Journal: Mucosal immunology

Article Title: A role for BATF3 in T H 9 differentiation and T-cell-driven mucosal pathologies.

doi: 10.1038/s41385-018-0122-4

Figure Lengend Snippet: Fig. 2 TL1A upregulates BATF and BATF3 expression during TH9 differentiation. a Transcriptional profiling of TH9 and TH9-TL1A cells by RNA sequencing. Heat map displaying RNA-sequencing data using the top 100 genes with the largest IQR (interquartile range). The dendrograms to the left and above the heat map represent clustering of genes (rows) and samples (columns). b Naive CD4+ T cells were differentiated into different TH subsets and Tregs for 3 days. Relative Batf mRNA expression was analyzed by qPCR. c Naive CD4+ T cells were differentiated under TH0- or TH9-polarizing conditions with or without TL1A for the indicated time periods. QPCR analysis of relative Batf mRNA expression. d Co- staining of BATF and IRF4 (top). Quantitative analysis of the percentages of BATF+IRF4+ cells (bottom). e Co-staining of BATF and IL-9. f–g Naive human CD4+ T cells were isolated from PBMCs of healthy volunteers and differentiated under TH9-polarizing conditions with or without TL1A. f Representative intracellular staining of IL-9 and BATF at 48 h. g Frequency of BATF+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual donor. N = 4. h Relative Batf3 mRNA expression in different murine TH subsets and Tregs was analyzed by qPCR. i Relative Batf3 mRNA expression at indicated time points. j Representative co-staining of BATF3 and IL-9. k Frequency of BATF3+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual experiment. N = 3. l–m Naive human CD4+ T cells were differentiated under TH9-polarizing conditions with or without TL1A. l Representative intracellular staining of IL-9 and BATF3 at 48 h. m Frequency of BATF3+IL-9+ T cells differentiated under TH9 and TH9-TL1A conditions. Means are shown. Each symbol represents an individual donor. N = 4. Data represent means ± SD of one independent experiment out of two (a) or at least three (b–d, i, k) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005 as determined by Student’s t test

Article Snippet: Intracellular staining Cells were restimulated with 50 ng/ml PMA (phorbol 12-myristate 13-acetate), 500 ng/ml ionomycin, and monensin (eBioscience) for 4 h, stained with anti-CD4 (RM4-5, eBioscience), fixed and permeabilized using the FoxP3 staining buffer set (eBioscience), and stained with antibodies against murine IL-9 (RM9A4, BioLegend), IL-10 (JES5-16E3), IL-13 (13A), IL-17A (eBio17B7), IL-17F (eBio18F10), IFN-γ (XMG1.2), IL-4 (BVD6-24G2), IL-22 (1H8PWSR), Ki67 (SolA15), BATF (MBM7C7), IRF4 (3E4), human IL-9 (MH9A4, all eBioscience), and BATF3 (841792, R&D Systems).

Techniques: Expressing, RNA Sequencing, Staining, Isolation

Journal: Mucosal immunology

Article Title: A role for BATF3 in T H 9 differentiation and T-cell-driven mucosal pathologies.

doi: 10.1038/s41385-018-0122-4

Figure Lengend Snippet: Fig. 4 Effects of BATF and BATF3 deficiencies on TH9 differentiation. a–d Naive CD4+ T cells from Batf−/−or WT mice were differentiated under TH0- or TH9-polarizing conditions with or without TL1A for 3 days. a Intracellular staining of IL-9 and IL-10. b ELISA analysis of IL-9 production. c ELISA analysis of IL-10 production. d ELISA analysis of IL-13 production. e–h Naive CD4+ T cells from Batf3−/−or WT mice were differentiated under TH9-polarizing conditions with or without TL1A for 3 days. e Intracellular staining of IL-9 and IL-10. f ELISA analysis of IL-9 production. g ELISA analysis of IL-10 production. h ELISA analysis of IL-13 production. i Transcriptional profiling of WT and Batf3−/−TH9-TL1A cells by RNA sequencing. Heat map displaying RNA-sequencing data of genes differentially expressed with p < 0.01. The dendrograms to the left and above the heat map represent clustering of genes (rows) and samples (columns). Data represent means ± SD of one independent experiment out of two (i) or three (a–h) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005 determined by Student’s t test

Article Snippet: Intracellular staining Cells were restimulated with 50 ng/ml PMA (phorbol 12-myristate 13-acetate), 500 ng/ml ionomycin, and monensin (eBioscience) for 4 h, stained with anti-CD4 (RM4-5, eBioscience), fixed and permeabilized using the FoxP3 staining buffer set (eBioscience), and stained with antibodies against murine IL-9 (RM9A4, BioLegend), IL-10 (JES5-16E3), IL-13 (13A), IL-17A (eBio17B7), IL-17F (eBio18F10), IFN-γ (XMG1.2), IL-4 (BVD6-24G2), IL-22 (1H8PWSR), Ki67 (SolA15), BATF (MBM7C7), IRF4 (3E4), human IL-9 (MH9A4, all eBioscience), and BATF3 (841792, R&D Systems).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, RNA Sequencing

Journal: Immunity

Article Title: Negative costimulation constrains T cell differentiation by imposing boundaries on possible cell states

doi: 10.1016/j.immuni.2019.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-BATF 154Sm , Fluidigm , Cat #:3154012A; Clone:D7C5.

