Optimization of cGMP purification and expansion of umbilical cord blood–derived T-regulatory cells in support of first-in-human clinical trials

David H McKenna, Darin Sumstad, Diane M Kadidlo, Bjorn Batdorf, Colin J. Lord, Sarah C. Merkel, Christine M. Koellner, Julie M Curtsinger, Carl H. June, James L. Riley, Bruce L. Levine, Jeffrey S Miller, Claudio G Brunstein, John E Wagner, Bruce R Blazar, Keli L Hippen

Research output: Contribution to journalArticlepeer-review

38 Scopus citations


Background aims Thymic-derived regulatory T cells (tTreg) are critical regulators of the immune system. Adoptive tTreg transfer is a curative therapy for murine models of autoimmunity, graft rejection, and graft-versus-host disease (GVHD). We previously completed a “first-in-human” clinical trial using in vitro expanded umbilical cord blood (UCB)-derived tTreg to prevent GVHD in patients undergoing UCB hematopoietic stem cell transplantation (HSCT). tTreg were safe and demonstrated clinical efficacy, but low yield prevented further dose escalation. Methods To optimize yield, we investigated the use of KT64/86 artificial antigen presenting cells (aAPCs) to expand tTreg and incorporated a single re-stimulation after day 12 in expansion culture. Results aAPCs increased UCB tTreg expansion greater than eightfold over CD3/28 stimulation. Re-stimulation with aAPCs increased UCB tTreg expansion an additional 20- to 30-fold. Re-stimulated human UCB tTreg ameliorated GVHD disease in a xenogeneic model. Following current Good Manufacturing Practice (cGMP) validation, a trial was conducted with tTreg. tTreg doses up to >30-fold higher compared with that obtained with anti-CD3/28 mAb coated-bead expansion and Foxp3 expression was stable during in vitro expansion and following transfer to patients. Increased expansion did not result in a senescent phenotype and GVHD was significantly reduced. Discussion Expansion culture with cGMP aAPCs and re-stimulation reproducibly generates sufficient numbers of UCB tTreg that exceeds the numbers of T effector cells in an UCB graft. The methodology supports future tTreg banking and is adaptable to tTreg expansion from HSC sources. Furthermore, because human leukocyte antigen matching is not required, allogeneic UCB tTreg may be a useful strategy for prevention of organ rejection and autoimmune disease.

Original languageEnglish (US)
Pages (from-to)250-262
Number of pages13
Issue number2
StatePublished - Feb 1 2017

Bibliographical note

Funding Information:
This work was supported in part by National Institutes of Health (NIH) National Heart, Lung, and Blood Institute contract HHSN268201000008C (JSM, DHM, KLH, JC, JEW) and grant R01 HL11879 (BRB), National Cancer Institute grants P01 CA65493 (CGB, JSM, DHM, KLH, BRB, JEW) and P30 CA77598 (JSM, JEW), Leukemia and Lymphoma Society Scholar in Clinical Research Award CDP-2417-11 (CGB), Leukemia and Lymphoma Translational Research grant R6029-07 (BRB) and the Earl E. Bakken Charitable Trust and Children's Cancer Research Fund. This work was also supported in part by NIH P30 CA77598 using the shared resource Flow Cytometry Core from the Masonic Cancer Center, University of Minnesota.

Publisher Copyright:
© 2017 International Society for Cellular Therapy


  • cGMP production
  • graft versus host disease
  • regulatory T cell


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