title: Restimulation After Cryopreservation and Thawing Preserves the Phenotype and Function of Expanded Baboon Regulatory T Cells.
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+/CD25hicells 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 forin vivoinfusion should be possible.
authors.affiliation: 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.
authors.forename: Joshua
authors.initials: J
authors.lastname: Weiner
authors.affiliation: 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.
authors.forename: Raimon
authors.initials: R
authors.lastname: Duran-Struuck
authors.affiliation: 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.
authors.forename: Jonah
authors.initials: J
authors.lastname: Zitsman
authors.affiliation: Department of Surgery, University Hospital of Geneva, 24 Rue Micheli du Crest, Geneva 141211, Switzerland, leo.buhler@hcuge.ch.
authors.forename: Leo
authors.initials: L
authors.lastname: Buhler
authors.affiliation: Department of Physiology, Maastricht University, Universiteitssingel 50 6229 ER, Maastricht, The Netherlands, hpsondermeijer@gmail.com.
authors.forename: Hugo
authors.initials: H
authors.lastname: Sondermeijer
authors.affiliation: Child and Family Research Institute, Room A4-186, 950 West 28 Avenue, Vancouver, BC V5Z 4H4, alicia.mcmurchy@gmail.com.
authors.forename: Alicia N
authors.initials: AN
authors.lastname: McMurchy
authors.affiliation: Child and Family Research Institute, Room A4-186, 950 West 28 Avenue, Vancouver, BC V5Z 4H4, mlevings@mail.ubc.ca.
authors.forename: Megan K
authors.initials: MK
authors.lastname: Levings
authors.affiliation: 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.
authors.forename: Megan
authors.initials: M
authors.lastname: Sykes
authors.affiliation: 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.
authors.forename: Adam
authors.initials: A
authors.lastname: Griesemer