Journal: Stem Cells International

Article Title: CD8 + γδ T Cells Are More Frequent in CMV Seropositive Bone Marrow Grafts and Display Phenotype of an Adaptive Immune Response

doi: 10.1155/2019/6348060

Figure Lengend Snippet: Enhanced response of CD8 + γδ T cells to TCR and cytokine stimulation. (a) representative histograms ( n = 4) of proliferating T cells from unstimulated or TCR/CD3 stimulated condition, gated on TCR γδ − CD8 + (blue), CD8 + γδ (green), and CD8 − γδ (orange). (b) Proportions of proliferating CD8 + γδ (green) and CD8 − γδ (orange) cells after TCR/CD3 stimulation. (c) Representative FACS plot of TIM3, CD69, and KIR2DL2/L3 gated on CD8 + γδ (upper) and CD8 − γδ (lower) T cells after TCR/CD3 stimulation. (d) Proportions of CD8 + γδ T cells (blue) and CD8 − γδ T cells (red) expressing TIM3+, CD69+, and KIR2DL2/L3+ after TCR/CD3 stimulation. Paired t -test used. (e) Representative histogram of proliferation of CD8 + γδ (A) and CD8 − γδ (B) cultured in the presence of medium only (green), IL-7 (orange), IL-15 (blue), and IL-18 (red). Proportions of proliferating cells after culture in a medium, IL-7, IL-15, and IL-18 in CD8 + γδ (f) and CD8 − γδ (g). Repeated measures ANOVA is used. Bar and whiskers represent the mean and S.E.M.

Article Snippet: Immunophenotyping was performed using fluorochrome-conjugated anti-human monoclonal antibodies (mAb) as follows: CD3-BV450 (UCHT1), CD3-BV510 (UCHT1), CD4-Alexa Fluor 700 (RPA-T4), CD8-APC-Cy7 (SK1), CD27-BV421 (M-T271), CD45RO-APC (UCHL1), CD197 (CCR-7)-PE-Cy7 (3D12), and CD69-FITC (L78) (BD Biosciences); CD158b-PE-Cy7 (DX27) and TCR V γ 9-FITC (B3) (BioLegend); TCR V δ 1-FITC (TS8.2) (Thermo Scientific); and TCR pan γδ -PE (REA591) and TIM3-APC (F38-2E2) (Miltenyi Biotec).

Techniques: Expressing, Cell Culture

Journal: Open Biology

Article Title: miR-149-3p reverses CD8 + T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells

doi: 10.1098/rsob.190061

Figure Lengend Snippet: Exhaustion marker upregulation in CD8 + T cells from spleens of 4T1 tumour-bearing mice. ( a ) Detection of mRNAs encoding PD-1, TIM-3, BTLA by RT–qPCR. Spleen cells were collected from naive mice and from tumour-bearing mice on days 16–18 after tumour cell injection. CD8 + T cells were purified from the collected splenocytes using Miltenyi magnetically labelled beads (Miltenyi Biotec). RT–qPCR was performed to detect PD-1, TIM-3, BTLA and Foxp1 mRNA levels in CD8 + T cells. ( b ) Detection of IRs on CD8 + T cells by flow cytometry. Spleen cells were collected from naive mice and from tumour-bearing mice. Flow cytometry was performed to detect PD-1, TIM-3 and BTLA expression level. Data are representative of three independent experiments. Unpaired Student's t -tests were performed to determine statistical significance (* p < 0.05, ** p < 0.01).

Article Snippet: Lymphocytes were stained with 0.2 µg of each of the following MAbs: FITC anti-mouse CD8, PerCP-eFluor 710 anti-mouse PD-1, PE-CY7 anti-mouse TIM-3, APC anti-mouse BTLA, PerCP-eFluor 710 Rat IgG2b Isotype Control, PE Mouse IgG2a κ Isotype Control, FITC Mouse IgG2a κ Isotype Control, PE-CY7 Mouse IgG2a κ Isotype Control, APC Mouse IgG1 κ Isotype Control, PE anti-mouse IL-2 PE anti-mouse TNF-α and PE anti-mouse IFN-γ- (eBioscience, San Diego, CA, USA).

