Journal: Immunity

Article Title: Central memory CD8 + T cells derive from stem-like Tcf7 hi effector cells in the absence of cytotoxic differentiation.

doi: 10.1016/j.immuni.2020.09.005

Figure Lengend Snippet: Figure 1. Effector-Stage Tcf7hi CD8+ T Cells Resemble Central Memory Cells B6 (CD45.1/2) mice were adoptively transferred with Tcf7GFP P14 cells (CD45.2) and infected with LCMV WE. (A) Tcf7GFP expression by splenic P14 cells at the indicated time points post-infection (p.i.).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Peptide: Ovalbumin amino acids 257-264 (OVA) (SIINFEKL) P. Romero, UNIL N/A Peptide: KL-SLP (KKKKKLEQLEAAYSIINFEKL) GenScript, NJ N/A Percoll GE Heathcare Cat# 17-0891-01 Polybrene Sigma-Aldrich Cat# TR-1003-G Recombinant human IL-2 Glaxo IMB, Genève, Switzerland gift from N. Rufer Sunflower seed oil Sigma-Aldrich Cat# S5007 Tamoxifen Sigma-Aldrich Cat# T5648 TDE1, Tagment DNA Enzyme Illumina Cat# 15027865 Trizol Life Technlogies Cat# 15596026 2xTD buffer Illumina Cat# 15027866 7-AAD (Viability dye) Biolegend Cat# 420404 Critical Commercial Assays Tumor Dissociation Kit Miltenyi Biotec Cat# 130-096-730 Mouse CD8+ T cell enrichment kit StemCell Technologies Cat# 19853 Direct–zol RNA Mini Prep Zymo Research Cat# R2050 Intracellular Fix & Perm Buffer set eBiosciences Cat# 88-8824 FoxP3/Transcription factor staining buffer set eBiosciences Cat# 00-5523 SMART-Seq v4 Ultra Low Input RNA reagents Clontech Cat# 634888 Illumina Nextera XT DNA Library reagents Illumina Cat# 15032354 MACS CD8+ positive selection Miltenyi Biotec Cat# 130-116-478 Annexin V - APC Apoptosis Detection Kit eBioscience RRID:AB_2575165 SuperScript III First-Strand Synthesis System ThermoFisher Scientific Cat# 18080051 KAPA SYBR FAST qPCR Kit Master Mix Kapabiosystems Cat# KR0389 Dynabeads Mouse T-Activator CD3/CD28 ThermoFisher Scientific Cat# 11452D CellTrace Violet Cell Proliferation Kit ThermoFisher Scientific Cat# C34557 MinElute PCR Purification kit QIAGEN Cat# 28004 Illumina’s Unique Dual (UD) Indexes Illumina Cat# 20027213 NEBNext High-Fidelity 2X PCR Master Mix New England Biolabs Cat# M0541 AMPure XP magnetic beads Beckman Coulter Cat# A63880 Zombie Aqua Fixable Viability kit Biolegend Cat# 423101 Deposited Data RNA-seq data This study GEO: GSE144383 ATAC-seq data This study GEO GSE144383 Experimental Models: Cell Lines Mouse: RMA RRID:CVCL_J385 Experimental Models: Organisms/Strains Mouse: C57BL/6 (B6) (CD45.2) Charles River Laboratoies Strain 027 Mouse: B6.SJL-Ptprc < a > (B6 CD45.1) Jackson Lab Strain 002014; RRID:MGI:6200621 Mouse: B6; D2-Tg(TcrLCMV)327Sdz P14 T cell receptor (TCR) transgenic (CD45.2) (Pircher et al., 1989) RRID:MGI:3810256 Mouse: B6.129-Tm(Tcf7)Cle (Tcf7 / ) (CD45.2) Verbeek et al., 1995 RRID:MGI:4360712 (Continued on next page) Immunity 53, 1–16.e1–e11, November 17, 2020 e3

