Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: (A) Deletion of Tet2 in allograft tumors reduced chemokine Cxcl9, Cxcl10, and Pdl1 expression. Total mRNA was extracted from WT or Tet2-KO tumor (n = 10 for each group), and mRNA levels of genes were determined by qPCR. Data represent mean ± SD. *P < 0.05, **P < 0.01 by unpaired Student’s t test. (B and C) Knocking out Tet2 blocked IFN-γ–induced chemokines and Pdl1 gene expression in B16-OVA (B) and THP-1 (C) cells. WT or Tet2-KO cells were treated with IFN-γ for 20 hours, and total RNA was extracted. The relative mRNA levels were determined by qPCR. Error bars represent ± SD for triplicate experiments. (D) Knocking out Tet2 decreased IFN-γ–induced CXCL9 and CXCL10 protein levels in B16-OVA cells. WT or Tet2-KO B16-OVA cells were treated with IFN-γ for 72 hours; then medium was collected and subjected to ELISA analysis. Error bars represent ± SD for triplicate experiments. **P < 0.01, ***P < 0.001 by unpaired Student’s t test. (E) TET2 catalytic activity was required for IFN-γ–induced CXCL10 and PD-L1 expression. TET2-WT and catalytic mutant R1896S were overexpressed in TET2-KO THP-1 cells; then cells were treated with IFN-γ for 20 hours as indicated, and total RNA was extracted. The relative mRNA levels were determined by qPCR. Error bars represent ± SD for triplicate experiments. (F) Deletion of Tet2 impaired T cell attraction by Transwell assay. WT or Tet2-KO B16-OVA cells were treated with IFN-γ for 48 hours, and CM was collected. Triplicate independent experiments were performed for each group. Error bars represent ± SD. Bonferroni-adjusted **P < 0.01, with raw P value derived from unpaired Student’s t test.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Activity Assay, Mutagenesis, Transwell Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: WT or TET2-KO THP-1 cells were treated with 100 ng/mL IFN-γ for 20 hours, and total RNA was extracted and subjected to RNA-Seq. (A) Venn diagram of affected genes (stimulated 2-fold or more) in the whole transcriptome is shown. (B) Global gene expression analysis of WT and TET2-KO THP-1 cells stimulated with IFN-γ by RNA-Seq is shown. The region (P < 0.001 and fold change > 5) is highlighted by dashed lines. Red dots represent CXCL9, CXCL10, and CXCL11; green dot represents PD-L1. (C) Heatmap depiction of differentially expressed genes (fold change ≥ 5) between control and TET2-KO THP-1 cells.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: RNA Sequencing, Gene Expression, Control

Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: (A) IFN-γ promoted TET2-STAT1 binding. THP-1 cells were treated with or without IFN-γ, and the interaction of TET2 and STAT1 was determined by IP–Western blot. (B) Y701F mutation disrupted TET2-STAT1 binding. STAT1-WT and Y701 mutant plasmids were transfected into THP-1 cells and treated with IFN-γ, and their binding to TET2 was determined by IP–Western blot. (C and D) IFN-γ promoted TET2 binding to the CXCL10 (C) and PD-L1 (D) promoters. THP-1 cells were treated with or without IFN-γ, and TET2 binding to the CXCL10 (C) and PD-L1 (D) promoters was determined by TET2 ChIP-qPCR. Error bars represent ± SD for triplicate experiments. (E and F) IFN-γ increased the 5hmC level of the CXCL10 (E) and PD-L1 (F) promoters. hMeDIP assays were performed in control and TET2-KO THP-1 cells treated or untreated with IFN-γ. 5hmC levels on the CXCL10 (E) and PD-L1 (F) promoters were determined by qPCR. Error bars represent ± SD for triplicate experiments. (G and H) JAK2 inhibitor blocked binding to (G) and demethylation of (H) the CXCL10 promoter by TET2 upon IFN-γ treatment. THP-1 cells were treated with IFN-γ and JAK2 inhibitor as indicated, and the ability of TET2 to bind to (G) and demethylate (H) the CXCL10 promoter was determined by TET2 (G) or 5hmC (H) ChIP-qPCR. Error bars represent ± SD for triplicate experiments. (I and J) JAK2 inhibitor blocked binding to (I) and demethylation of (J) the PD-L1 promoter by TET2 upon IFN-γ treatment. THP-1 cells were treated with IFN-γ and JAK2 inhibitor as indicated, and the ability of TET2 to bind to (I) and demethylate (J) the PD-L1 promoter was determined by TET2 (I) or 5hmC (J) ChIP-qPCR.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: Binding Assay, Western Blot, Mutagenesis, Transfection, ChIP-qPCR, Control

