超抗原SEA和SEB对正常人TCR V_β基因的限制性取用

金黄色葡萄球菌肠毒素 (SE )作为一种超抗原 ,以MHC非限制性及TCRVβ特异性的方式激活T细胞。本文用定量PCR方法 ,分析SEA和SEB刺激的正常人外周血淋巴细胞T细胞抗原受体Vβ的取用格局。揭示在不同HLA遗传背景下 ,SEA刺激的淋巴细胞均选择性地取用Vβ3和Vβ6两种基因片段 ,而SEB刺激的淋巴细胞则优势表达Vβ2、Vβ8和Vβ9。通过比较分析 ,Sigma产的SEB刺激淋巴细胞后则优势表达Vβ3、Vβ4、Vβ16和Vβ2 0 ,提示外周血淋巴细胞在超抗原作用下发生寡克隆扩增。     

Response to superantigen stimulation in peripheral blood mononuclear cells from children perinatally infected with human immunodeficiency virus and receiving highly active antiretroviral therapy

Our understanding of the pathogenesis of perinatal human immunodeficiency virus (HIV) infection is still evolving. We sought to characterize the response to the bacterial superantigen Staphylococcus enterotoxin B (SEB) of lymphocytes from HIV-infected children receiving treatment with highly active antiretroviral therapy (HAART). Using the flow cytometric methodology, we quantified apoptosis, proliferation, cytokine production, and activation antigen upregulation in CD4 and CD8 T lymphocytes following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with SEB. The levels of proliferation, CD4 interleukin-2 (IL-2) production, CD8 gamma interferon (IFN-gamma) production, and upregulation of CD69 expression by cells from HIV-infected children were indistinguishable from those by cells from controls. However, stimulation with SEB dramatically decreased the ratio of resting apoptotic cells to cycling apoptotic cells in the controls but not in the patients. In addition, unstimulated spontaneous apoptosis of CD4 T cells remained greater in the patients than in the controls. The percentages of IL-2-positive CD8 T cells and IFN-gamma-positive CD4 T cells following SEB stimulation were significantly lower in the patients than in the controls. Our multiparameter approach was able to demonstrate differences in lymphocyte superantigen responsiveness in HIV-infected children receiving HAART in comparison to that in uninfected controls, notably, an apoptotic versus a proliferative response to stimulation.     

Response to Superantigen Stimulation in Peripheral Blood Mononuclear Cells from Children Perinatally Infected with Human Immunodeficiency Virus and Receiving Highly Active Antiretroviral Therapy

Our understanding of the pathogenesis of perinatal human immunodeficiency virus (HIV) infection is still evolving. We sought to characterize the response to the bacterial superantigen Staphylococcus enterotoxin B (SEB) of lymphocytes from HIV-infected children receiving treatment with highly active antiretroviral therapy (HAART). Using the flow cytometric methodology, we quantified apoptosis, proliferation, cytokine production, and activation antigen upregulation in CD4 and CD8 T lymphocytes following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with SEB. The levels of proliferation, CD4 interleukin-2 (IL-2) production, CD8 gamma interferon (IFN-γ) production, and upregulation of CD69 expression by cells from HIV-infected children were indistinguishable from those by cells from controls. However, stimulation with SEB dramatically decreased the ratio of resting apoptotic cells to cycling apoptotic cells in the controls but not in the patients. In addition, unstimulated spontaneous apoptosis of CD4 T cells remained greater in the patients than in the controls. The percentages of IL-2-positive CD8 T cells and IFN-γ-positive CD4 T cells following SEB stimulation were significantly lower in the patients than in the controls. Our multiparameter approach was able to demonstrate differences in lymphocyte superantigen responsiveness in HIV-infected children receiving HAART in comparison to that in uninfected controls, notably, an apoptotic versus a proliferative response to stimulation.     

T cell- and perforin-dependent depletion of B cells in vivo by staphylococcal enterotoxin A.

