A reproducible immunopotency assay to measure mesenchymal stromal cell–mediated T-cell suppression

Abstract

Background The T cell suppressive property of bone marrow derived mesenchymal stromal cells (MSCs) has been considered a major mode of action and basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible assay to measure MSC-mediated T cell suppression. Methods At the University of Wisconsin-Madison Production Assistance for Cellular Therapy (PACT) Center we developed an in vitro quality control T cell suppression immunopotency assay (IPA) which utilizes anti-CD3 and anti-CD28 antibodies to stimulate T cell proliferation. We measured MSC-induced suppression of CD4+ T cell proliferation at various effector to target cell ratios using defined peripheral blood mononuclear cells and in parallel compared to a reference standard MSC product. We calculated an IPA value for suppression of CD4+ T cells for each MSC product. Results Eleven MSC products generated at three independent PACT centers were evaluated for cell surface phenotypic markers and T cell suppressive properties. Flow cytometry results demonstrated typical MSC cell surface marker profiles. There was significant variability in the level of suppression of T cell proliferation with IPA values ranging from 27% to 88%. However, MSC suppression did not correlate with HLA-DR expression. Discussion We have developed a reproducible immunopotency assay to measure allogeneic MSC-mediated suppression of CD4+ T cells. Additional studies may be warranted to determine how these in vitro assay results may correlate with other immunomodulatory properties of MSCs, in addition to evaluating the ability of this assay to predict in vivo efficacy.

Morphological features of IFN-γ–stimulated mesenchymal stromal cells predict overall immunosuppressive capacity

“…2 A and B). Similar to other studies, we observed that the magnitude of Tcell responses to stimulation can differ substantially between independent PBMC donors (27,28), and when multiple measurements of T-cell activation are considered, each independent PBMC donor shows a unique profile of T-cell activation. By using PCA to produce a composite activation variable specific to the PBMC donor in a given experiment, our method accounts for these unique profiles by considering each activation measurement in an unbiased manner and incorporating those with the most variation into the first principal component (PC1; Fig.…”

“…2C). This enhancement has been observed by others as well, and its incorporation into the AUC gives a broader measure of the immunosuppressive capacity of a given MSC line that may have biological relevance in some in vivo scenarios where MSCs present in small numbers could potentially increase rather than decrease inflammatory symptoms (27,29). The AUC for a given MSC line differed between experiments in which independent PBMC donors were used due to donor-specific differences in T-cell susceptibility to MSC-mediated immune suppression (Fig.…”

Potency Analysis of Mesenchymal Stromal Cells Using a Combinatorial Assay Matrix Approach

“…Several studies address the potential utility of a cellular universal ruler for MSC potency assays (Bloom et al, 2015; Deans, 2015; Salem et al, 2015; Tanavde et al, 2015; Viswanathan et al, 2014). However, MSCs’ mechanism of action in vivo may well involve the effect of a plurality of effector pathways with synergistic and overlapping functionalities whose integrative effects are not completely understood when examined for use in distinct clinical uses.…”

Mitochondrial Oxidative Stress Reduces the Immunopotency of Mesenchymal Stromal Cells in Adults With Coronary Artery Disease

“…50 Further we show that atherosclerotic-MSCs have higher levels of total intracellular ROS (a negative predictor of the MSCs expansion potential 51 ) and, in particular, elevated mitochondrial ROS levels that correlate with their reduced in vitro immunopotency. Considering that MSCs may play a role in inhibiting inflammation and stabilizing vulnerable atherosclerotic plaques, 52 and the implications of mitochondria to atherosclerosis development, 53 our findings provide the rationale for testing whether restoring mitochondrial dysfunction in atherosclerotic-MSCs could impact atherosclerosis progression reducing the risk of acute events.…”