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 divided into three groups according to their HLA (Human Leukocyte Antigens) phenotypes. A sensitive and reproducible limiting dilution analysis (LDA) based bioassay using CTLL-20 cells for detection of human IL-2 was set up and used 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 producing helper T cell precursors (HTLp) and amounts of IL-2 produced in each experiment, 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 reasonably tolerant, while the other ones evoked moderate to relatively strong alloimune responses. Finally, two groups with statistically significant difference 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 variable region F of the molecule could be formed, according to HTLp frequencies and amounts of IL-2 detected. Data presented are of great importance for the selection of unrelated as well as related bone marrow donors for haematological patients.