Advances in immunosuppressive management for transplantation (Tx) have improved graft survival, but lasting success will likely depend on induction of donor-specific unresponsiveness to avoid life-long immunosuppressive drug therapy. Tolerance strategies have been devised in rodents, but applicability to human Tx is uncertain, and critical factors predisposing to stable tolerance in outbred species are still poorly understood. We have established a preclinical kidney transplant (KTx) model in unrelated rhesus monkeys in which posttransplant administration of rabbit antithymocyte globulin (RATG) and infusion of a subpopulation of donor bone marrow cells (BMC) can induce enduring KTx survival without chronic immunosuppressive drugs. We propose to further investigate the mechanisms by which KTx tolerance is achieved in this model. The experiments are designed within the framework of a veto hypothesis of tolerance induction, postulating that distinct BMC precursor subpopulations are capable of inactivating donor-reactive CTL and Th. Using in vitro and in vivo experiments, we will examine the cellular mechanism(s) of donor BMC-induced specific CTLp and Th unresponsiveness and the role of hematopoietic chimerism in this model. Within this context, we will investigate a novel veto strategy using genetic engineering to induce unresponsiveness in KTx recipients. This supplemental strategy is expected to disable Th-dependent anti-class l IgG responses and allow more uniform lasting tolerance without chronic immunosuppressive drugs.
The specific aims of the proposal are: (1) To investigate in vivo induction of donor-specific CTLp unresponsiveness and promotion of KTx tolerance by a donor BMC subpopulation that is enriched for veto activity (VE-BMC). Using low dose infusion of donor VE-BMC, prepared by removal of DRbright APC and pretreatment with Leu-leu-OMe, we will investigate unresponsiveness in recipients given only a brief course of RATG. (2) To define the derivation and role of chimeric cells in this KTx tolerance model. We will clarify the lineage of chimeric cells and their role in promoting allograft tolerance, using donor VE-BMC that are transduced with the neomycin resistance gene. (3) To examine the cellular mechanism by which allogeneic donor BMC inactivate donor- directed CTLp. We will define the donor BMC population that inactivates normal allo-directed CTLp in vitro and study the mechanism. (4) To examine the inhibitory effect of VE-BMC on alloreactive T helper cells in vitro. We will determine if small resting B cells in donor VE-BMC inactivate Th cells by a veto effect that alters cytokine expression. (5) To investigate if 1 DR haplotype sharing facilitates stable KTx tolerance by a donor BMC veto effect on host Th cells that respond to alloantigen presented on DR + host or donor APC. (6) To clone specific genes. transduce the cDNAs into recipient VE-BMC and examine the effect of the transduced cells on tolerance induction in vivo. We will infuse the transduced recipient VE-BMC into KTx recipients with and without infusion of donor VE-BMC to investigate whether presentation of donor class l on recipient VE-BMC will prevent immunological escape from the donor- directed veto effect to facilitate stable KTx tolerance.
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