Tolerance to staphylococcal enterotoxin B initiated Th1 cell differentiation in mice infected with Candida albicans.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that specifically activates T cells bearing V beta 8 T-cell receptor domains, which eventually leads to a long-lasting state of clonal anergy accompanied by selective cell death in the targeted CD4+ subset. Because the superantigen is known to promote Th1 cell differentiation in vitro, we have investigated the effect of SEB treatment on the course of Th2-associated progressive disease in mice infected systemically with Candida albicans. On the basis of the kinetics of SEB-induced changes in CD4+ cells and production in sera of interleukin 4 (IL-4), IL-10, and gamma interferon, we obtained evidence that V beta 8+ cell anergy concomitant with infection abolished the early IL-4/IL-10 response of the host to the yeast, ultimately leading to a state of resistance characterized by gamma interferon secretion in vitro by antigen-specific CD4+ cells. In contrast, SEB administered near the time of challenge resulted in accelerated mortality. Significant resistance to infection was also afforded by exposure of mice to a retrovirally encoded endogenous superantigen. These data suggest that CD4+ V beta 8+ T cells play an important role in vivo in the initiation of a Th2 response to C. albicans and that suppression of their activity may alter the qualitative development of the T-cell response and the outcome of infection.     

Superantigen-induced anergy of V beta 8+ CD4+ T cells induces functional but non-proliferative T cells in vivo.

The response profile of staphylococcal enterotoxin B (SEB)-primed murine V beta 8+ CD4+ and V beta 8+ CD8+ T cells was analysed upon rechallenge in vitro. While in vitro responses to secondary stimulation with SEB were reduced to background levels, the in vivo reactivity after rechallenge with SEB was retained, in that SEB-primed mice succumbed to lethal T-cell shock, lymphokines [interleukin-1 (IL-1), IL-2, Il-4, IL-6, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)], and lymphokine-specific mRNA accumulation could be detected in V beta 8+ CD4+ and V beta 8+ CD8+ T cells. However, V beta 8+ CD4+ T cells failed to enter the cell cycle. While the phenotype of V beta 8+ CD8+ T cells was indistinguishable from that of their counterparts from naive mice, V beta 8+ CD4+ T cells exhibited in vivo an unusual phenotype as non-proliferative but functional T cells. We conclude that in vitro-defined anergy does not disclose the functional abilities of ligand-reactive V beta 8+ T cells in vivo, and that priming with superantigen (SAg) induces in vivo a differentiation of SEB-reactive V beta 8+ CD4+ T cells into a non-proliferative but functional phenotype.     

Clonal expansion precedes anergy and death of V beta 8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo

Staphylococcal enterotoxin B (SEB) selectively stimulates T cells bearing T cell receptor V beta 8 domains and hence provides a useful model to study immunity and tolerance in vivo. We show here that V beta 8+ T cells in both CD4+ and CD8+ subsets expand dramatically (fivefold) in lymphoid tissues of mice 2-4 days following injection with SEB. This initial clonal expansion, which is accompanied by a transient hyper-reactivity to SEB, is followed by a rapid decrease in V beta 8+ cells and a concomitant induction of specific non-responsiveness which persists for at least 30 days. Selective death of V beta 8+ cells occurs during this latter phase. Taken together, our data indicate that clonal expansion, anergy and death can occur as sequential stages of an immune response in vivo.     

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 CD4+ T-cell response to protein immunization is independent of accompanying IFN-gamma-producing CD8+ T cells.

By virtue of their strong bias towards production of interferon-gamma (IFN-gamma), CD8+ T cells have the potential to promote the development of type 1 immune responses. We have previously shown that the CD4+ T-cell response to immunization with the protein antigen keyhole limpet haemocyanin (KLH) has a mixed interleukin-4 (IL-4)/IFN-gamma production profile. Here we show that this immunization regimen also stimulates accumulation in the draining lymph nodes of CD8+ T cells, which preferentially contain IFN-gamma mRNA ex vivo and secrete IFN-gamma protein in vitro. This provides a model to test whether CD8+ cell-derived IFN-gamma participates in the normal control of the immune response to a non-viable exogenous antigen. To investigate regulation of the anti-KLH response by the CD8+ population or IFN-gamma produced by this or other cell types, mice were administered depleting antibodies. Depletion of CD8+ cells had no effect on the frequency of clonogenic KLH-specific CD4+ T cells, the IL-4/IFN-gamma profiles of their progeny, or the isotype profiles of the serum antibody response to KLH. In contrast, IFN-gamma neutralization diminished cell accumulation in the lymph nodes and reduced both the frequency of KLH-specific CD4+ T cells that gave rise to IFN-gamma-producing clones and serum titres of KLH-specific IgG2a and IgG3. Therefore, despite the potential for cross-regulation, the CD4+ T-cell response to this immunogen is independent of the IFN-gamma-skewed CD8+ response.     

