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

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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