Anti-Vbeta8 antibodies induce and maintain staphylococcal enterotoxin B-triggered Vbeta8+ T cell anergy

The mechanism involved in the maintenance of staphylococcal enterotoxin B (SEB)-induced T cell anergy is poorly understood. We demonstrated earlier that B cells play an important role in the maintenance of SEB-induced T cell anergy in vivo and in vitro. Here, we demonstrate that B cells are not essential in SEB-induced T cell activation, but are important for the maintenance of T cell memory phenotype and anergy in vivo. Studying the activated B cell repertoire, we observe that SEB treatment increases serum anti-Vbeta8 antibody titer as detected by enzyme-linked immunosorbent assay using soluble Vbeta8 chains as antigens, and by staining of a Vbeta8-expressing thymoma. These antibodies disappear gradually after immunization with SEB, whereas the capacity of the T cells to respond to SEB in vitro is restored. Anti-Vbeta8 monoclonal antibody treatment causes Vbeta8+ T cell unresponsiveness to SEB in vitro (anergy), without affecting CD4Vbeta8+ T cell frequency. Together, these results suggest a new mechanism to explain the maintenance of SEB-induced T cell anergy, which is dependent on B cells and on anti-Vbeta8 antibody that specifically interacts with Vbeta8+ T cells.     

SEB诱导的CD4+ T细胞无能、凋亡及MHC-I类分子表达下调

目的：探讨超抗原金黄色葡萄球菌肠毒素（SEB）体外诱导外周T细胞免疫耐受的作用机制。方法：采用SEB体外刺激C57BL/6J（B6）小鼠的脾细胞后，以MTT比色法检测脾细胞的增殖，并用PI染色后以流式细胞术（FCM）分析不同时间段处于S期，G0-G1期的细胞及无能T细胞的凋亡，测定T细胞亚群及MHC-I（H-2K^b)表达的变化。用琼脂糖凝胶电泳，观察不同时间段凋亡T细胞的DNA特征。结果：部分去除CD8^ T细胞后。SEB可刺激B6小鼠脾细胞中CD4^ T细胞大量增殖，在SEB刺激后第3天，CD4^ T细胞中处于S期的比率最大，此后开始下降；而处于G0-G1期的CD4^ T细胞变化则相反，在初次刺激后第3天，增殖的CD4^ T细胞出现无能，FCM检测及用琼脂糖凝胶电泳检查DNAladder证实，在第7天，无能CD4^ T细胞出现凋亡，且凋亡细胞的比率逐渐增多，不因加入抗CD3抗体或CoN A而逆转，在SEB刺激后，CD4^ T细胞表面MHC-I类分子（H-2K^b)的表达，随细胞无能的出现而明显下调。结论：SEB诱导的T细胞免疫耐受，可能与CD4^ T细胞的无能，凋亡及细胞表面分子MHC-I的表达下调有关。     

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.     

Tolerance to staphylococcal enterotoxin B initiated Th1 cell differentiation in mice infected with Candida albicans.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that specifically activates T cells bearing V beta 8 T-cell receptor domains, which eventually leads to a long-lasting state of clonal anergy accompanied by selective cell death in the targeted CD4+ subset. Because the superantigen is known to promote Th1 cell differentiation in vitro, we have investigated the effect of SEB treatment on the course of Th2-associated progressive disease in mice infected systemically with Candida albicans. On the basis of the kinetics of SEB-induced changes in CD4+ cells and production in sera of interleukin 4 (IL-4), IL-10, and gamma interferon, we obtained evidence that V beta 8+ cell anergy concomitant with infection abolished the early IL-4/IL-10 response of the host to the yeast, ultimately leading to a state of resistance characterized by gamma interferon secretion in vitro by antigen-specific CD4+ cells. In contrast, SEB administered near the time of challenge resulted in accelerated mortality. Significant resistance to infection was also afforded by exposure of mice to a retrovirally encoded endogenous superantigen. These data suggest that CD4+ V beta 8+ T cells play an important role in vivo in the initiation of a Th2 response to C. albicans and that suppression of their activity may alter the qualitative development of the T-cell response and the outcome of infection.     

Superantigen-induced anergy of V beta 8+ CD4+ T cells induces functional but non-proliferative T cells in vivo.

