Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: TIGIT but not DNAM1, CD112R or CD96—interacts with Nectin4. (A) FACS staining of IL-2 activated primary NK cells with Nectin4-Ig. Gray filled histogram represents background staining with secondary antibody only; black line histogram represents specific binding as indicated. (B–F) FACS staining of Raji cells transfected either with an empty vector as control (gray histograms) or with Nectin4 (black histograms). Cells were stained with commercial anti-Nectin4 mAb (B), TIGIT-Ig (C), DNAM1-Ig (D), CD112-Ig (E) or CD96-Ig (F). Figures show one representative experiment out of three performed. Graph depicting the mean fluorescence intensity values of the stainings appears in . (G) Direct binding of Nectin4 to TIGIT. The binding of fluorophore-labeled TIGIT-Ig and its ligands PVR-Ig (red) and Nectin4-Ig (green) was determined using MST. Measurements were repeated with at least three independent protein preparations. FACS, fluorescence-activated cell sorting; IL-2, interleukin-2; MST, microscale thermophoresis; NK, natural killer.

Article Snippet: Extracellular fluorescence-activated cell sorting (FACS) staining for Nectin4 (MAB2659, R&D Systems), Nectin2 (BLG-337402, BioLegend), PVR (MAB25301, R&D Systems) DNAM1 (BLG-338302, BioLegend) and TIGIT (MAB7898, R&D Systems) was performed with 0.2 μg antibody per 100 000 cells.

Techniques: Staining, Binding Assay, Transfection, Plasmid Preparation, Control, Fluorescence, Labeling, FACS, Microscale Thermophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity

doi: 10.1136/jitc-2019-000266

Figure Lengend Snippet: Nectin4 inhibits NK cytotoxicity via TIGIT. (A, C) IL2 secretion by parental BW (A, C), (A) BW-TIGIT and (C) BW-DNAM1 cells. IL2 secretion was determined by ELISA (od 650 nm) following incubation with control anti-TIGIT or anti-DNAM1 antibodies (left in a and C) or with PVR expressing cells (right in a and C). (B, D) IL2 secretion of parental BW (B, D), (B) BW-TIGIT and (D) BW-DNAM1 cells. IL2 secretion was determined by ELISA (od 650 nm) following incubation with Raji cells transfected either with an empty vector (Raji E) as a control, or with Nectin4 (Raji N4). Figure shows one representative experiment out of 3 performed. *P<0.05. (E) FACS staining of Raji cells overexpressing Nectin4 with TIGIT-Ig. TIGIT-Ig was preincubated with no antibody (left), with a control mAb (anti-CD99 mAb clone 12E7, middle) or with anti-TIGIT blocking antibody (mAb #4 generated as described previously, right). Black line histograms represent TIGIT-Ig binding. Gray filled histograms represent background staining of the secondary antibody only. (F) Mean fluorescence intensity (MFI) values of the TIGIT-Ig staining shown in (E) relative to NO antibody staining, *p<2×10 -4 . (G) [ 35 S] methionine-labeled Raji cells transfected either with an empty vector as control (Raji empty—gray) or with Nectin4 (Raji Nectin4—black), were incubated for 5 hours with NK cells. NK cells were preincubated with no antibody (left), with a control antibody (anti-CD99 mAb clone 12E7, middle) or with an anti-TIGIT antibody (mAb #4 generated as described previously, right). The effector to target (E:T) ratios are indicated on the x-axis. Figure shows one representative experiment out of three performed. Shown is the relative average killing ±SD, *p<0.05. FACS, fluorescence-activated cell sorting; IL2, interleukin-2; NK, natural killer; NS, not significant.

