Staphylococcal exotoxin activation of T cells. Role of exotoxin-MHC class II binding affinity and class II isotype

Staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 bind directly to class II molecules of the MHC and stimulate T cells based predominantly on the V beta segment used by the TCR. We investigated the relationship between the class II binding affinities of four of these exotoxins, SEA, SEB, SEC1, and toxic shock syndrome toxin-1 and their T cell signaling capabilities. Although the toxins stimulated T cells at concentrations that ranged over more than two orders of magnitude, their affinities for class II (DR1) differed by less than sixfold. The affinities of the toxins predicted their capacity to stimulate resting T cells to proliferate. The binding affinities of the toxins for class II molecules indicated that at concentrations required for T cell stimulation, as few as 0.1% of the class II molecules are complexed with toxin. Finally, the isotype of class II molecules affected the ability of the toxins to bind and use these MHC Ag to stimulate T cells. These data thus demonstrate that of the staphylococcal exotoxins studied, both their potency as T cell mitogens and their ability to function in the presence of single class II isotypes can be attributed in part to their characteristic abilities to bind class II molecules.     

T lymphocyte activation by staphylococcal enterotoxins: role of class II molecules and T cell surface structures

The enterotoxins produced by Staphylococcus aureus (SE) are the most potent mitogens known. Triggering of proliferation or cytotoxicity by SE requires the presence of MHC class II molecules on accessory or target cells. In this study we have investigated the role of HLA class II molecules in the activation of human T cells by SE and the nature of the target structure on the responding T lymphocyte for SE. This dependence on class II molecules is not due to an immunological "recognition" of SE since there is no restriction by polymorphic determinants of HLA molecules and since even xenogeneic class II molecules can reconstitute the human T cell response to SE. Furthermore, HLA class II-positive but not -negative cells absorb the mitogenic activity from SE solutions and significant binding of 125I-labeled SE can be demonstrated to class II-positive but not to class II-negative cells. Enterotoxin molecules react directly with T cells since they cause an increase in cytosolic Ca2+ concentration similar to anti-CD3 mAb. This increase is abrogated by prior modulation of the TCR/CD3 complex. Antibodies to CD2, CD3 and the TCR that block antigen-specific activation also block T cell activation by SE. Moreover, preincubation of purified resting accessory cell-free T cells with SE leads to modulation of the TCR/CD3 complex. Taken together these data indicate that SE interact selectively with HLA class II molecules on accessory or target cells and with a TCR-associated structure on the T cell.     

Staphylococcal enterotoxin B and toxic shock syndrome toxin-1 bind to distinct sites on HLA-DR and HLA-DQ molecules

Staphylococcal enterotoxins (SE) activate human T cells in vitro. This requires the presence of Ia+ accessory cells but does not require processing of the toxin by the accessory cell. We and others have recently demonstrated direct binding of SE to human MHC class II molecules. In this study, we have compared in detail the ability of class II molecules to bind two SE, toxic shock syndrome toxin-1 (TSST-1) and SEB. Scatchard analysis of equilibrium binding data indicate that SEB binds to Ia+ human cell lines with a 10-fold lower affinity than TSST-1. Likewise, SEB precipitates HLA-DR alpha- and beta-chains from detergent lysates of Ia+ cells less efficiently than TSST-1. The binding of TSST-1 and SEB to transfected L cells expressing HLA-DR and HLA-DQ gene products was differentially inhibited by anti-HLA-DR mAb. There was virtually no cross-inhibition of TSST-1 and SEB in competitive binding assays. We conclude that TSST-1 and SEB bind to two MHC class II sites which can be distinguished by their relative accessibility to blocking by anti-class II mAb and heterologous toxin.     

Staphylococcal enterotoxin-dependent lysis of MHC class II negative target cells by cytolytic T lymphocytes.

The enterotoxins of Staphylococcus aureus (SE) are extremely potent activators of human and mouse T lymphocytes. In general, T cell responses to SE are MHC class II dependent (presumably reflecting the ability of SE to bind directly to MHC class II molecules) and restricted to responding cells expressing certain T cell receptor beta-chain variable (TCR V beta) domains. Recently we demonstrated that CD8+ CTL expressing appropriate TCR V beta could recognize SE presented on MHC class II-bearing target cells. We now show that MHC class II expression is not strictly required for T cell recognition of SE. Both human and mouse MHC class II negative target cells could be recognized (i.e., lysed) in a SE-dependent fashion by CD8+ mouse CTL clones and polyclonal populations, provided that the CTL expressed appropriate TCR V beta elements. SE-dependent lysis of MHC class II negative targets by CTL was inhibited by mAb directed against CD3 or LFA-1, suggesting that SE recognition was TCR and cell contact dependent. Furthermore, different SE were recognized preferentially by CTL on MHC class II+ vs MHC class II- targets. Taken together, our data raise the possibility that SE binding structures distinct from MHC class II molecules may exist.     

