Selective induction of p38 mitogen-activated protein kinase activity following A6H co-stimulation in primary human CD4(+) T cells.

We have recently described the novel A6H antigen expressed on human peripheral blood T cells and on renal cell carcinoma cells. Cross-linking of the A6H antigen results in co-stimulation of human CD4(+) T cells, characterized by induction of the transcription factor activator protein-1 (AP-1), proliferation and prominent IFN-gamma production, but low levels of IL-2. The proximal signaling events associated with A6H ligation include protein tyrosine kinase phosphorylation and association of p56 Lck, ZAP-70 and the TCR zeta chain. In this study we show that A6H co-stimulation selectively induced activation of the p38 mitogen-activated protein kinase (MAPK) pathway, whereas no significant c-Jun N-terminal kinases (JNK) activity was observed. In contrast, CD28 co-stimulation resulted in both p38 and JNK MAPK activities. Human CD4(+) T cells co-stimulated with A6H up-regulated AP-1 binding proteins reactive with a proximal AP-1 binding site in the human IFN-gamma promoter and a consensus AP-1 binding site. Moreover, preincubation of the T cells with the specific p38 MAPK inhibitor SB203580 resulted in decreased AP-1 binding following A6H or CD28 co-stimulation. This suggests that the p38 MAPK pathway is required for induction of full AP-1 binding activity in human CD4(+) T cells co-stimulated with A6H or CD28. Blocking the p38 MAPK pathway by SB203580 completely inhibited IFN-gamma production from A6H co-stimulated T cells and radically reduced IFN-gamma production from T cells co-stimulated with anti-CD28. In contrast, no significant inhibition of IL-2 production was seen after blocking of the p38 MAPK in either A6H or CD28 co-stimulated T cells. Since the p38 MAPK recently has been shown to be critically involved in regulation of IFN-gamma production from T(h)1 cells, we propose that A6H co-stimulation induces a specific pathway, mediated via p38 and AP-1 activation, for induction of a T(h)1 profile in human CD4(+) T cells.

Cytokine-induced Src homology 2 protein (CIS) promotes T cell receptor-mediated proliferation and prolongs survival of activated T cells.

Members of the suppressor of cytokine signaling (SOCS) family were discovered as negative regulators of cytokine signaling by inhibition of the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway. Among them, cytokine-induced Src homology 2 (SH2) protein (CIS) was found to inhibit the interleukin 3- and erythropietin-mediated STAT5 signaling pathway. However, involvement of SOCS proteins in other signaling pathways is still unknown. This study shows that the expression of CIS is selectively induced in T cells after T cell receptor (TCR) stimulation. In transgenic mice, with selective expression of CIS in CD4 T cells, elevated CIS strongly promotes TCR-mediated proliferation and cytokine production in vitro, and superantigen-induced T cell activation in vivo. Forced expression of CIS also prolongs survival of CD4 T cells after TCR activation. Molecular events immediately downstream from the TCR are not changed in CIS-expressing CD4 T cells, but activation of mitogen-activated protein (MAP) kinase pathways by TCR stimulation is significantly enhanced. Together with the increased MAP kinase activation, a direct interaction of CIS and protein kinase Ctheta was also demonstrated. These results suggest that CIS is one of the important regulators of TCR-mediated T cell activation. The functions of CIS, enhancing TCR signaling and inhibiting cytokine signaling, may be important in the regulation of immune response and homeostasis.

IL-2 unresponsiveness in anergic CD4+ T cells is due to defective signaling through the common gamma-chain of the IL-2 receptor.

