Journal: Cell Genomics

Article Title: Proteomics of immune cells from liver tumors reveals immunotherapy targets

doi: 10.1016/j.xgen.2023.100331

Figure Lengend Snippet:

Article Snippet: Then, cells were stained with Zombie UV viability dye (Biolegend) to exclude dead cells and then surface markers were stained with the following antibodies: anti-CD8α Super Bright 702 (1/200, ThermoFisher), anti-CD45.1 Super Bright 436 (1/100, ThermoFisher), anti-CD45.2 PerCP-Cy5.5 (1/100, ThermoFisher), anti-PD1 PE (1/200, ThermoFisher), anti-Tim3 PE-Cy7 (1/200, ThermoFisher) and anti-LAG3 FITC (1/200, Thermofisher).

Techniques: Recombinant, Sequencing, Modification, Transfection, DNA Extraction, Enzyme-linked Immunosorbent Assay, Cell Isolation, Gene Expression, Mouse Assay, Software

Journal: Frontiers in Immunology

Article Title: Peripheral PD-1 + T Cells Co-expressing Inhibitory Receptors Predict SVR With Ultra Short Duration DAA Therapy in HCV Infection

doi: 10.3389/fimmu.2019.01470

Figure Lengend Snippet: Expression of inhibitory receptors on global T lymphocytes in DAA treated SVR and Relapse groups. (A) Gating strategy for identifying T cells expressing PD-1 and other inhibitory receptors using appropriate FMO controls. For B-E, left panels in each figure show change in indicated subset's frequency relative to baseline (0) after treatment initiation (week 4 and 16) in relapse and SVR groups; right panels show grouped averages in SVR, relapse (Rlps) and healthy Controls. (B) Baseline (D0) PD-1+CD160+ and PD-1+Tim-3+ CD8+ T cells, (C) D0 PD-1+CD160+, PD-1+Blimp-1+ CD4+ T cells, (D) Week 4 (W4 or EOT) PD-1hi, PD-1+2B4+ PD-1+KLRG1+, and PD-1+Blimp-1+ CD8+ T cells and (E) Week 4 (W4 or EOT) PD-1+CD39+, PD-1+Blimp-1+ CD4+ T cells in SVR, relapse and healthy controls. P- values were determined by 1 Way ANOVA with Bonferroni's-Dunn's post-hoc test for pairwise multiple comparisons, p ≤ 0.05 is significant. Controls, healthy control; D0, baseline; W4, week 4/End of treatment (EOT); W16, 12 weeks after EOT. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Phenotyping was performed to assess the expression of multiple T cell lineage, activation and inhibitory receptors by using the following monoclonal antibodies in two different panels containing: Fixable Aqua Live/dead (Invitrogen), anti-CD3 Alexa Fluor 700 (BD Biosciences), anti-CD4 APC-Cy7 (BD Biosciences), anti-CD8 Brilliant Violet 510 (Biolegend), anti-CD244 PercP-Cy5.5 (Biolegend), anti-CD39 FITC (Biolegend), anti-Tim-3 PE-CY7 (eBioscience), anti-PD-1 Brilliant Violet 421 (Biolegend), anti- Blimp-1 APC (eBioscience), anti-CCR7 FITC (Biolegend), anti-CD45RO APC-ef780 (Biolegend), anti-CD160 PE-Cy7 (Biolegend), anti-CTLA-4 PE-Cy5 (Biolegend).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Peripheral PD-1 + T Cells Co-expressing Inhibitory Receptors Predict SVR With Ultra Short Duration DAA Therapy in HCV Infection

doi: 10.3389/fimmu.2019.01470

Figure Lengend Snippet: HCV specific CD8+ T cells in PD-1+ CD8 T cell subsets. (A) Gating strategy to detect HCV tetramer (tet+) CD8+ T cells. PD-1 and 2B4 expression among global (gray) and HCV tet+ (red) CD8+ T cells show most tet+ cells in PD-1+2B4+ subset. (B) Example of CD8 T cell subsets distinguished by PD-1 and 2B4 expression (2B4+PD-1-, 2B4-PD-1-, 2B4-PD-1+, 2B4+PD-1+) show relative abundance of HCV tet+ cells in four subsets. (C) Frequency of HCV tet+ T cells in CD8 T cells subsets distinguished by PD-1/2B4, PD-1/CD160 and PD-1/Tim-3 show average tet+ cells in different subsets ( N = 5 HCV HLA-A*02 patients). Statistical significances determined by 1 Way ANOVA with Turkey's Multiple Comparison Test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Phenotyping was performed to assess the expression of multiple T cell lineage, activation and inhibitory receptors by using the following monoclonal antibodies in two different panels containing: Fixable Aqua Live/dead (Invitrogen), anti-CD3 Alexa Fluor 700 (BD Biosciences), anti-CD4 APC-Cy7 (BD Biosciences), anti-CD8 Brilliant Violet 510 (Biolegend), anti-CD244 PercP-Cy5.5 (Biolegend), anti-CD39 FITC (Biolegend), anti-Tim-3 PE-CY7 (eBioscience), anti-PD-1 Brilliant Violet 421 (Biolegend), anti- Blimp-1 APC (eBioscience), anti-CCR7 FITC (Biolegend), anti-CD45RO APC-ef780 (Biolegend), anti-CD160 PE-Cy7 (Biolegend), anti-CTLA-4 PE-Cy5 (Biolegend).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy

doi: 10.1007/s00262-020-02507-w

Figure Lengend Snippet: Increased mRNAs and proteins from PD-L1, galectin-9 and HVEM genes in spleen DCs from breast cancer-bearing mice. a Detection of miRNAs encoding PD-L1, galectin-9 and HVEM by RT-qPCR. DCs were isolated from spleens of wild type and breast tumor-bearing mice using CD11c magnetically-labeled beads (Miltenyi Biotec, USA). CD11c cDNA was used to perform RT-qPCR and normalized to β-action expression. b Detection of inhibitory receptors ligand on CD11c+ DCs by flow cytometry. Spleen cells were collected from naive mice and breast tumor-bearing mice. Flow cytometry was performed with FITC-labeled CD11c, PerCP-eFluor 710-labeled PD-L1, PE-CY7-labeled galectin-9, APC-labeled HVEM antibody staining. Data are representative of three mice from at least three independent experiments. Two-tailed unpaired Student’s t tests were performed to determine statistical significance. Error bars represent standard deviation (*p < 0.05; n.s., p > 0.05)

Article Snippet: To measure in vivo IR levels, secreted cytokine levels, and T reg detection, lymphocytes isolated from tumor-bearing mice treated with miR-5119 mimic/inhibitor or control miRNA-engineered DCs were stained with anti-CD8-FITC, anti-PD-1-PerCP-eFluor 710, anti-TIM-3-PE-CY7, anti-BTLA-APC, anti-IL-2-PE, anti-TNF-α-PE, anti-IFN-γ-PE, anti-CD4-PE-CY5, anti-CD25-PE, and anti-Foxp3-FITC (eBioscience, San Diego, CA).

Techniques: Quantitative RT-PCR, Isolation, Labeling, Expressing, Flow Cytometry, Staining, Two Tailed Test, Standard Deviation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: miRNA-5119 regulates immune checkpoints in dendritic cells to enhance breast cancer immunotherapy

doi: 10.1007/s00262-020-02507-w

Figure Lengend Snippet: miR-5119 mimic-transfected DC vaccines suppress T cell exhaustion in vivo. a miR-5119 mimic-engineered DC vaccines increased exhausted CD8+ T cell proliferation in vivo. Spleen cells were isolated from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA engineered DC vaccine-treated breast tumor-bearing mice. Isolated cells were labeled with CFSE and seeded in 96 well plate at 1 × 106 cells/well. Wild type BALB/c bone marrow DCs were pulsed with 4T1 freeze–thaw antigen (50 ng/ml) and co-cultured with spleen cells from three groups (DC: T cells = 1:10) at 37 °C in a humidified atmosphere and 5% CO2 for 3 days. T-Cell proliferation was detected by flow cytometry. b miR-5119 mimic-engineered DC vaccines reduced apoptosis in exhausted CD8+ T cells in vivo. Spleen cells were isolated from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA DC vaccine-treated breast tumor-bearing mice. CD8+ T cells were purified from the collected splenocytes using magnetically-labeled beads. Apoptosis was detected by flow cytometry using FITC-Annexin V/PI detection. c miR-5119 mimic-engineered DC vaccines inhibited immune checkpoint molecule levels in exhausted CD8+ T cells in vivo. Spleen cells were collected from miR-5119 mimic/inhibitor DC vaccine-treated or control miRNA DC vaccine-treated breast tumor-bearing mice. Flow cytometry was performed after FITC-labeled CD8, PerCP-eFluor 710-labeled PD-1, PE-CY7-labeled TIM-3, APC-labeled BTLA antibody staining. Data are representative of three mice per group. One-way ANOVA analysis was performed to determine statistical significance. Error bars represent standard deviation (*p < 0.05; n.s., p > 0.05)

Article Snippet: To measure in vivo IR levels, secreted cytokine levels, and T reg detection, lymphocytes isolated from tumor-bearing mice treated with miR-5119 mimic/inhibitor or control miRNA-engineered DCs were stained with anti-CD8-FITC, anti-PD-1-PerCP-eFluor 710, anti-TIM-3-PE-CY7, anti-BTLA-APC, anti-IL-2-PE, anti-TNF-α-PE, anti-IFN-γ-PE, anti-CD4-PE-CY5, anti-CD25-PE, and anti-Foxp3-FITC (eBioscience, San Diego, CA).

Techniques: Transfection, Vaccines, In Vivo, Isolation, Labeling, Cell Culture, Flow Cytometry, Purification, Staining, Standard Deviation

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