关键词: 外周血单个核细胞, 树突状细胞, 肿瘤新抗原, 反应性T细胞, 过继免疫治疗

Abstract: Objective To observe whether neoantigen peptides sequenced from tumor patients can induce allo-donor sourced PBMC to be specific reactive T cell. Methods PBMC were separated from healthy donors' buffy coat, then monocytes were collected using adhesion method, remaining PBL was kept frozen in -80℃. GM-CSF and IL-4 were added to induce monocytes into dendritic cells every three days. At Day 7 mature dendritic cells were harvested and plated in 24 well-plates, then these plates were loaded with 16 neoantigen peptides as one peptide in each well, 13/16 peptides were designed by our team. Five hours later PBLs recovered from -80℃ were added into these plates to be co-cultured with these treated DC in ratio of 4~10∶1. No peptide wells (DC only) were negative controls, and PBL only wells were prepared for adding PHA later as positive control. IL-2 was replenished in these plates every three days. Peptides were pulsed twice more at Day 14 and Day 21 respectively. Supernatant of each well was collected at Day 22 to measure IFN-γ concentration using ELISA methods, remaining cells were analyzed using Flowcytometry to measure the percentages of CD3⁺IFN-γ⁺ or CD8⁺IFN-γ⁺Tcells. Results 10 of 13 peptides can induce more than 3 kinds of PBL to proliferate into reactive CD3⁺ or CD8⁺ T cells, similar increases were also found in IFN-γ concentration（r=0.66, 0.58 for CD3⁺, CD8⁺ respectively, P<0.05) in supernatant by ELISA method. Conclusions Our research data demonstrate these neoantigen peptides sequenced from tumor patients can induce allo-blood donors'PBMC proliferate to neoantigen specific reactive T cells.

Key words: Peripheral blood mononuclear cell, Dendritic cells, Tumor neoantigens, Reactive T cell, Adoptive immunotherapy