Modified ELISPOT technique--highly significant inverse correlation of post-Tx donor-reactive IFNgamma-producing cell frequencies with 6 and 12 months graft function in kidney transplant recipients.

BACKGROUND: Upcoming trials for immunosuppression minimization and tolerance induction require the development of reliable in vitro assays for monitoring cellular alloimmunity in transplant patients. The IFN-gamma ELISPOT assay represents a promising tool for monitoring alloreactive memory/effector T cells. As T lymphopenia is a common finding during the early post-transplant (post-Tx) period, the IFN-gamma ELISPOT technique was here modified by using ELISPOT responder cells with enhanced percentage and standardized number of 200,000 T cells per well.
METHODS: Peripheral blood mononuclear cells (PBMNC) of kidney transplant recipients were depleted of CD14+ and CD15+ cells to increase the percentage of T cells from average 47.8% to 71.5% before transplantation (pre-Tx) and from 39.7% to 74.9% post-Tx. The assay was tested in a population of 23 de novo renal transplant patients for clinical relevance. Before and at 2-3 times during the first 6 months post-Tx, IFN-gamma-producing donor-reactive as well as recall antigen-reactive cell frequencies (Candida, tuberculin, tetanus) were determined and correlated with outcome.
RESULTS: Pre-Tx donor-reactive ELISPOT frequencies were enhanced in patients with acute rejection compared to non-rejectors. Moreover, mean post-Tx donor-reactive ELISPOT frequencies showed a highly significant inverse correlation with renal function at 6 and 12 months. In contrast, recall antigen-reactive ELISPOT frequencies did not correlate with outcome.
CONCLUSION: Our results suggest that the modified donor-reactive ELISPOT approach might provide a useful surrogate marker for renal transplant outcome with possible utility especially in T-lymphopenic patients.