Fig. 4

The deletion of CD244 on monocyte-lineage cells enhances antigen presentation and phagocytosis. (A-D) The population of tumor-infiltrating CD11b⁺ cells was isolated using magnetic sorting, and the expression of anti/pro-tumorigenic macrophage markers was evaluated using flow cytometry and real-time quantitative PCR. (A) MFI of CD80 in macrophages. (B) Relative mRNA expression level of anti-tumorigenic macrophage markers (IL6, NOS2, IFNB1). (C) MFI of CD206 in macrophages. (D) Relative mRNA expression level of pro-tumorigenic macrophage markers (ARG1, TGFB1, IL10). (E-F) Bone marrow cells were cultured with M-CSF and whole OVA protein. OVA presentation via MHC-I was measured using anti-H-2 kb-SIINFEKL antibody on day 3. Demonstrated are representative flow cytometry plots (left), proportion (right) (E) and absolute number (F) of MHC-I-OVA complex expressing cells in WT and CD244^−/− BMDMs. (G) MFI of costimulatory molecules (CD80, CD86, OX-40, CD40, and PD-L1) on WT and CD244^−/− BMDM on day 3. (H) Differentiated BMDMs and OT-1 cells were co-cultured for 48 h with whole OVA protein. Representative photographs (left) and the number of IFN-γ spots (right) were counted for OT-1 cells co-cultured with either WT or CD244^−/− BMDMs. (I-J) Differentiated BMDMs were co-cultured with CFSE-stained B16F10 cells, and phagocytic activity was assessed 24 h later by measuring CFSE fluorescence in macrophages. Presented are representative flow cytometry plots (left) and MFI of CFSE (right) in monocytes and macrophages (I) and the absolute number of CFSE⁺ macrophages (J) from WT and CD244^−/− BMDMs. *P < 0.05; **P < 0.01; ns, not significant; unpaired Student’s t-test. Data are representative of two (A-D, G, H) or three (E, F, I, J) independent experiments