肿瘤抗原肽的HLA限制性确认和诱导反应性T细胞杀瘤能力评估

Identification of HLA Restriction for Neoantigens and Evaluation of Anti-tumor Ability of Neoantigen Induced Reactive T cells

目的了解肿瘤抗原肽的HLA限制性以及其诱导的T细胞能否杀伤肿瘤细胞,探索无关供者来源细胞用于过继性抗肿瘤T细胞治疗。方法使用16个肿瘤抗原肽诱导18名无关供者外周血单个核细胞(PBMC)分化为反应性T细胞,并分析HLA型别;利用NetMHC数据库预测肽和HLA分子亲和力;选择HLA-A2限制性的肿瘤抗原肽诱导第二组17名无关供者的PBMC进行杀瘤实验,反应性T细胞作为效应细胞,T2细胞及肿瘤抗原肽同源的肿瘤细胞作为靶细胞,测量LDH(乳酸脱氢酶)释放量或者RTCA(实时无标记细胞分析仪)检测效应细胞杀瘤效率,比较HLA-A2+和A2-T细胞杀瘤效率。结果筛出和HLA-A2具有高亲和力的肿瘤抗原肽LM7,可诱导5/11 HLA-A2+为反应性T细胞,其中HLA-A2+纯合子则为3/3,而HLA-A2-者则为2/7。LM7诱导反应性T细胞杀伤肿瘤百分比A2+组明显强于A2-组(60.72±11.28 vs 47.2±4.46,P=0.03)。结论本研究显示NetMHC预测对于纯合子样品更有帮助,肿瘤抗原肽LM7具有HLA-A2限制性,可诱导部分HLAA2+PBMC分化为反应性T细胞,可杀伤肿瘤,应对供者进行HLA筛选并分析其细胞功能,其诱导的反应性T细胞可作为过继性T细胞抗肿瘤治疗的细胞来源。

Objectives To explore the possibility to utilize unrelated donors'cells as sources for adoptive T cell anti-tumor therapy,thus we need to identify HLA restriction for neoantigens and evaluate neoantigen induced reactive T cells'cytolysis ability.Methods 16 neoantigen peptides were used to induce 18 unrelated donors'(group 1)PBMC(peripheral blood mononuclear cells)into reactive T cells,and HLA typing was performed,the affinity between peptides'and HLA molecule was predicted by NetMHC database,then neoantigen with HLA-A2 restriction was selected to induce another group(group 2)of 17 unrelated donors'PBMC into reactive T cells,these cells would act as effectors,tumor cell line T2 cells or the tumor cells with the same source of this neoantigen would act as targets either,LDH release tests and RTCA(real time celluar analysis)were performed to evaluate the effectors'anti-tumor ability.Then these results were compared between HLA-A2+and HLA-A2-samples.Results NetMHC database predicted neoantigen LM7 showed high affinity with HLA-A2+antigens,5/11 of HLA-A2+samples can successfully be induced into reactive T cells,including 3/3 homozygous HLA-A2+samples,while 2/7 of HLA-A2-samples with more reactive T cells.Induced T cells from A2+samples showed higher percentages of tumor cells killed than those A2-samples(60.72±11.28 vs 47.2±4.46,P=0.03)。Conclusions Our study suggests prediction by NetMHC is more helpful in homozygous samples,neoantigens LM7 is confirmed as HLA-A2 restrictive,which can induce some HLA-A2+PBMC into reactive T cells which can kill tumor cells more efficiently,unrelated donors should be screened not only by HLAtyping but also cell function tested,thus induced reactive T cells can take the roles as the source for adoptive anti-tumor T cells therapy.