Targeting tumor-associated macrophages and granulocytic myeloid-derived suppressor cells augments PD-1 blockade in cholangiocarcinoma
Emilien Loeuillard, Jingchun Yang, EeeLN H. Buckarma, Juan Wang, Yuanhang Liu, Caitlin B. Conboy, Kevin D. Pavelko, Ying Li, Daniel R. O’Brien, Chen Wang, Rondell P. Graham, Rory L. Smoot, Haidong Dong, Sumera I. Ilyas
Abstract
Figure 1. TAMs are the predominant source of PD-L1 in CCA. (A) Representative images (left and middle panels) of PD-L1 (brown staining, black arrowhead) plus CD68 (red staining, red arrowhead) coimmunostaining (n = 33) and PD-L1 (brown staining) plus CK-19 (red staining) coimmunostaining (n = 18) in human resected CCA specimens. Percentage of patients with positive PD-L1/CD68 costaining and PD-L1/CK19 costaining, respectively (right panel). Scale bars: 40 m. (B) Histograms show expression of PD-L1 + macrophages in human CCA tumors. (C-F) Flow cytometry analysis of normal WT mouse livers (from WT mice without tumors) as well as adjacent livers and tumors of mice 28 days after orthotopic implantation of 1 10 6 SB (murine CCA) cells. (C) Percentage of PD-L1 + macrophages (M) of total macrophages (CD45 + CD11b + F4/80 + ) in WT mouse normal liver, tumor-adjacent liver, or tumor. Fluorescence Minus One (FMO) controls were used for each independent experiment to establish gates (See Supplemental Figure 1A for gating strategy) (n 8). Representative histograms show expression of PD-L1 + macrophages. (D) Percentage of CD206 + TAMs (left panel) and PD-L1 + CD206 + TAMs (middle panel) of F4/80 int macrophages (CD45 + CD11b + F4/80 int ) in WT mouse liver, tumor-adjacent liver, or tumor. Representative contour plots (right panel) show CD206 and PD-L1 expression of F4/80 int macrophages (n 7). (E) Percentage of PD-L1 + CD206 -macrophages or PD-L1 + CD206 + macrophages (CD11b + F4/80 + ) of CD45 + cells from SB tumors (n = 28). (F) Percentage of PD-L1 expression in myeloid cells from SB tumors. Macrophages, CD45 + PD-L1 + CD11b + F4/80 + ; MDSCs, CD45 + PD-L1 + CD11c -CD11b + F4/80 -GR-1 + ; DCs, CD45 + PD-L1 + CD11c hi ; (n = 11). Data are represented as mean SD. Unpaired Student's t test (E) and 1-way ANOVA with Bonferroni's post hoc test (C, D, and F) were used. *P < 0.05; **P < 0.01; ***P < 0.001.