Repeated administration of the superantigen staphylococcal enterotoxin A to mice transduces a state of anergy in the CD4+ T cell compartment, characterized by inhibition of IL-2 production and clonal expansion in vivo. In contrast to what has been reported on anergic T cell clones in vitro, culture of in vivo anergized CD4+ T cells in the presence of exogenous IL-2 did not overcome the block in responsiveness. In this study, we demonstrate that CD4+ T cells from mice anergized with staphylococcal enterotoxin A also exhibit a reduced proliferative capacity in response to IL-7 and IL-15, cytokines that share a common gamma-chain with the IL-2R. Flow-cytometric analysis revealed only modest changes in the expression of the different IL-2R chains. In a number of experiments, our results also provide evidence that excludes a major role of the IL-2R alpha-chain in this system. According to these results, the inability of anergic cells to respond to IL-2 is not mainly due to a down-regulation of the high affinity IL-2R, but to a perturbation in intracellular signaling. Our study confirmed that the activation and tyrosine phosphorylation of Janus-associated kinase 3 and STAT5 were considerably weaker after anergy induction. Moreover, anergic CD4+ T cells showed significantly reduced DNA-binding ability to STAT5-specific elements. Taken together, we suggest that the observed IL-2 unresponsiveness in anergic CD4+ T cells could be due to a defect in signaling through the common gamma-chain of the IL-2R.

The role of cytokines in immunological tolerance: potential for therapy.

Current immunosuppression protocols, although often effective, are nonspecific and therefore hazardous. Consequently, immunological tolerance that is antigen specific and does not globally depress the patient's immune system has become one of the Holy Grails of immunology. Since the discovery that cytokines have immunomodulatory effects, extensive research has investigated the potential of these molecules to induce and maintain specific immunological tolerance in the context of transplantation, allergy and autoimmunity. In this article, we review the possible mechanisms by which cytokines can modulate the immune response and the animal models that frequently confound the theory that a single cytokine, or group of cytokines, can induce tolerance in a predictable manner. Finally, we discuss the role of cytokines at a paracrine level, particularly in the context of inducing and maintaining antigen-specific, regulatory T cells with the clinical potential to suppress specific immune responses.

In vivo anergized CD4+ T cells have defective expression and function of the activating protein-1 transcription factor.

The transcription factor activating protein-1 (AP-1) contributes significantly to the regulation of IL-2 gene expression during T cell activation and has been suggested to play a unique role in T cell anergy in vitro. In this study we have used the superantigen staphylococcal enterotoxin A to investigate the regulation of AP-1 in T cell anergy in vivo. Repeated injections of staphylococcal enterotoxin A induce a state of anergy in CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. The perturbed IL-2 response in anergic T cells correlated with reduced DNA binding activity of the transcription factors AP-1 and Fos/Jun-containing NF-AT. Using AP-1-luciferase reporter transgenic mice, we now demonstrate the lack of AP-1-dependent transcription. AP-1 activity is controlled by synthesis of its subunits Fos and Jun and by posttranslational phosphorylations. Analysis of Fos and Jun protein levels revealed no major differences in the expression of Jun proteins, but a marked decrease in c-Fos in anergic T cells. Experiments in transgenic mice overexpressing c-Fos (H2-c-fos) showed reconstituted AP-1 DNA binding. In contrast, the AP-1-driven transcription and IL-2 production remained suppressed. The Jun N-terminal kinase is known to play a critical role in regulating AP-1 trans-activation. Analyses of Jun N-terminal kinase demonstrated normal protein amounts, but reduced enzymatic activity, in anergic compared with activated CD4+ T cells. This suggests that in vivo anergized T cells have defects in the AP-1 pathway due to both reduced protein expression and perturbed posttranslational modifications.

T cell- and perforin-dependent depletion of B cells in vivo by staphylococcal enterotoxin A.

