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: Journal of the American Society of Nephrology

Article Title: CD4+CD25+Regulatory T Cells Inhibit Experimental Anti-Glomerular Basement Membrane Glomerulonephritis in Mice

doi: 10.1681/asn.2004100837

Figure Lengend Snippet: Figure 4. Treg transfer inhibits the infiltration of interstitial macrophages and CD4 and CD8 T cells. The infiltration of inflammatory cells was analyzed by immunohistochemistry. Staining for macrophages (A) and CD4 and CD8 T cells (B) was performed 7 (n 14 per group) and 14 d (n 7 per group) after induction of anti-GBM nephritis in mice that received an injection of Treg () or CD4CD25 T cells (f). Interstitial macrophage accumulation was comparable in both groups at day 7 but significantly diminished at day 14 after transfer of Treg. CD4 T cell infiltration was significantly decreased 14 d after anti-GBM injection, whereas CD8 T cell infiltration was significantly decreased 7 and 14 d after anti-GBM injection in mice that received an injection of Treg (*P 0.05).

Article Snippet: For the detection of CD4 T cells, a rat anti-mouse CD4 mAb (clone YTS191.1; Serotec) was used, and for CD8 T cells, a rat anti-mouse CD8 mAb (clone KT15; Serotec) was used.

Techniques: Immunohistochemistry, Staining, Injection

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: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Differential gene expression in HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic individuals. ( a ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 and VP11/12 702–710 epitopes were sorted from HLA-A*0201-positive SYMP and ASYMP individuals, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 25,638 genes. ( b ) Frequencies of CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 and VP11/12 702–710 epitopes detected by FACS in SYMP vs. ASYMP individuals. ( c ) Heatmap is showing 772 differentially expressed genes among SYMP and ASYMP individuals. ( d ) Heatmap showing statistically significant pathways that are affected in HSV-specific CD8 + T cells from SYMP vs. SYMP individuals. Parametric Gene Set Enrichment Analysis (PSGEA) method was applied based on data curated in Gene Ontology and KEGG. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied. ( e ) Bulk RNA heatmap comparing differentially expressed CAM pathway associated T cell co-stimulatory and T cell exhaustion genes in HSV-specific CD8 + T cells from SYMP vs. SYMP individuals.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Gene Expression, Infection, Biomarker Discovery, RNA Sequencing, Expressing

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Single-cell RNA sequencing of trigeminal ganglia-resident CD45 + leukocytes from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Illustration of the experimental design and validation of differentially expressed genes in CD45 + leukocytes sorted on day 15 p.i. from the trigeminal ganglia (TG) of SYMP and ASYMP HLA Tg rabbits. ( b ) Heatmap expression of the most significant 140 differentially expressed genes among eight different clusters detected in TG-resident CD45 + leukocytes from HSV-1 infected SYMP and ASYMP HLA Tg rabbits (top two heatmap panels). Each cluster represents an individual immune cell population, determined on the basis of specific molecular markers: CD8 + T cells (CD8A), CD4 + T cells (CD4), NK cells (NKG7), B cells (CD19), macrophages (CD68), monocytes (CD14), granulocytes (FUT4) and dendritic cells (CD1c). The t-SNE dimensionality reduction, applied to single-cell RNA sequencing data revealed eight distinct clusters of immune cell populations among CD45 + leukocytes for the TG of HSV-1 infected ASYMP HLA Tg rabbits (middle panels). The total number of differentially expressed genes within each immune cell clusters (nCount) (lower panels). ( c ) Average frequencies of different immune cell populations detected within TG-resident CD45 + leukocytes of SYMP and ASYMP HLA Tg rabbits. ( d ) Volcano plot illustrates the total copy number reads observed for all the genes within one single cell (nFeature).

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: RNA Sequencing, Infection, Biomarker Discovery, Expressing

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Differential gene expression in HSV-specific CD8 + T cells from trigeminal ganglia of HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP HLA Tg rabbits. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 , VP11/12 702–710, and gD 53–61 epitopes were sorted from TG of HLA-A*0201-positive SYMP and ASYMP HLA Tg rabbits, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 23,669 rabbit genes (OryCun2.0 (GCA_000003625.1). ( b ) Frequencies of CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 , VP11/12 702–710, and gD 53–61 epitopes detected by FACS in TG of HLA-Tg rabbits. ( c ) The heatmap is showing the most significant 2,879 differentially expressed genes among SYMP and ASYMP HLA Tg rabbits. Genes with minimum count per million (CPM) ≥ 0.5 were used for obtaining the transformed counts data for clustering using regularized log (rlog). ( d ) Bulk RNA heatmap shows the pathways that are different among ASYMP and SYMP HLA Tg rabbits. Genes differentially expressed in both single-cell RNA sequencing and bulk RNA sequencing were considered for pathway analyses. Parametric gene set enrichment analysis (PSGEA) method based on data curated in Gene Ontology and KEGG was applied. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Gene Expression, Infection, Biomarker Discovery, RNA Sequencing, Expressing, Transformation Assay

