Pd l1 apc

T cells were re-stimulated with phorbol myristate acetate (Sigma, 50 ng/mL) and ionomycin (Sigma, 500 ng/mL). 90 minutes later, cells were treated with Brefeldin A to block cytokine secretion. 3 h later, cells were stained for surface markers and simultaneously labelled with Live/Dead Blue Viability Dye (Thermo Fisher) for 20 minutes at 4°C. Cells were washed twice and fixed overnight using a FoxP3 Fixation/Permeabilization kit (Thermo Fisher). The following day, cells were washed and stained for intracellular cytokines at room temperature for 1 h. They were then washed 3 times and analysed using an LSR Fortessa machine (Beckman Dickinson). Analysis of mean fluorescence intensity was performed using FlowJo v10.0. All experiments were performed at least two independent times. Antibodies used (at 1:100 unless otherwise noted) were TNF-PE (BioLegend, MP6-XT22, 506306), PD-1-PECy7 (BioLegend, RMP1–30, 109110) IFN-γ-FITC (BioLegend, XMG1.2, 505806), CD4-BV711 (BioLegend, RM4–5, 100550), CD8α-BV786 (BioLegend, 53–6.7, 100750), Tox-PE (Miltenyi, REA473, 130–120-716, 1:50), Tcf7-Alexa647 (Cell Signaling, C63D9, 6709), PD-L1-APC (BioLegend, 10F.9G2, 124312, 1:5000), LAG-3-PerCP-Cy5.5 (BioLegend, C9B7W, 125211), TIM-3-PE-Dazzle594 (BioLegend, B8.2C12, 134013).

For in vitro experiments assessing PD-L1, C1498FFDsR cells were washed with PBS then surface labeled with PD-L1-APC (BioLegend). Apoptosis was assessed in vitro with C1498 cells employing the FITC Annexin V apoptosis kit (BioLegend). Antibodies utilized for in vivo analyses were obtained from BioLegend, BD Horizon, and Thermo Fisher. For both in vitro and in vivo experiments, events were then collected using the FACS LSRII (BD Bioscience), and were gated on 15,000 events. FlowJo v10 was utilized to perform data analysis.

Mouse NK cells were isolated from single-cell suspensions of the dissociated spleens of 6- to 8-week-old C57BL/6 mice using the NK Cell Isolation Kit II according to the manufacturer's instructions (Miltenyi Biotec). In this protocol, T cells, dendritic cells, B cells, granulocytes, macrophages, and erythroid cells are indirectly magnetically labeled with a cocktail of biotin-conjugated antibodies and anti-biotin microbeads for 15 minutes. After depleting magnetically labeled cells, isolation and enrichment of unlabeled NK cells was confirmed by flow cytometry. Isolated NK cells were stained with CD3-FITC (catalog no. 100306; BioLegend), NK1.1-PE (catalog no. 108708; BioLegend), PD-1-Pe/Cy7 (catalog no. 109109; BioLegend), PD-L1-APC (catalog no. 124311; BioLegend) to distinguish enriched NK cells from CD3⁺ cells. Blood was taken either serially in an approximately 200 μL submandibular vein bleed or from cardiac puncture at the time of sacrifice. Blood was collected in heparinized tubes, washed twice with ACK lysis buffer, and resuspended in PBS for staining.
Flow cytometry analysis was carried out by the Mayo Microscopy and Cell Analysis core, and data were analyzed using FlowJo software. Enriched NK cells were identified by gating on NK1.1^hi CD49b^hi CD3ε^lo cells.

B16 or B16MRD tumor cells cocultured with isolated NK cells were seeded in DMEM containing 10% FBS and 1% penicillin/streptomycin containing anti–PD-1 (catalog no. BE0146; Bio X Cell), anti–PD-L1 (catalog no. BE0101; Bio X Cell), anti-CTLA4 (100 ng/mL; catalog no. BE0164; Bio X Cell), or isotype control (Chrome Pure anti-rabbit IgG; catalog no. 011-000-003; The Jackson Laboratory). Seventy-two hours postincubation, supernatants were harvested and analyzed for cytokine secretion using ELISAs for IFNγ and TFNα. Tumor cells were stained for CD45-PerCP (BD Bioscience) and PD-L1-APC (BioLegend). Flow cytometry analysis was performed as discussed.