Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Schematic diagram showing the establishment of humanized mice (humice) with human immune system reconstituted in NIKO mice. The presence of human CD45 + cells, NK cells, CD4 + and CD8 + T cells in the mice’s peripheral system was validated by flow cytometry. b Primary LM2 tumor size in humice (control, n = 14; Keytruda, n = 14; ENT, n = 12; PX478, n = 14; ENT + Keytruda, n = 16; PX478 + Keytruda, n = 16) and NIKO mice (control, n = 10; ENT + Keytruda, n = 10; PX478 + Keytruda, n = 10), at Day 21 of treatments. c Lung metastasis of humice (control, n = 6; Keytruda, n = 6; ENT, n = 6; PX478, n = 6; ENT + Keytruda, n = 7; PX478 + Keytruda, n = 7) and NIKO mice (control, n = 5; ENT + Keytruda, n = 5; PX478 + Keytruda, n = 5) bearing LM2 tumors at Day 35 assessed by bioluminescence (BLI) measurement. d Representative bioluminescence (BLI) images showing the lung metastasis of humice and NIKO mice. e Flow cytometric analysis of LM2 tumors harvested from humanized mice. IFNγ, TNFα, and granzyme B expression was examined in tumor-infiltrating human CD8 + T cells and NK cells. N = 5 for each group. f Flow cytometry analysis of LM2 tumors harvested from humanized mice. Expressions of human PD-L1 and PD-L2 were examined in total living cells dissociated from LM2 tumors. N = 5 for each group. Quantification data of flow cytometry ( e , f ) are presented as a box and whiskers, with median values and whiskers of minimum and maximum values. Data for b and c were presented as mean ± SD . P values were determined by one-way ( e , f ) or two-way ( b , c ) ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: The following antibodies were used for staining, anti-activated pimonidazole FITC antibody (Hypoxyprobe, CAT# HP2-200kit, dilution 1:200), anti-mouse HIF1α APC antibody (R&D Systems, CAT# IC1935A, dilution 1:50), anti-mouse CD3 BV421 antibody (BD Biosciences, CAT# 564008, dilution 1:100), anti-mouse CD45 Percp-Vio700 antibody (Miltenyi Biotec, CAT# 130-110-663, dilution 1:100) anti-mouse CD8 APC-Vio770 antibody (Miltenyi Biotec, CAT# 130-120-737, dilution 1:100), anti-mouse Nkp46 APC antibody (Miltenyi Biotec, CAT# 130-112-202, dilution 1:100), anti-mouse CD4 BV650 antibody (Biolegend, CAT# 563747, dilution 1:100), anti-mouse TIM-3 BV711 antibody (Biolegend, CAT# 119727, dilution 1:100), anti-mouse PD-1 PE-Vio770 (Miltenyi Biotec, CAT# 130-120-391, dilution 1:100), anti-mouse IFNγ PE (Miltenyi Biotec, CAT# 130-117-352, dilution 1:100), anti-mouse TNFα BV711 (BD Biosciences, CAT# 563944, dilution 1:100), anti-mouse/human granzyme B FITC (Miltenyi Biotec, Cat#130-118-430, dilution 1:100), anti-mouse PD-L1 BV786 antibody (BD Biosciences, CAT# 741014, dilution 1:100), anti-mouse PD-L2 FITC antibody (Miltenyi Biotec, Cat# 130-102-222, dilution 1:100), anti-human CD45 FITC antibody (BD Biosciences, CAT# 304006, dilution 1:100), anti-human CD3 PE antibody (Biolegend, CAT# 300308, dilution 1:100) anti-human CD8 APC-Cy7 antibody (BD Biosciences, CAT# 557834, dilution 1:100), anti-human CD56 BV711 antibody (Biolegend, CAT# 318336, dilution 1:100), anti-human CD4 APC antibody (Biolegend, CAT# 300514, dilution 1:100), anti-human IFNγ BV785 (Biolegend, CAT# 502542, dilution 1:100), anti-human TNFα BV650 (Biolegend, CAT# 502398, dilution 1:100), anti-human Granzyme B BV421 (BD Biosciences, Cat# 563389, dilution 1:100), anti-human PD-L1 PE-Cy7 antibody (Biolegend, CAT# 374506, dilution 1:100), anti-human PD-L2 PE antibody (Miltenyi Biotec, CAT# 130-098-530, dilution 1:100).