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Activation and exhaustion genes differentially expressed in trigeminal ganglia-resident HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Expression of T cell activation genes ( CD69 , CD62L , CD44 , CD107 , and IFN-γ ) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of T cell exhaustion genes ( PD-1 , LAG-3 , CTLA4 , ICOS , and BLIMP1 ) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). ( b ) Average frequencies of specific genes representing memory CD8 + T CM , CD8 + T EM , and CD8 + T RM cell subsets from TG of SYMP vs. ASYMP HLA Tg rabbits (left panel). Average frequencies of CD8 + T RM cells expressing various exhaustion genes in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (top right panel). Average frequencies of functional CD107 a/b+ IFN- γ + CD8 + T RM cells in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (lower right panel). ( c ) Bulk RNA sequencing showing expression of T-cell activation (left panel) and T-cell exhaustion genes (right panel) in HSV-specific T RM cells from SYMP vs. ASYMP HLA Tg rabbits. ( d ) Frequencies of memory CD8 + T CM , CD8 + T EM , and CD8 + T RM cell subsets detected by FACS in HSV-1 infected TG of SYMP vs. ASYMP HLA Tg rabbits. ( e ) Fluorescence microscopy images showing infiltration of CD8 + T cells in HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits. TG sections were co-stained using DAPI and mAb specific to rabbit CD8 + T cells (magnification, × 20). Blue, DAPI: DNA, green: CD8 + T cells.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Activation Assay, Infection, Expressing, RNA Sequencing, Functional Assay, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Genes of cytokines/chemokines and receptors differentially expressed in trigeminal ganglia-resident HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Expression of genes of chemokines and chemokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8 + T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of genes of cytokines and cytokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8 + T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). ( b ) Average frequencies of CD8 + T RM cells expressing genes of cytokines/chemokines and receptors in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. ( c ) Bulk RNA sequencing showing expression of genes of chemokines/chemokine receptors (left panel) and genes of cytokines/cytokine receptors (right panel) in HSV-specific T RM cells from SYMP vs. ASYMP HLA Tg rabbits. ( d ) Representative (left panels) and average (right panels) frequencies of CCR3 + CD8 + T RM cells in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. ( e ) Fluorescence microscopy images showing infiltration of HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits by CD8 + T cells expressing T cell-attracting chemokines and receptors (i.e., CXCR3, CXCL9, CXCL10, and CXCL11) (magnification, × 20). Blue: DAPI (DNA stain); red: CXCR3 cells.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Infection, Expressing, RNA Sequencing, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: TG-resident HSV-specific memory CD8 + T RM cells downregulate the T cell exhaustion associated pathway and confer protection from ocular herpes in HSV-1 infected asymptomatic humans and HLA transgenic rabbits. ( 1 ) Upon exposure to stressors, the HSV-1 enters into the cornea and travels through neurons to Trigeminal ganglia. ( 2 ) Following primary HSV-1 infection, the vast majority (up to 95%) of antiviral effector CD8 + T cells die, leaving behind only about 5% of CD8 + T cells destined to differentiate into a heterogeneous pool of memory CD8 + T cells. ( 3 ) The effector memory (T EM ) and tissue-resident memory (T RM ) CD8 + T-cell subsets are found mainly in the HSV-infected but "naturally protected" asymptomatic subjects, whereas the lymphoid organ-resident central memory (T CM ) CD8 + T cell subsets are mainly present in non-protected Symptomatic subjects. ( 4 ) Reduced viral reactivation was observed among asymptomatic subjects possessing a higher frequency of CD8 + T RM cells resulting in a less severe herpes disease. ( 5 ) The findings study suggests that by blocking immune checkpoints, there is a reduced expression of T cell exhaustion molecules ( PD-1 , LAG-3 , PSGL-1 , CTLA-4 , TIM3 , and TIGIT ) and T cell exhaustion associated Cell Adhesion Molecule pathway and increased retention of CD8 + T RM cell population in asymptomatic subjects. This memory CD8 + T cell population mediates recall responses and halts attempts of virus reactivation in the infected TG, thus accelerating viral clearance. More-so, reduced expression of T cell exhaustion pathway also gives rise to higher expression of genes associated with T cell function ( CD107 , IFN-γ ), T cell homing ( CXCR3 , CCR7 ), and T-cell keeping ( IL7R , IL15R ). This helps in reducing the ocular herpes infection and recurrent herpetic disease.

