Figure 4.

MSC-mediated immunomodulation of leukocytes. (A): In selected experiments, MSCs were exposed to IFN-γ and TNF-α for 48 hours (pMSCs). After priming, IDO expression was substantially upregulated as assessed by quantitative polymerase chain reaction compared with untreated MSCs. (B): Unstimulated MSCs and pMSCs affected viability of control or LPS-stimulated PMNs. (C): CD16 (FcγR-III) expression was used as a marker of PMN viability and matched with the percentage of viable PMNs in all culture conditions. Similarly, CD11b and CD54 expressions were associated with PMN activation status. The percentage of CD11b-positive PMNs was higher in the presence of MSCs and further enhanced by LPS treatment (D), and CD54 relative MFI was upregulated by LPS treatment, and this effect was enhanced by coculture with MSCs (E). The capacity of MSCs to promote the generation of myeloid-derived suppressor cells (MDSCs) was tested within our coculture model with PMNs. Coculture with MSCs led to a marked increase of mature CD11^bright/CD16^bright/CD62L^dim (N2-type) PMNs resembling granulocytic MDSCs (F). (G): MSC and PMN cocultures were stained with May-Grünwald-Giemsa dye to observe cell morphology after reciprocal interaction for 24 hours. According to the marked increase of mature CD11^bright/CD16^bright/CD62L^dim (N2-type) PMNs shown at this time by flow cytometry, PMNs displayed a hypersegmented nuclear morphology after coculture with both resting (PMN MSCs at 24 hours) and pMSCs (PMN pMSCs at 24 hours). Similarly, coculturing MSCs with healthy control PBMCs at different ratios promoted CD14⁺HLA-DR^low monocytes resembling monocytic MDSCs (H). MSCs were also capable of promoting the induction and expansion of immune suppressive CD4⁺CD25^highCD127^low T_Regs, when cocultured in different ratios with purified healthy control T cells (I). (J): Increased levels of circulating CD4⁺CD25^highCD127^low T_Regs were also observed up to 20 days after MSC administration (red, patient 1; black, patient 2). (K): This could in part have resulted from increased thymic output, indicated by an increased proportion of CD31⁺ RTEs among CD45RA⁺ naïve T_regs in both patients (red lines, patient 1; black lines, patient 2). Bars indicate the SEM. ∗, p ≤ .05; ∗∗, p ≤ .01; ∗∗∗, p ≤ .01. Abbreviations: IDO, indoleamine-2,3-dioxygenase; IFN-γ, interferon-γ; h, hour; LPS, lipopolysaccharide; MFI, mean fluorescence intensity; MSCs, mesenchymal stromal cells; PBMCs, peripheral blood mononuclear cells; PMNs, polymorphonuclear leukocytes; pMSCs, primed MSCs; RTEs, recent thymic emigrants; TNF-α, tumor necrosis factor-α; T_Regs, regulatory T cells.