Fig 2: Bcl11b-deficient CD4+ T-cells downregulate the Th2 program and upregulate Th17 and Th1 genes. a–c Box and whisker plots of normalized counts for specific Th2-associated (a), Th17-associated (b), and Th1-associated (c) mRNAs derived from RNA-Seq on total CD4+ T-cells from the mLNs of H. polygyrus bakeri infected Bcl11bfl/fl dLck-iCre mice (n = 4) and Bcl11bfl/fl control mice (n = 4). Normalized counts and statistical analyses were taken from DESeq2 analysis software. Data are derived from two independent experiments. p values have been adjusted for the FDR (false discovery rate) by the Benjamini–Hochberg adjustment. d–f Box and whisker plots of normalized counts for specific Th2-associated (d), Th17-associated (e), and Th1-associated (f) mRNAs derived from RNA-Seq on IL-4+-enriched CD4+ T-cells from the lung parenchyma of asthma-induced Bcl11bfl/fl dLck-iCre mice (n = 4) and Bcl11bfl/fl control mice (n = 4). g Box and whisker plots of normalized counts for Gfi1 and Zbtb7b mRNA from RNA-Seq on IL-4+-enriched CD4+ T-cells from the lung parenchyma of asthma-induced Bcl11bfl/fl dLck-iCre mice (n = 4) and Bcl11bfl/fl control mice (n = 4). Normalized counts and statistical analyses were taken from DESeq2 analysis software. p values have been adjusted for the FDR (false discovery rate) by the Benjamini–Hochberg adjustment. All error bars shown are of standard deviation

Fig 3: The in vitro minigene assay of the c.427 + 1G > A variant and the overexpression assay of the c.2461_2462insGAGCCACACCGGCG variant in HEK293T cells. (A) The identification of a shorter transcript with 286 bp deletion of the Exon 2 downstream, attributing to the activation of a novel cryptic 5'donor splice site in Exon 2. (B) A schematic diagram of the abnormal splice and Sanger sequencing of cDNA from transfected cells. (C) Quantitative analysis of mRNA expression of WT and frameshift mutant plasmids. (D) Western blot analysis of WT and frameshift mutant protein. (E) Quantitative analysis of protein expression of WT and frameshift mutant plasmids. (F) Fluorescent image (200px) of wild-type and mutant BCL11B-EGFP in HEK293T cells. Both wild-type and mutant types could be expressed, and there was no significant change in cell morphology and localization. M, DNA marker; WT, wild type; MT, mutant type.

Fig 4: Bcl11b-deficient Treg cells of Bcl11b/Treg KO mosaic female mice have decreased suppression markers at steady state.(A, C, and E) Representative histograms and average frequencies of CD25+ (A), CTLA4+ (C), and GITR+ (E) YFP+Foxp3+ (KO) and YFP-Foxp3+ (WT) Treg cells within the same Bcl11bF/F Foxp3YFP-Cre+/- (Bcl11b/Treg KO mosaic) female mice. (B, D, and F) Mean fluorescence intensities (MFIs) of surface CD25 (B), CTLA4 (D), and GITR (F) on YFP+Foxp3+ (KO) and YFP-Foxp3+ (WT) Treg cells of Bcl11b/Treg KO mosaic female mice at steady state (n = 6). (G) Representative histogram and average frequencies of Helios in YFP+Foxp3+ (KO) and YFP-Foxp3+ (WT) Treg cells from peLNs of Bcl11b/Treg KO mosaic female mice at steady state. (H) MFI of Helios in YFP+Foxp3+ (KO) and YFP-Foxp3+ (WT) Treg cells from peLNs of Bcl11b/Treg KO mosaic female mice (n = 4). Analysis was conducted on YFP+Foxp3+ (KO) and YFP-Foxp3+ (WT) Treg cells of Bcl11b/Treg KO mosaic female mice (A to H). (I) Representative contour plots depicting the YFP+Foxp3+ (KO) and YFP-Foxp3+ (WT) Treg cells in Bcl11bF/FFoxp3YFP-Cre+/- (Bcl11b/Treg KO mosaic) and Bcl11bF/WT Foxp3YFP-Cre+/- (HT mosaic) female mice. (J) Frequencies and absolute numbers of YFP+ Treg cells in Bcl11b/Treg KO mosaic and HT mosaic female mice. (K) Foxp3 and YFP MFIs in YFP+Foxp3+ Treg cells from Bcl11b/Treg KO mosaic and HT mosaic female mice (n = 7). Analysis was conducted on YFP+ Treg cells from Bcl11b/Treg KO mosaic and HT mosaic female mice (J and K). P values determined by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: A schematic diagram of primer design for a minigene assay of the BCL11B gene. The upstream and the downstream of Intron 1 and Intron 2 were retained, respectively, and the intermediate sequences were removed (NG_027894.1: from g.5780 to g.18155 of Intron 1; from g.20001 to g.44443 of Intron 2).