Figure 3. PD-L1⁺ TAMs are recruited from the BM in CCA.

(A–C and E–I) Tumor growth of 28 days after orthotopic implantation of 1 × 10⁶ SB (murine CCA) cells in WT or Pd-l1^–/– mouse livers. (A) Ratio of recruited TAMs (CD45⁺CD11b⁺F4/80^intCCR2⁺) to resident TAMs (CD45⁺CD11b⁺F4/80^hiClec4F⁺) in WT mouse liver (from mice without tumors) or SB tumor (n ≥ 11). (B) Percentage of PD-L1⁺CCR2⁺ recruited TAMs of F4/80^int TAMs (CD45⁺CD11b⁺F4/80^int) in WT mouse liver, tumor-adjacent liver, or tumor. Representative flow plots show expression of CCR2 and PD-L1 in F4/80^int TAMs (n ≥ 7). (C) Percentage of PD-L1⁺Clec4F⁺ resident TAMs of F4/80^hi TAMs (CD45⁺CD11b⁺F4/80^hi) in WT mouse liver, tumor-adjacent liver, or tumor. Representative flow plots show expression of Clec4F and PD-L1 in F4/80^hi TAMs (n ≥ 7). (D) Schematic of mouse BM transplantation. (E) Average tumor weights in mg of Pd-l1^–/–mice transplanted with WT BM (WT–Pd-l1^–/–) or WT mice transplanted with Pd-l1^–/– BM (Pd-l1^–/––WT) (n ≥ 8). (F) Representative photographs of livers from E. (G) Percentage of CCR2⁺ recruited TAMs of total TAMs (CD45⁺CD11b⁺F4/80⁺) in tumors from WT–Pd-l1^–/– or Pd-l1^–/––WT mice (n ≥ 7). (H) Percentage of CD8⁺CD11a⁺ reactive CTLs of CD45⁺CD3⁺ cells in tumors from WT–Pd-l1^–/– or Pd-l1^–/––WT mice (n ≥ 7). (I) Percentage of granzyme B expressed in CD8⁺CD11a⁺ reactive CTLs (CD45⁺CD3⁺CD8⁺ CD11a⁺) in tumors from WT–Pd-l1^–/– or Pd-l1^–/––WT mice (n ≥ 7). Data are represented as mean ± SD. Unpaired Student’s t test (A, E, and G–I) and 1-way ANOVA with Bonferroni’s post hoc test (B and C) were used. *P < 0.05; **P < 0.01; ***P < 0.001.