Fig 1: B cell–specific Ptpn2 deficiency increases JAK/STAT and TLR7 signaling and ABC differentiation.(A) In 24-week-old mice phosphorylation of STAT1 pY701 after IL-21 stimulation, with IL-21 receptor expression. Data shown are gated on B220+ cells (n = 5). (B) Western blot and quantification of STAT3 phosphorylation (pY705) after IL-21 stimulation. pSTAT3 pY705 level was normalized to total STAT3 (n = 5–7). (C) Phosphorylation of STAT1 pY701 after IFN-γ stimulation, with IFN-γ receptor expression. Data shown are gated on B220+ cells (n = 5). (D) CD69 and CD86 expression after R848 (TLR7) stimulation at indicated doses (n = 3). (E) ABC frequency (B220+, CD93–, CD21–, CD11c+) after 48 hours in vitro stimulation (n = 3). All experiments utilized 24-week-old mice. Data are shown as mean ± SEM and are representative of ≥ 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, 2-way ANOVA.
Fig 2: IL-21 and IL-18 play accessory roles in facilitating trNK and cNK activation, respectively(A) Flow cytometric analysis of IL-21R expression on trNK and cNK from liver, uterus, and SGs. The same gating strategy as Figure S1A is used.(B) Statistical calculation of IL-21 expression level on trNK and cNK from liver, uterus, and SGs, based on its mean fluorescence intensity (MFI) in (A) (n = 4 per group).(C) Flow cytometric analysis of IL-18R1 expression on trNK and cNK from liver, uterus, and SGs. The same gating strategy as Figure S1A is used.(D) Statistical calculation of IL-18R1 expression level on trNK and cNK from liver, uterus, and SGs, based on its mean fluorescence intensity (MFI) in (C) (n = 4 per group).(E) Flow cytometric analysis of IFN-γ and TNF-α expression in liver trNK and cNK kept unstimulated or stimulated by the indicated conditions.(F) Statistical calculation of IFN-γ and TNF-α expression changes in liver trNK and cNK under the indicated conditions, and relative to PMA/ionomycin stimulation alone induced IFN-γ and TNF-α expression changes in trNK and cNK, respectively (n = 4 per group). Data are shown as the mean ± SEM. P. values are calculated by unpaired t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Data are representative of at least three independent experiments.
Fig 3: Additional IL-21 stimulation enhances the activation features of trNK(A) Workflow of the RNA-seq analysis of ex vivo stimulated liver trNK and cNK. Sorting strategy is the same as Figure S4A.(B) Pearson correlation between trNK and cNK under the indicated stimulatory conditions, using trNK and cNK genesets.(C) Pearson correlation of trNK and cNK under the indicated stimulatory conditions according to their whole transcriptomes.(D) PCA of trNK (left) and cNK (right) under the indicated stimulatory conditions (n = 2 per group). Dashed arrows in purple and blue respectively indicate additional IL-21 (purple) and IL-18 (blue) stimulation induced changes to PMA/ionomycin-activated trNK (right) and cNK (left).(E) Heatmap profiling of upregulated (left) and downregulated (right) genes by additional IL-21 stimulation in trNK. The genes are screened based on, (1) reaching to the highest or lowest expression in trNK stimulated by additional IL-21, and (2) exhibiting at least 2-fold changes compared to either unstimulated or PMA/ionomycin-stimulated trNK.(F) Go analysis of the upregulated (top) and downregulated (bottom) genes in trNK by additional IL-21 stimulation in (E).
Fig 4: Bifunctional transcription factors are involved in mediating the transcriptome change in trNK induced by the additional IL-21 stimulation(A) Workflow of the ATAC-Seq analysis of ex vivo stimulated liver trNK.(B) Schematics showing screening of concordant ChARs for the upregulated or downregulated genes by additional IL-21 stimulation in trNK, and their related genes.(C) Heatmap profiling of concordant ChARs for the upregulated (left) or downregulated (right) genes by additional IL-21 stimulation in trNK.(D) Pie charts showing proportions of the concordant ChAR-related genes in upregulated (top) or downregulated (bottom) genes by additional IL-21 stimulation in trNK.(E) Go analysis of the concordant ChAR-related genes upregulated (top) or downregulated (bottom) by additional IL-21 stimulation in trNK.(F) Scatterplots of potential transcription factors for the concordant ChAR-related genes upregulated (left) or downregulated (right) by additional IL-21 stimulation in trNK, showing both sufficient expression and substantial regulatory roles.(G) Profiling of each potential transcription factor in (F), showing the percentage of their potentially regulated genes within the upregulated or downregulated genes by additional IL-21 stimulation in trNK in trNK (left) and their expression in trNK under the indicated stimulatory conditions (right).
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