DCs are less activated and express a tolerogenic PD-L1⁺phenotype in patients with MC. (A) The contribution and phenotype of cDC and pDC within the peripheral mononuclear cell compartment at 4 to 8 weeks after allo-HSCT was assessed by flow cytometry. The ratio of cDC:pDC was similar in patients with FC (2.5:1; n = 15) or MC (2.6:1; n = 15). (B) Expression of CD86 on DC in relation to chimerism status. A total of 81% of DC in FC expressed CD86 (n = 15) compared with only 58% of DC in MC (n = 15; **P = .0068). (C) Expression of PD-L1 and PD-L2 on DC in relation to chimerism status. A total of 41% of DC in MC expressed PD-L1 compared with 18% of DC in FC (**P = .0052; FC, n = 15; MC, n = 15). No difference was observed in the level of PD-L2 expression (FC, 4.3%; MC, 7% [not significant]). DCs therefore appear to have a tolerogenic phenotype in the setting of MC. (D) Expression of PD-L1 on DC subsets. Increased PD-L1 expression was most pronounced on the cDC subset in MC (47%; n = 15) compared with cDC in FC (20%; n = 15; **P = .0053), whereas a trend was observed toward increased PD-L1 expression on pDC in MC. (E) Expression of PD-L1 on host and donor DC in MC. This was determined by combining cell surface phenotyping with RNA probes specific for the KDM5D (SMCY) gene, derived from the Y chromosome. Host and donor cells were thereby distinguishable in the context of patients undergoing gender-mismatched allo-HSCT. PD-L1 expression was observed on a mean of 67% donor DC compared with only 29% of host DC (n = 10; ***P = .0002). For graphs with unpaired data, the mean and standard error of the mean are shown; analyses were undertaken with 2-tailed Mann-Whitney U tests. Paired analysis used a Wilcoxon matched pairs signed rank test.