Characterization of agretopes and epitopes involved in the presentation of beef insulin to T cells.
Beef insulin-specific I-Ad-restricted T cell hybridomas were derived from the fusion of antigen-primed (BALB/c X B6)F1 T cells with BW5147 thymoma. Specificity analysis revealed that the A-chain loop region is involved in antigen recognition. Hybridoma A20.2.15 is specific for beef insulin and cross-reacted with sheep insulin, but not with pork insulin. Using synthetic peptides we showed that the A-chain loop containing peptide A1-A14 jointed to the B7-B15 peptide by a disulfide bond can activate this hybridoma. Fragments generated by enzyme digest further suggest that the peptide recognized on beef insulin appears to involve A-chain loop residues A5-A12 and B-chain residues B7-B13 that are linked by the A7-B7 disulfide bridge. We found that beef insulin needs to be processed prior to T cell activation. Glutaraldehyde fixation and chloroquine treatment of presenting cells abolished their capacity to present insulin. Beef insulin denatured by pH changes cannot activate, thus suggesting that simple denaturation is not sufficient for presentation by antigen presenting cells. Finally, the agretope on beef insulin is comprised of two functional regions B7-B13 on the B chain and the A-chain loop in the A-chain, while residues A8 and A10 are probably involved in interaction with the T cell receptor.
Department of Immunology, University of Alberta, Edmonton, Canada.
A
A
Fotedar
W
W
Smart
M
M
Boyer
T
T
Dillon
E
E
Fraga
J
J
Lauzon
E M
EM
Shevach
B
B
Singh
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000938:Antigen-Presenting Cells / Q000502:physiology*; D002417:Cattle / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes; D006825:Hybridomas; D007328:Insulin / Q000276:immunology*; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011489:Protein Denaturation; D013552:Swine; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007328:Insulin; D007376:Interleukin-2; D010446:Peptide Fragments