Research Group in Immunobiology and Therapeutic Sciences, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Biomedical Research Incubator Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Graduate program in Immunology, Department of Immunulogy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
J Immunol Methods. 2018 Sep;460:17-25. doi: 10.1016/j.jim.2018.06.004. Epub 2018 Jun 9.
CD4 immunotherapy is potentially useful in immune reconstitution of CD4 T cells for HIV-infected patients. Transfusion of anti-CD3/28 expanded CD4 T cells is also proved to be safe and effective in both SIV-infected macaques and HIV-infected patients. However, there is no such standardized and practical protocol available for cell production in order to use in clinics. This study thus aimed to develop a closed-culture system for in vitro CD4 T lymphocyte expansion by using a commercially available GMP-grade culture bag and anti-CD3/28 activation. Freshly isolated CD4 T cells by immunorosette formation from healthy donors and cryopreserved CD4 T cells from HIV-infected patients with CD4 count over 500 cells/μL were stimulated with anti-CD3/28 coated beads. The activated cells were then expanded in conventional culture flasks and GMP-grade culture bags for three weeks. Fold expansion, cell viability, growth kinetic and phenotypic characters were observed. Results revealed that purified CD4 T cells from healthy individuals cultured in flasks showed better expansion than those cultured in bags (797-fold and 331-fold, respectively), whereas, their cell viability, growth kinetic and expanded CD4 T cell purity were almost similar. A large-scale production was also conducted and supported consistency of cell proliferation in the closed-culture system. Frozen CD4 T lymphocytes from the patients were able to remain their growth function and well expanded with a good yield of 415-fold, 85% viability and 96% purity of CD4 T cells at the end of a 3-week culture in bags. This developed closed-culture system using culture bags and anti-CD3/28 coated beads, therefore, can achieve a large number of expanded CD4 T lymphocytes with good reproducibility, suggesting a promising protocol required for adoptive immunotherapy.
CD4 免疫疗法有可能用于重建 HIV 感染患者的 CD4 T 细胞免疫功能。输注抗 CD3/28 扩增的 CD4 T 细胞已被证明在 SIV 感染的猕猴和 HIV 感染患者中是安全有效的。然而，目前尚无标准化且实用的细胞生产方案用于临床。因此，本研究旨在开发一种封闭培养系统，通过使用商业上可获得的 GMP 级培养袋和抗 CD3/28 激活，进行体外 CD4 T 淋巴细胞扩增。从健康供体的免疫玫瑰花环形成中分离新鲜的 CD4 T 细胞，以及从 CD4 计数超过 500 个细胞/μL 的 HIV 感染患者的冷冻 CD4 T 细胞中分离，并用抗 CD3/28 包被珠刺激。然后将激活的细胞在常规培养瓶和 GMP 级培养袋中扩增 3 周。观察细胞扩增倍数、细胞活力、生长动力学和表型特征。结果表明，在培养瓶中培养的来自健康个体的纯化 CD4 T 细胞比在培养袋中培养的细胞扩增更好（分别为 797 倍和 331 倍），但其细胞活力、生长动力学和扩增的 CD4 T 细胞纯度几乎相似。还进行了大规模生产，支持了封闭培养系统中的细胞增殖一致性。来自患者的冷冻 CD4 T 淋巴细胞能够保持其生长功能，并在培养袋中培养 3 周后，以 415 倍的高产量、85%的活力和 96%的 CD4 T 细胞纯度良好扩增。因此，该研究开发的使用培养袋和抗 CD3/28 包被珠的封闭培养系统能够实现大量具有良好可重复性的 CD4 T 淋巴细胞扩增，为过继免疫治疗提供了一种有前景的方案。
