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: Frontiers in Immunology

Article Title: TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

doi: 10.3389/fimmu.2019.01644

Figure Lengend Snippet: PD-L1, PD-L2, PD-1, CD8, and CD4 expression in p16-positive and p16-negative HNSCC. PD-L1, PD-L2, PD-1, CD8, and CD4 expression was assessed in tumor biopsy tissue from five p16-positive and four p16-negative HNSCC patients using immuno-histochemistry (details in Methods). Representative staining (scale bars, 100 μm) and cumulative data of marker expression (grading scale PD-L1/PD-L2: 1, low; 2, moderate; 3 high expression; grading scale PD-1, CD8, CD4: 1, <50 cells/field; 2, 50–150 cells/field; 3, >150 cells/field).

Article Snippet: The following primary antibodies were used: mouse monoclonal CD4 (#NCL-L-CD4-368); mouse monoclonal CD8 (#NCL-L-CD8-4B11); mouse monoclonal CD68 (#NCL-L-CD68) and mouse monoclonal CD163 (#NCL-L-CD163) (all from Novocastra, Leica Biosystems); rabbit polyclonal anti-inducible nitric oxide synthase (iNOS, Abcam; #ab3523); goat polyclonal PD-1 (#AF1086); and goat polyclonal PD-L2 (#AF1224) (both R&D Systems); and mouse monoclonal CD274 (PD-L1) (Biolegend; #329702).

Techniques: Expressing, Immunohistochemistry, Staining, Marker

Journal: Frontiers in Immunology

Article Title: TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

doi: 10.3389/fimmu.2019.01644

Figure Lengend Snippet: HPV-positive HNSCCs increase PD-L1 and PD-L2 on fibroblasts. PD-L1 and PD-L2 expression on primary BJ human fibroblasts, HPV-positive (SCC154), and HPV-negative (SCC099) HNSCC cell lines (HNSCCs) was detected by flow cytometry. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts (black histograms), HPV-positive (red histograms), or HPV-negative (blue histograms) HNSCCs (A) . Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 5) in fibroblasts, HPV-positive, and HPV-negative HNSCCs (B) . Fibroblasts were cultured alone or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs. Fibroblasts were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 ( C ; red histograms) or HPV-negative SCC099 ( D ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 5) on fibroblasts cultured alone (w/o) or co-cultured directly with HNSCC cells (E) . HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with fibroblasts. HNSCCs were identified in co-cultures by EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured directly with fibroblasts ( F ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on HPV-positive SCC154 cultured alone or co-cultured directly with fibroblasts (G) . Illustrative histograms show PD-L1 and PD-L2 expression on HPV-negative SCC099 cultured alone (black histograms) or co-cultured directly with fibroblasts ( H ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on HPV-negative SCC099 cultured alone or co-cultured directly with fibroblasts (I) . * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.

Article Snippet: The following primary antibodies were used: mouse monoclonal CD4 (#NCL-L-CD4-368); mouse monoclonal CD8 (#NCL-L-CD8-4B11); mouse monoclonal CD68 (#NCL-L-CD68) and mouse monoclonal CD163 (#NCL-L-CD163) (all from Novocastra, Leica Biosystems); rabbit polyclonal anti-inducible nitric oxide synthase (iNOS, Abcam; #ab3523); goat polyclonal PD-1 (#AF1086); and goat polyclonal PD-L2 (#AF1224) (both R&D Systems); and mouse monoclonal CD274 (PD-L1) (Biolegend; #329702).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

doi: 10.3389/fimmu.2019.01644

Figure Lengend Snippet: PD-L1 and PD-L2 expression in co-cultures of macrophages and HPV-positive or HPV-negative HNSCCs. Macrophages were cultured alone (w/o) or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCC cell lines (HNSCCs). Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone ( A ; black histograms). Macrophages were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 ( B ; red histograms) or HPV-negative SCC099 ( C ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on macrophages cultured alone (w/o) or co-cultured directly with HNSCCs (D) . HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with macrophages. HNSCCs were identified in co-cultures by EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured directly with macrophages ( E ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on HPV-positive SCC154 cultured alone or co-cultured directly with macrophages (F) . Illustrative histograms show PD-L1 and PD-L2 expression on HPV-negative SCC099 cultured alone (black histograms) or co-cultured directly with macrophages ( G ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on HPV-negative SCC099 cultured alone or co-cultured directly with macrophages (H) . * p < 0.05; ** p < 0.01; ns, not significant ( D : one-way ANOVA with Bonferroni correction for multiple comparisons; F,H : unpaired two-tailed Student's t -test) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.

Article Snippet: The following primary antibodies were used: mouse monoclonal CD4 (#NCL-L-CD4-368); mouse monoclonal CD8 (#NCL-L-CD8-4B11); mouse monoclonal CD68 (#NCL-L-CD68) and mouse monoclonal CD163 (#NCL-L-CD163) (all from Novocastra, Leica Biosystems); rabbit polyclonal anti-inducible nitric oxide synthase (iNOS, Abcam; #ab3523); goat polyclonal PD-1 (#AF1086); and goat polyclonal PD-L2 (#AF1224) (both R&D Systems); and mouse monoclonal CD274 (PD-L1) (Biolegend; #329702).