Article Snippet: The following antibodies were used: mouse anti-rabbit CD8 (clone MCA1576F, SEROTEC), mouse anti-human CD103 (clone H4A3) FITC, CD69 (clone H4B4) APC/Cy7 (BIOLEGEND), and rat anti-IFN- γ (clone XMG1.2) (BD Biosciences).

Techniques: Infection, Transgenic Assay, Blocking Assay, Expressing, Virus, Cell Function Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 3. CmAb-(IL10)2-Mediated Antitumor Effects Depend on Host Immunity (A and B) Tumor growth in C57BL/6J (A) or Rag1/ (B) mice (n = 5) bearing B16-cEGFR tumors treated by intratumoral (i.t.) injection of Cetuximab, CmAb-(IL10)2, or control IgG (indicated by arrows). (C) Quantification of OVA tetramer-positive (OVA-specific) CD8+ T cells in tumor tissues collected from B16-cEGFR-OVA tumor-bearing C57BL/6J mice (n = 4–5) treated twice by i.t. injection with control IgG or CmAb-(IL10)2 on days 11 and 14 after tumor cell inoculation. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (D) IFN-g ELISPOT assay of splenocytes collected from B16-cEGFR-OVA tumor-bearing C57BL/6J mice (n = 5–6) treated three times by i.t. injection of control IgG or CmAb-(IL10)2. The spleens were harvested 9 days after the first treatment. OT1 peptide, OVA-derived SIINFEKL peptide; SIY, a control peptide SIYRYYGL. (E) Tumor growth in C57BL/6J mice (n = 5) bearing B16-cEGFR tumors treated with control IgG or CmAb-(IL10)2 (i.t., indicated by arrows). a-CD8 or a-CD4 antibodies were administered for T cell depletion during the CmAb-(IL10)2 treatment. (F and G) Tumor growth in NSG-SGM3 (F) and NSG-SGM3 humanized (G) mice (n = 5) bearing A431 tumors treated with CmAb-(IL10)2 or Cetuximab on days 11, 14, 17, and 20 after tumor cell inoculation. (A–G) Data are shown as means ± SEM. **p < 0.01, ****p < 0.0001; ns, not significant. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Injection, Control, Cytometry, Enzyme-linked Immunospot, Derivative Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 4. DCs are Essential for the Antitumor Effects of CmAb-(IL10)2 by Preventing Apoptosis of Antigen-Specific CD8+ T Cells (A and B) Proliferation of CFSE-labeled CD8+ OT1 T cells co-cultured with BMDCs from C57BL/6J mice in the presence of OVA and treated with CmAb-(IL10)2, Cetuximab, or vehicle. The percentage (A) and the number (B) of proliferating CD8+T cells were assessed by flow cytometry at the indicated time points. (C) Apoptosis of proliferating CD8+ T cells at 72 h after co-culture as described in (A and B), assessed by flow cytometry.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Labeling, Cell Culture, Cytometry, Co-Culture Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 5. IL-10R Signaling on DCs Is Required for Preventing Apoptosis of Antigen-Specific CD8+ T Cells (A) Proliferation of CFSE-labeled CD8+ OT1 T cells co-cultured with BMDCs from WT (left) or Il10r/ (right) mice in the presence of OVA treated with CmAb-(IL10)2 or vehicle, assessed by flow cytometry at the indicated time points. (B) Cell number of proliferating CD8+ OT1 T cells co-cultured with BMDCs from Il10r/ mice in the presence of OVA and treated with CmAb-(IL10)2 or vehicle, assessed by flow cytometry at the indicated time points. (C) Apoptosis of proliferating CD8+ T cells at 72 h after co-culture as described in (B), assessed by flow cytometry. (D) Scheme of adoptive transfer of CD8+ T cells (2 3 104 OT1 CD8+ mixed with 2 3 106 WT CD8+ T cells) and CmAb-(IL10)2 treatment of Rag1/ or Il10r/ Rag1/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Labeling, Cell Culture, Cytometry, Co-Culture Assay, Adoptive Transfer Assay

