Stimulation of human naive and memory T helper cells with bacterial superantigen. Naive CD4+45RA+ T cells require a costimulatory signal mediated through the LFA-1/ICAM-1 pathway.

The role of the accessory molecule ICAM-1 in activation of subpopulations of human T cells was examined using the bacterial superantigen staphylococcal enterotoxin A (SEA) as a MHC class II and TCR-dependent polyclonal T cell activator. Human T cells responded with different sensitivity to SEA when presented on mouse accessory cells expressing a human transfected MHC class II gene product. Mouse L cells cotransfected with both MHC class II (DR2A or DR7) and ICAM-1-stimulated T cells at 100-fold lower concentrations of SEA as compared to the single transfected cells. mAb reacting with the CD11a, CD18, or ICAM-1 molecules efficiently inhibited T cell activation with the cotransfected HLA-DR2A/ICAM-1 cell but did not influence T cell activation with the HLA-DR2A single transfected cell. Analysis of the ICAM-1 requirement on CD4+ memory (CD4+45RO+) and naive (CD4+45RA+) T cells revealed that CD4+45RA+ naive Th cells were hyporesponsive to SEA-induced activation with the HLA-DR2A single transfectant. However, cotransfection of ICAM-1 enabled these cells to respond to low doses of SEA implicating that they are more dependent on accessory molecules than the CD4+45RO+ cells. rICAM-1 immobilized on a plastic surface, was able to strongly costimulate SEA-induced T cell activation with the HLA-DR2A single transfectant, suggesting that costimulatory signals mediated to the T cells through LFA-1 can be delivered physically separated from the TCR signal. CD4+45RO+ memory and CD4+45RA+ naive Th cells apparently differ in their capacities to be activated by SEA bound to HLA-DR. Although the TCR molecule densities are similar in these two subsets, costimulation with ICAM-1 is required for activation of the CD4+45RA+, but not the CD4+45RO+ T cell subset at 1 to 10,000 ng/ml concentrations of SEA. This observation indicates different activation thresholds of naive and memory Th cells when triggering the TCR over a wide dose interval of superantigen.     

Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells

Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1.     

Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease

Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.     

Staphylococcal toxins bind to different sites on HLA-DR

Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin 1 (TSST-1) bind to MHC class II molecules and the toxin-class II complexes induce proliferation of T cells bearing specific V beta sequences. We have previously reported that these toxins display varying binding affinities for HLA-DR1. We now investigated whether these differences simply reflected differences in binding affinity for a single class II binding site or, at least in part, the engagement of different binding sites on the HLA-DR complex. Through competitive binding studies we show that SEB and TSST-1, which are not closely related by their amino acid sequences, bind to two different sites on HLA-DR. Both of these sites are also occupied by staphylococcal enterotoxin A (SEA), enterotoxin D (SED), and enterotoxin E (SEE) which exhibit more than 70% amino acid sequence homology. SEB and TSST-1 failed to inhibit SEA binding to HLA-DR. These studies suggest that there may be three distinct, although perhaps overlapping, binding sites on HLA-DR for these toxins. Further, although SED and SEE are similar to SEA in structure, and appear to bind the same sites on HLA-DR as SEA, they displayed significantly lower binding affinities. T cell proliferative responses to SED required a higher concentration of the toxin than SEA, probably reflecting its lower binding affinity. SEE, however, elicited T cell responses at very low concentrations, similar to SEA, despite its much lower binding affinity. Therefore, although the affinities of these toxins to MHC class II molecules appear to significantly influence the T cell responses, the effective recognition of the toxin-class II complex by the TCR may also contribute to such responses.     

Crystal structure of a SEA variant in complex with MHC class II reveals the ability of SEA to crosslink MHC molecules

Although the biological properties of staphylococcal enterotoxin A (SEA) have been well characterized, structural insights into the interaction between SEA and major histocompatibilty complex (MHC) class II have only been obtained by modeling. Here, the crystal structure of the D227A variant of SEA in complex with human MHC class II has been determined by X-ray crystallography. SEA(D227A) exclusively binds with its N-terminal domain to the alpha chain of HLA-DR1. The ability of one SEA molecule to crosslink two MHC molecules was modeled. It shows that this SEA molecule cannot interact with the T cell receptor (TCR) while a second SEA molecule interacts with MHC. Because of its relatively low toxicity, the D227A variant of SEA is used in tumor therapy.     

