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

点击翻译按钮获取中文摘要

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.