Anti-Vbeta8 antibodies induce and maintain staphylococcal enterotoxin B-triggered Vbeta8+ T cell anergy

The mechanism involved in the maintenance of staphylococcal enterotoxin B (SEB)-induced T cell anergy is poorly understood. We demonstrated earlier that B cells play an important role in the maintenance of SEB-induced T cell anergy in vivo and in vitro. Here, we demonstrate that B cells are not essential in SEB-induced T cell activation, but are important for the maintenance of T cell memory phenotype and anergy in vivo. Studying the activated B cell repertoire, we observe that SEB treatment increases serum anti-Vbeta8 antibody titer as detected by enzyme-linked immunosorbent assay using soluble Vbeta8 chains as antigens, and by staining of a Vbeta8-expressing thymoma. These antibodies disappear gradually after immunization with SEB, whereas the capacity of the T cells to respond to SEB in vitro is restored. Anti-Vbeta8 monoclonal antibody treatment causes Vbeta8+ T cell unresponsiveness to SEB in vitro (anergy), without affecting CD4Vbeta8+ T cell frequency. Together, these results suggest a new mechanism to explain the maintenance of SEB-induced T cell anergy, which is dependent on B cells and on anti-Vbeta8 antibody that specifically interacts with Vbeta8+ T cells.     

In vivo responses of CD4+ and CD8+ cells to bacterial superantigens.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex (MHC) class II molecules and specifically activates T cells bearing V beta 8 T cell receptor domains. We have compared several aspects of the response of CD4+ and CD8+ T cell subsets to SEB in vivo. V beta 8+ cells in both subsets proliferated to a similar extent upon SEB injection. Furthermore, mRNA for interferon-gamma was induced in both subsets with similar kinetics and SEB dose-response. Finally CD8+ (but not CD4+) T cells from SEB-injected mice exhibited SEB-specific lysis of MHC class II-bearing target cells. Collectively, these data indicate that the CD4: MHC class II interaction confers no detectable selective advantage to CD4+ cells in the in vivo response to SEB. The observed effector functions of both subsets may contribute to SEB-induced immunopathology.     

Roles of CD4+ and CD8+ T cells in adjuvant activity of diesel exhaust particles in mice

Through an imbalance in Th1 and Th2 cytokine profiles, diesel exhaust particles (DEP) are thought to induce Th2-dominated IgE and IgG1 production. However, the roles of CD4+ and CD8+ T-cell subtypes in the increased immune responses to antigen in mice exposed to DEP are unclear. In the present study, we investigated whether treatment with anti-CD4 or anti-CD8 mAb abrogated the adjuvant activity of DEP. On day -1 and day 1, each group of mice was injected intraperitoneally with anti-CD4, anti-CD8, or rat IgG (vehicle). On day 0, the mice were immunized with ovalbumin (OVA) or OVA plus DEP. After 3 weeks, each mouse was boosted with 10 microg of OVA alone. On day 7 after the first injection with OVA+DEP or OVA alone, the numbers of total, IA+, CD80+/IA+ and CD86+/IA+ cells in peritoneal exudate cells (PEC) were higher in OVA+DEP-immunized mice than in OVA-immunized mice. Depletion of CD8+ cells resulted in a modulation of the production of granulocyte-macrophage colony-stimulating factor, IL-12 and PGE(2) in peritoneal exudate fluid from OVA+DEP-immunized mice. On day 28, DEP injection markedly increased IL-4 production in the culture supernatants of spleen cells from CD4+ or CD8+-depleted mice. Depletion of CD8+ cells in OVA+DEP-immunized mice resulted in a decrease in IFN-gamma production compared with that in OVA-immunized mice. Adjuvant activity of DEP was observed in anti-OVA IgE, anti-OVA IgG1, anti-OVA IgG3, and total IgE production. Depletion of CD4+ T cells abrogated the adjuvant effect of DEP on anti-OVA IgE, and anti-OVA IgG1 production in plasma. However, depletion of CD8+ T cell inhibited the upregulated anti-OVA IgG3 production. These findings suggest that DEP injection may affect not only the function of CD4+ cells but also that of CD8+ T-cell subsets to modulate the synthesis of proinflammatory cytokine in PEC and type-1 and type-2 cytokine production in spleens.     

