Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Anti-murine Tim-3 antibodies bind non-overlapping epitopes. Jurkat T cells expressing the Balb/c form of Tim-3 were incubated in the presence of unlabeled B8.2C12, 5D12, and RMT3-23 at the concentrations indicated prior to staining with PE-labeled 5D12 (A), B8.2C12 (B), or RMT3-23 (C).

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Expressing, Incubation, Staining, Labeling

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Anti-murine Tim-3 do not deplete Tim-3+ cells in vivo. B8.2C12 (n = 3), 5D12 (n = 3), RMT3-23 (n = 3) or matched isotype control antibodies were administered i.p. to Tim-3 Tg mice. After 48 h, the frequency of CD4+ and CD8+ T cells was determined by flow cytometry. A, Total number of CD4+ and CD8+ cells in treated mice. Error bars represent s.e.m. B, Down-modulation of Tim-3 expression was examined by staining with a non-competing Tim-3 antibody clone. Error bars represent s.e.m. *p = 0.03, t-test.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: In Vivo, Flow Cytometry, Expressing, Staining

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Anti-murine Tim-3 antibodies do not block galectin-9 binding. A, mTim-3-Ig was added at the indicated concentrations to ELISA plates coated with galectin-9. Similar results were obtained in an independent experiment. B, mTim-3-Ig (0.1 μg/ml) was added to ELISA plates coated with galectin-9 in the presence of lactose at the indicated concentrations. mTim-3-Ig was incubated with RMT3-23 (C), B8.2C12 (D), 5D12 (E) or matched isotype control antibody prior to addition to ELISA plates coated with galectin-9. Similar results were obtained in an independent experiment.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Effect of anti-murine Tim-3 antibodies on binding to phosphatidylserine. A, Dexamethasone-treated thymocytes were labeled with CellTracker Green and stained with Annexin V to detect phosphatidylserine expression. Representative flow cytometry data are shown. B, mTim-3-Ig was incubated with RMT3-23, B8.2C12, 5D12 or matched isotype control antibody prior to addition to dexamethasone treated thymocytes. Data are representative of 4 independent experiments.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Binding Assay, Labeling, Staining, Expressing, Flow Cytometry, Incubation

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Effect of anti-murine Tim-3 antibodies on binding to CEACAM1. A, Expression of murine CEACAM1 on transduced Jurkat T cells. B, Untransduced or murine CEACAM1-transduced Jurkat T cells were stained with mTim-3-Ig that was pre-incubated with no antibody, RMT3-23, 2C12, 5D12 or matched isotype control antibody. Data are representative of 3 independent experiments.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Binding Assay, Expressing, Staining, Incubation

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Ligand-blocking properties of an anti-human TIM-3 antibody. A, hTIM-3-Ig was incubated with F38.2E2 or matched isotype control antibody prior to addition to ELISA plates coated with galectin-9. B, hTim-3-Ig was incubated with F38.2E2 or matched isotype control antibody prior to addition to dexamethasone treated thymocytes. C, Untransduced or human CEACAM1-transduced Jurkat T cells were stained with hTim-3-Ig that was pre-incubated with no antibody, F38.2E2 or matched isotype control antibody. Data are representative of 6 independent experiments.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS Pathogens

Article Title: TIM-3 Does Not Act as a Receptor for Galectin-9

doi: 10.1371/journal.ppat.1003253

Figure Lengend Snippet: A) Characterization of T cell stimulator cells. Upper and middle panel: Expression of membrane-bound anti-CD3 antibody fragments (mb-aCD3) on T cell stimulator cells was detected with Dylight-649-labelled goat-anti-mouse IgG (H+L-chain specific) antibodies (grey histograms). Open histogram: reactivity with control Bw cells. Lower panel: T cell stimulator cells engineered to express human galectin-9 were probed with galectin 9 antibodies (grey histogram) or isotype control antibodies (open histogram). B) Human T cells derived from healthy donors were co-cultured with T cell stimulator cells expressing human galectin-9 or no human costimulatory molecule (control). 3 [H]-methyl-thymidine uptake was assessed following 3 days of co-culture (cpm, counts per minute). The results summarize 16 experiments with different donors. C) Culture supernatant derived from T cells activated with galectin-9 expressing stimulator cells or control stimulator cells was harvested after 48 h and subjected to multiplex cytokine measurement. Data show +/− SD of triplicates from one experiment representative for 16 independently performed.

