title: Quantitative activation suppression assay to evaluate human bone marrow-derived mesenchymal stromal cell potency.
abstract: OBJECTIVE
  With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantify the immune-modulatory capacity of bone marrow-derived mesenchymal stromal cells (BM-MSCs).
  
  
  METHODS
  Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third-party BM-MSCs. After stimulation with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression.
  
  
  RESULTS
  The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16-h) assay that was based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation between different responder T cells. Suppression by BM-MSCs on different responders correlated with suppression by K299. We therefore used K299 suppression as the reference to define suppression potency of BM-MSCs in K299 Suppression Units. We found that inter-donor variability, passage number, method of manufacture and exposure of BM-MSCs to steroids or interferon-γ all affected BM-MSC potency of suppression.
  
  
  CONCLUSIONS
  This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA; Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, and Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Bahey
authors.initials: B
authors.lastname: Salem
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Samantha
authors.initials: S
authors.lastname: Miner
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Nancy F
authors.initials: NF
authors.lastname: Hensel
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Minoo
authors.initials: M
authors.lastname: Battiwalla
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Keyvan
authors.initials: K
authors.lastname: Keyvanfar
authors.affiliation: Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: David F
authors.initials: DF
authors.lastname: Stroncek
authors.affiliation: Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, and Baylor College of Medicine, Houston, Texas, USA.
authors.forename: Adrian P
authors.initials: AP
authors.lastname: Gee
authors.affiliation: Program for Cell Enhancement and Technologies for Immunotherapy, Center for Cancer and Immunology Research, Sheikh Zayed Institute for Pediatric Surgical Innovation, and the Division of Blood and Marrow Transplantation, Children's National Health System, Washington, DC, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Hanley
authors.affiliation: Program for Cell Enhancement and Technologies for Immunotherapy, Center for Cancer and Immunology Research, Sheikh Zayed Institute for Pediatric Surgical Innovation, and the Division of Blood and Marrow Transplantation, Children's National Health System, Washington, DC, USA.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Bollard
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: itos2@mail.nih.gov.
authors.forename: Sawa
authors.initials: S
authors.lastname: Ito
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: A John
authors.initials: AJ
authors.lastname: Barrett
mesh_terms: D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001681:Biological Assay; D001854:Bone Marrow Cells / Q000166:cytology*; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology* / Q000276:immunology; D023201:CD40 Ligand / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000276:immunology; D018920:Coculture Techniques; D006801:Humans; D007165:Immunosuppression Therapy / Q000379:methods*; D007371:Interferon-gamma / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000276:immunology*; D059630:Mesenchymal Stem Cells / Q000166:cytology*
chemical_list: D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D023201:CD40 Ligand; D007371:Interferon-gamma
keywords: K299; immunosuppression; mesenchymal stromal cells; suppression potency assay