OPTIMIZING A LIMITING DILUTION CULTURE SYSTEM FOR QUANTIFYING THE FREQUENCY OF INTERLEUKIN-2-PRODUCING ALLOREACTIVE T HELPER LYMPHOCYTES1

Background. The development of sensitive, specific, and reproducible techniques to quantify T cells with direct allospecificity has potential applications in the selection of bone marrow donors and in the monitoring of the antidonor alloresponse in patients after organ transplantation. Such data may provide an objective basis for altering existing immunosuppression, monitoring novel antirejection therapies, and predicting long-term graft outcome. We have previously published a correlation between donor antirecipient T helper frequencies (HTLf) and the severity of acute graft-versus-host disease after bone marrow transplantation. Using the same assay protocol, we have described the development of donor-specific hyporesponsiveness in a proportion of renal transplant recipients. However, several imperfections existed in the protocols used in these studies. Cellular interactions within the stimulator and the responder cell populations, and back stimulation of T cells within the stimulator cell population, could give rise to extraneous interleukin-2 and alter the validity or estimation of derived recipient antidonor HTLf. Methods. Using peripheral blood mononuclear cells as the responding population and splenic mononuclear cells as the stimulating population, we have examined the possible effects of these cellular interactions on the results of limiting dilution analysis assays for HTLf measurement. Results. These interactions have the ability to alter the validity or estimation of HTLf. We show that by depleting the responder population of HLA class II+ cells and depleting T cells from the stimulating population, these interactions are effectively abrogated. Conclusions. On the basis of the findings reported here, we describe an optimized HTLf assay which is sensitive, specific, and reproducible. This has obvious applications in the analysis of alloimmune responses in transplantation.