AN ALTERNATIVE METHOD FOR T-CELL RECEPTOR REPERTOIRE ANALYSIS - CLUSTERING OF HUMAN V-BETA SUBFAMILIES SELECTED IN RESPONSES TO STAPHYLOCOCCAL ENTEROTOXINS-B AND ENTEROTOXINS-E

We have designed a convenient procedure for the analysis of Vbeta repe rtoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucl eotidyl transferase and submitted to one-side specificity PCR amplific ation with a constant region oligonucleotide primer. The amplified mat erial is then analysed by reverse spot-test hybridization: after P-32- labelling, the amplification product is put to hybridize on a membrane where specially designed Vbeta subfamily-specific probes are immobili zed. The radioactivity fixed on each probe can then be easily quantifi ed and the signal obtained is directly proportional to the initial amo unt of homologous RNA. We applied this technique to the study of Vbeta gene selection following T-cell stimulation by staphylococcal enterot oxins B and E. We show that with these toxins two almost non-overlappi ng sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth comple mentarity determining region of the TCR-beta chain. These residues con stitute two tandemly-conserved tripeptide sequences (Asp39Pro40Gly41)- (Val69Ser70Arg71) and (Arg66Phe67Ser68)-(Asp88Ser89Ala90) in the SEB- and the SEE-responsive Vbeta gene clusters respectively.