COMPLETE DISSECTION OF THE HB(64-76) DETERMINANT USING T-HELPER-1, T-HELPER-2 CLONES, AND T-CELL HYBRIDOMAS

We have generated cloned Th1 cells, Th2 cells, and T cell hybridomas specific for the single immunogenic peptide from the beta-chain of murine hemoglobin (Hb(64-76)). The availability of these various types of T cells provided us an unique opportunity to examine and dissect the T cell response to an immunogenic peptide. A panel of altered Hb peptides was made by replacing each amino acid in the Hb peptide (positions 64-76) with a conservative amino acid substitution or an alanine. Although none of the eleven T cell clones and hybridomas tested exhibited the same pattern of reactivity to the substituted Hb peptides, some general features were identified for all T cell responses. The primary T cell contact residue of Hb(64-76) was shown to be asparagine 72. For every Hb(64-76) specific T cell, no activation was observed using a peptide containing the conservative substitution of a glutamine for the asparagine at position 72. The flanking glutamic acid at position 73 was also required for a proliferative response for all of the Th1 and Th2 clones. The Th subtypes were not grossly unique in their responses to the substituted Hb peptides, but exhibited minor differences in fine specificity with the Thl cells identifying more critical amino acids then did the Th2 cells. For the Th1 cells and also the T cell hybridomas, the phenylalanine at position 71 was critical for a T cell response. Analysis of peptide affinity for IE(k) molecules indicated that position 71 played a role in peptide binding to MHC. Secondary T cell contact residues, which were important for many but not all of the T cells, were identified at positions 69, 70, and 76. Overall T cell responses were minimally affected by changes in the amino acid residues at positions 64-68, 74, and 75. We have also demonstrated that cloned Th1 cells, Th2 cells and T hybridomas can be generated against the same Hb(64-76) determinant.