Bacterial superantigens bind to major histocompatibility complex (MHC) class II and subsequently activate both CD4+ and CD8+ T lymphocytes expressing certain T-cell receptor (TCR)-Vbeta chains. In response to superantigen exposure these subsets proliferate, produce large amounts of proinflammatory cytokines and in addition CD8+ cytotoxic T lymphocytes (CTL) are induced. Previous studies in vitro have shown that these CTL effectively lyse MHC class II-expressing cells presenting the proper superantigen. However, it is unknown whether superantigens induce a similar response towards MHC class II+ antigen-presenting cells in vivo. In this study we demonstrate that administration of repeated injections of the superantigen staphylococcal enterotoxin A (SEA) to TCR-Vbeta3 transgenic mice results in a loss of MHC class II-expressing cells in the spleen. Analysis of different MHC class II+ subsets revealed a selective depletion of CD19+ B cells, while F4/80+ macrophages increased in number. Depletion of T cells with anti-CD4 or anti-CD8 monoclonal antibody indicated that CD8+ T cells were crucial for SEA-induced cytotoxicity in vivo. Repeated injections of SEA to perforin-deficient mice resulted in significantly less B-cell depletion compared with control mice. This suggests that superantigen-activated CD8+ T cells lyse MHC class II+ antigen-presenting cells in a perforin-dependent manner in vivo. It is suggested that this represents a novel bacterial immune escape mechanism, which may particularly impair local humoral immune responses.

Immunoregulatory role of IL-10 during superantigen-induced hyporesponsiveness in vivo.

Injection of the superantigen staphylococcal enterotoxin A (SEA) to mice rapidly elicits production of the Th1-related pro-inflammatory cytokines IL-2, TNF, and IFN-gamma, while repeated SEA challenges transduce a hyporesponsive state characterized by T cell deletions and anergy in the remaining SEA-reactive T cells. In the present study we show that exposure to SEA promotes the development of immunoregulatory IL-10-producing Th cells. Serum IL-10 was undetectable in mice given a priming injection of SEA, but rose to considerable levels when a SEA challenge was administered 4 days later. This coincided with maintained IL-2 production and superinduction of TNF, IFN-gamma, and IL-4. Interestingly, administration of a second SEA challenge resulted in high serum levels of IL-10, but the production of all other studied cytokines (IL-2, TNF, IFN-gamma, and IL-4) was impaired. The IL-10 production was sustained after a third and a fourth SEA challenge in the complete absence of IL-2, IFN-gamma, and IL-4. Pretreatment of mice with neutralizing anti-lL-10 mAb before the SEA challenges substantially enhanced IFN-gamma and TNF serum levels, but failed to rescue IL-2 production. Depletion of T cells with anti-CD4 or anti-CD8 mAb indicated that CD4+ T cells were crucial for SEA-induced IL-10 production. This finding was confirmed using CD4- and CD8- knockout mice. We conclude that repeated SEA challenges promote an IL-10-producing Th cell subset ("Th10" profile), which exerts an immunoregulatory effect by suppressing IFN-gamma and TNF production.

Immune response during tumor therapy with antibody-superantigen fusion proteins.

To engineer superantigens (SAg) to express tumor reactivity, we genetically fused the Fab-part of the tumor-reactive MAb C215 and the bacterial SAg staphylococcal enterotoxin A (SEA). Treatment of mice carrying established lung micrometastases of the C215-transfected syngeneic B16 melanoma with 3-4 daily injections of C215Fab-SEA resulted in strong antitumor effects, while only moderate effects were seen when treatment was given every 4th day (intermittent treatment). High serum levels of IL-2, TNF-alpha, IFN-gamma and strong induction of CTLs (cytotoxic T lymphocytes) were noted after priming with the fusion protein. T cells responded well to 3 daily injections of C215Fab-SEA and then gradually entered a hyporesponsive state, characterized by a reduced ability to produce IL-2, TNF-alpha and IFN-gamma and failure to mediate cytotoxicity in vitro. Intermittent treatment was characterized by increased levels of IL-10, concomitant with accentuated loss of IL-2, TNF-alpha and IFN-gamma production. A 10-fold increase in SEA-reactive TCR V(beta)3+ CD4+ cells was observed in the spleen, while a loss of TCR V(beta)3+ CD8+ and V(beta)11+ CD8+ cells was noted. This is in striking contrast to injections of native SEA which induced a marked deletion of TCR V(beta)3+ CD4+ T cells, but not of CD8+ cells. Recovery of the TH1 cytokine profile occurred within 1-2 weeks, while restoration of cytotoxicity required several months and correlated with recovery of TCR V(beta)3+ CD8+ and TCR V(beta)11+ CD8+ T cells. These results show that the temporal relationship of SAg stimulations dictates the cytokine profile. Moreover, different mechanisms appear to regulate hyporesponsiveness in CD4+ and CD8+ T cells.