Signals delivered via MHC class II molecules synergize with signals delivered via TCR/CD3 to cause proliferation and cytokine gene expression in T cells.

We examined the role of MHC class II molecules in transducing signals to activated human T cells. Cross-linking of MHC class II molecules synergized with submitogenic amounts of anti-CD3 mAb in causing proliferation and secretion of the cytokines IL-2, IL-3, IFN-gamma, and TNF-alpha by MHC class II-alloreactive T cell lines. Signaling via MHC class II molecules in T cells resulted in activation of tyrosine kinases, in generation of inositol phosphates, and in Ca2+ mobilization that was abrogated by the tyrosine kinase inhibitor herbimycin A. Thus, like signaling via TCR/CD3, signaling via MHC class II molecules involved tyrosine kinase-dependent activation of phospholipase C, resulting in phosphoinositol turnover and Ca2+ flux. However the signaling pathways coupled to MHC class II molecules and to TCR/CD3 differed, because engagement of the transmembrane phosphatase CD45 inhibited Ca2+ fluxes triggered via TCR/CD3 but not Ca2+ fluxes triggered via MHC class II molecules.     

Quantitative relationship between MHC class II-superantigen complexes and the balance of T cell activation versus death

The binding of bacterial superantigens (SAgs) is profoundly affected by the nature of the MHC class II-associated antigenic peptide. It was proposed that this limitation in the density of SAgs displayed at the surface of APCs is important for efficient TCR serial triggering as well as for preventing apoptosis of the responding T lymphocytes. Here, we have addressed quantitatively the size of this SAg-receptive pool of HLA-DR molecules that are available to bind and present staphylococcal enterotoxin A (SEA) at the surface of B lymphocytes. Our binding curves, depletion experiments, and quantitative immunoprecipitations show that about half the HLA-DR class II molecules on B cells are refractory to SEA binding. Yet, as compared with typical nominal Ags, an unusually high amount of class II-SAg complexes can be presented to T cells. This characteristic appears to be necessary for SAg-induced T cell apoptosis. When <0.3% of the total cell surface MHC class II molecules are occupied by SEA, T cells undergo a normal sequence of early activation events. However, presentation of a ligand density beyond this threshold results in T cell activation that is readily aborted by apoptosis but only after a few cell divisions. Thus, we confirm the existence of MHC class II subsets that are structurally unable to present SEA and provide a quantitative framework to account for the ability of bacterial SAgs to induce peripheral activation vs tolerance in the host.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.     

A regulatory role for the CD4 and CD8 molecules in T cell activation

The role of the CD4 and CD8 molecules in T cell activation is presently a matter of controversy. Although their role as associative binding elements to MHC class II or class I is well documented, their influence on the triggering process in unclear. Because antibodies to CD4 or CD8 block T cell activation in the absence of their respective ligands, a negative signaling by these molecules has been suggested. However, recent experimental evidence argues against a negative regulatory effect of these molecules, since, e.g., simultaneous cross-linking of TCR and CD4 leads to enhanced T cell activation. Therefore, a current model suggests that the association of TCR and CD4 in the membrane gives a positive signal essential for triggering. In this report we present evidence that this model is likely to be too simple. Anti-CD4 and CD8 antibodies inhibit alternative, nonreceptor pathways of T cell triggering via Tp103 and Tp44 in the absence of class II positive accessory or target cells. These antibodies also inhibit bypass activation of T cells by phorbol ester and calcium ionophore in an accessory cell-free system. Furthermore, if the CD4 or CD8 molecules are removed from the cell surface by antibody-induced modulation, the proliferative and cytotoxic response of T cell clones is enhanced. This enhancement is also observed if resting peripheral blood T cells are used as responder cells. These data show that the CD4 or CD8 molecules have a complex regulatory function in T cell activation beyond the requirement for co-cross-linking with the TCR.     

Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.     

