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 Heatmap showing Pearson’s correlation between hypoxic signature genes expression and immune-related genes expression in basal TNBC samples ( n = 98) in TCGA dataset. b Scatter plots (upper panel) and Pearson’s correlation coefficients (lower panel) showing the expression of hypoxic gene signatures and immune-related genes in breast cancers in TCGA dataset (Basal, n = 98; HER2, n = 58; Luminal A, n = 231; Luminal B, n = 129). Regression lines with a 95% confidence interval (gray fill) are shown in the scatter plots. c Images of fluorescent staining of human TNBC samples. Scale bar, 50 µm. Data were representative of 30 independent experiments. d Quantification of infiltrating IFNγ + CD8 + T cell number in HIF1α − and HIF1α + regions of human TNBC sample ( n = 30). P values were determined with paired two-tailed t -test. e Correlation between infiltrating IFNγ + CD8 + T cell count and HIF1α fluorescent intensity in human TNBC samples ( n = 30). The simple linear regression R 2 and P values (two-tailed) are calculated. Dot plot is shown with regression line and 95% confidence interval. f Representative images of fluorescent staining of mouse 4T1 tumor samples. Scale bar, 50 µm. Data represents three independent experiments. g Flow cytometry (left panel) demonstrating the gating strategy of activated-PIM high (H) and activated-PIM low (L) populations in living cells dissociated from 4T1 tumors. The CD8 + T cell percentage and IFNγ expression in CD8 + T cells was quantified (right panel, n = 6). Data were presented as box and whiskers, with median value and whiskers of minimum and maximum values. P values were determined with an unpaired two-tailed t -test. h Kaplan–Meier overall survival (OS) and distant metastasis-free survival (DMFS) analysis of the indicated gene signatures in TNBC patients. The publicly available data used in Fig. 1a, b are available in the TCGA database under accession code BRCA.exp.547.med.txt [ https://gdc.cancer.gov/about-data/publications/brca_2012 ]. The publicly available data used in h are available in the KM-Plotter-Breast Cancer [ https://kmplot.com/analysis/index.php?p=service&cancer=breast ]. For the remaining data, source data are provided in 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: Expressing, Staining, Two Tailed Test, Cell Counting, Flow Cytometry

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 graph demonstrating the coculture model. b Representative flow cytograms (upper panel) gated from human pan-T cell culture and quantification (lower panel, n = 3) of differentiated CD8 + T cell subtypes: Tn (naïve T cells), Tcm (central memory T cells), Tem (effector memory T cells), Teff (effector T cells). c Schematic graph demonstrating the normoxia (20% O 2 ) and hypoxia (1% O 2 ) culture condition of T cells coculturing with human TNBC cell line. d Heatmap of the differentially expressed genes (DEGs) in hypoxic cultured human T cells compared to normoxia group. DEGs were identified in edgeR (|logFC| > 1, adjusted P < 0.01). P values were adjusted using Benjamini–Hochberg method in edgeR. DEGs identified in the indicated GO gene clusters are marked in the heatmap. e GSEA analysis of human T cells in hypoxic versus normoxic conditions. Analysis was based on ranked logFC from edgeR. FDR and adjusted p value are shown in the graph. P values were adjusted using Benjamini–Hochberg method in GSEA analysis. f Flow cytometry quantifications of immune effector molecules and exhaustion markers in CD8 + T cells gated from human pan-T cells cultured under the indicated conditions ( n = 4). g Representative flow cytograms of PD-1 and TIM-3 expression in CD8 + T cells gated from human pan-T cells culture. h Flow cytometric quantification of terminally exhausted T cells (PD-1 + TIM-3 + ) in CD8 + T cells gated from human pan-T cells culture ( n = 3). i Flow cytometric quant i fication of proliferating cells (Ki76 + ) in CD8 + and CD4 + T cells gated from human T cells cocultured with TNBC ( n = 3). All flow cytometry data ( b , f , h , and i ) are presented as the mean ± SD of samples from three to four donors. For all flow cytometry data, P values were determined by one-way ANOVA ( f , h ) or two-way ANOVA ( b ) with Turkey’s test, or paired two-tailed t -test ( i ). Raw RNA-seq data i s available in the GEO database with accession number GSE179885 . For the remaining data, source data are provided in 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: Cell Culture, Flow Cytometry, Expressing, Two Tailed Test, RNA Sequencing