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 6. CmAb-(IL10)2 Prevents Antigen-Specific CD8+ T Cell Apoptosis through Regulating DC-Mediated IFN-g Production (A) Apoptosis assessment of re-stimulated CD8+ T cells by co-culturing antigen-activated CD8+ OT1 T cells with BMDCs from C57BL/6J mice in the presence of OVA and CmAb-(IL10)2 or vehicle, determined at 48 h after re-stimulation by flow cytometry. (B) IFN-g production from the indicated co-cultures of DCs and CD8+ OT1 T cells in the presence of OVA and treated with CmAb-(IL10)2 or vehicle. (C and D) Apoptosis of proliferating CD8+ T cells co-cultured with BMDCs from WT (C) or Il10r/ (D) mice in the presence of OVA and treated with CmAb-(IL10)2, IFN-g, or a-IFN-g (10 mg/mL), assessed by flow cytometry. (E) Apoptosis of CD8+ T cells in B16-cEGFR-OVA tumor tissues from Rag1/ mice (n = 5–6) i.t. treated with 1 3 106 antigen-activated CD8+ OT1 T cells plus control IgG, CmAb-(IL10)2 or anti-IFN-g (150 mg, i.p.) on day 11 after tumor cell inoculation. Tumor tissues were collected 2 days after treatment and analyzed by flow cytometry. (F) Apoptosis of OVA tetramer-positive CD8+ T cells in B16-cEGFR-OVA tumor tissues from Ifng/ mice (n = 7) i.t. treated with control IgG or CmAb-(IL10)2 on days 8 and 11 after tumor cell inoculation. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (A–F) Data are shown as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Cytometry, Cell Culture, Control

Journal: Cancer cell

Article Title: Targeting Tumors with IL-10 Prevents Dendritic Cell-Mediated CD8 + T Cell Apoptosis.

doi: 10.1016/j.ccell.2019.05.005

Figure Lengend Snippet: Figure 7. CmAb-(IL10)2 Can Prevent Antigen-Specific CD8+ TIL Apoptosis and Improve the Antitumor Effects of Immune Checkpoint Blockade in the Treatment of Advanced Tumors (A) Apoptosis of OVA tetramer-positive CD8+ T cells in B16-cEGFR-OVA tumor tissues from C57BL/6J mice (n = 7) treated twice by a-PD-L1 and a-CTLA-4 (immune checkpoint blockade [ICB]) in combination with control IgG or CmAb-(IL10)2. Tumor tissues were collected 7 days after first treatment and analyzed by flow cytometry. (B and C) Scheme (B) (top), tumor growth (B) (bottom), and survival curve (C) of the advanced B16cEGFR tumor-bearing (80–120 mm3) C57BL/6J mice (n = 6–7) treated with CmAb-(IL10)2, ICB, or the combination therapy as indicated. (D) Tumor growth after challenge with B16-cEGFR cells in treatment-naı¨ve mice or mice cured by the combination therapy for ICB and CmAb-(IL10)2. (E) Scheme of treatment (left) and tumor growth (right) of C57BL/6J mice (n = 5–7) bearing advanced B16-cEGFR tumors treated with CmAb-(IL10)2, ICB, or the combination therapy as indicated. (A–E) Data are shown as means ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies InVivoMAb anti-mouse CD4 (GK1.5) BioXcell Cat# BE0003-1 InVivoMAb anti-mouse CD8 (YTS 169.4) BioXcell Cat# BE0117 InVivoMAb anti-mouse PDL1 (10F.9G2) BioXcell Cat# BE0101 InVivoMAb anti-mouse CTLA-4 (9D9) BioXcell Cat# BE0164 InVivoMAb anti-mouse IFN g (R4-6A2) BioXcell Cat# BE0054 InVivoMAb anti-mouse IL-12 p40 (C17.8) BioXcell Cat# BE0051 Anti-CD45 (Flow cytometry, 30-F11) BioLegend Cat# 103126 Anti-CD8 (Flow cytometry, 53-6.7) BioLegend Cat# 100730 Anti-CD8a (Flow cytometry, KT15) Invitrogen Cat# MA5-16759 Anti- Active Caspase-3 (Flow cytometry, C92-605) BD Biosciences Cat# C92-605 Peroxidase AffiniPure Goat Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 109-035-088 AffiniPure Goat Anti-Human IgG, Fcg fragment specific Jackson ImmunoResearch Cat# 109-005-098 Annexin V (Flow cytometry) BioLegend Cat# 640912 Fixable Viability Dye eFluor 506 Thermo Fisher Cat# 65-0866-18 7-AAD Viability Staining Solution (Flow cytometry) BioLegend Cat# 420404 iTAg Tetramer/PE - H-2 Kb OVA (SIINFEKL) MBL Cat# TB-5001-1 Donkey Anti-Human IgG (H+L) Jackson ImmunoResearch Cat# 709-116-149 Anti-FcgIII/II receptor (clone 2.4G2) BD Biosciences Cat# 553141 Erbitux (Cetuximab) Pharmacy N/A Chemicals, Peptides, and Recombinant Proteins FTY720 (hydrochloride) Selleckchem Cat# S5002 Clophosome -A - Clodronate Liposomes (Anionic) FormuMax Scientific Cat# F70101C-A IRDye 800CW NHS Ester Fisher: LI-COR Cat# NC9690013 TMB Solution (1X) eBioscience Cat# 00-4201-56 Diphtheria toxin Sigma- Aldrich Cat# D0564 OVA257-264 (SIINFEKL) Invivogen Cat# vac-sin SIYRYYGL (SIY) peptide Sigma- Aldrich N/A Ovalbumin Sigma- Aldrich Cat# A2512 Sulfadiazine/ Trimethoprim (Aurora Pharmaceutical LLC) UTSW-Veterinary Drug Services Cat# 302 Dulbecco’s Modified Eagle’s Medium Sigma- Aldrich Cat# D6429 GE Healthcare Ficoll-Paque PLUS Media Fisher Cat# 45-001-750 Recombinant murine IFN-g Fisher Cat# 50-813-664 Recombinant murine IL-10 PeproTech Cat# 210-10 Recombinant mouse IL-12 BioLegend Cat# 577002 Recombinant mouse GM-CSF BioLegend Cat# 576306 Critical Commercial Assays BD Cytometric Bead Array (CBA) Mouse Inflammation Kit BD Biosciences Cat# 552364 BD Mouse IFN-g ELISPOT Sets BD Biosciences Cat# 551083 SsoAdvanced Uni SYBR Grn Supmix Bio-Rad Cat# 1725272 RNeasy Plus Mini Kit Qiagen Cat# 74134 iScript gDNA Clear cDNA Synthesis Kit Bio-Rad Cat# 1725035 True-Nuclear Transcription Factor Buffer Set BioLegend Cat# 424401 EasySep Mouse CD8+ T Cell Isolation Kit STEMCELL Cat# 19853 (Continued on next page) e1 Cancer Cell 35, 901–915.e1–e4, June 10, 2019