Article Snippet: Extracellular fluorescence-activated cell sorting (FACS) staining for Nectin4 (MAB2659, R&D Systems), Nectin2 (BLG-337402, BioLegend), PVR (MAB25301, R&D Systems) DNAM1 (BLG-338302, BioLegend) and TIGIT (MAB7898, R&D Systems) was performed with 0.2 μg antibody per 100 000 cells.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control, Expressing, Transfection, Plasmid Preparation, Staining, Blocking Assay, Generated, Binding Assay, Fluorescence, Labeling, FACS

Journal: Respiratory Research

Article Title: TIGIT/SHIP-1/RelB regulating Th1 inflammation in smoking induced COPD

doi: 10.1186/s12931-025-03400-9

Figure Lengend Snippet: High expression of TIGIT in CD4 + T cells from the lungs of COPD patients and CS mice. A Representative flow cytometry analysis of CD4 + T cells in the lungs of Control, Smoker, and COPD groups. B - D Statistical analysis bar graphs of IFN-γ, IL-17 A, and TNF-α in lung CD4 + T cells from the three groups (Control: n = 11, Smoker: n = 18, COPD: n = 16). E Diagram depicting the construction of a tobacco exposure model of emphysema in mice. F Representative images of H&E staining of lung tissue sections from Air and CS group mice. G Comparison of Lm values in the morphological analysis of lung tissues from Air and CS mice ( n = 5). H , I Representative flow cytometry analysis of CD4 + T cells in the lungs of Air and CS mice. J - L Statistical analysis bar graphs of IFN-γ, IL-17 A, and TNF-α in lung CD4 + T cells from the two groups of mice ( n = 10). Data are representative of three independent experiments and are presented as medians. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Wild-type C57BL/6J mice were obtained from the Animal Experiment Center of Guangxi Medical University, while Tigit flox/flox (Tigit-Flox) and Tigit flox/flox CD4-Cre (Tigit-Cre) mice were sourced from Suzhou Cyagen Biotechnology Co., Ltd.

Techniques: Expressing, Flow Cytometry, Control, Staining, Comparison

Journal: Respiratory Research

Article Title: TIGIT/SHIP-1/RelB regulating Th1 inflammation in smoking induced COPD

doi: 10.1186/s12931-025-03400-9

Figure Lengend Snippet: Specific knockout of CD4-Tigit significantly enhances Th1 inflammation in the lungs of CS mice. A Correlation analysis of the proportions of Th1 and Th17 cells in the lungs of COPD and CS mice with the proportion of TIGIT + CD4 + T cells (COPD: n = 16, CS: n = 10). B Construction of pathological models of pulmonary emphysema in Tigit-Cre and Tigit-Flox mice following tobacco exposure. C Representative images of H&E staining of lung tissue sections from CS-Tigit-Cre and CS-Tigit-Flox mice. D Comparison of Lm values in the morphological analysis of lung tissues from CS-Tigit-Cre and CS-Tigit-Flox mice ( n = 5). E - G Representative flow cytometry analysis images and comparative bar graphs of Th1 and Th17 cells in the lungs of CS-Tigit-Cre and CS-Tigit-Flox mice ( n = 8). H , I Representative flow cytometry analysis images and comparative bar graphs of CD4 + Tn, CD4 + Tcm, and CD4 + Tem cells in the lungs of CS-Tigit-Cre and CS-Tigit-Flox mice ( n = 8). Data are representative of three independent experiments and are presented as medians. * P < 0.05, *** P < 0.001

Article Snippet: Wild-type C57BL/6J mice were obtained from the Animal Experiment Center of Guangxi Medical University, while Tigit flox/flox (Tigit-Flox) and Tigit flox/flox CD4-Cre (Tigit-Cre) mice were sourced from Suzhou Cyagen Biotechnology Co., Ltd.

Techniques: Knock-Out, Staining, Comparison, Flow Cytometry

Journal: Respiratory Research

Article Title: TIGIT/SHIP-1/RelB regulating Th1 inflammation in smoking induced COPD

doi: 10.1186/s12931-025-03400-9

Figure Lengend Snippet: The impact of the TIGIT/SHIP-1 axis on pulmonary Th1 inflammation in CS mice. A , B Co-localization analysis of SHIP-1 and CD4 in the lungs of Air and CS mice using dual immunofluorescence staining ( n = 7). C , D Co-localization analysis of SHIP-1 and CD4 in the lungs of Air, CS-Tigit-Cre, and CS-Tigit-Flox mice using dual immunofluorescence staining ( n = 5). E Schematic representation of the SHIP-1 inhibitory tobacco exposure emphysema pathological mouse model. F - H Representative flow cytometry analysis images and statistical bar graphs of pulmonary Th1 and Th17 in CS + 3-AC and CS + Vehicle mice ( n = 7). Data are representative of three independent experiments and are presented as medians. * P < 0.05, ** P < 0.01

Article Snippet: Wild-type C57BL/6J mice were obtained from the Animal Experiment Center of Guangxi Medical University, while Tigit flox/flox (Tigit-Flox) and Tigit flox/flox CD4-Cre (Tigit-Cre) mice were sourced from Suzhou Cyagen Biotechnology Co., Ltd.