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 RT-qPCR analysis assessing IFNG expression in T/NK cells in an epigenetic-drug screening. Both T cells and NK cells were cultured under 1% O 2 with indicated treatments. Data were presented as the log2 fold change of IFNG mRNA level normalized to vehicle control, mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). b , c Representative histograms (left panel) and flow cytometric quantifications (right panel) of IFNγ expression in human CD8 + T cells ( b n = 4) and NK cells ( c n = 3) with indicated treatments. Quantification data were presented as the mean ± SD of samples from three to four donors. P values were determined by two-way ANOVA with Turkey’s test. d ChIP-qPCR analysis of HDAC1, HDAC2, HDAC3, EZH2, and SUZ12 occupancy on IFNG promoter of human T cells. Four primers were designed to span the promoters of IFNG , with P1 at −1448 to −1354b, P2 at −707 to −628b, P3 at −257 to −171b, P4 at +350 to +461b, relative to TSS. For ChIP analysis of EZH2 and SUZ12 occupancy, RPL30 serves as the negative control and CCND2 as the positive control. e , f ChIP-qPCR analysis of H3K27ac and H3K27me3 enrichment on IFNG promoter of human T cells under indicated conditions. All ChIP-qPCR data ( d – f ) are presented as fold enrichment relative to IgG and expressed as mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). For ChIP-qPCR data of d , e , statistics were performed to analyze bindings of indicated markers across different sites in IFNG promoter ( RPL30 and CCND2 excluded) between hypoxia and normoxia. P values were determined by two-way ANOVA analysis. g RT-qPCR analysis of human T cell with indicated gene knockdown. Data were presented as the fold change of mRNA level normalized to the control group under normoxia (1% O2), mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). 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: Quantitative RT-PCR, Expressing, Drug discovery, Cell Culture, Control, ChIP-qPCR, Negative Control, Positive Control, Knockdown

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 ChIP-qPCR analysis of HIF1α and HIF2α occupancy on IFNG promoter in human T cells. VEGFA served as a positive control. b Co-immunoprecipitation shows the physical interaction between HDAC1 and HIF1α, and the interaction between HDAC1 and SUZ12 in human T cells. Data is representative of two independent experiments ( n = 2). c Representative western blot images ( n = 2) to demonstrate knockdown of HIF1α in human T cells. d ChIP-qPCR analysis of HDAC1 occupancy on IFNG promoter in human T cells. e ChIP-qPCR analysis of H3K27ac and H3K27me3 enrichment on IFNG promoter in human T cells with indicated treatments. All ChIP-qPCR data ( a , d , e ) are presented as fold enrichment relative to IgG and expressed as mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). For ChIP-qPCR data of a , statistics were performed to analyze bindings of indicated markers across different sites in IFNG promoter ( VEGFA excluded) between hypoxia and normoxia. P values were determined by two-way ANOVA analysis. f Flow cytometric quantifications of IFNγ in CD8 + T cells gated from human pan-T cells cultured under the indicated conditions. Data were presented as the mean ± SD of three independent experiments ( n = 3). P values were determined by one-way ANOVA with Turkey’s test. g Representative western blot images ( n = 2) to demonstrate the inhibition of HIF1α level by indicated compounds in human T cells. h Representative histograms (left panel) and flow cytometric quantifications (right panel) of IFNγ expression in human CD8 + T cells with indicated treatments. Quantification data were presented as the mean ± SD of samples from four donors ( n = 4). P values were determined by two-way 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: ChIP-qPCR, Positive Control, Immunoprecipitation, Western Blot, Knockdown, Cell Culture, Inhibition, Expressing

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 Cell lysis of TNBC cells cocultured with human T cells from two different healthy donors. Human T cells were stimulated with TNBC cell lysate-primed DC cells. Data were presented as mean ± SD of three independent experiments ( n = 3). P values were determined by two-way ANOVA. b Western blot analysis of IFNγ–regulated proteins in TNBC cells cocultured with human T cells. Data were representative of two independent experiments ( n = 2). c Cell lysis of TNBC cells cocultured with human T cells. Human T cells were stimulated with TNBC cell lysate-primed DC cells and pretreated with indicated compounds. Data presented as mean ± SD of three independent experiments ( n = 3). P values were determined by one-way ANOVA with Dunnett’s test. d Western blot analysis of IFNγ–regulated proteins in TNBC cells cocultured with human T cells. Human T cells were stimulated with TNBC cell lysate-primed DC cells and pretreated with indicated compounds. Data were representative of two independent experiments ( n = 2). e Cell lysis of TNBC cells cocultured with human T cells. Data were presented as mean ± SD of three independent experiments ( n = 3). P values were determined by two-way ANOVA with Dunnett’s test. f Flow cytometric quantifications of immune effector molecules in human CD8 + T cells cultured under the indicated conditions. Data were presented as the mean ± SD of samples from three donors ( n = 3). P values were determined by two-way 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: Lysis, Western Blot, Cell Culture

