Alloreactive cytolytic T cell (CTL) lines and clones have been used to identify the sites of polymorphism of antigens of the major histocompatibility complex (MHC). Specific CTL were generated against wild-type H-2b products by cells from H-2b mutant mice that had one or a few amino acid changes in either the alpha 1 or alpha 2 domains of the Kb or Db class I molecules. These CTL populations, which might be expected to react with determinants expressed on single MHC domains, were examined for lytic activity on L cells expressing newly constructed hybrid class I molecules. Transformed cell lines expressing native class I molecules or hybrid class I molecules in which the alpha 1 and alpha 2 domains of H-2Kb had been substituted by those domains of H-2Db were lysed by H-2Db-specific CTL. Similarly, all H-2Kb-specific CTL recognized hybrid molecules in which the alpha 1 and alpha 2 domains of H-2Kb were inserted into the H-2Db molecule. In contrast, exchange of the alpha 1 domains of H-2Kb and H-2Db resulted in a total loss of recognition by Kb and Db-specific CTL. These results suggest that the allodeterminants recognized by H-2 mutant CTL are influenced by interactions between the alpha 1 and alpha 2 domains, findings similar to those seen using conventional alloreactive T cells (11). These results were compared to the binding of alloreactive mAbs, including 5 new mAbs specific for the Kb molecules. Finally, it was shown that primary and secondary CTL responses could be generated by direct sensitization against hybrid class I molecules, demonstrating that these molecules express neoantigenic determinants recognized by alloreactive CTL.

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