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

Background. The development of sensitive, specific, and reproducible t echniques to quantify T cells with direct allospecificity has potentia l applications in the selection of bone marrow donors and in the monit oring of the antidonor alloresponse in patients after organ transplant ation. Such data may provide an objective basis for altering existing immunosuppression, monitoring novel antirejection therapies, and predi cting long-term graft outcome. We have previously published a correlat ion between donor antirecipient T helper frequencies (HTLf) and the se verity of acute graft-versus-host disease after bone marrow transplant ation. Using the same assay protocol, we have described the developmen t of donor-specific hyporesponsiveness in a proportion of renal transp lant recipients. However, several imperfections existed in the protoco ls used in these studies. Cellular interactions within the stimulator and the responder cell populations, and back stimulation of T cells wi thin the stimulator cell population, could give rise to extraneous int erleukin-2 and alter the validity or estimation of derived recipient a ntidonor HTLf. Methods. Using peripheral blood mononuclear cells as th e responding population and splenic mononuclear cells as the stimulati ng 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 alte r the validity or estimation of HTLf. We show that by depleting the re sponder population of HLA class II+ cells and depleting T cells from t he stimulating population, these interactions are effectively abrogate d. Conclusions. On the basis of the findings reported here, we describ e an optimized HTLf assay which is sensitive, specific, and reproducib le, This has obvious applications in the analysis of alloimmune respon ses in transplantation.