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.     

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.     

Activation of murine T cells by staphylococcal enterotoxin E: requirement of MHC class II molecules expressed on accessory cells and identification of V beta sequence of T cell receptors in T cells reactive to the toxin

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.     

A recombinant C-terminal fragment of staphylococcal enterotoxin A binds to human MHC class II products but does not activate T cells.

Binding of staphylococcal enterotoxin A (SEA) to MHC class II encoded proteins is a prerequisite for its subsequent activation of a large fraction of T lymphocytes through interaction with variable segments of the TCR-beta chain. We cloned SEA in Escherichia coli and produced four recombinant fragments covering both the N- and C-terminal regions. These fragments were used to analyze the interaction between SEA and the human MHC class II products. A C-terminal fragment of SEA, representing amino acids 107-233 bound to HLA-DR and HLA-DP but did not activate T cells. The three other fragments (amino acids 1-125, 1-179 and 126-233) neither bound to MHC class II Ag nor activated T cells. SEA apparently bind to HLA-DR and HLA-DP through its C-terminal part, whereas T cell activation is dependent on additional parts of the protein.     

The crystal structure of staphylococcal enterotoxin H: implications for binding properties to MHC class II and TcR molecules

The X-ray structure of the superantigen staphylococcal enterotoxin H (SEH) has been determined at 1.69 A resolution. In this paper we present two structures of zinc-free SEH (apoSEH) and one zinc-loaded form of SEH (ZnSEH). SEH exhibits the conventional superantigen (SAg) fold with two characteristic domains. In ZnSEH one zinc ion per SEH molecule is bound to the C-terminal beta-sheet in the region implicated for major histocompatibility complex class II (MHC class II) binding in SEA, SED and SEE. Surprisingly, the zinc ion has only two ligating amino acid residues His206 and Asp208. The other ligands to the zinc ion are two water molecules. An extensive packing interaction between two symmetry-related molecules in the crystal, 834 A(2)/molecule, forms a cavity that buries the zinc ions of the molecules. This dimer-like interaction is found in two crystal forms. Nevertheless, zinc-dependent dimerisation is not observed in solution, as seen in the case of SED. A unique feature of SEH as compared to other staphylococcal enterotoxins is a large negatively charged surface close to the Zn(2+) site. The interaction of SEH with MHC class II is the strongest known among the staphylococcal enterotoxins. However, SEH seems to lack a SEB-like MHC class II binding site, since the side-chain properties of structurally equivalent amino acid residues in SEH and those in SEB-binding MHC class II differ dramatically. There is also a structural flexibility between the domains of SEH. The domains of two apoSEH structures are related by a 5 degrees rotation leading to at most 3 A difference in C(alpha) positions. Since the T-cell receptor probably interacts with both domains, SEH by this rotation may modulate its binding to different TcR Vbeta-chains.     

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.     

Monoclonal antibodies to hamster class II MHC molecules distinguish T and B cells

Monoclonal antibodies were prepared against cell surface antigens present on Syrian hamster lymphocytes and a hamster B cell lymphoma line, GD-36. One of these antibodies, S11, precipitated glycoproteins of 29,000 and 39,000 m.w. These glycoproteins were shown to be identical to or a subset of la-like glycoproteins precipitated by hamster alloantisera; however, molecules identified by S11 differed from the predominant hamster la homologues detected with a cross-reactive monoclonal antibody to murine la.7. The immunofluorescence pattern of both anti-la reagents, S11 and anti-la.7, on hamster lymphoid cells is similar by fluorescence-activated cell sorter analysis. A subpopulation of spleen and lymph node cells stains brightly with these antibodies. By two-color fluorescence, this peripheral lymphocyte subpopulation, identified with monoclonal anti-hamster la, also bears surface immunoglobulin (IgM). These data strongly suggest that hamster resting peripheral B cells, and not T cells, express la antigens and can be identified and isolated differentially by using this marker.     

