Induction of clonal anergy by oral administration of staphylococcal enterotoxin B

The staphylococcal enterotoxin is a major cause of food poisoning. The bacterial substance stimulates T cells expressing specific Vβ T cell receptors (TcR) and is termed “the superantigen”. We have previously demonstrated that intravenous injection of staphylococcal enterotoxin B (SEB) induces functional unresponsiveness (anergy) of reactive T cells as well as a partial deletion by activationinduced programmed cell death. In the present study, we examined the effect of oral administration of SEB in mice. Our results indicate that spleen T cells from SEB-primed mice are hyporesponsive to SEB stimulation in vitro, but the response to SEA was normal. Vβ8⁺ T cells purified from SEB-primed mice did not respond to stimulation of TcR. This SEB-specific unresponsiveness could not be reversed by exogenous interleukin-2, but was partially reversed by phorbol 12-myristate 13-acetate. Activation of mitogen-activated protein kinase during TcR-mediated stimulation was significantly inhibited in anergic T cells. Although the mechanisms of oral tolerance are not well understood, these results show that oral administration of SEB induce clonal anergy in peripheral T cells.     

Superantigens and conventional antigens induce different responses in alpha beta T-cell receptor transgenic mice.

While superantigens such as staphylococcal enterotoxin B (SEB) have been shown to induce both clonal deletion and clonal anergy, it is still not known why tolerance rather than memory is induced. To address this issue, we tested the proliferative capacity of T cells from ovalbumin (OVA)-specific alpha beta T-cell receptor transgenic mice primed with either SEB emulsified in complete Freund's adjuvant (CFA) or with OVA peptide, the specific antigen, in CFA. By contrast cells from mice primed with SEB in CFA appeared to be anergic in that they were hyporesponsive to OVA peptide as well as to SEB. The anergic cells could respond to phorbol myristate acetate (PMA) and ionomycin, suggesting that a proximal signal transduction step was affected. Cells from transgenic mice primed with OVA peptide and CFA were not anergic and in fact displayed an enhanced response when they were challenged with OVA in vitro. Thus, when the two antigens are emulsified in CFA and then injected subcutaneously, they behave very differently: the superantigen SEB induces anergy whereas the conventional antigen OVA induces a memory type of response.     

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.     

Effect of Treatments with Cyclosporin A and Anti-Interferon-γ Antibodies on the Mechanisms of Immune Tolerance in Staphylococcal Enterotoxin B Primed Mice

The authors were interested to investigate the effect of Cyclosporin A (CsA), known to block interleukin-2 (IL-2) production, or of anti-interferon-γ antibodies (anti-IFN-γ Abs) in a model of T cell tolerance induced by the injection of the superantigen Staphylococcal Enterotoxin B (SEB) in BALB/c mice. After SEB immunization, tolerance was mainly achieved through deletion and anergy of SEB-reactive Vβ8⁺ T cells. Association of CsA treatment with SEB led to a greater decrease of the percentage of Vβ8⁺ CD4⁺ lymphocytes in the spleen and an abolition of clonal anergy. In contrast, treatment of SEB primed mice with anti-IFN-γ Abs resulted in an increased percentage of Vβ8⁺ CD4⁺ cells without affecting the induction of clonal anergy. The authors found that 1–2 h after SEB priming, splenic mRNA levels of IFN-γ and IL-4 were decreased by either CsA and anti-IFN-γ Abs, whereas FasL, Bcl-2, p. 53, and c-myc levels were not influenced by either treatment. However, SEB-induced IL-2 and IL-10 mRNA expression was suppressed only by CsA, whereas tumour necrosis factor-α (TNF-α) was decreased only by anti-IFN-γ Abs. To investigate whether the effect of CsA on the tolerance mechanisms was related to suppression of IL-2, CsA was administered together with recombinant IL-2. Whereas anergy was not influenced, the decreased percentage of Vβ8⁺ CD4⁺ cells seen in CsA-treated animals in the second week after SEB injection was partially corrected by the administration of IL-2. Experiments involving bromodeoxiuridine incorporation revealed that the latter effect of IL-2 was mainly due to a correction of the defective proliferation of Vβ8⁺ T cells after SEB injection in CsA-treated mice. These results suggest that the effect of CsA and anti-IFN-γ Abs on tolerance mechanisms are in part explained by their action on cytokines.     

