Sequence analysis of T-cell receptor participating in rejection of renal allografts.

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In order to know what kind of T cell population participate in rejection of renal allografts, in this research project, I carried out sequence analysis of T-cell receptor cDNA expressed by renal infiltrating lymphocytes (RIL). First, I established the method for TCR sequence analysis, called Linker-Ligated PCR, in which synthetic anchor ligated at the upstream of TCR cDNA is utilized as target sequence for PCR-amplification. By utilizing the method established, I next analyzed TCR involved in recognition of HLA-DR molecules in mixed lymphocyte reaction (MLR), a in vitro model for allogeneic immune reaction. I found that diversity of TCR does not depend on the number of amino acid difference of DR molecules between responder and stimulator cells and even for a single amino acid difference of DR, enormously diverse set of TCR involved in MLR. Finally, I analyzed TCR in RIL isolated from rejected and nephrectomized allograft by treatment with collagenase and density-gradient centrifugation in Percoll. CD25^+(activated T marker) fraction was isolated and subjected to TCRbeta sequence analysis. Seven different Vbeta gene families and 8 Jbeta segments were detected among 31 cDNA clones sequenced. Several cDNA clones were found to have completely identical CDR3 sequences, i. e., 6 clones with Vbeta6.9-Jbeta1.2, 3 clones with Vbeta3.1-Jbeta2.1, 2 clones with Vbeta3.1-Jbeta2.7, 2 clones with Vbeta5.6-Jbeta2.5, 2 clones with Vbeta12b-Jbeta2.3. Considering that cDNA with identical CDR3 is rarely observed in the analysis of around 30 TCR cDNA clones obtained from in vitro MLR, CD25^+ RIL was thought to composed of T cell population expressing relatively limited TCR diversity.