Response to Superantigen Stimulation in Peripheral Blood Mononuclear Cells from Children Perinatally Infected with Human Immunodeficiency Virus and Receiving Highly Active Antiretroviral Therapy

Our understanding of the pathogenesis of perinatal human immunodeficiency virus (HIV) infection is still evolving. We sought to characterize the response to the bacterial superantigen Staphylococcus enterotoxin B (SEB) of lymphocytes from HIV-infected children receiving treatment with highly active antiretroviral therapy (HAART). Using the flow cytometric methodology, we quantified apoptosis, proliferation, cytokine production, and activation antigen upregulation in CD4 and CD8 T lymphocytes following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with SEB. The levels of proliferation, CD4 interleukin-2 (IL-2) production, CD8 gamma interferon (IFN-γ) production, and upregulation of CD69 expression by cells from HIV-infected children were indistinguishable from those by cells from controls. However, stimulation with SEB dramatically decreased the ratio of resting apoptotic cells to cycling apoptotic cells in the controls but not in the patients. In addition, unstimulated spontaneous apoptosis of CD4 T cells remained greater in the patients than in the controls. The percentages of IL-2-positive CD8 T cells and IFN-γ-positive CD4 T cells following SEB stimulation were significantly lower in the patients than in the controls. Our multiparameter approach was able to demonstrate differences in lymphocyte superantigen responsiveness in HIV-infected children receiving HAART in comparison to that in uninfected controls, notably, an apoptotic versus a proliferative response to stimulation.     

SEB活化的人外周血T细胞CD25,CD69的表达

本文用超抗原葡萄球菌肠毒素B(SEB)诱导外周血淋巴细胞增殖。结果显示出微量超抗原能诱导外周血淋巴细胞的增殖,大量的增殖反应发生在SEB刺激后的第5天,增殖的细胞是CD4~+T细胞,它们由刺激前的27％增加到42％。外周血活化的T细胞不依赖外源性IL-2:大量内源性IL-2的存在抑制T细胞的增殖反应。伴随CD4~+T细胞的增殖,CD25和CD69分子表达明显增加。提示SEB能改变外周血T细胞表面的分子表达。     

Normalized CD8+ but not CD4+ lymphocyte IL-2 expression is associated with early treatment with highly active antiretroviral therapy

CD4+ and CD8+ lymphocyte cytokine production in patients with HIV/AIDS and Controls, in response to stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin was assessed using single cell flow cytometric methods. Sixty-eight patients with HIV were divided into those on no antiretroviral therapy and those on highly active antiretroviral therapy (HAART). Patients on HAART were analyzed further on the basis of gender, ethnicity, viral load (> or 100 or <100 cells/mm(3)) and CD4 count (>200 or <200 cells/mm(3)). Interferon gamma (IFNgamma) expression by CD4+ and CD8+ lymphocytes was elevated in HIV-infected groups as compared to Controls. This elevation was statistically significant for patients on HAART but not for those not on HAART. The most significant difference was seen when the CD4+ count reached >200 cells/mm(3) (p=0.018 for CD4+ IFNgamma production and p=0.004 for CD8+ IFNgamma production). CD4+ interleukin-2 (IL-2) expression was significantly lower in HIV patients as compared to Controls but did not significantly improve however good the response to HAART. IL-2 expression by CD8+ lymphocytes was also lower in HIV patients as compared to Controls. IL-2 expression by CD8+ lymphocytes significantly improved in all patients on HAART as compared to HIV patients on no HAART. IL-2 expression was not significantly different from that of the Controls when the HIV viral load was less than 50 copies/ml. These results demonstrate improvements in both CD4+ and CD8+ responsiveness with HAART. IFNgamma production was elevated in response to HAART and was maximal only with significant CD4 count recovery. In contrast, normalization of IL-2 production by CD8+ lymphocytes was seen early in patients receiving HAART even when there was only a small increase in CD4+ lymphocyte numbers.     

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.     

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.     

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.     

