The induction of specific unresponsiveness to allografts in large animals remains the central objective of these studies. Dogs treated with a minimal course of antilymphocyte serum (ALS) and post-transplant injection of either fresh or previously frozen donor bone marrow cells (BMC) show significant allograft prolongation. Percoll fractionation of donor BMC has resolved the graft prolonging cell to a low density population (F-BMC) representing 20% of the original BMC. One dog injected with F-BMC is still healthy after 300 days and is specifically unresponsive in vitro to its donor's antigens. Studies in B6AF1 mice treated with ALS and C3H/He BMC show significant and donor specific skin graft prolongation. By selective cell killing and cell separation methods it was determined that the active BMC are largely FcGammaR+, Ia-, Thy 1.2-, C'R-, Ig-, and Ly-. Mouse BMC active in graft prolongation are donor age-independent, survive frozen and refrigerated storage but do not survive in vitro when maintained by standard culture techniques. Proposed renal allograft experiments in dogs will include (i) the combination of F-BMC injection with other immunosuppressive agents such as cyclosporin A and with pre-transplant blood transfusions, (ii) the effect of additional BMC pulses during rejection periods and (iii) the effect of post-transplant injection of peripheral blood cells on graft prolongation. Studies in the mouse will include the further purification of graft prolonging BMC by continuous Percoll centrifugation and by antibody affinity methods, and assay of surface receptors and functional properties. Attempts will be made to activate graft prolonging BMC via their FcGammaR with ag-ab complexes prior to injection and to culture and clone their BMC. Mouse experiments will also include a continuation of studies which showed that neonatal skin survives longer than adult skin when transplanted to histoincompatible recipients. This prolongation difference appears to be related to the presence of immature epidermal dendritic cells (EDC) in neonatal skin. Experiments are designed to isolate neonatal EDC and to study their effect as direct stimulators and/or as potential suppressors in vitro. Results may lead to methods which can lessen the immunogenicity of adult tissue prior to transplantation. Optimal protocols derived from studies in the mouse will initially be applied to dogs and ultimately to monkeys. Parallel to these experiments an in vitro study of human BMC will be initiated to isolate and characterize potentially graft prolonging cells. Future application of this ALS/F-BMC model to man is anticipated.
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