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

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

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.     

T cells made deficient in interleukin-2 production by exposure to staphylococcal enterotoxin B in vivo are primed for interferon-γ and interleukin-10 secretion

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor Vβ domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-γ and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 μg SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-γ and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-γ levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4⁺ or CD8⁺ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4⁺ and CD8⁺ cells from SEB-pretreated mice were primed for IL-10 and IFN-γ production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-γ was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-γ monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-γ and IL-10 production, and that IFN-γ up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.     

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.     

CD3-mediated apoptosis of human medullary thymocytes and activated peripheral T cells: Respective roles of interleukin-1, interleukin-2, interferon-γ and accessory cells

Clonal deletion represents an important mechanism for the establishment of tolerance, by the elimination of autoreactive T cells. Deletion is accomplished by programmed cell death, termed apoptosis, induced by mobilization of the T cell receptor (TCR) on both thymocytes and mature T cells. The mechanism which drives T cells towards cell death or cell proliferation after TCR mobilization remains unclear. We show here that the mobilization of the CD3/TCR complex of both CD4⁺ and CD8⁺ single-positive medullary human thymocytes and human mature activated T cells, in the absence of accessory cells, leads to an activation-induced cell death process by apoptosis. In both cases, apoptosis was associated with interferon (IFN)-γ gene expression and secretion in the absence of interleukin (IL)-2 gene expression; and the addition of anti-IFN-γ antibody prevented cell death. Apoptosis could also be prevented by cyclosporin A (CsA) treatment and could be re-induced by the addition of IFN-γ to CsA-treated cells. Addition of IL-2 had two different effects, it prevented apoptosis and also allowed proliferation in response to CD3 monoclonal antibody. Addition of IL-1, which induces IL-2 gene expression and secretion or addition of accessory cells, had the same preventive effect. These results suggest that the uncoupling of IFN-γ and IL-2 gene expression following CD3/TCR mobilization initiates apoptosis of human T cells at several different stages during development and activation. We propose that co-signals provided by accessory cells allow a coupling of IL-2 gene and IFN-γ gene expression, and that an essential role for IL-2 secretion in T cell activation involves the inhibition of a death program induced by IFN-γ secretion.     

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

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

Polyethylene Glycol-Modified IL-2 Abrogates Superantigen-Induced Anergy without Affecting Peripheral Clonal Deletionin Vivo

As compared with the native molecule, recombinant human interleukin-2 that is modified by covalently attached polyethylene glycol residues (IL-2-PEG) exhibits a markedly enhanced half-lifein vivo, thus facilitating its biological evaluation. We have characterized the effect of IL-2-PEG on theStaphylococcus aureusenterotoxin B (SEB)-induced tolerance of peripheral SEB-reactive (Vβ8⁺) T cells. Treatment with sublethal doses of IL-2-PEG does not modulate (inhibit or enhance) the SEB-triggered apoptosis and deletion of Vβ8⁺T cells. In contrast,in vivotreatment with IL-2-PEG partially abolishes the SEB-triggered anergy of Vβ8⁺T cells, i.e., the failure to proliferate in response to SEBin vitro. To abolish SEB-triggered anergy, IL-2-PEG must act for an extended periodin vivo; short term treatmentin vivo(2 days) or exposure of anergic T cells to IL-2in vitrofails to reconstitute proliferative responses. Moreover, the effect ofin vivotreatment with IL-2-PEG on lymphokine production by anergic T cells is partial. IL-2-PEG restores IL-4-dependent autocrine proliferation in response to SEB but does not reestablish defective IL-2 production. These data are compatible with the notion that IL-2 is a regulator of postdeletional rather than deletional T cell tolerance.     

Staphylococcal Enterotoxin B Promotes Deletion and Functional Inactivation of CD4Vβ8-Positive Cells in the Absence of CD8 T Cells

The staphylococcal enterotoxins stimulate discrete subsets of T cells depending on their expression of particular V genes. Among these, staphylococcal enterotoxin B (SEB) vigorously stimulates Vβ8⁺ cells. This stimulation results in proliferation of both CD4 ⁺ Vβ8 ⁺ and CDS ⁺ T cells and eventually to anergy and clonal deletion in the former subset. We have examined the possible role of CD8⁺ T cells in the response of CD4⁺ cells to SEB, by in vivo CD8⁺ T-cell-depletion. We found no qualitative difference in the responses of untreated and CD8⁺ T-cell depleted mice to SEB; however, a small quantitative difference in deletion was observed. Thus it appears that on the whole the response of CD4 ⁺Vβ8 ⁺ T cells to SEB is independent of CD8⁺ T-cell effector function, although the latter may play a partial role.     