Techniques: Flow Cytometry, Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 3. Infection of DCs with HHV-8 is blocked by anti DC-SIGN mAb. A, Immunofluorescence results on DCs that were left untreated or treated with either anti-DC-SIGN mAb (clone 120507) or anti-CD11a mAb, infected with HHV-8, incubated, and stained with anti-K8.1A/B mAb (red) at 24 h. Uninfected DCs were used as controls. B, Immunoflu- orescence results on DCs that were either treated with anti-DC-SIGN mAb (clone 120507) or left untreated, infected, and stained at 24 h with anti- DC-SIGN mAb (green) and anti-ORF 59 mAb (red). The overlay of com- bined colors for anti-DC-SIGN and ORF 59 is shown. Cells were coun- terstained with DAPI (blue) (600). Data are from one experiment representative of eight independent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Infection, Incubation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 4. DC-SIGN expression renders resistant cells susceptible to HHV-8 infection. A, Immunofluorescence results on K562 and K562-DC- SIGN cells that were infected with HHV-8 and stained with anti-K8.1A/B mAb at 24 h (red). B, Immunofluorescence results on B-LCL and B-LCL DC-SIGN that were infected with HHV-8 and stained after 24 h with anti- K8.1A/B mAb (red). Cells were counterstained with DAPI (600). Data are from one experiment representative of five independent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Expressing, Infection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 5. HHV-8 binds to DC-SIGN. A, Inhibition of binding of radio- actively labeled HHV-8 by treatment of target cells with anti-DC-SIGN mAb. DCs or B-LCL-DC-SIGN were pretreated with anti-DC-SIGN mAb (clone 120507) or mannan, or left untreated. Each bar represents the mean percent of binding inhibition ( SE) (compared with untreated cells) from two duplicate determinations. B, Inhibition of binding of radioactively labeled HHV-8 by treatment of virus with soluble DC-SIGN. Results are the mean ( SE) per- centage of inhibition of binding of soluble DC-SIGN-treated HHV-8 com- pared with binding of radiolabeled untreated virus to each cell type from two determinations. C, Dose response of inhibition of virus binding to DCs by treatment with anti-DC-SIGN mAb. Each bar represents the mean of duplicate reactions ( SE) from duplicate determinations. Data are from one experiment representative of three independent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Inhibition, Binding Assay, Labeling, Virus

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 6. HHV-8 infection of IL-13-activated macrophages is related to DC-SIGN expression. A, Flow cytometric analysis showing expression of DC-SIGN on HHV-8-infected (empty histogram, broken line) or un- infected (empty histogram, solid line) IL-13-activated macrophages. Full histogram, isotype controls. B, Im- munofluorescence results on IL-13-activated macro- phages that were infected with HHV-8 for 24 h and stained for ORF 59 (red) and DC-SIGN (green). The overlay of combined colors for anti-DC-SIGN and ORF59 is shown. C, Immunofluorescence results on IL- 13-activated macrophages that were pretreated with anti- DC-SIGN mAb (clone 120507) or mouse IgG, infected with HHV-8 for 24 h and stained for anti-K8.1 mAb (red). Cells were counterstained with DAPI (600). Data are from one experiment representative of four in- dependent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Infection, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages.

doi: 10.4049/jimmunol.176.3.1741

Figure Lengend Snippet: FIGURE 7. Effect of HHV-8 in- fection of DCs on expression of DC- SIGN and costimulatory molecules. A, DC-SIGN expression on unin- fected or HHV-8 infected DCs. Data are mean MFI (SE) from seven in- dependent experiments. B, Expres- sion of HLA-ABC, HLA-DR, CD83, and DC-SIGN on infected DCs. Blue histogram, HHV-8-infected DCs; yellow histogram, uninfected DCs; empty histogram, fine line, and bro- ken line isotype controls for the infected and uninfected DCs, respec- tively. Data are from one experiment representative of 10 independent ex- periments. C, Confocal microscopy of HHV-8-infected DCs stained with anti-DC-SIGN (green) and anti-ORF 59 (red) mAbs at 24 h (left panel) and 48 h (center panel) after infection. Uninfected DCs served as controls (right panel). Data are from one ex- periment representative of two inde- pendent experiments.

Article Snippet: For blocking studies, cells were pretreated with 20 g/ml anti-human-DC-SIGN mAb (clone 120507; R&D Systems, or clone DCN46; BD Biosciences), anti-CD11a mAb (BD Biosciences), mouse IgG (Sigma-Aldrich), or 100 g/ml mannan (Sigma-Aldrich), for 1 h at 4°C before exposure to HHV-8.

Techniques: Expressing, Infection, Confocal Microscopy, Staining