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.     

An evolutionary conserved mechanism of T cell activation by microbial toxins. Evidence for different affinities of T cell receptor-toxin interaction.

The enterotoxins produced by Staphylococcus aureus are the most potent mitogens known. They belong to a group of distantly related mitogenic toxins that differ in other biologic activities. In this study we have compared the molecular mechanisms by which these mitogens activate human T lymphocytes. We used the staphylococcal enterotoxins A to E, the staphylococcal toxic shock syndrome toxin, the streptococcal erythrogenic toxins A and C (scarlet fever toxins, erythrogenic toxins (ET)A, ETC), and the soluble mitogen produced by Mycoplasma arthritidis. We found that all these toxins can activate both CD4+ and CD8+ T cells and require MHC class II expression on accessory and target cells. However, T cells could be activated in the absence of class II molecules if the toxins ETA or SEB were co-cross-linked on beads together with anti-CD8 or anti-CD2 antibodies. Enterotoxins, toxic shock syndrome toxin and scarlet toxins stimulate a major fraction of human T cells, and show preferential, but not exclusive, stimulation of T cells carrying certain TCR V beta. In contrast, the mitogen of M. arthritidis, a pathogen for rodents stimulates only a minority of human T cells but activates a major fraction of murine T cells. Analysis of human T cell clones expressing V beta 5 or V beta 8 TCR showed that these clones responded also to those toxins that did not stimulate V beta 5+ and V beta 8+ T cells in bulk cultures. These results indicate that different TCR bind to these toxins with different affinities and that the specificity of the TCR-V beta-toxin interaction is quantitative rather than qualitative in nature. Taken together our findings suggest that these toxins use a common mechanism of T cell activation. They are functionally bivalent proteins crosslinking MHC class II molecules with variable parts of the TCR. Besides V beta, other parts of the TCR must be involved in this binding. The finding that murine T cells responded more weakly to the toxins produced by the human-pathogenic bacteria than to the Mycoplasma mitogen could indicate that the toxins have been adapted to the host's immune system in evolution.     

H2-O inhibits presentation of bacterial superantigens, but not endogenous self antigens

H2-O/HLA-DO are MHC class II accessory molecules that modulate exogenous Ag presentation. Most class II accessory molecules are expressed in all professional APC; however, H2-O is only expressed in B cells and medullary thymic epithelial cells. Because B cells present exogenous Ags and superantigens (SAgs), and medullary thymic epithelial cells are specialized APC for self Ags during negative selection in the thymus, we have hypothesized that H2-O might play a role in MHC class II-restricted SAg and self Ag presentation. In this study, we demonstrate that H2-O expression inhibits presentation of the bacterial SAgs staphylococcal enterotoxins A and B to four SAg-reactive T hybridoma cells. In contrast, H2-O has no effect on presentation of endogenous self Ags, as measured by tumorigenicity in vivo and Ag presentation to three self Ag-specific T hybridoma cells. Additional experiments suggest that H2-O inhibits presentation of exogenous Ags by both newly synthesized and recycling MHC class II molecules. These data suggest H2-O may have a physiological role in tolerance induction and SAg-mediated toxic shock.     

Superantigens interact with MHC class II molecules outside of the antigen groove

Superantigens, including the staphylococcal enterotoxins and the minor lymphocyte stimulatory antigens, are highly potent immunostimulatory molecules, capable of activating virtually all T cells that express particular T cell receptor (TCR) variable regions. Superantigen stimulation of T lymphocytes depends on major histocompatibility complex (MHC) class II molecules, so there has been some debate as to whether superantigens interact with the antigen binding "groove" on class II complexes, just like conventional peptide antigens, or whether they bind elsewhere and serve as TCR coligands. We compared the presentation of peptide antigens and superantigens by a panel of mutant-presenting cell lines, each displaying an A kappa alpha chain with a single alanine replacement along the alpha helix proposed to form one face of the groove. The negligible effect of these 30 mutations on superantigen presentation, versus their drastic consequences for peptide presentation, prompts us to conclude that superantigens interact with MHC class II molecules outside the groove.     