In susceptible mice,Leishmania major induce very rapid interleukin-4 production by CD4+ T cells which are NK1.1−

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-γ (IFN-γ) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-γ dependent. Treatment of resistant C57BL/6 mice with anti-IFN-γ allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in Vβ8, 7, 2^? CD4⁺ cells in BALB/c mice and NK1.1^? CD4⁺ cells in anti-IFN-γ treated C57BL/6 mice. These results show that the NK1.1⁺ CD4⁺ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.     

Superantigen-reactive T cells that display an anergic phenotype in vitro appear functional in vivo

Clonal deletion and/or inactivation establishes tolerance toself antigens. Endogenous and exogenous (bacterial) superantigens,like the staphylococcal enterotoxlns, induce ligand-specificclonal anergy in vivo and thus are believed to mirror aspectsof post-thymic tolerance mechanisms in mature peripheral T cells.Here we analyzed the level of anergy of ligand-responsive V_ß8⁺T cells from staphylococcal enterotoxin B (SEB)-primed micein vivo and in vitro. Upon in vitro restimulation with SEB,CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells failed toproduce IL-2. However, functional IL-2 receptors were triggered,since supplementation with IL-2 induced clonal growth in virtuallyall CD4⁺V_ß8⁺ and CD8⁺V_ß8⁺ T cells as determinedby limiting dilution analyses. Thus in vitro unresponslvenessof lymphocytes from SEB-primed mice reflects the inability ofSEB-reactlve V_ß8⁺ T cells to produce IL-2. Surprisingly,anergy as defined in vitro was at variance with that in vivo.Following further challenge with SEB, systemic and acute lymphokineproduction (Including IL-2 and tumor necrosis factor) occurredwith almost identical peak values and kinetics to primary invivo responses, and D-galactosamlne-sensltlzed mice succumbedto lethal shock. Polymerase chain reaction analyses revealedthat CD4⁺V_ß8⁺ expressed IL-2-specific mRNA in vivoupon restimulatlon with SEB. While lymphokine production andexpression of the IL-2 receptor was similar to the responseto in vivo primary stimulation, only CD8⁺V_ß8⁺ T cellsexpanded clonally upon reintroductlon of SEB in vivo. Henceprimed V_ß8⁺ T cells challenged with SEB display invitro anergy yet in vivo responsiveness, at least in part. Weconclude that the state of anergy is reversible, dependent uponthe quality of activation signals provided in in vivo ratherthan in in vitro culture conditions.     

Manipulation of the superantigen-induced lymphokine response. Selective induction of interleukin-10 or interferon-γ synthesis in small resting CD4+ T cells

The production of several lymphokines by freshly isolated CD4⁺ T cells has been analyzed at the single-cell level, after stimulation with staphylococcal enterotoxin B (SEB). High frequencies of cells producing interleukin-2 (IL-2) and interferon-γ (IFN-γ) were induced, but very low frequencies of CD4⁺ T cells produced IL-4, IL-5 or IL-10 in response to SEB. Exogenously added IL-4 markedly altered the lymphokine profile induced during primary SEB stimulation. IFN-γ production was reduced, while a high fraction of cells contained IL-10 and IL-4 after activation in the presence of IL-4. We further demonstrate that IL-4 and IL-10 or IFN-y production was selectively induced in resting, high-density CD4⁺ T cells during primary stimulation, by SEB + IL-4 or SEB. Under conditions where both IL-10 and IFN-γ were produced, most cells contained only one of the two lymphokines.     

