Journal: iScience
Article Title: PTEN acts as a crucial inflammatory checkpoint controlling TLR9/IL-6 axis in B cells
doi: 10.1016/j.isci.2024.110388
Figure Lengend Snippet: Loss of PTEN in B2 B cells promotes TLR9-mediated IL-6 production (A) Overlaid curves showing the expression levels of IL-6, IL-9, IL-17, CCL5, G-CSF, IL-4, IL-10, IFN-γ, and TNF-α in splenic B cells from the indicated mice ( n = 3 male mice/group) at 25–33 weeks was determined by flow cytometry. Cells were left unstimulated or stimulated with CpG, LPS, P+I, CpG+P+I, or LPS+P+I, in vitro for 24 h. (B) FACS profiles of IL-6 versus CD19 in splenic B cells from the indicated mice ( n = 9 male mice/group) at 10 w. (C) Percentages of IL-6 + B cells in splenic B cells as described in (B). (D) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells, which were left unstimulated (resting) or stimulated with CpG, CL307, LPS, and poly(I:C) in vitro for 24 h, as determined by ELISA. Each group containing 3 male mice. (E) Histograms showing IL-6 produced by FO (left), MZ (middle), and B1a (right) B cells from the indicated mice that were left untreated (resting) or stimulated with CpG in vitro for 24 h, as determined by ELISA. Each group containing 4 male mice. (F) Histograms showing IL-6 produced by FO (left) and MZ (right) B cells, which were left untreated (resting) or stimulated with CpG in the absence or presence of inhibitors (inh.) targeting NF-κB p65 or p50, respectively, for 24 h, as determined by ELISA. Each group containing 4 male mice. (G) Kaplan-Meier curves of female CD23-control mice (black line, n = 16), female CD23-cKO mice (red line, n = 24), and female CD23-cKO/IL-6 −/− double knockout mice (blue line, n = 19). The samples were compared using an unpaired two-tailed t test; ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.0005, and the data are presented as mean ± SEM.
Article Snippet: BV421 Rat Anti-Mouse IL-4 (clone 11B11) , BD Biosciences , Cat#562915; RRID: AB_2737889.
Techniques: Expressing, Flow Cytometry, In Vitro, Produced, Enzyme-linked Immunosorbent Assay, Control, Double Knockout, Two Tailed Test
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![( A and B ) Overall survival of NSCLC (left, total cases; right, tumor size ≥ 5 cm) or CRC (left, total cases; right, pT3 microsatellite-stable). ( C to K ) Analysis of publicly available scRNA-seq data on the TILs in human NSCLCs and CRCs. (C) and (D) Uniform manifold approximation projection (UMAP) visualization of the TILs in the NSCLCs and CRCs. (E) and (F) Volcano plots comparing FOXP3 low CD4 + and FOXP3 high CD4 + TILs in the NSCLCs and CRCs. (G) and (H) Heatmap showing the gene set variation analysis (GSVA) scores in the TILs from the NSCLCs and CRCs. H, Hallmark; K, Kyoto Encyclopedia of Genes and Genomes; G, Gene Ontology; R, Reactome. TCA, tricarboxylic acid. (I) and (J) Heatmap showing the mitochondrial biogenesis-related gene expressions in the NSCLCs and CRCs. (K) Venn diagram showing down-regulated genes in the heatmap. ( L to S ) Flow cytometric analysis of the CD4 + T cell subpopulations in human NSCLCs. (L) and (M) Five fractions were defined ( n = 27). (N) Frequencies of <t>IFN-γ</t> + ( n = 38), IL-4 + ( n = 52), or IL-17A + ( n = 59) cells. (O) and (P) Mitochondrial biogenesis in FOXP3 low and FOXP3 high CD4 + T cells ( n = 18). MFI, mean fluorescence intensity. (Q) and (R) CRIF1 expression in FOXP3 low and FOXP3 high CD4 + T cells ( n = 22). (S) Correlation between CRIF1 and FOXP3 expression in CD4 + T cells (NSCLCs, n = 16; CRCs, n = 18). Dots represent individual cases. The data are pooled from at least three independent experiments and are presented as means ± SEM of biological replicates. Flow cytometry plots are representative of at least two independent experiments. ns, not significant; *** P < 0.001 and **** P < 0.0001. Statistical testing was conducted with the Kaplan-Meier method and the log-rank test (A), Wilcoxon test [(N), (P), and (R)], and Pearson correlation test (S).](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1410/pmc10971410/pmc10971410__sciadv.adj9600-f1.jpg)
