Efficient Capture and Traceless Release of Functional CD8+ T Cells with a Microfluidic Chip for Enhanced In Vitro and In Vivo CD4-CAR Transduction,Analytical Chemistry

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Efficient Capture and Traceless Release of Functional CD8+ T Cells with a Microfluidic Chip for Enhanced In Vitro and In Vivo CD4-CAR Transduction
Analytical Chemistry ( IF 6.7 ) Pub Date : 2024-09-13 , DOI: 10.1021/acs.analchem.4c03135
Aynur Abdulla _{1,

2} , Tuersunayi Abudureheman ₃ , Kaiming Chen ₃ , Behafarid Ghalandari ₂ , Haoni Yan ₁ , Hang Zhou ₃ , Hengxing Su ₄ , Yunqian Zhang ₁ , Cai-Wen Duan ₃ , Xianting Ding _{1,

2}

Affiliation

The chimeric antigen receptor (CAR) T cells targeting CD4 expressed cells in acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) could reduce the risk of off target effects in normal tissues. However, the efficacy of adoptive cell therapy is predominantly attributed to CD8⁺ T cells, necessitating their purification before lentivirus transfection to enhance the production of CD4-CAR-T cells. In this study, we developed a microfluidic chip functionalized with an optimized CD8 aptamer, A3t-MU, to facilitate the enrichment and purification of CD8⁺ T cells. The presented chip showed efficient capture and seamless release of CD8⁺ T cells from cultured T cells and peripheral blood mononuclear cells (PBMCs). The purity of the released CD8⁺ T cells reached 98.1%, representing a 13% improvement over the conventional magnetic bead separation method. CD4-CAR was efficiently transduced into the purified CD8⁺ T cells to construct CAR-T cells. We evaluated the antitumor capability of the CD4-CAR transduced CD8⁺ T cells (anti-CD4 CD8-CAR T cells) both in vitro and in vivo. The anti-CD4 CD8-CAR T cells exhibited significant cancer-cell-killing capacity across multiple tumor cell lines, including CEM, Jurkat, and MV4-11. Meanwhile, anti-CD4 CD8-CAR T cells significantly inhibited tumor growth in vivo. In conclusion, the presented microfluidic chip offers a cost-effective and high-purity approach for CD8⁺ T cell separation, enhancing CD4-CAR transduction and achieving efficient antitumor capability both in vitro and in vivo.

中文翻译：

使用微流控芯片有效捕获和无痕释放功能性 CD8+ T 细胞，增强体外和体内 CD4-CAR 转导

针对急性淋巴细胞白血病（T-ALL）和急性髓性白血病（AML）中CD4表达细胞的嵌合抗原受体（CAR）T细胞可以降低正常组织中脱靶效应的风险。然而，过继细胞疗法的功效主要归因于CD8 ⁺ T细胞，因此需要在慢病毒转染之前对其进行纯化，以增强CD4-CAR-T细胞的产生。在本研究中，我们开发了一种具有优化的 CD8 适体 A3t-MU 功能的微流控芯片，以促进 CD8 ⁺ T 细胞的富集和纯化。该芯片显示出从培养的 T 细胞和外周血单核细胞 (PBMC) 中有效捕获和无缝释放 CD8 ⁺ T 细胞。释放的CD8 ⁺ T细胞纯度达到98.1%，比传统磁珠分离方法提高了13%。 CD4-CAR被有效转导到纯化的CD8 ⁺ T细胞中，构建CAR-T细胞。我们在体外和体内评估了 CD4-CAR 转导的 CD8 ⁺ T 细胞（抗 CD4 CD8-CAR T 细胞）的抗肿瘤能力。抗 CD4 CD8-CAR T 细胞在多种肿瘤细胞系（包括 CEM、Jurkat 和 MV4-11）中表现出显着的癌细胞杀伤能力。同时，抗CD4 CD8-CAR T细胞在体内显着抑制肿瘤生长。总之，所提出的微流控芯片为 CD8 ⁺ T 细胞分离提供了一种经济高效且高纯度的方法，增强了 CD4-CAR 转导并在体外和体内实现高效的抗肿瘤能力。

更新日期：2024-09-13

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