title: Optimization of cGMP purification and expansion of umbilical cord blood-derived T-regulatory cells in support of first-in-human clinical trials.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA. Electronic address: mcken020@umn.edu.
authors.forename: David H
authors.initials: DH
authors.lastname: McKenna
authors.affiliation: Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Darin
authors.initials: D
authors.lastname: Sumstad
authors.affiliation: Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Diane M
authors.initials: DM
authors.lastname: Kadidlo
authors.affiliation: Department of Laboratory Medicine and Pathology, Division of Transfusion Medicine, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Bjorn
authors.initials: B
authors.lastname: Batdorf
authors.affiliation: Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Lord
authors.affiliation: Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Merkel
authors.affiliation: Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Koellner
authors.affiliation: Department of Medicine, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Curtsinger
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA; Abramson Family Cancer Center Research Institute, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA.
authors.forename: Carl H
authors.initials: CH
authors.lastname: June
authors.affiliation: Abramson Family Cancer Center Research Institute, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA; Department of Microbiology, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA.
authors.forename: James L
authors.initials: JL
authors.lastname: Riley
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA; Abramson Family Cancer Center Research Institute, University of Pennsylvania Cancer Center, Philadelphia, Pennsylvania, USA.
authors.forename: Bruce L
authors.initials: BL
authors.lastname: Levine
authors.affiliation: Department of Medicine, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Miller
authors.affiliation: Department of Medicine, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Claudio G
authors.initials: CG
authors.lastname: Brunstein
authors.affiliation: Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Wagner
authors.affiliation: Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Blazar
authors.affiliation: Department of Pediatrics, Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis/Saint Paul, Minnesota, USA. Electronic address: hippe002@umn.edu.
authors.forename: Keli L
authors.initials: KL
authors.lastname: Hippen
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000637:transplantation; D002138:Calibration; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards*; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods / Q000592:standards*; D002478:Cells, Cultured; D002986:Clinical Trials as Topic; D036101:Cord Blood Stem Cell Transplantation / Q000379:methods / Q000592:standards*; D005260:Female; D005312:Fetal Blood / Q000166:cytology* / Q000276:immunology; D006086:Graft vs Host Disease / Q000276:immunology / Q000628:therapy; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods / Q000592:standards; D006801:Humans; D020014:K562 Cells; D066192:Manufacturing Industry / Q000592:standards; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008822:Mice, Transgenic; D017410:Practice Guidelines as Topic; D011786:Quality Control; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000502:physiology
keywords: cGMP production; graft versus host disease; regulatory T cell