Interleukin-2, a pro-autoimmune lymphokine that interferes with post-deletional tolerance.

Various complementary mechanisms arranged in a fail-safe hierarchy impede the immune system from launching auto-aggressive attacks, namely intrathymic or post-thymic clonal deletion, anergy, and immunosuppression. Both epidemiological and clinical evidence demonstrates that interleukin-2 (IL-2) may exert a pro-autoimmune effect. Thus, IL-2 reverses immunological tolerance in determined in vitro or in vivo circumstances, is produced at abnormally high levels in certain autoimmune diseases, and induces organ-specific autoimmune lesions when administered to patients. To unravel the molecule and cellular mechanisms responsible for the pro-autoimmune nature of IL-2, our group has employed a recombinant hIL-2 vaccinia virus construct (IL-2. VV) as a self-replicating IL-2-releasing device that may be administered as a source of exogenous IL-2 to experimentally manipulated mice. IL-2 does not interfere with clonal deletion of potentially autoreactive T cells in the thymus since application of IL-2. VV to young mice fails to augment the frequency of T cells bearing 'forbidden' T cell receptor (TCR) V beta gene products (i.e. T cells that recognize endogenous retroviral superantigens). Along the same line, IL-2. VV does not reverse the clonal deletion in male mice bearing a male-specific transgenic alpha/beta TCR that undergo depletion of CD4+ CD8+ thymocytes, nor does it abolish post-thymic clonal deletion of V beta 8+ T cells reactive with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB). In contrast with its incapability to abolish intra- and post-thymic elimination of T cells with unwarranted specificities, IL-2 abrogates anergy of T cells. Non-deleted, 'forbidden' T cells from congenitally athymic or neonatally thymectomized mice, under normal circumstances anergic, become responsive to TCR-triggering and mediate a systemic autoimmune syndrome upon IL-2. VV treatment. Thus, IL-2 appears to interfere with T cell tolerance at a post-deletional stage, reversing functional non-responsiveness and enabling non-deleted T cells that bear a potentially autoreactive TCR repertoire to cause manifest autoimmunity.     

Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB-reactive T cells bearing T cell receptor (TcR) V_β8 gene products. Given that interleukin 2 (IL-2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL-2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL-2 modulate SEB responses in vivo. Surprisingly, neither IL-2 nor CsA were able to change the in vivo kinetics and magnitude of SEB-induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB-reactive TcR V_β8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing “anergy” and apoptotic cell death of V_β8⁺ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V_β8⁺ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)α chains in vivo, although SEB induces IL-2R α in vitro. Altogether, these results indicate that the IL-2/IL-2R-mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of V_β8⁺ T cells from SEB-primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL-2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.     

Interleukin-2 allows in vivo induction of anti-erythrocyte autoantibody production in nude mice associated with the injection of rat erythrocytes.

Mice injected with rat red blood cells developed anti-erythrocyte autoantibodies detectable by a direct Coombs' test. Nude mice injected with rat red blood cells did not develop a Coombs-positive state, but nude mice injected with rat red blood cells plus the T cell helper factor, interleukin-2, produced autoantibodies to autologous mouse erythrocytes. The simultaneous injection of rat red blood cells and allogeneic spleen cells induced an early and vigorous autoantibody response in athymic nude mice as well as in euthymic control mice. These results are interpreted as indicating the possibility of an interleukin-2-stimulated in vivo differentiation (or clonal expansion) of helper T cells in nude mice in response to heterologous erythrocytes which could mediate an autoimmune B cell response.     

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.     

