Limiting dilution analysis: quantification of IL-2 producing allospecific lymphocytes after renal and cardiac...

A rapid and robust limiting dilution assay was developed to measure the frequency of donor-reactive, IL-2 (interleukin 2) producing, helper T lymphocytes in the peripheral T cell population of organ allograft recipients. The IL-2 bioassay was performed using two methodologies to assess the response of CTLL-2 indicator cells. The first depended on spectrophotometric detection of bioreduced XTT whilst the second involved measurement of [3H]thymidine incorporation. The radioisotopic method was slightly more sensitive but both assays could be used for analysis of limiting dilution culture supernatants after primary incubation of recipient lymphocytes with donor splenic cells for 48 hours. All the assays produced results which conformed to single hit kinetics, indicating that IL-2 was production was dependent on a single limiting cell type. The frequency of allospecific helper lymphocytes in the peripheral T cell population of normal volunteers did not vary significantly during a 28-day period. It was found that immunosuppressed allograft recipients had a significantly reduced proportion of T cells in their peripheral blood mononuclear cell population. However, it was possible to measure the frequency of donor-reactive helper cells in the T cell population of transplant patients. These frequency values were very low in two renal allograft recipients who were HLA-DR matched to their donor organs. Three of four HLA-DR mismatched cardiac recipients showed a significant decrease in the frequency of their donor-reactive helper lymphocytes during the period of monitoring. The fourth patient, who received antilymphocyte antibodies for the first three days after transplantation, showed significant fluctuations in the frequency of these cells. The four cardiac recipients showed little histopathological evidence of acute graft rejection with only one patient experiencing a brief episode of moderate rejection; this patient showed a high frequency of donor-reactive helper cells when assayed immediately after this episode but the frequency subsequently declined.