Bacterial superantigens bind to major histocompatibility complex (MHC) class II and subsequently activate both CD4+ and CD8+ T lymphocytes expressing certain T-cell receptor (TCR)-Vbeta chains. In response to superantigen exposure these subsets proliferate, produce large amounts of proinflammatory cytokines and in addition CD8+ cytotoxic T lymphocytes (CTL) are induced. Previous studies in vitro have shown that these CTL effectively lyse MHC class II-expressing cells presenting the proper superantigen. However, it is unknown whether superantigens induce a similar response towards MHC class II+ antigen-presenting cells in vivo. In this study we demonstrate that administration of repeated injections of the superantigen staphylococcal enterotoxin A (SEA) to TCR-Vbeta3 transgenic mice results in a loss of MHC class II-expressing cells in the spleen. Analysis of different MHC class II+ subsets revealed a selective depletion of CD19+ B cells, while F4/80+ macrophages increased in number. Depletion of T cells with anti-CD4 or anti-CD8 monoclonal antibody indicated that CD8+ T cells were crucial for SEA-induced cytotoxicity in vivo. Repeated injections of SEA to perforin-deficient mice resulted in significantly less B-cell depletion compared with control mice. This suggests that superantigen-activated CD8+ T cells lyse MHC class II+ antigen-presenting cells in a perforin-dependent manner in vivo. It is suggested that this represents a novel bacterial immune escape mechanism, which may particularly impair local humoral immune responses.     

Comparison of activation marker and TCR V beta gene product expression by CD4+ and CD8+ T cells in peripheral blood and lymph nodes from HIV-infected patients.

Since lymphoid organs constitute the site of active and progressive HIV disease, analysis of their lymphocytes may provide more accurate information on T cell abnormalities than that obtained from studying peripheral blood lymphocytes. The objective of this study was to compare the expressions of activation markers and T cell receptor (TCR) V beta gene products by CD4+ and CD8+ T cells in lymph nodes (LN) and peripheral blood (PB) from healthy individuals and asymptomatic HIV-infected patients to determine whether anomalies that could be identified at the HIV replication site could support the hypothesis of T cell activation by HIV-encoded antigens or superantigens. CD4+ and CD8+ T cells in paired LN and PB obtained from six healthy controls and five asymptomatic HIV-infected individuals were analysed by flow cytometry, using anti-CD38, anti-HLA-DR and 13 anti-V beta MoAbs that cover, approximately, 45% of the T cell repertoire. Analysis of T cell activation marker expression indicated that the percentages of CD4+ and CD8+ T cells bearing CD38 or CD38 and HLA-DR molecules were higher in patients than in controls and, in patients, higher in LN than in PB. Comparison between the V beta repertoires of CD4+ and CD8+ T cells in LN and PB showed that, in each healthy individual, a limited number of V beta families expressed by CD4+ or CD8+ T cells had different repartition in LN and PB, whereas in each HIV+ patient, more V beta families exhibited different distributions and these differences recurred among certain V beta segments, such as V beta 5.3 and V beta 21 in the CD4+ T cell population and V beta 5.2/5.3, V beta 12 and V beta 21 in the CD8+ T cell population. Taken together, these data argue for a skewed TCR repertoire in HIV infection and sustained activation of T cells by HIV-encoded antigens at the site of HIV replication, and further demonstrate that a high proportion of CD4+ T cells are in an activation state that may, indirectly, participate in their functional abnormalities.     

Vβ Specificity of Superantigen TSST-1 Plus CD28 Costimulation Without APCs

Little has so far been known about the role of antigen-presenting cells (APCs) in the activation of T cells by superantigens. Recently several studies showed that superantigens could directly activate purified T cells in the presence of CD28 costimulation. Here we investigate Vβ expression of T cells activated by the superantigen toxic shock syndrome toxin-1 (TSST-1), plus CD28 costimulation, in the absence of APCs. The aim of this study was to ask if TSST-1 activated purified T cells in the presence of CD28 costimulation with the same specificity as in the presence of APCs. We provide evidence that the specificity of TSST-1 to human Vβ, in the presence of CD28 costimulation, is identical to that in the presence of APCs, with Vβ2 being significantly expanded. The results indicate that the main role of APCs in the superantigen-mediated T cell activation may be to provide T cells with CD28 costimulation.     

