超抗原SEA诱导T细胞无能的作用机制探讨

目的 探讨超抗原诱导T细胞无能的分子机制。方法 采用ELISA的FACS，分别检测超抗原对诱导T细胞无能的过程中，IL－2，IL－10的产生和IL－2Rα链（CD25）的表达，并以^3H-TdR掺入法，测定无能T细胞对rhIL-2,PMA及ionomycin的应答能力，而L－10的产生则逐渐升高；CD25的表达与活化组相比较无显著差异。rhIL-2的加入可恢复T细胞增殖。PMA单独作用能诱导无能T细胞的部分增殖能力，但PMA＋ionomycin则能更大程度地恢复T细胞的增殖。结论 超抗原SEA对T细胞无能的诱导，可能与降低IL－2的水平和升高IL－10的水平有关。超抗原SEA的反复刺激对T细胞无能的诱导，可能是干扰了TCR信号途径的近端事件，导致Ras/MAPK途径和Ca/calcineurin途径受阻，而使IL－2基因不能转录所致。     

Reversal of in vitro T cell clonal anergy by IL-2 stimulation.

Stimulation of a normal type I mouse T helper cell clone (TH1) with concanavalin A in the absence of antigen presenting cells (APC) in vitro results in the induction of a hyporesponsive state known as T cell clonal anergy. This state is characterized by a decrease in proliferation following stimulation with antigen and APC resulting from a decrease in the production of IL-2. Production of the lymphokines IL-3/granulocyte macrophage colony stimulating factor and IFN-gamma is also reduced, although to a lesser degree. Stimulation of such anergic cells with IL-2 results in proliferation and a complete reversal of the state. We demonstrate that this reversal is not due to the outgrowth of a subpopulation of cells that had escaped anergy induction, but rather occurs in all the cells. Anergy also dissipated spontaneously, although much more slowly, in the absence of T cell antigen receptor occupancy. Finally, we show that a similar state can be produced by normal activation with antigen and APC if IL-2 and other factors are removed at 16-20 h. These results indicate that the anergic state is not a permanent change in the TH1 cell. Anergy induction appears to be a consequence of the inability of the cell to divide extensively following stimulation through the antigen-specific receptor. We propose a model to explain these results in terms of a relatively stable negative regulatory factor.     

Stimulation of a shorter duration in the state of anergy by an invariant natural killer T cell agonist enhances its efficiency of protection from type 1 diabetes

We have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist α-galactosylceramide C26:0 (α-GalCer) or its T helper type 2 (Th2)-biasing derivative α-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to α-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of α-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with α-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by α-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than α-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.     

Suppressor activity of anergic T cells induced by IL-10-treated human dendritic cells: association with IL-2- and CTLA-4-dependent G1 arrest of the cell cycle regulated by p27Kip1

We have previously shown that human IL-10-treated dendritic cells (DC) induce an antigen-specific anergy in CD4+ T lymphocytes. These anergic T cells are characterized by an inhibited proliferation, a reduced production of IL-2, and additionally display antigen-specific suppressor activity. In this study we investigated the mechanisms underlying the anergic state and regulatory function of these T cells. We did not observe enhanced rates of programmed cell death of anergic CD4+ suppressor T cells compared to T cells stimulated with mature DC. Cell cycle analysis by DNA staining and Western blot experiments revealed an arrest of anergic CD4+ T suppressor cells in the G1 phase. High levels of the IL-2-dependent cyclin-dependent kinase (cdk) inhibitor p27Kip1 were found in anergic CD4+ suppressor T cells resulting in an inhibited activation of retinoblastoma protein and an arrest of cell cycle progression in the G1 phase. Addition of IL-2, but not blocking of the CTLA-4 pathway restored the proliferation of the suppressor T cells. In contrast, both treatments induced a down-regulation of p27Kip1 and acomplete inhibition of the antigen-specific regulatory function as demonstrated by high proliferation and enhanced IFN-gamma production of co-cultured T cells. Further experiments demonstrated that p27Kip-expressing regulatory CD4+CD25+ T cells did not contribute to induction of T cell anergy in this model. Our data show that regulatory function of anergic CD4+ suppressor T cells is associated with an arrest in the G1 phase of the cell cycle mediated by increased levels of the IL-2- and CTLA-4-dependent cdk inhibitor p27Kip1.     