Induction of resistance to active experimental allergic encephalomyelitis by myelin basic protein-specific Th2 cell lines generated in the presence of glucocorticoids and IL-4

We have produced T cell lines with a Th2 phenotype in the presence of IL-4 and the glucocorticoid dexamethasone (DEX). IL-4 and DEX together were more effective in inducing a Th2 response than IL-4 alone. Myelin basic protein (MBP)-specific Th2 lines were obtained and their ability to induce experimental allergic encephalomyelitis (EAE) was studied. Lines treated with IL-4 and DEX did not transfer passive EAE and did not induce cellular infiltration into the central nervous system as opposed to the encephalitogenic Th1 lines. Lines treated with IL-4 and DEX did not protect animals from the effect of encephalitogenic Th1 lines when the two were injected together. However, a high proportion of animals injected with IL-4 + DEX-treated lines became refractory to the development of EAE after immunization with MBP; that is, it was possible to induce resistance to active EAE without prior episodes of disease. Interestingly, animals injected with T cell lines had accelerated antibody responses against MBP and the predominant isotype was dependent on the cytokines synthesized by the T cell line injected. There was not evidence that the resistance to active EAE was due to anergy of MBP-reactive cells or the action of CD8+ cells. Our data suggest that MBP-specific T cell lines prevent the induction of disease by deviating the reactivity to MBP from a cell-mediated to a humoral one and not merely from a Th1 to a Th2 response.     

Chronic exposure to superantigen induces regulatory CD4(+) T cells with IL-10-mediated suppressive activity

The repeated injection of bacterial superantigens (SAg), such as staphylococcus enterotoxin (SE) A or B, has been shown in mice to induce a state of unresponsiveness characterized by the lack of secretion of Th1 lymphokines, such as IL-2 and IFN-gamma, following subsequent SAg challenge. We made the observation, in vivo as well as in vitro, that unresponsiveness to SAg could be transferred from SEA- to SEB-reactive T cells (and reversibly from SEB- to SEA-specific T cells) in C57BL/6 mice but not in BALB/c mice. Since C57BL/6 mice, unlike BALB/c mice, possess TCR V(beta)3+ and V(beta)11+ T cells able to react with both SEA and SEB, we hypothesized that SAg-unresponsive V(beta)3(+) and V(beta)11+ T cells could mediate linked suppression of other SAg-reactive T cells. To analyze further this possibility, spleen cells from BALB/c mice made unresponsive to SEB were tested for their capacity to suppress the response of normal BALB/c cells to SEB. The production of both IFN-gamma and IL-2 following SEB stimulation was greatly impaired in co-cultures containing CD4(+) T cells, but not CD8(+) T cells, isolated from unresponsive animals. In vivo, the production of both IFN-gamma and IL-2 responses to SEB was dramatically reduced in animals adoptively transferred with unresponsive spleen cells. This suppression was abrogated in recipients injected with neutralizing anti-IL-10 antibodies. Moreover, in animals made unresponsive to SEB, SAg-reactive CD4(+) T cells were found to express high levels of CTLA-4, a molecule recently described to play an essential role in the suppressive function of regulatory T cells. Taken together these results demonstrate that the repetitive injection of SAg induces the differentiation of regulatory CD4(+) T cells capable of suppressing SAg-reactive naive T cells.     

Regulation of secretion of IL-4 and IgG1 isotype by melatonin-stimulated ovalbumin-specific T cells

In the present study, we describe the potential role of melatonin, a pineal hormone, in regulating the activation of the antigen-specific T cell response. Melatonin encouraged the proliferation of Th cells and improved their ability to secrete IL-4, but down-regulated the levels of IL-2 and interferon-gamma (IFN-γ). Melatonin, however, could not exert any influence on the T cells of unprimed mice. On studying the regulation of subclass of IgG isotype, melatonin specifically enhanced the secretion of antigen-specific IgG1 antibodies and decreased the yield of IgG2a isotype. The results suggest that melatonin possibly acts by selectively activating a Th2-like immune response.     

Similar co-stimulation requirements of CD4+ and CD8+ primary T helper cells: role of IL-1 and IL-6 in inducing IL-2 secretion and subsequent proliferation.

