Analysis of the T-cell receptor expressed by renal allograft infiltrating cells

为了研究肾移植浸润性T细胞（赖特）中T细胞的克隆性，本研究通过cDNA序列测定和V基因使用半定量分析了赖特中T细胞受体的多样性。通过锚定PCR扩增TCR β cDNA并克隆，随机选择30-40个cDNA克隆进行序列分析。在本研究分析的4例病例中，有3例获得了多对cDNA克隆，其CDR 3序列彼此相同，表明这些赖特具有寡克隆性。其中一例显示出特别显著的寡克隆性。2. TCRV基因半定量检测从活检标本中的赖特中合成TCR cDNA，用生物素标记的引物进行锚定PCR扩增，与大量膜结合的V β片段杂交，并用过氧化物酶缀合的链霉亲和素进行显色，用于V β使用半定量。在5名经历急性或慢性排斥反应的患者中，4名患者表现出PBL中未观察到的Vbeta使用偏态。在1名反复出现排斥反应并接受抗OKT 3治疗的患者中，Vbeta偏态通过这种治疗显著降低。因此，我们的方法是有用的T细胞克隆性分析肾移植浸润细胞活检标本。

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 TCRCD25^+-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 usageTCR 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.