Salem Bahey, Miner Samantha, Hensel Nancy F, Battiwalla Minoo, Keyvanfar Keyvan, Stroncek David F, Gee Adrian P, Hanley Patrick J, Bollard Catherine M, Ito Sawa, Barrett A John
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.
Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Cytotherapy. 2015 Dec;17(12):1675-86. doi: 10.1016/j.jcyt.2015.08.008. Epub 2015 Sep 28.
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).
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.
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.
This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay.
随着涉及免疫调节细胞的细胞疗法使用日益增加，需要一种简单的标准化方法来评估和比较不同细胞产品的抑制效力。我们使用卡尔帕斯299（K299）细胞系作为参考抑制细胞，开发了一种标准化抑制试验，以量化骨髓来源的间充质基质细胞（BM-MSC）的免疫调节能力。
将健康供体的CD4 T细胞与K299细胞系或第三方BM-MSC共同培养。用抗CD3/CD28磁珠刺激后，测量CD154的激活情况以及CD4 T细胞的增殖，以计算抑制率。
K299细胞系可重复性地以剂量依赖方式抑制健康供体CD4 T细胞的激活和增殖。我们选择了一种基于激活-抑制的快速（16小时）试验进行开发。在重复测试中，不同反应性T细胞之间抑制的固有变异性为变异系数11%。BM-MSC对不同反应细胞的抑制作用与K299的抑制作用相关。因此，我们以K299的抑制作用为参考，用K299抑制单位来定义BM-MSC的抑制效力。我们发现供体间变异性、传代次数、制造方法以及BM-MSC接触类固醇或干扰素-γ均会影响BM-MSC的抑制效力。
该方法为标准化抑制功能提供了一个平台，便于实验室之间进行比较，并用作细胞产品放行试验。