Techniques: Immunofluorescence, Staining, Flow Cytometry

Journal: Respiratory Research

Article Title: TIGIT/SHIP-1/RelB regulating Th1 inflammation in smoking induced COPD

doi: 10.1186/s12931-025-03400-9

Figure Lengend Snippet: PI3K/AKT signaling enhances the activation of CD4 + T cells in CS mice and promotes activation in a Th1 polarizing environment. A - D Flow cytometric analysis of pPI3K and pAKT1 in lung CD4 + T cells from Air and CS mice, with representative images and statistical analysis bar graphs ( n = 6). E Schematic diagram of the initial CD4 + T cell isolation and culture model. F - I Flow cytometric analysis of IFN-γ, pPI3K, pAKT1, and TIGIT in initially cultured CD4 + Tn cells after in vitro Th1 polarization, with representative images and statistical analysis bar graphs ( n = 3). J - K Flow cytometric analysis of TIGIT and IFN-γ double-positive cells in initially cultured CD4 + Tn cells after in vitro Th1 polarization, with representative images and statistical analysis bar graphs ( n = 3). Data are representative of three independent experiments and are presented as medians. * P < 0.05, ** P < 0.01, **** P < 0.0001

Article Snippet: Wild-type C57BL/6J mice were obtained from the Animal Experiment Center of Guangxi Medical University, while Tigit flox/flox (Tigit-Flox) and Tigit flox/flox CD4-Cre (Tigit-Cre) mice were sourced from Suzhou Cyagen Biotechnology Co., Ltd.

Techniques: Activation Assay, Cell Isolation, Cell Culture, In Vitro

Journal: Respiratory Research

Article Title: TIGIT/SHIP-1/RelB regulating Th1 inflammation in smoking induced COPD

doi: 10.1186/s12931-025-03400-9

Figure Lengend Snippet: In vitro cell culture reveals that TIGIT/SHIP-1 regulates Th1 differentiation by influencing the activation of the PI3K/AKT signaling pathway. ( A , B ) After co-culturing CD4 + T cells with different concentrations of the PI3K inhibitor LY294002 under Th1 polarization conditions, flow cytometry analysis of IFN-γ, pPI3K, and pAKT1 is shown with representative graphs and statistical analysis dot plots ( n = 3). C - D Following co-culture of CD4 + T cells with the TIGIT ligand CD155 recombinant protein and CD155 + 3-AC under Th1 polarization conditions, flow cytometry analysis of IFN-γ, pPI3K, and pAKT1 is shown with representative graphs and statistical analysis dot plots ( n = 3). Data are representative of three independent experiments and are presented as medians. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Wild-type C57BL/6J mice were obtained from the Animal Experiment Center of Guangxi Medical University, while Tigit flox/flox (Tigit-Flox) and Tigit flox/flox CD4-Cre (Tigit-Cre) mice were sourced from Suzhou Cyagen Biotechnology Co., Ltd.

Techniques: In Vitro, Cell Culture, Activation Assay, Flow Cytometry, Co-Culture Assay, Recombinant

Journal: Respiratory Research

Article Title: TIGIT/SHIP-1/RelB regulating Th1 inflammation in smoking induced COPD