In vivo anergized CD4+ T cells express perturbed AP-1 and NF-kappa B transcription factors.

Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell receptor (TCR) variable region beta-chain families and induces strong and rapid production of interleukin 2 (IL-2). In contrast, repeated injections of SEA cause CD4+ T-cell deletion and anergy in the remaining CD4+ T cells, characterized by reduced expression of IL-2 at mRNA and protein levels. We analyzed expression of AP-1, NF-kappa B, NF-AT, and octamer binding transcription factors, which are known to be involved in the regulation of IL-2 gene promoter activity. Large amounts of AP-1 and NF-kappa B and significant quantities of NF-AT were induced in SEA-activated CD4+ spleen T cells, whereas Oct-1 and Oct-2 DNA binding activity was similar in both resting and activated T cells. In contrast, anergic CD4+ T cells contained severely reduced levels of AP-1 and Fos/Jun-containing NF-AT complexes but expressed significant amounts of NF-kappa B and Oct binding proteins after SEA stimulation. Resolution of the NF-kappa B complex demonstrated predominant expression of p50-p65 heterodimers in activated CD4+ T cells, while anergic cells mainly expressed the transcriptionally inactive p50 homodimer. These alterations of transcription factors are likely to be responsible for repression of IL-2 in anergic T cells.

Superantigen-induced anergy in cytotoxic CD8+ T cells.

This study describes the use of bacterial superantigens to investigate the mechanisms governing peripheral tolerance in CD8+ T cells. Administration of superantigens to mice activates T cells to proliferation, cytokine production, and cytotoxicity, but responding cells subsequently enter a state of hyporesponsiveness or are deleted. Superantigen-induced inactivation has so far mainly been demonstrated for CD4+ T cells. Injection of amounts of the superantigen staphylococcal enterotoxin A (SEA) that are optimal for T cell activation and which induce anergy in CD4+ T cells result in preserved responsiveness in CD8+ CTLs. In contrast, we found that intravenous injection of low concentrations of SEA induced a profound down-regulation of the cytotoxic function in SEA-reactive CD8+ TCR V beta 11+ T cells. No reduction in the number of CD8+V beta 11+ T cells was found, suggesting that anergy and not deletion is the main mechanism for the observed cytotoxic hyporesponsiveness. The cytotoxic anergy was evident 2 days after low-dose priming and remained present 4 wk later, indicating a rapid induction phase and long-lasting persistence. The anergized CD8+ T cell subset expressed lower levels of the alpha-(CD11a) chain of the cell adhesion molecule lymphocyte function-associated Ag 1 (LFA-1) and failed to mediate cytotoxicity, but retained the capacity to proliferate, express IL-2R, produce IFN-gamma, and express granzyme mRNA, which imply a partial defect in TCR-transduced signals. Taken together, these findings suggest that there is a biphasic stimulus-dependent threshold for acquiring responsiveness or anergy in CD8+ T cells.

Superantigens anergize cytokine production but not cytotoxicity in vivo.