Induction of global anergy rather than inhibitory Th2 lymphokines mediates posttrauma T cell immunodepression

Depressed mitogen-induced IL-2 and IFN-gamma responses after severe mechanical or thermal injury are postulated to result from an expansion of Th2 lymphocytes with concomitant excessive production of IL-4 and/or IL-10. Here, we simultaneously assessed proliferation and Th1 (IFN-gamma) versus Th2 (IL-10, IL-4) lymphokine production in trauma patients' isolated T cells stimulated in a costimulation sufficient, antigen presenting cell independent system (anti CD3 + anti-CD4). T cells with depressed proliferation and IL-2 production simultaneously lost IL-4, IL-10, and IFN-gamma protein and mRNA responses. Exogenous IL-12 addition did not restore IFNgamma responses, but exogenous IL-2 partially restored IL-4, IFN-gamma, and IL-10 production. Although initially partially restored by exogenous IL-2 or stimulation with PMA + ionomycin, patient T cells with persisting anergy progressively lost even these lymphokine and proliferative responses. Development of global T cell anergy was not a result of lost T cell viability or protein synthesis, since it corresponded to predominance of anergic T cells with upregulated expression of CD11b, but downregulated CD28 and CD3 expression. Thus, the subset of posttrauma patients whose isolated T cells become unresponsive experienced progressively worsening global anergy, mediated not by an increased production of Th2 lymphokines, but possibly by T cell incapacity to be activated through TCR triggering or Ca(2+) mobilization.     

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 are not essential in T cell anergy

Recent evidence suggests that the cyclin-dependent kinase (Cdk) inhibitors p27Kip1 and p21Cip1 are important factors in T cell anergy, but it has remained unclear whether anergy can be induced in their absence. We therefore induced anergy by stimulation of purified T cells from wild-type, p21Cip1-/-, and p27Kip1-/- mice with anti-CD3 antibodies. Anergic wild-type T cells were arrested in the G1 phase of the cell cycle with a high p27Kip1 protein level and low Cdk2 activity. In p27-/- and p21-/- T cells, the pattern of protein expression was preserved, but Cdk2 activity was increased. To confirm the in vivo relevance of these data, anergy was induced by repeated injection of mice with staphylococcal enterotoxin B (SEB), which leads to partial deletion of the responsive Vbeta8+ T cell population and anergy in the remaining T cells. p21-/- mice and wild-type mice reacted similarly to this treatment. p27-/- mice showed reduced deletion of SEB-responsive T cells, but persisting T cells were anergic. These data indicate that other cell cycle regulators contribute to the cell cycle arrest of anergic T cells, as neither Cdk inhibitor is required for the induction of anergy.     

The ability of antigen,but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors

The ability of the cell cycle inhibitor n-butyrate to induce T helper 1 (Th1) cell anergy is dependent upon its ability to block the cell cycle progression of activated Th1 cells in G1. Results reported here show that although both interleukin (IL)-2 and antigen (Ag) push Th1 cells into G1 where they are blocked by n-butyrate, only the Ag-activated Th1 cells demonstrate functional anergy once the n-butyrate has been removed from the culture. Because n-butyrate-induced Th1 cell anergy has been linked to increased expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, mechanistic experiments focused on the role of these inhibitors. It was found that when Th1 cells were reincubated in Ag-stimulated secondary cultures, the Th1 cells previously exposed to Ag and n-butyrate (anergic Th1 cells) demonstrated a cumulative increase in p21Cip1 and p27Kip1 when compared with Th1 cells previously exposed to recombinant (r)IL-2 and n-butyrate (non-anergic Th1 cells). p27Kip1 in the anergic Th1 cells from the secondary cultures was associated with cyclin-dependent kinases (cdks). In contrast, p21Cip1 in the anergic Th1 cells, although present at high levels, did not associate significantly with cdks, suggesting that p21Cip1 may target some other protein in the anergic Th1 cells. Taken together, these findings suggest that Th1 cell exposure to Ag and n-butyrate, rather than IL-2 and n-butyrate, is needed to induce the cumulative increase in p21Cip1 and p27Kip1 that is associated with the proliferative unresponsiveness in anergic Th1 cells. In addition, p21Cip1 may inhibit proliferation in the anergic Th1 cells by some mechanism other than suppression of cdks that is unique to the induction of Th1 cell anergy.     