Journal: The Journal of Clinical Investigation

Article Title: Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy

doi: 10.1172/JCI129317

Figure Lengend Snippet: (A) Infiltrating lymphocyte numbers including CD3+ T cells, CD8+ cytotoxic T lymphocytes (CTLs), and CD56+ NK cells were decreased along with the loss of 5hmC in adenoma of colon. Two representative pictures show multicolor, fluorescently labeled inflammatory cells in adenomas expressing high 5hmC levels and adenomas with decreased 5hmC expression, separately. Scale bars: 100 μm. (B) Quantification of CD3+ T cells, CD8+ CTLs, and CD56+ NK cells in colon adenomas classified by high and low 5hmC staining. Four cases of adenoma with high 5hmC expression and 8 cases of adenoma with low 5hmC expression were used to count cytotoxic T cells (CD3+ and CD8+) and NK cells (CD56+). For each case, 3 areas highly infiltrated with inflammatory cells were selected. **P < 0.01 by Student’s t test. Data represent mean ± SEM. (C) Intratumoral CXCL9, CXCL10, and CXCL11 levels were correlated with 5hmC levels in colon adenomas. Representative photographs show the expression of CXCL9, CXCL10, and CXCL11 in samples with high 5hmC and in samples with low 5hmC in the same fields, on serial sections in adenoma with low-grade dysplasia, adenoma with high-grade dysplasia, and adenocarcinoma specimens. Scale bars: 50 μm. (D–F) Quantification of CXCL9 (D), CXCL10 (E), and CXCL11 (F) expression classified by high and low 5hmC staining. Five samples representing cases with low 5hmC expression and 5 with high 5hmC expression were selected separately in adenoma with low-grade dysplasia, adenoma with high-grade dysplasia, and adenocarcinoma. For each case, 5 fields were randomly selected to calculate the integrated staining density by i-Solution image analysis software. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t test. Data represent mean ± SEM.

Article Snippet: The bottom well was filled with RPMI medium or conditioned medium (CM) derived from B16-OVA control or Tet2 -KO cells, with or without 100 ng/mL recombinant murine CXCL10 (R&D Systems, catalog 466-CR-010).

Techniques: Labeling, Expressing, Staining, Software

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon signaling in hematopoietic cells is required for survival in mouse polymicrobial sepsis by regulating CXCL10

doi: 10.1084/jem.20091959

Figure Lengend Snippet: Type I IFN signaling does not play a role in inflammation associated with CLP. (A) SEV129 and IFNAR −/− mice underwent CLP surgery and were sacrificed at 0, 6, and 12 h after surgery. Serum cytokine levels from peripheral blood were determined by MILLIPLEX MAP Mouse Cytokine/Chemokine–Premixed 22 Plex kits. Select cytokines in this figure include TNF, KC, IL-6, IL-1β, and IP-10. Each time point was performed once ( n = 3 per group per time point; *, P < 0.05 using the Student’s t test). Error bars indicate SD. (B) SEV129 and IFNAR −/− mice underwent CLP surgery and were sacrificed at 12, 48, and 96 h after surgery. Bacteremia was determined from peritoneal lavage fluid plated on sheep blood agar. Each point represents CFUs from one mouse. The experiment was performed three times ( n = 3 per group; P < 0.05 using the Student’s t test). Horizontal bars indicate means.