Techniques: Marker, Quantitative RT-PCR, Injection, Purification, Flow Cytometry, Expressing

Journal: Open Biology

Article Title: miR-149-3p reverses CD8 + T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells

doi: 10.1098/rsob.190061

Figure Lengend Snippet: Global miRNA level profile analysis and potential targeting of exhaustion-associated IR mRNAs by miR-149-3p. ( a ) Heat map of miRNAs with differential levels between CD8 + PD1− and CD8 + PD1+ samples. Total RNA was labelled using a Flash Tag Biotin HSR kit (Genisphere) and hybridized to Affymetrix miRNA 3.0 arrays. A total of 117 miRNAs with significant changes were found (electronic supplementary material, table S1). ( b ) Predicting miRNAs that target IR mRNAs by miRNA walk, Targetscan, miRanda and other databases. Among 117 miRNAs, 5 candidate miRNAs, including miR-149-3p, miR-146a-3p, miR-122-5p, miR-211-5p and miR-721, were selected on the basis of their potential to bind the 3′UTRs of mRNAs encoding PD-1, TIM-3, BTLA and Foxp1. ( c ) miR-149-3p levels were confirmed by RT–qPCR. Total RNA generated from naive and tumour-bearing mice was used to detect the level of miR-149-3p. Data are representative of three independent experiments. Unpaired Student's t -test analyses were performed to determine statistical significance (* p < 0.05).

Article Snippet: Lymphocytes were stained with 0.2 µg of each of the following MAbs: FITC anti-mouse CD8, PerCP-eFluor 710 anti-mouse PD-1, PE-CY7 anti-mouse TIM-3, APC anti-mouse BTLA, PerCP-eFluor 710 Rat IgG2b Isotype Control, PE Mouse IgG2a κ Isotype Control, FITC Mouse IgG2a κ Isotype Control, PE-CY7 Mouse IgG2a κ Isotype Control, APC Mouse IgG1 κ Isotype Control, PE anti-mouse IL-2 PE anti-mouse TNF-α and PE anti-mouse IFN-γ- (eBioscience, San Diego, CA, USA).

Techniques: Quantitative RT-PCR, Generated

Journal: Open Biology

Article Title: miR-149-3p reverses CD8 + T-cell exhaustion by reducing inhibitory receptors and promoting cytokine secretion in breast cancer cells

doi: 10.1098/rsob.190061

Figure Lengend Snippet: Altered expression of markers of exhaustion in T cells after treatment with miR-149-3p mimic or miR-149-3p inhibitor. ( a ) Detection of mRNAs encoding PD-1, TIM-3, BTLA and Foxp1 by RT–qPCR analysis. CD8 + T cells were purified from the tumour-bearing mice splenocytes using Miltenyi magnetically labelled beads and transfected with control miRNA, miR-149-3p mimics or miR-149-3p inhibitors for 48 h. After transfection, RT–qPCR was performed to detect PD-1, TIM-3, BTLA and Foxp1 mRNA levels in CD8 + T cells. ( b ) Examination of IRs on CD8 + T cells by flow cytometry. CD8 + T cells were purified from tumour-bearing mice splenocytes and transfected with control miRNA, miR-149-3p mimics or miR-149-3p inhibitors for 48 h. Flow cytometry was performed after transfection to detect PD-1, TIM-3 and BTLA expression level. Data are representative of three independent experiments. One-way ANOVA analyses were performed to determine statistical significance (* p < 0.05, ** p < 0.01).

Article Snippet: Lymphocytes were stained with 0.2 µg of each of the following MAbs: FITC anti-mouse CD8, PerCP-eFluor 710 anti-mouse PD-1, PE-CY7 anti-mouse TIM-3, APC anti-mouse BTLA, PerCP-eFluor 710 Rat IgG2b Isotype Control, PE Mouse IgG2a κ Isotype Control, FITC Mouse IgG2a κ Isotype Control, PE-CY7 Mouse IgG2a κ Isotype Control, APC Mouse IgG1 κ Isotype Control, PE anti-mouse IL-2 PE anti-mouse TNF-α and PE anti-mouse IFN-γ- (eBioscience, San Diego, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Purification, Transfection, Flow Cytometry

