Human cytotoxic T lymphocytes. I. Limiting-dilution analysis of alloreactive cytotoxic T-lymphocyte precursor frequencies.

A limiting-dilution system was established to measure the frequency of alloreactive cytotoxic T-lymphocyte precursors (CTL-p) in human peripheral blood T cells. Culture medium supplemented with recombinant interleukin-2 enabled clonal expansion of all CTL-p stimulated by allogeneic peripheral blood or spleen cells. The range of CTL-p frequencies in fully HLA-mismatched responder-stimulator combinations was 1:240 to 1:1230. Split-well analysis of individual microwells showed that the cytotoxic T-cell clones generated under limiting-dilution conditions showed exquisite specificity for the stimulating alloantigens. Alloreactive CTL-p were enriched in the OKT4- T-cell subset. This limiting-dilution system was highly reproducible and can thus be applied to investigate human cytotoxic T-cell precursor frequencies in various clinically relevant situations.