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Mol Ther Methods Clin Dev. 2016 Dec 24;4:178-191. doi: 10.1016/j.omtm.2016.12.003. eCollection 2017 Mar 17.

Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy.

Author information

1
Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
2
Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
3
Division of Hematology and Oncology, Department of Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
4
ViaCord, LLC, Waltham, MA 02451, USA.
5
Cbr Systems, Inc., San Bruno, CA 94066, USA.
6
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.
7
Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.

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 × 109 Tregs 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 the FOXP3 Treg-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.

KEYWORDS:

T cell receptor; adoptive cell transfer; autoimmunity; cord blood; good manufacturing practices; regulatory T cell; type 1 diabetes

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