Identification of two binding sites in staphylococcal enterotoxin B that confer specificity for TCR V_β gene products

The enterotoxlns produced by Staphytococcua af1reus are potent mitogens. They stimulate T cells in an oligocional fashion that Is dependent on the expression of particular variable region gene elements in the β-chaln of the TCR (V_β). The fourth hypervarlable loop of the TCR β-chaln Is generally regarded as the site of contact for both viral and mlcroblal superantigens. Recently, residues 60 and 61 of staphylococcal enterotoxin B (SEB) have been highlighted as central to the interaction of this toxin with the TCR. We have, therefore, analysed a series of toxins with mutations at these positions to investigate how amlno add substitutions affect the ability of mutant toxins to stimulate both human and mouse T cells. Each of the variant toxins induced proliferation in a murine V_β8.3 T cell clone, whereas a V_β8.1 T cell clone only responded to native toxin. A panel of nine human T cell clones expressing six different V_β elements, all of which responded to native SEB, was tested for reactivity to the variant toxins. Only one V_β19.1⁺ T cell clone was found to be sensitive to substitution at positions 60 and 61 In a manner analogous to the murine V_β8.1 T cell done. Seml-quantitatlve analysis of the TCR V_β expression of human T cell lines expanded with native and mutant SEB revealed that none of the variant toxins could stimulate T cells that expressed V_β19.1. Taken together, these results suggest that the interaction of mouse V_β8.1 and human V_β19.1 TCRs with SEB differs from other TCRs. Sequence comparisons of the different TCR V_β chains indicated that residues in the second complementarity determining region (CDR2) interact with the 60-61 loop of SEB. Therefore, a minimum of two distinct binding modules confer specificity to the interaction of the TCR with SEB.