This study will attempt to establish the optimal (reproducibility and utility) in vitro assay/s for quantification and characterization of anti-donor alloimmune responses for monitoring the efficacy of tolerance promoting immunosuppressive regimens. Three assays will be compared: A. The refined limiting dilution analysis (LDA) assay that we have described previously for quantitation of proliferating, IFNγ-, and IL-5-secreting cells; B. ELISPOT assays for the same cytokines; C. CFSE staining for estimating frequencies of proliferating alloreactive T cells. The regulatory role of CD4+CD25+ T cells will be examined by depletion of this population from responder cells using magnetic beads prior to assays of anti-donor immunity. To be able to properly address the hypothesis of this study it is divided into 3 protocols: Donor-specific hyporesponsiveness occurs selectively in recipient memory cells. We will try to define the optimal surrogate donor cell for the study of participants for whom viable donor cells are unavailable. To address this we will study 10 recipients of living related, haplotype matched grafts from group 1. Direct pathway responses against different stimulator cells will be measured by ELISPOT and by CFSE labelling for proliferation, on enriched naïve and memory. The surrogate donor cell type that most faithfully reproduces the anti-donor frequencies will be used in subsequent assays in which donor cells are unavailable. We will then go on to study two groups of 12 participants from groups 1 and group 3. Direct pathway responses against donor and equally mismatched third party stimulator cells will be measured by IL-2 ELISPOT and by CFSE labelling on enriched CD4+ naïve and memory responder populations. On enriched CD8+ naïve and memory responder subsets IFNγ, ELISPOT and CFSE labelling will also be used. Group 2 participants (MMF and Rapamycin treated) should have a more marked decrease in the frequency of donor-specific RO+ T cells. Raised frequencies of T cells with indirect anti-donor allospecificity are associated with chronic transplant rejection. Fifteen participants per group from groups 1 and 2 of will be investigated. Direct pathway frequencies (IFNγ and IL-5) will be measured in total CD4+ and CD8+ T cells by ELISPOT analysis. Indirect pathway frequencies (IFNγ and IL-5) on T cells will be measured by ELISPOT using the most sensitive method as defined below. Allo-antibodies in serum will be detected by ELISA. Donor-specific regulatory T cells (CD4+CD25+ T cells) are a feature of transplantation tolerance. Twelve participants per group from groups 1-4 will be recruited for these assays. Three assays will be performed using CD4+T cells before and after CD25 depletion as responder cells. Direct pathway LDA, direct pathway CFSE-estimated proliferation, and indirect pathway ELISPOT analysis. As donor cells are likely to be scarce in group 4 the assays outlined in protocol 1 will determine which source of surrogate donor cells to use for these assays.

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Development of antigen-specific assays indicative of donor-specific tolerance in renal transplant recipients

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