Techniques: Control, Cytometry

Journal: Journal of Veterinary Internal Medicine

Article Title: Can levamisole upregulate the equine cell‐mediated macrophage (M1) dendritic cell (DC1) T‐helper 1 (CD4 Th1) T‐cytotoxic (CD8) immune response in vitro?

doi: 10.1111/jvim.15404

Figure Lengend Snippet: Staining combinations used for immune phenotype analysis

Article Snippet: Cells were resuspended and stained with optimized concentrations of cell surface antibodies CD21 (BD557327), CD4 (Abd Serotec MCA1078F), CD8 (Abd Serotec MCA1080PE), CD14 (Wagner laboratory clone 105), CD172a (Kingfisher Biotech WS0567B‐100) and activation markers major histocompatibility complex (MHC) class II (Abd Serotec MCA1085F), CD86 (BD Biosciences 555 665), and FoxP3 (ebioscience 53‐4776) were added to the samples in different combinations (Table ) to allow determination of subset‐specific activation and cytokine production and were incubated at 4°C for 20 minutes.

Techniques: Staining, Marker, Activation Assay

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 1. Flow cytometric evaluation of Nef-mediated CD8 down-regulation in retrovirally transduced cells. (A) Bivariate dot plots (CD8- allophycocyanin, CD8-phycoerythrin versus EGFP) of flow cytometric measurement of Nef (control) and Nef (NA-7 allele) transduced peripheral blood mononuclear cells, gated on CD8 cells, at day 3 after transduction. (B) Bivariate dot plots of flow cytometric measurement (CD8-allophycocyanin, CD8-phycoerythrin versus EGFP) of Nef NA-7 wild-type and NA-7 LLAA transduced SupT1 cells (left) and SupT1 cells overexpressing CD8 (right), at day 2 after transduction. (C) The solid and open histograms show the CD8 expression profile of SupT1 CD8 cells and SupT1 CD8 cells (CD8-transduced population), respectively, gated as shown in the inset. (D) Daudi CD8 cells and Daudi CD8 cells were transduced with control, HIV-1 (NL4-3, LAI and NA-7), SIV (mac239), and HIV-2 (Rod) Nef. Percent down-regulation is shown with white bars for CD8 in Daudi CD8, with gray bars for CD8 in Daudi CD8, and with black bars for CD8 in Daudi CD8. All percentages were calculated, as described in Materials and Methods, using the ranges E and E, as indicated in A.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Control, Transduction, Expressing

