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Determination of the Functional Status of Alloreactive T Cells by Interferon-&ggr; Kinetics

Background. A fundamental limitation of in vitro immunologic tests in the field of transplantation is that existing functional tests poorly correlate with in vivo immune responses such as rejection, tolerance, or absence of rejection due to immunosuppression. It would be helpful to have a measure of T lymphocyte responsiveness that reliably reflects these conditions. Methods. C57BL/6J mice received skin transplants from BALB/c donors with: a) no treatment, b) treatment with CsA, or c) treatment with CTLA-4Ig, &agr;-CD40L mAb, and &agr;-CD25 mAb. Syngeneic skin transplants served as controls. Recipient splenocytes were co-cultured with irradiated donor splenocytes and culture supernatant was harvested once a day for 5 consecutive days. IFN-&ggr; levels were measured by ELISA. Results. Splenocytes obtained from non-transplanted mice responded to specific alloantigen stimulation (primary response) at least 2 days later than the splenocytes from mice which had rejected skin grafts (effector/memory response). Splenocytes from mice treated with CsA after skin transplants had no response to third-party alloantigen, but showed an effector/memory pattern of IFN-&ggr; elaboration with donor cell stimulation (immunosuppression), although the IFN-&ggr; levels were not as high as those mice with unmodified graft rejection. Mice treated with combined CTLA4Ig, &agr;-CD40L and &agr;-CD25 accepted skin grafts without further immunosuppression. Splenocytes from these tolerant mice showed a primary response to the third-party and failed to secrete detectable IFN-&ggr; in the presence of donor cells (tolerance). Conclusion. This assay clearly differentiated the functional status of the alloreactive T cells, including primary alloimmune response, effector/memory response, immunosuppressed T cell response, and donor specific tolerance.