CD127 expression and regulation are altered in the memory CD8 T cells of HIV-infected patients--reversal by highly active anti-retroviral therapy (HAART)

HIV infection activates abnormally the immune system and the chronic phase is accompanied by marked alterations in the CD8 compartment. The expression of CD127 (IL-7R alpha chain) by memory CD8 T lymphocytes in HIV-infected patients is analysed and reported. The memory CD8 T cell subset was characterized by expression of CD45RA and CD27 markers, and CD127 cell surface expression was measured ex vivo by four-colour flow cytometry. HIV infection was associated with a fall in the proportion of CD127(+) cells among memory CD8 lymphocytes that resulted in a higher CD127(-) CD45RA(-)CD27(+) CD8 T cell count in HIV-infected patients. Diminished CD127 cell surface expression [mean fluorescence intensity (MFI)] by positive cells was also observed in this subset. The data suggest that these defects were reversed by highly active anti-retroviral therapy (HAART). The regulation of CD127 expression was also studied in vitro. Down-regulation of CD127 by interleukin (IL)-7 was observed in memory CD8 lymphocytes from healthy donors and HAART patients. Expression of CD127 by memory CD8 lymphocytes cultured in the absence of IL-7 confirmed that IL-7R regulation is altered in viraemic patients. Under the same experimental conditions, memory CD8 lymphocytes from HAART patients were shown to express CD127 at levels comparable to cells from healthy individuals. Altered CD127 cell surface expression and defective CD127 regulation in the memory CD8 T lymphocytes of HIV-infected patients are potential mechanisms by which these cells may be impeded in their physiological response to endogenous IL-7 stimulatory signals. Our data suggest that these defects are reversed during the immune reconstitution that follows HAART.     

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.     

Differential regulation of cytokine production by CD1d-restricted NKT cells in response to superantigen staphylococcal enterotoxin B exposure

NKT cells are a heterogeneous population characterized by the ability to rapidly produce cytokines, such as interleukin 2 (IL-2), IL-4, and gamma interferon (IFN-gamma) in response to infections by viruses, bacteria, and parasites. The bacterial superantigen staphylococcal enterotoxin B (SEB) interacts with T cells bearing the Vbeta3, -7, or -8 T-cell receptors, inducing their expansion and cytokine secretion, leading to death in some cases due to cytokine poisoning. The majority of NKT cells bear the Vbeta7 or -8 T-cell receptor, suggesting that they may play a role in regulating this response. Using mice lacking NKT cells (CD1d(-/-) and Jalpha18(-/-) mice), we set out to identify the role of these cells in T-cell expansion, cytokine secretion, and toxicity induced by exposure to SEB. We find that Vbeta8(+) CD4(+) T-cell populations similarly expand in wild-type (WT) and NKT cell-null mice and that NKT cells did not regulate the secretion of IL-2. By contrast, these cells positively regulated the secretion of IL-4 and IFN-gamma production and negatively regulated the secretion of tumor necrosis factor alpha (TNF-alpha). However, this negative regulation of TNF-alpha secretion by NKT cells provides only a minor protective effect on SEB-mediated shock in WT mice compared to mice lacking NKT cells. These data suggest that NKT cells may regulate the nature of the cytokine response to exposure to the superantigen SEB and may act as regulatory T cells during exposure to this superantigen.     

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).     

Bacterial superantigens reactivate antigen-specific CD8+ memory T cells

Superantigens stimulate naive CD4+ and CD8+ T cells in a TCR V beta- specific manner. However, it has been reported that memory T cells are unresponsive to superantigen stimulation. In this study, we show that staphylococcal enterotoxins (SE) can activate influenza virus-specific CD8+ memory cytotoxic T cells. In vivo SEB challenge of mice that had recovered from influenza virus infection (memory mice) resulted in the generation of vigorous influenza-specific cytotoxic T lymphocyte (CTL) activity and in vitro SEA or SEB stimulation of splenic T cells from memory mice, but not naive mice, also induced influenza-specific CTL. Analysis of the mechanism of activation suggested that although there may be a component of cytokine-mediated bystander activation, the CTL activity is largely generated in response to direct TCR engagement by superantigen. Moreover, influenza-specific CTL could be generated from purified CD8+ CD62L loCD44hi (memory phenotype) T cells cultured in the presence of T cell-depleted splenic antigen-presenting cells and SE. Purified CD8+ memory T cells also secreted lymphokines and synthesized DNA in response to superantigen. These results definitively demonstrate that CD8+ memory T cells respond to SE stimulation by proliferating and developing appropriate effector function. Furthermore, the data raise the possibility that otherwise inconsequential exposure to bacterial superantigens may perturb the CD8+ T cell memory pool.      