Activation of murine T cells by toxic shock syndrome toxin-1. The toxin-binding structures expressed on murine accessory cells are MHC class II molecules

Toxic shock syndrome toxin-1 (TSST-1)-binding structures present on murine lymphoid tissues were investigated by using 125I-TSST-1. T-depleted C57BL/6 spleen cells incubated with TSST-1 for 3 h at 0 degree C were mitogenic to splenic T cells, indicating that the former cells bind and present TSST-1 to T cells. TSST-1-binding activity was observed in C57BL/6 splenic B cells and L cells transfected with I-Ab genes, but not in splenic T cells and control L cells. Scatchard plot analysis showed that these B cells and transfectants bound TSST-1 with similar binding affinity. SDS-PAGE analysis showed that lysates of C57BL/6 spleen cells and the I-Ab-positive transfectants contain a single band which bound TSST-1 and comigrated with I-Ab heterodimers. TSST-1-binding activity observed clearly in C57BL/6. BALB/c, and C3H/HeN spleen cells and L cells transfected with I-Ab or I-Ak genes was not reduced by paraformaldehyde fixation. Binding of 125I-TSST-1 to the three spleen cells was markedly reduced by anti-I-A antibodies, but not by anti-I-E antibodies. C57BL/6, C3H/HeN, and (C3H/HeN x C57BL/6) F1 T cells were activated by TSST-1 to proliferate and produce IL-2 in the presence of FT6.2 cells, LT1-30-3 cells and either of them, respectively, but not in the presence of control L cells. These results indicate that I-A molecules function as the structures via that accessory cells directly bind TSST-1 on the cell surface and present a triggering signal of TSST-1 to T cells.     

MHC class II molecules on the move for successful antigen presentation

Major histocompatibility complex class II (MHC II) molecules are targeted to endocytic compartments, known as MIIC, by the invariant chain (Ii) that is degraded upon arrival in these compartments. MHC II acquire antigenic fragments from endocytosed proteins for presentation at the cell surface. In a unique and complex series of reactions, MHC II succeed in exchanging a remaining fragment of Ii for other protein fragments in subdomains of MIIC before transport to the cell surface. Here, the mechanisms regulating loading and intracellular trafficking of MHC II are discussed.     

MHC class II molecules: transport pathways for antigen presentation

During biosynthesis, MHC class II molecules travel through the endocytic pathway and interact with antigenic peptides before their stable insertion in the plasma membrane. The process of class II association with these peptides and their final deposition at the cell surface are essential steps in boosting specific antibody responses. Therefore, the study of class II molecules is important in understanding how cell-biological events can direct an immune response.     

Direct binding of a myasthenia gravis related epitope to MHC class II molecules on living murine antigen-presenting cells.

MHC gene products present antigenic epitopes to the antigen receptor on T cells. Nevertheless, direct binding of such epitopes to MHC class II proteins on normal living antigen-presenting cells (APCs) has not yet been demonstrated. We have previously shown a significant difference in the ability of T cells of myasthenia gravis (MG) patients to proliferate in response to the synthetic peptide p195-212 of the human acetylcholine receptor (AChR) alpha-subunit in comparison to healthy controls. The observed proliferative responses correlated significantly with HLA-DR5. Moreover, lymph node cells of various mouse strains that were primed with the T cell epitope, p195-212, were found to proliferate to different extents. To investigate these observations further, we designed an assay for direct binding of p195-212 to MHC class II proteins on the surface of freshly prepared splenic adherent cells. Binding of a biotinylated p195-212 was monitored using phycoerythrin-avidin by flow cytometry. Fifteen to sixty per cent of the cells were labeled following incubation with the biotinylated peptide. Binding was observed only to splenic adherent cells derived from mouse strains of which T cells were capable of proliferating in response to p195-212. The binding specificity, in terms of epitope structure and its site of interaction on the cells, was shown by its inhibition with an excess of the unlabeled peptide or with the relevant monoclonal anti-I-A antibodies. These results constitute the first direct evidence for the specific binding of a T cell epitope to live APC.     

Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A.

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram-positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C-terminus and at selected sites in the N-terminal domain. Four amino acids in the C-terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000-fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10-fold reduction in MHC class II affinity. The combination of these mutations in the N- and C-terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II-dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab-SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II-independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

MHC class II molecules control murine B cell responsiveness to lipopolysaccharide stimulation

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHC(b) haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHC(d) haplotype. Furthermore, B cells from MHC class II(-/-) mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.     

