NK cells and IFN-γ are required to polarize TAMs toward an M1-type phenotype. Regressor cell line 2 was transplanted into syngeneic RAG2^−/− mice (injected with isotype control, anti-NK1.1, or anti–IFN-γ monoclonal antibodies) or RAG2^−/−x γc^−/− mice. Tumor masses were harvested 15 d after transplantation, disaggregated into single-cell suspensions, and analyzed by IHC (A) or FACS (B) to measure the percentage of M1 and M2 macrophages as defined by MHC class II and CD206 expression of CD68⁺ events (for IHC) or MHC class II and Ly6C expression of CD11b⁺ populations (for FACS), respectively. (C and D) An example of the flow cytometry gating to quantitate M1 and M2 macrophages. M1 macrophages are 7AAD⁻, CD45⁺, Ly 6C^lo, MHC class II^hi, F4/80⁺, CD206^lo cells. M2 macrophages are 7AAD⁻, CD45⁺, Ly6C^lo, MHC classII^lo, F4/80⁺, CD206^hi cells. (E) Cultured supernatant from single-cell suspensions were assessed for production of the indicated cytokines after 24 h of culture. (F) TAMs were sorted from harvested tumors at day 15, and genes associated with classically activated M1 type genes, such as iNOS and TNF, or alternatively activated M2 type genes, such as Arginase, eCAD, and Gas3, were measured by quantitative PCR. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars are represented by ± SEM. Each symbol represents a different mouse. Results were reproduced at least once.