HIV-infected children with moderate/severe immune-suppression: changes in the immune system after highly active antiretroviral therapy

The objective of this study was to monitor the changes in the immune system of HIV-infected children with moderate or severe immunodeficiency after highly active antiretroviral therapy (HAART), comprising a follow-up study in 14 HIV-infected children on HAART at two time points separated approximately by 11.8 +/- 0.4 (9.9; 15.4) months. HIV-infected children had significantly lower TREC levels than the control group, but 1 year after HAART the levels increased significantly (P < 0.05). In contrast, viral load (VL) did not change significantly. A positive correlation between T cell receptor excision circle (TREC) levels and both CD4(+) T cell absolute counts (r = 0.558; P = 0.05) and percentages (r = 0.625; P = 0.030) was found. During follow-up on HAART, the percentages and absolute counts of naive CD4(+) and CD8(+) T cell subsets were increased significantly (P < 0.05). CD4(+) CD45RA(hi+) CD62L(+), CD4(+) CD45RA(+) and CD4(+) CD38(+) percentages, and the CD8(+) CD45RA(hi+) CD62L(+) counts reached similar values to the control group. Also, CD8(+) CD45RO(+) CD38(+) and CD8(+) CD45RO(+) percentages, and CD8(+) CD45RO(+) CD38(+) absolute counts (P < 0.05) decreased with respect to the baseline. Lymphoproliferative responses to pokeweed mitogen (PWM) before HAART were lower in HIV-infected children than the control group, but they recovered to normal levels after a year on HAART. Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma production by PHA-activated peripheral blood mononuclear cells (PBMC) was lower before HAART (P < 0.001), but reached similar levels to the control group 1 year after HAART. In HIV-infected children IgG, IgG(1) and IgG(3) plasma levels decreased significantly after HAART. The immune system reconstitution induced by HAART in HIV-infected children seems to be the consequence of decreased immune system activation and naive T cell reconstitution, mainly of thymic origin.     

Adrenaline-induced mobilization of T cells in HIV-infected patients

The present study aimed to investigate lymphocyte mobilization from peripheral cell reservoirs in HIV-infected patients. Nine HIV-infected patients on stable highly active anti-retroviral therapy (HAART), eight treatment-naive HIV-infected patients and eight HIV- controls received a 1-h adrenaline infusion. The adrenaline infusion induced a three-fold increase in the concentration of lymphocytes in all three groups. All HIV-infected patients mobilized significantly higher numbers of CD8+ cells but less CD4+ cells. All subjects mobilized CD45RA+CD62L+ and CD8+CD28+ cells to a lesser extent than CD45RO+CD45RA- and CD8+CD28-cells. Furthermore, high numbers of CD8+CD38+ cells were mobilized only in the HIV-infected patients. It was therefore predominantly T cells with an activated phenotype which were mobilized after adrenaline stimulation. It is concluded that the HIV-associated immune defect induced an impaired ability to mobilize immune-competent cells in response to stress stimuli. Furthermore, the study does not support the idea that CD4+ T cells are trapped in lymph nodes by HIV antigens, because untreated and HAART-treated HIV-infected patients mobilized similar numbers of CD4+ T cells. Finally, no evidence was found for the existence of a HAART-induced non-circulating pool of CD4+ T cells.     

Increased Th1 and Th2 allergen-induced cytokine responses in children with atopic disease

BACKGROUND: Polyclonal cytokine responses following stimulation of T cells with mitogens or superantigens provides information on cytokine production from a wide range of T cells. Alternatively allergen-induced T cell responses can provide information on cytokine production by allergen-reactive T cells. While there is evidence of increased Th2 and reduced Th1 cytokine production following T cell stimulation with non-specific mitogens and superantigens, the evidence that Th1 cytokine production to allergens is decreased in line with a postulated imbalance in Th1/Th2 responses is unclear, with studies finding decreased, no difference or increased IFN-gamma responses to allergens in atopic subjects. OBJECTIVE: To examine childhood polyclonal and allergen-induced cytokine responses in parallel to evaluate cytokine imbalances in childhood atopic disease. METHODS: PBMC cytokine responses were examined in response to a polyclonal stimulus, staphylococcal superantigen (SEB), in parallel with two inhalant allergens, house dust mite (HDM) and rye grass pollen (RYE), and an ingested allergen, ovalbumin (OVA), in (a) 35 healthy children (non-atopic) and (b) 36 children with atopic disease (asthma, eczema and/or rhinitis) (atopic). RESULTS: Atopic children had significantly reduced IFN-gamma and increased IL-4 and IL-5 but not IL13 production to SEB superantigen stimulation when compared with non-atopic children. HDM and RYE allergens stimulated significantly increased IFN-gamma, IL-5 and IL-13, while OVA stimulated significantly increased IFN-gamma production in atopic children. CONCLUSION: We show that a polyclonal stimulus induces a reduced Th1 (IFN-gamma) and increased Th2 (IL-4 and IL-5) cytokine pattern. In contrast, the allergen-induced cytokine responses in atopic children were associated with both increased Th1 (INF-gamma) and Th2 (IL-5 and IL-13) cytokine production. The increased Th1 response to allergen is likely to reflect prior sensitization and indicates that increases in both Th1 and Th2 cytokine production to allergens exists concomitantly with a decreased Th1 response to a polyclonal stimulus in atopic children.     

