An alternative method for T-cell receptor repertoire analysis: clustering of human V-beta subfamilies selected in responses to staphylococcal enterotoxins B and E.
We have designed a convenient procedure for the analysis of V beta repertoire expression in polyclonal T-cell populations. In this procedure T-cell RNA is converted to cDNA, polydC-tailed with terminal deoxynucleotidyl transferase and submitted to one-side specificity PCR amplification with a constant region oligonucleotide primer. The amplified material is then analysed by reverse spot-test hybridization: after 32P-labelling, the amplification product is put to hybridize on a membrane where specially designed V beta subfamily-specific probes are immobilized. The radioactivity fixed on each probe can then be easily quantified and the signal obtained is directly proportional to the initial amount of homologous RNA. We applied this technique to the study of V beta gene selection following T-cell stimulation by staphylococcal enterotoxins B and E. We show that with these toxins two almost non-overlapping sets of T-cells are recruited and that this selection is likely to be dependent on specific amino acid residues shaping the fourth complementarity determining region of the TCR-beta chain. These residues constitute two tandemly-conserved tripeptide sequences (Asp39Pro40Gly41)-(Val69Ser70Arg71) and (Arg66Phe67Ser68)-(Asp88Ser89Ala90) in the SEB- and the SEE-responsive V beta gene clusters respectively.
Champagne
E
E
Centre de Recherches sur le Polymorphisme Génétique des Populations Humaines, CNRS, Toulouse, France.
Huchenq
A
A
Sevin
J
J
Casteran
N
N
Rubin
B
B
D000595:Amino Acid Sequence; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D002478:Cells, Cultured; D004768:Enterotoxins / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D005810:Multigene Family; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000276:immunology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*
D004768:Enterotoxins; D007135:Immunoglobulin Variable Region; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D016693:Receptors, Antigen, T-Cell, alpha-beta; C049463:enterotoxin E, Staphylococcal; C031818:enterotoxin B, staphylococcal