T cell clonality of TIL (tumor-infiltrating lymphocytes) and PBL in vivo, and analysis of tumor-specific T cell clonotype

一些报告表明，在人类实体瘤中，存在大量的肿瘤浸润淋巴细胞（TIL），据信它们代表了宿主对肿瘤的特异性免疫反应的一个子集。一般认为，每个T细胞具有不同的T细胞受体（TCR）克隆型，并且TCR的连接区在抗原识别中起重要作用。然而，由于难以在体内分离这些细胞，因此迄今为止还不可能分析TIL的直接表征。我们通过RT-PCR-SSCP方法在连接CDR 3（V-D-J-C）区域的异质性评价的发展使我们能够直接检测体内积累的某些T细胞克隆。结果如下：手术时获得肿瘤和PBL。PBL由于不同的CDR 3区域而表现出拖尾模式。TIL具有明显的T细胞克隆型聚集，但不受V β区的影响。累积T的数量和位置 ...更多信息 细胞克隆型似乎与肿瘤的分期有关。越是高级，积累就越多。将肿瘤细胞与TIL分离后，将其共同培养3 ~ 4周，比较其克隆形成情况。培养的TIL中聚集的克隆性T细胞显示，部分TIL在体外抗原刺激下保持不变，特异性克隆型条带变得清晰。剩下的部分消失了。确定存在相同的克隆性。与TIL类似，认为PBL和腹膜渗出液淋巴细胞（PEL）中发生对自体肿瘤应答的特异性T细胞克隆的增加。通过传代培养的恶性肿瘤细胞刺激同一患者的PBL和PEL，我们正在研究被认为是作为结果积累的克隆性。克隆型的成功体内检测和随后的治疗增强这些克隆型将导致有效的肿瘤特异性免疫治疗在未来。少

Several reports have suggested that, within human solid tumors, there are numbers of tumor-infiltrating lymphocytes (TIL) and they are believed to represent a subset of specific host immune responses to tumors. It is generally believed that each T cell bears a distinct clonotype of T cell receptor (TCR) and that the junctional regions of TCRs play important roles in antigen recognitions. Direct characterization of TIL,however, has not been hitherto possible to analyze because of difficulties in separating these cell in vivo. Our development of heterogeneity evaluation in the junctional CDR3 (V-D-J-C) regions by the RT-PCR-SSCP method has enabled us to directly detect certain T cell clones accumulated in vivo. This has resulted in the followings.Tumor and PBL were obtained at surgery. PBL exhibited a smear pattern because of the diverse CDR3 regions. TIL bore distinct T cell clonotype accumulations but there were not bias by V beta region. The numbers and locations of the accumulated T … More cell clonotypes seemed to correlate with the stage of tumor. The more advanced, the larger the accumulation. These results support the idea that specific immune response by tumor antigen occur in vivo in the tumor site.After TIL and tumor cells were separated, they were cultured together for three to four weeks, and clonatlities of this TIL and that of the initially obtained TIL were compared. The accumulated clonal T cells in the cultured TIL showed that some parts of it were maintained unchaged because of antigen stimulation in vitro and specific clonotype bands became clearer. The remaining parts disappeared. The existence of same clonality was determined. To confirm this, the DNA sequencing is being performed.Similar to TIL,the increase of specific T cell clones responsive to the autologous tumors are believed to occur in PBL and peritoneal exudate lymphocytes (PEL). Stimulating PBL and PEL of the same patient by subcultured malignant tumor cells, we are studying the clonality that are considered to accumulate as the result. The successful in vivo detection of clonotypes and the subsequent therapeutic augumentation of these clonotypes will result in effctive tumor-specific immune treatments in future. Less