Journal: Nature Communications

Article Title: TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma

doi: 10.1038/s41467-017-02358-7

Figure Lengend Snippet: Anti-PD-1 triggers TIM-3 expression on CD8+ TILs in a TNF-dependent manner. WT and TNF-deficient mice were injected as described in the legend to Fig. . TIM-3 and PD-1 expression on CD8+ TILs was determined by flow cytometry at day 10. a Representative staining; values indicate the proportion of cells in the different quadrants. b Quantification of the proportion of TIM-3+ (left panel) and PD-1+ (right panel) among CD8+ TILs. c Quantification of the proportion of CD8+ TILs expressing or not TIM-3 and PD-1 (left panel), and proportion of cell death among CD8+ T cells of the indicated populations (right panel). Values in 5–6 mice per group from one experiment are represented as Tukey boxes (b: Student’s t -test: ** p < 0.01; c: two-way Anova: *** p < 0.001). d Representative histograms of live/dead staining in CD8+ TILs expressing both TIM-3 and PD-1. Values are percentages of dead cells among the TIM-3+ PD-1+ CD8+ TILs

Article Snippet: Additional antibodies used in this study were anti-mouse CD45 (BD Biosciences, BUV395, clone 30-F11; 1/200), anti-mouse Thy1 (Biolegend, APC-Cy7, clone 30-H12; 1/400), anti-mouse CD8 (BD Biosciences, BV605, clone 53-6.7; 1/200), anti-mouse CD4 (BD Biosciences, BUV496, clone GK1.5; 1/200), anti-mouse TIM-3 (eBioscience, PE-Cy7, clone RMT3-23; 1/200), anti-mouse LAG3 (Biolegend, PE, clone C9B7W; 1/100), anti-mouse PD-1 (eBioscience, FITC, clone J43; 1/200), anti-mouse CTLA-4 (eBioscience, APC, clone UC10-4B9; 1/100), anti-mouse TIGIT (Biolegend, Pe-Cy7, clone 1G9; 1/100), anti-mouse CD11c (eBioscience, PE-Cy7, clone N418; 1/100), anti-mouse CD3 (Biolegend, FITC, clone 145-2C11; 1/200), anti-mouse PD-L1 (BD Biosciences, PE, clone MIH5; 1/100), anti-mouse PD-L2 (BD Biosciences, APC, clone TY25; 1/100), anti-mouse Ki67 (BD Biosciences, FITC, clone B56; 1/10), anti-human TIM3 (BD Biosciences, BV421, clone 7D3; 1/50) and anti-human CD8 (BD Biosciences, BV605, clone SK1; 1/50).

Techniques: Expressing, Injection, Flow Cytometry, Staining

Journal: Nature Communications

Article Title: TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma

doi: 10.1038/s41467-017-02358-7

Figure Lengend Snippet: TNF induces TIM-3 expression on CD8+ T cells ex vivo. a WT or TNFR1-deficient CD8+ T cells were incubated with murine TNF for 2 days. TIM-3 expression was next analysed by flow cytometry. Upper panels: representative histograms. Lower panel: data are means ± s.e.m. of three independent experiments. b and c TILs from two human metastatic melanoma patients were cultured with or without autologous melanoma cells for two days in the presence 200 U ml −1 IL-2 +/−50 ng ml −1 human TNF. TIM-3 expression was next analysed by flow cytometry: histograms showing TIM-3 staining on TILs from patient 1 ( b ); bar graph depicting the fold increase in TIM-3 expression on TILs from patients 1 and 2 ( c )

Article Snippet: Additional antibodies used in this study were anti-mouse CD45 (BD Biosciences, BUV395, clone 30-F11; 1/200), anti-mouse Thy1 (Biolegend, APC-Cy7, clone 30-H12; 1/400), anti-mouse CD8 (BD Biosciences, BV605, clone 53-6.7; 1/200), anti-mouse CD4 (BD Biosciences, BUV496, clone GK1.5; 1/200), anti-mouse TIM-3 (eBioscience, PE-Cy7, clone RMT3-23; 1/200), anti-mouse LAG3 (Biolegend, PE, clone C9B7W; 1/100), anti-mouse PD-1 (eBioscience, FITC, clone J43; 1/200), anti-mouse CTLA-4 (eBioscience, APC, clone UC10-4B9; 1/100), anti-mouse TIGIT (Biolegend, Pe-Cy7, clone 1G9; 1/100), anti-mouse CD11c (eBioscience, PE-Cy7, clone N418; 1/100), anti-mouse CD3 (Biolegend, FITC, clone 145-2C11; 1/200), anti-mouse PD-L1 (BD Biosciences, PE, clone MIH5; 1/100), anti-mouse PD-L2 (BD Biosciences, APC, clone TY25; 1/100), anti-mouse Ki67 (BD Biosciences, FITC, clone B56; 1/10), anti-human TIM3 (BD Biosciences, BV421, clone 7D3; 1/50) and anti-human CD8 (BD Biosciences, BV605, clone SK1; 1/50).

