Partial T cell activation with an altered superantigenic ligand

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.     

Neutralization of staphylococcal enterotoxin B by soluble, high-affinity receptor antagonists

Exotoxins of Staphylococcus aureus belong to a family of bacterial proteins that act as superantigens by activating a large subset of the T-cell population, causing massive release of inflammatory cytokines. This cascade can ultimately result in toxic shock syndrome and death. Therapeutics targeting the early stage of the pathogenic process, when the superantigen binds to its receptor, could limit the severity of disease. We engineered picomolar binding affinity agents to neutralize the potent toxin staphylococcal enterotoxin B (SEB). A single immunoglobulin-like domain of the T-cell receptor (variable region, Vbeta) was subjected to multiple rounds of directed evolution using yeast display. Soluble forms of the engineered Vbeta proteins produced in Escherichia coli were effective inhibitors of SEB-mediated T-cell activation and completely neutralized the lethal activity of SEB in animal models. These Vbeta proteins represent an easily produced potential treatment for diseases mediated by bacterial superantigens.     

Analysis of the in vivo dendritic cell response to the bacterial superantigen staphylococcal enterotoxin B in the mouse spleen

To investigate the in vivo effects of Staphylococcal enterotoxin B (SEB) on dendritic cells (DCs) in the spleen, a single dose of SEB (50 microg/kg) was administered to BALB/c mice by intraperitoneal injection. Afterwards, the mice were sacrificed at 2, 6 and 24 hr, 2, 4, 7 and 15 days, and the spleens were removed. The immunocytochemical characterization of the cells was carried out using various monoclonal antibodies in cryostat-cut sections. The distribution patterns of DCs and their major costimulatory molecules, CD80, CD86 and CD40 in the spleen were identified, and the evidence for maturation of DCs in vivo in response to SEB was obtained. It was found that systemic administration of SEB induced the migration of most of the immature, splenic DCs from the marginal zone to the periarterial lymphatic sheath within 6 hr. This movement paralleled a maturation process, as assessed by upregulation of CD40, CD80 and CD86 expression in the interdigitating dendritic cells (IDCs). The upregulation of costimulatory molecule expression was conspicuous only in DCs in contrast to other antigen-presenting cells (APCs) such as macrophages and B cells which did not show any significant alterations in their costimulatory molecule expression. We also demonstrated the temporal expression pattern of these costimulatory molecules on the activated DCs. The upregulation of costimulatory molecules on DCs reached a peak level 6 hr after SEB injection, while the increase in number of T cells expressing T cell receptor V138 reached a peak level on day 2 after SEB treatment. In conclusion, we demonstrated the in vivo DC response to SEB in the mouse spleen, especially a potent stimulative effect of SEB on DCs in vivo, a temporal distribution pattern of DCs as well as T cells including TCR Vbeta8+ T cells, and a differential expression pattern of costimulatory molecules on the activated DCs. The results of the present study indicate that DCs are the principal type of APCs which mediate T cell activation by SAg in vivo, and that each costimulatory molecule may have different role in the activation of DCs by SAg. Thus, it is plausible to speculate that DCs play a critical role in the T cell clonal expansion by SAgs and other SAg-induced immune responses in vivo.     

Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells

Microbial superantigens can alter host immunity through aberrant activation and subsequent anergy of responding naive T cells. We show here that the superantigen, staphylococcal enterotoxin B (SEB), directly induces tolerance in memory CD4 T cells. Murine naive and memory CD4(+) T cells were labeled with the fluorescent dye CFSE and the cells were exposed to SEB before they were cultured with specific peptide antigen. Memory, but not naive, T cells became anergic and did not respond to their cognate peptide antigen. The extent and duration of T cell receptor (TCR) clustering was similar to promote naive T cell activation and memory T cell anergy, suggesting similar TCR-SEB interactions led to distinct intracellular signaling processes in the two cell types. Like SEB, soluble anti-CD3 mAb does not stimulate memory cell proliferation. However, unlike SEB, soluble anti-CD3 mAbs did not induce anergy to cognate peptide. Anergy was directly visualized in vivo. CD4(+) memory T cells were identified in mice that had been administered SEB. The cells failed to proliferate in response to subsequent immunization with their cognate recall antigen. Hence, one mode of pathogen survival is the modulation of host immunity through selective elimination of memory T cell responses.     