Amino acid substitutions in the putative MHC class II "dimer of dimers" interface inhibit CD4+ T cell activation

Activation of T lymphocytes is dependent on multiple ligand-receptor interactions. The possibility that TCR dimerization contributes to T cell triggering was raised by the crystallographic analysis of MHC class II molecules. The MHC class II molecules associated as double dimers, and in such a way that two TCR (and two CD4 molecules) could bind simultaneously. Several subsequent studies have lent support to this concept, although the role of TCR cross-linking in T cell activation remains unclear. Using DRA cDNAs modified to encode two different C-terminal tags, no evidence of constitutive double dimer formation was obtained following immunoprecipitation and Western blotting from cells transiently transfected with wild-type DRB and tagged DRA constructs, together with invariant chain and HLA-DM. To determine whether MHC class II molecules contribute actively to TCR-dependent dimerization and consequent T cell activation, panels of HLA-DR1beta and H2-E(k) cDNAs were generated with mutations in the sequences encoding the interface regions of the MHC class II double dimer. Stable DAP.3 transfectants expressing these cDNAs were generated and characterized biochemically and functionally. Substitutions in either interface region I or III did not affect T cell activation, whereas combinations of amino acid substitutions in both regions led to substantial inhibition of proliferation or IL-2 secretion by human and murine T cells. Because the amino acid-substituted molecules were serologically indistinguishable from wild type, bound antigenic peptide with equal efficiency, and induced Ag-dependent CD25 expression indicating TCR recognition, the reduced ability of the mutants to induce full T cell activation is most likely the result of impaired double dimer formation. These data suggest that MHC class II molecules, due to their structural properties, actively contribute to TCR cross-linking.     

T cell receptor-independent CD4 signalling: CD4-MHC class II interactions regulate intracellular calcium and cyclic AMP

CD4 is a coreceptor on T helper (Th) cells that interacts with MHC class II molecules (MHCII). The mechanisms mediating the effects of CD4 on responses by T helper cells to stimulation of the antigen-specific T cell receptor (TCR) are still poorly understood. Here, we demonstrate T cell costimulation via CD4 signalling independent of T cell receptor-mediated signals. Incubation of T helper cells with peptide mimetics of the CD4-binding region on the MHC class II beta2 domain caused intracellular calcium mobilization in the absence of antigen or other T cell receptor stimuli. Engagement of CD4 by peptide mimetics or wild-type MHC class II, but not by mutant MHC class II molecules incapable of engaging CD4, inhibited the T cell receptor-mediated increase in cyclic AMP (cAMP) concentrations in T helper cells. CD4-mediated signals activated cyclic AMP phosphodiesterases (PDEs) and inhibited adenylyl cyclase. Full activation and clonal expansion of antigen-stimulated T helper cells required the CD4-mediated regulation of cyclic AMP. Our results suggest a costimulatory mechanism of CD4 function that acts on the second messengers, calcium and cyclic AMP.     

Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders, including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules, we investigated whether ASM can present SAg to resting CD4(+) T cells, and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg, staphylococcal enterotoxin A (SEA), elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface, indicative of immunological synapse formation, in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine, IL-13, into the coculture medium, which was MHC class II dependent. Moreover, when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues, the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively, these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells, and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that, in turn, evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus, a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and, accordingly, may underlie the reported association between microbial SAg exposure, T cell activation, and severe asthma.     

Superantigen and HLA-DR ligation induce phospholipase-C gamma 1 activation in class II+ T cells.

