To assess the role of HLA Class I molecules in the indirect presentation of alloantigen, we have investigated the fine specificity and MHC restriction of in vitro primary alloreactive cytotoxic T-lymphocytes (CTL), using limiting dilution analysis of CTL precursor frequencies in HLA-mismatched responder-stimulator pairs. By employing split-well analysis of limiting dilution (LD) microcultures and third-party target cells 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 common feature of the primary in vitro alloresponse. Higher frequencies of alloantigen-specific CTL precursors in the presence of self-HLA antigens were only detected in 5 of 31 limiting dilution assays established from seven different responder-stimulator pairs. In two cases, the higher precursor frequencies could be explained on the basis of Class II-restricted presentation of Class I-derived antigenic peptide and are supported by flow cytometric analysis of HLA antigen expression on target 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-specific CTLp frequency was not detected when targets shared three HLA Class I antigens with the responder, demonstrating that we had not biased the responses by selecting single HLA antigen-sharing targets in the other assays. Analysis of reactivity against PHA blast targets at the single cell per well level demonstrated that CTL reactive only with the original stimulator comprised the majority of lytic reactions. Heteroclitic CTL (i.e., CTL that recognize single HLA targets only and not the original stimulator) formed only a small fraction of total reactivity. Our results confirm the role of Class II antigens in the indirect presentation of alloantigen in vitro but suggest that HLA Class I antigens play a limited role in this phenomenon.
- Flow Cytometry
- HLA Antigens
- Histocompatibility Antigens Class I
- Indicator Dilution Techniques
- T-Lymphocytes, Cytotoxic