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: Acta Pharmaceutica Sinica. B

Article Title: Arsenic trioxide-based nanoparticles for enhanced chemotherapy by activating pyroptosis

doi: 10.1016/j.apsb.2025.08.003

Figure Lengend Snippet: Immunomodulatory effects of AsMn/Dz@BSA-FA. Analysis of the proportion of (A) CD11c + CD86 + cells, (B) CD3 + CD4 + cells, and (C) CD3 + CD8 + cells in tumor tissues of each group of mice using flow cytometry. (D) Immunofluorescence detection of changes in CD86, CD11c, CD8, and CD4 in tumor tissue after treatment with different formulations, Scale bar: 100 μm. (E) Expression levels of CD86, CD8, and CD4 in tumor tissue after treatment with different formulations. Changes in (F) TNF- α and (G)IL-6 levels in tumor tissue after treatment with different formulations. Data are presented as mean ± SD ( n = 5). ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: APC Anti-Mouse CD11c Antibody, PE Anti-Mouse CD86 Antibody, FITC Anti-Mouse CD3 Antibody, APC Anti-Mouse CD4 Antibody, PE Anti-Mouse CD8a Antibody and Purified Anti-Mouse CD16/32 Antibody were from Elabscience (Wuhan, China).

Techniques: Flow Cytometry, Immunofluorescence, Expressing

Journal: CytoJournal

Article Title: The mechanism of prostaglandin E2 upregulation of programmed death ligand 1 expression promoting immune escape in non-small cell lung cancer

doi: 10.25259/Cytojournal_129_2025

Figure Lengend Snippet: PGE2 upregulates PD-L1 expression in NSCLC and promotes immune escape response. (a-c) PD-L1 expression detected after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (d and e) Cytotoxicity tested by LDH kit assay after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (f and g) CD8 + T cell viability tested after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (h and i) CD8 + T cell apoptosis examined after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). (j-m) IFN-γ, TNF-α, granzyme B, and perforin quantification by ELISA after PTGES overexpression (OE-PTGES) and knockdown (sh-PTGES), compared with respective negative controls (OE-NC or sh-NC). n = 6; ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001. PEG2: Prostaglandin E2, PD-L1: Programmed death ligand 1, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control, LDH: Lactate dehydrogenase, OE-PTGES: Overexpression prostaglandin E synthase, sh-PTGES: Short hairpin prostaglandin E synthase, IFN-γ: Interferon-gamma, TNF-α: Tumor necrosis factor-alpha, ELISA: Enzyme-linked immunosorbent assay.

Article Snippet: First, a single-cell suspension was prepared and incubated with CD3 (E-AB-F1013E, Elabscience, Wuhan, China) and CD8 (E-AB-F1104Q, Elabscience, Wuhan, China) antibodies in the dark.

Techniques: Expressing, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: CytoJournal

Article Title: The mechanism of prostaglandin E2 upregulation of programmed death ligand 1 expression promoting immune escape in non-small cell lung cancer

doi: 10.25259/Cytojournal_129_2025

Figure Lengend Snippet: PGE2 promotes immune escape in NSCLC in vivo by upregulating PD-L1 expression. (a) Isolated tumor images after PTGES overexpression and knockout. (b and c) Changes in tumor weight and volume after PTGES overexpression and knockout (significant difference markers marked with ✶ represent OE-NC versus OE-PTGES, and those marked with # represent sh-NC vs. sh-PTGES). (d-f) WB analysis of PTGES and PD-L1 after PTGES overexpression and knockout in vivo . (g and h) IHC analysis of CD8 after PTGES overexpression and knockout in vivo (scale bar: 20 μm, magnification, 400×). (i-l) IFN-γ, TNF-α, granzyme B, and perforin quantification by ELISA after PTGES overexpression and knockout in vivo . n = 5; ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ## P < 0.01. OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control. PEG2: Prostaglandin E2, PD-L1: Programmed death ligand 1, NSCLC: Non-small cell lung cancer, PTGES: Prostaglandin E synthase, OE-NC: Overexpression negative control, sh-NC: Short hairpin negative control, OE-PTGES: Overexpression prostaglandin E synthase, sh-PTGES: Short hairpin prostaglandin E synthase, IHC: Immunohistochemistry, IFN-γ: Interferon-gamma, TNF-α: Tumor necrosis factor-alpha, ELISA: Enzyme-linked immunosorbent assay.

Article Snippet: First, a single-cell suspension was prepared and incubated with CD3 (E-AB-F1013E, Elabscience, Wuhan, China) and CD8 (E-AB-F1104Q, Elabscience, Wuhan, China) antibodies in the dark.

