Macs isolation system

For purification of CD11b⁺Ly6C⁺ monocytes/macrophages from tumor tissues, single-cell suspensions from tumor tissues were prepared after 24 h of intratumoral treatment with control, anti-CD47 mAb, cGAMP, or a combination. CD45⁺ cells were isolated from the single-cell suspensions using the MACS isolation system (Miltenyi Biotech) according to the manufacturer’s protocol, and then CD45⁺CD11b⁺Ly6C⁺Ly6G⁻ monocytes/macrophages were sorted using FACSAria II (BD Biosciences). The sorted monocytes/macrophages were cocultured with OT-I cells in a MAHAS4510 plate (Millipore) for 24 h. IFN-γ–producing T cell numbers were visualized by anti-mouse IFN-γ enzyme-linked immunospot assay (ELISpot, catalog no. 3321-2A; Mabtech) according to the manufacturer’s protocol. BCIP/NBT plus substrate (Mabtech) was used for detection. Plates were scanned with an automated ELISpot plate reader (Autoimmun Diagnostika). Spots were counted and analyzed using the AID ELISpot plate reader software (Autoimmun Diagnostika).

Naïve CD4⁺CD62L^hi T cells were isolated from the spleen and lymph nodes using a MACS isolation system (Miltenyi Biotec) or sorted by flow cytometry for CD4⁺CD44^lowCD62L^high naïve cell population. Naïve CD4 T cells were activated with plate‐bound anti‐CD3 (2 μg/ml, 145‐2C11) and soluble anti‐CD28 (37.51, 0.5 μg/ml) with additional cytokines and antibodies to generate Th1 cells (5 ng/ml IL‐12; 50 U/ml human IL‐2; and 10 μg/ml anti‐IL‐4, 11B11), Th17 cells (20 ng/ml IL‐6; 5 ng/ml human TGF‐β; 10 ng/ml IL‐1β, 10 U/ml human IL‐2 on d3; 10 μg/ml anti‐IL‐4, 11B11; and 10 μg/ml anti‐IFN‐γ, XMG), and Treg cells (2.5 ng/ml human TGF‐β; and anti‐IL‐4, 11B11). Cells were expanded in the fresh medium after 3 days with half concentration (Th17) of the original cytokines or additional 50 U/ml human IL‐2 in Treg culture. In long‐term culture conditions, IL‐17a‐producing cells were sorted based on GFP marker. Cells were plated at 0.5 × 10⁶ cells/ml and activated with 1 μg/ml plate‐bound anti‐CD3 alone or with the addition of Th17 cytokines (IL‐6 and TGF‐β). Recombinant cytokines and antibodies are from BD Biosciences, Bio X Cell, or R&D. In some experiments, Stat5 inhibitor (Millipore, Stat5 Inhibitor III, Pimozide) or anti‐mouse CD25 (BioLegend, PC61) were added to the culture on day 0, and cells were analyzed on day 5.

Naive CD4⁺CD62L^hi T cells were isolated from spleen and lymph nodes using a MACS isolation system (Miltenyi Biotec) or sorted by flow cytometry for CD4⁺CD44^lowCD62L^high naive cell population. Naive CD4⁺ T cells were activated with plate-bound anti-CD3 (2 ug/ml, 145–2C11) and soluble anti-CD28 (37.51, 0.5 ug/ml) for Th0 with additional cytokines and antibodies to generate Th17 cells (20 ng/ml IL-6; 5 ng/ml human TGF-β; 10 ng/ml IL-1 β, 10 ug/ml anti-IL-4, 11B11; and 10 ug/ml anti-IFN-γ, XMG). 20 ng/ml IL-6 was added to Th0 in some culture conditions (Th0+IL-6). Cells were expanded after 3 days without additional cytokines (Th0, Th0+IL-6) or half concentration (Th17) of the original cytokines in fresh medium. Recombinant cytokines and antibodies are from BD Biosciences or R&D.