Techniques: Expressing, Ex Vivo, Incubation, Flow Cytometry, Cell Culture, Staining

Journal: Nature Communications

Article Title: TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma

doi: 10.1038/s41467-017-02358-7

Figure Lengend Snippet: TNF blockade prevents TIM-3 up-regulation on TILs in response to anti-PD-1. WT mice were injected as described in the legend to Fig. . a – c TIM-3 expression level on CD8+ TILs and CD4+ TILs was determined by flow cytometry on tumors from WT mice at day 10 after B16K1 graft following injection of vehicle (PBS), anti-PD-1 (αPD-1), anti-TNF (αTNF) or a combination of both. Representative stainings ( a ) mean fluorescence intensity (MFI) of TIM-3 on CD8+ ( b ) and CD4+ TILs ( c ) measured in at least 5 tumors per group from one experiment are represented as Tukey boxes. b , c : Mann–Whitney U test: * p < 0.05; ** p < 0.01; *** p < 0.001). d and e C57BL/6 WT mice were intradermally and bilaterally grafted with 3 × 10 B16K1 melanoma cells prior to injection with vehicle, anti-PD-1 alone or combined with anti-TNF and/or anti-TIM-3 (10 mg kg −1 of each antibody) at days 6, 10, and 13 (d) (PBS ( n = 12), αPD-1 ( n = 9), αPD-1 + αTNF ( n = 14), αPD-1 + αTIM-3 ( n = 13), αPD-1 + αTNF + αTIM-3 ( n = 15), data from two experiments) or at days 13, 16, and 19 ( e ) ( n = 10 mice per group, data from one representative experiment out of two). Tumor volumes were determined with a calliper. Data are means ± s.e.m. (d) and e, two-way Anova: ** p < 0.01, *** p < 0.001 at day 35 ( d ) and day 21 ( e ); p = 0.08 at day 35 when comparing anti-PD-1 alone and anti-PD-1 plus anti-TIM-3 ( d )

Article Snippet: Additional antibodies used in this study were anti-mouse CD45 (BD Biosciences, BUV395, clone 30-F11; 1/200), anti-mouse Thy1 (Biolegend, APC-Cy7, clone 30-H12; 1/400), anti-mouse CD8 (BD Biosciences, BV605, clone 53-6.7; 1/200), anti-mouse CD4 (BD Biosciences, BUV496, clone GK1.5; 1/200), anti-mouse TIM-3 (eBioscience, PE-Cy7, clone RMT3-23; 1/200), anti-mouse LAG3 (Biolegend, PE, clone C9B7W; 1/100), anti-mouse PD-1 (eBioscience, FITC, clone J43; 1/200), anti-mouse CTLA-4 (eBioscience, APC, clone UC10-4B9; 1/100), anti-mouse TIGIT (Biolegend, Pe-Cy7, clone 1G9; 1/100), anti-mouse CD11c (eBioscience, PE-Cy7, clone N418; 1/100), anti-mouse CD3 (Biolegend, FITC, clone 145-2C11; 1/200), anti-mouse PD-L1 (BD Biosciences, PE, clone MIH5; 1/100), anti-mouse PD-L2 (BD Biosciences, APC, clone TY25; 1/100), anti-mouse Ki67 (BD Biosciences, FITC, clone B56; 1/10), anti-human TIM3 (BD Biosciences, BV421, clone 7D3; 1/50) and anti-human CD8 (BD Biosciences, BV605, clone SK1; 1/50).