doi: 10.1186/s12931-025-03400-9

Figure Lengend Snippet: RelB is a key transcription factor regulating T-bet in CD4 + T cells, while TIGIT/SHIP-1 can influence the expression levels of pRelB and T-bet in CD4 + T cells through the PI3K/AKT signaling pathway. A , B Co-localization analysis of SHIP-1 and CD4 in double immunofluorescence staining of lung tissues from Air and CS mice ( n = 3). C Electrophoresis of DNA binding sites in agarose gel. D PCR results analysis of TBX21 precipitated by anti-RelB antibody and IgG ( n = 3). E , F Flow cytometry analysis of pRelB and T-bet after co-culturing CD4 + T cells with different concentrations of the PI3K inhibitor LY294002 under Th1 polarization conditions, with representative images and statistical analysis dot plots ( n = 3). G , H Flow cytometry analysis of pRelB and T-bet after co-culturing CD4 + T cells with the TIGIT ligand CD155 recombinant protein, CD155 + 3-AC under Th1 polarization conditions, with representative images and statistical analysis dot plots ( n = 3). Data are representative of three independent experiments and are presented as medians. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Wild-type C57BL/6J mice were obtained from the Animal Experiment Center of Guangxi Medical University, while Tigit flox/flox (Tigit-Flox) and Tigit flox/flox CD4-Cre (Tigit-Cre) mice were sourced from Suzhou Cyagen Biotechnology Co., Ltd.

Techniques: Expressing, Double Immunofluorescence Staining, Electrophoresis, Binding Assay, Agarose Gel Electrophoresis, Flow Cytometry, Recombinant

Journal: European journal of immunology

Article Title: TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

doi: 10.1002/eji.201545480

Figure Lengend Snippet: Figure 1. TIGIT expression on human NK cells shows wide variation among health individuals. (A) Representative FACS histograms showing the expression of TIGIT on peripheral blood CD4+ T cells, CD8+ T cells, CD8+CD45RO+ T cells, CD4+CD25high T cells, and NK cells. The MFI of TIGIT is also shown as mean + SEM (n = 10 subjects per group, right). Data are from a single experiment representative of three. **p < 0.01, ***p < 0.001 (Mann–Whitney U test), relative to the MFI of TIGIT on CD4+ T cells. (B) Representative FACS plots showing the expression of TIGIT on peripheral blood CD19+ B cells, CD14+ monocytes, CD11c+ DC, and CD13+ neutrophils. (C) Representative FACS plots showing the expression of TIGIT on peripheral blood CD4+ T cells, CD8+ T cells, and NK cells. The percentages of TIGIT+ cells are shown as the mean + SEM (n = 10–14 subjects per group). Data are from a single experiment representative of three. (D) PBMCs isolated from healthy individuals were stimulated with IL-12 or LPS for 24 h. The percentages of TIGIT+ NK cells are shown as mean + SEM (n = 16–23 subjects per group). Data are from a single experiment representative of three. (E) Scatter plots showing TIGIT expression on NK cells in 199 healthy individuals. Horizontal bar indicates the median. (F) Frequency distribution analysis of the percentages of TIGIT+ NK cells in 199 healthy individuals. (G) FACS plots showing the expression of CD226 on NK cells from two representative healthy individuals. Correlation between TIGIT and CD226 expression on NK cells is also shown (Spearman’s rank correlation test). Each symbol represents an individual donor.

Article Snippet: For cell stimulation studies, PBMCs, monocyte-depleted PBMCs, or purified NK cells were stimulated with IL-2 (1000 U/mL, R&D Systems), IL-12 (100 U/mL, BioLegend), or LPS (10 μg/mL, Sigma) for 24 h. In some experiments, recombinant human TIGIT Fc Chimera protein (5 μg/mL, R&D Systems), functional antihuman TIGIT antibody (5 μg/mL, eBioscience), or IgG control (R&D Systems) were added to the culture medium for blocking TIGIT pathway.

Techniques: Expressing, MANN-WHITNEY, Isolation

Journal: European journal of immunology

Article Title: TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

doi: 10.1002/eji.201545480

Figure Lengend Snippet: Figure 2. The relationship between TIGIT expression level and NK- cell phenotype. (A, B) PBMCs isolated from healthy individuals were analyzed by flow cytometry. Correlation (A) between TIGIT and CD25 expression or (B) between TIGIT and CD69 expression on NK cells is shown (Spearman’s rank correlation test). The expressions of (A) CD25 and (B) CD69 on TIGIT−or TIGIT+ NK cells are expressed as the mean + SEM (n = 8–12 subjects per group) and are from a single experiment rep- resentative of three. (C) Correlation between TIGIT and CD107a expres- sion on NK cells (Spearman’s rank correlation test). (D) FACS plot show- ing the expressions of TIGIT and CD107a on NK cells from a represen- tative healthy individual. CD107a expression on TIGIT−or TIGIT+ NK cells is shown as the mean + SEM (n = 9 subjects per group). Data are from a single experiment representative of three. **p < 0.01 (Student’s t-test). (E) FACS histograms showing the expression of perforin in NK cells from two representative healthy individuals. Correlation between TIGIT and perforin expression in NK cells (Spearman’s rank correlation test). (A–C, E) Each symbol represents an individual donor.