Superantigen enhancement of specific immunity: antibody production and signaling pathways

Superantigens are microbial proteins that induce massive activation, proliferation, and cytokine production by CD4+ T cells via specific Vbeta elements on the TCR. In this study we examine superantigen enhancement of Ag-specific CD4+ T cell activity for humoral B cell responses to T-dependent Ags BSA and HIV gp120 envelope, type I T-independent Ag LPS, and type II T-independent Ag pneumococcal polysaccharides. Injection of BSA followed by a combination of superantigens staphylococcal enterotoxin A and staphylococcal enterotoxin B (SEB) 7 days later enhanced the anti-BSA Ab response in mice approximately 4-fold as compared with mice given BSA alone. The anti-gp120 response was enhanced approximately 3-fold by superantigens. The type II T-independent Ag pneumococcal polysaccharide response was enhanced approximately 2.3-fold by superantigens, whereas no effect was observed on the response to the type I T-independent Ag LPS. The superantigen effect was completely blocked by the CD4+ T cell inhibitory cytokine IL-10. SEB-stimulated human CD4+ T cells were examined to determine the role of the mitogen-activated protein (MAP) kinase signal transduction pathway in superantigen activation of T cells. Inhibitors of the mitogen pathway of MAP kinase blocked SEB-induced proliferation and IFN-gamma production, while an inhibitor of the p38 stress pathway had no effect. Consistent with this, SEB activated extracellular signal-regulated kinase/MAP kinase as well as MAP kinase-interacting kinase, a kinase that phosphorylates eIF4E, which is an important component of the eukaryotic protein synthesis initiation complex. Both kinases were inhibited by IL-10. Thus, superantigens enhance humoral immunity via Ag-specific CD4+ T cells involving the stress-independent pathway of MAP kinase.     

T cell activation by coxsackievirus B4 antigens in type 1 diabetes mellitus: evidence for selective TCR Vbeta usage without superantigenic activity.

Numerous clinical and epidemiological studies link enteroviruses such as the Coxsackie virus group with the autoimmune disease type 1 diabetes mellitus (DM). In addition, there are reports that patients with type 1 DM are characterized by skewing of TCR Vbeta chain selection among peripheral blood and intraislet T lymphocytes. To examine these issues, we analyzed TCR Vbeta chain-specific up-regulation of the early T cell activation marker, CD69, on CD4 T cells after incubation with Coxsackievirus B4 (CVB4) Ags. CD4 T cells bearing the Vbeta chains 2, 7, and 8 were the most frequently activated by CVB4. Up-regulation of CD69 by different TCR families was significantly more frequent in new onset type 1 DM patients (p = 0.04), 100% of whom (n = 8) showed activation of CD4 T cells bearing Vbeta8, compared with 50% of control subjects (n = 8; p = 0.04). T cell proliferation after incubation with CVB4 Ags required live, nonfixed APCs, suggesting that the selective expansion of CD4 T cells with particular Vbeta chains resulted from conventional antigen processing and presentation rather than superantigen activity. Heteroduplex analysis of TCR Vbeta chain usage after CVB4 stimulation indicated a relatively polyclonal, rather than oligo- or monoclonal response to viral Ags. These results provide evidence that new-onset patients with type 1 DM and healthy controls are primed against CVB4, and that CD4 T cell responses to the virus have a selective TCR Vbeta chain usage which is driven by viral Ags rather than a superantigen.     

A serine/threonine phosphorylation site in the ectodomain of a T cell receptor beta chain is required for activation by superantigen

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post-translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR alpha and beta chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR alpha and beta chains was accomplished using site-directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL-2 in response to substrate-immobilized antibody to TCR. However, mutation of serine-88 in the protein kinase A phosphorylation site of the TCR beta chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR-associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine-88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three-dimensional structure of the complex of SEB and TCR, are consistent with the view that serine-88 is important for the contact of the TCR beta chain with SEB.     

