Fig. 8

Experimental validation of novel candidate molecules regulating FOXP3+ Tregs. a–c 37 novel candidate genes with a putative role in Treg induction were chosen for a targeted shRNA validation screen. Transduced or ‘untransduced’ primary CD4+ T cells were cultured under iTreg conditions (TGF-β + ATRA), or left unstimulated (‘unstim’), and then stained for FOXP3 and other markers. a FOXP3 histograms for T cells transduced with shRNA targeting FOXP3, IKZF4, or the candidate gene TRIM22 (red lines). Black and blue lines: negative control shRNA (shScr), empty vector (pLKO.1 empty). A representative donor of 3 (IKZF4) or 6 (FOXP3, TRIM22) is shown. b FOXP3 expression (gated on live CD4+ cells) in iTregs transduced with shRNA targeting candidate genes (grey bars; ‘–1’ and ‘–2’ indicate two independent shRNA pools). Blue, red bars: negative, positive controls. Displayed are mean ± SEM values, each dot represents an individual T cell donor (n = 3–6 donors from two independent experiments). FDR-adjusted p values (two-sided t test shRNA vs. shScr paired within a donor) are labeled as follows: ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. c FOXP3 and other flow cytometry markers in shRNA-transduced iTregs as in (b). The ratio of each marker relative to shScr-transduced cells of the same donor was calculated, and mean values of n = 3–6 donors are indicated by the color scale. It was pre-gated on live lymphocytes except for % lymphocyte gate and % live parameters. MFI median fluorescence intensity. Hierarchical clustering: complete linkage based on Euclidian distance