Costimulatory molecule requirement for bovine WC1+gammadelta T cells' proliferative response to bacterial superantigens

We have previously shown that the proliferation of freshly isolated bovine WC1+gammadelta T cells to superantigens (SAgs) including staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) or toxic shock syndrome type-1 (TSST-1) required the presence of antigen-presenting cells (APC) and the addition of exogenous interleukin (IL)-2. The costimulatory activity provided by molecules expressed on professional APC for the proliferation of gammadelta T cells has not been addressed hitherto. In the present study, we investigated the ability of two selected APC populations, the dendritic cells (DCs) highly expressing CD80 and CD86 molecules (CD80highCD86high) and the monocytes expressing the same molecules at a rather low level (CD80lowCD86low), to stimulate the proliferation of purified bovine WC1+gammadelta T cells to SAgs. DCs were more efficient than monocytes in inducing gammadelta T-cell proliferation, and this response was dependent on exogenous IL-2 in both presentation modes. Stimulating gammadelta T cells with gradual doses of SAgs or concanavalin A (ConA) resulted in similar dose-dependent reaction profiles suggesting a minimal role of the major histocompatibility complex (MHC). However, significant proliferation was already obtained with the starting doses in the presence of DC compared with monocytes, and higher proliferation was reached with DC at optimal doses. Finally, the addition of monoclonal antibody (MoAb) anti-CD86 markedly inhibited SAgs- and ConA-mediated proliferation, whereas MoAb anti-CD80 had no effect. The combination of both anti-CD80 and anti-CD86, however, suppressed this response. These results suggest that bovine gammadelta T-cell proliferation response requires indubitably CD86 costimulation. The role of CD80 molecule seems less clear.     

A role for antigen-presenting cells and bacterial superantigens in reversal of human T lymphocyte anergy

The induction of anergy in T lymphocytes generates T cells incapable of proliferation in response to a conventional antigenic stimulus. To investigate the induction and maintenance of anergy in human T cells, we used T cell-T cell presentation of myelin basic protein (MBP) or MBP synthetic peptides to induce anergy in vitro. Although anergic T cells responded normally to interleukin-2 (IL-2), these T cells did not produce IL-2 or IL-4 when peripheral blood mononuclear cells presented MBP or MBP peptides. Proliferation of anergic T cells was reduced by greater than 95% compared to nonanergic, control T cells. However, when autologous B cell lines were used to present MBP, anergy was partially reversed with a proliferation response about 50% of nonanergic levels. Bacterial superantigens also partially restored proliferation in anergic T cells following presentation by either B cell lines or macrophage isolated from peripheral blood mononuclear cells. Anergic, MBP-reactive T cells fully retained antigen-specific cytolytic activity against both B cell and T cell targets presenting MBP. These results suggest that T cell proliferative anergy may be reversible with both the type of antigen-presenting cell and superantigens potentially contributing to the initiation or maintenance of an autoimmune response.     

Mechanisms of age-related decline in antigen-specific T cell proliferative response: IL-2 receptor expression and recombinant IL-2 induced proliferative response of purified Tac-positive T cells

Proliferative response of T cells from aged persons was significantly reduced to a specific antigen tuberculin-active peptide (TAP) determined by [3H]TdR uptake and FCM in comparison to that from the young. Cytokinetic analysis for the proliferative response to TAP showed that, in the aged, the clonal size or the number of the first generation responding cells to TAP was not significantly reduced but the ability to repeat replication was more profoundly affected. Neither the delayed entry into the cell replication nor prolongation of the cell cycle time could explain these results. Similar results have been reported on the proliferative response of T cells to mitogen: PHA (Phytohemagglutinin). Expression of Tac-antigen on T cells determined by anti-Tac antibody binding with FACS after stimulation with either TAP or PHA was found to be reduced significantly in the aged. Both the numbers of high and low affinity IL-2 receptors determined by radiolabelled IL-2 binding assay were also reduced in the aged, but the degree of reduction in number of high affinity ones was more pronounced than that in low affinity ones. Tac-positive T cells were isolated with the use of anti-Tac rosette methods and stimulated with recombinant IL-2 (r-IL-2). Their proliferative response was significantly lower in the aged than that in the young at any concentration of r-IL-2 examined. The number of the first generation responding cells to r-IL-2 in purified Tac-positive T cells from the aged was 82% of that from the young whereas the proliferative response by aged T cells was 39% of that by young ones when the cells were allowed to repeat replication for 3 days. The mechanisms of these multifactorial defects in proliferation of T cells from aged persons were discussed.     

