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

同种异体移植肾浸润细胞表达的T细胞受体分析

• 批准号: 06671613
• 负责人: OBATA Fumiya
• 金额: $ 1.28万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06671613/
• 关键词: renal transplantation; infiltrating cell; clonality; T-cell receptor (TCR); V-gene usage; CDR3-diversity; 移植腎浸潤細胞; バイオプシー; PCR; 移植腎拒絶反応

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.

为了研究肾移植浸润t细胞（RAIT）中t细胞的克隆性，本文采用cDNA序列测定和v基因半定量分析了RAIT中t细胞受体的多样性。TCRCD25^+-RAIT从排斥和肾切除的同种异体移植物中分离，作为cDNA合成的mRNA来源。采用锚定PCR扩增TCRbeta cDNA并进行克隆，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例给出了多对CDR3序列相同的cDNA克隆，表明这些RAIT具有低克隆性。单细胞分析进一步证实。其中一个病例显示出特别显著的寡克隆性。通过RT-PCR分析每个单细胞的CD4/8表达，证实本例中的寡克隆为CD8^+细胞。tcrv基因半定量从活检标本的RAIT中合成etcr cDNA，用生物素标记引物锚定PCR扩增，与大量膜结合的Vbeta片段杂交，用过氧化物酶结合的链亲和素显色进行Vbeta半定量。在经历急性或慢性排斥反应的5名患者中，4名患者表现出在PBL中未见的vβ使用偏差。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vβ偏度。因此，我们的方法可用于活检标本中同种异体肾移植浸润细胞的t细胞克隆分析。

项目成果

Akiyama K,Yoshii T,Obata F,Kashiwagi N,Ishiyama I.: "Polymerase chain reaction (PCR) /single strand conformation polymorphism (SSCP) analysis of the human HLA-DQB region." Japn.J.Legal Medicine. 48. 38-43 (1994)

Akiyama K、Yoshii T、Obata F、Kashiwagi N、Ishiyama I.：“人类 HLA-DQB 区域的聚合酶链反应 (PCR)/单链构象多态性 (SSCP) 分析。”

Obata F.et al.: "Sequence analysis of the T-cell receptor expressed by renal-allograft infiltrating cells." Transpl.Proc.26. 861-863 (1994)

Obata F.等人：“同种异体移植肾浸润细胞表达的 T 细胞受体的序列分析。”

Akiyama K.et al.: "Polymerase chain reaction(PCR)/single strand conformation polymorphism(SSCP)analysis of the human HLA-DOB reqion." Jap.J.Legal Medicine. 48. 38-43 (1994)

Akiyama K.et al.：“人类 HLA-DOB 要求的聚合酶链式反应 (PCR)/单链构象多态性 (SSCP) 分析。”

Onda K,Kashiwagi N,and Obata F: "T-Cell Receptor (TCR) diversity expressed by CD4+ T cells activated by primary allogeneic HLA-DR stimulation : Estimation of the degree of the CDR3 diversity." Scand J Immunol. 43. 519-524 (1996)

Onda K、Kashiwagi N 和 Obata F：“初级同种异体 HLA-DR 刺激激活的 CD4 T 细胞表达的 T 细胞受体 (TCR) 多样性：CDR3 多样性程度的估计。”

Kaneko T and Obata F: "Allogeneic recognition of HLA-DRB1^<**>0406 by T cells with HLADRB1^<**>0403 : Role of amino acid residue 37 of the beta sheet in T cell recognition." Immunobiology. 195 (3). 261-270 (1996)

Kaneko T 和 Obata F：“具有 HLADRB1^<**>0403 的 T 细胞对 HLA-DRB1^<**>0406 的同种异体识别：β 片层的氨基酸残基 37 在 T 细胞识别中的作用。”