and stained for cell-surface and intracellular molecules as described above. Cells were analysed using a BD FACSymphony A5. FlowJo v.10.0 was used for data analysis. Production of recombinant PLA2G2D protein Recombinant human or mouse PLA2G2D proteins were generated by fusing the cDNA of human (NM_012400.4) or mouse (NM_011109.3) PLA2G2D with the Fc region of human IgG1 on the C terminus in the pcDNA3.4 expression vector (Thermo Fisher Scientific). An enzymatic-dead mutant of PLA2G2D–Fc was generated by introduc- ing an H47Q mutation into the human PLA2G2D cDNA. An effector-less Fc-tag variant was generated by introducing L234A, L235A and P329G mutations into the human IgG1 fusion tag. PLA2G2D–Fc proteins were expressed by transiently transfecting Expi293 cells using the Expi293 Expression System Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 5 days of culture, supernatants were collected and recombinant proteins were purified by protein A affinity chromatography using a HiTrap PrismA column (Cytiva) on a Bio-Rad NGC FPLC system. In vivo tumour models C57BL/6 and BALB/c mice were purchased from the Jackson Labora- tory or Envigo, housed in specific pathogen-free conditions and fed ad libitum. Pla2g2d −/− mice were generated by CRISPR–Cas9-medi- ated gene editing in which deletion of exon 2 of the Pla2g2d gene results in loss of function through a frameshift from exon 1 to exon 3. Proximal (5′-CGAAAACCTAAGAGCCTGCG-3′) and distal (5′-TAGG TGGCTGGAACAACCAT-3′) Pla2g2d guide RNAs and Cas9 protein were injected into C57BL/6NTac zygotes using established methods. PLA2G2D -humanized mice on a C57BL/6 background were generated by Ozgene using gene targeting by homologous recombination. In brief, the mouse Pla2g2d genomic locus from residues L21 within exon 2 to the stop codon in exon 4 was replaced with human cDNA encoding residues I22 through to the stop codon. Gene targeting was performed on C57BL/6 ES cells. LN6-987AL and B16F0 cells 19 were provided by N. Reticker-Flynn and cultured in Dulbecco’s modified Eagle’s medium (DMEM). MC38 and B16F10 (ATCC) tumour-cell lines were cultured in DMEM, and E.G7-OVA and CT26 (ATCC) tumour-cell lines were cul- tured in RPMI medium. All media were supplemented with Glutamax, gentamycin (50 μ g ml −1 ) and 10% FBS. Cell lines were not subjected to further authentication but were routinely tested for mycoplasma contamination. To prepare mice for tumour inoculation, they were shaved on the right flank. MC38, B16F10, CT26 or E.G7-OVA (10 6 cells) and LN6-987AL or B16F0 (2 × 10 5 cells) were collected and washed twice with PBS before subcutaneous inoculation (in 100 μ l PBS) into mice. Mice were monitored daily, and tumours were measured by calliper every 3–4 days. When tumours reached 50–150 mm 3 and were palpable, mice were randomized into groups and antibodies or proteins were administered i.p. every 3–4 days for a total of four doses. Mice with pal- pable tumours were injected with anti-PLA2G2D, anti-IFNγ (BioXCell) or isotype control antibodies (all 10 mg kg −1 ) in 200 μ l PBS. Anti-mouse PD-1 antibody (clone RMP1-14, Leinco) was dosed at 5 mg kg −1 using the same schedule. Recombinant PLA2G2D–Fc protein was administered i.p. at 50 μ g in 100 μ l PBS per dose every 4 days for a total of four doses. For CD8 + T cell depletion experiments, anti-CD8 depleting antibodies (200 μ g) were administered i.p. one day before the initiation of anti- PLA2G2D treatment. Antibody administration (mouse IgG2a isotype control; anti-mouse CD8a, clone YTS169, BioXCell) was repeated every 3–4 days and continued throughout the duration of the experiment. In some experiments, blood, LNs, TDLNs and tumours were collected at the indicated time points after tumour inoculation. Single-cell suspen- sions of LNs and TDLNs were prepared by mechanically dispersing the LNs through a 100-μm nylon-mesh cell strainer. For tumours, single cells were prepared using the Tumour Dissociation Kit (Miltenyi). After staining with antibodies as described above, cell suspensions were analysed by flow cytometry. Mice were killed when tumours