Fig 3. miR-142 ablation impairs suppressive function and homeostasis of T_reg cells.

(A) in vitro T_reg suppression assay. Purified CD4⁺ T_conv cells were loaded with CTV dye and incubated with FACS-sorted T_reg cells from Foxp3^Cre (red dots) and Foxp3^CremiR-142^fl/fl (blue dots) spleens (n = 3) in the presence of beads coated with anti-CD3 and anti-CD28 specific antibodies. After 3 days, the rate of T_conv cell proliferation was determined by flow cytometry as dilution of the CTV dye. Several T_reg to T_conv cell ratios were analyzed as indicated in the graph. Unstimulated T_conv cells were used as control. (B) Survival of mice in aGVHD model. Lethally irradiated BALB/c recipient mice were transplanted with T-cell–depleted bone marrow from C57BL/6J mice (TCD-BM), Thy1.2⁺CD4⁺ T cells from C57BL/6J spleens (WT T), and CD4⁺YFP⁺ T_reg cells derived from either Foxp3^Cre (WT) or Foxp3^CremiR-142^fl/fl (KO) spleens (P = 0.0096; n = 5 per group). (C) Body weight analysis of mice in aGVHD model. Mouse grouping as in B (n = 5 per group). (D) Frequency of donor Foxp3^Cre (WT) or Foxp3^CremiR-142^fl/fl (KO) CD4⁺YFP⁺ T_reg cells in spleens of BALB/c recipient mice on day 17 after BMT (n = 4 for all groups). (E) Left panel, FACS analysis of activation-induced cell death in splenic Foxp3^Cre and Foxp3^CremiR-142^fl/fl T_reg cells. Apoptotic T_reg cells were detected by Annexin V staining upon stimulation with anti-CD3 (1 μg/ml) specific antibodies for 48 hours. Numbers indicate percentage of Annexin V⁺ cells. Right panel, the frequency of Annexin V⁺ Foxp3^Cre (WT) and Foxp3^CremiR-142^fl/fl (KO) T_reg cells (n = 3 per group). (F) FACS analysis of CD4⁺Foxp3⁺ T_reg cells (left panels), YFP-Cre expression in CD4⁺Foxp3⁺ T_reg cells (middle panels), and Foxp3 expression in CD4⁺YFP⁺ miR-142–sufficient and miR-142–deficient T_reg cells (right panels) from female Foxp3^Cre/WTmiR-142^+/+ and Foxp3^Cre/WTmiR-142^fl/fl mice. (G) Left panel, intracellular FACS analysis of BrdU incorporation into T_reg cells isolated from Foxp3^Cre and Foxp3^CremiR-142^fl/fl spleens. FACS plots were pregated on CD4⁺YFP⁺ T_reg cells, and numbers indicate percentage of cells in the gate. Right panel, the frequency of BrdU⁺ T_reg cells in Foxp3^Cre (WT) and Foxp3^CremiR-142^fl/fl (KO) spleens 16 hours after BrdU injection (1mg i.p.) (n = 3 per group). (H) FACS analysis of cell surface markers on T_reg cells isolated from Foxp3^Cre (red) and Foxp3^CremiR-142^fl/fl (blue) spleens with anti-GITR, anti-CD25, anti-CD103, anti-PD-1, anti-CTLA-4, and anti-ICOS specific antibodies. Histograms were pregated on CD4⁺YFP⁺ T_reg cells. Results are shown as mean ± SD. P values were calculated using log-rank test (B), 1-way ANOVA (C), and 2-tailed Student t test (D, E, G). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; NS, not significant. The underlying numerical raw data can be found in S1 Data file. The underlying flow cytometry raw data can be found at the Figshare repository. aGVHD, acute graft-versus-host disease; BMT, bone marrow transplantation; CTV, cell trace violet; FACS, fluorescence activated cell sorting; KO, knockout; SD, standard deviation; T_reg, regulatory T; YFP, yellow fluorescent protein; WT, wild-type.