Techniques: Injection, Expressing, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma

doi: 10.1038/s41467-017-02358-7

Figure Lengend Snippet: TNF expression is associated with an immune escape gene signature in human metastatic melanoma. a Heatmap for a selected list of genes encoding immune escape proteins in metastatic melanoma patients from the TCGA melanoma cohort ( n = 342), exhibiting high (80th percentile) and low (20th percentile) TNF expression in melanoma samples. Genes were clustered using a Euclidean distant matrix and average linkage clustering. b and c Correlation analysis between the expression of HAVCR2 (encoding TIM-3), PDCD1LG1 (encoding PD-L1), PDCD1LG2 (encoding PD-L2) and TNFA (encoding TNF) in melanoma samples from metastatic melanoma patients from the TCGA cohort ( n = 342) ( b ) and from patients treated with anti-PD-1 (our analysis of data published in ref. ) ( n = 13) ( c )

Article Snippet: Additional antibodies used in this study were anti-mouse CD45 (BD Biosciences, BUV395, clone 30-F11; 1/200), anti-mouse Thy1 (Biolegend, APC-Cy7, clone 30-H12; 1/400), anti-mouse CD8 (BD Biosciences, BV605, clone 53-6.7; 1/200), anti-mouse CD4 (BD Biosciences, BUV496, clone GK1.5; 1/200), anti-mouse TIM-3 (eBioscience, PE-Cy7, clone RMT3-23; 1/200), anti-mouse LAG3 (Biolegend, PE, clone C9B7W; 1/100), anti-mouse PD-1 (eBioscience, FITC, clone J43; 1/200), anti-mouse CTLA-4 (eBioscience, APC, clone UC10-4B9; 1/100), anti-mouse TIGIT (Biolegend, Pe-Cy7, clone 1G9; 1/100), anti-mouse CD11c (eBioscience, PE-Cy7, clone N418; 1/100), anti-mouse CD3 (Biolegend, FITC, clone 145-2C11; 1/200), anti-mouse PD-L1 (BD Biosciences, PE, clone MIH5; 1/100), anti-mouse PD-L2 (BD Biosciences, APC, clone TY25; 1/100), anti-mouse Ki67 (BD Biosciences, FITC, clone B56; 1/10), anti-human TIM3 (BD Biosciences, BV421, clone 7D3; 1/50) and anti-human CD8 (BD Biosciences, BV605, clone SK1; 1/50).

Techniques: Expressing

Journal: Immunology

Article Title: T‐bet‐mediated Tim‐3 expression dampens monocyte function during chronic hepatitis C virus infection

doi: 10.1111/imm.12686

Figure Lengend Snippet: T‐bet is up‐regulated and positively associated with Tim‐3 expression in monocyte/macrophages (M/Mφ) from patients with chronic hepatitis C virus (HCV) infection. (a) Cells were gated and then analysed for the percentage of T‐bet and Tim‐3. Peripheral blood mononuclear cells (PBMCs) were stained with anti‐CD14 and anti‐T‐bet antibodies, followed by flow cytometric analysis. The representative histograms for the expression of T‐bet in CD14+ M/Mφ from HCV patients and healthy participants are displayed, isotype controls (filled grey area) are used to identify the level of background staining. Summary data of each group (n = 25) are shown, and the horizontal bar represents the median value. ***P < 0·001. (b) Representative dot plots of T‐bet+ and Tim‐3+ CD14+ M/Mφ, and their correlation are shown. The open circles represent data from chronically HCV‐infected patients, and the filled circles represent data from healthy participants.

Article Snippet: Cell surface staining was carried out by using allophycocyanin‐conjugated anti‐Tim‐3 (R&D, Minneapolis, MN), followed by intracellular staining with phycoerythrin‐conjugated anti‐IL‐12 (BD Biosciences) and phycoerythrin‐Cy7‐conjugated anti‐T‐bet.

Techniques: Expressing, Infection, Staining

Journal: Immunology

Article Title: T‐bet‐mediated Tim‐3 expression dampens monocyte function during chronic hepatitis C virus infection

doi: 10.1111/imm.12686

Figure Lengend Snippet: T‐bet expression induced by hepatitis C virus (HCV) core requires activation of the c‐Jun N‐terminal kinase (JNK) pathway. (a–b) Peripheral blood mononuclear cells (PBMCs) from healthy participants were treated with HCV core in the presence of mitogen‐activate protein kinase (MAPK) kinase 1/2 (MEK1/2) inhibitor PD98059, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580 or DMSO (1 : 1000), respectively, for 24 hr. T‐bet (a) and Tim‐3 (b) expressions and summary data in monocyte/macrophages (M/Mφ) are shown. *P < 0·05, **P < 0·05, NS, not significant. (c) Representative dot plots for T‐bet and Tim‐3 expressions were detected in CD14+ M/Mφ treated with HCV core in the presence of specific inhibitors for different signal pathways.