Control of CD4 T cell fate by antigen re-stimulation with or without CTLA-4 engagement 24 h after priming

After two consecutive inoculations with Staphylococcus enterotoxin B (SEB) at 24 h intervals in vivo, CD4 T cells became anergic to the antigen challenge in vitro. Administration of anti-CTLA-4 mAb in conjunction with the second SEB inoculation 24 h after antigen priming interfered with anergy and CD4 T cells became T(h)2 cells. However, the anergy induction was not ablated when SEB and anti-CTLA-4 mAb were administered 48 or 72 h after antigen priming. Moreover, anti-CTLA-4 mAb without SEB did not interfere with anergy nor promoted the T(h)2 differentiation. T-antigen-presenting cell (APC) interaction in vitro in the presence of high doses of antigen and anti-CTLA-4 mAb induced a T(h)2-polarizing cytokine IL-6 and IL-10. IL-10 then down-modulated a T(h)1-polarizing cytokine IL-12. The results demonstrate that 24 h after the initial antigen stimulation, CD4 T cells enter the critical activation phase where antigen re-stimulation with or without CTLA-4 engagement alters the fate of the cell, anergy or differentiation respectively. Once anergy is interfered with, T(h)2-polarizing cytokines produced upon prolonged T-APC interaction favor the T(h)2 differentiation.     

Invariant NKT cell anergy is induced by a strong TCR-mediated signal plus co-stimulation

Vα14 TCR expressing invariant NK T (iNKT) cells recognize α-galactosylceramide (αGC)/CD1d complex and produce large amounts of various cytokines before the onset of the adaptive immunity. After stimulation with a high dose (2-5 μg) of αGC in vivo, iNKT cells in the spleen and liver become anergic in terms of the proliferation and cytokine production to subsequent stimulation. In this study, we monitor how iNKT anergy is induced. Anergized iNKT cells dramatically reduced the expression of IL-2Rα, and exogenous IL-2 restored the ability to proliferate and produce IL-4 but not to produce IFN-γ. Anergized iNKT cells expressed high levels of programmed death-1 (PD-1). However, iNKT cells in PD-1-deficient mice became anergic as a result of αGC injection, as do normal mice. Furthermore, anti-PD-1 blocking mAb was unable to restore their responsiveness. When iNKT cells were stimulated with immobilized anti-CD3 in the presence or absence of anti-CD28, they produced cytokines in a dose-dependent manner. Unlike in naive CD4 T cells, the strong TCR-mediated signaling with co-stimulation renders them anergic to any subsequent stimulation with αGC and spleen dendritic cells (DCs). Moreover, iNKT cells also became anergic after stimulation with phorbol-12-myristate-13-acetate + ionophore. Finally, the injection of αGC-pulsed DCs was more potent in inducing anergy than B cells. These results indicate that strong TCR-mediated activation with co-stimulation provides signals that induce the anergic state in iNKT cells.     

Induction of antigen-specific human CD4(+) T cell anergy by peripheral blood DC2 precursors

Dendritic cells (DC) are antigen (Ag)-presenting cells that are essential for initiation of T cell-dependent immunity, and distinct DC subsets are known to direct different classes of immune responses. DC2 precursors (pDC2) or plasmacytoid DC were recently identified as a Th2-skewing and IFN-alpha-producing human DC subset. Here, we demonstrate that pDC2 enriched from human peripheral blood have a capacity to induce an anergic state in human Ag-specific CD4(+) T cell lines. Tetanus toxoid-specific T cell lines incubated with tetanus toxoid-pulsed autologous pDC2 failed to proliferate in secondary cultures with optimal Ag stimulation. T cell anergy induction required TCR engagement with Ag/MHC complex presented on pDC2. T cells rendered anergic lost IL-2 production but produced IFN-gamma and IL-10 upon stimulation. The pDC2-induced unresponsiveness was completely or partially reversible when a high concentration of exogenous IL-2 was added in the secondary cultures. Autoreactive CD4(+) T cell clones specific for topoisomerase I derived from a patient with scleroderma were also rendered anergic after co-culture with topoisomerase I-pulsed autologous pDC2,resulting in failure to proliferate or provide help to B cells. These results suggest that pDC2 are involved in maintenance of peripheral T cell tolerance and have potential for use in the suppression of pathogenic T cell responses in autoimmune diseases and organ transplantation.     

