SPECTRATYPING OF TCRS EXPRESSED BY CTL-INFILTRATING MINOR HISTOCOMPATIBILITY ANTIGEN-DISPARATE ALLOGRAFTS

Minor histocompatibility Ags (HA) play prominent roles in stimulating allograft rejection and are recognized by CTLs that mediate this proce ss, However, there is no information on the diversity of TCRs that are specific for single minor histocompatibility Ag peptides and expresse d by CTLs in vivo. We have used the technique of spectratyping to stud y the diversity of V beta usage and beta complementarity-determining r egion 3 (CDR3) length of TCRs expressed by CTL-infiltrating skin allog rafts expressing the immunogenic H4 peptide during the process of reje ction. Spectratyping revealed overall reduction in diversity of both V beta usage and CDR3 length, with sequential application of primary, s econd-set, and third-set H4-incompatible grafts. This dissection of th e array of beta-chains expressed by graft-infiltrating CTLs allowed th e direct sequencing of individual beta-chain PCR products, beta CDR3s were characterized by a net negative charge, as we have observed previ ously with CDR3s expressed by H4-specific CTL clones selected in vitro , Identical and closely related CDR3 amino acid sequences could be ide ntified that were shared by TCRs that 1) utilized different V beta gen es, 2) derived from different mice, or 3) derived from different seque ntial sets of allografts on individual mice, Furthermore, a number of CDR3 sequences expressed by graft-infiltrating CTLs were identical or closely related to sequences we have identified previously in in vitro selected CTL clones that were specific for the H4 peptide.