In vivo anti-PD-L1 treatment resolves systemic inflammation and repairs CLL-induced myeloid skewing. (A) Blood was collected by cardiac puncture, and serum levels (pg/mL) of IL-10, CCL2, TNF-α, granulocyte-macrophage colony-stimulating factor, MIP-2 (CXCL2), CCL4, CXCL9, CXCL16, and CXCL5 were analyzed in isotype (n = 5), aPD-L1 (n = 7), and hWT (n = 5) mice using multiplex bead arrays or enzyme-linked immunosorbent assay. (B) Spleen single-cell suspensions were analyzed by flow cytometry. Monocytes were defined as Lin–(CD19, CD3, Ly6G, and NK1.1)CD11b+F4/80intCD11clow-intMHC-IIlowSSCint cells, and their percentages of total viable spleen CD19– mononuclear cells (MNCs) were compared between isotype (n = 9), aPD-L1 (n = 14), and hWT (n = 5) mice. (C) Spleen monocyte subsets were defined on the basis of expression of Ly6C and CD43 as inflammatory (INF; Ly6ChiCD43low), intermediate (INT; Ly6ChiCD43hi), and patrolling (PAT; Ly6ClowCD43hi) monocytes. A representative example of the gating is depicted (left panel, numbers indicate percentage of populations), and a quantification after gating on total monocytes of at least 9 animals per group are shown (right panel). (D) Expression of adhesion molecules PECAM-1 and ICAM-1 was analyzed in splenic monocytes, and mean fluorescence intensities (MFIs) were compared. (E) Percentage of macrophages, defined as Lin–CD11blowF4/80hi cells, and conventional dendritic cells (cDCs), defined as CD11chiCD11b+ cells, of CD19– cells in spleens were compared. (F) In vivo proliferation of cDCs (CD11chiCD11b+) in spleen was assessed as percentage of EdU-positive cells of at least 10 mice per group injected with 100 µg per gram body weight EdU 20 hours before being euthanized. (G) MHC-II expression was analyzed on splenic CD11chiCD11b+ cDCs. A representative histogram is depicted (left panel), and MFIs of at least 9 mice per group are shown (right panel). All graphs depict mean ± SD. FMO, fluorescence-minus-one; Mac, macrophage; ns, not significant; *P < .05; **P < .001; ***P < .0001.