Article Snippet: Mouse and human TIM-3 immunoglobulin (Ig) fusion proteins, human ILT5 Ig, human TROP2 Ig, human TREML2 Ig, mouse CTLA4 Ig fusion proteins - all expressed in NSO-cells – and recombinant human (rh) and recombinant mouse (rm) galectin-9 were purchased from R&D systems.

Techniques: Expressing, Membrane, Derivative Assay, Cell Culture, Co-Culture Assay, Multiplex Assay

Journal: PLoS Pathogens

Article Title: TIM-3 Does Not Act as a Receptor for Galectin-9

doi: 10.1371/journal.ppat.1003253

Figure Lengend Snippet: A, D) Bw control cells (open histograms) or Bw cells transduced to express human (A) or murine (D) galectin-9 (grey histograms) were probed with galectin-9 antibodies or with immunoglobulin fusion proteins representing human TIM-3 (TIM3-Ig; A) or murine TIM-3 (m-TIM-3-Ig; D) or a control fusion protein (B7-H3-Ig). Bound antibodies were detected with PE-labelled donkey-anti-goat antibodies. Immunoglobulin fusion proteins were detected with PE-labelled goat antibodies to the Fc-part of human IgG. B, E) Bw control cells (open histogram) or Bw cells transduced to express human (B) or murine (E) TIM-3 (grey histograms) were probed with appropriate anti-TIM-3 mAbs or with biotin-labelled recombinant human (B) or murine (E) galectin-9. Bound anti-human TIM-3 mAb and anti-murine TIM-3 mAb were detected with PE-labelled goat-anti-mouse IgG and APC-labelled goat-anti-rat IgG, respectively. SA-PE was used as secondary reagent for biotin-labelled recombinant human and murine galectin-9. C, F) Recombinant human (C) or murine (F) galectin-9 was immobilized on ELISA plates and probed with immunoglobulin fusion proteins (Ig) representing the extra-cellular domain of human TIM-3 (TIM-3-Ig; C) or murine TIM-3 (m-TIM-3-Ig; F) at the indicated concentrations. HRP-conjugated goat-anti-human IgG-Fc-specific antibodies were used for detection. Binding buffer only (PBS) or the indicated immunoglobulin fusion proteins were used as controls. All binding experiments were repeated three times with similar outcome.

Article Snippet: Mouse and human TIM-3 immunoglobulin (Ig) fusion proteins, human ILT5 Ig, human TROP2 Ig, human TREML2 Ig, mouse CTLA4 Ig fusion proteins - all expressed in NSO-cells – and recombinant human (rh) and recombinant mouse (rm) galectin-9 were purchased from R&D systems.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: PLoS ONE

Article Title: Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

doi: 10.1371/journal.pone.0147186

Figure Lengend Snippet: (A) The expression of Galctin-9 in HTR8 and K562 cells was determined by RT-PCR. (B) The expression of Galectin-9 in human early pregnant trophoblasts cells was detected by immunohistochemical staining (×400). Formalin fixed paraffin-embedded trophoblasts tissue sections were stained with Goat anti-human Galectin-9 and biotinylated rabbit anti-goat Ig followed by treptavidin-conjugated peroxidase. (C) Knock down Galectin-9 expression by siRNA. The knock down efficacy was determined by RT-PCR. (D) Galectin-9 siRNA significantly increased NK cytotoxicity toward trophoblasts. HTR-8 cells, transfected with NC control or Galectin-9 siRNA, were used as targets and co-cultured with TGF-β1 pretreated pNK cells. The cytotoxicity of NK cells was measured using a Cell Counting Kit-8. (E) HTR-8, transfected with Galectin-9 siRNA, and co-cultured with TGF-β1 pretreated pNK cells at E:T ratio (10:1) with or without rhGalectin-9 (rhGal-9) for 4 hours. The cytotoxicity of NK cells was measured using a Cell Counting Kit-8.