Article Snippet: Cell surface staining was carried out by using allophycocyanin‐conjugated anti‐Tim‐3 (R&D, Minneapolis, MN), followed by intracellular staining with phycoerythrin‐conjugated anti‐IL‐12 (BD Biosciences) and phycoerythrin‐Cy7‐conjugated anti‐T‐bet.

Techniques: Expressing, Activation Assay

Journal: Immunology

Article Title: T‐bet‐mediated Tim‐3 expression dampens monocyte function during chronic hepatitis C virus infection

doi: 10.1111/imm.12686

Figure Lengend Snippet: T‐bet knockdown regulates hepatitis C virus (HCV) core‐induced Tim‐3 and interleukin‐12 (IL‐12) expression in THP‐1 cells. (a) THP‐1 cells were transfected with T‐bet small interfering RNA (siRNA) or a control siRNA for 6 hr, and stimulated with lipopolysaccharide (LPS)/R848 and HCV core for another 72 hr. T‐bet expression was measured by both flow cytometry (left panel) and RT‐PCR (right panel). (b) Representative dot plots showing Tim‐3 and IL‐12 expressions in THP‐1 cells transfected with T‐bet siRNA and control siRNA, respectively. (c) IL‐12 levels (left panel) were measured by ELISA from the culture supernatant, and the phosphorylation of signal transducer and activator of transcription 1 (STAT‐1) (right panel) was assessed by Western blot.

Article Snippet: Cell surface staining was carried out by using allophycocyanin‐conjugated anti‐Tim‐3 (R&D, Minneapolis, MN), followed by intracellular staining with phycoerythrin‐conjugated anti‐IL‐12 (BD Biosciences) and phycoerythrin‐Cy7‐conjugated anti‐T‐bet.

Techniques: Expressing, Transfection, Small Interfering RNA, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Immunology

Article Title: T‐bet‐mediated Tim‐3 expression dampens monocyte function during chronic hepatitis C virus infection

doi: 10.1111/imm.12686

Figure Lengend Snippet: Schematic model for hepatitis C virus (HCV) core/gC1qR interaction‐induced T‐bet/Tim‐3 expression via the c‐Jun N‐terminal kinase (JNK) pathway. Our results indicate that HCV core/gC1qR interaction induces T‐bet and Tim‐3 expression in monocyte/macrophages (M/Mφ). Blocking the JNK pathway may abrogate the HCV core/gC1qR‐induced up‐regulation of T‐bet and Tim‐3 expressions in these cells. T‐bet silencing significantly reduces the HCV core‐induced Tim‐3 expression, and increases interleukin‐12 (IL‐12) expression, suggesting that T‐bet is an essential transcriptional factor controlling Tim‐3‐mediated inhibition of M/Mφ functions.

Article Snippet: Cell surface staining was carried out by using allophycocyanin‐conjugated anti‐Tim‐3 (R&D, Minneapolis, MN), followed by intracellular staining with phycoerythrin‐conjugated anti‐IL‐12 (BD Biosciences) and phycoerythrin‐Cy7‐conjugated anti‐T‐bet.

Techniques: Expressing, Blocking Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Natural Killer Cells Induce CD8 + T Cell Dysfunction via Galectin-9/TIM-3 in Chronic Hepatitis B Virus Infection

