anti batf  (Cell Signaling Technology Inc)

Journal: Neoplasia (New York, N.Y.)

Article Title: OSMR coordinates a self-perpetuating circuit linking chemoresistance and neutrophil-driven immunosuppression in gastric cancer

doi: 10.1016/j.neo.2026.101279

Figure Lengend Snippet: Tumor induced PD-L1 expression in neutrophils enhances IL31 secretion of CD8 + T cells. (A) IL31 and OSM mRNA level in CD8 + T cells after co-cultured neutrophils and GC cells. (B) TIM-3, PD-1 and IL31 mRNA level in CD8 + T cells treated with IL2 and Anti-CD3/CD28 antibody. (C) IL31 mRNA and protein level in CD8 + T cells co-cultured with neutrophils treated with BMP5 and PD-L1 inhibitor (Atezolizumab). (D) Schematic of PD-1/PD-L1 signaling pathway in CD8 + T cells. (E) BATF mRNA and protein level in CD8 + T cells treated with IL2 and Anti-CD3/CD28 antibody for 0 day and 14 days. (F) BATF mRNA and protein level in CD8 + T cells after co-cultured neutrophils and GC cells. (G) IL31 mRNA and protein level in CD8 + T cells when BATF was up-regulated or down-regulated. (H) Dual-Luciferase Reporter Assay analysis for BATF-binding IL31 promoter. (I) p-PI3K, PI3K, p-AKT, AKT, p-P27, P27, CCNE2, PD-L1, and PD-L2 expression in MKN28 cells treated with supernatant of CD8 + T cells (J) mIF staining analysis of correlation of OSMR, CD15, CD8, and IL31 in tumor samples from patients in Cohort 2. (**, P < 0.01).

Article Snippet: The membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, followed by an overnight incubation at 4°C with primary antibodies (OSMR (Abcam, #ab315388), CCNE2 (Abcam, #ab40890), β-actin (CST, #4967), PI3K (P85) (CST, #4292), p-PI3K (p-P85) (Abcam, #ab278545), AKT (CST, #9272), p-AKT (CST, #4060), P27 (CST, #2552), p-P27 (Abcam, #ab62364), FLAG (CST, #14793), BMP5 (Proteintech, #13253-1-AP), Ub (CST, #20326), TRIM2 (Proteintech, #20356-1-AP), GATA3 (CST, #5852), p-SMAD1/5 (CST, #9516), SMAD5 (CST, #12534), PD-L1 (CST, #13684), BATF (CST, #8638), IL-31 (abcam, #ab62579)), which were diluted in accordance with the manufacturer's instructions.

Techniques: Expressing, Cell Culture, Luciferase, Reporter Assay, Binding Assay, Staining

Journal: Nature Communications

Article Title: Chromatin remodeling enhances MAP3K8 expression in HAM: a key pathogenesis for therapeutic intervention

