Cells with extra centrosomes show enhanced T cell activation and cytokine secretion. (a) Left: Representative images of 2N BMDCs sorted for CETN2-GFP^low and CETN2-GFP^high expressing cells. Sorted BMDC subpopulations were immunostained against γ-tubulin (white) and CEP135 (red) to assess centrosome numbers. Left graph: Quantification of percentage of cells with ≥2 centrosomes in CETN2-GFP^low (2N1C, blue) and CETN2-GFP^high (2N2C, red) expressing cells. Centrosome numbers of sorted DC subpopulations were determined by confocal microscopy according to CETN2-GFP/γ-tubulin⁺ foci. Each data point represents one experiment. ****, P < 0.0001 (two-tailed, paired Student’s t test). Right graph: Ratio of cells with multiple centrosomes between 2N2C and 2N1C cells. (b) Quantification of IL-2 levels after co-culture of OVA-pulsed 2N BMDC subpopulations with OT-II CD4⁺ T cells. Graph displays mean values ± SD of one representative out of six independent experiments. Data points represent technical replicates. 2N1C, cells enriched for one centrosome (blue); 2N2C, cells enriched for multiple centrosomes (red). (c) Left: Representative histograms of T cell proliferation of co-cultured OT-II CD4⁺ T cells with enriched BMDC subpopulations pulsed with OVA. Unprimed T cells (−OVA) served as negative control and are displayed as gray filled line. Right: Graph shows quantification of OT-II CD4⁺ T cells that divided after co-culture with OVA-pulsed enriched BMDC subpopulations and displays mean values ± SD of one representative out of four independent experiments. Data points represent technical replicates. Black bars indicate gates for proliferating (left) and non-proliferating (right) cells. (d) Quantification of cell-surface expression levels of co-stimulatory molecules on sorted 2N1C and 2N2C BMDC subpopulations. Left: Representative histograms of co-stimulatory molecules. Unstained controls were included in the histograms as gray filled lines. Right: Graph displays mean values of mean fluorescence intensities (MFI) ± SD. Each data point represents one independent experiment. *, P = 0.027 (all two-tailed, paired Student’s t test). (e) Levels of indicated cytokines in conditioned medium harvested from sorted DC subpopulations were quantified by ELISA. Graphs show mean values ± SD of at least nine independent experiments. **, P = 0.002 (CCL17; Wilcoxon test); ***, P = 0.0005 (IL-6); ****, P < 0.0001 (CCL5) and **, P = 0.0057 (CXCL1; all two-tailed, paired Student’s t test). (f) mRNA levels of indicated cytokines in sorted 2N1C and 2N2C DC subpopulations. Graph displays mean values ± SD of at least three independent experiments. *, P = 0.012 (two-tailed, paired Student’s t test). (g) Transmigration of CD4⁺ T cells toward supernatant harvested from 2N1C and 2N2C cells and toward control media. Graph displays mean values ± SD from 12 independent experiments. *, P = 0.033 (one-way ANOVA followed by two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).