doi: 10.3389/fimmu.2022.884290

Figure Lengend Snippet: CD8 + T cells are immune exhausted through the Gal-9/TIM-3 axis in active CHB patients. (A) Comparison of the levels of TIM-3 expressed in circulating CD8 + T cells in active CHB patients and HCs. (B) The percentages of PD-1 expressed in TIM-3 + CD8 + T cells and TIM-3 - CD8 + T cells in peripheral blood from active CHB patients. (C) The percentages of TIGIT expressed on TIM-3 + CD8 + T cells and TIM-3 - CD8 + T cells in peripheral blood from active CHB patients. (D) The percentage of TIM-3 expressed on CD8 + T cells in peripheral blood from active CHB patients with or without antiviral treatment, respectively. (E–I) Gal-9 + NK, Gal-9 - NK, and CD8 + T cells were sorted from active CHB patients, cocultured in vitro , and stimulated with anti-CD3/anti-CD28 for 3 days. The frequency of tetramer + CD8 + T cells (E) and the production of the cytokines IFN-γ, TNF-α, CD107a, and granzyme B (F-I) by CD8 + T cells were analyzed. Results are expressed as the mean ± SEM, and the number of samples (n) in each group was ≥ 3. Unpaired t-test was used to compare two independent groups. A paired t-test was used to compare paired samples. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: The following mouse anti-human monoclonal antibodies were used: BV510-CD3, BV605-CD3, FITC-CD56, PE-CD56, BV421-CD56, APC-H7-CD8, APC-CY7-CD8, PE-CY7-CD8, BV421-TIM-3, PE-PD-1, FITC-CD158a, 647-NKP30, APC-NKP46, APC-NKG2D, FITC-CD226, PE-CY7-CD69, FITC-IFN-γ, PE- IFN-γ, PerCP-IFN-γ, FITC-TNF-α, PE-TNF-α, APC-TNF-α, PE-CY7-CD107a, BV510-CD107a, PE-Granzyme B, V450-Granzyme B, BV510-Granzyme B, 647-Perforin (BD Biosciences); FITC-NKG2A, PE-CD158b (Miltenyi Biotec); PE-CY7-NKG2A (BECKMAN); 660-Galectin-9, PE-HLA-E (eBioscience); PE-TIM-3, PE-KIR3DL1 (R&D); APC-TIGIT, BV605-TIGIT (BioLegend), FITC-CD69 (Thermo fisher).

Techniques: Comparison, In Vitro

Journal: Frontiers in Immunology

Article Title: Natural Killer Cells Induce CD8 + T Cell Dysfunction via Galectin-9/TIM-3 in Chronic Hepatitis B Virus Infection

doi: 10.3389/fimmu.2022.884290

Figure Lengend Snippet: Exogenous Gal-9 induces circulating CD8 + T cell exhaustion in active CHB patients. (A–G) Coculture of CD8 + T cells from active CHB patients and rhGal-9 for 3 days and stimulation with anti-CD3/anti-CD28. (A) The percentage of TIM-3 + CD8 + T cells after culture with or without rhGal-9. (B) The percentage of CD69 + CD8 + T cells after culture with or without rhGal-9. (C) Comparison of CD8 + T cell early apoptosis after culture with and without rhGal-9. (D–G) Comparison of the expression of IFN-γ, TNF-α, CD107a, and granzyme B in CD8 + T cells from active CHB patients after culture with and without rhGal-9. Results are expressed as the mean ± SEM, and the number of samples (n) in each group was ≥ 3. A paired t-test was used to compare paired samples. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following mouse anti-human monoclonal antibodies were used: BV510-CD3, BV605-CD3, FITC-CD56, PE-CD56, BV421-CD56, APC-H7-CD8, APC-CY7-CD8, PE-CY7-CD8, BV421-TIM-3, PE-PD-1, FITC-CD158a, 647-NKP30, APC-NKP46, APC-NKG2D, FITC-CD226, PE-CY7-CD69, FITC-IFN-γ, PE- IFN-γ, PerCP-IFN-γ, FITC-TNF-α, PE-TNF-α, APC-TNF-α, PE-CY7-CD107a, BV510-CD107a, PE-Granzyme B, V450-Granzyme B, BV510-Granzyme B, 647-Perforin (BD Biosciences); FITC-NKG2A, PE-CD158b (Miltenyi Biotec); PE-CY7-NKG2A (BECKMAN); 660-Galectin-9, PE-HLA-E (eBioscience); PE-TIM-3, PE-KIR3DL1 (R&D); APC-TIGIT, BV605-TIGIT (BioLegend), FITC-CD69 (Thermo fisher).