doi: 10.1038/s41467-025-64836-7

Figure Lengend Snippet: A Fosl2 and HTLV-1 Tax enrichment (ChIP-seq) in HCT-4 cells and chromatin accessibility (ATAC-seq) in HTLV-1-infected CD4 + T cells in patients with HAM, highlighting the HAM-characteristic OCR. Genomic DNA from HCT-4 cells prior to immunoprecipitation served as input control. ChIP-seq and ATAC-seq peaks were visualized with IGV (data range: ChIP-seq, 0-30; ATAC-seq, 0-100). B Enrichment analysis of transcription factor motifs in ChIP-seq with Fosl2 using the HOMER algorithm. Motifs are ranked by -log10(P). Statistical analysis was performed using a one-sided hypergeometric test. Nominal P values are reported. C Enhancer assays using the MAP3K8 promoter and HAM-characteristic OCR at the MAP3K8 locus. Relative luciferase activity for Fosl2 with partner factors (c-Jun, JunB, BATF, and BATF3) was examined. Data are presented as mean values + SD. Statistical significance was assessed by one-way ANOVA followed by two-sided Tukey’s honestly significant difference (HSD) test for multiple comparisons. Asterisks indicate statistical significance (*** P < 0.001). n = 3, 4, or 5 biologically independent experiments, each performed on separate days using independently transfected HEK293 cells. D Enhancer assays using the MAP3K8 promoter and HAM-characteristic OCR, with Fosl2, c-Jun, and HTLV-1 Tax. Yellow arrows indicate OCR orientation (5’ → 3’ or 3’ → 5’). Data are presented as mean values + SD. Statistical significance was assessed by one-way ANOVA followed by two-sided Tukey’s HSD test for multiple comparisons. Exact P value are as follows: MAP3K8 promoter with Fosl2, c-Jun, and HTLV-1 Tax vs MAP3K8 promoter and HAM-characteristic OCR (5’ → 3’) with Fosl2, c-Jun, and HTLV-1 Tax, P = 0.000010, MAP3K8 promoter with Fosl2, c-Jun, and HTLV-1 Tax vs MAP3K8 promoter and HAM-characteristic OCR (3’ → 5’) with Fosl2, c-Jun, and HTLV-1 Tax, P = 0.000021. Asterisks indicate statistical significance (*** P < 0.001). n = 4 or 7 biologically independent experiments, each performed on separate days using independently transfected HEK293 cells. E Representative histograms depicting chromatin accessibility at the MAP3K8 locus, as determined by ATAC-seq analysis, across various CD4⁺ T cell populations: resting normal CD4⁺ T cells, HAM_D subset, CD4⁺ T cells transiently expressing HTLV-1 Tax for 7 days via a lentiviral system (Tax-cell), and CD4⁺ T cells activated with anti-CD3/CD28 antibodies ( GSM5554060 ). The highlighted region shows HAM-characteristic OCR. ATAC-seq peaks were visualized with IGV (data range: 0-100).

Article Snippet: Anti-c-Jun antibody (9165S, Cell Signaling Technology) Anti-JunB antibody (sc-8051, Santa Cruz Biotechnology) Anti-BATF antibody (8638 T, Cell Signaling Technology) Anti-BATF3 antibody (GTX130125, Gene Tex) Anti-Tax antibody (LT-4, kindly gifted by Dr. Yuetsu Tanaka) Anti-Rabbit IgG (whole molecule)-Peroxidase antibody (A9169, Sigma-Aldrich) Anti-Mouse IgG (whoke molecule)-Peroxidase antibody (A5278, Sima-Aldrich) ECL Prime Western Blotting Detection Reagent (Cytiva, Little Chalfront, England)

Techniques: ChIP-sequencing, Infection, Immunoprecipitation, Control, Luciferase, Activity Assay, Transfection, Expressing

Journal: iScience

Article Title: An interleukin-27-centered cytokine circuit regulates macrophage and T cell interactions in autoimmune diabetes

doi: 10.1016/j.isci.2025.113537

Figure Lengend Snippet: IL-27 signaling in CD4 T cells impacts CD8 T cells (A) Schematic diagram of CD4 and CD8 T cell co-transfer into NOD. Rag1 −/− recipients for analyzing Venus (IL-21)-expressing CD4 T cells. (B and C) IL-27 signaling in CD4 T cells promotes their IL-21 expression. Islet-infiltrating CD4 T cells were analyzed by flow cytometry for Venus (IL-21) expression at 8–9 weeks post-transfer. (B) Representative flow cytometry profiles of Venus (IL-21) and CXCR6 expression. (C) Summarized percentages of Venus (IL-21) + cells among CD4 T cells (upper) and CXCR6 + cells among Venus (IL-21) + CD4 T cells (lower). Combined results from two independent experiments are shown ( n = 5 per group). ∗∗ p < 0.01, unpaired t test. (D) Schematic diagram of CD4 and CD8 T cell co-transfer into NOD. Rag1 −/− recipients for analyzing the phenotype of CD8 T cells. (E) Representative histograms showing BATF expression in CD44 high CD8 T cells in the spleens (dotted lines) and pancreatic islets (shaded areas). gMFI, geometric mean fluorescent intensity. (F) Summarized BATF gMFI of spleen and islet CD44 high CD8 T cells. Combined results from two independent transfer experiments are shown (n = 6–7 per group). ∗∗∗ p < 0.005, unpaired t test. (G) The BATF regulon activity in IGRP 206-241 -specific CD8 T cells isolated from pancreatic islets and spleens. The BATF regulon activity was computed by SCENIC using a previously published scRNA-seq dataset ( GSE200608 ). The p value is determined by Wilcoxon test. (H and I) IFNγ and TNFα expression in islet-infiltrating CD8 T cells co-transferred with WT or Il27ra −/− CD4 T cells. (H) Representative flow cytometry profiles of IFNγ and TNFα expression. (I) Summarized percentages of IFNγ + (left) and TNFα + (right) CD44 high CD8 T cells. Combined results from two independent transfer experiments are shown (n = 5–6 per group). (J) Granzyme B expression in islet-infiltrating CD8 T cells co-transferred with WT or Il27ra −/− CD4 T cells. Representative histograms of granzyme B expression (left). Summarized relative granzyme B expression (right). Granzyme B gMFI of CD44 high CXCR6 + CD8 T cells co-transferred with Il27ra −/− CD4 T cells is normalized to those co-transferred with WT CD4 T cells in each experiment. Combined results from three independent transfer experiments are shown ( n = 10 per group). ∗ p < 0.05, unpaired t test.

