display strong Ag-specific effector functions, which enables these cells to cause severe skin lesions under lymphopenic conditions that favor cytokine-driven homeostatic expansion. Our findings also implicate SOCS1 in regulating the Blimp-1, an important regulator of CD8+ T-cell differentiation.
Pmel-1 cells are widely used to study antitumor CTL responses in the murine B16-F10 melanoma model (
). Pmel-1 cells expanded in vitro using hgp10025–33 and adoptively transferred to C57Bl/6 mice induce regression of subcutaneously implanted melanoma. However, such tumor regression can be achieved only upon re-stimulation of the donor cells in vivo with hgp10025–33 along with innate immune stimuli (
Recovery from cyclophosphamide-induced lymphopenia results in expansion of immature dendritic cells which can mediate enhanced prime-boost vaccination antitumor responses in vivo when stimulated with the TLR3 agonist poly(I:C).
The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD8+ T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu.
). Effective antitumor response against B16-F10 melanoma is often accompanied by vitiligo, indicating that normal melanocytes become targets of cytokine-stimulated, Ag-activated Pmel-1 cells. Our findings show that SOCS1 deficiency enables Pmel-1 cells to acquire the capacity to attack normal cells even without exogenous Ag stimulation, and at physiological levels of cytokines available during lymphopenia. These observations highlight the key role played by SOCS1 in preventing potentially autoreactive CD8+ T-cell clones from gaining the capacity to become auto-aggressive CTLs, leading to initiation and perpetuation of autoimmune tissue destruction.
Development of inflammatory lesions in multiple organs of SOCS1-deficient mice has been variously attributed to hyperactivation of NKT cells, macrophages, and dendritic cells, and deregulation of Th1, Th17, and T regulatory cells (
). Even though all these inflammatory cells would contribute to the chronic inflammatory lesions observed in SOCS1 null mice, it is also possible that inflammatory cytokines could facilitate activation of potentially self-reactive T cells (
have shown that SOCS1 deficiency in T cells alone is not sufficient to induce inflammatory manifestations, which occurred only upon simultaneous deletion of SOCS1 in myeloid cells. This study postulated that increased sensitivity of Socs1−/- macrophages to inflammatory stimuli and abnormally high responsiveness of Socs1−/- T cells to pro-inflammatory cytokines could establish a self-perpetuating, Ag-nonspecific inflammatory loop between these cells. CD8+ T cells are the most affected leukocyte population in Socs1−/- mice, characterized by CD44hi-activated/memory phenotype, which occurs even in the absence of IFN-γ (
). We have shown that this phenotypic conversion is driven by IL-15, and SOCS1 attenuates Ag-nonspecific activation of CD8+ T cells by IL-15 and IL-21 (
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.
). Even though IFN-γ hastens inflammation, leading to overt disease in Socs1−/- mice at a very young age, development of inflammatory lesions in older Socs1−/-Ifng−/- mice indicates that IFN-γ is dispensable to initiate and perpetuate the inflammatory loop in SOCS1-deficient mice. Our findings show that Socs1−/- Pmel-1 cells display potent Ag-specific cytolytic activity following cytokine or Ag stimulation, which indicate that increased CTL activity could be a key factor contributing to the development of autoimmune manifestations in Socs1−/- mice. This notion is also supported by the destruction of pancreatic islets by Socs1−/- CD8+ T cells in TCR transgenic mouse models of autoimmune diabetes (
Even though Socs1−/- Pmel-1 cells mount efficient Ag-specific CTL activity, they display reduced Ag-induced proliferation, as we have previously reported for P14 cells (
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.
). In our previous study on Socs1−/-P14 cells, we have argued that the Ag-specific proliferative unresponsiveness of Socs1−/- CD8+ T cells is not related to “split anergy” (
) on the grounds that Socs1−/- CD8+ T cells express normal levels of costimulatory receptors and their proliferative defect cannot be reversed even after cytokine-driven expansion (
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.
). Our findings indicate that the Ag-specific proliferative defect in Socs1−/- Pmel-1 cells, without the loss of effector functions, could be related to the increased level of Blimp-1 expression. It has been shown that Blimp-1 is upregulated in terminally differentiated CD8+ T effector cells, whereas central memory cells downmodulate Blimp-1 (
). Accordingly, Blimp-1 deficiency leads to the generation of memory CD8+ T cell subsets with increased proliferative capacity, whereas overexpression of Blimp-1 in P14 cells attenuated Ag-induced proliferation (
). Further investigation will reveal how SOCS1 regulates Ag-induced Blimp-1 expression, and whether SOCS1 deficiency also modulates the signaling pathways (NFAT, mTOR, AMPK, DGKα) and expression of genes (Cbl-b, GRAIL, Otubain1, Ikaros, Egr2/3, CREM) implicated in the induction and/or maintenance of the anergic state (
Autoreactive T cells that escape negative selection in thymus are regulated by peripheral tolerance mechanisms, which include “ignorance” due to tissue-restricted expression of autoantigens, induction of tolerogenic Ag-presenting cells and regulatory T cells, and clonal anergy and deletion of autoreactive cells (
). Inflammatory conditions can breach these safety mechanisms by enhancing the immunogenic properties of Ag-presenting cells. For instance, in transgenic mice expressing ovalbumin-derived peptide in skin, adoptive transfer of ovalubumin-specific OT-I TCR transgenic CD8+ T cells did not cause immunopathology unless physical skin inflammation was also induced (
). Collectively, these studies suggest that autoreative CD8+ T cells with low-avidity TCR can be activated under inflammatory conditions. Therefore, the inflammatory status of Socs1−/- mice might have facilitated activation of Pmel-1 cells, however, breakdown of peripheral tolerance and development of tissue damage required a lymphopenic setting.