A Serine/Threonine Phosphorylation Site in the Ectodomain of a T Cell Receptor β Chain is Required for Activation by Superantigen

Abstract

The presence of consensus phosphorylation sites in the ectodomains of cell surface proteins suggests that such post‐translational modification may be important in regulation of surface receptor activity. To date, the only cell surface receptor for which such ectodomain phosphorylation has been conclusively demonstrated is the clonally expressed T cell antigen receptor (TCR). Attempts to conclusively identify individual phosphorylated residues in TCR α and β chains and determine their functional significance by biochemical approaches failed due to insufficient quantities of purified molecules. Here we present the results of an alternative approach where survey of phosphorylation sites in the TCR α and β chains was accomplished using site‐directed mutagenesis and retroviral vector expression, as well as in vitro phosphorylation of synthetic peptide substrates. All mutants studied directed the cell surface expression of normal amounts of TCR, and all transfectants could be stimulated to produce IL‐2 in response to substrate‐immobilized antibody to TCR. However, mutation of serine‐88 in the protein kinase A phosphorylation site of the TCR β chain resulted in a complete lack of response to the superantigen staphylococcal enterotoxin B (SEB). In addition, this mutation abolished TCR‐associated tyrosine phosphorylation, consistent with the impairment of cell signaling. Reversion of the serine‐88/alanine mutation with phosphorylatable threonine completely restored the SEB recognition by TCR. These results, interpreted in the context of the known three‐dimensional structure of the complex of SEB and TCR, are consistent with the view that serine‐88 is important for the contact of the TCR β chain with SEB.     

Differential susceptibility to staphylococcal superantigen (SsAg)-induced apoptosis of CD4+ T cells from atopic dermatitis patients and healthy subjects: the inhibitory effect of IL-4 on SsAg-induced apoptosis

This study had two aims: 1) to determine whether there are differences between atopic dermatitis (AD) patients and healthy subjects in staphylococcal superantigen (SsAg)-induced CD4(+) T cell activation, cytokine production, chemokine receptor expression, and apoptosis; and 2) to investigate the effect of IL-4 on SsAg-induced apoptosis. By using immunofluorescence and annexin V staining, we analyzed PBMC with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of rIL-4 or anti-IL-4-neutralizing Abs in 15 healthy subjects and 27 AD patients. We found that SEB preferentially induced production of Th1 cytokine in SEB-reactive (TCRVbeta3(+) or Vbeta12(+) or Vbeta17(+)) CD4(+) T cells from healthy subjects and Th2 cytokine in those from AD patients. SEB induced up-regulation of CXCR3(+) cells in SEB-reactive CD4(+) T cells from healthy subjects and CCR4(+) cells in those from AD patients. SEB-reactive CD4(+) T cells from AD patients were more resistant to SEB-induced apoptosis than those from healthy subjects. There was no significant difference between AD and healthy subjects in SEB-induced activation of CD4(+) T cells. CXCR3(+) CD4(+) T cells were more susceptible to SEB-induced apoptosis than CCR4(+) CD4(+) T cells in healthy subjects. Exogenously added IL-4 inhibited SEB-induced apoptosis of SEB-reactive CD4(+) and CXCR3(+) CD4(+) T cells but not of CCR4(+) CD4(+) T cells in healthy subjects. Inhibition of endogenous IL-4 increased SEB-induced apoptosis of SEB-reactive CD4(+) T cells from AD patients. These results might provide new clues to the mechanism that SsAgs contribute to the persistence and exacerbation of allergic skin inflammation in AD.     