Article Snippet: For cell stimulation studies, PBMCs, monocyte-depleted PBMCs, or purified NK cells were stimulated with IL-2 (1000 U/mL, R&D Systems), IL-12 (100 U/mL, BioLegend), or LPS (10 μg/mL, Sigma) for 24 h. In some experiments, recombinant human TIGIT Fc Chimera protein (5 μg/mL, R&D Systems), functional antihuman TIGIT antibody (5 μg/mL, eBioscience), or IgG control (R&D Systems) were added to the culture medium for blocking TIGIT pathway.

Techniques: Expressing, Isolation, Cytometry

Journal: European journal of immunology

Article Title: TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

doi: 10.1002/eji.201545480

Figure Lengend Snippet: Figure 3. TIGIT expression levels correlate with differential cytokine secretion and degranulation of NK cells. (A) PBMCs isolated from healthy individuals were stimulated with IL-12 for 24 h. The percentages of IL-12-stimulated IFN-γ in TIGIT−or TIGIT+ NK cells were measured by flow cytometry. Data are shown as the mean + SEM (n = 21 subjects per group) and are from a single experiment representative of three. ***p < 0.001 (Student’s t-test). (B) FACS plots showing the expressions of TIGIT and IL-12-stimulated IFN-γ in NK cells from two representative healthy individuals. Correlation between TIGIT expression and percentages of IL-12-stimulated IFN-γ in NK cells is also shown (Spearman’s rank correlation test). (C) The percentages of IFN-γ+ NK cells before and after stimulation with IL-12 in different TIGIT expression groups were evaluated by flow cytometry. Data are expressed as the mean + SEM (n = 8–15 subjects per group) and are from a single experiment representative of three. *p < 0.05, ***p < 0.001 (Mann–Whitney U test). (D–H) PBMCs were stimulated with LPS in the (D–F) absence or presence (G, H) of K562 cells for 24 h. (D) FACS plots showing the expressions of TIGIT and CD107a on NK cells from two representative healthy individuals. (E) Correlation between TIGIT and LPS-stimulated CD107a expression on NK cells (Spearman’s rank correlation test). (F, H) The percentages of CD107a+ NK cells in the (F) absence or (H) presence of K562 in different TIGIT expression groups were evaluated by flow cytometry. Data are shown as the mean + SEM (n = 5–8 subjects per group) and are from a single experiment representative of three. **p < 0.01 (Mann–Whitney U test). Individual 1: representing low-level TIGIT expression donor; Individual 2: representing high-level TIGIT expression donor. (B, E) Each symbol represents an individual donor.

Article Snippet: For cell stimulation studies, PBMCs, monocyte-depleted PBMCs, or purified NK cells were stimulated with IL-2 (1000 U/mL, R&D Systems), IL-12 (100 U/mL, BioLegend), or LPS (10 μg/mL, Sigma) for 24 h. In some experiments, recombinant human TIGIT Fc Chimera protein (5 μg/mL, R&D Systems), functional antihuman TIGIT antibody (5 μg/mL, eBioscience), or IgG control (R&D Systems) were added to the culture medium for blocking TIGIT pathway.