Effect of isotypes and allelic polymorphism on the binding of staphylococcal exotoxins to MHC class II molecules

Interaction of staphylococcal exotoxins (SE) with MHC class II molecules plays a central role in the activation of immune cells by SE. We have recently demonstrated directly that toxic shock syndrome toxin-1 (TSST-1) binds to MHC class II molecules with high affinity, and similar results have been reported for SEA and SEB. The ability of T cells to respond to individual SE is associated with the expression of particular TCR-V beta gene elements. In the present study we have examined the effect of polymorphism on the ability of MHC class II molecules to bind SEB and TSST-1. We have used a panel of L cell transfectants that express different allelic forms of each of the three human class II isotypes. Radioligand binding assays detected binding of SEB and TSST-1 to most, but not all of the MHC class II molecules examined. Toxin-driven MHC class II-dependent T cell proliferation occurred with all transfectants examined even in the absence of detectable toxin binding. These results indicate that SE can bind to human MHC class II molecules of diverse phenotypes.     

Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM

Major histocompatibility complex (MHC) class II molecules bind and present to CD4(+) T cells peptides derived from endocytosed antigens. Class II molecules associate in the endoplasmic reticulum with invariant chain (Ii), which (i) mediates the delivery of the class II-Ii complexes into the endocytic compartments where the antigenic peptides are generated; and (ii) blocks the peptide-binding site of the class II molecules until they reach their destination. Once there, Ii must be removed to allow peptide binding. The bulk of Ii-class II complexes reach late endocytic compartments where Ii is eliminated in a reaction in which the cysteine protease cathepsin S and the accessory molecule H-2DM play an essential role. Here, we here show that Ii is also eliminated in early endosomal compartments without the intervention of cysteine proteases or H-2DM. The Ii-free class II molecules generated by this alternative mechanism first bind high molecular weight polypeptides and then mature into peptide-loaded complexes.     

Cysteinylation of MHC class II ligands: peptide endocytosis and reduction within APC influences T cell recognition

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4(+) T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.     

Activation of MyD88 signaling upon staphylococcal enterotoxin binding to MHC class II molecules

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88(-/-) mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II(+) cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1β. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1β. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.     

Stimulator cell-dependent requirement for CD2- and LFA-1-mediated adhesions in T lymphocyte activation by superantigenic toxins.

The staphylococcal enterotoxins and related microbial T cell mitogens stimulate T cells by cross-linking variable parts of the T cell receptor (TCR) with MHC class II molecules on accessory or target cells. We have used cloned human T cells and defined tumor cells as accessory cells (AC) to study the requirements for T cell activation by these toxins. On AC expressing high levels of CD54 (intercellular adhesion molecule-1, ICAM-1) and CD58 (lymphocyte function-associated antigen-3, LFA-3), mAb to CD2 were relatively ineffective in inhibiting the response to the toxins and antibodies to the lymphocyte function-associated antigen-1 (LFA-1) did not inhibit at all. If added together, however, these mAb inhibited the response completely. Similar results were obtained using antibodies to the target structures of CD2 and LFA-1. In contrast, on cells expressing low levels of LFA-3, mAb to LFA-1 but not to CD2 were strongly inhibitory. The same pattern of inhibition was found when these same cells were used as presenters of specific antigen to the T cells. These data show that adhesions via CD2 or LFA-1 are alternatively required for the stimulation of the T cells by superantigenic toxins and demonstrate another similarity between T cell stimulation by superantigens and by specific antigen recognition.     

Immunosuppressive activity of staphylococcal enterotoxin B. II. Activation of suppressor-effector cells by a staphylococcal enterotoxin B-induced suppressor factor

The capacity of staphylococcal enterotoxins to stimulate all T cells bearing certain T cell receptors has recently generated a great deal of interest. These toxins are believed to bind directly both to the TCR:CD4 complex via its V beta domains and to class II MHC molecules on accessory cells prior to T cell activation. Previous studies from this laboratory have demonstrated that staphylococcal enterotoxin B (SEB) is capable of inducing multiple T suppressor cell populations which can inhibit in vitro antibody responses. Additional studies have demonstrated that the suppressive activity of these cells is mediated, at least in part, by an I-J-restricted suppressor factor. Efforts to characterize the inhibitory activity of this factor have demonstrated that the suppressive element is capable of activating both early and late acting suppressor cell populations in vitro. Analysis by both positive and negative selection shows that cells bearing the Lyt1-2+ surface marker phenotype are active early, whereas Lyt1+2+ cells are active both early and late in the antibody response. Additional experiments using various strains of mice as sources of suppressor factor and of naive splenocyte populations have demonstrated that activation of suppressor-effector cells by this suppressor factor is restricted at the I-J, but not Igh, gene locus. These studies suggest that this SEB-induced suppressor factor alone provides the signals necessary for the induction and activation of suppressor-effector cell activity.     