Primary murine CD4+ and CD8+ T helper (Th) cells provide help for various immune responses by secreting lymphokines which activate effector cells. The purpose of the present study was to investigate the co-stimulatory signals that, together with T cell receptor (TCR) cross-linking, induce phenotypically distinct primary Th cells to secrete IL-2 and proliferate. We isolated highly purified populations of primary CD4+ or CD8+ T cells and stimulated them in vitro with platebound anti-CD3 mAb. TCR cross-linking by anti-CD3 mAb induced both IL-2 receptor expression and responsiveness to exogenous IL-2, but was not sufficient to induce either IL-2 secretion or T cell proliferation. Rather, for both CD4+ and CD8+ primary Th cells, IL-2 secretion and proliferation required both TCR cross-linking and antigen presenting cell (APC)-derived co-stimulatory signals. Based on G-10 adherence and sensitivity to gamma-irradiation, the APC populations able to induce primary CD4+ Th cells and primary CD8+ Th cells to secrete IL-2 were indistinguishable. In addition, we found that either IL-1 or IL-6 could replace the requirement for APC-derived co-stimulatory signals for IL-2 secretion and proliferation by both primary CD4+ Th cells and primary CD8+ Th cells. Thus, the present study has examined and compared the co-stimulatory requirements of rigorously purified subsets of IL-2-secreting primary CD4+ and primary CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Profound effect of the absence of IL-4 on T cell responses during infection with Schistosoma mansoni.

T cell responses of interleukin (IL)-4(-/-) and wild-type (WT) mice infected with the helper T cell 2 (Th2) response-inducing pathogen Schistosoma mansoni were compared. As expected, given the important role of IL-4 in Th2 response induction, the absence of IL-4 resulted in diminished Th2 responses, apparent as reduced production of IL-4, -5, and -10 by CD4(+) cells isolated from the spleens of infected IL-4(-/-) mice. Surprisingly, these cells produced significantly less interferon (IFN)-gamma and proliferated less than did those from infected WT mice after T cell receptor ligation. CD8(+) cells isolated from infected IL-4(-/-) mice also produced less IFN-gamma than WT CD8 cells, although there was no difference in the proliferative responses of these cell populations. After infection, spleens of infected IL-4(-/-) mice did not enlarge to the same extent as those of WT mice, and attrition of the CD8(+) cell population within this lymphoid organ was noted. Taken together, the data indicate that in addition to inhibiting Th2 response development, the lack of IL-4 during schistosomiasis significantly affects additional aspects of T cell responses.     

Constitutive expression of interleukin 4 in vivo does not lead to the development of T helper 2 type CD8+ T cells secreting interleukin 4 or interleukin 5.

Interleukin 4 (IL-4) has been shown to commit CD8+ T cells to a T helper (Th) 2 functional phenotype in vitro. To study the effects of IL-4 on CD8+ T cell development in vivo we analysed the CD8+ T cell phenotype in mice constitutively expressing IL-4. Purified CD8+ T cells from uninfected or flu infected IL-4 transgenic (tg) animals produced no detectable IL-4 or IL-5 after in vitro stimulation on anti-CD3 coated plates. However, CD8+ T cells from IL-4 tg mice could be converted into IL-4 and IL-5 producers in vitro in the presence of exogenous added IL-4, showing that these cells were still responsive to IL-4. IL-4 tg mice also showed a delay in influenza virus clearance from the lung, which was probably due to the observed reduction of total CD8+ T cell numbers in the IL-4 tg animals since IL-4 tg CD8+ T cells showed normal levels of influenza-specific cytotoxicity in comparison to controls. Taken together these results suggest that CD8+ T cells are not necessarily switched to a Th2 phenotype by the presence of IL-4 and that some other factor(s) may be important in the switch process of CD8+ T cells in vivo, since the addition of IL-4 during CD8+ T cell activation in vitro leads to Th2 type CD8+ T cells secreting IL-4 and IL-5.     

A new T cell subset expressing B220 and CD4 in lpr mice: defects in the response to mitogens and in the production of IL-2.

Autoimmune-prone mice homozygous for the lpr gene develop prominent lymphadenopathy composed mainly of Thy-1+ CD8- CD4- B220+ cells. Expression patterns of B220 vs CD4 on lymph node cells from lpr mice were analysed using two-colour flow microfluorometry. B220+CD4+ cells, which were hardly seen in lymph nodes of B6-+/+ mice, increased significantly in B6-lpr mice with ageing. Functional analysis of purified B220+ CD4+ cells from lpr mice revealed that these cells scarcely responded to T cell mitogens with or without rIL-2. Furthermore, B220+ CD4+ cells were defective in IL-2 production when cultured with Con A. On the other hand, B220-CD4+ cells from B6-lpr mice showed an ability to respond to T cell mitogens similar to that of B220- CD4+ cells from B6-+/+ mice. These results indicate that an unusual T cell subset expressing both B220 and CD4 in lpr mice is functionally defective, but the intrinsic ability of B220-CD4+ cells is almost intact as compared with the counterpart in normal mice.     

