Bahey Salem1, Samantha Miner2, Nancy F Hensel2, Minoo Battiwalla2, Keyvan Keyvanfar2, David F Stroncek3, Adrian P Gee4, Patrick J Hanley5, Catherine M Bollard5, Sawa Ito6, A John Barrett2. 1. 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. 2. Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. 3. Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland, USA. 4. Center for Cell and Gene Therapy, Texas Children's Hospital, Houston Methodist Hospital, and Baylor College of Medicine, Houston, Texas, USA. 5. 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. 6. Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA. Electronic address: itos2@mail.nih.gov.
Abstract
BACKGROUND AIMS: 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. Published by Elsevier Inc.
BACKGROUND AIMS: 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 donorCD4 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 donorCD4 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. Published by Elsevier Inc.
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