Techniques: Expressing, Isolation, Cytometry, MANN-WHITNEY

Journal: European journal of immunology

Article Title: TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

doi: 10.1002/eji.201545480

Figure Lengend Snippet: Figure 4. The relationship between TIGIT expression level and the proliferation, apoptosis, and cytotoxicity of NK cells. (A) Purified NK cells labeled with CFSE were stimulated with IL-2 for 5 days. FACS histograms showing the prolifer- ation of NK cells from two represen- tative individuals (M1: representing the percentage of NK-cell proliferation). The percentages of NK-cell proliferation in dif- ferent TIGIT expression groups were eval- uated by CFSE dilution by flow cytome- try. Data are shown as the mean + SD (n = 5 subjects per group) and are from a single experiment representative of three. (B) PBMCs isolated from healthy individu- als were stimulated with or without LPS for 24, 48, and 72 h. FACS plots showing the apoptosis of NK cells from two repre- sentative individuals. The apoptosis of NK cells in different TIGIT expression groups was analyzed at different time points and shown as the mean ± SD (n = 4 subjects per group) and from a single experiment representative of three experiments per- formed. *p < 0.05 (Mann–Whitney U test). (C) Purified NK cells stimulated with or without IL-12 for 24 h were collected as effector cells. K562 cells labeled with CFSE were used as target cells. Representative FACS plots showing the purity of effec- tor cells and CFSE-labeled target cells. (D) Effector cells and target cells were coincu- bated at different (effector-to-target) E:T ratios for 6 h. CFSEhigh target cells were gated for analysis of PI intensities. FACS histograms showing the cytotoxicity of NK cells from two representative individ- uals (M1: representing the percentage of dead target cells). The cytotoxicity of NK cells in different TIGIT expression groups was analyzed and shown as the mean ± SD (n = 5 subjects per group) and from a single experiment representative of three experiments performed. **p < 0.01 (Mann– Whitney U test).

Article Snippet: For cell stimulation studies, PBMCs, monocyte-depleted PBMCs, or purified NK cells were stimulated with IL-2 (1000 U/mL, R&D Systems), IL-12 (100 U/mL, BioLegend), or LPS (10 μg/mL, Sigma) for 24 h. In some experiments, recombinant human TIGIT Fc Chimera protein (5 μg/mL, R&D Systems), functional antihuman TIGIT antibody (5 μg/mL, eBioscience), or IgG control (R&D Systems) were added to the culture medium for blocking TIGIT pathway.

Techniques: Expressing, Labeling, Isolation, MANN-WHITNEY

Journal: European journal of immunology

Article Title: TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals.

doi: 10.1002/eji.201545480

Figure Lengend Snippet: Figure 6. Variation in TIGIT expression levels on NK cells from patients with cancer and autoimmune disease. (A) Patients with LTBI (n = 22), inactive HBV carriers (n = 23), and patients with SLE and RA (n = 14), and gastric and colon cancer (n = 22) were recruited for analysis of TIGIT expression by flow cytometry. The expression of TIGIT on NK cells from patients with above diseases was compared with TIGIT expression on NK cells from healthy individuals (n = 199). Each symbol represents an individual donor, and horizontal bars indicate the median. (B) FACS plots showing the expression of IFN-γ in NK cells from a representative healthy individual, a representative RA/SLE patient, and a representative gastric/colon cancer patient after IL-12 stimulation. (C) The percentages of IFN-γ+ NK cells before and after IL-12 stimulation in healthy individuals, RA/SLE patients, and gastric/colon cancer patients are shown. The percentages of IFN-γ+ NK cells after IL-12 stimulation are shown as the mean + SEM (n = 14–20 subjects per group) and are from a single experiment representative of three experiments performed. (D) Correlation between TIGIT expression and IL-12-stimulated IFN-γ production in NK cells from healthy individuals, RA/SLE patients, and gastric/colon cancer patients (Spearman’s rank correlation test). (A–C) **p < 0.01, ***p < 0.001 (Mann–Whitney U test).

Article Snippet: For cell stimulation studies, PBMCs, monocyte-depleted PBMCs, or purified NK cells were stimulated with IL-2 (1000 U/mL, R&D Systems), IL-12 (100 U/mL, BioLegend), or LPS (10 μg/mL, Sigma) for 24 h. In some experiments, recombinant human TIGIT Fc Chimera protein (5 μg/mL, R&D Systems), functional antihuman TIGIT antibody (5 μg/mL, eBioscience), or IgG control (R&D Systems) were added to the culture medium for blocking TIGIT pathway.

Techniques: Expressing, Cytometry, MANN-WHITNEY