Figure 3.

MiR130b is essential for SLFN4⁺-MDSC activity. (A) TG-elicited PCs collected from WT mice were transfected with MiR130b mimic, MiR130b antisense (a-130b), Slfn4 siRNA or scrambled control (Scr) and then treated with IFNα (800 U/mL) or PBS for 24 hours. Differential gene expression was evaluated by qPCR. N=5 expts. (B) Protocol to collect SLFN4⁺ cells for coculture with T cells. TG-elicited PCs from Slfn4-tdT mice were treated with IFNα for 24 hours and then flow-sorted for SLFN4⁺ and SLFN4^– cells. Sorted cells were then transfected with MiR130b mimic, antisense or Scr. These sorted cells and NT peritoneal cells were cocultured with activated T cells for 72 hours. (C) CFSE-based T cell suppression assay was quantified by flow cytometry. The top four representative histograms show proliferation of control groups: NO CFSE, without anti-CD3/28 microbeads activation, with CD3/28 only and cocultured with Tregs (CD4⁺CD25⁺). The median percentage of proliferating T cells is shown in the representative histograms and plotted for n=4 expts in the (D) bar graph. One-way ANOVA followed by Tukey’s multiple comparisons test on log-transformed values. The mice infected with Helicobacter felis for 4 months were treated with MIR130b antisense or scrambled control using Invivofectamine. Three weeks postinjection, (E) CD45⁺CD8⁺ cytotoxic T cells in the stomach were detected by flow cytometry and (F) metaplastic change was shown by H&E, CD44 variant 9 (red), GSII (green) and clusterin (green) staining. N=3 expts. *P values are relative to IFNα-treated group. #P values are relative to CON or scrambled. * or #p<0.05, ** or ##p<0.01, *** or ###p<0.001, ####p<0.0001. NS, not significant. The median and IQR is shown. ANOVA, analysis of variance; NT, non-treated; PCs, peritoneal myeloid cells; TG, thioglycollate; UI, uninfected.