Limiting dilution analysis of human IL-2 producing T-cell precursors. Determination of hierarchical alloantigenic potential of human leukocyte antigens and evaluation of alloimune responses to single HLA-DP molecules

Twenty nine healthy unrelated individuals were carefully selected and divid ed into three groups according to their HLA (Human Leukocyte Antigens) phen otypes. A sensitive and reproducible limiting dilution analysis(LDA) based bioassay using CTLL-20 cells for detection of human IL-2 was set up and use d to assess the hierarchical impact of highly polymorphic HLA molecules on individual's alloreactivity. Our main interest was to evaluate the role of HLA-DP molecules in this process. By calculating frequencies of IL-2 produc ing helper T cell precursors (HTLp) and amounts of IL-2 produced in each ex periment, we were able to confirm that HLA-DR molecules are the most potent alloantigens. In 29 different combinations where a single HLA-DP mismatch between stimulating and responding cells was evaluated, some were reasonabl y tolerant, while the other ones evoked moderate to relatively strong alloi mune responses. finally, two groups with statistically significant differen ce in alloimune responses to stimulating HLA-DP molecules carrying D,E,A,V or G,G,P,M amino acid sequences at positions 84,85,86 and 87 in the sixth v ariable region F of the molecule could be formed, according to HTLp frequen cies and amounts of IL-2 detected. Data presented are of great importance f or the selection of unrelated as well as related bone marrow donors for hae matological patients.