The response profile of staphylococcal enterotoxin B (SEB)-primed murine V beta 8+ CD4+ and V beta 8+ CD8+ T cells was analysed upon rechallenge in vitro. While in vitro responses to secondary stimulation with SEB were reduced to background levels, the in vivo reactivity after rechallenge with SEB was retained, in that SEB-primed mice succumbed to lethal T-cell shock, lymphokines [interleukin-1 (IL-1), IL-2, Il-4, IL-6, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)], and lymphokine-specific mRNA accumulation could be detected in V beta 8+ CD4+ and V beta 8+ CD8+ T cells. However, V beta 8+ CD4+ T cells failed to enter the cell cycle. While the phenotype of V beta 8+ CD8+ T cells was indistinguishable from that of their counterparts from naive mice, V beta 8+ CD4+ T cells exhibited in vivo an unusual phenotype as non-proliferative but functional T cells. We conclude that in vitro-defined anergy does not disclose the functional abilities of ligand-reactive V beta 8+ T cells in vivo, and that priming with superantigen (SAg) induces in vivo a differentiation of SEB-reactive V beta 8+ CD4+ T cells into a non-proliferative but functional phenotype.     

Clonal expansion precedes anergy and death of V beta 8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo

Staphylococcal enterotoxin B (SEB) selectively stimulates T cells bearing T cell receptor V beta 8 domains and hence provides a useful model to study immunity and tolerance in vivo. We show here that V beta 8+ T cells in both CD4+ and CD8+ subsets expand dramatically (fivefold) in lymphoid tissues of mice 2-4 days following injection with SEB. This initial clonal expansion, which is accompanied by a transient hyper-reactivity to SEB, is followed by a rapid decrease in V beta 8+ cells and a concomitant induction of specific non-responsiveness which persists for at least 30 days. Selective death of V beta 8+ cells occurs during this latter phase. Taken together, our data indicate that clonal expansion, anergy and death can occur as sequential stages of an immune response in vivo.     

T‐cell tolerance induced by repeated antigen stimulation: Selective loss of Foxp3− conventional CD4 T cells and induction of CD4 T‐cell anergy

Repeated immunization of mice with bacterial superantigens induces extensive deletion and anergy of reactive CD4 T cells. Here we report that the in vitro proliferation anergy of CD4 T cells from TCR transgenic mice immunized three times with staphylococcal enterotoxin B (SEB) (3× SEB) is partially due to an increased frequency of Foxp3⁺ CD4 T cells. Importantly, reduced number of conventional CD25^? Foxp3^? cells, rather than conversion of such cells to Foxp3⁺ cells, was the cause of that increase and was also seen in mice repeatedly immunized with OVA (3× OVA) and OVA—peptide (OVAp) (3× OVAp). Cell‐transfer experiments revealed profound but transient anergy of CD4 T cells isolated from 3× OVAp and 3× SEB mice. However, the in vivo anergy was CD4 T‐cell autonomous and independent of Foxp3⁺ Treg. Finally, proliferation of transferred CD4 T cells was inhibited in repeatedly immunized mice but inhibition was lost when transfer was delayed, despite the maintenance of elevated frequency of Foxp3⁺ cells. These data provide important implications for Foxp3⁺ cell‐mediated tolerance in situations of repeated antigen exposure such as human persistent infections.     

Increased c-Fos/activator protein-1 confers resistance against anergy induction on antigen-specific T cell

We have studied the contribution of c-Fos/activator protein-1 (AP-1) to antigen-specific T cell response with reference to T cell anergy by increasing c-Fos/AP-1 in vivo and in vitro. First, after injection of a high dose of staphylococcus enterotoxin B (SEB), clonal deletion of SEB-reactive V(beta)8(+) CD4 T cells occurred both in control B6 and H2-c-fos transgenic (fos) mice, whereas proliferation of T cells against SEB was profoundly depressed in B6 but not in fos mice. Second, the keyhole limpet hemocyanin-specific CD4 T(h)1 cell clone produced decreasing amounts of IL-2 in response to increasing amounts of concanavalin A (Con A) in vitro, whereas the decrease was less significant in the T(h)1 clones stably transfected with c-fos gene. Electrophoretic mobility shift assay with nuclear protein from the transformants showed that overexpression of the c-fos gene compensated the amounts of AP-1 in the nuclei of Con A-treated T(h)1 clones. Thus, increased c-Fos/AP-1 confers resistance against anergy induction on antigen-specific T cells.     

