Fig 2: PD-L1 expression is increased in Lal–/– CD11c+ cells.(A) Percentage of PD-L1+ cells in the blood of Lal+/+ and Lal–/– mice by flow cytometry analysis. (B) PD-L1 expression in blood CD11c+, MHCII+, F4/80+, CD11b+, Ly6C+, and Ly6G+ cells of Lal–/– versus Lal+/+ mice by flow cytometry analysis. (C) PD-L1 expression in CD11c− or CD11c+ double-gated myeloid cells of Lal–/– versus Lal+/+ blood by flow cytometry analysis. (D) Percentage of CD11c+ cells in the blood of Lal+/+ and Lal–/– mice by flow cytometry analysis. (E) Cytokine expression in Lal–/– versus Lal+/+ CD11c+ cells by flow cytometry analysis. Data are expressed as mean ± SD. Experiments were independently repeated, n = 6 for A–D, n = 5 for E. *P < 0.05, **P < 0.01, unpaired Student’s t test.

Fig 3: Metabolic reprogramming in Lal–/– CD11c+ cells.(A and B) Gene expression of glycolysis (A) and citrate cycle (B) across cell clusters in t-SNE plots of CD11c+ cells from Lal–/– versus Lal+/+ mice. (C) Statistical analysis of ROS MFI in Lal–/– versus Lal+/+ CD11c+ cells by flow cytometry. (D) Expression of gene response to ROS across cell clusters in t-SNE plots of CD11c+ cells from Lal–/– versus Lal+/+ mice. (E) Extracellular acidification rate (ECAR) of glycolysis and oxygen consumption rate (OCR) in mitochondrial respiration in Lal–/– versus Lal+/+ CD11c+ cells. (F) ATP production in mitochondrial respiration in Lal–/– versus Lal+/+ CD11c+ cells. (G) MFI of G6PD, PDH, LDH, and GLUD expression in Lal–/– versus Lal+/+ CD11c+ cells by flow cytometry. (H) Percentage of CD11c+ cells in the blood and percentage of PD-L1+ cells in CD11c+ blood cells after CPI-613 treatment by flow cytometry analysis. (I) CPI-613–pretreated CD11c+ cells were cocultured with CFSE-labeled Lal+/+ CD4+ T cells for T cell proliferation assay. (J) CPI-613–pretreated CD11c+ cells (2 × 105) were coinjected with B16 melanoma cells (2 × 105) into the flank region of Lal+/+ recipient mice for tumor growth assay. Data are expressed as mean ± SD. Experiments were independently repeated, n = 4 for C, G, and I, n = 6–8 for E and F, n = 7 for H, n = 12 for J. *P < 0.05, **P < 0.01, unpaired Student’s t test for C, F, and G, 2-way ANOVA for H and I, 1-way ANOVA for J.

Fig 4: CSF1R expression and function in Lal–/– CD11c+ cells.(A) Csf1r expression across cell clusters in t-SNE plots of CD11c+ cells from Lal–/– versus Lal+/+ mice. (B) Percentage of CSF1R+ cells in blood CD11c+ cells of Lal–/– versus Lal+/+ mice by flow cytometry analysis. (C) Isolated Lal+/+ or Lal–/– CD11c+ cells were pretreated with IgG or anti-CSF1R antibody (5 μg/mL) and cocultured with CFSE-labeled Lal+/+ CD4+ T cells (at 1:1 ratio). The proliferation of labeled CD4+ T cells was analyzed by flow cytometry. (D) Isolated Lal+/+ or Lal–/– CD11c+ cells (2 × 105) were pretreated with IgG or anti-CSF1R antibody (5 μg/mL) and coinjected with B16 melanoma cells (2 × 105) into the flank region of Lal+/+ recipient mice. The tumor size was measured at 14 days after cell injection. (E) Percentage of PD-L1+ cells in CD11c+ cells after anti-CSF1R antibody treatment (5 μg/mL) by flow cytometry analysis. (F) Percentages of CSF1R+ cells, PD-L1+ cells, PD-L1+CSF1R+ cells, and IFN-γ+ cells and MFI of IFN-γ in blood CD11c+ cells and percentage of blood CD11c+ cells in Lal+/+, Lal–/–, untreated (–DOX), and DOX-treated (+DOX) c-fms–Tg/KO (Tg/KO) mice by flow cytometry analysis. Data are expressed as mean ± SD. Experiments were independently repeated, n = 6 for B, n = 5 for C and E, n = 10 for D, n = 6–7 for F. **P < 0.01, unpaired Student’s t test for B, 2-way ANOVA for C and E, 1-way ANOVA for D and F.

Fig 5: Identification and gene expression of Lal–/– versus Lal+/+ CD11c+ cell clusters by scRNA-Seq.(A) t-SNE plot of CD11c+ cell clusters from Lal–/– versus Lal+/+ mice. Each dot represents a single cell colored by cluster assignment. The dashed blue line circles cluster 158, and the dashed red line circles cluster 0236. (B) Feature plot of Cd274 (PD-L1) expression across cell clusters identified in A. (C) Percentages of cells for expressed genes were increased in cluster 158 and relatively unchanged in cluster 0236 of Lal–/– versus Lal+/+ CD11c+ cells. The percentage was calculated using the number of expressed cells for the gene divided by the number of cells for this sample. (D) Percentages of cells for expressed genes were increased in cluster 158 but decreased in cluster 0236 of Lal–/– versus Lal+/+ CD11c+ cells. (E) Percentages of cells for expressed genes were increased in cluster 0236 but undetectable or increased in cluster 158 of Lal–/– versus Lal+/+ CD11c+ cells.