Anti cd14 clone 63d3

Cells were washed once in MACS buffer (containing PBS, 1% BSA, 0.5 mM EDTA), centrifuged at 550 g for 5 min and re-suspended in 200 μL MACS buffer. Cells were stained in 100 μL of staining buffer containing anti-CD137, conjugated with phycoerythrin (PE, clone 4B4-1; BD Biosciences, USA), anti-CD8-PC5 (clone B9.11; Beckman Coulter 1:100), anti-CD4 (clone SK3; BioLegend, 1:200), anti-CD14 (clone 63D3; BioLegend, 1:200), and anti-CD19 (clone HIB19; BioLegend,1:200), all conjugated to Fluorescein isothiocyanate (FITC) for exclusion gates, for 30 min on ice. Samples were covered and incubated for 30 min on ice, washed twice in PBS, and resuspended in 1 mL PBS prior to analysis.

Fresh tumor tissue was obtained for CITE-seq. Tumor cells were dissociated, washed, and resuspended. Antibodies for CITE-seq were anti-CD34 (clone 581), anti-CD31 (clone WM59), anti-CD45RA (clone HI100), anti-CD3 (clone UCHT1), anti-CD8A (clone RPAT8), anti-CD4 (clone RPAT4), anti-CD14 (clone 63D3), and anti-CD19 (clone HIB19) TotalSeq-A antibodies (BioLegend, San Diego, CA). Cells from one tumor were used to optimize preparation by adding variable antibody concentrations to 1 million cells in 50 mcl, washing with 200 mcl FACS buffer, resuspending in 200 mcl FACS buffer, and analyzing by flow cytometry. Otherwise, cells were used for CITE-seq using 3′ v3 Single Cell Immune Profiling technology according to the manufacturer’s protocol (10× Genomics, San Diego, CA).

Positive results in the ELISpot assay were validated by ICS for IFN‐γ as described previously.³⁵ The pre‐cultured cells were restimulated with the peptides showing a positive result at a concentration of 10 μg mL⁻¹ for 16 h at 37°C and 5% CO₂. After one hour, Brefeldin A (Sigma‐Aldrich, St. Louis, MO, USA) in a final concentration of 5 μg mL⁻¹ was added to inhibit cytokine secretion.
The cells were stained with Zombie NIR fixable viability dye (BioLegend, San Diego, CA, USA) and the following fluorochrome‐conjugated monoclonal antibodies on the cell surface: anti‐CD3 (clone UCHT1, Alexa Fluor 700), anti‐CD4 (clone SK3, BV510), anti‐CD8 (clone RPA‐T8, PerCP‐Cy5.5), anti‐CD14 (clone 63D3, APC‐Cy7) and anti‐CD19 (clone HIB19, APC‐Cy7). After fixation and permeabilisation using the FoxP3 transcription factor staining buffer set (eBioscience, Thermo Fisher Scientific), the cells were stained for intracellular IFN‐γ using a monoclonal anti‐IFN‐γ antibody (clone 4S.B3, PE‐Dazzle594). All antibodies were purchased from BioLegend. The cells were acquired on a LSRFortessa II cytometer (BD) using FACSDiva version 8 for Windows (BD). The full gating strategy is reproduced in Supplementary figure 14.