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 3. Flow cytometric analysis of Nef-mediated receptor down- regulation and internalization in retrovirally transduced SupT1 cells. SupT1 cells were transduced with an inducible NA-7.ER construct. At time zero, 4-hydroxytamoxifen (1 M) was added to the culture me- dium. (A) Percent down-regulation was calculated, as described in the Materials and Methods. CD4-allophycocyanin (solid line) and CD8- phycoerythrin (dashed line) were measured as a function of time. (B) The figure shows the percentage of CD4, CD8, and CD28 molecules internalized by HIV-1 NA-7.ER, calculated as described in Materials and Methods. In each graph, Nef-positive (EGFP expressing Nef, solid line) and Nef-negative (EGFP not expressing Nef, dashed line) cells are depicted. The EGFP ranges used for calculation are indicated in Fig. 1A.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Transduction, Construct, Expressing

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 4. Mutations in the CD8 -chain and their effect on endocytosis and down-regulation. Daudi cells were cotransduced with CD8, wild-type or mutant CD8, and control or wild-type Nef. (A and C) Alignment of amino acid sequences of wild-type (210*) and mutant CD8 -chain cytoplasmic tails. An asterisk indicates a stop codon. (A) The bar chart shows the percent down-regulation of (mutant) CD8 by HIV-1 Nef alleles NA-7, LAI, and NL4-3. In both B and D the percentage of (mutant) CD8 molecules internalized by wild-type HIV-1 NA-7 is shown, including in both the same data for a Nef-negative construct as a control (wild-type Nef ). (C) The bar chart represents the percentage of (mutant) CD8 down-regulation after transduction with either control virus or wild-type Nef NL4-3. Each bar represents a (mutant) CD8 -chain, as indicated by the changed amino acid sequence compared with CD8. Percent down-regulation and internalization were calculated as described in Materials and Methods, using the ranges indicated in Fig. 1A. In A, B, and D, mean values are shown and standard deviations are calculated from the data generated from three independent experiments. In B the results for NL4-3 are representative of the results with HIV-1 alleles NA-7 and LAI.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Mutagenesis, Control, Construct, Transduction, Virus, Sequencing, Generated

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 5. Chimeric constructs. Daudi cells were retrovirally trans- duced with the CD8(EC-TM)-CD8(IC) chimera (cyt tail) or a CD8(EC-TM)-CD8(IC) chimera (cyt tail), using bicistronic con- structs with NGFR as the reporter. Bivariate dot plots are gated on NGFR-positive cells, at day 2 after transduction of these cells with control virus, HIV-1 Nef (NA-7 allele), and SIV Nef (mac239), using bicistronic constructs with EGFP as the reporter. CD8-phycoerythrin versus EGFP expression is shown.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Construct, Transduction, Control, Virus, Expressing

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 6. Confocal images of 293T cells. Nef.EGFP was detected by direct fluorescence (green, left panels) and CD8.HA or CD8 by monoclonal antibodies as indicated in Materials and Methods (red, middle panels). Nuclei were visualized by DAPI staining (blue). Right panels show the merged images from Nef.EGFP and CD8. Areas of colocalization of Nef.EGFP/CD8 are shown in yellow. As indicated, the upper panels show cells expressing wild-type LAI, the middle panels show the LLAA mutant, and alower panels show the PPAA mutant. Scale bars represent 5 m.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Bioprocessing, Staining, Expressing, Mutagenesis

Journal: Journal of Virology

Article Title: Human Immunodeficiency Virus Nef Induces Rapid Internalization of the T-Cell Coreceptor CD8αβ

doi: 10.1128/jvi.79.17.11422-11433.2005

Figure Lengend Snippet: FIG. 7. Blocking Nef-mediated internalization and down-regulation by ikarugamycin and RNA interference. (A) The figures show the percentage of CD8 molecules internalized in Daudi CD8 cells by HIV-1 NA-7.ER. Cells were incubated for 2 h with (IKA) or without (IKA ) ikarugamycin (2 M) prior to the internalization experiment. At time zero, 4-hydroxytamoxifen (1 M) was added to NA-7.ER- transduced Daudi cells. (B) Western blot, performed as indicated in Materials and Methods, shows protein expression levels of AP-2 2 subunit, clathrin heavy chain (Chc), and dynamin 2 (Dyn-2, arrowhead) in control and RNAi-transduced SupT1 cells, with equal amounts of protein loaded. (C) Bivariate dot plots of flow cytometric measurement of SupT1 cells transduced with AP-2i and HIV-1 Nef (LAI). CD8 (phycoerythrin) versus NGFR (allophycocyanin) expression is shown, gated on EGFP-negative and EGFP-positive cells. (D) The bar charts represent the effect of AP-2, clathrin heavy chain (Chc), and dynamin 2 RNAi on HIV-1 Nef (LAI) and SIV (mac239) Nef-induced CD4 (left panel) and CD8 (right panel) down-regulation in transduced SupT1 cells, gated on EGFP-negative and EGFP-positive cells. Percent down-regulation was calculated, as described in Materials and Methods, using the ranges N and N, as indicated in C. Mean values and standard deviations are shown, calculated from data generated from three independent experiments.