Techniques: Virus, Recombinant, Adjuvant, Polymer, Conjugation Assay, Staining, Mass Cytometry, Sequencing, Transgenic Assay, Double Knockout, Software

Journal: Scientific Reports

Article Title: Immune system development-related signature predicts prognosis and sorafenib-treatment resistance of hepatocellular carcinoma by intergrating machine learning and single-cell analyses

doi: 10.1038/s41598-025-93187-y

Figure Lengend Snippet: BATF in relation to prognosis and sorafenib response in MCHH cohort. ( A ) Boxplot shows the distribution of BATF expression between resistant and sensitive samples. ( B , C ) IHC images and BATF-score based on IHC images showing the distribution of BATF expression between sorafenib resistant and sensitive samples (Red arrows: BATF + immune cells; black arrows: BATF- immune cells). ( D ) Paired plot shows the distribution of BATF expression between HCC and paracancer samples. ( E , F ) IHC images and BATF-score based on IHC images shows the distribution of BATF expression between HCC and paracancer samples (Black arrows: immune cells). ( G ) Kaplan-Meier curves of OS with high- and low-BATF expression groups. ( H ) Time-dependent ROC curve of BATF expression analysis for predicting OS at 1-, 2-, and 3-years. ( I ). Univariate and multivariate Cox regression analysis of BATF expression in HCC samples. ( J ) Kaplan-Meier curves of OS with high- and low-BATF score groups. ( K ) Time-dependent ROC curve of BATF-score analysis for predicting OS at 1-, 2-, and 3-years. ( M ) Univariate and multivariate Cox regression analysis of BATF-score expression in HCC samples. MCHH: Mengchao Hepatobiliary Hospital; IHC: Immunohistochemistry; HCC: Hepatocellular carcinoma; OS: Overall survival; ROC: Receiver operating characteristic; *: P < 0.05; **: P < 0.01.

Article Snippet: IHC staining were performed using the antibodies against BATF (1:15000, CSB-PA619074LA01HU, CUSABIO) as described previously.

Techniques: Expressing, Immunohistochemistry

Journal: Mucosal immunology

Article Title: Protein Kinase 2 (CK2) Controls CD4 + T-cell Effector Function in the Pathogenesis of Colitis

doi: 10.1038/s41385-020-0258-x

Figure Lengend Snippet: RNA sequencing of CK2α fl/fl CD4 + T-cells (WT) and CK2α fl/fl dLck-Cre CD4 + T-cells (KO) from the colon at 6 weeks after colitis induction was performed (three to five mice were combined per experiment, two experiments in each group). (A) Summary of genes differentially regulated by CK2α using the following cutoffs are shown: p < 0.05, fold change >1.5. (B) GSEA plot shows enrichment of RNA sequencing data compared with KEGG pathway dataset. (C) Heat map shows TCR signaling relative gene expression. (D) Selected gene expression was validated in CD4 + T-cells from colitis Rag1 −/− mice by qRT-PCR. CK2α fl/fl , n=5; CK2α fl/fl dLck-Cre, n=4. (E) Naive CD4 + T-cells from CK2α fl/fl and CK2α fl/fl dLck-Cre mice were activated with 0.5 μg/ml of anti-CD3 and 1.0 μg/ml of anti-CD28 Abs for 24 h, and expression of NFAT2 was detected by flow cytometry. Representative line graphs and quantitation are shown. n=3 in each group. (F) Naïve CD4 + T-cells from CK2α fl/fl and CK2α fl/fl dLck-Cre mice were activated and transduced with caNFAT2 or empty control vector. GFP + transduced cells were sorted and stimulated with 0.1 μg/ml of anti-CD3 and 1.0 μg/ml of anti-CD28 Abs for 48 h, and expression of ICOS, CD40L and BATF was detected by flow cytometry. Representative line graphs are shown. (G) Quantitation of relative MFI of ICOS, CD40L and BATF is shown. n=3 in each group. (H) GFP + cells were sorted and stimulated with 0.1 μg/ml of anti-CD3 and 1.0 μg/ml of anti-CD28 Abs for 48 h, and proliferating cells detected by Ki-67 staining. Representative line graphs and quantitation are shown. (I) Quantitation of absolute number of Ki-67 + CD4 + T-cells is shown. n=3 in each group. Bars represent the mean ± SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: The following antibodies were used in this study (all Biolegend except where noted otherwise): anti-CD3ε PerCP-Cy5.5/PE-Cy7/APC (clone 145-2C11); anti-CD4 Pacific Blue/FITC /Alexa Fluor 647/APC-Cy7/ PE (clone RM4-5); anti-CD11b FITC (clone M1-70); anti-CD11b BUV395 (clone M1-70, BD Bioscience), anti-CD45 APC-Cy7 (clone 30-F11); anti-CD45.1 Alexa Fluor 488/PerCP-Cy5.5 (clone A20); anti-CD45.2 Alexa 647/APC-Cy7 (clone 104); anti-CD45RB FITC (clone C363.16A), anti-CD25 Alexa Fluor 647/PE-Cy7 (clone PC61.5); anti-IFN-γ Pacific Blue/PE-Cy7 (clone XMG1.2); anti-IL-17A APC (clone TC11-18H10); anti-Ki-67 Brilliant Violet 421 (clone 16A8); anti-CD40L PE-Cy7 (clone 24-31); anti-ICOS APC (clone 15F9); anti-BATF PE (clone S39-1060, BD Bioscience); NFAT2 (Cell Signaling Technology); Alexa Fluor 647 Anti-Rabbit IgG (H+L) (Jackson Immuno Research Labs); anti-CD212 (IL-12Rβ1) PE (Clone 114, BD Bioscience); and anti-IL-12Rβ2 Alexa Fluor 488 (R&D System).

Techniques: RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Quantitation Assay, Transduction, Plasmid Preparation, Staining