Photodynamic Virus Inactivation of Blood Components

Staphylococcal enterotoxin B (SEB) can activate specific T cell clones bearing specific TcR Vβ domains together with MHC class II ligands on accessory cells. The release of proinflammatory cytokines is the consequence of this activation as well as the main pathological aspect involved in SEB infection. This current study looked at the active role of both T and B cells during the induction of anergy by SEB in vivo Euthymic and nude BALB/c mice were injected with SEB and over a period of 8 days, cells from the spleen and sera from the blood were collected. After a single injection with SEB (50 μg/mouse), a transient increase of CD4⁺Vβ8⁺ T cells were detected after 2 days followed by a decrease after 4 days, which persisted until day 8. These clones were rendered anergic upon restimulation in vitro with SEB. Interestingly, cells taken out 2 days after SEB injection, exhibited reduced proliferation in response to Con A. However, this response gradually recovered on days 4, 6 and 8. Furthermore, early IgM antibody production (day 2) was observed after SEB injection. SEB-induced IgM antibody production in euthymic BALB/c was found to have specificity against SEB, cardiolipin (CL) and phosphatidylethanolamine (PE). SEB-treated nude mice did not produce antibody secreting cells in response to SEB, indicating that this process is T cell dependent.     

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.     

Superantigen and endotoxin synergize in the induction of lethal shock

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-γ (IFN-γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D -galactosamine (D -GalN). Opposed to D -GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.     

Interleukin-2: a possible trigger for autoimmunity.

High doses of recombinant interleukin-2 (IL-2) may induce autoimmune lesions in patients receiving experimental cancer treatment. In most cases, the manifestation of autoaggression is transient and organ-specific, predominantly affecting the thyroid gland. Only a fraction of the patients are concerned; most individuals (around 90%) do not develop any signs of autoimmunity. Apparently, endogenously hyperproduced IL-2 may also be implicated in the pathogenesis of autoaggression, since active phases of such disparate autoimmune diseases, like multiple sclerosis and systemic lupus erythematosus, are accompanied by elevated IL-2 serum levels. Taking into account that immunological self-tolerance is maintained by several distinct mechanisms, we investigated whether IL-2 would interfere with clonal deletion or clonal anergy in vivo. In several experimental systems, IL-2 failed to abolish clonal deletion in the murine thymus or in the peripheral T-cell compartment. IL-2 did not affect the clonal deletion of self-reactive B cells in the bone marrow either. In contrast, IL-2 was found to be effective in abrogating clonal anergy of non-deleted self-specific T cells. Only in the presence of high frequencies of self-specific, potentially autoreactive T cells, IL-2 induces autoimmune lesions. Thus, IL-2 interferes with a mechanism of self-tolerance that guarantees the inactivation of T cells that for some reason have 'escaped' clonal deletion. If these data, obtained in the murine system, are extrapolated to man, then it may be stated that the T-cell repertoire of most individuals has been completely purged from self-reactive cells. Only in the presence of a non-deleted, anergic, potentially auto-reactive T-cell population, could organ-specific disease be induced by IL-2.     

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     

Thymus-derived cytokine(s) including interleukin-7 induce increase of T cell receptor α/β+ CD4−CD8− T cells which are extrathymically differentiated in athymic nude mice

Extrathymic T cell differentiation pathways have been reported, although the thymus is the main site of T cell differentiation. The thymus is also known to produce several cytokines that induce proliferation of thymocytes. In the present study, we investigated the influence of thymus-derived cytokines on extrathymic T cell differentiation by intraperitoneal implantation with a diffusion chamber which encloses fetal thymus (we named it fetal thymus-enclosed diffusion chamber, FTEDC) in athymic BALB/c nu/nu mice. Increase in number of T cells bearing T cell receptor (TcR) α/β was detected in lymph nodes and spleens of FTEDC-implanted nude mice 1 week after implantation, whereas no such increase was detected in control nude mice implanted with a diffusion chamber without thymus. The FTEDC-induced increase of T cells was suppressed by intraperitoneal injection of anti-interleukin-7 monoclonal antibody (mAb). The TcR α/β T cells in FTEDC-inplanted BALB/c nu/nu mice preferentially expressed Vβ11, although Vβ11-positive T cells are deleted in the thymus of euthymic BALB/c mice by clonal elimination of self-superantigen Dvb 11-specific T cells. TcR α/β T cells in FTEDC-implanted nude mice were of CD4^?CD8^? phenotype and showed no proliferative response against anti-TcR monoclonal antibody stimulation. These results suggest that the thymus can induce extrathymic T cell differentiation through the influence of thymus-derived cytokine(s) including interleukin-7, and that such extrathymically differentiated T cells have acquired only a little or no ability for proliferation when they recognize antigen by their TcR.     