Non-antigen specific CD8+ T suppressor lymphocytes

Abstract. The homeostasis of peripheral immune system function is maintained by the activity of regulatory lymphocytes. Among these cells, a subset of CD8+CD28- T suppressor lymphocytes has recently been characterized for the capacity to mediate their effects without antigen restriction. These non-antigen-specific CD8+ T suppressor lymphocytes originate from circulating CD8+CD28- T lymphocytes after stimulation with interleukin-2 and interleukin- 10. CD8+ suppressor cells inhibit both antigen-specific CD4+ T cell proliferation and cellular cytoxicity through secretion of cytokines such as interferon-, interleukin-6, and interleukin-10. The function of CD8+ suppressor cells is impaired in patients with systemic lupus erythematosus in relapse as well as in patients with systemic sclerosis with disease progression, suggesting the involvement of CD8+ suppressor cells in the pathogenesis of autoimmune diseases. Interestingly, CD8+ suppressor cells have been found among tumor-infiltrating lymphocytes, which could be related to tumor-induced-immunosuppression. Failure to generate CD8+ suppressor cells from the peripheral blood is frequently observed in HIV-infected patients. It remains to be clarified whether this phenomenon is due to depletion and/or functional impairment of this cell subset or to their compartmentalization in peripheral tissues and immunocompetent organs where they could contribute to the induction of immunodeficiency.     

Active suppression induced by cutaneous exposure to bacterial superantigen is prevented by interleukin-12 treatment in vivo.

Exposure to the bacterial superantigen staphylococcal enterotoxin B (SEB) leads to inhibition of several immune responses and the induction of regulatory cells. The aim of this study was to characterize these regulatory cells further and to investigate the effect of interleukin-12 (IL-12) on superantigen-induced suppression. For this purpose BALB/c mice were injected subcutaneously with low doses of SEB that did not deplete the SEB-reactive V beta T cells. Intravenous transfer of unseparated local-draining lymph node cells from these SEB-treated animals suppressed the proliferative response of mononuclear spleen cells of naive syngeneic recipients for at least 3 weeks. The regulatory cells did not produce the type 2 cytokines, interleukin-4 (IL-4) or interleukin-10 (IL-10), or increased amounts of transforming growth factor-beta (TGF-beta). Depletion of CD8+ or SEB-reactive V beta 7+ and V beta 8+ T cells, prior to transfer, abrogated the suppressive effect. Intraperitoneal injections of IL-12 into donors, prior to SEB treatment, prevented the induction of functional regulatory cells, and treatment of recipients with IL-12, prior to receipt of cells from SEB-treated donors, prevented the suppressive effect of regulatory cells that were already induced. The data indicate that exposure to minute amounts of superantigens directly induces superantigen-reactive and CD8+ regulatory T cells and that superantigen-induced suppression can be prevented and reversed by IL-12 treatment in vivo.     

Apoptosis in T-Lymphocyte Subsets in Human Immunodeficiency Virus-Infected Children Measured Immediately Ex Vivo and following In Vitro Activation

Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.     

Apoptosis in T-lymphocyte subsets in human immunodeficiency virus-infected children measured immediately ex vivo and following in vitro activation

Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.     

The viral superantigen Mls-1a induces interferon-gamma secretion by specifically primed CD8+ cells but fails to trigger cytotoxicity.

