Efficient Capture and Traceless Release of Functional\nCD8<sup>+</sup> T Cells with a Microfluidic Chip for Enhanced <i>In Vitro</i> and <i>In Viv</i><i>o</i> CD4-CAR\nTransduction

The\nchimeric antigen receptor (CAR) T cells targeting CD4 expressed\ncells in acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia\n(AML) could reduce the risk of off target effects in normal tissues.\nHowever, the efficacy of adoptive cell therapy is predominantly attributed\nto CD8<sup>+</sup> T cells, necessitating their purification before\nlentivirus transfection to enhance the production of CD4-CAR-T cells.\nIn this study, we developed a microfluidic chip functionalized with\nan optimized CD8 aptamer, A3t-MU, to facilitate the enrichment and\npurification of CD8<sup>+</sup> T cells. The presented chip showed\nefficient capture and seamless release of CD8<sup>+</sup> T cells\nfrom cultured T cells and peripheral blood mononuclear cells (PBMCs).\nThe purity of the released CD8<sup>+</sup> T cells reached 98.1%,\nrepresenting a 13% improvement over the conventional magnetic bead\nseparation method. CD4-CAR was efficiently transduced into the purified\nCD8<sup>+</sup> T cells to construct CAR-T cells. We evaluated the\nantitumor capability of the CD4-CAR transduced CD8<sup>+</sup> T cells\n(anti-CD4 CD8-CAR T cells) both <i>in vitro</i> and <i>in vivo</i>. The anti-CD4 CD8-CAR T cells exhibited significant\ncancer-cell-killing capacity across multiple tumor cell lines, including\nCEM, Jurkat, and MV4-11. Meanwhile, anti-CD4 CD8-CAR T cells significantly\ninhibited tumor growth <i>in vivo</i>. In conclusion, the\npresented microfluidic chip offers a cost-effective and high-purity\napproach for CD8<sup>+</sup> T cell separation, enhancing CD4-CAR\ntransduction and achieving efficient antitumor capability both <i>in vitro</i> and <i>in vivo</i>.