T cell clones interacting with accessory and T helper cells for a proliferative response

The requirement for cell interactions in T cell activation has been studied with two continuously in vitro growing T cell clones. These clones are specific for minor histocompatibility antigens, are H-2K restricted, and one clone is functionally a cytolytic T lymphocyte. Both can proliferate when interleukin 2 is added to the cultures, but for continuous growth they require irradiated spleen cells carrying the specific minor histocompatibility antigen and the restricting H-2. In this study we show that for proliferation the clones require at least two cell populations in the stimulator spleen, one is a splenic-adherent cell (SAC), the other a T cell. The SAC are plastic adherent, Thy-1-, Ia+. The T cells are nylon wool nonadherent, Thy-1+, Lyt-1+2- and Ia-. Cell mixing experiments of stimulator cells (all were done with H-2-syngeneic cells), depleted of either SAC or T cells confirm the requirement for a specific interaction between these two cell types and the T clone. Neither SAC, syngeneic with the T clone when mixed with T cells of the stimulator type, nor T cells syngeneic with the clone added to stimulator SAC, can induce an optimal proliferative response. Such a response is obtained only if both cell types, SAC and T cells, are of the stimulating genotype. This suggests that, in addition to an interaction of clonal T cells with SAC, a specific recognition at the T cell level between T stimulator and T clone is necessary. The interaction of the T clones with stimulator SAC and T cells leads to an activation, mediated by antigen recognition, of all three cell populations. Since we also show that each of the stimulator cell types are impaired by ultraviolet light irradiation, we conclude that factor production by SAC and T helpers is the final prerequisite for clonal expansion.     

T regulatory cells in atopic dermatitis and subversion of their activity by superantigens

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease involving colonization by superantigen (SAg)-secreting Staphylococcus aureus. CD4+CD25+ T regulatory (Treg) cells are thought to play an important role in controlling inflammatory responses. OBJECTIVE: In this study we examined whether Treg cells might be deficient in patients with AD. METHODS: CD4+CD25+ and CD4+CD25- T cells were isolated from PBMCs by using immunomagnetic beads. Cells were cultured with anti-CD3 or SAg, staphylococcal enterotoxin B (SEB), for 72 hours. Proliferation was measured by means of tritiated thymidine incorporation. CD4, CD8, CD25, and cutaneous lymphocyte-associated antigen expression on PBMCs was assessed by means of flow cytometry. RNA was extracted from isolated subsets of T cells, and the results of real-time PCR for FoxP3 mRNA were determined. RESULTS: Surprisingly, CD4+CD25+ T cells were significantly (P <.01) increased in patients with AD (6.68%+/-0.99%, n=15) compared with in asthmatic patients (3.42%+/-0.58%, n=12) or nonatopic healthy control subjects (3.34%+/-0.43%, n=14). Patients with AD also had a higher expression of CD25+ in skin-homing, CD4+, cutaneous lymphocyte-associated antigen-positive T cells than asthmatic and nonatopic subjects, with values of 35.95% versus 22.44% versus 23.03%, respectively (P <.006). Only CD4+CD25+ cells expressed FoxP3, whereas CD4+CD25- T cells and CD4- cells did not. Consistent with known properties of Treg cells, CD4+CD25+ cells were anergic to anti-CD3 stimulation. When CD4+CD25+ cells from each study group were mixed with CD4+CD25- cells, proliferative responses were equally suppressed after anti-CD3 stimulation. In contrast, after SEB stimulation, CD4+CD25+ cells were no longer anergic. Furthermore, when CD4+CD25+ cells were mixed with CD4+CD25- cells and stimulated with SEB, the suppressive function of Treg cells was reversed. CONCLUSION: Patients with AD have significantly increased numbers of peripheral blood Treg cells with normal immunosuppressive activity. However, after SAg stimulation, Treg cells lose their immunosuppressive activity. These data suggest a novel mechanism by which SAgs could augment T-cell activation in patients with AD.     

Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets

Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear cells (PBMC) from 47 donors of different genetic background induced generalized T cell activation and anti-apoptotic effects. Most effects were dependent upon interactions among immune cells. Specialized functions of CD4 and CD8 T cells were less dependent upon and often dampened by the presence of other PBMC populations. In particular, cytotoxic T cell effector function was variably affected with a component strictly dependent upon the direct stimulation of CD8 T cells in the absence of other PBMC. This observation may provide a roadmap for the interpretation of the discrepant biological activities of rIL-2 observed in distinct pathological conditions or treatment modalities.     

Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: enhanced T cell stimulatory capacity despite IDO

The exclusive ability of dendritic cells (DCs) to stimulate primary and secondary immune responses favors the use of antigen-loaded human monocyte-derived DCs (MoDCs) in vaccinations against tumors. Previous studies demonstrated that PGE(2) is fundamental during MoDC maturation to facilitate migration toward lymph node-derived chemokines. A recent study challenged the use of PGE(2), as PGE(2) induced IDO in mature MoDCs. In MoDCs compatible for clinical use, we now demonstrate that PGE(2) is responsible for IDO induction if matured by soluble CD40 ligand, LPS, or cytokines. In contrast, IDO expression in MoDCs matured by TLR3 triggering occurs independently of PGE(2). It is surprising that despite active IDO protein, MoDCs matured with PGE(2) display a greater potential to stimulate na?ve CD4(+) and CD8(+) T cell proliferation, which is not increased further by IDO inhibition. Moreover, we found elevated levels of tryptophanyl-tRNA-synthetase (TTS) in T cells cultured with PGE(2)-matured MoDCs. Our data demonstrate that PGE(2) induces IDO in MoDCs but that T cell-stimulating capacities of PGE(2)-matured MoDCs overcome IDO activity, probably through TTS induction. As PGE(2) is critical for DC migration and enhances the capability of MoDCs to induce T cell proliferation, we highly recommend supplementing DC maturation stimuli with PGE(2) for use in clinical trials.     

Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE₂, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO^?CD31⁺ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE₂ primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and TNF-β. PGE₂ does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-γ. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE₂, prevents the development of IL-4-producing cells but does not abolish the effects of PGE₂ on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE₂ on naive T cell maturation. Thus PGE₂ does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE₂ suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.     

The role of regulatory T lymphocytes in the induced immune response mediated by biological vaccines

Immunotherapy has become a novel therapeutic alternative for various kinds of tumours. Recently, we have finalized the first phase I clinical study in Chile for the treatment of advanced malignant melanoma, using dendritic cells (DCs) loaded with allogeneic melanoma cell lysate. This study included 20 patients and the obtained results, pioneer in Latin America, showed that DC-based immunotherapy is innocuous, even provided in combination with IL-2. In addition, immunological responses were detected in 50% of the treated patients, establishing a positive correlation between the delayed type hypersensitivity (DTH) reaction, which indicates induction of in vivo immunological memory, and patients surviving. Nevertheless, objective clinical responses in vaccinated patients are still insufficient. Only sporadic objective metastasis regressions have been registered and an important proportion of the treated patients did not respond, or their responses were weak. Several strategies have been described to be used by tumours to escape from the immune response. Actually, we have demonstrated that IL-10 inhibits antigen presentation in melanoma, reducing tumour sensitivity to melanoma-specific cytotoxic T lymphocytes (CTLs). Regulation of the immunological response by inhibitory cells could be another possible cause of clinical unresponsiveness. Lately, the existence of subpopulations of regulatory T lymphocytes (RTL) able to limit the immune response in a specific form has been established, specially inhibiting the proliferation and activity of CD4+ and CD8+ effector T lymphocytes. These cellular subpopulations, mostly CD4+/CD25+/Foxp3+ T lymphocytes (Treg) of thymic origin, or TR1 lymphocytes able to release IL-10, and tumour growth factor beta (TGF-beta) producing TH3 lymphocytes, would be accumulated in the body during tumour growth, inhibiting the immune response. In relation to RTL and cancer, evidence indicates that Treg cell numbers are increased in blood and other tissues in different types of cancer. Additionally, it has been demonstrated that in patients with refractory metastatic melanoma, the adoptive transference of anti-tumour CD8+ T lymphocytes after non-myeloablative chemotherapy was able to induce important tumour regressions that would be due to elimination of RTL populations. Additionally, chemotherapeutical drugs like decarbazine, besides their effect on tumour proliferation, also have an immunosuppressive effect on T lymphocyte populations, as well as on accumulated RTL. In this article, a novel strategy for the study of RTL is proposed, including potential therapeutic innovations, which is being pioneered in current clinical trials.     