Superantigens can be operationally defined by their ability to stimulate CD4+ and CD8+ T cells via the T cell receptor beta chain variable domain (TcR V beta). We show here that effector functions of CD8+ T cells specific for superantigens differ depending upon the nature of the superantigen involved. Hence, activated CD8+ T cells bearing TcR V beta specific for the superantigen Mls-1a [encoded in the open reading frame of the 3' long terminal repeat of endogenous mouse mammary tumor virus (MMTV)] are unable to lyse Mls-1a-bearing target cells despite the fact that they release interferon-gamma (IFN-gamma) upon Mls-1a stimulation. In contrast CD8+ T cells specific for the exogenous superantigen staphylococcal enterotoxin B (SEB) readily mediate both lysis and IFN-gamma secretion when exposed to SEB-bearing target cells. This dissociation between lysis and IFN-gamma production by Mls-1a-specific CD8+ T cells is independent of the initial stimulus used for activation and appears not to be simply explained by a low Mls-1a determinant density. We suggest that this phenomenon reflects differing TcR affinity thresholds for lymphokine secretion and cytolysis. Such differences may be exploited by retroviruses such as MMTV in order to escape immunosurveillance.     

Broad-spectrum immunity against superantigens is elicited in mice protected from lethal shock by a superantigen antagonist peptide

Bypassing the restricted presentation of conventional antigens, superantigens trigger an excessive cellular immune response leading to toxic shock. Antagonist peptides that inhibit the induction of human Th1 cytokine gene expression by a variety of bacterial superantigens protect mice from lethal toxic shock. We show that the surviving mice rapidly develop a broad-spectrum, protective immunity against further lethal toxin challenges with the same superantigen and even with superantigen toxins that they have not encountered before. By blocking the induction of a cellular immune response leading to toxic shock, the antagonist peptide allows the superantigen to induce a vigorous humoral immune response directed against itself, resulting in anti-toxin IgM and IgG that are broadly protective. Adoptive transfer of these antibodies to na?ve mice rendered them resistant to lethal superantigen challenge. The appearance of these antibodies does not require immunization with an adjuvant and is not elicited by the antagonist peptide. Our results show that superantigens are potent immunogens when given the opportunity to induce a B cell response, in conditions where a deleterious Th1 response is prevented by the superantigen antagonist peptide.     

In vitro influence of Mycoplasma penetrans on activation of peripheral T lymphocytes from healthy donors or human immunodeficiency virus-infected individuals.

Mycoplasma penetrans is a mycoplasma species newly isolated from the urine of human immunodeficiency virus (HIV)-infected individuals and presents the only case in which an association has been found between antibodies against a mycoplasma and HIV infection. To further explore the effects of M. penetrans on the immune system, we studied the influence of this mycoplasma on peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-infected individuals. M. penetrans induced, in addition to blastogenesis of PBMCs, a significant proliferative response associated with the expression of some activation markers such as CD69, HLA-DR, and CD25. This M. penetrans-dependent lymphocyte activation was observed not only in healthy donors but also in HIV-infected persons at different stages of the disease. In addition, our study revealed that both CD4+ and CD8+ T lymphocytes were responsive to M. penetrans. Interestingly, the mitogenic activity of M. penetrans was associated with mycoplasma cells but not with the supernatants of mycoplasma culture. The potent stimulating activity of M. penetrans on T lymphocytes from HIV-infected individuals is of particular interest in view of the supposed contribution of immune activation to HIV replication and disease progression.     

Evidence that induction of tolerance in vivo involves active signaling via a B7 ligand-dependent mechanism: CTLA4-Ig protects Vβ8+ T cells from tolerance induction by the superantigen staphylococcal enterotoxin B

Co-stimulation through CD28 is thought to be necessary for the activation of unprimed CD4⁺ T cells, which are otherwise rendered tolerant. However, we previously found that CD4⁺ T cell priming was normal or augmented in mice which overexpressed a soluble form of CTLA4 where co-stimulation through CD28 was abrogated. To investigate this CD4⁺ T cell response, we exploited the capacity of the superantigen staphylococcal enterotoxin B to stimulate T lymphocytes bearing Vβ8⁺, which represent ?30% of all CD4⁺ T cells. In littermate controls of CTLA4-Ig transgenic mice, immunization with staphylococcal enterotoxin B leads to expansion, followed by deletion of Vβ8⁺ T cells, and the remaining cells are tolerant when stimulated in vitro. Comparable expansion and deletion of Vβ8⁺ T cells occurs in CTLA4-Ig transgenic mice. However, in contrast to normal mice, the remaining Vβ8⁺ T cells from CTLA4-Ig transgenic mice are not anergic and remain responsive to superantigen in vitro.     

