Anti pd l1

The immortalized immature DC line JAWS II was purchased from ATCC. JAWS II cells were seeded onto a cover slip (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Baden-Württemberg, Germany) and incubated with 250 μg/mL of rhodamine-preloaded MSNs for two days. Cells were fixed with 4% formaldehyde and permeabilized with 0.01% Triton X-100. Cells were stained with Alexa-Fluor488-conjugated phalloidin (Life Technologies, Eugene, OR, USA) and DAPI (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence images were acquired using a Zeiss Observer D1 fluorescence microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). The activation of JAWS II cells by CM-MSNs was determined by flow cytometry. JAWS II cells (5 × 10⁶ cells) were seeded in 12-well plates and incubated with 100 or 200 μg/mL of CM-MSNs for two days. Cells were stained with anti-CD40 (BD562846), anti-CD80, anti-CD86, anti-MHC I (BD742859), anti-MHC II and anti-PD-L1 (12-5982-82, eBioscience, San Diego, CA, USA). Stained cells were analyzed by a BD FACSVerse flow cytometry.

Cells were thawed, washed and reconstituted in AIM V serum-free medium (Life Technologies, Oslo, Norway) containing 0.1% human serum albumin. After an overnight rest, cells were pulse-labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE, Life Technologies) at a concentration of 2 μM for 5 minutes. The following blocking antibodies were added to parallel culture wells at a final concentration of 10 μg/mL: anti-IL-10 (R&D Systems, MN, USA; clone 23738), anti-TGF-β (R&D; clone 1D11), anti-PD-L1 (eBioscience, CA, USA; clone MIH1), and anti-HVEM (R&D; clone 94801).
After a 30 minute incubation, cultures were stimulated with either Gag or Env 15-mer overlapping peptide panels (NIH AIDS Research and Reference Reagent Program, MD, USA) at a final concentration of 2 μg/mL/peptide. Staphylococcal Enterotoxin B (SEB, Sigma-Aldrich, MO, USA) at a final concentration of 0.5 μg/mL was used as a positive control.
Cells were cultured at 37°C in 5% CO₂ for 5 days, harvested, and stained with the following fluorochrome-conjugated antibodies: CD3 V450, CD8 APC-H7, HLA-DR BV605, CD45RA APC (all BD) and CD25 PE (Biolegend). 7-aminoactinomycin D (7-AAD, BD) was added for dead cell exclusion. Flow cytometry data were acquired on a BD FACS Canto II with BD Diva 6.1 software, and analyzed in FlowJo X (FlowJo LLC, OR, USA).

Whole blood cell cultures were performed to determine the in vitro responses to antigens. Briefly, whole blood was diluted 1:1 with RPMI1640 medium supplemented with penicillin/streptomycin (100 U/100 mg/ml), L-glutamine (2 mM), and HEPES (10 mM; all from Invitrogen) and distributed in 12-well tissue culture plates (Costar). The cultures were then stimulated with ESAT-6, CFP-10, or anti-CD3 or with medium alone in the presence of CD49d/CD28 at 37°C for 18 h. Brefeldin A (10 µg/mL) was added after 12 h. After 18 h, centrifugation, washing, and red blood cell lysis was performed. The cells were fixed using cytofix/cytoperm buffer (BD Biosciences, San Jose, CA) and stored at −80°C. For neutralization experiments, whole blood was cultured in the presence of anti-IL-27 (5 μg/ml), anti-TGFβ (5 μg/ml) (R&D Systems, Minneapolis, MN), anti-PDL-1 (5 μg/ml) (eBiosciences, San Diego, CA), and anti CLTA-4 (5 μg/ml) (Ancell), or isotype control antibody (5 μg/ml) (R&D Systems) at 37°C for 6 h following which PPD was added and Brefeldin A (10 µg/ml) was added after 1 h. The cells were then cultured for a further 16 h.

The peripheral blood mononuclear cells were isolated by centrifuging the whole blood with an EZ-Sep TM (Dakewe, Shenzhen, China) lymphocyte separation tube. The cells were stained with corresponding anti-human FCM antibodies which include anti-PD-1 (eBioscience), anti-PD-L1 (eBioscience), anti-CD8 (eBioscience), anti-CD4 (eBioscience), anti-CD3 (BioLegend), anti-CD14 (eBioscience), and anti-human CD56 (eBioscience). One test contains 10,000 cells which were incubated for 30 min with corresponding antibodies and analyzed by FACSAria II. The gated strategy is shown in Figure S1.

Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (mAbs) to CD86, CD1a, PD-L1 (CD274), IFN-γ (all anti-human), phycoerythrin (PE)-conjugated mAbs to CD80, CD83 and CD25, APC-conjugated HLA-DR (all anti-human), anti-human Alexa Fluor 700-CD4 were from BD Biosciences, and PE-conjugated mAb to anti-human CD40 was from Becton Dickinson. Human Foxp3-APC, anti-PD-L1 and isotype control were from eBioscience. CD14 magnetic beads, T cell isolation kit II, GM-CSF and IL-4 were from Miltenyi Biotec. Anti-β-ACTIN and anti-PGE₂ antibodies were purchased from Sigma-Aldrich. Anti-SHH, anti-GLI1, anti-NUMB, anti-Ser9 phospho-GSK-3β, anti-NICD (Cleaved Notch1), anti-Ser2448 phospho mTOR, anti-Tyr458 phospho p85 and anti-Ser536 phospho NF-κB p65 were purchased from Cell Signaling Technology. Anti-COX-2 was from Calbiochem. Anti-Notch 2 intracellular domain and anti-Notch 4 -C-terminal antibodies were from Abcam. HRP conjugated anti-rabbit IgG and anti-mouse IgG was obtained from Jackson ImmunoResearch.

Cells were stained with antibodies from Biolegend (anti-CD4, RM4-5; anti-CD8, 53-5.8; anti-CD11b, M1/70; anti-CD11c, N418; anti-Ly6G, 1A8; anti-Ly6C, HK1.4; anti-Gr1, RB6-8C5; anti-MHC-II, M5/114.15.2; anti-CD40, 3/23; anti-CD80, 16-10A1; anti-CD86, GL-1; anti-CX3CR1, SA011F11; anti-PDL1, MIH7; anti-IL4, 11B11; anti-CD44, IM7; anti-CD62L, MEL-14; anti-Ki67, 11F6; anti-CD25, PC61; anti-H2Kb, AF6-88.5; anti-H2Kd, SF1-1.1), eBioscience (anti-FoxP3, FJK-16S). For intracellular staining, FoxP3/Transcription Factor Staining Buffer Set was used (eBioscience, 00-5523-00). Flow cytometry was determined by Canto-II instrument (BD) and analyzed by FlowJo v10.
For cell sorting, CD11b⁺ cells were pre-enriched with anti-CD11b microbeads (Miltenyi Biotec, 130-097-142). Enriched CD11b⁺ cells were stained with anti-CD11b and anti-Gr-1 for FACS by using an MoFlo Astrios system (Beckman Coulter).
Detailed reagents information is listed in Table S2.