What is the role of regulatory T cells in transplantation tolerance?

There has been considerable recent progress in the characterization of the regulatory T cells that mediate tolerance in a number of transplantation models. The conditions that facilitate the generation of regulatory T cells point to the thymus, the nature of immune suppression and the dose of immunosuppressive agent(s) being important. Putative mechanisms of immune regulation by regulatory T cells, particularly in the 'infectious' tolerance pathway, include Th2-type cytokines (IL-4, IL-10 and transforming growth factor beta) that may play a direct role as an indispensable requirement or may contribute indirectly as a favorable milieu for acquisition of tolerance. Anergic T cells may suppress immune responses via either cytokine competition or antigen-presenting cells. Models of autoimmune disease, in which regulatory T cells were shown to represent a distinct thymus-derived T cell subset, also suggest the role of antigen-presenting cells in mediating immune suppression. Progress has also been made in generating and characterizing regulatory T cells in vitro.     

Bacterial superantigens and T cell receptor beta-chain-bearing T cells in the immunopathogenesis of ulcerative colitis

Ulcerative colitis (UC) is a chronic relapsing-remitting inflammatory bowel disease (IBD) that affects the colon and the rectum producing debilitating symptoms, which impair ability to function and quality of life. The aetiology of IBD is incompletely understood, but within the lymphocyte population, specific T cell subsets are known to be major factors in the development of intestinal immune pathology while different subsets are essential regulators, controlling IBD. Hence, IBD is thought to reflect dysregulated T cell behaviour. This study was to investigate if the normal molecular configuration of the T cell receptor (TCR) repertoire is compromised in patients with UC. The percentage of T cell-bearing beta-chain 4 (TCRBV4) was high in patients with UC, and T cells showed polyclonal expansion in the presence of bacterial superantigens (SA) such as streptococcal mitogenic exotoxin Z-2 (SMEZ-2), indicating that bacterial SA promote specific TCRBV family expansion. Further, in patients with UC, the duration of UC was significantly longer in patients with skewed TCRBV4 compared with patients without TCRBV4 skewing, suggesting that long-term exposure to bacterial SA such as SMEZ-2 might promote systemic immune disorders like the remission-relapsing cycles seen in patients with UC. In conclusion, our observations in this study support the perception that the systemic activation of T cells by enteric bacterial SA might lead to a dysregulated, but exuberant immune activity causing the remission and flare-up cycle of mucosal inflammation in patients with UC. Future studies should strengthen our findings and increase understanding on the aetiology of IBD.     

Prostaglandin E2 is a major soluble factor produced by stromal cells for preventing inflammatory cytokine production from dendritic cells

Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC-MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor-alpha production. Using molecular-size selection and HPLC, we determined that prostaglandin E2 (PGE2) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE2 is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules.     

Epitope specificity of the T cell proliferative response to lysozyme: proliferative T cells react predominantly to different determinants from those recognized by B cells

The fine specificity of murine B 10.A/SgSn (B 10.A) T cells reactive with hen egg-white lysozyme (HEL) has been studied through the use of reduced, carboxymethylated HEL, a set of peptides encompassing the entire molecule, and a set of variant lysozymes from other species. Cells were taken from the lymph nodes draining the site of immunization at the base of the tail, and were restimulated in vitro with immunogen or analogue to measure T cell reactivity. Unlike B cell reactivity, which we have shown to be mainly associated with an epitope preserved in the N-C peptide (residues 1--17, Cys6--Cys 127, 120--129) most T cell reactivity appears to be directed towards a limited number of determinants on cyanogen bromide cleavage fragment II of HEL (LII) (13--105). This was confirmed by a cell-dilution assay in which antigen-reactive units are measured; reactivity was highest to LII, intermediate to N-C, and low but significant to cyanogen bromide cleavage fragment III (LIII) (106--129). Furthermore, priming with LII is as effective as immunization with HEL and results in the same extensive cross-reactivities to variant lysozymes. Although LII reactivity predominates in the response to HEL, injection of LIII and N-C reveals sizeable reactivity to the homologous peptides and to HEL. By cross-stimulation studies, specific epitopes could be defined in certain regions of HEL. B 10.A is clearly responsive to the overlap between N-C and LII (residues 13--17), and to an epitope in the region 106--121, but is poorly responsive to the C-terminal portion (120--129). The response to 106--121 is characterized by an exquisite specificity in which as little as a single amino acid substitution (Asn for Gln) is recognized.     

