|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1996 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1995 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1994 : ¥800,000 (Direct Cost : ¥800,000)
In order to investigate the T-cell clonality in renal-allograft infiltrating T-cells (RAIT), I analyzed the T-cell receptor diversity of RAIT by both cDNA sequence determination and V-gene usage semi-quantitation.
LcDNA sequence determination of RAIT TCR
CD25^+-RAIT were isolated from rejected and nephrectomized allografts and used as the source of mRNA for cDNA synthesis. TCRbeta cDNA was amplified by anchored PCR and cloned, and randomly selected 30-40 cDNA clones were subjected to sequence analysis. Among four cases analyzed in this study, three gave many pairs of cDNA clones that had CDR3 sequences identical to each other, indicating oligoclonality in these RAIT.Single cell analysis was carried out for further confirmation. In of one case that showed particularly remarkable oligoclonality. The oligoclone in this case was shown to be CD8^+ cells by RT-PCR analysis for CD4/8 expression of each single cells.
2.Semi-quantitation of TCRV-gene usage
TCR cDNA was synthesized from the RAIT in biopsy specimens and amplified by anchored PCR using biotin labeled primers, hybridized with a large number of membrane bound Vbeta segments, and subjected to color development with peroxidase conjugated streptavidin for Vbeta usage semi-quantitation. Among the five patients who experienced acute or chronic rejection, four showed skewed Vbeta usage that were not seen in their PBL.In one patient who showed repeated rejection and received anti-OKT3 treatment, Vbeta skewness was dramatically reduced by this treatment. Thus, our method was useful for T-cell clonality analysis of renal allograft infiltrating cells in biopsy specimens.