Vbeta14(+) T cells mediate the vaccine-enhanced disease induced by immunization with respiratory syncytial virus (RSV) G glycoprotein but not with formalin-inactivated RSV

Mice immunized with respiratory syncytial virus (RSV) G glycoprotein or with formalin-inactivated RSV (FI-RSV) exhibit severe disease following RSV challenge. This results in type 2 cytokine production and pulmonary eosinophilia, both hallmarks of vaccine-enhanced disease. RSV G-induced T-cell responses were shown to be restricted to CD4(+) T cells expressing Vbeta14 in the T-cell receptor (TCR), and the deletion of these T cells resulted in less severe disease. We therefore examined the role of Vbeta14(+) T cells in FI-RSV-induced disease. BALB/c mice were immunized with vaccinia virus expressing secreted RSV G (vvGs) or with FI-RSV. At the time of challenge with live RSV, mice were injected with antibody to the Vbeta14 component of the TCR. vvGs-immunized mice treated with anti-Vbeta14 had reduced cytokine levels in the lung. Eosinophil recruitment to the lung was also significantly reduced. In contrast, depletion of Vbeta14(+) T cells in FI-RSV-immunized mice had little impact on cytokine production or pulmonary eosinophilia. An analysis of TCR Vbeta chain usage confirmed a bias toward Vbeta14 expression on CD4(+) T cells from vvGs-immunized mice, whereas the CD4(+) T cells in FI-RSV-immunized mice expressed a diverse array of Vbeta chains. These data show that although FI-RSV and vvGs induce responses resulting in similar immunopathology, the T-cell repertoire mediating the response is different for each immunogen and suggest that the immune responses elicited by RSV G are not the basis for FI-RSV vaccine-enhanced disease.     

CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery

BACKGROUND: CD69 is a surrogate marker of T-cell responsiveness to mitogen and Ag stimulus and can be used as a measure of T-lymphocyte activation. Quantitative flow cytometric determination of CD69 expression on T lymphocytes has several advantages over traditional lymphocyte proliferation assays, but this method has not yet been standardized for clinical applications. METHODS: We qualified a commercially available assay using the manufacturer's procedures for measurement of T-cell response to a mitogen (PHA), superantigen (Staphylococcus endotoxin B; SEB) and Ca(2+) ionophore (phorbyl myristate acetate; PMA) with peripheral blood from healthy volunteers. Following this, we tested the usefulness of the assay in determining T-cell responses to PHA and SEB for six immunocompromised patients. RESULTS: Healthy volunteers showed 17-fold increases in T-cell CD69 Ab bound per cell (ABC) with PHA stimulation compared with the baseline. SEB was also an effective T-cell activating agent, increasing CD69 ABC by 5-fold, comparable with results obtained with PMA stimulation. PHA- and SEB-stimulated T-cell CD69 ABC for patients 100 days post-BM transplant were generally below 1 SD of that from healthy volunteers. SEB-stimulated T-cell CD69 expression was significantly depressed for CD8(+) T cells while CD4(+) T-cell responses to SEB were generally within 1 SD of the mean for healthy volunteers. DISCUSSION: These results suggest that quantitative measurement of CD69 surface expression by flow cytometry is a useful diagnostic tool for detailed assessment of T-lymphocyte and subset activation.     

Overexpressed Bcl-x(L) prevents bacterial superantigen-induced apoptosis of thymocytes in vitro

bcl-x, a homologous gene of bcl-2, has an anti-apoptotic function and appears to play a critical role in the development of lymphoid systems. To investigate the effect of overexpressed Bcl-x(L) on the development of T lymphocytes, we established two lines of transgenic mice by using Emu-chicken bcl-x(L) (cbcl-x(L)) transgene, where the cBcl-x(L) protein was expressed mainly in lymphoid cells. Although thymocytes and splenocytes from cbcl-x(L) transgenic mice are resistant to apoptosis in vitro, clonal deletion of thymocytes, recognizing endogenous self-superantigens in the thymus, still normally proceeded and no self-reactive T cells were found in the spleen of the transgenic mice. To dissect clonal deletion, we utilized two in vitro models, thymocytes/antigen presenting cells co-culture system and fetal thymus organ culture system. In both, bacterial superantigen staphylococcus aureus enterotoxin B (SEB) induces apoptosis of T cells with Vbeta8+ T cell receptor (TCR) reacting to SEB, which mimics clonal deletion of self-reactive thymocytes in vivo. SEB-induced depletion of Vbeta8+ T cells from thymocytes when taken from the transgenic mice was effectively inhibited. The data might raise the possibility that cell death process involved in clonal deletion in the thymus is a form of apoptosis inhibited by Bcl-x(L).     