Article Snippet: Goat anti-human TGF-β1, goat anti-human Galectin-9 and Tim-3-Fc fusion protein were purchased from R&D Systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Transfection, Cell Culture, Cell Counting

Journal: PLoS ONE

Article Title: Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

doi: 10.1371/journal.pone.0147186

Figure Lengend Snippet: NK cell alone or co-cultured with HTR-8, HTR-8 treated with Tim-3-Fc or HTR-8 transfected with Galectin-9 siRNA for 3h. Then the co-cultures were harvested and incubated with CD56-FITC. The level of CD107a on CD56 + cells was determined by FACS, one representative result was shown (A). (B) Statistical analysis of CD107a expression (n = 5). *, p < 0.05 compared with no HTR-8 stimulation group, #, p < 0.05 compared with HTR-8 stimulation group.

Article Snippet: Goat anti-human TGF-β1, goat anti-human Galectin-9 and Tim-3-Fc fusion protein were purchased from R&D Systems.

Techniques: Cell Culture, Transfection, Incubation, Expressing

Journal: bioRxiv

Article Title: Distinct Single-cell Immune Ecosystems Distinguish True and De Novo HBV-related Hepatocellular Carcinoma Recurrences

doi: 10.1101/2022.06.02.494526

Figure Lengend Snippet: Distinct Cell-cell Interaction Patterns in De Novo versus Truly Recurrent HCC Microenvironments. (A) Correlations of immune cell subsets in their cellular proportions with the exhaustion score of tumor-specific CD8 + T cells in recurrent tumor samples. (B) Ligand and receptor (L-R) pairs between TAM and tumor-specific CD8+ T cells (left panel), and between mDC and tumor-specific CD8+ T cells (right panel) in de novo recurrent tumors and truly recurrent tumors. The L-R pairs below the diagonal line indicated its superiority in de novo recurrent tumors while those above the diagonal line indicated its superiority in truly recurrent tumors. (C) Violin plot showing the LGALS9 expression levels in six myeloid cell subtypes for truly recurrent tumors (green) and de novo recurrent tumors (red). Comparison was performed by Wilcoxon test. (D) Representative multiplexed immunofluorescent images showing that LGALS9 + CD68 + myeloid cells resided near HAVCR2 + CD8 + T cells. (E) Pathways enriched in de novo recurrent versus truly recurrent malignant cells, based on gene set enrichment analysis (GSEA) using HALLMARK gene set. (F) Violin plot showing the expression score of proliferation and immune escape signatures in truly recurrent and de novo recurrent tumors. Comparison was performed by Wilcoxon test. (G) Differentially expressed genes of malignant cells between true recurrent and de novo recurrent tumors. The most significant genes with p values=0 were all shown in the plot. (H) The expression level of GDF15 was confirmed to be higher in truly recurrent tumors than in de novo recurrent tumors in validation cohort 1. (I) Dotplot showing the significance and strength of specific interactions between malignant cells and DC subtypes (DC1, DC2 and mDC) in truly recurrent (green) and de novo recurrent samples (red). (J) Representative multiplexed immunofluorescent images showing that GDF15 + CD45 - malignant cells resided near CD44 + CD11c + DCs. (K) Kaplan-Meier secondary recurrence-free survival curve in recurrent HCC patients with high or low GDF15 expression in validation cohort 1 ( n =26 patients with 3 patients having RNA-seq data in two times of recurrence). (L) Dotplot showing the significance and strength of specific interactions between tumor-specific CD8 + T cells and DC subtypes (DC1, DC2 and mDC) in truly recurrent (green) and de novo recurrent samples (red).

Article Snippet: Slides were stained to enable the simultaneous visualization of five markers– Panel 1: anti-CD8 antibody (clone ab93278, Abcam); Panel 2: anti-CD8 antibody (clone 70306S, CST), anti-CD68 antibody (clone 76437S, CST), anti-Galectin 9 antibody (clone 54330S, CST), anti-Tim3 antibody (clone 45208S, CST); Panel 3: anti-CD8 antibody (clone ab93278, Abcam), anti-CD68 antibody (clone 76437S, CST), anti-PD1 antibody (clone ab237728 Abcam), anti-PD-L1 antibody (clone 13684S, CST); Panel 4: anti-GDF15 antibody (clone ab206414, Abcam), anti-CD45 antibody (clone 13917S, CST), anti-CD44 antibody (clone ab206414, Abcam), anti-CD11c antibody (clone ab52632, Abcam)– on the same slide using PANO 7-plex IHC kit, cat 0004100100 (Panovue, Beijing, China).

