Figure 8. Combination of miR-21^−/− macrophages and PD-1 antibody confers superior anti-tumor efficiency.

(a, b) Flow cytometry analysis of PD-L1 expression in TAMs isolated from tumors as described in Figure 7a. (a) The portion of PD-L1-positive TAMs in total macrophages. (b) Mean fluorescence intensity (MFI) of the PD-L1 signal from TAMs. (c) Immunoblotting analysis of the protein levels of JAK2, pSTAT1, and PD-L1 in immortalized WT and miR-21^−/− BMDMs treated with IFN-γ. (d–i) Mice implanted with B16 and macrophages as described in Figure 7a were treated with IgG or antibodies against PD-1. N = 6–7 per group. (d) Representative images of tumors at 16 days post inoculation. (e) Tumor weights. (f) Tumor volumes. Cell populations within tumors were analyzed by flow cytometry analysis; (g) M1 and M2 TAMs; (h) M1:M2 ratio; and (i) CD8⁺ T cells within tumors. (j) The role of miR-21 in macrophage polarization of TAMs. miR-21 suppresses the expression of STAT1, JAK2, and PDCD4 to inhibit STAT1 and NF-κB activation and prevent TAMs towards M1 polarization. Yet elevated STAT1 activation mediated by miR-21 deficiency promotes PD-L1 expression in TAMs, which can be mitigated by PD-1 antibody blockade. *P ≤ 0.05, ***P ≤ 0.001; n.s., not significant.