Bacterial enterotoxin superantigens bind directly to HLA class II molecules (HLA-DR) expressed on both APC and activated human T cells, and simultaneously bind to certain V beta chains of the TCR. In this report, we compared early T cell signaling events in human alloantigen-stimulated T cells when activated by HLA-DR ligation through antibody cross-linking or by direct enterotoxin superantigen binding. Both types of stimuli induced tyrosine phosphorylation of phosphatidylinositol-specific phospholipase C gamma 1 (PLC gamma 1) and an increase in intracellular calcium concentration; however, superantigen-induced signaling was stronger than class II ligation alone. Antibody-mediated ligation of HLA-DR with CD3 resulted in augmented PLC gamma 1 activation and increased calcium mobilization, consistent with a mechanism of superantigen activity through a combination of class II and CD3/Ti signals. In addition, down-modulation of CD3 receptors with antibody demonstrated that superantigen-induced signaling events were CD3-dependent. Superantigen signaling was also class II-dependent, in that resting T cells were not responsive to direct enterotoxin stimulation. To address how early signal transducing activity correlated with T cell responsiveness, alloantigen-primed T cells were activated with immobilized class II-specific mAb or soluble superantigen. Both HLA-DR mAb-stimulated T cells and enterotoxin-treated T cells proliferated strongly in response to co-stimulation by a combination of CD28 receptor engagement and PMA addition. In addition, superantigen-induced growth was induced by CD28 receptor ligation with antibody or the B7 counter-receptor expressed on Chinese hamster ovary cells. Taken together, these results indicate that class II molecules expressed on activated T cells are directly coupled to the PLC gamma 1 signal transduction pathway, and that coligation of HLA-DR with CD3 augments T cell signaling comparable to that induced by enterotoxin superantigen. Thus, we suggest that superantigen-induced early signaling responses in activated T cells may be due in part to class II transmembrane signals induced when HLA-DR and V beta are ligated in cis.     

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.     

Signal transduction via CD4, CD8, and CD28 in mature and immature thymocytes. Implications for thymic selection.

The positive and negative selection of immature thymocytes that shapes the mature T cell repertoire appears to occur at an intermediate stage of development when the cells express low levels of TCR/CD3. These cells are also CD4+CD8+ and CD28+ (dull), and signals delivered by these three accessory molecules have been implicated in the selection process. We have examined the regulatory function of these accessory molecules on responses of immature thymocytes stimulated through the TCR/CD3 complex. Cross-linking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosine phosphorylation and calcium mobilization. Subsequent cell proliferation could be induced by soluble anti-CD28 mAb, which was comitogenic for cells stimulated with CD3 x CD4 or CD3 x CD8 cross-linking, but was without effect on cells stimulated with CD3 x CD3 cross-linking. A potential role for CD28 signal transduction in thymic maturation is suggested by the demonstration that the BB-1 molecule, a natural ligand for CD28, is expressed on thymic stromal cells. Taken together, our data suggest a model of thymic development in which CD4 or CD8 may enhance TCR/CD3 signaling upon coligation by an MHC molecule. If the CD28 surface receptor is simultaneously stimulated by a BB-1 expressing stromal cell, this set of interactions could lead to proliferation and positive selection. In the absence of CD28 stimulation the enhanced TCR/CD3 signals might lead to apoptosis and negative selection.     

Staphylococcal enterotoxin H displays unique MHC class II-binding properties

Staphylococcal enterotoxin H (SEH) has been described as a superantigen by sequence homology with the SEA subfamily and briefly characterized for its in vivo activity. In this study, we demonstrate that SEH is a potent T cell mitogen and inducer of T cell cytotoxicity that possesses unique MHC class II-binding properties. The apparent affinity of SEH for MHC class II molecules is the highest affinity ever measured for a staphylococcal enterotoxin (Bmax1/2 approximately 0.5 nM for MHC class II expressed on Raji cells). An excess of SEA or SEAF47A, which has reduced binding to the MHC class II alpha-chain, is able to compete for binding of SEH to MHC class II, indicating an overlap in the binding sites at the MHC class II beta-chain. The binding of SEH to MHC class II is like SEA, SED, and SEE dependent on the presence of zinc ions. However, SEH, in contrast to SEA, binds to the alanine-substituted DR1 molecule, betaH81A, believed to have impaired zinc-bridging capacity. Furthermore, alanine substitution of residues D167, D203, and D208 in SEH decreases the affinity for MHC class II as well as its in vitro potency. Together, this indicates an MHC class II binding site on SEH with a different topology as compared with SEA. These unique binding properties will be beneficial for SEH to overcome MHC class II isotype variability and polymorphism as well as to allow an effective presentation on APCs also at low MHC class II surface expression.     

Apoptosis observed in murine peritoneal macrophages treated with interferon gamma through staphylococcal enterotoxin-dependent cell-mediated cytotoxicity

The concept of superantigens is well-known and widely accepted. In this brief communication, we analyze the behaviour of antigen-presenting cells after T-cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T-cell activation was observed. We, therefore, analyzed surface-specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon gamma. Interferon gamma-primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T-cell activation.