Fig 3. MSCs differentially affect cytokine production in effector CD4⁺ T cells in vitro and in vivo.

For in vitro experiments, naïve CD62L⁺CD4⁺ T cells were cultured in the absence or presence of MSCs at a T cell: MSC ratio of 4:1. CD4+ T cells were activated with plate-bound α-CD3 and soluble α-CD28 ± polarizing cytokines and neutralizing antibodies. After 72 hours, T cells were harvested, re-stimulated with cell stimulation cocktail for 5 hours, stained, and analyzed by flow cytometry. Shown are representative flow cytometric plots, gated on CD4⁺ T cells, of T_H17 cells (A) and activated CD4⁺ T cells and T_H1 cells (B), in the absence and presence of MSCs. Compiled bar graph data are from three separate experiments. Roman numerals refer to specific polarization conditions as shown. For (B), IFNγ mean fluorescence intensity (MFI) values are shown and were calculated from CD4-subgated IFNγ⁺ cells. Compiled bar graph data are from two independent experiments. For in vivo experiments (C), female C57BL/6 mice were immunized with MOG_35–55 and complete Freund’s adjuvant for EAE induction as previously described (n = 5/treatment group). On days 3 and 8 post-immunization, mice received either murine MSCs or phosphate-buffered saline vehicle as previously described. Brains were harvested 14 days post-immunization and processed for analysis of infiltrating lymphocytes, which were then analyzed by flow cytometry for frequencies of IFNγ⁺ T_H1 cells and IL-17A⁺ T_H17 cells. Shown are compiled bar graph data from two separate experiments. Significance was measured by Student’s t-test, with *p<0.05.