Article Snippet: After a 10-min rehydration in phosphate-buffered saline, cells were blocked for 30 min at room temperature (0.4% fish skin gelatin [Sigma-Aldrich] in phosphate-buffered saline), followed by incubation for 60 min with a mouse anti-human CD8 primary antibody (5F2 [15], Serotec, Oxford, United Kingdom) or mouse anti-HA antibody (HA.11, Covance), both 1:100 diluted in blocking solution.

Techniques: Blocking Assay, Incubation, Western Blot, Expressing, Control, Transduction, Generated

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A IOD of ACAT2 expression in CC tissues and adjacent tissues was examined using immunohistochemical staining ( n = 47 biologically independent samples). IOD of DHCR7 B and MSMO1 C expression in CC patients with high ( n = 27 biologically independent samples) or low ( n = 20 biologically independent samples) expression of ACAT2 was examined using immunohistochemical staining. The number of activated CD8 T cells (CD8A + GZMB + ) D or activated NK cells (CD56 + GZMB + ) E infiltrated in the tumor tissues of patients with high ( n = 27 biologically independent samples) and low ACAT2 ( n = 20 biologically independent samples) expression was detected. Data represent mean ± SEM. Statistical analysis was performed using the paired A or unpaired ( B – E ) t-test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: ACAT2 expression in HCeEpiC and CC cell lines was examined using RT-qPCR A and Western blot analysis B ( n = 5 independent experiments). C ACAT2, DHCR7, and MSMO1 expression in CC cells after infection with Scramble-sh, ACAT2-sh #1, and ACAT2-sh #2 was examined using Western blot analysis ( n = 5 independent experiments). D Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in CC cells ( n = 5 independent experiments). The proliferation of CC cells was examined using CCK8 ( E ) and colony formation assays F (n = 5 independent experiments). G CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 T cells for 6 h, respectively, and the death of CC cells was detected ( n = 5 independent experiments). H IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , B ) or two-way ( C - H ) ANOVA, followed by Tukey’s multiple comparisons test ( A – H ).

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Infection, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A ACAT2 knockdown efficiency in U14 cells was examined using western blot analysis ( n = 10 independent experiments). B Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells (n = 10 animals). C The images and weight of the tumors harvested on day 21 ( n = 10 animals). D Protein expression of ACAT2, MSMO1, DHCR7, and PCNA in transplanted tumors was examined using western blot analysis ( n = 10 animals). E Detection of total cholesterol, free cholesterol, and cholesteryl ester levels in transplanted tumors ( n = 10 animals). The gating strategy for GZMB + NK cells and CD8 + T cells F and quantification G were analyzed using flow cytometry ( n = 10 animals). H Survival of mice over 60 days after subcutaneous inoculation of U14 cells was analyzed using the log-rank test ( n = 20 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( A , C , E , G ) or two-way ( B , D ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Knockdown, Western Blot, Expressing, Flow Cytometry

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: The proliferation of CC cells was examined using CCK8 A and colony formation assays B ( n = 5 independent experiments). C CC cells were co-cultured with (E: T = 3:1) with NK cells or CD8 + T cells, and the death of CC cells was detected ( n = 5 independent experiments). D IFN-γ and GZMB released from immune cells in a co-culture system with CC cells were examined using ELISA ( n = 5 independent experiments). E TGF-β1 released by CC cells was examined using ELISA ( n = 5 independent experiments). F PD-L1 expression levels in CC cells were observed using immunofluorescence staining ( n = 5 independent experiments). Data represent mean ± SEM. Statistical analysis was performed using the two-way ( A – F ) ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining

Journal: Communications Biology

Article Title: SREBF2 enhances lipid metabolism and represses anti-tumor immune responses in cervical cancer by increasing ACAT2

doi: 10.1038/s42003-026-09678-9

Figure Lengend Snippet: A Volume changes of transplanted tumors in mice subcutaneously inoculated with U14 cells ( n = 5 animals). B The images and weight of the tumors harvested on day 21 ( n = 5 animals). The gating strategy for GZMB + NK cells and CD8 + T cells C and quantification D were analyzed using flow cytometry ( n = 5 animals). Data represent mean ± SEM. Statistical analysis was performed using the one-way ( B , D ) or two-way A ANOVA, followed by Tukey’s multiple comparisons test.