Techniques: In Vivo, Expressing, Isolation, Over Expression, Knock-Out, Enzyme-linked Immunosorbent Assay, Negative Control, Immunohistochemistry

Journal: Journal of Virology

Article Title: A novel nanoparticle vaccine, based on S1-CTD, elicits robust protective immune responses against porcine deltacoronavirus

doi: 10.1128/jvi.00674-25

Figure Lengend Snippet: CTDnps vaccine elicited robust antibody and T cell responses in mice. ( A ) Schematic diagram of the mice immunization procedure. Five mice per group received prime/boost vaccination with various vaccines at Weeks 0 and 4. Serum samples were collected every 2 weeks. ( B ) Titers of antigen-specific IgG antibodies were measured in serum samples by iELISA. ( C ) Titers of NAbs were measured in serum samples from mice at Week 8 by VNT. ( D ) Titers of antigen-specific IgG1 and IgG2a for each immunization group. Serum samples from mice at Week 8 were tested. IL-4 ( E ) and IFN-γ ( F ) concentrations were measured by double-antibody sandwich ELISA. ( G, H ) The percentage of CD3 + CD4 + and CD3 + CD8 + T lymphocytes among spleen lymphocytes was determined by flow cytometry. ( I ) The percentage of CD4 + α4β7 + T lymphocytes among spleen lymphocytes was determined by flow cytometry. ( J, K ) The percentage of CD4 + and CD8 + Tcm among spleen lymphocytes was determined by flow cytometry. Data are mean ± SD. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; and ns, P > 0.05).

Article Snippet: At week 8, spleen lymphocytes of mice were isolated, cell concentration was adjusted to 1 × 10 7 cells/mL, and 10 μL of FITC anti-mouse CD3 antibody, 10 μL of Violet 450 anti-mouse CD4 antibody, and 10 μL of PerCP/Cyanine5.5 anti-mouse CD8a antibody (Elabscience, Wuhan, China) were added to 200 μL of splenocytes for analysis of CD4 + and CD8 + T cells.

Techniques: Vaccines, Sandwich ELISA, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 2.5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) five times at three-day intervals. Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05 and ** p < 0.01. Two-Way ANOVA test ( n = 4). B The resected tumors were weighed on Day 30. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. D The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). E The mRNA levels of Cd86 and Cd163 in resected tumors were analyzed by qRT‒PCR ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001. One-Way ANOVA test ( n = 3). F The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry ( n = 3-4). * p < 0.05 and *** p < 0.001. One-Way ANOVA test. G The quantification of the M1/M2 ratio is shown ( n = 3). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). H The frequencies of CD4 (CD4 + CD3 + CD45 + 7AAD - ) and CD8 (CD8 + CD3 + CD45 + 7AAD - ) tumor-infiltrating T cells were analyzed by flow cytometry ( n = 3-4). * p < 0.05. One-Way ANOVA test. I The quantification of CD8 + TILs is shown. * p < 0.05. One-Way ANOVA test ( n = 3). J The frequency of regulatory CD4 T cells (CD25 + CD127 - CD4 + CD3 + CD45 + 7AAD - ) was analyzed by flow cytometry ( n = 3-4). K The quantification of the CD8/Treg ratio is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). L The densities of dendritic cells (CD11c + ) and cytotoxic T cells (CD8 + ) in resected tumors were analyzed by immunofluorescence staining. The density of CD11c + DCs is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3). M The density of CD8 + TILs is shown. * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3).

Article Snippet: The cells were stained with different antibodies against combinations of surface markers: (1) CD4 and CD8 T cells: ViaDyeRed, CD45-BV785 (E-AB-F1136UD, Elabscience, Texas, USA), CD3-PE-Fir700, CD4-PE/Cy7, CD8a-BV570 (E-AB-F1104UF, Elabscience, Texas, USA), CD127-BV711, CD25, IFNγ-PE and GzmB-AF647; (2) Tumor-associated macrophages: ViaDyeRed, CD3-PE-Fir700, CD19-PE-Fir700, CD45-BV785, CD11b-BV421, F4/80-PE, CD11c-AF488, CD206-APC.

