Using solubel peptide-MHC tetramer, the trial of staining and cloning the melanoma antigen-specific cytotoxic lymphocytes

本项目的目的是构建可溶的I类MHC-多肽复合体(“四聚体”)，并利用所产生的四聚体可视化黑色素瘤抗原特异性细胞毒性淋巴细胞(CTL)。我们已经生产了人类白细胞抗原A2/gp100；(209-217)和人类白细胞抗原A2/mART-1；(27-35)和人类白细胞抗原-A24/EBV-P四聚体。在大肠杆菌中分别表达了HLA2-微球蛋白(HLA2M)及其羧基末端连接的可溶性β-A2和A24重链。表达的HLAA2或A24-BIRA底物多肽和β2M亚基在人工合成的抗原肽存在下体外折叠在一起，对应于HLAA2限制性黑色素瘤免疫原性表位(ITDQVPFSV)和MART-1(27-35)&gt；(AAGIGILTV)和HLAA24限制性病毒免疫原性表位(TYGPVFMCL)。折叠后的材料用Bira酶进行生物素化。HLAA2或A24/多肽复合物首先经过GET过滤和…纯化更多随后在Mono Q离子交换柱上。将纯化的生物素化杂二聚体与链霉亲和素-藻红蛋白(PE)以4：1.2的摩尔比混合，最终得到生物素化的人类白细胞抗原A2或A24/多肽的四聚体复合体。分别对相应的多肽类肿瘤浸润性T淋巴细胞(TILs)、TIL1520和TIL1235进行染色，并用流式细胞仪进行分析，以评价HLA-A2/gp100_&gt；(209-217)&gt；和(27-35)&gt；四聚体的特异性结合。人类白细胞抗原-A2/gp100；(209-217)&gt；四聚体对gp100；(209-217)&gt；(209-217)&gt；四聚体特异地染色了90%以上的TIL1520，但对不相关的gp100；(154-162)&gt；(G154：KTWGQYWQQQQQV)多肽没有特异地染色TIL1370。此外，我们还利用HLA-A24/EBV-P四聚体分析了从健康供者外周血单个核细胞(PBMC)体外刺激产生的EBV-P特异性CTL，发现该CTL与四聚体染色，这些结果表明，所产生的多肽-MHC四聚体可以与HLA-A2和HLA-A24共同限制的抗原特异性CTL特异性结合，并可能在异种人群中丰富特异性CTL。较少

The purpose of this project is to construct the soluble class I MHC-peptide complex ("tetramers") and to visualize the melanoma antigen-specific cytotoxic lymphocytes (CTL) using the produced tetramers.1. We have produced HLA-A2/gp100_<(209-217)>, HLA-A2/MART-1_<(27-35)>, and HLA-A24/EBV-P tetramers. The β2-microglobulin (β2m) and soluble HLA-A2 and A24 heavy chains linked at its carboxyl terminus to a BirA substrate peptide were expressed separately in Escherichia coli. The expressed HLA-A2 or A24-BirA substrate peptide and β2m subunits were refolded together in vitro in the presence of synthetic antigenic peptides, corresponding to the HLA-A2-restricted melanoma-immunogenic epitopes of gp100_<(209-217)> (ITDQVPFSV) and MART-1_<(27-35)> (AAGIGILTV), and HLA-A24 restricted viral-immunogenic epitope of EBV-P (TYGPVFMCL). Folded materials were then subjected to enzymatic biotinylation with BirA enzyme. The HLA-A2 or A24/peptide complexes were purified first on a get filtration column and … More subsequently on a Mono Q ion exchange column. Tetrameric complexes of the biotinylated HLA-A2 or A24/peptide were finally produced by mixing the purified biotinylated heterodimers with Streptavidine-phycoerythrin (PE) conjugates at a molar ratio of 4 : 1.2. The specific binding of the HLA-A2/gp100_<(209-217)> and HLA-A2/MART-1_<(27-35)> tetramers were assessed by staining the relevant peptide-specific tumor-infiltrating T-lymphocytes (TILs), TIL1520 and TIL1235 respectively, and analyzing them on flow cytometry. The HLA-A2/gp100_<(209-217)> tetramer specifically stained more than 90% of TIL1520 specific for the gp100_<(209-217)> peptide, but, did not stain TIL1370 specific for irrelevant gp100_<(154-162)> (G154 : KTWGQYWQV) peptide. In addition, using HLA-A24/EBV-P tetramer, we have analyzed EBV-P specific CTL that was generated by in vitro stimulation with the peptide from peripheral blood mononuclear cells (PBMCs) of HLA-A24 healthy donor and the CTL were found to stain with the tetramer.These results indicate that the produced peptide-MHC tetramers can be used to specifically bind to antigen-specific CTL restricted by both HLA-A2 and HLA-A24, and possibly to enrich specific CTL among a heterogeneous population. Less