We have investigated the effect of the superantigen staphylococcal enterotoxin A (SEA) on the balance between T-cell response and non-responsiveness in T-cell receptor (TcR) V beta 3 transgenic mice. One injection of SEA resulted in a substantial activation of TcR V beta 3+ cells, whereas T cells from mice injected with repeated doses of SEA displayed a diminished response to a subsequent in vitro challenge. The reduced responsiveness became apparent when SEA was injected multiple times with short intervals. Proliferation and cytokine production in anergized T cells were severely reduced when stimulated with SEA in vitro, whereas cytotoxic T lymphocyte (CTL) activity remained unaffected. The dichotomy between these functions was examined in vitro with respect to different T-cell subsets. The total number of CD4+ T cells was reduced in the hyporesponsive spleens, compatible with cell deletion. The remaining CD4+ TcR V beta 3+ T cells showed anergy of all tested functions and did not respond to exogenous interleukin-2 (IL-2). In contrast, there was an expansion of CD8+ TcR V beta 3+ T cells with an intact cytotoxic activity. The in vitro proliferation and production of cytokines in the CD8+ compartment was impaired, but could be partially restored in the presence of exogenously added IL-2. Analysis of the cytokine response to SEA in vivo showed that IL-2 and tumour necrosis factor (TNF) were mainly produced by CD4+ T cells, while interferon-gamma (IFN-gamma) was predominantly released by CD8+ T cells. Induction of anergy resulted in a reduction of IL-2 and TNF mRNA levels, frequencies of producing cells as well as serum protein content. In contrast, there was only a moderate influence on the IFN-gamma level in vivo. The results suggest that SEA-induced hyporesponsiveness involves CD4+ cell deletion and a failure to produce cytokines in the remaining CD4+ T-cell compartment, while IFN-gamma production and cytotoxicity in the CD8+ T-cell compartment stay relatively intact.

Immunopharmacology of the superantigen staphylococcal enterotoxin A in T-cell receptor V beta 3 transgenic mice.

The response of mouse T cells to the superantigen staphylococcal enterotoxin A (SEA) requires 1000-fold higher concentrations compared to human T cells. In order to develop a sensitive model for SEA studies in mice, the immunopharmacology has been studied in T-cell receptor (TcR) V beta 3 transgenic (TGV beta 3) and non-transgenic (non-TG) C57Bl/6 mice. The frequency of SEA-responsive T cells in the TGV beta 3 mice exceeded 90%, whereas a 10-fold lower frequency was seen in normal C57Bl/6 mice. Nanograms of SEA injected intravenously into TGV beta 3 mice induced strong cytolytic T lymphocyte (CTL) activity against SEA-coated major histocompatibility complex (MHC) class II+ B-lymphoma cells, whereas administration of 1000-fold higher amounts of SEA to non-TG littermates or normal C57Bl/6 mice induced only a moderate response. Kinetic analysis demonstrated that the CTL activity was more rapidly detectable in TG mice, but substantial levels were seen 2 days after SEA injection in both TGV beta 3 and non-TG mice. The cytotoxic T-cell response induced by SEA in TGV beta 3 and non-TG mice was completely MHC class II dependent, as SEA-coated MHC class II-transfected syngeneic B16 melanoma cells but not untransfected B16 cells were sensitive to lysis. Large amounts of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) accumulated in serum of TGV beta 3 mice after injection of 10 ng SEA, whereas only marginal amounts were recorded in non-TG even after injection of 100 micrograms SEA. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that SEA-induced TNF-alpha and IFN-gamma mRNA reached maximal levels 1 hr after SEA administration in TGV beta 3 mice, whereas peak serum levels of TNF-alpha and IFN-gamma proteins were recorded after 2 hr. Comparison of the mRNA levels of a panel of cytokines in the TGV beta 3 and non-TG mice revealed that almost similar amounts of interleukin-1 (IL-1) were induced in both strains, whereas IL-4 was only detected at significant levels in the TGV beta 3 mouse. The results suggest that TGV beta 3 mice are suitable for studying in vivo immune responses to superantigens at concentrations comparable to the potent effects elicited in humans. Moreover, this model is useful for detailed studies on the dynamic regulation of T-cell activation and anergy induced by superantigens.

