Fig 2: aCML neutrophils show distinct morphologic changes and reduced expression of surface CD16, CD15, fMLPR, CXCR1 and CXCR2. a The first frame of image sequences acquired during video microscopy of neutrophils from an age- and gender-matched control (top) and the aCML patient before therapy (bottom). From left to right, the cells were treated with PBS as a control, fMLP [10 nM], CXCL1 [100 ng/ml] and CXCL8 [100 ng/ml]. Black arrows in the lower panel indicate prominently enlarged cell bodies. Magnification: 20x. b Statistical summary of the cell size in μm2 of aCML neutrophils before therapy (left) and the relative number of neutrophils with a cell size of > 225 μm2 (right). Both parameters were compared to age- and gender-matched controls (n = 6). On average, 41 and 56 cells per condition were analyzed in age- and gender-matched controls and the aCML patient, respectively. Bars are given as median ± interquartile range and the given p-values were calculated using Mann-Whitney U test. The cutoff of 225 μm2 (grey dashed line, left) was chosen as assuming a perfect circle equals a diameter of 16 μm and is thus close to a neutrophil’s normal diameter in cell culture [29]. c Representative contour plots and histograms of purified neutrophils. Analyses of CD16 (FITC) and CD15 (VioBlue) (left) and fMLPR, CXCR1 and CXCR2 (right) expressions are shown. An age- and gender-matched control (control, left of left panel; dotted light grey line of right panel) and aCML neutrophils before ruxolitinib therapy (before therapy, right of left panel; solid dark grey line of right panel) are depicted. d Statistical summary of expression levels for CD16, CD15, fMLPR, CXCR1 and CXCR2 on purified neutrophils from age- and gender-matched controls (controls; black dots, white bars; n = 5) and aCML neutrophils before treatment (before therapy; black triangles, grey bars; n = 1). Expression levels are given as the mean fluorescent intensity (mfi) and bars are given as median ± interquartile range

Fig 3: Expression of ANKRD55 and neighboring genes in immature moDC. (A–D) Monocytes were cultivated for 6 days in IL-4/GM-CSF medium for differentiation into immature moDC in the absence (colored curves) or presence (gray curves) of AM-580, and expression levels of the indicated genes were measured by qPCR at the start of the cultivation (day 0) and on days 1, 2, 4, and 6. Mean ± SEM of 3 independent measurements; Friedman test (followed by Dunn’s multiple comparison test) for comparison of data points in each curve with day 0 and Wilcoxon test for comparison of both curves (Control vs. AM580). (E–H) Effect of tolerogenic compounds on gene expression in immature moDC by qPCR. Concentrations of compounds are provided in the Materials and Methods. Mean ± SEM of maximum 5 independent measurements; Friedman test (followed by Dunn’s multiple comparison test). (I) Analysis of ALDH1A2, RARα, and RARγ expression by qPCR. Mean ± SEM; n = 6; Friedman test (followed by Dunn’s multiple comparison test). (J) Expression levels of three discrete ANKRD55 splice variants 201, 202, and 204 in immature moDC. Mean ± SEM; n = 10; Wilcoxon test. (K) Classical (Cla; CD14hi/CD16-), intermediate (Int; CD14hi/CD16+), and non-classical (NC; CD14+/CD16+) monocytes were isolated and separately cultivated for 6 days to differentiate into moDC. Gene expression levels were measured in the original monocyte subsets (M) and derived moDC (D). Mean ± SEM; n = 3; Mann–Whitney test for comparison between monocytes and moDC for each gene and subset. *p ≤ 0.05, **p ≤ 0.01.