Article Snippet: For in vivo chemokine treatment, each mouse was injected i.p. with 100 ng of mouse recombinant IP-10 (R&D Systems) or PBS 6 h after surgery, and if experiments lasted longer than 3 d, again on day 3 ( ).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon signaling in hematopoietic cells is required for survival in mouse polymicrobial sepsis by regulating CXCL10

doi: 10.1084/jem.20091959

Figure Lengend Snippet: CXCL10 improves outcome by decreasing bacteremia in IFNAR −/− mice. (A) SEV129 wild-type mice ( n = 10), IFNAR −/− mice ( n = 11), or IFNAR −/− mice with 100 ng CXCL10 6 h and on day 3 after CLP ( n = 11). Survival was monitored for 7 d. There was a 50% survival advantage in SEV129 wild-type compared with IFNAR −/− mice, and a 54% survival advantage in IFNAR −/− + CXCL10 compared with IFNAR −/− mice. *, P = 0.01 using Fisher’s exact test. Data are from a single experiment with 10 mice in the SEV129 group and 11 mice in the IFNAR −/− and IFNAR −/− + CXCL10 groups. Two independent survival experiments were performed with consistent results. (B) IFNAR −/− mice underwent CLP surgery, and 6 h after surgery mice were injected with PBS or 100 ng of recombinant IP-10. Mice were sacrificed 48 h after CLP, and bacteremia was determined from dilutions of peritoneal lavage fluid obtained aseptically. Each point represents CFUs from one mouse. P < 0.05 using the Student’s t test. Horizontal bars indicate means. The figure represents data from two independent experiments using four mice per group with similar results. (C) SEV129 wild-type and IFNAR −/− mice were injected with 100 ng CXCL10. Peritoneal cells were harvested 18 h later and were examined by flow cytometry for phagocytic neutrophils (Gr1 + CD11b + cells containing FITC + latex beads). *, P = 0.001 using one-way ANOVA; *, P < 0.05 using the Tukey post hoc analysis. Error bars indicate SD. The figure represents data from three independent experiments using at least four mice per group with similar results.

Article Snippet: For in vivo chemokine treatment, each mouse was injected i.p. with 100 ng of mouse recombinant IP-10 (R&D Systems) or PBS 6 h after surgery, and if experiments lasted longer than 3 d, again on day 3 ( ).

Techniques: Injection, Recombinant, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon signaling in hematopoietic cells is required for survival in mouse polymicrobial sepsis by regulating CXCL10

doi: 10.1084/jem.20091959

Figure Lengend Snippet: CXCL10 treatment improves human neutrophil phagocytic function in vitro . Whole-blood leukocytes were collected using Histopaque 1119. 10 5 unstimulated cells, cells treated with 100 ng CXCL10 for 4 or 2 h followed by an 18-h stimulation with 1 µg/ml LPS, or cells stimulated with LPS alone for 18 h were plated in round-bottom 96-well plates. FITC beads were added and cells were incubated at 37°C for 30 min. Cells were washed and stained for neutrophil markers (CD66b + CD16 hi ). (A) Representative histograms of the percentage of phagocytic neutrophils from unstimulated (top) or CXCL10 treatment for 4 h (bottom). (B) Graphical analysis of the percentage of phagocytic neutrophils from one healthy control (P = 0.019 using the Student’s t test). (C) Representative histograms of the percentage of phagocytic neutrophils from unstimulated (top), LPS-stimulated (middle), and CXCL10-treated + LPS-treated leukocytes (bottom). (D) Graph of the percentage of phagocytic neutrophils from one healthy control (P < 0.001 using one-way ANOVA). For all panels, treatment was performed in triplicate and the experiment was performed on three healthy controls with similar results. Error bars indicate SD.

Article Snippet: For in vivo chemokine treatment, each mouse was injected i.p. with 100 ng of mouse recombinant IP-10 (R&D Systems) or PBS 6 h after surgery, and if experiments lasted longer than 3 d, again on day 3 ( ).

Techniques: In Vitro, Incubation, Staining