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

一些报道表明，在人类实体瘤中，存在大量的肿瘤浸润性淋巴细胞(TIL)，它们被认为是对肿瘤的特异性宿主免疫反应的一个子集。一般认为，每个T细胞都有不同的T细胞受体(TCR)克隆型，TCR的连接区在抗原识别中起着重要作用。然而，由于在体内分离TIL细胞的困难，到目前为止还不可能分析TIL的直接特征。我们利用RT-PCR-SSCP方法对连接CDR3(V-D-J-C)区域的异质性进行了评估，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：肿瘤和PBL是在手术中获得的。由于CDR3区域的多样性，PBL呈现出一种涂片模式。TIL具有明显的T细胞克隆性蓄积，但不存在Vβ区偏倚。积累的T…的数量和位置更多的细胞克隆型似乎与肿瘤的分期有关。越先进，积累的越大。分离TIL和肿瘤细胞后，将TIL与肿瘤细胞共同培养3~4周，比较该TIL与初步获得的TIL的克隆性。培养的TIL中积累的克隆性T细胞在体外因抗原刺激而部分保持不变，特异性克隆带变得更加清晰。剩下的部分消失了。确定了相同克隆性的存在。为了证实这一点，正在进行DNA测序。与TIL类似，对自体肿瘤有反应的特异性T细胞克隆的增加被认为发生在PBL和腹膜渗出液淋巴细胞(PEL)中。通过传代培养的恶性肿瘤细胞刺激同一患者的PBL和PEL，我们正在研究克隆性，这被认为是累积的结果。克隆型在体内的成功检测和随后这些克隆型的治疗性增强将导致未来有效的肿瘤特异性免疫治疗。较少

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