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.     

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.     

Highly active anti-retroviral therapy (HAART) is associated with a lower level of CD4+ T cell apoptosis in HIV-infected patients

HAART may increase CD4+ T cell counts despite a persistently detectable HIV load. The impact of HAART on apoptosis, which may play a role in the disease process in HIV-infected patients, has not been extensively studied. We performed a study to compare the level of spontaneous T cell apoptosis and anti-retroviral treatments in a cohort of HIV-1-infected patients. Data were obtained from a computerized medical record. Quantification of apoptotic cells was by cytofluorometric technique. From November 1995 to December 1997 we studied T cell apoptosis in 112 HIV-infected patients. Forty patients were classified A, 36 B and 36 C. Thirty patients were naive and 82 received an anti-retroviral treatment, 49 including a protease inhibitor (PI). The median plasma viraemia determined in 63 patients was 3.6 (range 1.3-5.6) log10. The median apoptotic cell count was 22% (range 2-73%) and 12% (range 2-60%) for CD4+ and CD8+ T cells, respectively. We did not observe any correlation between the HIV viraemia and the level of apoptosis of T cell subsets. Patients with HAART showed a lower percentage of apoptotic CD4+ T cells only: 16% (range 2-61%) versus 25% (range 5-73%) for patients receiving two nucleoside analogues (P = 0.02). This effect was significant in stage A patients and remained observable during the whole course of HIV disease. In conclusion, HAART, without any relation to plasma viraemia, is able to reduce apoptosis of CD4+ T cells.     

A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.     

The bacterial superantigen Staphylococcal enterotoxin B stimulates lymphocyte locomotor capacity during culture in vitro.

The bacterial superantigen Staphylococcal enterotoxin B (SEB) was investigated for its effects on lymphocyte locomotion in vitro. Culture of peripheral blood mononuclear cells (PBMC) for 24-72 hr in SEB (1-100 micrograms/ml) increased the proportion of lymphocytes in locomotor (polarized) morphology and capable of invading collagen gels, to the same extent as the established locomotor activator, anti-CD3 (alpha-CD3), though the conventional antigen, tetanus toxoid was ineffective. The cells responding to SEB were predominantly T cells. SEB had no effect on lymphocyte locomotion in short-term (45 min) assays, thus its effect is to stimulate growth-related locomotor capacity and it does not act as a chemoattractant. During culture of PBMC in SEB, the chemokines interleukin-8 (IL-8) and macrophage chemotactic protein-1 (MCP-1) were released into the culture medium. The presence of anti-IL-8, but not of anti-MCP-1, either during culture or added to SEB culture supernatants and tested in short-term assays, inhibited the development of polarization suggesting that IL-8, which is a lymphocyte chemoattractant, also plays a key role in SEB-induced locomotor activation. Among SEB-activated lymphocytes, CD45RO+CD45RA- lymphocytes showed enhanced locomotor responses, but a relation was not found between locomotor activity and the presence of cell surface CD69.     

In vivo and in vitro death of mature T cells induced by separate signals to CD4 and alpha beta TCR.

To investigate whether a clonal deletion mechanism is responsible for the mature T cell tolerance that may be induced in vivo by TCR signal to anti-CD4 (H129.19 mAb) coated cells, we analyzed the T cell repertoire in anti-CD4 mAb treated BALB/c mice by flow cytometry following TCR signals through anti-alpha beta TCR mAb or SEB superantigen. Lymph nodes showed a strong reduction in the CD4+/CD8+ cell ratio, and a selective clonal loss of CD4+ V beta 8+ cells 4d following anti-alpha beta TCR or SEB injection, respectively. Following lymph node cell activation in a short-term in vitro assay with SEB or anti-V beta 8 mAb, a selective elimination of CD4+ V beta 8+ cells was again detected, and DNA fragmentation analysis disclosed a cell death by apoptosis. These findings suggest that TCR triggering transduces an apoptotic signal into CD4+ mAb saturated cells that in turn leads to specific holes in the mature T cell repertoire.