Blockade of the TNFα/TNFR2 interaction reduces Foxp3 and activation markers expressions in Tregs used to prevent GVHD. GVHD experiments were reproduced using “anti-TNFR2” mAb treatment (A) or T cells collected from TNFα-deficient mice (B). Splenocytes from grafted animals were harvested at day 13 posttransplantation and enriched in CD4⁺ and CD8⁺ T cells through positive magnetic selection using large selection columns (Miltenyi Biotec). Depending on the marker evaluated, Tregs were stained with CD4-FITC, CD4-allophycocyanin or CD4-Vioblue, Foxp3-PE-Cy5 or Foxp3-V450, and CD25-PE-Cy7, ICOS-PE, CTLA4-biotin. Intracellular Foxp3 staining was performed using the Foxp3 staining buffer set from eBioscience. Cells were gated on CD4⁺ Foxp3⁺ T cells except for the percentage of Foxp3 (up), which is gated on CD4⁺ T cells. For each marker, the strategy of gating is indicated on the left of the figure. Each dot represents a single mouse. For each group of mice, horizontal lines represent mean value and standard error of the mean. MFI values are represented as ratio of the measured value for each sample to the mean value of the control group (ie, the group of mice receiving BM cells plus T cells and Treg cells). We have normalized the MFI values with T-cell + Treg control group. Then we used unpaired, 2-tailed Student t tests for generation of P values. *P < .05; **P < .01; ***P < .001; ****P < .0001.