Control of T cell responses to staphylococcal enterotoxins by stimulator cell MHC class II polymorphism

The bacterial toxic mitogens or superantigens are a family of related proteins that elicit potent T cell proliferative responses. These responses require APC that express MHC class II proteins, but they are not MHC restricted and they do not depend on a processing step, presumably because these mitogens bind directly to MHC class II molecules. These mitogens stimulate T cells by interacting in an unknown way with the portion of the TCR encoded by certain V beta gene segments. In this paper, we explore the importance of MHC class II polymorphism in T cell responses to staphylococcal enterotoxins. We find that certain MHC molecules present SEB to V beta 8-bearing T cells far better than others. These data suggest that one route of host defence against bacterial toxic mitogens may be to alter MHC class II molecules so that stimulation is inhibited.     

Stimulation of B10.BR T cells with superantigenic staphylococcal toxins

The Staphylococcus aureus enterotoxins are known to be potent T cell activators, stimulating cell proliferation and lymphokine production. Two additional S. aureus proteins, exfoliating toxin and toxic shock syndrome toxin, share these properties. Recently these molecules have been termed "super-antigens" because of their ability to bind to class II MHC molecules and thus form ligands that interact with TCR in an unconventional manner. In this paper we show that each toxin stimulates mouse T cells bearing receptors that include particular V beta regions, almost regardless of the other variable receptor components. In addition, different toxins have different specificities for V beta.     

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.     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

Characterization of the signaling function of MHC class II molecules during antigen presentation by B cells.

In addition to their role as peptide binding proteins, MHC class II proteins can also function as signal transducing molecules. Recent work using B cells expressing genetically engineered truncated MHC class II molecules has suggested that signaling through the cytoplasmic domains of these proteins plays an important role in the generation of signals required for the activation of some T cell hybrids. Treatment of truncated Ia-expressing B cells with cAMP-elevating agents corrects the deficiency in Ag presentation by these cells. We report that the MHC class II-mediated signal appears to act by a mechanism that increases the efficiency of Ag presentation by B cells thereby lowering the amount of specific Ag required for T cell activation. We further show that the induction of the cAMP-induced signal in B cells is inhibited by cycloheximide and cytochalasin A, implicating protein synthesis as well as cytoskeletal rearrangements in Ag presentation to accessory signal- dependent hybrids. In contrast, these agents do not block Ag presentation to a T cell hybrid previously shown not to require the cAMP-induced signal for activation. The signal-dependent T hybrid is additionally dependent on LFA-1-ICAM-1 interaction for activation, whereas the signal-independent hybrid is not. These observations suggest the existence of two types of T cell hybrid with respect to their requirements for activation: those that require only the recognition of MHC class II-peptide complexes without accessory signals, as shown by their ability to respond to purified Ia on planar membranes, and those that, in addition to recognition of MHC II/Ag, require LFA-1-ICAM-1 interaction and the delivery of additional signal(s) induced in the B cell via signal transduction through MHC class II molecules.     

Disruption of MHC class II-restricted antigen presentation by vaccinia virus

Vaccinia virus (VV), currently used in humans as a live vaccine for smallpox, can interfere with host immunity via several discrete mechanisms. In this study, the effect of VV on MHC class II-mediated Ag presentation was investigated. Following VV infection, the ability of professional and nonprofessional APC to present Ag and peptides to CD4+ T cells was impaired. Viral inhibition of class II Ag presentation could be detected within 1 h, with diminished T cell responses dependent upon the duration of APC infection and virus titer. Exposure of APC to replication-deficient virus also diminished class II Ag presentation. Virus infection of APC perturbed Ag presentation by newly synthesized and recycling class II molecules, with disruptions in both exogenous and cytoplasmic Ag presentation. Virus-driven expression of an endogenous Ag, failed to restore T cell responsiveness specific for this Ag in the context of MHC class II molecules. Yet, both class II protein steady-state and cell surface expression were not altered by VV. Biochemical and functional analysis revealed that VV infection directly interfered with ligand binding to class II molecules. Together, these observations suggest that disruption of MHC class II-mediated Ag presentation may be one of multiple strategies VV has evolved to escape host immune surveillance.