Joshua Weiner1, Raimon Duran-Struuck2, Jonah Zitsman3, Leo Buhler4, Hugo Sondermeijer5, Alicia N McMurchy6, Megan K Levings7, Megan Sykes8, Adam Griesemer9. 1. Columbia Center for Translational Immunology, Columbia University Medical Center, 650 West 168 Street, Black Building 15 Floor, New York, NY 10032, jiw2106@cumc.columbia.edu. 2. Columbia Center for Translational Immunology, Columbia University Medical Center, 650 West 168 Street, Black Building 15 Floor, New York, NY 10032, rd2605@cumc.columbia.edu. 3. Columbia Center for Translational Immunology, Columbia University Medical Center, 650 West 168 Street, Black Building 15 Floor, New York, NY 10032, jsz2105@cumc.columbia.edu. 4. Department of Surgery, University Hospital of Geneva, 24 Rue Micheli du Crest, Geneva 141211, Switzerland, leo.buhler@hcuge.ch. 5. Department of Physiology, Maastricht University, Universiteitssingel 50 6229 ER, Maastricht, The Netherlands, hpsondermeijer@gmail.com. 6. Child and Family Research Institute, Room A4-186, 950 West 28 Avenue, Vancouver, BC V5Z 4H4, alicia.mcmurchy@gmail.com. 7. Child and Family Research Institute, Room A4-186, 950 West 28 Avenue, Vancouver, BC V5Z 4H4, mlevings@mail.ubc.ca. 8. Columbia Center for Translational Immunology, Columbia University Medical Center, 650 West 168 Street, Black Building 15 Floor, New York, NY 10032, ms3976@cumc.columbia.edu. 9. Columbia Center for Translational Immunology, Columbia University Medical Center, 650 West 168 Street, Black Building 15 Floor, New York, NY 10032, adg2101@cumc.columbia.edu.
Abstract
BACKGROUND: Regulatory T cells (Treg) are being explored for their tolerance-inducing capabilities. Freezing and banking Treg for future use makes this strategy more clinically applicable. We aimed to devise an improved method of expanding and cryopreserving Treg to maximize yield, purity, and function for use in xenotransplantation. METHODS: Baboon peripheral blood mononuclear cells (PBMC) were isolated from whole blood. CD4+/CD25hi cells were isolated by flow cytometric sorting and expanded for 26 days in culture with IL-2, anti-CD3 antibody, artificial APCs transfected with human CD58, CD32, and CD80, and rapamycin with weekly restimulations. Expanded Treg were frozen for 2 months then thawed and cultured for 48 hours in medium plus 1) no additives, 2) IL-2, 3) anti-CD3 antibody, 4) IL-2 + anti-CD3 antibody, and 5) IL-2 + anti-CD3 antibody + L cells. Phenotype and suppression were assessed after expansion, immediately after thawing, and after culturing. RESULTS: We expanded purified baboon Treg more than 10,000-fold. Expanded Treg exhibited excellent suppression in functional assays. Cryopreservation decreased suppressive function without changing phenotype, but increasing amounts of reactivation after thawing produced significantly better viability and suppressive function with a trend towards greater Treg purity. CONCLUSIONS: We produced numbers of expanded Tregs consistent with clinical use. In contrast to some previous reports, both Treg phenotype and suppressive function were preserved or even enhanced by increasing amounts of restimulation after thawing. Thus, banking of expanded recipient Tregs for in vivo infusion should be possible.
BACKGROUND: Regulatory T cells (Treg) are being explored for their tolerance-inducing capabilities. Freezing and banking Treg for future use makes this strategy more clinically applicable. We aimed to devise an improved method of expanding and cryopreserving Treg to maximize yield, purity, and function for use in xenotransplantation. METHODS:Baboon peripheral blood mononuclear cells (PBMC) were isolated from whole blood. CD4+/CD25hi cells were isolated by flow cytometric sorting and expanded for 26 days in culture with IL-2, anti-CD3 antibody, artificial APCs transfected with humanCD58, CD32, and CD80, and rapamycin with weekly restimulations. Expanded Treg were frozen for 2 months then thawed and cultured for 48 hours in medium plus 1) no additives, 2) IL-2, 3) anti-CD3 antibody, 4) IL-2 + anti-CD3 antibody, and 5) IL-2 + anti-CD3 antibody + L cells. Phenotype and suppression were assessed after expansion, immediately after thawing, and after culturing. RESULTS: We expanded purified baboonTreg more than 10,000-fold. Expanded Treg exhibited excellent suppression in functional assays. Cryopreservation decreased suppressive function without changing phenotype, but increasing amounts of reactivation after thawing produced significantly better viability and suppressive function with a trend towards greater Treg purity. CONCLUSIONS: We produced numbers of expanded Tregs consistent with clinical use. In contrast to some previous reports, both Treg phenotype and suppressive function were preserved or even enhanced by increasing amounts of restimulation after thawing. Thus, banking of expanded recipient Tregs for in vivo infusion should be possible.
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