Techniques: Expressing, Cell Culture, Two Tailed Test, Control, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

doi: 10.3389/fimmu.2019.01644

Figure Lengend Snippet: Conditioned medium from HPV-positive HNSCCs up-regulates PD-L1 and PD-L2 on fibroblasts. Fibroblasts were cultured alone or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured with conditioned medium from HPV-positive SCC154 ( A ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 13) on fibroblasts cultured alone (w/o), co-cultured directly with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM) (B) . HPV-positive (SCC154) HNSCCs were cultured alone or co-cultured in direct contact with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured with conditioned medium from fibroblasts ( C ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on HPV-positive SCC154 cultured alone (w/o), co-cultured directly with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM) (D) . Macrophages were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on macrophages for the indicated conditions (E) . HPV-positive SCC154 HNSCCs were cultured alone (w/o) or co-cultured in direct contact with macrophages (MFs direct) or with conditioned medium from macrophages (MFs CM). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 2) on HPV-positive SCC154 HNSCCs for the indicated conditions (F) . ** p < 0.01; **** p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI = mean fluorescence intensity.

Article Snippet: The following primary antibodies were used: mouse monoclonal CD4 (#NCL-L-CD4-368); mouse monoclonal CD8 (#NCL-L-CD8-4B11); mouse monoclonal CD68 (#NCL-L-CD68) and mouse monoclonal CD163 (#NCL-L-CD163) (all from Novocastra, Leica Biosystems); rabbit polyclonal anti-inducible nitric oxide synthase (iNOS, Abcam; #ab3523); goat polyclonal PD-1 (#AF1086); and goat polyclonal PD-L2 (#AF1224) (both R&D Systems); and mouse monoclonal CD274 (PD-L1) (Biolegend; #329702).

Techniques: Cell Culture, Expressing, Control, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

doi: 10.3389/fimmu.2019.01644

Figure Lengend Snippet: Blockade of IFN-γ, TNF-α, or CD81 does not affect PD-L1 and PD-L2 up-regulation by HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) as indicated. Graphs (A) show IFN-γ and TNF-α levels in culture supernatants (mean ± SEM; n = 4). The dashed red line indicates the lowest value (15.6 pg/ml) of the dynamic range for the ELISA assays used. Neutralizing antibodies anti-IFN-γ (B,C) , anti-TNF-α (D,E) , or anti-CD81 (F,G) were added to the cultures as indicated. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 or with conditioned medium from HPV-positive SCC154 alone (red histograms) or in the presence of blocking antibodies (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 3) on fibroblasts for the indicated treatments. ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.

Article Snippet: The following primary antibodies were used: mouse monoclonal CD4 (#NCL-L-CD4-368); mouse monoclonal CD8 (#NCL-L-CD8-4B11); mouse monoclonal CD68 (#NCL-L-CD68) and mouse monoclonal CD163 (#NCL-L-CD163) (all from Novocastra, Leica Biosystems); rabbit polyclonal anti-inducible nitric oxide synthase (iNOS, Abcam; #ab3523); goat polyclonal PD-1 (#AF1086); and goat polyclonal PD-L2 (#AF1224) (both R&D Systems); and mouse monoclonal CD274 (PD-L1) (Biolegend; #329702).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Blocking Assay, Control, Staining, Fluorescence

Journal: Frontiers in Immunology

Article Title: TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2

doi: 10.3389/fimmu.2019.01644

Figure Lengend Snippet: The TLR9 antagonists ODN TTAGGG and chloroquine inhibit PD-1 ligands up-regulation on fibroblasts co-cultured with HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) in the presence or absence of the TLR9 antagonists ODN TTAGGG (ODN) or chloroquine (CHQ). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 (red histograms) or co-cultured directly with HPV-positive SCC154 in the presence of ODN (A) or CHQ (H) (green histograms). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), cultured with conditioned medium from HPV-positive SCC154 (red histograms) or with conditioned medium from HPV-positive SCC154 in the presence of ODN (B) or CHQ (I) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 6) on fibroblasts for the indicated treatments (C,J) . Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or in the presence of ODN (D) or CHQ (K) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 4) on fibroblasts for the indicated treatments (E,L) . HPV-positive SCC154 were cultured alone or co-cultured in direct contact (direct) with fibroblasts in the presence or absence of TLR9 inhibitor ODN TTAGGG (ODN) or chloroquine (CHQ). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms), co-cultured directly with fibroblasts alone (red histograms), or co-cultured directly with fibroblasts in the presence of ODN (F) or CHQ (M) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean ± SEM; n = 6) on HPV-positive SCC154 cultured alone or for the indicated co-culture conditions (G,N) . * p < 0.05; ** p < 0.01; **** p < 0.0001; ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.

Article Snippet: The following primary antibodies were used: mouse monoclonal CD4 (#NCL-L-CD4-368); mouse monoclonal CD8 (#NCL-L-CD8-4B11); mouse monoclonal CD68 (#NCL-L-CD68) and mouse monoclonal CD163 (#NCL-L-CD163) (all from Novocastra, Leica Biosystems); rabbit polyclonal anti-inducible nitric oxide synthase (iNOS, Abcam; #ab3523); goat polyclonal PD-1 (#AF1086); and goat polyclonal PD-L2 (#AF1224) (both R&D Systems); and mouse monoclonal CD274 (PD-L1) (Biolegend; #329702).

Techniques: Cell Culture, Expressing, Co-Culture Assay, Control, Staining, Fluorescence