Structure-based selection of small molecules to alter allele-specific MHC class II antigen presentation

Class II major histocompatibility molecules are the primary susceptibility locus for many autoimmune disorders, including type 1 diabetes. Human DQ8 and I-A(g7), in the NOD mouse model of spontaneous autoimmune diabetes, confers diabetes risk by modulating presentation of specific islet peptides in the thymus and periphery. We used an in silico molecular docking program to screen a large "druglike" chemical library to define small molecules capable of occupying specific structural pockets along the I-A(g7) binding groove, with the objective of influencing presentation to T cells of the autoantigen insulin B chain peptide consisting of amino acids 9-23. In this study we show, using both murine and human cells, that small molecules can enhance or inhibit specific TCR signaling in the presence of cognate target peptides, based upon the structural pocket targeted. The influence of compounds on the TCR response was pocket dependent, with pocket 1 and 6 compounds inhibiting responses and molecules directed at pocket 9 enhancing responses to peptide. At nanomolar concentrations, the inhibitory molecules block the insulin B chain peptide consisting of amino acids 9-23, endogenous insulin, and islet-stimulated T cell responses. Glyphosine, a pocket 9 compound, enhances insulin peptide presentation to T cells at concentrations as low as 10 nM, upregulates IL-10 secretion, and prevents diabetes in NOD mice. These studies present a novel method for identifying small molecules capable of both stimulating and inhibiting T cell responses, with potentially therapeutic applications.     

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.     

Some cloned murine CD4+ T cells recognize H-2Ld class I MHC determinants directly. Other cloned CD4+ T cells recognize H-2Ld class I MHC determinants in the context of class II MHC molecules.

Murine T lymphocytes recognize nominal Ag presented by class I or class II MHC molecules. Most CD8+ T cells recognize Ag presented in the context of class I molecules, whereas most CD4+ cells recognize Ag associated with class II molecules. However, it has been shown that a proportion of T cells recognizing class I alloantigens express CD4 surface molecules. Furthermore, CD4+ T cells are sufficient for the rejection of H-2Kbm10 and H-2Kbm11 class I disparate skin grafts. It has been suggested that the CD4 component of an anti-class I response can be ascribed to T cells recognizing class I determinants in the context of class II MHC products. To examine the specificity and effector functions of class I-specific HTL, CD4+ T cells were stimulated with APC that differed from them at a class I locus. Specifically, a MLC was prepared involving an allogeneic difference only at the Ld region. CD4+ clones were derived by limiting dilution of bulk MLC cells. Two clones have been studied in detail. The CD4+ clone 46.2 produced IL-2, IL-3, and IFN-gamma when stimulated with anti-CD3 mAb, whereas the CD4+ clone 93.1 secreted IL-4 in addition to IL-2, IL-3, and IFN-gamma. Cloned 46.2 cells recognized H-2Ld directly, whereas recognition of Ld by 93.1 apparently was restricted by class II MHC molecules. Furthermore, cytolysis by both clones 46.2 and 93.1 was inhibited by the anti-CD4 mAb GK1.5. These results demonstrate that CD4+ T cells can respond to a class I difference and that a proportion of CD4+ T cells can recognize class I MHC determinants directly as well as in the context of class II MHC molecules.     

Assembly and transport of MHC class II molecules.

At the surface of antigen-presenting cells MHC class I and class II molecules present peptides to respectively CD8+ and CD4+ T cells. MHC class I molecules acquire peptides right after synthesis in the endoplasmic reticulum. MHC class II molecules do not acquire peptides in the endoplasmic reticulum but instead associate with a third chain, the invariant chain which impedes peptide binding. Subsequently the invariant chain takes MHC class II molecules to the endosomal/lysosomal compartment thanks to a targeting signal retained in its cytoplasmic tail. It then dissociates from the MHC class II dimer to allow it to bind peptides.     

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.     

Comparison of the capacity of murine and human class I MHC molecules to stimulate T cell activation.

The mechanism underlying the apparent differences in the capacity of murine and human class I MHC molecules to function as signal transducing structures in T cells was examined. Cross-linking murine class I MHC molecules on splenic T cells did not stimulate an increase in intracellular calcium ([Ca2+]i) and failed to induce proliferation in the presence of IL-2 or PMA. In contrast, modest proliferation was induced by cross-linking class I MHC molecules on murine peripheral blood T cells or human class I MHC molecules on murine transgenic spleen cells, but only when costimulated with PMA. Moreover, cross-linking murine class I MHC molecules or the human HLA-B27 molecule on T cell lines generated from transgenic murine splenic T cells stimulated only modest proliferation in the presence of PMA, but not IL-2. On the other hand, cross-linking murine class I MHC molecules expressed by the human T cell leukemic line, Jurkat, transfected with genes for these molecules, generated a prompt increase in [Ca2+]i, and stimulated IL-2 production in the presence of PMA. The results demonstrate that both murine and human class I MHC molecules have the capacity to function as signal transducing structures, but that murine T cells are much less responsive to this signal.     

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.