Techniques: Expressing, RNA Sequencing Assay

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Anti-murine Tim-3 antibodies bind non-overlapping epitopes. Jurkat T cells expressing the Balb/c form of Tim-3 were incubated in the presence of unlabeled B8.2C12, 5D12, and RMT3-23 at the concentrations indicated prior to staining with PE-labeled 5D12 (A), B8.2C12 (B), or RMT3-23 (C).

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Expressing, Incubation, Staining, Labeling

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Anti-murine Tim-3 do not deplete Tim-3+ cells in vivo. B8.2C12 (n = 3), 5D12 (n = 3), RMT3-23 (n = 3) or matched isotype control antibodies were administered i.p. to Tim-3 Tg mice. After 48 h, the frequency of CD4+ and CD8+ T cells was determined by flow cytometry. A, Total number of CD4+ and CD8+ cells in treated mice. Error bars represent s.e.m. B, Down-modulation of Tim-3 expression was examined by staining with a non-competing Tim-3 antibody clone. Error bars represent s.e.m. *p = 0.03, t-test.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: In Vivo, Flow Cytometry, Expressing, Staining

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Anti-murine Tim-3 antibodies do not block galectin-9 binding. A, mTim-3-Ig was added at the indicated concentrations to ELISA plates coated with galectin-9. Similar results were obtained in an independent experiment. B, mTim-3-Ig (0.1 μg/ml) was added to ELISA plates coated with galectin-9 in the presence of lactose at the indicated concentrations. mTim-3-Ig was incubated with RMT3-23 (C), B8.2C12 (D), 5D12 (E) or matched isotype control antibody prior to addition to ELISA plates coated with galectin-9. Similar results were obtained in an independent experiment.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Blocking Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Effect of anti-murine Tim-3 antibodies on binding to phosphatidylserine. A, Dexamethasone-treated thymocytes were labeled with CellTracker Green and stained with Annexin V to detect phosphatidylserine expression. Representative flow cytometry data are shown. B, mTim-3-Ig was incubated with RMT3-23, B8.2C12, 5D12 or matched isotype control antibody prior to addition to dexamethasone treated thymocytes. Data are representative of 4 independent experiments.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Binding Assay, Labeling, Staining, Expressing, Flow Cytometry, Incubation

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Effect of anti-murine Tim-3 antibodies on binding to CEACAM1. A, Expression of murine CEACAM1 on transduced Jurkat T cells. B, Untransduced or murine CEACAM1-transduced Jurkat T cells were stained with mTim-3-Ig that was pre-incubated with no antibody, RMT3-23, 2C12, 5D12 or matched isotype control antibody. Data are representative of 3 independent experiments.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Binding Assay, Expressing, Staining, Incubation

Journal: Oncoimmunology

Article Title: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy

doi: 10.1080/2162402X.2017.1385690

Figure Lengend Snippet: Ligand-blocking properties of an anti-human TIM-3 antibody. A, hTIM-3-Ig was incubated with F38.2E2 or matched isotype control antibody prior to addition to ELISA plates coated with galectin-9. B, hTim-3-Ig was incubated with F38.2E2 or matched isotype control antibody prior to addition to dexamethasone treated thymocytes. C, Untransduced or human CEACAM1-transduced Jurkat T cells were stained with hTim-3-Ig that was pre-incubated with no antibody, F38.2E2 or matched isotype control antibody. Data are representative of 6 independent experiments.

Article Snippet: 0.1 µg/mL murine Tim-3-Ig (Chimerigen) or human Tim-3-Ig (Sino Biological) was incubated with either anti-Tim-3 antibody or isotype control for 30 minutes at room temperature prior to addition to the galectin-9 coated plate.

Techniques: Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Staining