Cytotoxic activity of V beta 8+ T cells in Crohn's disease: the role of bacterial superantigens.

In Crohn's disease, disease-related stimuli could alter the T cell receptor (TCR) repertoire. To examine the possibility that changes in function may occur in T cell subsets without obvious changes in expression of TCR, we analysed the TCR repertoire of cytotoxic T lymphocytes in Crohn's disease peripheral blood. Furthermore, we examined the effect of bacterial superantigens, staphylococcal enterotoxin B (SEB) and E (SEE) on the cytotoxic function of T cell subsets bearing different TCR V genes using MoAbs specific for CD3 and TCR V gene products in a redirected cytotoxicity assay. There was no difference between patients and controls in the cytotoxicity measured in concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 or with six of seven anti-TCR V gene MoAbs. However, the cytotoxicity of V beta 8 T cells was decreased in Crohn's disease patients. This was not due to a decrease in total or CD8+ T cells expressing V beta 8. Furthermore, in normal subjects, PBMC stimulation with SEE and SEB selectively expanded and increased the cytotoxicity of V beta 8 and V beta 12 T cells, respectively. In Crohn's disease, although SEB stimulation increased the number and cytolytic function of the V beta 12 subset, SEE stimulation failed to increase cytolytic activity of V beta 8+ T cells in spite of the expansion of V beta 8+ T cells. These results suggest that the changes in cytotoxic function observed in V beta 8 T cells in Crohn's patients may reflect previous exposure to a V beta 8-selective superantigen.     

Interferon gamma: a crucial role in the function of induced regulatory T cells in vivo

Interferon (IFN)gamma can have paradoxical functions, eliciting inflammatory T helper 1 (Th1)-driven immune responses in some circumstances, and enabling induced regulatory T (Treg) cells to control immune responses in others. Here, we propose a model in which IFNgamma produced rapidly and only transiently by induced Treg cells is crucial to their function in vivo. This early production of IFNgamma by induced Treg cells during an immune response can directly inhibit the activation and proliferation of IFNgammaR1- and IFNgammaR2-bearing T cells. Furthermore, it can indirectly prevent further T-cell activation by creating a microenvironment that influences the function of antigen-presenting cells (APCs) as a result of IFNgamma-induced inducible nitric oxide synthase (iNOS), indoleamine-2,3-dioxygenase (IDO) and heme oxygenase (HO)-1 expression.     

Prostaglandin D2 affects the differentiation and functions of human dendritic cells: impact on the T cell response

The local environment in which dendritic cells (DC) differentiate is important for the acquisition of their immunostimulatory properties. Since prostaglandin D(2) (PGD(2)), a major prostanoid produced during inflammatory reactions, is involved in the control of immune responses, its effect on the differentiation and functions of human monocyte-derived dendritic cells (MDDC) was studied. We show that DC differentiated in the presence of PGD(2) (PG/DC) have an unusual phenotype, with modifications in the expression of molecules involved in antigen (Ag) capture and presentation, leading to higher endocytic and Ag-processing activities. However, under conditions that necessitated Ag processing and presentation, PG/DC have an impaired ability to stimulate naive T cells, whereas superAg-pulsed DC efficiently promote their proliferation. Upon lipopolysaccharide or TNF-alpha/IL-1beta stimulation, PG/DC phenotypically mature but produce abnormal amounts of immunoregulatory cytokines (decreased IL-12p70/IL-10 ratio). Moreover, mature PG/DC fail to up-regulate the chemokine receptor CCR7 and show an impaired migration towards its ligand CCL19. Finally, PG/DC favor the differentiation of naive T cells toward Th2 cells, an effect dependent on IL-10 and inducible costimulator ligand expression by DC. Most of the herein described effects of PGD(2) on MDDC can be reproduced, usually with a higher efficacy, with a selective D prostanoid receptor (DP)1, but not DP2, agonist. Taken as a whole, these results demonstrate that PGD(2) impacts DC differentiation and functions, and extend the concept that it exerts important roles in immunity.     

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.