Complete x vivo 15 media

After confirming the post sort purity of >98%, 1 × 10⁵ BDCA-3 DCs were plated in 96 well v-bottom plate in complete X-VIVO 15 media (Lonza). TLR agonists, Poly I:C, and LPS (Invivogen, San Diego, CA, USA) were added at a final concentration of 10 μg/mL. Plates were incubated at 37°C for 18 h. Cells were then harvested and stained with ILT4 (R&D Systems, Minneapolis, MN, USA). After staining, BDCA-3 DCs were sorted based on their expression of ILT4 for subsequent analysis.

Healthy PBMCs were isolated from whole blood (StemCell) using Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation at 300 g for 20 min. The interphase containing PBMC was harvested and stimulated in complete X-VIVO 15 media (Lonza) at a cell density of 1 × 10⁷/mL with immobilized anti-CD3 antibody (10 μg/mL, eBioscience) and soluble anti CD28 antibody (4 μg/mL, eBioscience) at 37 °C, 5% CO₂ for 48 h. The CD8 T cells then were enriched by magnetic bead-conjugated anti-CD8 antibody (Miltenyi-Biotec) prior to SCBC assay and ICS assay.

M2c macrophage polarization was performed in 96-well plates by differentiating 50,000 monocytes/well in complete X-Vivo 15 media (Lonza) with 100 ng/mL of M-CSF for 5 days. Differentiated macrophages were polarized to an M2c phenotype with 50 ng/mL of IL-10 for 2 days. Alternatively, differentiated macrophages were polarized to an M1 phenotype with 100 ng/mL of IFNγ and 1 ng/mL of LPS for a positive control. Macrophages were characterized by flow cytometry using 1:100 dilutions of antibodies specific for human CD80, CD86, CD163, CD206, MHC class II, and PD-L1 (Supplementary Fig. 5). (Fig. 4a–d) Polarized macrophages in each well were co-cultured with 5 × 10⁵ autologous CellTrace™ Violet (CTV; Thermo Fisher Scientific)-labeled T cells with different concentrations of test articles and 100 ng/mL anti-CD3 (clone OKT3). Cell culture supernatants were harvested at 24 h for IL-2 quantitation by HTRF (Cisbio). Dilution of CTV was used to measure T cell proliferation at 72 h. Supernatants were also harvested at end of assay to measure IFNγ, IL-2, TNFα, GM-CSF and IL-21 using a multiplex bead-based magnetic MAGPIX™ assay (Luminex). (Fig. 4e) M2c polarized macrophages, E6 TCR⁺ T cells, and PD-L1⁺ SCC-152 tumor cells (25,000 each) were co-cultured in triplicate wells for 24 h in the presence of test articles. Supernatants were collected and soluble IFNγ, IL-2, and TNFα were quantitated.