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: FLT3L combined with GM-CSF induced dendritic cells drive broad tumor-specific CD8 + T cell responses and remodel the tumor microenvironment to enhance anti-tumor efficacy

doi: 10.3389/fimmu.2025.1649891

Figure Lengend Snippet: FL/GM-DCs enhance the anti-tumor immune response. (a) BM cells, FL/GM-DCs, and GM-DCs were cultured in equal numbers, and OVA-FITC was added for 4 h at 37 °C. OVA uptake was quantified using flow cytometry by first gating on cells based on MHCII and CD11c expression, and the results are displayed as a histogram. Each group contained at least n = 3 biological replicates. (b, c) FL/GM-DCs (b) and GM/IL4-DCs (c) were cultured in equal numbers and co-cultured with OVA at concentrations of 1μg/ml, 10μg/ml, 100μg/ml, 500μg/ml, and 1000μg/ml at 37 °C for 24 hours. The expression levels of H - 2Kb OVA257 – 264 on DCs were measured by flow cytometry. Each group contained at least n = 3 biological replicates. (d) Flow cytometric analysis of expression levels of the chemokine receptor CCR7, gated within MHCII+CD11c+ cells. The analyzed cells were stimulated with polyI:C and R484 prior to analysis. Each group contained at least n = 3 biological replicates. (e) Flow cytometric analysis of expression levels of the Integrin CD103, gated within MHCII + CD11c + cells. Each group contained at least n = 3 biological replicates. (f) Flow cytometric analysis of expression levels of the costimulatory molecule CD86, gated within MHCII + CD11c + cells. Each group contained at least n = 3 biological replicates. (g) Cytokine and chemokine levels, including IL - 12p70, IL - 12p40, IL - 10, IL - 6, TNF-α, IFN-γ, CCL2, CCL4, CCL5, and CXCL1, were measured in the supernatants of FL/GM-DC and GM/IL4-DC cultures after antigen stimulation and maturation for 72 h (n=3). (h) Schematic of T cell proliferation assays using CFSE-labeled OT-I and OT-II T cells co-cultured with B16-OVA tumor lysate-pulsed DCs. (i) Flow cytometric analysis of CD8 + T cell proliferation and activation after 96 h of co-culture. (j) Flow cytometric analysis of CD4 + T cell proliferation and activation after 96 h of co-culture.

Article Snippet: After pretreatment, tissues were blocked, and primary antibodies were applied at the following dilutions: CD4 ((Abcam Cat#ab183685, RRID: AB_2686917)), 1:3000; CD8a (Cell Signaling Technology Cat# 98941, RRID: AB_2756376)), 1:2000; CD11c (Abcam Cat# ab254183, RRID: AB_3096928), 1:3000; and Foxp3 (R and D Systems Cat#MAB8214, RRID: AB_2929004), 1:2000.

Techniques: Cell Culture, Flow Cytometry, Expressing, Labeling, Activation Assay, Co-Culture Assay

Journal: Frontiers in Immunology

Article Title: FLT3L combined with GM-CSF induced dendritic cells drive broad tumor-specific CD8 + T cell responses and remodel the tumor microenvironment to enhance anti-tumor efficacy

doi: 10.3389/fimmu.2025.1649891

Figure Lengend Snippet: FL/GM-DCs exhibit migration and long-term survival in vivo . (a) Experimental protocol for tracking the fate of adoptively transferred FL/GM-DCs and GM/IL4-DCs in tumor-free mice (above) and MC38 tumor-bearing mice (below). In this experiment, each group consisted of 3 mice. (b) Flow cytometric analysis of CD45.1 + CD8 + T cell infiltration in the lymph nodes of tumor-free mice at 7 or 14 days after adoptive transfer of DCs and T cells. (c) Flow cytometric analysis of the proportion of CD45.1 + cells in the lymph nodes of MC38 tumor-bearing mice on days 7, 14, and 30 post-transfer. (d) Flow cytometric analysis of the proportion of CD45.1 + cells in MC38 tumor tissue on days 7, 14, and 30 post-transfer. (e) Flow cytometric analysis of infiltrating CD45.1 + DCs (MHCII + CD86 + ) in MC38 tumor tissue after adoptive transfer of CD45.1 + DCs. (f) Flow cytometry histogram showing XCR1 expression on MHCII + CD86 + DCs.

