title: Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy.
abstract: Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of theFOXP3Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Howard R
authors.initials: HR
authors.lastname: Seay
authors.affiliation: Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Putnam
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Judit
authors.initials: J
authors.lastname: Cserny
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Posgai
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Emma H
authors.initials: EH
authors.lastname: Rosenau
authors.affiliation: Division of Hematology and Oncology, Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Wingard
authors.affiliation: ViaCord, LLC, Waltham, MA 02451, USA.
authors.forename: Kate F
authors.initials: KF
authors.lastname: Girard
authors.affiliation: ViaCord, LLC, Waltham, MA 02451, USA.
authors.forename: Morey
authors.initials: M
authors.lastname: Kraus
authors.affiliation: Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
authors.forename: Angela P
authors.initials: AP
authors.lastname: Lares
authors.affiliation: Cbr Systems, Inc., San Bruno, CA 94066, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Brown
authors.affiliation: Cbr Systems, Inc., San Bruno, CA 94066, USA.
authors.forename: Katherine S
authors.initials: KS
authors.lastname: Brown
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Kristi T
authors.initials: KT
authors.lastname: Balavage
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Leeana D
authors.initials: LD
authors.lastname: Peters
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Ashley N
authors.initials: AN
authors.lastname: Bushdorf
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA; Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Atkinson
authors.affiliation: Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Bluestone
authors.affiliation: Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Haller
authors.affiliation: Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Todd M
authors.initials: TM
authors.lastname: Brusko
keywords: T cell receptor; adoptive cell transfer; autoimmunity; cord blood; good manufacturing practices; regulatory T cell; type 1 diabetes