The main goal of this project is to investigate the immunopathogenetic mechanisms of chronic allograft rejection in human renal transplant recipients. Principal hypotheses in this project are: 1) that clinical chronic rejection processes are mediated by ongoing indolent specific immune reactivity against the mismatched MHC antigens of the graft, and that the immune response is driven by CD4+ T cell clones primed to donor allo-epitopes presented as peptides by recipient APCs (indirect allorecognition pathway); 2) that the expression of tissue injury is modified by inherited genetic polymorphisms that alter the level of response in genes that are known to be immune modifiers, including some that are particularly known to function in T cell activation and death, and in development of fibrosis; and 3) that prediction of susceptibility to chronic rejection can be defined by quantitative analysis of pro-inflammatory and pro-fibrotic gene activation in the graft and/or peripheral blood mononuclear cells prior to and during the course of transplantation. Therefore, the specific aims of the project are: 1) Study the role of cell mediated immune responses in chronic rejection. An array of synthetic HLA allopeptides will be used to define the presence/absence of primed cells to donor HLA. We will use novel assays (ELISPOT, CFSE labeling of peripheral blood lymphocytes) to determine the frequency- and phenotype of alloreactive T cells serially after transplantation, and to define patterns of overall responses and responses to individual donor MHC epitopes (epitope spreading/shifting) for comparison with anti-donor IgG responses, graft morphology, and graft function over time. 2) Investigate the role of polymorphic variants of the angiotensin system and TGF-beta genes in chronic rejection. Based on our preliminary data showing an association of chronic rejection with inheritance in the recipient of single nucleotide polymorphisms of genes of the angiotensin system, we plan to prospectively define transplant recipients genotypes for the angiotensin system: angiotensinogen, angiotensin converting enzyme, angiotensin receptor type 1 and type 2, as well as TGF-D genotype. We will explore the relationship between tines . genetic polymoprhisms and the alloimmune response to HLA peptides above. 3) Study intragraft and peripheral blood mononuclear cell expression patterns by real time quantitative PCR (TaqMan) of key cellular markers cytokines, chemokines and growth factors at the time of transplantation and prospectively at different time points post-transplant to define the level of association between intragraft events and the recipient's immune system. In this Project we plan to prospectively study a total of approximately 90 renal transplant recipients (30 patients per year over 3 years) followed for at least 2 years post-transplantation. The uniqueness of this project is that we plan to study prospectively in humans indirect alloreactivity, serum humoral immune responses, intragraft and peripheral blood mononuclear cell expression Patterns of functionally important molecules, and novel gene polymorphisms, all in the same patient population. The data from these experiments will provide the first comprehensive detailed prospective mechanistic studies in human transplant recipients during the course of development of chronic rejection.
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