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 whether the Ags recognized reside in allogeneic MHC proteins themselves regardless of the structure of the associated peptides or whether specific peptides presented by allogeneic MHC proteins determine each epitope. We developed HLA-A*0201-specific alloreactive human CD8+ CTL lines and clones to address this issue. Acid treatment of HLA-A*0201+ target cells resulted in the loss of Ab-defined epitopes as well as recognition by all alloreactive CTL. In the presence of brefeldin A, no class I molecules were re-expressed at the surface of the acid-treated cells. Addition of a mixture of synthetic peptides corresponding to known, naturally processed, HLA-A*0201-associated peptides together with exogenous human beta2m restored binding by specific Ab but not recognition by alloreactive CTL. However, addition of a more complex mixture of peptides directly extracted from HLA-A*0201 reconstituted CTL recognition. This demonstrates that these alloreactive CTL recognize specific peptides and not a common peptide-dependent conformation of HLA-A*0201. Reverse phase HPLC fractionation of the extracted peptides resulted in the loss of recognition by CTL lines from three individuals. This was not due to the loss of specific peptide species because repooling of the HPLC fractions led to a recovery of recognition. Furthermore, three HLA-A*0201-alloreactive CTL clones recognized single distinct peptide peaks from the same HPLC fractionation. These data suggest that the epitopes recognized in allogeneic responses to HLA-A*0201 are complex, and the response is a result of recognition of multiple unique peptide-MHC complexes.