Figure 7.

MDSCs infusion increases induction of FoxP3⁺ Treg cells and anti-inflammatory cytokine and decreases pro-inflammatory cytokine production in xenogeneic GVHD model. The mice were bled and sacrificed at day 60 post human PBMCs infusion and (A) the human cytokine levels in the serum were measured by ELISA kits. (B-C) Splenocytes were stained with fluorochrome-conjugated anti-human CD3 (PE-Cy7), anti-human CD4 (FITC) and anti-human CD25 (APC) and then intracellular stained with anti-human Foxp3 (PE), anti-human IL-17 (PerCP-Cy5.5), anti-human IFN-γ (Pacific Blue) and anti-human IL-4 (APC-Cy7) antibodies using fixation and permeabilization buffer. CD3⁺CD4⁺ T cells gated contour plots were presented. (D) The ratio of Th1/Th2 on CD3⁺CD4⁺ T cells. The ratio was calculated as the ratio of IL-4⁺ CD3⁺CD4⁺ T cells divided by the percentage of IFN-γ⁺ CD3⁺CD4⁺ T cells. (E) The ratio of Treg/Th17 on CD3⁺CD4⁺ T cells. The ratio was calculated as the ratio of IL-17⁺ CD3⁺CD4⁺ T cells divided by the percentage of CD25⁺FoxP3⁺CD3⁺CD4⁺ T cells. (F) Serum cytokine levels were estimated semi-quantitatively with a multi-cytokine (human) membrane array. The data were presented a decrease in the pro-inflammatory cytokine levels, including C reactive protein (CRP), IL-1β, IL-6, TNF-α, IL-17, macrophage inflammatory protein-3β (MIP-3β), matrix metallopeptidase 9 (MMP9), Regulated on Activation Normal T cell Expressed and Secreted (RANTES) and stromal cell-derived factor-1α (SDF-1α) in the MDSCs mice compared with GVHD mice (n = 3 mice/group). Data are mean ± S.E.M. of three independent experiments, each performed in triplicate. *p < 0.05, ***p < 0.001, ****p < 0.0001.