肿瘤细胞治疗研究
英文原题：Distinct effector functions and synergy of CAR mRNA-engineered T cells and macrophages in the clearance of CD19(+) leukemia cells.
我们的研究结果凸显了 CAR-T 细胞优越的肿瘤细胞杀伤能力。
引言：嵌合抗原受体（CAR）技术是基因治疗领域的重大进展。CAR-T细胞已获批用于某些白血病，但CAR巨噬细胞的治疗潜力尚未得到充分探索。此外，CAR-T细胞与CAR巨噬细胞在清除癌细胞方面是否具有协同作用，也仍不清楚。 目的：本研究通过共培养实验，探讨人CAR-T细胞和CAR巨噬细胞清除癌细胞的效率及机制。 方法：研究者使用不同方法将第二代CD19 CAR信使RNA或增强型绿色荧光蛋白（EGFP）对照信使RNA包载至脂质纳米颗粒（LNP）中。利用这些LNP转染人原代T细胞或巨噬细胞，并通过流式细胞术或共聚焦显微镜分析CAR表达。通过杀伤和吞噬实验，评估CAR-T细胞和CAR巨噬细胞对CD19阳性NALM6白血病细胞及CD19阴性对照细胞的作用。另利用活细胞成像分析巨噬细胞、T细胞和癌细胞的运动能力。 结果：活化T细胞上调低密度脂蛋白受体（LDLR），因此转染后CAR表面表达高于未活化T细胞。CAR-T细胞可高效诱导淋巴瘤细胞死亡。CAR巨噬细胞主要通过吞噬清除癌细胞，CAR信使RNA还改变了M1及M2相关基因表达。有趣的是，CAR巨噬细胞也会上调CD47配体受体信号调节蛋白α（SIRPA）；采用siRNA敲低SIRPA可有效抵消这一现象。在含有CAR转染效应细胞的共培养体系中，观察到巨噬细胞、T细胞和癌细胞运动能力下降。CAR-T细胞与CAR巨噬细胞共培养可协同增强肿瘤细胞清除。 结论：研究结果凸显CAR-T细胞强大的癌细胞杀伤能力。此外，CAR巨噬细胞通过吞噬清除癌细胞发挥重要作用，从而协同支持CAR-T细胞活性。
INTRODUCTION: Chimeric antigen receptor (CAR) technology represents a groundbreaking advancement in gene therapy. While CAR-T cells are approved for specific types of leukemia, the therapeutic potential of CAR-macrophages remains largely unexplored. Additionally, potentially synergistic effects between CAR-T cells and CAR-macrophages in cancer cell clearance are still elusive. OBJECTIVES: This study aimed to investigate the efficiency and mechanisms of human CAR-T cells and CAR-macrophages for clearing cancer cells using co-culture experiments. METHODS: We encapsulated a second generation CD19 CAR mRNA or enhanced green fluorescent protein (Egfp) encoding control mRNA into lipid nanoparticles (LNP) using different methods. We transfected human primary T cells or macrophages with these LNP and studied their CAR expression using flow cytometry or confocal microscopy. Killing and phagocytosis assays were conducted to evaluate the impact of CAR-T cells and CAR-macrophages on CD19 + NALM6 leukemia and CD19 - control cells. Motility analyses of macrophages, T cells, and cancer cells were done using live-cell imaging. RESULTS: Activated T cells upregulated the low-density lipoprotein receptor (LDLR), resulting in higher CAR surface expression post-transfection than in non-activated T cells. CAR-T cells efficiently induced death of lymphoma cells. CAR-macrophages cleared cancer cells mainly via phagocytosis and CAR mRNA altered the expression of both M1 and M2-associated genes. Interestingly, CAR macrophages also upregulated the Signal regulatory protein alpha (SIRPA), a receptor of the CD47 ligand expressed by cancer cells, and knockdown of SIRPA by siRNA efficiently counteracted this phenomenon. We noted a decrease in the motility of macrophages, T cells, and cancer cells in co-cultures containing CAR-transfected effector cells. Co-culturing CAR-T cells with CAR-macrophages synergistically enhanced tumor cell eradication. CONCLUSION: Our findings underscore the superior cancer cell-killing capability of CAR-T cells. Additionally, CAR-macrophages play a crucial role in clearing cancer cells through phagocytosis, thereby synergistically supporting the activity of CAR-T cells.
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