The role of IL-4 in the staphylococcal enterotoxin B-triggered immune response: increased susceptibility to shock and deletion of CD8Vbeta8+ T cells in IL-4 knockout mice

Administration of superantigens in vivo triggers responding T cells into clonal expansion and subsequent activation of the programmed cell death pathway, as well as into anergy. We examined the possibility that Th1 cytokines are involved in rescue from superantigen-induced programmed cell death and prevention of anergy by studying the Staphylococcus aureus enterotoxin B (SEB) immune response in mice in which the IL-4 gene was deleted (IL-4-/-). In these mice, Th1 cell activation triggers increased IFN-gamma and reduced IL-5 production as compared to IL-4+/+ mice. The primary anti-SEB antibody response in IL-4-/- mice is thus dominated by immunoglobulins of the IgG2a isotype, whereas the IgG1 isotype prevails in IL-4+/+ mice. Our results also show that, in contrast to expectations, IL4-/- mice are more susceptible to SEB plus low-dose D-galactosamine-induced shock and that this response is TNF-alpha-dependent. In vivo treatment induces partial deletion and anergy of remaining SEB-reactive T cells. During the SEB-induced response, CD4Vbeta8+ T cells are deleted in IL-4-/- mice, but not in IL-4+/+ mice, suggesting a function for IL-4 in CD8+ T cell rescue from apoptosis. We show that IL-4 efficiently protects CD8+ T cells from in vitro starvation-induced apoptosis, and conclude that IL-4 has an important role in Th1 immune response regulation.     

In vivo responses of CD4+ and CD8+ cells to bacterial superantigens.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex (MHC) class II molecules and specifically activates T cells bearing V beta 8 T cell receptor domains. We have compared several aspects of the response of CD4+ and CD8+ T cell subsets to SEB in vivo. V beta 8+ cells in both subsets proliferated to a similar extent upon SEB injection. Furthermore, mRNA for interferon-gamma was induced in both subsets with similar kinetics and SEB dose-response. Finally CD8+ (but not CD4+) T cells from SEB-injected mice exhibited SEB-specific lysis of MHC class II-bearing target cells. Collectively, these data indicate that the CD4: MHC class II interaction confers no detectable selective advantage to CD4+ cells in the in vivo response to SEB. The observed effector functions of both subsets may contribute to SEB-induced immunopathology.     

Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: Dose-response relationship

Ligand-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. T cell receptor (TcR) transgenic mice offer a clear advantage for studying post-thymic tolerance mechanisms in vivo that are operational in a monoclonal T cell population with preselected antigen specificity. Yet it is unclear whether the rules defined in monoclonal T cells of genetically manipulated mice reflect those operative in clonally diverse peripheral T cells of normal mice. To analyze acute tolerance mechanisms in unselected peripheral T cells, we challenged normal mice with the superantigen staphylococcal enterotoxin B (SEB) and analyzed ligand-reactive Vβ8⁺ T cells for TcR-triggered tolerance mechanisms such as anergy, TcR down-regulation, or apoptosis. Upon challenge with graded doses of SEB (0.001–10 μg) Vβ8^? T cells become anergic within 6–16 h. Importantly, a dosage effect of SEB in regard to the level of anergy induced was observed. Anergy induced by low concentrations of SEB (0.001–0.1 μg) is transient and is overcome by clonal growth, while higher concentrations of SEB (0.1–10 μg) cause long-lasting anergy resistant to cell cycle progression. At high SEB concentrations (1–10 mg) about 50% of the anergic Vβ8⁺ T cells additionally down-regulate their TcR-CD3 complex, followed by a loss of CD2, CD4, CD8 accessory molecules. In parallel, T cell phenotypenegative but genotypically Vβ8⁺ T cells are generated. The T cell phenotypenegative cells reacquire their Vβ8⁺ T cell phenotype upon culture in vitro. In vivo, a subset of Vβ8⁺ cells, defined by an intermediate stage of TcR down-regulation, i.e. Vβ8^lowCD3⁺ cells, but not T cell phenotype-negative cells are selectively programmed for apoptosis, which occurs within 1 h. These data suggest that SEB triggers distinct tolerance pathways which operate in a hierarchical fashion in clonally diverse ligand-reactive T cells. Specifically, the results illustrate the power of exogenous superantigens to exploit these distinct tolerance pathways, thereby achieving distinct levels of immunosuppression.     