Article Snippet: After pretreatment, tissues were blocked, and primary antibodies were applied at the following dilutions: CD4 ((Abcam Cat#ab183685, RRID: AB_2686917)), 1:3000; CD8a (Cell Signaling Technology Cat# 98941, RRID: AB_2756376)), 1:2000; CD11c (Abcam Cat# ab254183, RRID: AB_3096928), 1:3000; and Foxp3 (R and D Systems Cat#MAB8214, RRID: AB_2929004), 1:2000.

Techniques: Migration, In Vivo, Adoptive Transfer Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: FLT3L combined with GM-CSF induced dendritic cells drive broad tumor-specific CD8 + T cell responses and remodel the tumor microenvironment to enhance anti-tumor efficacy

doi: 10.3389/fimmu.2025.1649891

Figure Lengend Snippet: The anti-tumor effect of FL/GM-DC in tumor models. (a) Experimental protocol for studying the anti-tumor effects of FL/GM-DCs and GM/IL4-DCs in the tumor models. Mice with tumors were vaccinated on days 3, 6, 9, 12, 15, and 21 with FL/GM-DCs or GM/IL4-DCs (Each mouse received 2.5 x 10 6 cells per administration). Measured the size of the tumor every 3 days. (b) Tumor size in MC38 tumor-bearing mice measured over time (mean ± SEM; NC, n=8; FL/GM-DC, n=8; GM/IL4-DC, n=8). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. (c) Flow cytometric analysis of the proportion of infiltrating DCs (MHCII + CD11c + ) in MC38 tumor tissue. (d) Representative fluorescence images of MC38 tumors from treated mice, showing CD11c (green), CD8a (orange), CD4 (pink), Foxp3 (red), and DAPI nuclear staining (blue). Scale bar, 100 μm. The bar chart represented the positive rate of CD8a + , CD4 + , and Foxp3 + cells in the tumors of each group of MC38 mice. (e) Experimental research protocol on the anti-tumor effects of FL/GM-DCs and GM/IL4-DCs in an antibody-blockaded MC38 tumor model. Mice with tumors were vaccinated on days 3, 6, 9, 12 with FL/GM-DCs or GM/IL4-DCs (Each mouse received 2.5 x 106 cells per administration) and received antibody blocking therapy (Each mouse received 200ug antibodies per administration) on the next day. Measured the size of the tumor every 3 days. (f) Tumor size in MC38 tumor-bearing mice measured over time (mean ± SEM; NC, n=6; FL/GM-DC+IgG2b, n=6; FL/GM-DC+antiCD8a, n=6; FL/GM-DC+antiCD4, n=6; FL/GM-DC+antiF4/80, n=6). Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparisons test. (g) Each subplot displayed the tumor volume over time for individual mice in different treatment groups (NC, FL/GM-DC+IgG2b, FL/GM-DC+antiCD8a, FL/GM-DC+antiCD4, and FL/GM-DC+antiF4/80).

Article Snippet: After pretreatment, tissues were blocked, and primary antibodies were applied at the following dilutions: CD4 ((Abcam Cat#ab183685, RRID: AB_2686917)), 1:3000; CD8a (Cell Signaling Technology Cat# 98941, RRID: AB_2756376)), 1:2000; CD11c (Abcam Cat# ab254183, RRID: AB_3096928), 1:3000; and Foxp3 (R and D Systems Cat#MAB8214, RRID: AB_2929004), 1:2000.

Techniques: Fluorescence, Staining, Blocking Assay

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Oncolytic adenovirus in treating malignant ascites: A phase II trial and longitudinal single-cell study.

doi: 10.1016/j.ymthe.2024.04.029

Figure Lengend Snippet: Figure 4. Characteristics and dynamics of CD8+ and CD4+ T cell subsets during H101 treatment (A) Distinct cell subtypes of CD8+ T cells demonstrated by UMAP plot. (B) Cell proportion of each CD8+ T cell subtype. Black asterisks represent the statistical significantly increased proportion on day 7 compared with that at baseline. (C) Fraction of CD8_MKI67 cells in two response groups on days 0, 7, and 14. Wilcoxon test. *p < 0.05. (D) Representative double-color immunofluorescence staining of CD8+Ki67+ cells in MA samples (P04 [T-long patient] and P07 [T-short patient]) on days 0, 7, and 14, with

Article Snippet: For double-color immunofluorescence of CD8A and Ki67, mouse anti-human CD8a (CST, Cat#70306) and rabbit anti-human Ki67 (Abcam, Cat#15580), were applied.

