Allotransplant rejection is a T-cell-dependent reaction. Functional in vitro T-cell assays are being used widely for donor-recipient matching in bone marrow transplantation and have recently been used in some centres for transplant monitoring. In order to assess tolerance induction after clinical transplantation, we measured the T-cell response of the host against donor spleen cells of 33 kidney transplant patients before and every 3 months after transplantation over a period of 18 months. The T-cell reactivity before transplantation was not significantly different in any of the assays in rejecting and non-rejecting patients. In the classical mixed lymphocyte culture (MLC), a donor-specific loss of reactivity was seen only in a patient with a CMV-associated irreversible transplant rejection. One patient with chronic rejection acquired a very high MLC response against donor spleen cells and a high response against third-party cells. Little or nonspecific changes were seen in the MLCs of all other patients. Using the method of limiting dilution analysis (LDA), we found a significant reduction of donor-specific cytotoxic T-cell precursors (CTL-p) within the first 3 months after transplantation in most patients with high antidonor CTL-p frequencies before transplantation. The reduction of donor-specific CTL-p was seen in patients with rejection episodes as well as in patients without. Thus we conclude, in contrast to others, that MLC and CTL-p LDA have no predictive value on the outcome of clinical transplantation.

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We investigated the in vitro immunosuppressive effect of BT 563, a monoclonal antibody, against the achain of the human interleukin-2 (IL-2) receptor (p 55), which has been used to prevent transplant rejection in several clinical trials. We also measured the proliferative T cell alloresponse and pCTL frequencies of BT 563treated kidney transplant patients. In mixed lymphocyte cultures BT 563 caused a reduction ofT cell proliferation to about 50%. This could not be reversed by the addition of exogenous IL-2. A more effective reduction (80%) was seen in the generation of cytotoxic T cells from CML cultures and at the clonal level. The specific T cell response after preincubation with antigen and BT 563 was not reduced so that BT 563 did not induce tolerance. The in vitro findings indicated that BT 563 had a significant but incomplete immunosuppressive effect. This correlated with the clinical course and ex vivo analysis of PBL from BT 563-treated patients after kidney transplantation.The human interleukin-2 (IL-2} receptor consists of two glycoproteins, p55 and p75, which by themselves display low and intermediate affinity for IL-2 and combine to form a high affinity receptor complex.BT 563 is an IgG 1 mouse monoclonal antibody, developed by Wijdenes [6], which is directed against the p55 chain of the IL-2 receptor. It blocks ligand binding to both low and high affinity receptors. Since only activated T lymphocytes express p55 following organ transplantation, the suppressive effect should concentrate on transplant reactive cells. In several clinical trials BT 563 has been used to prevent rejection after solid organ transplantation and to treat graft-versus-host disease after bone marrow transplantation.Offprint requests to: Jorg Steinmann, Inst.Initial experiments have revealed that BT 563 does not deplete target cells and that the blocking of IL-2 binding is noncompetitive. To increase our understanding of the clinically observed immunosuppressive effect, we investigated the mode of action of BT 563 in various T cell assays in vitro. Additionally, anti-donor T cell reactivity in kidney transplant patients was measured before, during and after BT 563 rejection prophylaxis. Materials and methodsBT 563. BT 563 is a mouse IgG 1 monoclonal antibody against the human IL-2 receptor. It is produced and purified by Biotext Pharma according to the guidelines of the European Communities: "On the production and quality control of monoclonal antibodies of murine origin intended for usc in man".Cells. Peripheral blood lymphocytes (PBL) from healthy donors were obtained by density gradient centrifugation of heparinized blood.MLR. One-way mixed lymphocyte reactions (MLR) were carried out with PBL from different donors. We seeded 5 x 104 responder cells and 5 x 104 irradiated stimulator cells per well into 96-well round-bottomed microculuture plates in 200111 RPMI 1640, supplemented with 15% AB serum, glutamine and penicillin/streptomycin. After 96 h the microcultures were pulsed with 37 kBq/well JH-methylthymidine for 24 hand har...

Numerous studies have demonstrated that the generation of alloreactive effector cells depends on cytokines. Conversely, there is evidence that cytokine metabolism is altered at the clonal level in tolerant chimaeras. This has led to preclinical and clinical studies using antibodies that antagonize interleukin-2 (IL-2), with the hope of achieving immunosuppression and inducing tolerance. Monoclonal antibodies against the alpha-chain (p55) of the human IL-2 receptor are being applied to prevent transplant rejection and graft-versus-host disease in several clinical trials. The antibodies that have been applied clinically so far antagonize the binding of IL-2 to the IL-2 receptor alpha-chain which is part of the high affinity IL-2 receptor, but they do not deplete the receptor-bearing cells. Our study investigates the immunosuppressive effect of monoclonal antibodies against the alpha-chain (p55) and beta-chain (p75). In mixed lymphocyte cultures the p55 antibody causes a reduction in T-cell proliferation to about 50%. The generation of cytotoxic T cells is reduced more effectively (up to 80%). By additional blocking of the IL-2 receptor beta-chain we achieved an additional but still incomplete immunosuppressive effect. Moreover we show that IL-2 receptor-blocked alloreactive T cells escape suppression by using IL-4 as an alternative stimulating signal. To prevent T lymphocytes benefiting from this alternative and thwarting the immunosuppressive effect, cytotoxic IL-2 receptor antibodies that deplete the high affinity receptor-bearing cells are needed.

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