Article were washed, counted and resuspended in PBS at 1 × 10 6 cells per 100 μl. Recipient mice received bone-marrow cells by retro-orbital injection. Donor chimerism was assessed 6 weeks later by flow cytometry. After chimerism was confirmed, mice were subcutaneously implanted with MC38 tumour cells for downstream analysis of tumour growth. Generation of anti-PLA2G2D antibodies Antibodies against PLA2G2D were generated by immunizing BALB/c mice with recombinant human PLA2G2D–Fc-tagged proteins. Mice were immunized at multiple sites using complete Freund’s adjuvant for multiple boosts. Test bleeds were performed by saphenous vein lancing seven days after the last boost. When the antibody titre was high enough, mice were given a final intravenous boost in the lateral tail vein, after which immunized mice were killed and spleens isolated. Hybridomas were generated by electrofusion of splenocytes with Sp2/0 myeloma cells. Fused cells were plated into 96-well plates in HAT selec- tive medium and grown for 10–14 days to generate hybridoma clones, which were assayed for binding to human PLA2G2D proteins by ELISA. Positive binders were expanded and antibodies in the supernatants were purified by protein G (HiTrap Protein G HP, GE Healthcare). The specificity of anti-PLA2G2D antibodies was determined by verifying their lack of binding to other related sPLA2 family members by ELISA. In brief, recombinant PLA2G1B, PLA2G2A, PLA2G2E, PLA2G2F, PLA2GV and PLA2G2X Fc-tagged proteins were generated as described above for PLA2G2D–Fc. Binding to sPLA2 family members was tested by ELISA. Purified proteins were coated at 0.5 μ g ml −1 on 96-well plates and titrations of anti-PLA2G2D antibodies or isotype control antibodies were applied, followed by HRP-conjugated secondary detection and visualization with TMB-Ultra reagent (Thermo Fisher Scientific). The specificity of the antibodies was further tested by evaluating their ability to inhibit the enzymatic activity of PLA2G2D–Fc or the various sPLA2 family members in a phospholipase enzyme assay as previously described 37 . Isolation and culture of human peripheral blood cells Human white blood cell concentrate was obtained from Stanford Blood Center or the Sanquin Blood Bank, and peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll density gradient. Pan T cells, CD8 + T cells and monocytes were isolated from human PBMCs using Pan T, CD8 + T Cell and Pan Monocyte isolation kits (Miltenyi) according to the manufacturer’s instructions, respectively. Stimulation of T cells in PBMC-based assays PBMCs were labelled with CellTrace CFSE (Invitrogen) and T cells were stimulated with 1 μ g ml −1 anti-CD3 and 0.2 μ g ml −1 anti-CD28 antibodies in the presence of recombinant PLA2G2D–Fc, PLA2G2D(H47Q)–Fc or Fc control in RPMI. After 48 h of incubation, cell-culture supernatants and cells were collected for cytokine production and flow-cytometry analysis, respectively. IL-2 and IFN-γ levels were measured using Meso Scale Discovery Human or Mouse V-Plex/U-Plex kits. T cell prolifera- tion was measured by multicolour flow cytometry on a BD LSRFortessa X-20 flow cytometer using CD3, CD4, CD8 and live–dead markers for live T cell gating, followed by CFSE dilution analysis for proliferation analysis according to the manufacturer’s instructions. The binding of PLA2G2D to T cells was determined by incubating various concentra- tions of PLA2G2D–Fc or Fc control proteins to resting or activated T cells for 30 min, followed by washing and secondary detection with fluorophore-labelled anti-human IgG antibodies and subsequent analy- sis on a flow cytometer. Stimulation of isolated T cells for surface–PLA2G2D binding kinetics CD8 + cells were isolated and cultured as described above and stimulated with T cell TransAct (Miltenyi). At the indicated time points, cells were collected, washed and stained with PLA2G2D–His for 30 min at 4 °C in PBS. Next, the cells were stained with anti-His-APC together with other extracellular antibodies and live–dead markers as described above. All cells were then washed and resuspended in FACS buffer before analysis on a BD FACSymphony A5 flow cytometer. Stimulation of isolated T cells for intracellular cytokine staining and proliferation assays CD8 + cells were isolated and cultured as described above and stimulated with T cell TransAct (Miltenyi) in the presence or absence of 10 μ g ml −1 PLA2G2D protein, which was added again after 48 h. For proliferation assays, isolated CD8 + cells were labelled with the CellTrace Violet Cell Proliferation Kit (0.05 μ M, Thermo Fisher Scientific) before starting the culture. After 72 h of incubation, cells were collected, washed and stained with antibodies and live–dead markers as described above. For intracellular cytokine staining, cells were then fixed with 2% para- formaldehyde for 20 min at room temperature and permeabilized with 0.5% saponin for 10 min at 4 °C, after which the cells were stained for intracellular cytokines in 0.5% saponin for 60 min at 4 °C. All cells were then washed and resuspended in FACS buffer before analysis on a BD FACSymphony A5 flow cytometer. Bulk RNA-seq of stimulated T cells CD8 + cells were isolated and cultured as described above and stimulated with T cell TransAct (Miltenyi) in the presence or absence of 10 μ g ml −1 PLA2G2D protein. After 24 h of incubation, cells were collected and washed, and RNA was isolated using the RNeasy Micro kit (QIAGEN) according to the manufacturer’s instructions. Samples for bulk RNA-seq were sequenced and analysed as described before 70 . In brief, raw counts and reads per kilobase million (RPKM) were determined on the exons of RefSeq-annotated genes using HOMER’s (v.5.1) 71 analyzeRepeats.pl command. The R package DESeq2 v.1.48.2 was used for the identifica- tion of DEGs, sample scaling and statistical analysis. Genes with an RPKM < 1 in 50% of replicates in one condition were excluded. DEGs were determined by an absolute log 2 -transformed fold change value > 0.5 and an adjusted P value < 0.05. Pathway enrichment analysis on DEGs was performed using Metascape 58 (v.3.5). Mixed lymphocyte reactions For mouse allogeneic mixed lymphocyte reactions, human monocyte- derived DCs were differentiated from monocytes using the DC differ- entiation toolbox (Miltenyi) according to the manufacturer’s instruc- tions. Monocyte-derived DCs were matured in the presence of TNF where indicated. DCs were then treated with mitomycin-C for 1 h at 37 °C, followed by three washes with RPMI, then added to a 96-well round-bottomed plate. T cells were added to DCs at a DC-to-T cell ratio of 1:10 in RPMI, and were incubated for 5 days before analysis of cytokine levels by MSD or collection of conditioned medium. Where indicated, 0.5 μ g ml −1 recombinant PLA2G2D–Fc, PLA2G2D(H47Q)–Fc, Fc control or anti-PD-1 (pembrolizumab; 5 mg ml −1 or 10 mg ml −1 ) were added during the allogeneic coculture. Quantitative PCR Total RNA was isolated from in vitro differentiated macrophages, MDMs and DCs using the RNeasy Mini Kit (QIAGEN) according to the manu- facturer’s instructions. Total RNA was synthesized into cDNA using the High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems). Quantitative PCR with reverse transcription (qRT–PCR) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) per the manufacturer’s instructions. The amplification condition was set at 40 cycles at 95 °C for 15 s and 60 °C for 60 s using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). β-actin ( ACTB ) and hypoxanthine phosphoribosyl transferase ( HPRT1 ) were used for nor- malization. Cq values for the two reference genes were determined by ABI SDS software (v.1.5.1) and used to calculate the geometric mean of ACTB and HPRT1 as the normalization factor using GeNorm. The