Superantigen-induced cytokines suppress growth of human colon-carcinoma cells.

We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) conjugated to colon-carcinoma-reactive monoclonal antibodies (MAbs) directs T cells to lyse human colon-carcinoma cells, representing a potential novel tumor therapy. To further analyze the mechanism of antitumor effects of superantigen-activated T cells, we compared the activity of free and MAb-conjugated SEA in a long term in vitro co-culture assay of human peripheral-blood mononuclear cells (PBMC) and colon-carcinoma cell lines. Activation of resting T lymphocytes with SEA conjugated to the colon-carcinoma-reactive MAb C215 or free SEA resulted in strong inhibition of the growth of all studied colon-carcinoma cell lines. The growth of WiDr colon-carcinoma cells was unaffected by the presence of unactivated mononuclear cells, whereas addition of pM concentrations of SEA or C215-SEA conjugate completely suppressed tumor-cell growth. The suppressive effect was mediated by both CD4+ and CD8+ T cells and required the presence of MHC-Class II+ monocytes. The inhibition of tumor-cell growth was to a large extent mediated by soluble factors present in supernatants from SEA- or C215-SEA-activated mononuclear cells. Quantitation of cytokine mRNA in SEA-activated mononuclear cells by the reverse transcriptase-polymerase chain reaction (RT-PCR) revealed strong induction of mRNA encoding the cytokines IL-1 alpha, IL-1 beta, IL-2, IL-6, TNF-alpha, TNF-beta and IFN-gamma. The use of cytokine-specific MAb demonstrated that IFN-gamma was of major importance for the tumor-growth-inhibitory activity in supernatants of SEA-activated lymphocytes. Addition of recombinant cytokines to WiDr colon-carcinoma cells showed that TNF-alpha was able to act synergistically with IFN-gamma to suppress tumor-cell growth. The local production of tumor-suppressive cytokines induced by MAb-targeted superantigens is likely to be of particular relevance for inhibition of the growth of tumor cells not expressing the targeted tumor-associated antigen.

Targeting of superantigens.

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on MHC class II antigens. In order to induce T lymphocytes to reject a tumor, we substituted the specificity of SEA for MHC class II molecules with specificity for tumor cells by combining SEA with a MAb recognizing colon carcinomas. Chemical conjugates or recombinant fusion proteins of the MAb C215 and SEA retained excellent antigen binding properties whereas the binding to MHC class II was markedly reduced. The hybrid proteins directed SEA responsive T cells to tumors with specificity determined by the specificity of the MAb. Significant tumor cell killing was obtained at picomolar concentrations of the hybrid proteins and was the result of direct cell mediated by cytotoxicity as well as production of tumoricidal cytokines by T cells. Targeting of superantigens represents a novel approach to specific immunomodulation and deserves further study as a potential therapy for malignant disease.

Monoclonal antibodies and superantigens: a novel therapeutic approach.

We have developed a monoclonal antibody (mAb) based therapy intended for the treatment of solid tumors utilizing both main arms of the immune system by incorporating the colon carcinoma recognizing mAb C215 and the T cell activating bacterial staphylococcal enterotoxin A (SEA) in a single hybrid molecule. The recombinant tumor specific superantigen C215-SEA retained excellent antigen binding properties while the binding to MHC class II was markedly reduced and should allow targeting of a large fraction of T cells to tumors in vivo. C215-SEA mediated T cell killing of C215 expressing tumor cells irrespective of their expression of MHC class II antigens and induced levels of IFN-gamma and TNF in mononuclear cells sufficient to completely suppress the growth of colon carcinoma cells in vitro. In initial studies of anti-tumor effects, C215Fab-SEA was found to markedly inhibit the growth of colon carcinoma cells transplanted to Scid mice adoptively transferred with human mononuclear cells.