Genetic control of T and B lymphocyte activation in nonobese diabetic mice.

Type 1 diabetes in nonobese diabetic (NOD) mice is characterized by the infiltration of T and B cells into pancreatic islets. T cells bearing the TCR Vbeta3 chain are disproportionately represented in the earliest stages of islet infiltration (insulitis) despite clonal deletion of most Vbeta3(+) immature thymocytes by the mammary tumor virus-3 (Mtv-3) superantigen (SAg). In this report we showed that a high frequency of NOD Vbeta3(+) T cells that escape deletion are activated in vivo and that this phenotype is linked to the Mtv-3 locus. One potential mechanism of SAg presentation to peripheral T cells is by activated B cells. Consistent with this idea, we found that NOD mice harbor a significantly higher frequency of activated B cells than nondiabetes-prone strains. These activated NOD B cells expressed cell surface molecules consistent with APC function. At the molecular level, the IgH repertoire of activated B cells in NOD mice was equivalent to resting B cells, suggesting a polyclonal response in vivo. Genetic analysis of the activated B cell phenotype showed linkage to Idd1, the NOD MHC haplotype (H-2(g7)). Finally, Vbeta3(+) thymocyte deletion and peripheral T cell activation did not require B cells, suggesting that other APC populations are sufficient to generate both Mtv-3-linked phenotypes. These data provide insight into the genetic regulation of NOD autoreactive lymphocyte activation that may contribute to failure of peripheral tolerance and the pathogenesis of type I diabetes.     

T cell receptor revision does not solely target recent thymic emigrants

CD4(+)Vbeta5(+) T cells enter one of two tolerance pathways after recognizing a peripherally expressed superantigen encoded by an endogenous retrovirus. One pathway leads to deletion, while the other, termed TCR revision, results in cellular rescue upon expression of an alternate TCR that no longer recognizes the tolerogen. TCR revision requires the rearrangement of novel TCR beta-chain genes and depends on recombinase-activating gene (RAG) expression in peripheral T cells. In line with recent findings that RAG(+) splenic B cells are immature cells that have maintained RAG expression, it has been hypothesized that TCR revision is limited to recent thymic emigrants that have maintained RAG expression and TCR loci in a recombination-permissive configuration. Using mice in which the expression of green fluorescent protein is driven by the RAG2 promoter, we now show that in vitro stimulation can drive reporter expression in noncycling, mature, peripheral CD4(+) T cells. In addition, thymectomized Vbeta5 transgenic RAG reporter mice are used to demonstrate that TCR revision can target peripheral T cells up to 2 mo after thymectomy. Both sets of experiments strongly suggest that reinduction of RAG genes triggers TCR revision. Approximately 3% of CD4(+)Vbeta5(+) T cells in thymectomized Vbeta5 transgenic reporter mice have undergone TCR revision within the previous 4-5 days. TCR revision can also occur in Vbeta5(+) T cells from nontransgenic mice, illustrating the relevance of this novel tolerance mechanism in unmanipulated animals.     

Murine Vbeta3+ and Vbeta7+ T cell subsets are specific targets for the HERV-K18 Env superantigen

