Premrutai ThitilertdechaPornpichaya SuwannachodPoonsin PoungpairojVarangkana TantithavornLadawan KhowawisetsutPalanee AmmaranondNattawat OnlamoonChulalongkorn UniversityFaculty of Medicine, Siriraj Hospital, Mahidol University2019-08-232019-08-232018-09-01Journal of Immunological Methods. Vol.460, (2018), 17-2518727905002217592-s2.0-85048317219https://repository.li.mahidol.ac.th/handle/20.500.14594/45977© 2018 Elsevier B.V. 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.Mahidol UniversityImmunology and MicrobiologyMedicineArticleSCOPUS10.1016/j.jim.2018.06.004