Regulation of Human Cytotoxic T Lymphocytes Development by the Synergistic Effect of IL-7 and sCD23

The present study was undertaken to investigate the interaction of IL-7 and sCD23 on human peripheral blood T cell activation and CTL differentiation. Purified T lymphocytes were stimulated with mitogen plus IL-2 and subcultured for 7 days with IL-7 and/or sCD23. The combination of IL-7 and sCD23 synergistically enhanced the proliferation of both CD4⁺and CD8⁺T cells. CD8⁺T cells, however, were usually more responsive to IL-7 and sCD23. This synergy was observed on both subsets of T cells. Furthermore, these cytokines synergistically augment the CTL activity of CD8⁺T cells in both mitogen- and antigen-activated T cells. MAbs anti-IL-2 or anti-IL-2R (CD25) and anti-IL-12 had no effect on T cell proliferation and CD8⁺cytotoxic activity induced by IL-7 and sCD23. We analyzed the effect on IFN-γ induction by CD8⁺T cells and found that IL-7 alone was incapable of inducing detectable levels of IFN-γ production, but together with sCD23 it enhanced the production of IFN-γ. We also found that IFN-γ was not required for enhanced CTL activity of CD8⁺T cells, because rabbit anti-IFN-γ did not block the synergistic effects of either cytokine. The data demonstrate that the synergistic stimulatory activity of IL-7 and sCD23 may be of significance in the human CTL development and provide an alternative mechanism of stimulating T cells for use in immunotherapy.     

Clonal deletion of thymic mature T cells induced by staphylococcal enterotoxin B in murine fetal thymus organ culture

The present study aims at investigating the intrathymic maturational stage of T cells at which clonal deletion can be induced. Staphylococcal enterotoxin B (SEB) was added to organ cultures of murine fetal thymus lobes at various time points of culture, and Vβ8-expressing cells were assayed on day 14. Vβ8 low-expressing (Vβ8^′°) cells were reduced to 40-60 % of the control receiving no SEB, though the reduction was ambiguous when SEB was given on day 13. In marked contrast, Vβ8 high-expressing (Vβ8^hi) cells were virtually completely deleted in all groups including the group given SEB on day 13. Most of the Vβ8^hl cells that were deleted by 24 h of treatment with SEB were shown to be of the CD4⁺8 mature phenotype, though CD4^?8⁺Vβ8^hi cells were also deleted. It was further shown that the thymic Vβ8^hi CD4⁺8^? cells recovered from organs cultured for 14 days without SEB responded to immobilized anti-Vβ8 monoclonal antibody, indicating that Vβ8^hi cells, which were highly sensitive to clonal deletion, were functionally competent mature T cells. These results strongly suggest that the thymus is capable of eliminating all T cells recognizing antigen present in the thymus regardless of the maturational stage of T cells.     

Role of transforming growth factor-βthe preferential induction of T helper cells of type 1 by staphylococcal enterotoxin B

Stimulation of murine CD4⁺ T cells with staphylococcal enterotoxin B (SEB) results in the preferential development of T helper (Th) 1 cells [i.e. high interferon (IFN)-γ and low interleukin (IL)-4, IL-5 and IL-10]; whereas in response to plate-bound anti-CD3 or anti-T cell receptor-αβ, Th1 as well as Th2 cells develop. In the present study, we examined the mechanism which is responsible for the selective Th1 development in the SEB system. The addition of IL-4 resulted in a strong development of Th2 cells showing that SEB stimulation can result inTh2 differentiation. Co-stimulation with anti-CD28 was insufficient in this regard. Lack of Th2 development in the SEB system was in part due to the inhibitory effect of endogenously produced transforming growth factor-β (TGF-β), because anti-TGF-β allowed the development of Th2 cells. Similarly, TGF-β inhibited Th2 development and stimulated Th1 development in the anti-CD3 system. This shift was only partially prevented by also including IL-4 in the cultures. The effects of TGF-β could only partially be explained by stimulation of IFN-γ or inhibition of IL-4 as intermediatory cytokines: (1) TGF-β stimulated Th1 development even in the presence of anti-IL-4 and anti-IFN-γ, and (2) a strong inhibitory effect of anti-TGF-β on Th1 development was still observed when anti-IL-4 and IFN-γ were simultaneously added to the cultures. It is concluded that SEB favors Th1 development by stimulation of TGF-β production. Inhibition of Th2 development by TGF-β is due, in part, to inhibition of IL-4 and stimulation of IFN-γ, and, in part, to a direct effect of TGF-β on the responding T cells.     

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.     

