ISOLATION OF A KIDNEY-SPECIFIC PEPTIDE RECOGNIZED BY ALLOREACTIVE HLA-A3-RESTRICTED HUMAN CTL

The molecular nature of tissue-specific Ags involved in MHC-restricted CTL responses is as yet undefined. To determine the specificity of th ese peptides, their function, and their possible relationship to allog raft rejection, we have utilized human kidney-specific CD8(+) CTL clon es to screen reversed-phase HPLC (RP-HPLC)-separated self peptides pre sented by allo-class I molecules: One of these clones is HLA-A3-restri cted and the other HLA-B62-restricted, lysing human kidney cell lines but not MHC identical B lymphoblastoid cells which express the appropr iate HLA molecules. We have identified a biologically active RP-HPLC f raction containing self peptides eluted from affinity-purified MHC mol ecules from HLA-A3(+) kidney. This peptide is not expressed in HLA-A3( +) spleen. Similarly, a HLA-B62-associated peptide fraction was identi fied in kidney but not in spleen using the HLA-B62-restricted CTL clon e. Sequence analysis of the biologically active fraction from HLA-A3 k idney revealed multiple peptides. Because of the ambiguity of the pept ide sequence, a mixed peptide library corresponding to this sequence w as synthesized that included the HLA-A3 binding motif. The biologicall y active peptide library was RP-HPLC fractionated and the fraction con taining HLA-A3-restricted CTL activity was sequenced. The resulting se quence of the alloreactive HLA-A3-restricted peptide epitope is GPPGVT IVK. By using this unique strategy, we describe the successful isolati on and sequencing of an antigenic peptide that is recognized by a huma n alloreactive kidney-specific CTL clone.