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

为了研究同种异体肾移植浸润性T细胞(RAIT)中T细胞的克隆性，本研究通过基因序列测定和V基因利用半定量分析了RAIT中T细胞受体的多样性。用锚定聚合酶链式反应扩增并克隆TCRbeta基因，随机选取30-40个克隆进行序列分析。在本研究分析的4例中，有3例给出了多对CDR3序列相同的克隆，表明这些RAIT具有寡克隆性。单细胞分析进一步证实。其中一例表现出特别显著的寡克隆性。2.TCRV基因用途的半定量从活检标本中提取的RAIT合成TCRcDNA，用生物素标记的引物进行锚定PCR扩增，与大量膜结合的Vbeta片段杂交，经过氧化物酶标记的链霉亲和素显色后进行Vbeta用途半定量。在经历过急性或慢性排斥反应的5名患者中，4名患者表现出外周血中未见的Vbeta偏斜使用。在一名反复出现排斥反应并接受抗OKT3治疗的患者中，这种治疗显著减少了Vbeta偏斜。因此，我们的方法是有用的T细胞克隆性分析的肾移植浸润性细胞活检标本。

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.