Techniques: Infection, Expressing

Journal: Immunity

Article Title: Central memory CD8 + T cells derive from stem-like Tcf7 hi effector cells in the absence of cytotoxic differentiation.

doi: 10.1016/j.immuni.2020.09.005

Figure Lengend Snippet: Figure 5. Vaccination of Mice and Humans Generates Effector-Phase Tcf1hi CD8+ T Cells (A–H) B6 Tcf7GFP mice were vaccinated as indicated with a modified Ovalbumin peptide and Pam3CSK4 in Montanide (Ova) or with Pam3CSK4 in Montanide (Ø). (B, C, and E) Abundance and (D and F) Tcf7GFP and CD62L expression by KbOva+ CD8+ T cells in the blood on d21 and d35 (B–D) and in the spleen on d35 post- vaccination (E–G). (H) IL-2 and TNF-a production by d35 splenic IFN-g+ cells. (I–K) Healthy volunteers received a yellow fever vaccine, and peripheral blood CD8+ T cells were analyzed 14 days later. (I) TCF1 expression by A2/LLW tetramer+

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Peptide: Ovalbumin amino acids 257-264 (OVA) (SIINFEKL) P. Romero, UNIL N/A Peptide: KL-SLP (KKKKKLEQLEAAYSIINFEKL) GenScript, NJ N/A Percoll GE Heathcare Cat# 17-0891-01 Polybrene Sigma-Aldrich Cat# TR-1003-G Recombinant human IL-2 Glaxo IMB, Genève, Switzerland gift from N. Rufer Sunflower seed oil Sigma-Aldrich Cat# S5007 Tamoxifen Sigma-Aldrich Cat# T5648 TDE1, Tagment DNA Enzyme Illumina Cat# 15027865 Trizol Life Technlogies Cat# 15596026 2xTD buffer Illumina Cat# 15027866 7-AAD (Viability dye) Biolegend Cat# 420404 Critical Commercial Assays Tumor Dissociation Kit Miltenyi Biotec Cat# 130-096-730 Mouse CD8+ T cell enrichment kit StemCell Technologies Cat# 19853 Direct–zol RNA Mini Prep Zymo Research Cat# R2050 Intracellular Fix & Perm Buffer set eBiosciences Cat# 88-8824 FoxP3/Transcription factor staining buffer set eBiosciences Cat# 00-5523 SMART-Seq v4 Ultra Low Input RNA reagents Clontech Cat# 634888 Illumina Nextera XT DNA Library reagents Illumina Cat# 15032354 MACS CD8+ positive selection Miltenyi Biotec Cat# 130-116-478 Annexin V - APC Apoptosis Detection Kit eBioscience RRID:AB_2575165 SuperScript III First-Strand Synthesis System ThermoFisher Scientific Cat# 18080051 KAPA SYBR FAST qPCR Kit Master Mix Kapabiosystems Cat# KR0389 Dynabeads Mouse T-Activator CD3/CD28 ThermoFisher Scientific Cat# 11452D CellTrace Violet Cell Proliferation Kit ThermoFisher Scientific Cat# C34557 MinElute PCR Purification kit QIAGEN Cat# 28004 Illumina’s Unique Dual (UD) Indexes Illumina Cat# 20027213 NEBNext High-Fidelity 2X PCR Master Mix New England Biolabs Cat# M0541 AMPure XP magnetic beads Beckman Coulter Cat# A63880 Zombie Aqua Fixable Viability kit Biolegend Cat# 423101 Deposited Data RNA-seq data This study GEO: GSE144383 ATAC-seq data This study GEO GSE144383 Experimental Models: Cell Lines Mouse: RMA RRID:CVCL_J385 Experimental Models: Organisms/Strains Mouse: C57BL/6 (B6) (CD45.2) Charles River Laboratoies Strain 027 Mouse: B6.SJL-Ptprc < a > (B6 CD45.1) Jackson Lab Strain 002014; RRID:MGI:6200621 Mouse: B6; D2-Tg(TcrLCMV)327Sdz P14 T cell receptor (TCR) transgenic (CD45.2) (Pircher et al., 1989) RRID:MGI:3810256 Mouse: B6.129-Tm(Tcf7)Cle (Tcf7 / ) (CD45.2) Verbeek et al., 1995 RRID:MGI:4360712 (Continued on next page) Immunity 53, 1–16.e1–e11, November 17, 2020 e3

Techniques: Expressing

Journal: Immunity

Article Title: Central memory CD8 + T cells derive from stem-like Tcf7 hi effector cells in the absence of cytotoxic differentiation.

doi: 10.1016/j.immuni.2020.09.005

Figure Lengend Snippet: Figure 6. Tcf1 Is Essential for the Stemness of d8 Tcf7GFPhi CD8+ T Cells (A–D) B6 mice (CD45.1/2) were transplanted with WT or Tcf7/ (KO) Tcf7GFP P14 cells (CD45.2) and infected with LCMV WE. (A) Abundance of P14 cells in the spleen at d8 p.i. and (B) expression of Tcf7GFP. Phenotype (C) and cytokine production (D) by WT and KO Tcf7GFPhi P14 cells. (E–G) Recall response of sorted d8 WT and KO Tcf7GFPhi P14 cells. (E) Abundance of P14 cells in the spleen and (F) Tcf7GFP expression 8 days later (d8+8). (G) Abundance of secondary Tcf7GFPhi cells compared to input. Data are representative of 3 experiments with 4 mice per group (A–C), representative of 2 experiments with 5–6 mice per group (D), or compiled from 2 ex- periments with 5–7 mice per group (E–G). Mean ± SD are shown. Statistics: non-paired t test (A–D and F) or one-way ANOVA with Tukey’s test (E and G) with *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; and (ns) p > 0.05. See also Figure S7.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Peptide: Ovalbumin amino acids 257-264 (OVA) (SIINFEKL) P. Romero, UNIL N/A Peptide: KL-SLP (KKKKKLEQLEAAYSIINFEKL) GenScript, NJ N/A Percoll GE Heathcare Cat# 17-0891-01 Polybrene Sigma-Aldrich Cat# TR-1003-G Recombinant human IL-2 Glaxo IMB, Genève, Switzerland gift from N. Rufer Sunflower seed oil Sigma-Aldrich Cat# S5007 Tamoxifen Sigma-Aldrich Cat# T5648 TDE1, Tagment DNA Enzyme Illumina Cat# 15027865 Trizol Life Technlogies Cat# 15596026 2xTD buffer Illumina Cat# 15027866 7-AAD (Viability dye) Biolegend Cat# 420404 Critical Commercial Assays Tumor Dissociation Kit Miltenyi Biotec Cat# 130-096-730 Mouse CD8+ T cell enrichment kit StemCell Technologies Cat# 19853 Direct–zol RNA Mini Prep Zymo Research Cat# R2050 Intracellular Fix & Perm Buffer set eBiosciences Cat# 88-8824 FoxP3/Transcription factor staining buffer set eBiosciences Cat# 00-5523 SMART-Seq v4 Ultra Low Input RNA reagents Clontech Cat# 634888 Illumina Nextera XT DNA Library reagents Illumina Cat# 15032354 MACS CD8+ positive selection Miltenyi Biotec Cat# 130-116-478 Annexin V - APC Apoptosis Detection Kit eBioscience RRID:AB_2575165 SuperScript III First-Strand Synthesis System ThermoFisher Scientific Cat# 18080051 KAPA SYBR FAST qPCR Kit Master Mix Kapabiosystems Cat# KR0389 Dynabeads Mouse T-Activator CD3/CD28 ThermoFisher Scientific Cat# 11452D CellTrace Violet Cell Proliferation Kit ThermoFisher Scientific Cat# C34557 MinElute PCR Purification kit QIAGEN Cat# 28004 Illumina’s Unique Dual (UD) Indexes Illumina Cat# 20027213 NEBNext High-Fidelity 2X PCR Master Mix New England Biolabs Cat# M0541 AMPure XP magnetic beads Beckman Coulter Cat# A63880 Zombie Aqua Fixable Viability kit Biolegend Cat# 423101 Deposited Data RNA-seq data This study GEO: GSE144383 ATAC-seq data This study GEO GSE144383 Experimental Models: Cell Lines Mouse: RMA RRID:CVCL_J385 Experimental Models: Organisms/Strains Mouse: C57BL/6 (B6) (CD45.2) Charles River Laboratoies Strain 027 Mouse: B6.SJL-Ptprc < a > (B6 CD45.1) Jackson Lab Strain 002014; RRID:MGI:6200621 Mouse: B6; D2-Tg(TcrLCMV)327Sdz P14 T cell receptor (TCR) transgenic (CD45.2) (Pircher et al., 1989) RRID:MGI:3810256 Mouse: B6.129-Tm(Tcf7)Cle (Tcf7 / ) (CD45.2) Verbeek et al., 1995 RRID:MGI:4360712 (Continued on next page) Immunity 53, 1–16.e1–e11, November 17, 2020 e3

Techniques: Infection, 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 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: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: Ascites blocks NK cell degranulation but not TRAIL-dependent HPMC apoptosis (A and B) TAL were pre-cultured in RPMI/5% AB media or 100% ascites pool (+/− α-CD3 Ab stimulation) prior to co-culture with HPMC. (A) Apoptosis of HPMC is shown as percentage of annexin V+ cells after gating on CD45 − cells (n = 7 patients). (B) Degranulating NK cells in response to HPMC were measured via flow cytometry and depicted as percentage of CD335+/CD107a+ cells (n = 6 patients). (C and D) TAL were pretreated with α-TRAIL blocking Ab prior to HPMC co-culture. An irrelevant mouse IgG was included as control. Experiments were conducted with TAL cultured in RPMI/5% AB media (n = 11 patients) (C) and 100% ascites pool (n = 5 patients) (D). The amount of Annexin V+ HPMC was determined as described previously. (E) Schematic representation of co-culture experiments applying T cell CM for NK cell activation. CM were collected from CD3 + , CD3+/CD4+, and CD3+/CD8+ T cell subsets cultured in media +/− α-CD3 Ab stimulation. Purified NK cells were then stimulated with T cell CM prior to HPMC co-cultures. (F) The amount of apoptotic HPMC induced by NK cells activated with CM of CD3 + T cells was compared with CM of CD3+/CD4+ and CD3+/CD8+ T cells (n = 4 matched pairs of different patients). (G) Analysis of TRAIL signaling was performed by applying an α-TRAIL blocking Ab to NK cells stimulated with CD3 + T cell CM (α-CD3 Ab activated) prior to the co-culture with HPMC (n = 5 patients). An irrelevant mouse IgG was included as control. The mean is shown by horizontal bars or boxes; vertical error bars represent the standard deviation in A–D, F, and G. ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; determined by paired t test and Benjamini-Hochberg adjustment (ns, not significant).

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Cell Culture, Co-Culture Assay, Flow Cytometry, Blocking Assay, Control, Activation Assay, Purification, Standard Deviation

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet:

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Control, Recombinant, Polymer, Software

Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Representative anamnestic vaccine-induced immune responses in the vaccinees. All vaccinees made CD8+ T-cell responses to several epitopes, including some restricted by the MHC-I molecule Mamu-A*02. In the interest of space, we are showing only two of the vaccinees in this figure; data from the remaining vaccinees are shown elsewhere (see Fig. S3 in the supplemental material). Whole-PBMC responses are indicated by blue bars, with responses postchallenge indicated in dark blue and responses observed immediately prior to challenge indicated in light blue. Responses observed in PBMCs depleted of CD8+ cells (red) are likely mediated by CD4+ T cells. CD8+ cell depletion was typically 99% complete (data not shown). Dark red bars indicate responses observed postchallenge, whereas light red bars indicate responses present immediately prior to the challenge. Green bars represent responses to minimal optimal peptides that bind to Mamu-A*02. As indicated in Table S1 in the supplemental material, some of these epitopes are conserved between SIVmac239 and SIVsmE660, whereas others have several substitutions, some of which could affect T-cell recognition. Again, light green bars indicate responses observed prior to the challenge, whereas dark green bars indicate anamnestic responses. Most anamnestic response analyses were performed at 14 to 15 days postinfection. For a couple of the animals (r00061, r02103), these assays were delayed to 21 days postinfection due to the very low viral loads observed.

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Frequency of anamnestic cellular immune responses in PBMC and PBMC depleted of CD8 + cells

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Breadth of anamnestic cellular immune responses in PBMC and PBMC depleted of CD8 + cells

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: Total CD8 + T cells from individuals without HIV were treated with medium, vehicle (Veh.) control, or the GSK3 inhibitor (inh), followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( A and B ) Analysis of CD8 + T cell subpopulations in unstimulated cells ( n = 4). ( C ) Fold change of CD8 + T cell subpopulations upon vehicle control or GSK3 inhibitor treatment, relative to the medium alone condition ( n = 4). ( D ) Expression of TCF-1 in CD8 + T cell subsets ( n = 4). ( E ) Fold change in the expression of the indicated markers induced by anti-CD3/anti-CD28 antibody stimulation relative to unstimulated cells ( n = 5). ( F ) Analysis of dead cells by Aqua LIVE/DEAD + staining (Aqua L/D) among total and T-bet + CD8 + T cells, and fold change in dead CD8 + T cells induced by anti-CD3/anti-CD28 stimulation relative to the unstimulated (Unstim.) condition ( n = 5). ( G ) Frequencies of granzyme B + (GZMB + ), IL-2 + , IFN-γ + , and TNF-α + CD8 + T cells after anti-CD3/anti-CD28 stimulation. ( H ) Expression of 1 to 4 functions in CD8 + T cells ( n = 5). * P < 0.05, by Dunn’s test ( B ) and Wilcoxon test ( C , D , and F – H ). Data obtained from 2 ( A – F ) or 3 ( G and H ) independent experiments are shown.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Incubation, Expressing, Staining

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: ( A and B ) Total CD8 + T cells from individuals without HIV were treated with vehicle control or the GSK3 inhibitor, followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( A ) Analysis of the expression of 2-NBDG, BODIPY, MitoTracker Green, and CellROX among CD8 + T cell subsets. ( B ) Frequencies of 2-NBDG + , BODIPY + , MitoTracker Green + , and CellROX + cells among CD8 + T cell subpopulations after stimulation ( n = 5). ( C and D ) Total CD8 + T cells from individuals without HIV were treated with vehicle control or the GSK3 inhibitor, followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( C ) Flow cytometric analysis of the expression of p-S6 and p-AKT in total CD8 + T cells, and frequencies of p-S6 + p-AKT + , p-S6 – p-AKT + , and p-S6 – p-AKT – subsets in total CD8 + T cells ( n = 7). ( D ) Flow cytometric analysis of IL-2 and IFN-γ expression among TNF-α + CD8 + T cells, after anti-CD3/anti-CD28 stimulation. Histograms show the expression of p-S6 in the indicated cell subsets, in reprogrammed and nonreprogrammed cells. Frequency of p-S6 + cells among TNF-α + IFN-γ + IL-2 + or TNF-α + IFN-γ – IL-2 – subsets ( n = 6 individuals without HIV). * P < 0.05 and ** P < 0.01, by Wilcoxon test ( B and C ) and Šidák’s multiple-comparison test ( D ). Data are from 2 independent experiments.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Incubation, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: After treatment with the GSK3 inhibitor, CD8 + T cells from people with HIV were stained with HLA-matched HIV dextramers for analysis of the phenotype of HIV dextramer + cells. ( A ) UMAP plots generated from HIV dextramer + CD8 + T cells after data concatenation ( n = 4). Vehicle control and GSK3 inhibitor treatments as well as CD8 + T cell subpopulations were identified by manual gating and projected into the UMAP space. ( B ) Analysis of the expression of CCR7, CD27, and TCF-1 expression in HIV dextramer + CD8 + T cells ( n = 7). ( C ) Frequencies of CD8 + T cell subpopulations among HIV dextramer + CD8 + T cells ( n = 7). ( D – G ) After reprogramming, cells from people with HIV were stimulated for 6 hours with Gag peptides for analysis of the total frequency (IFN-γ + or CD107a + or IL-2 + or TNF-α + ) of antigen-specific CD8 + T cells ( n = 5) ( D ), the proportion of memory T cell subpopulations ( n = 5) ( E ), the expression of CD107a, IFN-γ, granzyme B, IL-2, and TNF-α on a per-cell basis ( n = 5–11) ( F ), and the frequency of polyfunctional cells ( n = 6) ( G ). ( H and I ) Cells from people with HIV were stimulated for 6 days with Gag peptides and restimulated with the same peptides for another 12 hours, followed by analysis of the viability of proliferating HIV Gag–specific CD8 + T cells ( n = 6) ( H ) and frequencies of the total (live IFN-γ + , IL-2 + , or TNF-α + ) HIV Gag–specific response ( n = 8) ( I ). ( J and K ) After reprogramming, cells from people with HIV ( n = 6) were stimulated for 6 hours with Gag peptides, followed by analysis of p-S6 + p-AKT – , p-S6 + p-AKT + , p-S6 – p-AKT + , and p-S6 – p-AKT – cell subsets ( J ) and the intensity of expression of p-S6 and p-AKT ( K ) in HIV Gag–specific (IFN-γ + and/or IL-2 + ) CD8 + T cells. * P < 0.05 and *** P < 0.001, by Wilcoxon test. Data are from 3 independent experiments.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Staining, Generated, Control, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: HIV-1 BaL–superinfected CD4 + T cells from people with HIV were cultured alone or in the presence of autologous nonreprogrammed or reprogrammed CD8 + T cells. After 7 days, the levels of infection were measured by flow cytometry (KC57 anti-Gag antibody) or ELISA (p24 in culture supernatant). ( A ) Representative flow cytometric analysis of the frequency of infected CD4 + T cells (from a total of 4 donors). ( B ) HIV-suppressive capacity of nonreprogrammed and reprogrammed CD8 + T cells (log 10 decrease of p24 levels in culture supernatant; n = 5 individuals, with the median of triplicates for each experiment). ( C and D ) The frequency of IFN-γ + HIV-specific CD8 + T cells ( C ) and expression of CCR7, PD-1, and LAG-3 in HIV-specific CD8 + T cells ( D ) were measured after 7 days of coculturing. * P < 0.05 and ** P < 0.01, by Wilcoxon test.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Cell Culture, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: ( A ) Total CD8 + T cells from people without HIV were treated with vehicle control or the GSK3 inhibitor, followed by evaluation of Eomes and CD122 expression in CD8 + T cell subsets ( n = 5). ( B ) After vehicle control or GSK3 inhibitor treatment, CD8 + T cells from people without HIV were left unstimulated or stimulated with IL-7 or IL-15 for 6 days, followed by analysis of cell proliferation ( n = 5; data are from 2 independent experiments). ( C and D ) CD8 + T cells from people with HIV were treated with vehicle control or the GSK3 inhibitor, followed by stimulation with IL-15 for 6 days, for analysis of the proliferation of HIV dextramer + CD8 + T cells ( n = 4; data from 3 independent experiments). * P < 0.05, by Wilcoxon test.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Expressing