In vivo and in vitro death of mature T cells induced by separate signals to CD4 and alpha beta TCR.

To investigate whether a clonal deletion mechanism is responsible for the mature T cell tolerance that may be induced in vivo by TCR signal to anti-CD4 (H129.19 mAb) coated cells, we analyzed the T cell repertoire in anti-CD4 mAb treated BALB/c mice by flow cytometry following TCR signals through anti-alpha beta TCR mAb or SEB superantigen. Lymph nodes showed a strong reduction in the CD4+/CD8+ cell ratio, and a selective clonal loss of CD4+ V beta 8+ cells 4d following anti-alpha beta TCR or SEB injection, respectively. Following lymph node cell activation in a short-term in vitro assay with SEB or anti-V beta 8 mAb, a selective elimination of CD4+ V beta 8+ cells was again detected, and DNA fragmentation analysis disclosed a cell death by apoptosis. These findings suggest that TCR triggering transduces an apoptotic signal into CD4+ mAb saturated cells that in turn leads to specific holes in the mature T cell repertoire.     

超抗原SEB在同种异体细胞移植小鼠中诱导的免疫耐受及其特征

目的 探讨超抗原葡萄球菌肠毒素B（SEB）体内诱导的免疫耐受性及其特征。方法 采用MHC不同的两种小鼠进行淋巴细胞移植。用流式细胞术和混合淋巴细胞培养技术，检测受体鼠T细胞亚群的变化，供体鼠H－2K^d分子在受体鼠体内表达的阳性率与免疫应答性。结果 （1）注射SEB可选择性地降低CD4^ T细胞和CD4^ T/H-2K^b 细胞的百分率，而不影响CD8^ T细胞的数量。在SEB注射的21d，抑制作用最强，其对CD4^ T细胞和CD4^ T/H-2K^b 细胞的抑制率分别是6.24%和23.38%。（2）在进行异源性MHC淋巴细胞移植时注射SEB，于移植细胞后21d，受体小鼠肝脏淋巴细胞上开始表达供体小鼠H－2K^d分子，至移植后40d，表达率达到高峰7.90%。与此同时，受体鼠外周淋巴细胞对供体鼠淋巴细胞的免疫应答能力也明显降低。结论 SEB能诱导小鼠产生免疫耐受，免疫耐受的形成与受体鼠内CD4^ T细胞克隆的明显减少有关。     

Clonal anergy to staphylococcal enterotoxin B in vivo: selective effects on T cell subsets and lymphokines

Injection of bacterial superantigens such as staphylococcal enterotoxin B (SEB) in adult mice results in initial proliferation of SEB-responsive Vβ8⁺ T cells followed by induction of a state of non-responsiveness frequently referred to as clonal anergy. We show here that SEB-induced anergy involves selective changes in lymphokine production and that it affects CD4⁺ Vβ 8⁺ and CD8⁺ Vβ 8⁺ T cells in different fashions. Whereas both CD4⁺ Vβ 8⁺ and CD8⁺ Vβ 8⁺ cells from anergic mice exhibit strongly reduced proliferative capacity and interleukin(IL)-2 production upon restimulation with SEB either in vivo or in vitro the CD8⁺ subset from SEB-injected mice produces other lymphokines (such as interferon(IFN)-γ) at normal or slightly increased levels in response to SEB. Changes in the levels of production of IL-2 and IFN-γ protein correlated well with mRNA accumulation both in vivo and in vitro. Collectively these data suggest that superantigen-induced anergy involves selective changes in signal transduction and/or gene regulation in T lymphocytes.     