T cell anergy and costimulation

Summary: T lymphocytes play a key role in immunity by distinguishing self from nonself peptide antigens and regulating both the cellular and humoral arms of the immune system. Acquired, antigen‐specific unresponsiveness is an important mechanism by which T cell responses to antigen are regulated in vivo. Clonal anergy is the term that describes T cell unresponsiveness at the cellular level. Anergic T cells do not proliferate or secrete interleukin (IL)‐2 in response to appropriate antigenic stimulation. However, anergic T cells express the IL‐2 receptor, and anergy can be broken by exogenous IL‐2. Anergy can be induced by submitogenic exposure to peptide antigen in the absence of a costimulatory signal provided by soluble cytokines or by interactions between costimulatory receptors on T cells and counter‐receptors on antigen‐presenting cells. The molecular events that mediate the induction and maintenance of T cell anergy are the focus of this review. The molecular consequences of CD28–B7 interaction are discussed as a model for the costimulatory signal that leads to T cell activation rather than the induction of anergy.     

CTLA-4与超抗原对SEA诱导T细胞无能的关联研究

在建立超抗原SEA诱导T细胞无能的体外模型基础上 ,观察了无能T细胞受SEA刺激时共刺激分子CD2 8和CTLA 4的表达。结果发现 ,与活化组相比 ,在SEA加入后的不同时相点无能T细胞上CD2 8的表达都是正常的 ,而CTLA 4分子在SEA加入的第 60小时细胞表面有高水平的表达。这些结果表明 ,超抗原SEA诱导的这种无能状态与CTLA 4所介导的抑制作用增强有关     

Unimpaired clearance of Mycobacterium bovis BCG infection in selectively T-cell anergic TCR-V beta 8.2 transgenic mice.

The anergy induced in mice with staphylococcal enterotoxin B (SEB) has been shown to involve selective unresponsiveness in cytokine expression. While interleukin-2 (IL-2), IL-3 and IL-4 mRNA levels are substantially reduced in anergic T cells upon restimulation with SEB, mRNA for interferon-gamma (IFN-gamma) is expressed normally. On the other hand, infection with Nippostrongylus brasiliensis is known to break an established T-cell anergy. This knowledge prompted us to examine the effect of infection with an intracellular microbe, bacillus Calmett-Guérin (BCG), on the expression of anergy induced with SEB. We have demonstrated that while the SEB-induced anergy was not abrogated by BCG infection, the V beta 8.2 transgenic mice, in which almost all T cells were anergized with SEB, were capable of developing the effective acquired protective immunity, possibly through the preserved capacity to induce IFN-gamma leading to induction of nitric oxide synthase.     

Bolus injection of aqueous antigen leads to a high density of T-cell-receptor ligand in the spleen, transient T-cell activation and anergy induction.

In vivo anergy can be modelled by administration of soluble peptide to T-cell receptor (TCR) transgenic mice specific for the moth cytochrome c peptide 88-103 (MCCp). Two weeks after initial peptide treatment, T cells were present in normal numbers but were unresponsive to antigen stimulation in vitro. Only bolus injections of peptide, either subcutaneous or intravenous, were effective at inducing tolerance, while slowly released antigen administered via mini-osmotic pump failed to result in anergy. Examination of T cells soon after bolus peptide administration revealed that anergy induction was preceded by a transient hyperactivation of T cells in vivo. Within 2 hr of peptide treatment, interleukin-2 was detectable in the plasma of the transgenic mice. Interestingly, only bolus injections of peptide led to high levels of T-cell activation, while adjuvant emulsified and pump-administered peptide resulted in very low stimulation in vivo. When the dose of bolus-injected peptide used for tolerization was titrated, the extent of anergy induction directly correlated with the intensity of early T-cell activation. Indirect measurements of TCR-ligand density on the surface of antigen-presenting cells following peptide administration revealed that aqueous peptide delivered via bolus injection generated a large number of major histocompatibility complex-peptide complexes, while pump-delivered and adjuvant-emulsified peptide did not. These data suggest that high levels of TCR ligand are required for in vivo T-cell hyperactivation and induction of anergy.     

