B cells enhanced the anti-U251/U373 activity of EphA2-CAR T cells

Chimeric antigen receptor (CAR) T cell therapy is a powerful adoptive immunotherapy against blood cancers, but the therapeutic effect was not efficient enough on solid tumors. B cells have been reported to play a critical role in regulating memory T differentiation and cytotoxic T development. However, as of yet the influence of such B cells on CAR T cells has not been discussed. In this study, using ephrin type-A receptor 2 (EphA2) specific CAR T cells, we cultured B cells successfully to stimulate T cells in vitro, and investigated the cell differentiation and anti-tumor efficiency. We observed that EphA2-CAR T cells stimulated by B cells performed enhanced anti-tumor ability with more interferon γ (IFN γ) production and higher OX40 expression. The differentiation of CAR T cells was arrested after B cells stimulation for more than 7 days with the percentage of central memory T cells (Tcm) increasing. In addition, next generation sequencing was performed. The top expressed genes clustered in activation, leukocyte migration and chemokine signaling pathway contributed to the anti-glioblastoma (GBM) activity of CAR T cells stimulated by B cell. In conclusion, these results indicated the importance of B cells in retarding CAR T cells differentiation and enhancing anti-tumor activity, which paves the way for the rapid exploitation of EphA2-CAR T cells against GBM in the future. EphA2-CAR T cells stimulated with or without B cells were co-cultured with U251 or U373 (1:1). The suspended cells were collected and washed with PBS for three time. CD3+ T cells were isolated using Dynabeads™ CD3 (Invitrogen). Total RNA was extracted from cells attached on the beads using TRIzol Reagent (Invitrogen). The culture dish was washed with PBS for three times and RNAs of adherent cells were collected using TRIzol reagents. Samples containing 2 µg total RNA each were used as input material for generating the sequencing libraries by company.

嵌合抗原受体（CAR）T细胞疗法是一种针对血液系统恶性肿瘤的强效过继性免疫治疗手段，但该疗法对实体瘤的治疗效果仍不够理想。已有研究表明，B细胞在调控记忆性T细胞分化以及细胞毒性T细胞发育过程中发挥关键作用。然而，截至目前，此类B细胞对CAR T细胞的影响尚未被探讨。本研究使用靶向ephrin type-A receptor 2（EphA2）的CAR T细胞，成功培养B细胞并在体外刺激T细胞，对细胞分化及抗肿瘤效能展开研究。我们观察到，经B细胞刺激的EphA2-CAR T细胞抗肿瘤能力显著增强，其干扰素γ（IFN-γ）分泌量更高，且OX40的表达水平也显著提升。经B细胞刺激超过7天后，CAR T细胞的分化过程被阻滞，同时中枢记忆性T细胞（central memory T cells, Tcm）的占比有所升高。此外，本研究开展了下一代测序（next generation sequencing），结果显示，显著上调的基因富集于激活通路、白细胞迁移及趋化因子信号通路，这些通路与B细胞刺激后的CAR T细胞抗胶质母细胞瘤（glioblastoma, GBM）活性密切相关。综上，本研究结果证实了B细胞在阻滞CAR T细胞分化、增强其抗肿瘤活性方面的重要作用，为未来快速开发针对胶质母细胞瘤的EphA2-CAR T细胞疗法铺平了道路。将经B细胞刺激或未经B细胞刺激的EphA2-CAR T细胞与U251或U373细胞以1:1的比例共培养。收集悬浮细胞并用磷酸盐缓冲液（phosphate buffered saline, PBS）洗涤三次。使用Dynabeads™ CD3磁珠（Invitrogen公司）分离CD3阳性T细胞。使用TRIzol试剂（Invitrogen公司）从磁珠结合的细胞中提取总RNA。用磷酸盐缓冲液洗涤培养皿三次，使用TRIzol试剂收集贴壁细胞中的RNA。以每份含2 µg总RNA的样本作为起始材料，由合作公司完成测序文库的构建。