Recurrent superantigen exposure in vivo leads to highly suppressive CD4+CD25+ and CD4+CD25‐ T cells with anergic and suppressive genetic signatures

Staphylococcal enterotoxin B (SEB) activates T cells via non‐canonical signalling through the T cell receptor and is an established model for T cell unresponsiveness in vivo. In this study, we sought to characterize the suppressive qualities of SEB‐exposed CD4⁺ T cells and correlate this with genetic signatures of anergy and suppression. SEB‐exposed CD25⁺ and CD25^‐Vβ8⁺CD4⁺ T cells expressed forkhead box P3 (FoxP3) at levels comparable to naive CD25⁺ T regulatory cells and were enriched after exposure in vivo. Gene related to anergy in lymphocytes (GRAIL), an anergy‐related E3 ubiquitin ligase, was up‐regulated in the SEB‐exposed CD25⁺ and CD25^‐FoxP3⁺Vβ8⁺CD4⁺ T cells and FoxP3^‐CD25^‐Vβ8⁺CD4⁺ T cells, suggesting that GRAIL may be important for dominant and recessive tolerance. The SEB‐exposed FoxP3⁺GRAIL⁺ T cells were highly suppressive and non‐proliferative independent of CD25 expression level and via a glucocorticoid‐induced tumour necrosis factor R‐related protein‐independent mechanism, whereas naive T regulatory cells were non‐suppressive and partially proliferative with SEB activation in vitro. Lastly, adoptive transfer of conventional T cells revealed that induction of FoxP3⁺ regulatory cells is not operational in this model system. These data provide a novel paradigm for chronic non‐canonical T cell receptor engagement leading to highly suppressive FoxP3⁺GRAIL⁺CD4⁺ T cells.     

A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.     

T cell anergy is programmed early after exposure to bacterial superantigen in vivo

We have investigated the effects of the protein synthesis inhibitor,cycloheximide (CHX), on the induction of post-thymic T celltolerance in mice primed with the bacterial superantigen, Staphylococcusaureus enterotoxin B (SEB). A single injection of 1 mg CHX preventedprotein synthesis in splenic cells for <6 h in vivo. Theconcomitant administration of SEB and CHX prevented inductionof SEB-specific anergy, but did not interfere with the deletionof SEB-speclfic V_ß8⁺ T cells by activation-induced,programmed cell death. When CHX was given 24 h after SEB administrationthe expression of anergy was not affected. These findings suggestthat anergy and deletion represent independent processes. Furthermore,these observations, together with the fact that SEB retainsthe potential to induce anergy in specific T cells 8 h afterpriming in vivo, imply that the determination of alternate fates(anergy or death) occurs at early time points after SEB injection     

T Cell-Dependent Antibody Response to Staphylococcal Enterotoxin B

Treatment of mice with staphylococcal enterotoxin B (SEB) induces specific T-cell tolerance to this superantigen, characterized by partial deletion of Vβ8⁺ T cells in vivo and T cell anergy in vitro . In this study we examined the humoral response to SEB in BALB/c mice. Immunization of mice with SEB results in a detectable anti-SEB antibody response. Upon further treatment of mice with SEB, specific antibody levels increase significantly and the response is accelerated—characteristics of a secondary humoral response. The secondary antibody response is T cell dependent, can be transferred to T cell deficient mice with splenocytes and is composed mainly of IgM, IgG1 and IgG2b isotypes, suggesting that Th2 cells provide B cell help in this response. These data demonstrate that at the same time as inducing in vitro unresponsiveness, SEB primes SEB-specific T helper cells to provide help for B cells in a secondary antibody response.     

CD8+ Cells are the Main Producers of IL10 and IFNγ after Superantigen Stimulation

Purified CD8⁺ T cells were recently shown to produce TH1 as well as TH2 types of cytokines upon restimulation, indicating an important role for these cells in regulation of immune responses. However, it is not known if the CD8⁺ cells would contribute to cytokine production in the presence of cytokine secreting CD4⁺ cells. In the present study the authors have investigated the proportion of cytokine-producing CD4⁺ and CD8⁺ cells in the spleen after in vitro or in vivo stimulation. They found that stimulation of spleen cells with the superantigen Staphylococcal Enterotoxin B (SEB) in the presence of IL4 promoted production of IL10 and IFNγ predominately by CD8⁺ cells. In contrast, the production of IL4 was almost exclusively confined to the CD4⁺ subset. When priming with SEB in vivo before subsequent restimulation in vitro , a protocol previously shown to induce anergy, up to 80% of the IL10 and IFNγ positive cell expressed the CD8 marker. Taken together, these results emphasize the important role of cytokine-producing CD8⁺ cells and indicate that CD4⁺ and CD8⁺ T cells may, in a given situation, produce distinct cytokines.