Article Snippet: The cell suspension (100 μL) was incubated with BeyoFC Fc Receptor Blocking Solution (C1755, Beyotime) for 10 min at 4 °C and with primary antibodies, including FITC-coupled CD3 antibody (1:100, FITC-65077, ProteinTech, RRID: AB_2883763), PE-coupled NK1.1 antibody (1:100, PE-65138, ProteinTech, RRID: AB_2883920), and APC-coupled CD8A antibody (1:100, APC-65069, ProteinTech, RRID: AB_2882970) for 1 h at 4 °C.

Techniques: Flow Cytometry

Journal: Nature Communications

Article Title: p140Cap inhibits β-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response

doi: 10.1038/s41467-023-37824-y

Figure Lengend Snippet: a Effect of p140Cap over-expression on tumor growth and metastasis in TuBo and 4T1 BC cell models. TuBo (10 5 ) or 4T1 (10 4 ) mock and p140Cap cells were inoculated into the fat pad of 8-weeks-old female BALB/c mice. Tumor growth was monitored and tumor size was measured every two days from tumor onset (TuBo, n = 11; 4T1, n = 5; data are represented for n = x mices, two-tailed unpaired t test). For metastasis analysis in TuBo mock and p140Cap tumor-bearing mice, tumors were surgically removed when they reached 10 mm diameter and mice were kept alive. After 5 weeks, mice were sacrified and lungs were explanted and analyzed. For metastasis analysis of 4T1 mock and p140Cap tumors-bearing mice, lungs were analyzed 22 and 30 days post-injection for mock and p140Cap cells, respectively. Representative dot plots show the number of lung metastasis as mean ± SEM (Standard Error of the Mean; TuBo, n = 5; 4T1, n = 10; 2way ANOVA). b , c Flow cytometry analysis for M1- and M2-macrophages, CD4 + and CD8 + T-Lymphocytes, Natural Killer cells in tumor-bearing mice. Representative dot plots show the percentage (%) of tumor infiltrated M1- and M2-macrophages, CD4 + and CD8 + T-Lymphocytes, Natural Killer (NK) cells, normalized on CD45 + cells in TuBo mock and p140Cap tumor-bearing mice in panel ( b ) ( n = 8/M1, CD4 + and CD8 + and n = 9/NK, n = 5/M2) and 4T1 mock and p140Cap tumor-bearing mice in panel ( c ) ( n = 5/group). Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test). For TuBo mock and p140Cap tumors, the analysis was performed at day 26 or 32, respectively, while for 4T1 mock and p140Cap tumors at day 12. d Flow cytometry analysis for PMN-MDSCs (CD11b + Ly6G + Ly6C low ) and for M-MDSCs (CD11b + Ly6G − Ly6C + ) normalized on CD45 + cells, in tumor-bearing mice. Representative dot plots show the percentage of tumor infiltrated PMN-MDSCs and M-MDSCs cells in TuBo and 4T1 mock and p140Cap tumor-bearing mice, as described in panels ( b , c ) (TuBo n = 8/group; 4T1 n = 7/PMN-MDSCs and n = 8/M-MDSCs). Data are represented for n = x mice as mean ± SEM; two-tailed unpaired t test.

Article Snippet: The following antibodies were used (at a dilution of 1:200): CD45-VioGreen (Cat#130-110-803), CD11b-FITC (Cat#130-110-803), Ly6G-VioBlue (Cat#130-119-986), Ly6C-APC-Vio770 (Cat#130-121-439), F4/80-PE-Vio770 (Cat#130-118-320), MHC-II-APC (Cat#130-102-139), CD3-FITC (Cat#130-119-135), CD4-APC-Vio770 (Cat#130-119-134), CD8-VioBlue (Cat#130-123-865), CD49b-PE (Cat#130-123-702) all from Miltenyi Biotec (Miltenyi Biotec B.V. & Co. Bologna, Italy), while CD206-PE (Cat#141706) was from BioLegend (BioLegend, San Diego CA, USA).

Techniques: Over Expression, Two Tailed Test, Injection, Flow Cytometry