At low precursor frequencies,the T‐cell response to chronic self‐antigen results in anergy without deletion

The behavior of self‐reactive T cells in the peripheral immune system has often been studied by following the fate of adoptively transferred antigen‐specific T cells in antigen expressing mice. In most cases, after a period of expansion, such cells undergo a slow clonal deletion, accompanied by the onset of anergy and/or suppression in the remaining cells. Here, we demonstrate that at initial frequencies approaching those found in normal repertoires, it is possible to completely avoid deletion and still maintain peripheral tolerance. At starting numbers of <1000 T cells, stimulation by chronic self‐antigens resulted in a period of robust clonal expansion, followed by a steady plateau phase extending beyond 4 months. Despite their stable persistence, the self‐reactive T cells did not convert to a Foxp3+ fate. However, they displayed a considerable block in their ability to make IL‐2, consistent with the onset of anergy — in a precursor frequency or deletion independent fashion.     

Mycobacterium bovis BCG-infected mice are more susceptible to staphylococcal enterotoxin B-mediated toxic shock than uninfected mice despite reduced in vitro splenocyte responses to superantigens

Type 1 T-cell responses against intracellular pathogens play a crucial role in mediating protection. We examined whether the induction of a strong type 1 T-cell response during a chronic bacterial infection influences responses to superantigens capable of inducing acute shock. Intravenous infection of mice with Mycobacterium bovis BCG appeared to induce a progressive anergy towards staphylococcal enterotoxin B (SEB) and towards antigen preparation of BCG (BCG-Ag) itself, based on diminished gamma interferon (IFN-gamma) production by SEB- and BCG-Ag-stimulated splenocytes from infected mice. In contrast to these in vitro results, injection of SEB into BCG-infected mice led to a dramatic increase in the serum IFN-gamma levels and the death of infected but not of control mice. In vitro hyporesponsiveness towards SEB and BCG-Ag occurred only with unfractionated splenocyte cultures, as purified T cells from infected mice produced higher levels of IFN-gamma. Hyporesponsiveness towards SEB and BCG-Ag in unfractionated splenocyte cultures was not due to suppressive antigen-presenting cells (APCs), as APCs from infected mice stimulated higher levels of IFN-gamma from purified T cells. The diminished IFN-gamma levels observed with bulk splenocytes appear to be due to changes in the T-cell-to-APC ratio that result in a decreased proportion of T cells, coupled to reduced proliferative responses and an increased susceptibility of effector T cells to activation-induced cell death in vitro. Our results indicate that the reported phenomena of T-cell anergy during mycobacterial infection may be an in vitro consequence of the development of a strong type 1 response in vivo.     

Aged mice exhibit in vivo defective peripheral clonal deletion of D(b)/H-Y reactive CD8(+) T cells

We previously reported that T cells from aged mice were resistant to activation-induced cell death (AICD) in vitro. To determine whether the presence of AICD-resistant T cells is associated with defects in age-related peripheral clonal deletion in vivo, congenic male SCID mice were reconstituted with T cells from aged or young female D(b)/H-Y TCR (Tg71) transgenic mice. Compared with recipients of young cells, the recipients of T cells from aged mice exhibited a 3-fold increase in the percentage of autoreactive CD8(+) H-Y antigen-reactive T cells as defined by the clonotypic antibody, M33. There were significantly increased sera levels of interferon-gamma, a significantly decreased expression of FasL by M33(+)CD8(+) T cells, and significantly decreased apoptosis by DNA fragmentation staining of the spleen of mice reconstituted with T cells from aged mice compared to those from young mice. By day 21, the recipients of T cells from aged mice but not young mice, exhibited infiltration of CD3(+) cells into the non-lymphoid organs. These results indicate that there is defective peripheral deletion of the self-reactive T cells derived from aged female Tg71 mice, and that failure to delete these cells is associated with the defective T-cell clonal deletion in the recipient mice.     

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.     

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.