![Comparison of Crif1 fl/fl Foxp3 YFP-Cre and control Foxp3 YFP-Cre mice. ( A to H ) Foxp3-YFP + T reg phenotypes. (A) and (B) T reg frequencies in the splenic and LN CD4 + T cell population [left two plots in (B)] and absolute Treg numbers [right two plots in (B)]. (C) Foxp3-YFP expression intensity in splenic and LN Foxp3-YFP + T regs . (D) and (E) Ability of T regs to suppress effector T cell proliferation. (F) and (G) Frequencies of splenic T regs that spontaneously produce IFN-γ, IL-4, and IL-17A. (H) Cytokine production of T regs that underwent TCR stimulation for 2 days, as shown by enzyme-linked immunosorbent assay of the supernatants. ( I to N ) Adoptive transfer of Foxp3-YFP + T regs into Rag1- KO mice that did or did not lack Ifng and IL4 expression. (I) Schematic depiction of the experiment. (J) Foxp3-YFP expression intensity in the total CD4 + T cells in the spleen and LNs. (K) Frequencies of T regs in the spleen and LNs that produced IFN-γ, IL-4, or IL-17A. (L) Gross appearance of the spleen and LNs. (M) Absolute cell numbers in the spleen and LNs. (N) Representative histological images of the ear skin, lung, and liver. Scale bars, 200 μm (for the lungs) and 100 μm (for the ear skin and liver). Dots represent individual mice ( n = 4 to 13 per group). The data are pooled from at least two independent experiments and are presented as means ± SEM of biological replicates. Gross and histological images and flow cytometry plots are representative of at least two independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Statistical testing was conducted with an unpaired two-tailed t test [(B), (C), (E), (G), and (H)], and one-way analysis of variance (ANOVA) [(J), (K), and (M)]. i.v., intravenous.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1410/pmc10971410/pmc10971410__sciadv.adj9600-f2.jpg)
![Foxp3-YFP + T regs from Foxp3 YFP -Cre and Crif1 fl/fl Foxp3 YFP - Cre mice were assessed. ( A to E ) Integration of RNA-seq and ATAC-seq data of TCR-stimulated and unstimulated T regs . (A) Left: Venn diagram showing the number of ATAC peaks that corresponded to genes that were up-regulated (top) or down-regulated (bottom) in the TCR-stimulated CRIF1-deficient (red) and control (blue) T regs . (Right) Gene Ontology analysis. The size and color density of circles represent the number of genes and P values, respectively. (B) and (C) ATAC-seq peaks in and around the Ifng (B) and (C) Il4 genes in TCR-stimulated and unstimulated T regs . (D) and (E) Modeling of the binding of transcription factors to the Ifng (D) and Il4 (E) genes. ( F to H ) Relationship between EOMES/SATB1 and IFN-γ/IL-4 expression. (F) Flow cytometry of EOMES and SATB1 expression in TCR-stimulated and unstimulated T regs . Rapamycin was added to some TCR-stimulated T regs . Dashed line indicates immunoglobulin G (IgG) control. (G) ChIP-qPCR analysis of the binding of EOMES and SATB1 to the Ifng and Il4 regulatory regions in TCR-stimulated T regs . Rapamycin was added to some cells. (H) Flow cytometry analysis of the cytokine production of TCR-stimulated T regs after Eomes or Satb1 were knocked down. The data are presented as means ± SEM of biological replicates and are representative of at least two independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Statistical testing was conducted with two-way ANOVA [(F) to (H)].](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1410/pmc10971410/pmc10971410__sciadv.adj9600-f6.jpg)
![( A to E ) Solid tumors were induced in WT mice with TC-1 tumor cells and the infiltrating CD4 + T cells were subjected to flow cytometry. (A) Foxp3 expression of CD4 + T cells. (B) and (C) Mitochondrial biogenesis. (D) and (E) CRIF1 expression. ( F to P ) Solid tumors were induced in Foxp3 EGFP-cre-ERT2 and Crif1 fl/fl Foxp3 EGFP-cre-ERT2 mice with TC-1 cells and the mice were treated intraperitoneally with tamoxifen. (F) Treatment schedule. (G) and (H) Foxp3 expression intensity of the tumor-infiltrating T regs . (I) and (J) Cytokine production of the tumor-infiltrating T regs . (K) and (L) Foxp3 expression intensity (K) and cytokine production (L) of splenic and LN T regs . (M) Tumor volume. (N) Tumor weight at day 17. (O) Gross tumor images at day 17. (P) Flow cytometric frequencies of immune cells in the TC-1 tumor model. Myeloid cells (left) and lymphoid cells (right). Myeloid and lymphoid cells are gated from live CD45 + cells and lymphoid cells, respectively. ( Q ) Cytokine production of tumor-infiltrating CD4 + and CD8 + T cells. ( R to V ) Solid tumors were induced in Foxp3 EGFP-cre-ERT2 and Crif1 fl/fl Foxp3 EGFP-cre-ERT2 mice with TC-1 cells and the mice were treated intraperitoneally with tamoxifen with or without blocking anti–IFN-γ, anti–IL-4, or isotype-control antibodies. (R) Tumor volume. (S) Tumor weight at day 17. (T) Gross tumor images at day 17. (U) and (V) Cytokine production of tumor-infiltrating T regs (T) and CD4 + and CD8 + T cells (U). Dots represent individual mice ( n = 5 to 16 per group). The data are pooled from at least two independent experiments and are presented as means ± SEM of biological replicates. Flow cytometry plots are representative of at least two independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Statistical testing was conducted with Wilcoxon test [(C) and (E)], an unpaired two-tailed t test [(H) to (Q)], and one-way ANOVA [(R), (S), (U), and (V)]. PMN, polymorphonuclear.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1410/pmc10971410/pmc10971410__sciadv.adj9600-f7.jpg)