CLASS I-RESTRICTED ALLOREACTIVE CYTOTOXIC T-LYMPHOCYTES RECOGNIZE A COMPLEX ARRAY OF SPECIFIC MHC-ASSOCIATED PEPTIDES

A major issue in understanding alloreactive T cell responses is whethe r the Ags recognized reside in allogeneic MHC proteins themselves rega rdless of the structure of the associated peptides or whether specific peptides presented by allogeneic MHC proteins determine each epitope, We developed HLA-A0201(+)-specific alloreactive human CD8(+) CTL lin es and clones to address this issue, Acid treatment of HLA-A0201(+) t arget cells resulted in the loss of Ab-defined epitopes as well as rec ognition by all alloreactive CTL, In the presence of brefeldin A, no c lass I molecules were re-expressed at the surface of the acid-treated cells, Addition of a mixture of synthetic peptides corresponding to kn own, naturally processed, HLA-A0201-associated peptides together with exogenous human beta(2)m restored binding by specific Ab but not reco gnition by alloreactive CTL. However, addition of a more complex mixtu re of peptides directly extracted from HLA-A0201 reconstituted CTL re cognition, This demonstrates that these alloreactive CTL recognize spe cific peptides and not a common peptide-dependent conformation of HLA- A0201. Reverse phase HPLC fractionation of the extracted peptides res ulted in the Loss of recognition by CTL lines from three individuals, This was not due to the loss of specific peptide species because repoo ling of the HPLC fractions led to a recovery of recognition, Furthermo re, three HLA-A0201-alloreactive CTL clones recognized single distinc t peptide peaks from the same HPLC fractionation, These data suggest t hat the epitopes recognized in allogeneic responses to HLA-A0201 are complex, and the response is a result of recognition of multiple uniqu e peptide-MHC complexes.