Fig 2: 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-?+ cells and MFI of IFN-? 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 3: CB Expression Is Required for Immune Checkpoint Blockade Response(A) Representative ultrasound images of pancreatic tumors in B6 mice orthotopically implanted with KPC2a (CB-eGFP+), KPC2 parental, or KPC2-eGFP tumor cells. Pancreas mass, yellow cross section; K, kidney; S, spleen.(B) Mean tumor volume ± SEM. n = 5 mice per cohort. *p < 0.05 and **p < 0.005 (unpaired two-tailed Student’s t test).(C) Kaplan-Meier survival curve of mice bearing orthotopic tumors. Significance was determined using a log rank (Mantel-Cox) test. n = 5 per group.(D) Mean tumor weight in grams ± SEM at day 22 or at endpoint (EP; tumors > 500 mm3). Each dot is an independent mouse. n = 5 mice per group. *p < 0.05 (unpaired two-tailed Student’s t test).(E) Mean tumor volume ± SEM of KPC2 parental or KPC-eGFP+ cells. n = 3–6 mice per group.(F) Kaplan-Meier survival curve of mice bearing KPC2 parental or KPC-eGFP+ orthotopic tumors ± aPD-L1. n = 3–6 mice per group.(G) Mean tumor weight in grams ± SEM at day 22 or at endpoint (EP; tumors > 500 mm3). Each dot is an independent mouse. n = 3–6 mice per group.(H) Representative histograms of the indicated cell surface proteins in KPC2 parental and KPC2a clones ± 48 h pre-treatment with recombinant mouse IFN?.(I) Mean fluorescence intensity (MFI) of H-2Db, PD-L1, and mesothelin (Msln) expression by independent KPC parental tumor cells (n = 3) and their respective CB+ clones (KPC-CB) ± IFN?. Each dot is an independent clone. Statistical significance was determined using Student’s t test to compare the induction of protein expression following IFN?. *p < 0.05, **p < 0.005, and ***p < 0.005. Representative of two independent experiments.

Fig 4: PGRN promotes CD8+ T cell exclusion and inhibits tumor immunity. a. The expression of CD4, CD8 and Granzyme B in WT and PGRN KO mice breast cancer tissue sections was tested by immunohistochemical staining. b. Colocalization of CK19 (red), F4/80 (red), CD206 (red), PD-L1 (red) and CD8 (green) in WT and PGRN KO mice breast cancer tissue sections was examined by immunofluorescence analysis, and the nucleus was stained with DAPI (blue). c-d WT and PGRN-/- peritoneal macrophages were co-cultured with mouse spleen lymphocytes activated by aCD3/CD28. Here CM stands for control medium, without aCD3/CD28 stimulation. c. Flow cytometry was used to measure the proportion of activated CD8+ T cells (IFN-?+ CD8+ T cells) and (D) Ki-67+ CD8+ T cells. *p < 0.05; **p < 0.01

Fig 5: PD-L1 decreases chemotherapy sensitivity in BC cells. MTT viability assay of control and PD-L1KO MB49 (A) and RT4 (B) cells. Treatments are a 48-hour incubation in drug-free medium, 10 µM cis-platinum (cis), or 2 ng/mL gemcitabine (gem). Viability assessment (C) in MB49 cells in response to cis-platinum for 14 h prior to flow cytometry staining for Annexin V and propidium iodide (PI). P, (A-C) unpaired t-test