Thymic and peripheral apoptosis of antigen-specific T cells might cooperate in establishing self tolerance

Aside from CD4⁺CD8⁺ double-positive (DP) thymocytes, the subpopulations of T lineage cells affected by negative selection are unknown. To address whether this process occurs in more mature cell types, we have compared the responses of purified single-positive (SP) murine thymocytes and peripheral T cells to the superantigen staphylococcal enterotoxin B (SEB) utilizing as antigen-presenting cells (APC) a fibroblast cell line expressing transfected I-E^k class II molecules. Whereas ∽ 70% of SEB-reactive SP thymocytes, either CD4⁺ or CD8⁺, undergo programmed cell death (apoptosis) and, therefore, negative selection, CD4⁺ and CD8⁺ antigen-specific peripheral T cells are predominantly activated and proliferate to APC+SEB. Thus, mature thymocytes and peripheral T cells, with identical patterns and levels of expression of CD4, CD8 and T cell receptor (TCR), are programmed to elicit different responses followingTCR stimulation. Unexpectedly, however activation of peripheral T cells was preceded by deletion of a large fraction of Vβ8⁺ T lymphocytes (SEB specific). This surprising phenomenon was also observed in in vivo studies: in fact, administration of SEB to adult mice resulted in depletion of the majority of antigen-specific T cells from the peripheral lymphoid tissues analyzed (lymph nodes and spleen). This depletion is the consequence of deletion as indicated by program cell death of Vβ8⁺ T cells and is followed by proliferation of the remaining SEB-reactive T cells. Clonal elimination of peripheral T cells may represent a mechanism by which tolerance to self antigens never expressed in and/or exported to the thymus is achieved.     

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.     

Mechanisms of peripheral T cell deletion: anergized T cells are Fas resistant but undergo proliferation-associated apoptosis

The complementary receptor pair Fas ligand: Fas controls apoptosis during activation-induced cell death (AICD) of peripheral T cells sensitized for the Fas signal pathway by interleukin-2 (IL-2). In the present study, we used the bacterial superantigen staphylococcal enterotoxin B (SEB) to anergize ligand-reactive peripheral T cells in wild-type and Fas-defective lpr mice. In a second step, we investigated whether apoptosis in anergized and thus operationally IL-2-defective peripheral T cells is triggered via the Fas signal pathway. We report here that SEB-driven anergy induction and deletion of anergized peripheral Vβ8⁺ T cells is similar in wild-type and healthy C3H/lpr mice. In monitoring SEB-driven Vβ8⁺ T cell apoptosis in situ, we observe in both wild-type and lpr mice an intimate association between proliferation and apoptosis of anergized Vβ8⁺ T cells. We further show that Vβ8⁺ T cells activated in vitro from wild-type mice express a Fas-sensitive phenotype determined by Fas cross-linking which causes apoptosis. In contrast, Vβ8⁺ T cells anergized in vivo from wild-type mice are Fas resistant. As expected, T cells from lpr mice activated in vitro or anergized in vivo are Fas resistant. Taken together, these data indicate that both in wild-type and Fas-defective C3H/lpr mice, anergized T cells become deleted via a Fas-independent, proliferation-associated apoptosis signal pathway.     

Opposite role for interleukin-4 and interferon-γ on CD30 and lymphocyte activation gene-3 (LAG-3) expression by activated naive T cells

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene-3 (LAG-3) and CD30, respectively. In this study we have examined the LAG-3 and CD30 expression in relation to the lineage commitment of human naive CD4⁺ T cells, as assessed at the single-cell level of committed T cells. Purified CD45RA⁺ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)-2 in the absence or presence of interleukin IL-4 or IL-12 and assessed for CD30 and LAG-3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30⁺ cells were only found in CD4⁺ and CD8⁺ T lymphocytes of cultures primed with IL-4, which developed into cells able to produce IL-4 and IL-13 in addition to interferon (IFN)-γ. By contrast, LAG-3 expression was strongly up-regulated in CD4⁺ and CD8⁺ T cells from cultures primed with IL-12, which developed into high numbers of IFN-γ producers. The addition of a neutralizing anti-IFN-γ antibody to IL-12-primed CD4⁺ T cell cultures virtually abolished the development of LAG-3-expressing CD4⁺ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL-4, whereas LAG-3 expression is dependent on the production of IFN-γ during the lineage commitment of human naive T cells.     

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

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