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 process. However, there is no information on the diversity of TCRs that are specific for single minor histocompatibility Ag peptides and expressed by CTLs in vivo. We have used the technique of spectratyping to study the diversity of Vβ usage and β complementarity-determining region 3 (CDR3) length of TCRs expressed by CTL-infiltrating skin allografts expressing the immunogenic H4 peptide during the process of rejection. Spectratyping revealed overall reduction in diversity of both Vβ usage and CDR3 length, with sequential application of primary, second-set, and third-set H4-incompatible grafts. This dissection of the array of β-chains expressed by graft-infiltrating CTLs allowed the direct sequencing of individual β-chain PCR products. β CDR3s were characterized by a net negative charge, as we have observed previously with CDR3s expressed by H4-specific CTL clones selected in vitro. Identical and closely related CDR3 amino acid sequences could be identified that were shared by TCRs that 1) utilized different Vβ genes, 2) derived from different mice, or 3) derived from different sequential 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.