The overall goal of this research proposal is to further define the biochemical, molecular and functional nature of the human kidney specific non-MHC alloantigens and evaluate their clinical significance in renal transplantation. We will employ antibodies specific for human kidney alloantigens eluted from rejected renal allografts, heteroantisera defining the non-MHC kidney antigen and T lymphocyte clones specific for human kidney antigens developed in our laboratories to achieve the above goals. Our results have shown that eluates from rejected kidneys, including HLA identical transplants, define a polymorphic 90kD alloantigen expressed on human kidneys. Affinity purified 90kD kidney alloantigen will be sequenced and compared to the sequences of 2 unique CDNA clones isolated by immunoscreening a human kidney expression vector library with eluates. Transfection experiments will also be carried out to establish whether the CDNA codes for molecules detected by eluates, heteroantisera and cytotoxic T lymphocyte clones. CD8+ human T lymphocyte clones developed in our laboratories are cytotoxic to kidney cell lines in an MHC class I restricted manner but not to other MHC identical cells indicating that the T cell clones are recognizing an organ specific non-MHC peptide in the context of MHC class I antigens. We will define isolated peptide antigen(s) from kidney cells for their ability to sensitize B- lymphoblastoid cell lines (B-LCL) for T cell mediated lysis including cyanogen bromide cleaved cytoplasmic extract from kidney cells, eluted peptides from HLA class I molecules from kidney cell lines, and peptides made from the sequenced 90kD protein. Sensitizing fractions will then be sequenced in order to define the HLA class I restricted peptide motif as well as determine the protein origin of the kidney specific antigen. In addition to defining the kidney specificity of 21 clones already available in our laboratories, we will produce additional T cell clones, using kidney cells as stimulatory cells, from normal peripheral blood and from kidney biopsies from renal transplant recipients undergoing acute, late and chronic rejections. These clones will be analyzed for cytotoxic specificity using kidney cell lines and B-LCL as targets. T cell receptor variable region of kidney cell specific clones will also be analyzed by Northern analysis or immunofluorescence in order to determine any predominant T cell receptor variable region usage. Thus, studies proposed in this application will provide important new information about the mechanism of chronic allograft rejection by establishing the functional significance of organ specific cytotoxic T lymphocytes in chronic rejection biopsies, and will lead to new avenues to treat chronic rejection which is not amenable to current protocols of immunosuppressive therapy.
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