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: 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 neuroendocrinology

Article Title: GHR−/− Mice are Protected from Obesity-Related White Adipose Tissue Inflammation

doi: 10.1111/jne.12854

Figure Lengend Snippet: Flow Cytometry Antibodies (24 Months)

Article Snippet: Results were analyzed with FlowJo V10 software. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Target Conjugate Manufacturer Catalog # RRID CD206 AF488 BioRad MCA2235A488 AB_324891 Ly-6C PerCP-Cy5.5 Thermo Fisher 45–5932-80 AB_1518762 MHC-II Biotin Thermo Fisher 13–5321-81 AB_466661 F4/80 PE-Cy7 Thermo Fisher 25–4801-82 AB_469653 CD11c APC Thermo Fisher 117–0114-81 AB_469345 CD45 APC-eFluor 780 Thermo Fisher 47–0451-82 AB_1548781 CD11b Alexa Fluor 700 Thermo Fisher 56–0112-80 AB_657586 CCR2 Phycoerythrin R&D Systems FAB5538P AB_10718414 NK1.1 FITC Thermo Fisher 11–5941-81 AB_465317 NKT Phycoerythrin BD Biosciences 550082 AB_393552 CD44 Biotin Thermo Fisher 13–0441-81 AB_466441 CD4 PerCP-Cy5.5 Thermo Fisher 45–0042-80 AB_906231 CD3 PE-Cy7 Thermo Fisher 25–0031-81 AB_469571 CD25 APC Thermo Fisher 17–0251-81 AB_469365 CD62L Alexa Fluor 700 Thermo Fisher 56–0621-80 AB_494004 CD45 Alexa Fluor 700 Thermo Fisher 56–0451-80 AB_891456 CD16/CD32 Thermo Fisher 14–0161-82 AB_467133 Streptavidin eFluor 615 Thermo Fisher 42–4317-80 AB_11218079 Open in a separate window Flow Cytometry Antibodies (8 Months) table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 Target Conjugate Manufacturer Catalog # RRID CD11b VioGreen Miltenyi Biotec 130–113-811 AB_2726328 CD11c APC-Vio770 Miltenyi Biotec 130–110-841 AB_2654715 CD206 APC BioLegend 141707 AB_10896057 CD25 PE Miltenyi Biotec 130–108-996 AB_2656655 CD3 APC-Vio770 Miltenyi Biotec 130–109-840 AB_2657087 CD38 PE-Vio770 Miltenyi Biotec 130–109-258 AB_2657842 CD4 VioGreen Miltenyi Biotec 130–109-413 AB_2657964 CD44 PE-Vio770 Miltenyi Biotec 130–110-085 AB_2658157 CD45 VioBlue Miltenyi Biotec 130–110-802 AB_2658222 CD62L PerCP-Vio700 Miltenyi Biotec 130–107-046 AB_2660523 CD80 PE Miltenyi Biotec 130–116-460 AB_2727557 CD8a FITC Miltenyi Biotec 130–102-490 AB_2659883 F4/80 PerCP-Vio700 Miltenyi Biotec 130–102-161 AB_2651711 GITR APC Miltenyi Biotec 130–116-427 AB_2727531 Open in a separate window Flow Cytometry Antibodies (24 Months) 2.5.

Techniques: Flow Cytometry