Techniques: Injection, Immunofluorescence, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Targeting ENO1 reprograms macrophage polarization to trigger antitumor immunity and improves the therapeutic effect of radiotherapy

doi: 10.1038/s41419-026-08416-7

Figure Lengend Snippet: A A total of 5 × 10 5 CT26 cells were subcutaneously injected into the left legs of BALB/c mice for 5 days and then intraperitoneally administered with anti-ENO1 antibodies (HuL001, 40 mg/kg) or clodronate liposomes (50 μL/mouse) on the indicated days ( n = 5). Local radiotherapy was given on Day 10. The tumor volume was recorded every three days. * p < 0.05. Two-Way ANOVA test ( n = 4). B The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05 and *** p < 0.001. One-Way ANOVA test ( n = 3). C 4T1 cells (5 ×10 4 ) were subcutaneously injected into the left legs of BALB/c mice for 4 days and then intraperitoneally administered anti-ENO1 antibodies (HuL001, 20 mg/kg) six times on the indicated days ( n = 5). Local radiotherapy was given on Days 10 and 12. The tumor volume was recorded every three days. * p < 0.05 and *** p < 0.001. Two-Way ANOVA test ( n = 4). D The resected tumors were weighed on Day 40. * p < 0.05. One-Way ANOVA test ( n = 4). E The densities of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors were analyzed by immunofluorescence staining. F The quantification of M1 (CD80 + ) and M2 (CD206 + ) macrophages in resected tumors. * p < 0.05. One-Way ANOVA test ( n = 3). G The frequencies of M1 (CD11c + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) and M2 (CD1206 + CD11b + F4/80 + CD45 + 7AAD - CD3 - CD19 - ) tumor-infiltrating macrophages were analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). H The quantification of the M1/M2 ratio is shown. * p < 0.05. One-Way ANOVA test ( n = 3). I A representative image of flow cytometric analysis of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells. J The frequency of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells was analyzed by flow cytometry. * p < 0.05. One-Way ANOVA test ( n = 3-4). K The density of GzmB + (GzmB hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown. * p < 0.05. One-Way ANOVA test ( n = 3-4). L The density of IFNγ + (IFNγ hi CD8 + CD3 + CD45 + 7AAD - ) T cells is shown ( n = 3-4). * p < 0.05 and ** p < 0.01. One-Way ANOVA test ( n = 3-4). M The proposed mechanism of TGFβ1/TGFβR/Smad3/PRMT5-mediated ENO1 translocation for lactate release via MCT4.

Article Snippet: The cells were stained with different antibodies against combinations of surface markers: (1) CD4 and CD8 T cells: ViaDyeRed, CD45-BV785 (E-AB-F1136UD, Elabscience, Texas, USA), CD3-PE-Fir700, CD4-PE/Cy7, CD8a-BV570 (E-AB-F1104UF, Elabscience, Texas, USA), CD127-BV711, CD25, IFNγ-PE and GzmB-AF647; (2) Tumor-associated macrophages: ViaDyeRed, CD3-PE-Fir700, CD19-PE-Fir700, CD45-BV785, CD11b-BV421, F4/80-PE, CD11c-AF488, CD206-APC.

Techniques: Injection, Liposomes, Immunofluorescence, Staining, Flow Cytometry, Translocation Assay

Journal: Frontiers in Medicine

Article Title: Analysis and prediction of immune cell infiltration characteristics in COPD: Folium isatidis and its active ingredients are able to combat lung lesions caused by COPD by correcting immune cell infiltration

doi: 10.3389/fmed.2025.1584411

Figure Lengend Snippet: IDR and FI modulated changes in immune cell infiltration caused by COPD. Flow cytometric analysis of (A) CD4 + T lymphocytes, (B) CD8 + T lymphocytes, (C) Treg cells, (D) MDSCs, (E) B lymphocytes, (F) NK cells, and (G) Eos. Proportion of (H) CD4 + T lymphocytes, (I) CD8 + T lymphocytes. (J) CD4/CD8 index. Proportion of (K) Treg cells, (L) MDSCs, (M) B lymphocytes, (N) NK cells, and (O) Eos. Data were expressed as mean ± SD, ### p < 0.001 vs. CON group, * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. MOD group ( n = 3).

Article Snippet: The Anti-Mouse antibodies CD3-APC (E-AB-F1013E), CD4-FITC (E-AB-F1097C), CD8a-PC5.5 (E-AB-F1104I), CD25-PE (E-AB-F1102D), Foxp3-APC (E-AB-F1238E), CD11b-FITC (E-AB-F1081C), Ly-6G/Ly-6C-APC (E-AB-F1120E), CD19-PE (E-AB-F0986D), NK1.1-PE (E-AB-F0987D) and F4/80-APC (E-AB-F0995E) were purchased from Elabscience (Wuhan, China).

Techniques: