Sorted Teff and Tregs from healthy donors were activated with anti-CD3/CD28 beads (1:5 ratio) (Life Technologies) and 1000 UI IL-2 for 5 days a 37°C. Then, 100 uL of OSCC and control secretomes were added to 2x105 Teff or 2x105 Tregs (in 100uL) in XVIVO-15 serum-free medium 48h a 37°C. After the incubation, the supernatants were stored for further cytokine production measurement using the Cytokine Bead Array Th1/2/17 Kit (BD) and the cells were counted (CountBright Absolute Counting Beads), stained with Live/Dead dye (Life Technologies), anti-CXCR3, anti-CCR4, anti-CCR6, anti-CCR8, anti-PD-1 and anti-TIGIT (all BioLegend) and analyzed by flow cytometry. For the analysis of cells after secretome co-culture, cells were washed after co-culture with secretome and cultured in new media X-VIVO15 (LONZA) serum-free medium for 48 h at 37°C with anti-CD3/CD28 beads (1:5 ratio) (Life Technologies) and 1000 UI IL-2. After the incubation, the supernatants were stored for further cytokine production measurement and the cells were stained with Live/Dead dye (Life Technologies), anti-CCR6, anti-CCR8, anti-PD-1 and anti-TIGIT (all BioLegend).
Anti pd 1
Manufactured by BioLegend
Sourced in United States, Germany
Anti-PD-1 is a lab equipment product that functions as an antibody targeting the PD-1 receptor. It is used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
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Anti pd l1, by BioLegend (6 mentions)
Anti tigit, by BioLegend (5 mentions)
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Anti gr1, by BioLegend (4 mentions)
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51 protocols using anti pd 1
1
OSCC Secretome Effects on Teff and Tregs
2
CD4+ T Cell Subpopulation Isolation and Phenotyping
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CD4+ T cells were enriched by magnetic activated cell sorting (MACS) using 20 µl of anti-CD4 MicroBeads (anti-CD4 MicroBeads, human, Miltenyi Biotec, Germany) and 80 µl MACS buffer (PBS with 0.5% BSA and 2 mM ethylenediaminetetraacetic acid) per 107 cells. CD4+ T cells were stained for surface expression in MACS buffer at a concentration of 2 × 108 cells/ml. Cells were washed and stained with 4′,6-diamidino-2-phenylindole (1:250) and then sorted using the flow cytometer BD FACSAria II (BD Biosciences, Germany) into the following CD4+ T-cell subpopulations: Treg (CD25highCD127low) and non-Treg: TN (CD45RA+ CCR7+), TCM (CD45RA-CCR7+), TEM (CD45RA− CCR7−), and TEMRA (CD45RA+ CCR7−).
Antibodies used for surface staining were anti-CCR7 (GO43H7, 1:50), anti-CD25 (M-A251, 1:100), anti-CD45RA (HI100, 1:50), anti-CD127 (A019D5, 1:100), anti-CD3 (UCHT1, 1:200), anti-CD4 (OKT4, 1:200), anti-PD-1 (EH12.2H7, 1:20), anti-TIGIT (A15153G, 1:20), anti-TIM-3 (F38–2E2, 1:20), and LAG-3 (11C3C65, 1:20) from BioLegend, and anti-KLRB1 (191B8, 1:10), anti-KLRF1 (4A4.D10, 1:50), and anti-KLRG1 (REA261, 1:10, 1:50 since 2018/06 due to more concentrated formulation) from Miltenyi Biotec. See also Supplementary Fig.5 for the pre-gating strategy.
Antibodies used for surface staining were anti-CCR7 (GO43H7, 1:50), anti-CD25 (M-A251, 1:100), anti-CD45RA (HI100, 1:50), anti-CD127 (A019D5, 1:100), anti-CD3 (UCHT1, 1:200), anti-CD4 (OKT4, 1:200), anti-PD-1 (EH12.2H7, 1:20), anti-TIGIT (A15153G, 1:20), anti-TIM-3 (F38–2E2, 1:20), and LAG-3 (11C3C65, 1:20) from BioLegend, and anti-KLRB1 (191B8, 1:10), anti-KLRF1 (4A4.D10, 1:50), and anti-KLRG1 (REA261, 1:10, 1:50 since 2018/06 due to more concentrated formulation) from Miltenyi Biotec. See also Supplementary Fig.
3
CXCR2 Inhibition in Murine Cancer Models
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For drug treatments, mice were randomly assigned to cohorts. Treatments used were: CXCR2 pepducin (X1/2pal-i3, Genscript) or scrambled pepducin at 2.5 mg/kg by subcutaneous injection daily; gemcitabine (LC Laboratories) at 100 mg/kg by intraperitoneal (i.p.) injection twice weekly; CXCR2 SM (AstraZeneca) at 100 mg/kg per os (p.o.) twice daily; vehicle p.o. twice daily; anti-PD1 (Biolegend) or isotype control at 10 mg/kg by i.p. injection twice weekly; 1A8 antibody or 2A3 isotype control (BioXcell) at 10 mg/kg by i.p. injection thrice weekly. Efficacy testing of CXCR2 SM is described in Supplemental Experimental Procedures .
4
Impact of PD-1 and TIM-3 Blockade on NK Cells
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Blocking monoclonal antibodies were used to analyse the impact of PD-1 and TIM-3 signalling on the anti-tumour response of activated NK cells. Considering the direct effect of ICIs on NK cell receptors, purified NK cells were incubated with 20 µg/ml of anti-PD1 (pembrolizumab) and/or 20 µg/ml of anti-TIM-3 (clone F382E2; BioLegend, San Diego, CA, USA) for 20 min at 4°C, before the co-culture with target cells. Human IgG1 isotype (Enzo, Farmingdale, NY, USA) measuring 5 µg/ml was used as control.
The efficacy of blockade was assessed by flow cytometry. Total PBMCs were incubated with the blocking antibodies before staining with the labelled flow cytometry antibody targeting PD-1 or TIM-3.
The efficacy of blockade was assessed by flow cytometry. Total PBMCs were incubated with the blocking antibodies before staining with the labelled flow cytometry antibody targeting PD-1 or TIM-3.
5
Purification and Characterization of B Cells
6
Generating Luciferase-Expressing Mouse Pancreatic Tumor Cells
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Pan02 cells, a SMAD4-null chemically induced mouse pancreatic tumour line, was from the National Cancer Institute [45 (link), 46 (link)]. Pan02-CAG-luc2 cells were generated by lentiviral transduction. Briefly, DNA encoding the luciferase 2 gene (luc2, Promega) was cloned into a lentiviral vector (pCDH-CAG-IRES-puro, System Biosciences). Lentivirus was generated using the pPack lentiviral packaging system (SBI), and used to transduce Pan02 cells. Following antibiotic selection, a pool of stably transduced cells (Pan02-CAG-luc2) was obtained and used for in vivo studies. Luc2 expression in the Pan02-CAG-luc2 cell line pool was stable up to passage 16, as determined by luciferase assay (Promega, data not shown).
The following antibodies from eBioscience, BD Bioscience and Biolegend were used for flow cytometry: anti-CD45 (clone 30-F11), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-CD3 (clone 145-2C11), anti-CD4 (Clone RM4.5), anti-CD8 (clone 53-6.7), anti-F4/80 (clone BM8), anti-MHCI (clone 28-8-6), anti-CD80 (clone 16-10A), anti-CD86 (clone GL-1), anti-MHCII (clone M5/114.15.2), anti-Gr-1 (clone RB6-8C5), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone6D5), anti-PD-L1 (clone 10F.9G2), anti-PD-1 (clone 29F.1A12), anti-FoxP3 (FJK-16S).
The following antibodies from eBioscience, BD Bioscience and Biolegend were used for flow cytometry: anti-CD45 (clone 30-F11), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-CD3 (clone 145-2C11), anti-CD4 (Clone RM4.5), anti-CD8 (clone 53-6.7), anti-F4/80 (clone BM8), anti-MHCI (clone 28-8-6), anti-CD80 (clone 16-10A), anti-CD86 (clone GL-1), anti-MHCII (clone M5/114.15.2), anti-Gr-1 (clone RB6-8C5), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone6D5), anti-PD-L1 (clone 10F.9G2), anti-PD-1 (clone 29F.1A12), anti-FoxP3 (FJK-16S).
7
Comprehensive Immune Cell Profiling
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Prior to fluorochrome staining, FcRIII/II blocking was performed using the TrueStain fcX™ antibody (Biolegend, London, UK). Cell surface staining was done with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone 6D5), anti-CD26 (clone H194–112), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD172a (clone P84), anti-CD206 (clone C068C2), anti-EpCAM (clone G8.8), anti-F4/80 (clone BM8), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8), anti-MHC-I (clone AF6–88.5), anti-MHC-II (clone AF6–120.01), anti-NK1.1 (clone PK136), anti-PD-1 (clone 29F.1A12), anti-PD-L1 (clone 10F.9G2), anti-CD86 (clone GL-1), anti-CD40 (clone 3/23), anti-XCR1 (clone ZET; all BioLegend, London, UK) and anti-CD204 (clone 2F8, Biorad, Munich, Germany) antibodies, and Fixable Viability Dye (Thermo Fisher Scientific, Karlsruhe, Germany) was used to exclude dead cells. The gating strategy is depicted in Additional file 1 : Figure S1. Intracellular staining was done for arginase-1 (Polyclonal Sheep IgG; R&D Systems, Minneapolis, USA) using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific, Karlsruhe, Germany). Data were acquired on a BD LSRFortessa system (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo X software (FLOWJO LLC, Ashland, OR, USA).
8
Comprehensive Immune Cell Profiling
9
Quantifying IL-2 Production in NY-ESO-1-HLA-A2+ Cell Lines
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Target cell lines expressing NYESO-HLA-A2 were plated on 24-well plates at a concentration of 1 × 105 per well. Twelve to 16 hours later, media were changed, and Jk T cells expressing TCR (1G4) or human PBMCs (ATCC, PCS-800-011) were added to the culture wells at a concentration of 1 × 106/mL (400 μL) for 24 hours. Anti-CD3 (Invitrogen, 16-0037-81, RRID:AB_468854) and anti-CD28 (Invitrogen, 16-0289-81, RRID:AB_468926) were added into the culture media at a final concentration of 1 μmol/L (when using human PBMCs). Anti–PD-1 (BioLegend, 329925, RRID:AB_11147369) and anti–PD-L1 (BioLegend, 329715, RRID:AB_11149486), when applicable, were added in the culture media at a final concentration of 10 μmol/L. Media were harvested after coculture and diluted from 1/10 to 1/50 and subjected to ELISA to detect IL2 production (BioLegend, 431804).
10
Tumor-infiltrating T cell profiling by multiparameter flow cytometry
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For flow cytometry and FACS purification of tumor-infiltrating T cell subsets, the following fluorochrome-conjugated antibodies were used. anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-PD-1 were purchased from Biolegend (San Diego, CA). For western-blot anti-PBRM1 (D3F7O, CST) and anti-β-actin (13E5, CST) were used. For multiplex immunohistochemistry assay anti-PBRM1 (D3F7O, CST), anti-CD4 (SP35, Maixin), anti-CD8 (SP16, Maixin) were used.
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