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 × 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.

Human Regulatory T Cells From Umbilical Cord Blood Display Increased Repertoire Diversity and Lineage Stability Relative to Adult Peripheral Blood

“…Our prior work has demonstrated that tTregs can be isolated from human CB and expanded with exceptional purity and lineage stability (32). Here, we extend our prior studies optimizing Treg expansion protocols to further characterize the transcriptional profile and repertoire characteristics of human Tregs from CB in comparison to those isolated from APB.…”

Tracing human stem cell lineage during development using DNA methylation

“…An ongoing concern in adoptive cell transfer therapies is the paucity of informative markers reflecting epigenomic stability of expanded cell populations, as for example, in the expansion of umbilical cord blood derived T-regulatory cells (Seay et al 2017). …”

Reversal of Hyperglycemia and Suppression of Type 1 Diabetes in the NOD Mouse with Apoptotic DNA Immunotherapy™ (ADi™), ADi-100

“…Therefore, it is important to develop more regulatory-specific and potent TDSs such as soluble or particulate tolerance vehicles (e.g., nanoparticles, microspheres, and liposomes) containing different tolerogenic agents such as rapamycin, aryl-hydrocarbon receptor ligands, retinoic acid, vitamin D3, and cytokines such as interleukin (IL)-10 and transforming growth factor (TGF)-β [31,34]. Other TDSs are of a cellular nature in which tol-DCs or Tregs produced ex vivo are reintroduced in vivo [35,36], or are genetically modified gastrointestinal bacterial strains expressing autoantigen and tolerogenic cytokines [37]. Note that apoptotic tolerance vehicles are in this cellular class of TDSs.…”