(A) Tumor growth of 4T1 cells in BALB/c mice following treatment with lipopolysaccharide (LPS), polycytidylic acid (poly(I:C)), or PBS was measured at the indicated time points and dissected at the endpoint (n = 7 mice per group).

(B) SPADE tree derived from CyTOF (11-marker) analysis of tumor-infiltrating lymphocytes (TILs) from 4T1 tumors. Cell populations were identified as CD4 T cells (CD45+CD3+TCRβ+CD4+), CD8 T cells (CD45+CD3+TCRβ+CD8+), natural killer (NK) cells (CD45+NK1.1+), dendritic cells (DC; CD45+CD11c+), monocytes and macrophages (CD45+CD11b+Gr1-), B cells (CD45+B220+CD19+), and granulocytes (CD45+CD11b+Gr1+). The tree is colored by the intensity of CD8 marker shown to highlight CD8+ T cells.

(C) Percentages of immune cell populations within CD45+ TILs from 4T1 tumors, assessed with CyTOF (11-marker) and analyzed with SPADE (n = 3).

(D) Macrophage (CD11b+F4/80+) population in CD45+ TILs from 4T1 tumors (n = 7). Results are presented as mean ± SD from one representative experiment.

(E) Intracellular cytokine staining of IFN-γ+CD8+ in CD3+ T cell populations from 4T1 tumors (n = 7). Results are presented as mean ± SD from one representative experiment.

(F) Immunofluorescence staining of the protein expression pattern of F4/80 and granzyme B (GB) in 4T1 tumors (Nuc, nuclear). Scale bar, 100 μm.

(G) Western blot analysis of PD-L1 expression in various cancer cells treated with macrophage-conditioned medium (MP) for 12 hr.

(H) Time-lapse microscopy image showing the association of cancer cells with PD-1 at the 12-hr time point. Green fluorescent (green fluorescent-labeled PD-1/Fc protein) merged images of PD-L1-expressing cells are shown at the top, and a kinetic graph showing quantitative binding of PD-1/Fc protein on BT459 cells expressing PD-L1 at hourly time points is shown at the bottom. Scale bar, 100 μm.

(I) PD-1 binding affinity of various cancer cell lines examined using the PD-L1/PD-1 binding assay with MP or regular culture medium as a control.

∗p < 0.05, Student's t test. Error bars represent SD of three independent experiments.