Techniques: Staining

Journal: Immunity

Article Title: Negative costimulation constrains T cell differentiation by imposing boundaries on possible cell states

doi: 10.1016/j.immuni.2019.03.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-CD8a 146Nd , Fluidigm , Cat #:3146003B; Clone:53-6.7.

Techniques: Virus, Recombinant, Adjuvant, Polymer, Conjugation Assay, Staining, Mass Cytometry, Sequencing, Transgenic Assay, Double Knockout, Software

Journal: Research in Veterinary Science

Article Title: Relationship between rate of infection and markers of inflammation/immunity in Holy Birman cats with feline coronavirus

doi: 10.1016/j.rvsc.2014.08.009

Figure Lengend Snippet: Results recorded in Birman cats and in cats from other breeds.

Article Snippet: Immunophenotyping by flow cytometry was performed on aliquots of 50 μL of the cell suspension to identify lymphocyte subpopulations as previously described ( ) using the following panel of antibodies for feline surface antigen: 2.5 μL of mouse anti feline CD4 (specific for T helper cells, clone MCA1350, Serotec, Oxford, UK), 1 μL of mouse anti feline CD8 alpha/beta (specific for T cytotoxic cells, clone MCA1347G, Serotec, Oxford, UK), 50 μL of mouse anti feline CD5 (specific for T cells, clone MCA2038S, Serotec, Oxford, UK), and 1 μL of mouse anti canine CD21, specific for B cells, that cross reacts with feline species (clone MCA1781R, Serotec).

Techniques:

Journal: Nature Communications

Article Title: Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas

doi: 10.1038/s41467-024-44932-w

Figure Lengend Snippet: a Flow cytometry analysis of CT2A CRISPR control, Atrx KO-A or KO-B tumor-bearing hemispheres harvested 14 days post-intracranial implant showing percent live cell density of CD3 + , CD4+ and CD8 + T-cells and macrophages (CD45 + , CD3/19neg, NK1.1neg, CD11bhi, CD45hi). Gating strategy is provided in Supplementary Fig. . n = 10 for CRISPR control – CD3 + , CD4 + , CD8+ markers; n = 9 for CRISPR control - macrophage markers; n = 7 for Atrx KO-A - all markers; n = 5 for Atrx KO-B – all markers; n = 9 for normal brain - CD3 + , CD4 + , CD8+ markers; n = 8 for normal brain – macrophage markers. Data are presented as mean ± SEM. Asterisks denote significant one-way ANOVA with Dunnett’s post-hoc test comparing CRISPR control with Atrx KO tumor-bearing hemispheres (* p < 0.05, ** p < 0.01). CD3 + : Control vs Atrx KO-A – p = 0.0296; CD4 + : Control vs Atrx KO-A – p = 0.0117; Macrophages: Control vs Atrx KO-A – p = 0.0167, Control vs Atrx KO-B – p = 0.0086. b Representative IHC images for CD45 (A, B, C) and F4/80 (D, E, F) expression (brown) in CT2A CRISPR Control ( Atrx WT ) (A, D), Atrx KO-A (B, E) and KO-B (C, F) tumors, with hematoxylin counter-staining (blue). Scale bar: 50μm. Number of brains subjected to IHC per group: CRISPR Control – n = 3; Atrx KO-A – n = 2; Atrx KO-B – n = 3. c Representative IHC images for CD45 (A, B) and CD3 (C, D) expression (brown) in RCAS/ Ntv-a control (A, C) and Atrx -KO (B, D), with hematoxylin counter-staining. Scale bar: 50μm. Number of brains subjected to IHC per group: RCAS/ Ntv-a control – n = 6; Atrx KO – n = 4. d , e Kaplan–Meir survival curves for C57BL/6 mice bearing intracranial CT2A CRISPR ctrl (Control) or Atrx KO-B tumors treated with 250 μg of isotype control IgG, anti-CD4 antibody ( d ) or anti-CD8 antibody ( e ) as per schema in Supplementary Fig. . N Number of mice per group; MS median survival in days. P -values represent group comparisons calculated using log-rank test. Source data are provided as a Source Data file.

Article Snippet: For CD4+ and CD8 + T-cell depletion experiments, C57BL/6 mice ( n = 10 mice per group) received 250 μg per dose of rat IgG2B isotype control (BioXcell, Cat. no. BE0090), anti-mouse CD4 antibody (BioXcell, Cat. no. BE00003) or anti-mouse CD8 antibody (BioXcell, Cat no. BE0061) intraperitoneally 4 days prior to implant.

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