Figure 4

TAM subsets in subcutaneous LLC tumors from WT versus Cav-2 KO mice defined by flow cytometric analysis based on high and low MHC II expression levels. Tumors were extracted from WT and Cav-2 KO mice at day 8 after s.c. injection of LLC cells and single cell suspensions were prepared and stained with fluorescence-conjugated antibodies for flow cytometry as described in Methods. (A) Representative dot plots depicting gating strategy distinguishing MHC II^hi and MHC II^lo TAM subsets from LLC tumors implanted into WT (top panels) and Cav-2 KO mice (bottom panels). MHC II expressing cells, gated on CD11b⁺F4/80⁺ macrophages (left panels), were subsequently divided into Ly6C^lo MHC II^hi and Ly6C^lo MHC II^lo subsets, and defined as MHC II^hi TAM and MHC II^lo TAM, respectively (right panels). (B) Percentage of MHC II^hi TAM (left panel) and MHC II^lo TAM (central panel) within CD11b⁺F4/80⁺cells as well as combined percentages of MHC II^hi TAM versus MHC II^lo TAM within CD11b⁺F4/80⁺ cells (right panel) from WT and Cav-2 KO mice. (C) Percentage of MHC II^hi TAM (left panel) and MHC II^lo TAM (central panel) among total cells in LLC tumors  as well as combined percentage of MHC II^hi TAM versus MHC II^lo TAM among total cells (right panel) in LLC tumors from WT and Cav-2 KO mice. Data are from distinct samples and presented as the mean ± SEM. *p < 0.05, **p < 0.01; ***p < 0.001 compared with WT mice by unpaired t-test; n = 4.