Techniques: Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: Natural Killer Cells Induce CD8 + T Cell Dysfunction via Galectin-9/TIM-3 in Chronic Hepatitis B Virus Infection

doi: 10.3389/fimmu.2022.884290

Figure Lengend Snippet: The function of anti-HBV CD8 + T cells in active CHB patients was restored after blocking the Gal-9/TIM-3 pathway. (A-E) PBMCs isolated from active CHB patients were cultured in vitro with anti-human Gal-9 antibody, anti-human TIM-3 antibody, or control IgG and stimulated with HBV peptide (core18-27) for 10 days. The function of HBV-specific CD8 + T cells was analyzed. (F) PBMCs depleted of NK cells (PBMCs-ΔNK) were cultured in vitro with anti-Gal-9 or control IgG and stimulated with HBV peptide (core18-27) for 10 days. The production of the cytokines IFN-γ, TNF-α, CD107a, granzyme B, and perforin by CD8 + T cells was analyzed. (G–L) NK and CD8 + T cells were sorted from active CHB patients, cocultured in vitro with anti-human Gal-9 antibody, anti-human TIM-3 antibody, or control IgG and stimulated with anti-CD3/anti-CD28 for 3 days. The production of the cytokines IFN-γ, TNF-α, CD107a, granzyme B, and perforin by CD8 + T cells (G–K) and early CD8 + T cell apoptosis (L) were analyzed. Results are expressed as the mean ± SEM, and the number of samples (n) in each group was ≥ 3. One-way ANOVA test was conducted for three-group comparisons. A paired t-test was used to compare paired samples. *P < 0.05; **P < 0.01; n.s., not significant.

Article Snippet: The following mouse anti-human monoclonal antibodies were used: BV510-CD3, BV605-CD3, FITC-CD56, PE-CD56, BV421-CD56, APC-H7-CD8, APC-CY7-CD8, PE-CY7-CD8, BV421-TIM-3, PE-PD-1, FITC-CD158a, 647-NKP30, APC-NKP46, APC-NKG2D, FITC-CD226, PE-CY7-CD69, FITC-IFN-γ, PE- IFN-γ, PerCP-IFN-γ, FITC-TNF-α, PE-TNF-α, APC-TNF-α, PE-CY7-CD107a, BV510-CD107a, PE-Granzyme B, V450-Granzyme B, BV510-Granzyme B, 647-Perforin (BD Biosciences); FITC-NKG2A, PE-CD158b (Miltenyi Biotec); PE-CY7-NKG2A (BECKMAN); 660-Galectin-9, PE-HLA-E (eBioscience); PE-TIM-3, PE-KIR3DL1 (R&D); APC-TIGIT, BV605-TIGIT (BioLegend), FITC-CD69 (Thermo fisher).

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

Journal: Oncoimmunology

Article Title: Langerhans-type dendritic cells electroporated with TRP-2 mRNA stimulate cellular immunity against melanoma: Results of a phase I vaccine trial

doi: 10.1080/2162402X.2017.1372081

Figure Lengend Snippet: CD8 T cell subset analysis and Treg content after LC vaccines. PBMCs were analyzed by flow cytometry to assess fold-changes in lymphocyte composition at one and three months post-vaccination, compared with pre-vaccine baseline. (A) CD8 T-cell subsets: naïve (TN; CCR7+CD45ROneg), central memory (TCM; CCR7+CD45RO+), effector (TEF; CCR7negCD45ROneg), and effector memory (TEF; CCR7negCD45RO+). (B) Expression of inhibitory receptors (CTLA-4, LAG-3, PD-1, and TIM-3) by CD8 T cells. (C) ICOS expression by CD4 and CD8 T cells. (D) Regulatory T cell (Treg; CD3+CD4+CD25bright CD127neg) to CD3+CD8+CD25+ effector T cell ratios. For all panels (A-D), pooled data (mean ± SD; P = NS) from seven patients are shown.

Article Snippet: FITC-, PE-, PE-Texas Red-, ECD-, APC-, PE-Cy5-, PE-Cy7-, PerCP-Cy5.5-, Pacific Blue-, and AF700-conjugated mouse anti-human mAbs included anti-CD3, anti-CD4, anti-CD8, anti-CD14, anti-CD25, anti-CD45RA, anti-CD45RO, anti-CD80, anti-CD86, anti-CTLA-4, anti-HLA-DR, anti-IL-2, anti-CD83, anti-Mip-1-β (Beckman Coulter), anti-CD127, anti-IFN-γ, anti-LAG-3, anti-PD-1, anti-TNF-α (eBioscience), anti-CCR7, and anti-TIM-3 (R&D Systems).

Techniques: Vaccines, Flow Cytometry, Expressing