背景:同种异体反应性肿瘤特异性T细胞是对抗肿瘤的适应性免疫系统的重要武器。然而,干细胞样记忆T细胞(Tscm)是有效消除肿瘤细胞的关键。用于产生这些T细胞亚群的方法已经存在。然而,他们可以变得更有效率。Further,它们价格昂贵,资源匮乏的实验室无法获得。在这方面,我们在此描述了一种新的体外同种异体共培养方法,用于培养我们开发的同种异体限制性肿瘤特异性Tscm细胞。
方法:我们首先从健康供体获得筛选HLA-A2分子阴性的PBL,然后与T2/AFP细胞共培养以产生AFP肽特异性肿瘤反应性T细胞。控件,将IL-21和/或雷帕霉素应用于24孔板中的样品。收集样品并用抗人CD3,CD8,CD44,CD62L,和HLA-A2/AFP二聚体,然后进行流式细胞术分析。通过台盼蓝排除测定法测量细胞活力。使用单向ANOVA和独立t检验来比较组间和组间的平均差异,其中P值小于0.05被认为是显著的。
结果:我们的结果表明,雷帕霉素可以阻止分化,并扩增AFP特异性Tscm细胞。Further,在IL-21的存在下,Tscm细胞的扩增被增强。
结论:IL-21和雷帕霉素可以同时用于体外培养和维持抗原特异性Tscm细胞,以增强针对癌症的免疫治疗策略。
BACKGROUND: Alloreactive tumor specific T cells are important arsenals of the adaptive immune system in the fight against tumors. However, stem cell-like memory T cells (Tscm) provide the key to effective elimination of tumor cells. Methods for generating these T cell subsets already exist. However, they could be made more efficient. Further, they are expensive and unattainable to the resource poor laboratories. In this regard, we are hereby describing a novel in vitro allogeneic co-culture method for raising allo-restricted tumor specific Tscm cells that we developed.
METHODS: We started by obtaining PBLs that screened negative for HLA-A2 molecules from healthy donors followed by co-culture with T2/AFP cells to generate AFP peptide specific tumor-reactive T cells. Controls, IL-21 and/or rapamycin were applied to samples in 24 well plates. Samples were harvested and stained with anti-human CD3, CD8, CD44, CD62L, and HLA-A2/AFP dimer followed by flow cytometry analysis. Cell viability was measured by Trypan blue exclusion assay. One Way ANOVA and independent t test were used to compare the mean differences among and between groups where P values less than 0.05 were considered significant.
RESULTS: Our results show that rapamycin arrests the differentiation of, and expands AFP specific Tscm cells. Further, the expansion of Tscm cells is augmented in the presence of IL-21.
CONCLUSIONS: IL-21 and Rapamycin can be used concurrently to raise and maintain antigen specific Tscm cells in vitro for purposes of augmenting immunotherapy strategies against cancers.