Coculture of Th cells with interleukin (IL)-7 in the absence of antigenic stimuli induced T-cell anergy reversed by IL-15

Interleukin-7 (IL-7) is an important survival factor for T cells. We report here for the first time that it has another important role, facilitating T-cell clonal unresponsiveness, or anergy. The anergy was induced by a 20-day coculture of activated-human CD4(+) T-cell clones with IL-7 and irradiated peripheral blood mononuclear cells without antigenic stimuli. T-cell survival, but not T-cell anergy induction, was dependent on direct cell contacts between T cells and irradiated peripheral blood mononuclear cells. The anergic T cells exhibited no or very low expression of IL-7 receptor alpha chain (IL-7Ralpha), IL-2 receptor alpha chain (IL-2Ralpha), and common gamma chain (gammac), and did not express cytotoxic T-lymphocyte-associated protein 4, but expressed IL-15Ralpha. Coculture for 3 to 9 days of anergic T cells with a T-cell-activating cytokine IL-15, but not IL-2, restored the responsiveness of IL-7-induced anergic T cells together with reexpressions of IL-7Ralpha, IL-2Ralpha, and gammac. The anergy induction by IL-7 and restoration of responsiveness by IL-15 suggest novel mechanisms for regulation of helper T-cell responses, induction of peripheral tolerance, and breakdown of T-cell self-tolerance.     

Cross-linking HLA-DR molecules on Th1 cells induces anergy in association with increased level of cyclin-dependent kinase inhibitor p27(Kip1)

HLA class II molecules play pivotal roles in antigen presentation to CD4+ T cells. We investigated signaling via HLA-DR molecules expressed on CD4+ T cells. When HLA-DR or CD3 molecules on cloned CD4+ T cells were cross-linked by solid-phase mAbs, T cells proliferated, and this resulted in anergy. Whereas cross-linking of HLA-DR and CD3 resulted in secretion of the same levels of IFN-gamma and IL-8, secretion of IL-10 induced by cross-linking of HLA-DR was less than that induced by cross-linking of CD3 on CD4+ T cells. Interestingly, expression of p27(Kip1) but not p21(Cip1) increased after stimulation by either anti-HLA-DR or anti-CD3 mAb. This was indeed the case, when T cells were rendered anergic using a soluble form of antigenic peptide. In contrast, T cells stimulated by peptide-pulsed PBMC expressed little p27(Kip1). We propose that signaling via HLA-DR molecules on CD4+ T cells at least in part contributes to the induction of T cell anergy, through the upregulated expression of the p27(Kip1). The implication of our finding is that HLA-DR molecules play a role in human T cell anergy induced by a soluble form of antigenic peptide.     

Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect

The molecular basis of T cell anergy is not completely understood. We show that in antigen-primed anergic murine CD4(+) T cells the linker for activation of T cells (LAT) is hypophosphorylated upon CD3/CD28 restimulation. Signaling events downstream of LAT (PLCgamma1 phosphorylation and p85 [PI3-K] association) were impaired, whereas upstream events (CD3zeta and ZAP-70 phosphorylation) remained intact. LAT recruitment to the immunological synapse and its localization in detergent-resistant membrane (DRM) fractions were defective in anergic T cells. These defects resulted from impaired palmitoylation of LAT and were selective since the DRM localization and palmitoylation of Fyn were intact. This LAT defect was independent of Cbl-b and did not reflect enhanced LAT degradation. These results identify LAT as the most upstream target of anergy induction; moreover, they suggest that regulation of the amount of LAT in the immunological synapse and DRM by posttranslational palmitoylation contributes to the induction of T cell anergy.