Abstract
The superantigen SEA causes non-specific hyperactivation of T and B cells at low concentrations. Studies of mutants or soluble proteins suggest SEA is bivalent for its ligand, MHC class II. However, the interaction between these molecules on intact cells is unknown. On primary mouse B cells expressing the MHC class II allele HLA-DR1, measurements of Förster Resonance Energy Transfer between HLA-DR1 molecules on SEA-treated cells indicated specific clustering, not observed in untreated or monovalent superantigen treated cells. Tomographic visualization and electron microscopy of immunogold-labeled SEA-treated B cells revealed small clusters of surface HLA-DR1 (≤4 gold labels). These results present direct visual evidence of SEA-mediated clustering of MHC class II molecules on treated antigen presenting cells, and provide a new structural approach to addressing problems of this nature.
Highlights
The term ‘‘Superantigen’’ is used to define endogenous or exogenous factors that can stimulate T cells whose T Cell Receptors (TCR) bear specific Vb domains, irrespective of the composition of the rest of the receptor [1], resulting in the stimulation of a large fraction of the T cell population [2]
Soluble peptide-DR1 complexes that were SDS-unstable were pre-incubated with Staphylococcal Enterotoxin A (SEA) or the control Staphylococcal Enterotoxin H (SEH), incubated with SDS for an additional 2 minutes without boiling, and the solution was run on a gel in the presence of SDS under non-reducing conditions (Fig. 1A)
In a size-exclusion experiment where equal amounts of soluble SEA or SEH and soluble DR1 were incubated in the presence of excess ZnCl2 or EDTA and run on a pre-equilibrated sizeexclusion column, no high molecular weight peaks corresponding to SEA-DR1 multimers were observed
Summary
The term ‘‘Superantigen’’ is used to define endogenous or exogenous factors that can stimulate T cells whose T Cell Receptors (TCR) bear specific Vb domains, irrespective of the composition of the rest of the receptor [1], resulting in the stimulation of a large fraction (up to 20%) of the T cell population [2]. Studies have suggested a cooperative model where the binding of one SEA to MHC class II favors the binding of the second SEA molecule [13,17,18], and MHC class II - (SEA) trimers have been isolated in solution [18] These results have led to the speculation that SEA could crosslink multiple MHC class II molecules on the surface of APCs. when MHC class II expressing cell lines were treated with SEA, but not with mutants missing either binding site or toxins with one MHC class II binding site, downstream signaling [19,20], inflammatory cytokine gene upregulation [19] and homotypic aggregation [21] was observed, even in the absence of T cells. The actual membrane reorganization of MHC class II on the surface of a cell in response to SEA treatment has not been directly probed, and as such, remains unknown
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