Role of the T cell receptor α-chain in superantigen recognition

Superantigens bind to antigen-presenting cells on the outside of the major histocompatibility complex (MHC) class II molecule and to T cells via the external face of the T cell receptor (TCR) Vβ element. As a consequence, superantigens stimulate populations of T cells in a Vβ-specific, non-MHC-restricted manner. However, accumulating evidence has shown an additional contribution of the TCR α-chain and polymorphic residues of the MHC molecule to superantigen recognition by some T cells. These data suggest that the TCR and MHC come into contact during superantigen engagement and indirectly modulate the superantigen reactivity. Thus, additional interactions between non-Vβ elements of the TCR and MHC play a role in the overall stability of the superantigen/MHC/TCR complex, explaining the influence of the TCR α-chain. It is likely that this additional interaction is of greater consequence for weakly reactive T cells. This modulation of superantigen reactivity in individual T cells may have physiological consequences, for example, in the induction of autoimmunity.     

Acute activation of CD8+ T lymphocytes in interleukin-2-treated HIV-infected patients. ANRS-048 IL-2 Study Group. Agence Nationale de Recherches sur le SIDA.

CD8+ T lymphocytes play a key role in the control of HIV infection, through both cytotoxic and noncytotoxic mechanisms. To study in vivo effects of interleukin-2 (IL-2) treatment on this cell compartment, the level of activation of CD8+ T lymphocytes was evaluated before and just after 5-day administration of IL-2 in 16 HIV-infected patients. The serum level of soluble CD25 and of soluble CD8 significantly increased following IL-2 administration. The number of mRNA molecules coding for perforin and granzyme B, two enzymes that are contained in granules of cytotoxic cells, also significantly increased in peripheral blood mononuclear cells and in purified CD8+ cells (p < .001). Variations of plasma HIV viremia and perforin gene expression following IL-2 administration were inversely correlated (p = .023), suggesting that IL-2-induced activation of CD8+ T lymphocytes contributes to limit HIV replication in vivo. In contrast to perforin and granzyme B gene expression, IL-2 administration did not increase the expression of macrophage inhibitory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated-on-activation normal T-expressed and secreted (RANTES) genes. These findings indicate that CD8+ T lymphocytes in HIV-infected patients are acutely activated by IL-2 treatment, which may improve long-term control of HIV infection.     

Vα domain modulates the multiple topologies of mouse T cell receptor Vβ20/staphylococcal enterotoxins A and E complexes

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major histocompatibility complex (MHC) class II molecules on two sites located on the α and β chains. We have investigated the role of the T cell receptor (TCR) α chain in the modulation of the various topologies of TCR/SEA (or SEE)/class II complexes. For this purpose, we have used three mouse Vβ20 T cell lines expressing different Vα domains and two T cell hybridomas expressing mouse Vβ1 or Vβ11 segments. The response of these T cells to SEA and SEE was studied in the context of presentation by wild-type human MHC class II molecules; or by mutants on MHC, in each of the two superantigen binding sites (position α39K and β81H) to which the superantigens can still bind but with an altered conformation. Although Vβ20 T cell lines are efficiently stimulated using SEA and SEE presented by wild-type HLA-DR1 molecules, our results show that the nature of the TCR Vα domain can affect differently the recognition of the toxins bound to mutant class II molecules. This suggests that various functional topologies exist for both SEA and SEE/class II complexes and that the T cell response to each of these complexes can be modulated by the Vα domain of the TCR. Interestingly, the recognition of SEA and SEE is achieved in different fashions by a given Vβ20 T cell line.