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

Significance Substantial evidence exists demonstrating the immunosuppressive function of mesenchymal stromal cells (MSCs), but inconsistent clinical results suggest that better understanding of MSC-mediated immunosuppression and identification of features predictive of immunosuppressive capacity would advance MSC-based therapeutics. In this work, we present a robust analytical approach to quantify the immunosuppressive capacity of MSCs by integrating high-dimensional flow cytometry data from multiple experimental conditions into a single measure of immunosuppressive capacity. Additionally, we identified morphological features of MSCs that predicted immunosuppressive capacity, as well as the magnitude of IFN-γ–mediated immunosuppression enhancement. These improved methods of MSC characterization could be used to identify MSC preparations with desired immunosuppressive capacity, as well as screen for pretreatments that enhance their immunosuppressive function. Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSC-based therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ–enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.