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Using soluble HLA-peptide tetramer, the studies on the cloning and immune-response monitoring of melanoma antigen-specific cytotoxic lymphocyte

利用可溶性HLA肽四聚体进行黑色素瘤抗原特异性细胞毒性淋巴细胞克隆及免疫反应监测的研究

基本信息

批准号：
13670901
负责人：
SAKURAI Toshiharu
金额：
$ 2.3万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670901/
关键词：
HLA tetramer melanoma-antigen peptide HLA-A2.1V HLA-A2.6 TIL CMV-antigen peptide HLA-A^*0201 HLA-A^*2402 CTL HLA-A2.1 HLA-A2.6

项目摘要

The purpose of this project is the studies on the cloning and immune-response monitoring of the melanoma antigen-specific cytotoxic T lymphocytes using soluble peptide-HLA tetramers produced.1. The tumor-infiltrating T-lymphocyte (TIL) 620 line (recognized gp-100-209) was double-stained with anti-CD8 mAb-PC5 and HLA-A2.1/gp100-209 PE-tetramer. The tetramer-binding CD8+ T cells (14 %) were sorting from TIL 620 line by fluorescence-activated cell sorting (FACS) and expanded in vitro. The cultured tetramer-binding CD8+ T cells were stained 98.4 % with HLA-A2.1/gp 100-209 PE-tetramer and released IFN-γ more than 7 fold of that from original TIL 620 line. It become is able to cloning the antigen-specific active T lymphocytes using HLA tetramers.2. HLA-A2.6 and A2.7 heavy chains were constructed from HLA-A2.1 by mutagenesis kit, synthesized and purified using a prokaryotic expression system. HLA-A2.6/gp100-280 tetramer was produced, and stained 4-5 % of TIL 660 (recognized A2.l/gp-100-280 epitope) and 7.97 % of the induced T cell line from PBMC of HLA-A2.6+ healthy donor by gp100-280 peptide. These results suggested that the HLA-A2.1 restricted melanoma-antigen peptide might be cross-recognized by other A2-supertype molecules.3. As the model of monitoring tumor-specific T lymphocytes using HLA tetramers, fluorescent HLA-CMV peptide tetramer were used to monitor the recovery of CMV-specific T lymphocytes in recipients of allogeneic stem cell transplants, (1) CMV pp65-495 specific T cells were induced, using CMV pp65-495 peptide, in 7 of 8 PMBC from A2.1+ healthy donor, and stained with HLA-2.1/ CMV pp65-495 PE-tetramer. Then CMV pp65-495 is immunodominant peptide. (2) The use of HLA-peptide tetramer to quantify CMV specific T cells is valuable for monitoring and studying T-cell responses after allogeneic stem cell transplantation.

本课题的目的是利用制备的可溶性肽-HLA四聚体进行黑色素瘤抗原特异性细胞毒性T淋巴细胞的克隆和免疫反应监测的研究.肿瘤浸润性T淋巴细胞（TIL）620系（识别gp-100-209）用抗CD 8 mAb-PC 5和HLA-A2.1/gp 100 -209 PE-四聚体双重染色。通过荧光激活细胞分选（FACS）从TIL 620细胞系中分选出四聚体结合的CD 8 + T细胞（14%），并在体外扩增。培养的四聚体结合的CD 8 + T细胞对HLA-A2.1/gp 100-209 PE-四聚体的染色率为98.4%，并且释放的IFN-γ是原始TIL 620细胞系的7倍以上。利用HLA四聚体克隆抗原特异性活性T淋巴细胞成为可能.用突变试剂盒从HLA-A2.1中构建HLA-A2.6和A2. 7重链，用原核表达系统合成和纯化。产生HLA-A2.6/gp 100 -280四聚体，并通过gp 100 -280肽染色4- 5%的TIL 660（识别A2.1/gp-100-280表位）和7.97%的来自HLA-A2.6+健康供体的PBMC的诱导T细胞系。提示HLA-A2.1限制性黑色素瘤抗原肽可能与其他A2超型分子交叉识别.作为用HLA四聚体监测肿瘤特异性T淋巴细胞的模型，我们用荧光HLA-CMV肽四聚体监测异基因造血干细胞移植受体中CMV特异性T淋巴细胞的恢复，（1）用CMV pp 65 - 495肽在8例A2.1+健康供体的PMBC中诱导了7例CMV pp 65 - 495特异性T细胞，并用HLA-2.1/ CMV pp 65 -495 PE-四聚体染色。CMV pp 65 -495为免疫显性肽。(2)使用HLA-肽四聚体定量CMV特异性T细胞对于监测和研究异基因干细胞移植后的T细胞应答是有价值的。