Evidence for B cell participation in the in vitro and in vivo maintenance of in vivo staphylococcal enterotoxin B-induced T cell anergy

The mechanisms involved in the maintenance of staphylococcal enterotoxin B (SEB)-induced T cell anergy are poorly understood. Here, we demonstrate that CD4+ T cell anergy induced by SEB treatment is under partial B cell control. This effect is not mediated by anti-SEB antibodies or any in vitro B cell-produced suppresser factor. At day 13 after SEB immunization, T cells from B cell-deficient mice proliferate upon in vitro stimulation with SEB. These results suggest that SEB- induced T cell anergy is reversible and that B cells have an important function in anergy maintenance in CD4+ T cells, both in vivo and in vitro.      

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.     

Natural killer T cells are required for the development of a superantigen-driven T helper type 2 immune response in mice

We show, here, that one single injection or weekly injections of staphylococcal enterotoxin B (SEB), starting in 1-day-old newborn mice, induced a powerful immune response with a T helper type 2 (Th2) pattern, as judged by the isotype and cytokine profile, with the production of large amounts of SEB-specific immunoglobulin G1 (IgG1), detectable levels of SEB-specific IgE and increased production of interleukin-4 by spleen cells. These protocols also induced an increase in the levels of total IgE in the serum. Memory of SEB was transferred to secondary recipients by using total spleen cells from primed animals. The secondary humoral response in transferred mice was diminished if spleen cells from SEB-treated mice were previously depleted of CD3+ or Vbeta8+ T cells or NK1.1+ cells. In vivo depletion of NK1.1+ cells in adult mice resulted in a marked reduction in the SEB-specific antibody response in both the primary and secondary immune responses. Additionally, purified NK1.1+ T cells were able to perform SEB-specific helper B-cell actions in vitro and in vivo. These results suggest that NK1.1+ T cells are required for the full development of humoral immunological memory, whilst making neonatal tolerance to SEB unachievable.     

Control of CD4 T cell fate by antigen re-stimulation with or without CTLA-4 engagement 24 h after priming

After two consecutive inoculations with Staphylococcus enterotoxin B (SEB) at 24 h intervals in vivo, CD4 T cells became anergic to the antigen challenge in vitro. Administration of anti-CTLA-4 mAb in conjunction with the second SEB inoculation 24 h after antigen priming interfered with anergy and CD4 T cells became T(h)2 cells. However, the anergy induction was not ablated when SEB and anti-CTLA-4 mAb were administered 48 or 72 h after antigen priming. Moreover, anti-CTLA-4 mAb without SEB did not interfere with anergy nor promoted the T(h)2 differentiation. T-antigen-presenting cell (APC) interaction in vitro in the presence of high doses of antigen and anti-CTLA-4 mAb induced a T(h)2-polarizing cytokine IL-6 and IL-10. IL-10 then down-modulated a T(h)1-polarizing cytokine IL-12. The results demonstrate that 24 h after the initial antigen stimulation, CD4 T cells enter the critical activation phase where antigen re-stimulation with or without CTLA-4 engagement alters the fate of the cell, anergy or differentiation respectively. Once anergy is interfered with, T(h)2-polarizing cytokines produced upon prolonged T-APC interaction favor the T(h)2 differentiation.     

Frequencies of T cells secreting IL-2 and/or IL-4 among unprimed CD4+ populations. Evidence that clones secreting IL-2 and IL-4 give rise to clones which secrete only IL-4

We have utilized a limiting dilution assay, involving a minimum of in vitro culture, to determine the frequencies of CD4+ T cells which have the capacity to secrete IL-2 (Th1), IL-4 (Th2) or IL-2 and IL-4 (Th0). CD4+ lymph node cells obtained from unimmunized mice were found to contain 40% Th1, 34% Th2, and 26% Th0 cells. The fact that Th0 cells could be identified in cell populations obtained from unimmunized mice suggested a possible role for Th0 in the differentiation pathways of Th1 and Th2. Antigen-specific Th0 clones were thus established and observed to convert to a Th2 phenotype after prolonged culture. The relevance of these findings is discussed in the context of current models of CD4+ T cell subsets.