greater than 2,000 mm 3 developed or when tumour ulceration was observed. Mice (only females) were 8–12 weeks old when used in experiments. Sample sizes were determined on the basis of previous experience with the tumour models (for example, variability in growth kinetics in vivo), and on the basis of the practical availability of specific mice. Blinding was not applied in this study. Source data for all in vivo experiments are provided. Single-cell RNA-seq and data analysis C57BL/6 mice were inoculated with B16F0-tdTomato or LN6-987AL- tdTomato cells as described above and treated with isotype or anti- PLA2G2D antibodies at days 8 and 11. At day 12, tumours, TDLNs and non-draining LNs were collected and single-cell suspensions were prepared and stained for extracellular antibodies as described above. Using a FACSAria III (BD), cells were pooled from four sorted LN (or TDLN) populations that were mixed in equal ratios: (1) alive cells; (2) CD45 − cells; (3) CD45 + CD3 + CD44 + cells; and (4) combined CD45 + CD3 − CD11b + + CD45 + CD3 − CD11c + cells. From processed tumours, cells were pooled and mixed from three sorted fractions: (1) alive cells; (2) CD45 + CD3 + CD44 + cells; and (3) CD45 + CD3 − CD11b + + CD45 + CD3 − CD11c + cells. Next, pooled cell fractions were encapsulated for cDNA synthesis and barcoded with multiplexed runs on a Chro- mium X using the GEM-X 3′ OCM Chip (on-chip multiplexing). Final sequencing libraries were generated following the instructions of the GEM-X Universal 3′ Gene Expression v4 4-plex user guide (CG000768). Sequencing was performed on a Novaseq 6000 platform (Illumina) using an S2 (100 cycle) flow cell with a 28-10-10-90 cycle setting. The target sequencing depth of each library was >25,000 reads per cell. Sequencing reads were aligned to the mouse mm39 (GRCm39-2024-A) reference genome and quantified using CellRanger (10X Genomics, v.9.01). CellRanger output was loaded into R using Seurat (v.5.3.0) and filtered (200–6,000 features per cell, less than 15% mitochon- drial DNA). Next, counts were log-normalized, variable features were called (nfeatures = 3000) and the data were scaled. Next, a principal component analysis was run and a sample batch correction was per- formed using Harmony (v.1.2.3). Clusters were identified on the basis of shared-nearest-neighbour clustering using the first 30 principal components (PCs), with a resolution of 0.3 and k = 30. The first 30 PCs were then used to generate UMAP projections with a minimum distance of 0.3. Clusters were annotated using known gene markers: T cells ( Cd3e , Cd3g , Cd3d , Cd4 , Cd8a and Cd8b ), B cells ( Ms4a1 and Cd19 ), DCs ( Cst3 and Flt3 ), macrophages ( Cd68 and Csf1r ), neutrophils ( S100a9 ), NK cells ( Klrb1c ) and tumour ( Stmn1 ). In addition, FindMark- ers with a Wilcoxon rank-sum test and an absolute log 2 -transformed fold change cut-off of 1 was used to identify cluster marker genes and further stratify cell annotation. Previously published gene signatures of DCs and macrophage populations 68,69 were used to validate the identity of myeloid subsets. Published breast cancer (GSE167036) and lung adenocarcinoma (GSE131907) datasets containing both tumour and LN (or TDLN)-derived cells were processed, clustered and anno- tated as described in the original papers, with some simplification of complex annotations to improve comparisons between datasets. PLA2G2D expression was projected onto a UMAP of cells from the LNs (or TDLNs) using Seurat. Bone-marrow chimera experiments Pla2g2d +/+ (CD45.1 + ) and Pla2g2d −/− (CD45.2 + ) mice (8–12 weeks old) were lethally irradiated with two doses of 500 cGy administered 4–6 h apart (total 1,000 cGy) to induce myeloablation. After irradiation, mice received antibiotics in their drinking water for 2–3 weeks. Bone marrow was collected from the tibiae and femora of congenic Pla2g2d +/+ C57BL/6 (CD45.1 + ) and Pla2g2d −/− (CD45.2 + ) donor mice (6–8 weeks old). Cells were flushed with RPMI supplemented with 10% FBS, filtered through a 70-μm strainer and subjected to ACK lysis to remove erythrocytes. Cells