Superantigens are a class of proteins that are derived from microorganisms and have the unique characteristic of stimulating T cells in a TCR Vbeta-specific manner, causing massive T cell proliferation and immune deregulation. For this reason, superantigens have been implicated in the development of multiple diseases. We have previously identified and cloned an EBV-associated superantigen, human endogenous retrovirus (HERV)-K18 envelope protein (Env). This superantigen is transactivated upon IFN-alpha treatment and EBV infection and stimulates human Vbeta13+ T cells. Due to the limited scope of work that can be conducted with human samples and the complexity of HERVs in general, we set out to study the physiological effects of HERV-K18 Env in a murine model. In this report, we demonstrate the superantigen activity of HERV-K18 Env in mice and describe the generation of HERV-K18 transgenics, using a bacterial artificial chromosome as transgenes that allow the faithful reproduction of the expression pattern of this human provirus. From our in vitro and in vivo results we conclude that HERV-K18 Env stimulates Vbeta3+ and Vbeta7+ T cells in mice. The definition of the murine Vbeta specificity and the establishment of a transgenic model will permit the investigation of the role of this superantigen in the life cycle of EBV and its implicated diseases.     

Distribution of cycling T lymphocytes in blood and lymphoid organs during immune responses

Proliferation of murine T lymphocytes in blood, lymph nodes, and spleen was studied in four in vivo stimulation systems, using BrdU pulse-labeling of DNA-synthesizing cells. The T cell response to the superantigen Staphylococcus enterotoxin B (SEB) was studied in detail. Vbeta8+ T cells showed a peak of DNA synthesis 16-24 h after SEB injection, and the percentage of BrdU+ CD4 and CD8 T cells was higher in blood than in lymph nodes and spleen. DNA synthesis was preceded by massive migration of Vbeta8+ cells from blood to lymphoid organs, in which the early activation marker CD69 was first up-regulated. SEB-nonspecific Vbeta6+ cells showed minimal stimulation but, when cycling, also expressed a high level of CD69. The other systems studied were injection of the IFN-gamma inducer polyinosinic:polycytidylic acid, infection by the BM5 variants of murine leukemia virus (the causative agent of murine AIDS), and T cell expansion after transfer of normal bone marrow and lymph node cells into recombinase-activating gene-2-deficient mice. In each case, a peak of T cell proliferation was observed in blood. These data demonstrate the extensive redistribution of cycling T cells in the first few hours after activation. Kinetic studies of blood lymphocyte status appear crucial for understanding primary immune responses because cycling and redistributing T lymphocytes are enriched in the circulating compartment.     

Generated single point-mutations can considerably dismantle the lymphocyte overstimulation induced by Yersinia pseudotuberculosis superantigen

The superantigenic Yersinia pseudotuberculosis-derived mitogen (YPM) may contribute to severe complications in Y. pseudotuberculosis infections. Since the pathogenic mechanism of a superantigen (SAg) is based on its capability for T-cell overstimulation, by introducing point mutations into YPM an attempt was made to abrogate this effect. Six mutants studied exhibited a variety of T-cell proliferating responses. Two had activity reduced by 80-90%, three had activity reduced by approximately 50%, and one mutant showed almost no attenuation. The SAg-associated in vitro pathogenic functions, cytotoxic activation and the production of proinflammatory cytokines, were also diminished, in parallel. Since these mutants were confirmed to be defective in TCR Vbeta binding, it was possible to compare them with native YPM. Our results suggested that the intensity of TCR Vbeta binding is a crucial factor determining the severity of pathogenesis and that single amino acid alterations might be useful for producing immunotherapeautical agents from native YPM.     

Cutting edge: apoptosis of superantigen-activated T cells occurs preferentially after a discrete number of cell divisions in vivo.

Staphylococcal enterotoxins are bacterial products that display superantigen activity in vitro as well as in vivo. For instance, staphylococcal enterotoxin B (SEB) polyclonally activates T cells that bear the Vbeta8 gene segment of the TCR. SEB-activated T cells undergo a burst of proliferation that is followed by apoptosis. Using an in vivo adaptation of a fluorescent cell division monitoring technique, we show here that SEB-activated T cells divide asynchronously, and that apoptosis of superantigen-activated T cells is preferentially restricted to cells which have undergone a discrete number of cell divisions. Collectively, our data suggest that superantigen-activated T cells are programmed to undergo a fixed number of cell divisions before undergoing apoptosis. A delayed death program may provide a mechanistic compromise between effector functions and homeostasis of activated T cells.