Article Snippet: Anti-BATF (D7C5) antibodies were from Cell Signaling Technology.

Techniques: Expressing, Flow Cytometry, Activity Assay, Isolation

Journal: iScience

Article Title: An interleukin-27-centered cytokine circuit regulates macrophage and T cell interactions in autoimmune diabetes

doi: 10.1016/j.isci.2025.113537

Figure Lengend Snippet: IL-21 controls the effector functions of CD8 T cells (A) Schematic diagram of generating MBMCs. (B) Representative flow cytometry profiles showing the gating strategy for analyzing BATF expression in CD44 high CD8 T cells. Cells were stained with purified rabbit anti-mouse BATF primary antibody followed by fluorochrome-conjugated anti-rabbit IgG secondary antibody. The solid lines and shaded areas in the histograms, respectively, represent staining without and with the primary antibody. WT, wild type. gMFI: geometric mean fluorescent intensity. (C) Summarized BATF gMFI of spleen and islet CD44 high CD8 T cells. Combined results from two independent experiments are shown ( n = 6 per group). ∗ p < 0.05, paired t test. (D and E) The ability to produce IFNγ and TNFα is reduced in islet-infiltrating CD8 T cells in the absence of IL-21 signaling. (D) Representative flow cytometry profiles of IFNγ and TNFα expression. (E) Summarized percentages of IFNγ + (left) and TNFα + (right) CD44 high CD8 T cells. Combined results from three to four independent experiments are shown (n = 9–12 per group). ∗ p < 0.05, ∗∗ p < 0.01, paired t test. (F) Granzyme B expression in islet-infiltrating CD8 T cells is reduced in the absence of IL-21 signaling. Representative histogram of granzyme B expression (left). Summarized relative granzyme B expression (right). Granzyme B gMFI of Il21r −/− CD44 high CXCR6 + CD8 T cells is normalized to that of the WT counterpart in each MBMC experiment. Combined results from four independent experiments are shown ( n = 12 per group). ∗∗∗∗ p < 0.0001, paired t test. (G and H) Forced expression of BATF rescues the diabetogenic activity of CD8 T cells in the absence of CD4 T cell-derived IL-21. NY8.3 CD8 T cells were activated in vitro and transduced with either empty MIG or MIG-BATF retroviral vector (see ). A mixture of 5 × 10 6 transduced NY8.3 CD8 T cells along with 5 × 10 6 WT or Il21 −/− NOD CD4 T cells was transferred into NOD. Rag1 −/− female recipients. Mice were monitored for diabetes development. (G) The percentages of mice that developed diabetes over time are shown. Data are combined from two independent experiments with eight to nine total mice per group. ∗ p < 0.05, ∗∗∗ p < 0.001 by log rank test. (H) Representative islet images and the summary of insulitis in mice that remained nondiabetic at termination of the incidence study. Arrows indicate infiltrating immunocytes. Scale bars, 100 μm. ∗∗ p < 0.005, unpaired t test.

Article Snippet: Anti-BATF (D7C5) antibodies were from Cell Signaling Technology.

Techniques: Flow Cytometry, Expressing, Staining, Purification, Activity Assay, Derivative Assay, In Vitro, Transduction, Retroviral, Plasmid Preparation