PRIMARY ALLOREACTIVE CYTOTOXIC T-LYMPHOCYTES ARE NOT COMMONLY RESTRICTED BY SELF-HLA CLASS-I ANTIGENS

To assess the role of HLA Class I molecules in the indirect presentati on of alloantigen, we have investigated the fine specificity and MHC r estriction of in vitro primary alloreactive cytotoxic T-lymphocytes (C TL), using limiting dilution analysis of CTL precursor frequencies in HLA-mismatched responder-stimulator pairs. By employing split-well ana lysis of limiting dilution (LD) microcultures and third-parry target c ells bearing a stimulatory HLA Class I antigen alone or in combination with a single responder HLA antigen, we demonstrate that self-Class I restriction of HLA-A- or HLA-B-specific CTL precursors is not a commo n feature of the primary in vitro alloresponse. Higher frequencies of alloantigen-specific CTL precursors in the presence of self-HLA antige ns were only detected in 5 of 31 limiting dilution assays established from seven different responder-stimulator pairs. In two cases, the hig her precursor frequencies could be explained on the basis of Class II- restricted presentation of Class I-derived antigenic peptide and are s upported by flow cytometric analysis of HLA antigen expression on targ et cells. The remaining 3 assays of this type were suggestive of Class I restriction but revealed only marginally higher frequency estimates . All other LD assays revealed lower CTL precursor frequency estimates in the presence of self-HLA Class I antigens. A higher antigen-specif ic CTLp frequency was not detected when targets shared three HLA Class I antigens with the responder, demonstrating that we had not biased t he responses by selecting single HLA antigen-sharing targets in the ot her assays. Analysis of reactivity against PHA blast targets at the si ngle cell per well level demonstrated that CTL reactive only with the original stimulator comprised the majority of lytic reactions. Heteroc litic CTL (i.e., CTL that recognize single HLA targets only and not th e original stimulator) formed only a small fraction of total reactivit y. Our results confirm the role of Class II antigens in the indirect p resentation of alloantigen in vitro but suggest that HLA Class I antig ens play a limited role in this phenomenon.