Lymphopenia and infections are implicated as important triggers of autoimmunity (
). In fact, lymphopenia and inflammation may collude to trigger autoimmunity. We have shown that homeostatic cytokines synergize with inflammatory cytokines to stimulate Ag-nonspecific proliferation of naive CD8+ T cells, increase their responsiveness to weak TCR ligands and elicit their pathogenic potential (
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.
) and by the present study on Pmel-1 cells. Even though the Socs1 gene has not been directly linked to autoimmunity, it is epigenetically repressed via promoter methylation and by microRNA miR155 (
). It will be worthwhile to investigate the methylation status of Socs1 gene promoter and miR155 expression in clonal populations of CD8+ T cells in autoimmune disease patients.
Materials and Methods
Mice
Socs1+/-Ifng−/-, Rag1−/-Il15−/-, Rag1−/-H-Ytg and Socs1−/-Rag1−/-H-Ytg mice in C57BL/6 background had been previously described (
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.
) were purchased from the Jackson Laboratory (Bar Harbor, ME). Socs1−/-Pmel-1 mice were generated in our animal facility. The Université de Sherbrooke Ethics Committee on Care and Use of Animals has approved all experiments using mice.
Reagents
Fluorochrome-conjugated antibodies against mouse cell surface molecules TCR vβ13 (recognizes the Pmel-1 TCR) were from BD Pharmingen Biosciences (Palo Alto, CA) or eBiosciences (San Diego, CA). Recombinant IL-2, IL-7, IL-15, and IL-21 were from R&D Systems (Minneapolis, MN). CFSE and the calcium indicator dye Fluo-4 were from Molecular Probes (Eugene, OR). Antigenic peptides were custom synthesized to >95% purity by GenScript (Scotch Plains, NJ). Antibodies for western blot were purchased from Cell Signaling Technology (Beverly, MA) or Santa Cruz Biotechnology (Santa Cruz, CA).
Flow cytometry and cell sorting
Cell staining, flow cytometry data acquisition, and analysis were carried out as described elsewhere (
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.
). Briefly, total lymph node cells (1 × 105cells per well) or purified CD8+ T cells (2.5 × 104cells per well) were stimulated in 96-well culture plates with cytokines, Dynabeads Mouse T-activator CD3/CD28 (Life Technologies), or antigenic peptides. Irradiated wild-type splenocytes (105cells per well) were used as Ag-presenting cells to stimulate purified cells with Ag. One μCi of methyl-[3H]-thymidine (NEN Life Sciences, Boston, MA) was added during the last 8hours of culture and radioactivity incorporation was measured. Statistical significance was calculated by Student’s t-test.
In vivo proliferation
Splenocytes labeled with CFSE were injected into 6- to 8-week-old Rag1−/- or Rag1−/-Il15−/- mice (10 × 106 cells in 200μl phosphate-buffered saline) and analyzed as described elsewhere (
Purified 1 × 106 CD8+ T cells were incubated with anti-CD3 mAb 2C11 in cold for 15minutes, washed, thawed to 37°C, and exposed to goat anti-hamster IgG (Jackson Laboratory) to cross-link the CD3–TCR complex. At indicated time points, cells were lysed in SDS-PAGE sample buffer and analyzed by western blot as described previously (
). To measure TCR-induced calcium flux, purified CD8+ T cells, loaded with the calcium indicator dye Fluo-4, were stimulated using 2C11 Ab followed by its cross-linking. Ca2+ flux was recorded by flow cytometry as detailed elsewhere (
Target EL-4 cells, prepared by incubation with 400μCiml−1 of 51Cr (NEN Life Sciences) and peptides for 2hours at 37°C, were washed and cultured with activated Pmel-1 cells at different effector to target cell ratios. After 7hours at 37°C, released radioactivity was measured and specific lysis was calculated as previously described (
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.
Recovery from cyclophosphamide-induced lymphopenia results in expansion of immature dendritic cells which can mediate enhanced prime-boost vaccination antitumor responses in vivo when stimulated with the TLR3 agonist poly(I:C).
The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD8+ T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu.
Self-class I MHC molecules support survival of naive CD8 T cells, but depress their functional sensitivity through regulation of CD8 expression levels.
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