sCYLD confers a stimulatory phenotype in BMDCs that leads to T-cell expansion and suppression of tolerance upon α-DEC-205:OVA administration. (A) BMDCs from WT, CYLD^ko, and CYLD^ex7/8 mice were differentiated with GM-CSF for 6 days in culture and stimulated (100 ng/mL LPS, 12 hours) and submitted to fluorescence-activated cell sorting (FACS) analysis of the CD11c⁺ population and cell surface expression of CD86, MHC II, and Ox40L. Mean fluorescence intensity (MFI) values of indicated cell surface expression markers are representative of more than 10 BMDCs preparations with triplicates and SEM shown as error bars. (B) Supernatants from stimulated BMDCs cultures measured for IL-10, TNF-α, and IL-6 cytokines with Becton Dickinson CBA Flex Set System. *P < .05 using t test for WT compared with CYLD^ex7/8 and for CYLD^ko compared with CYLD^ex7/8 stimulated BMDCs. Values shown are means of triplicates with SEMs. (C) Day 3 analysis of blood T-cell expansion of adoptively transferred TCR tg (St42) donor splenocytes into WT recipient mice immunized with day 6–differentiated and LPS-stimulated WT, CYLD^ko, or CYLD^ex7/8 BMDCs SGP peptide loaded and injected intraperitoneally to recipient WT mice. Percentages indicate Thy1.1⁺ CD8⁺ cells. Values are representative of more than 4 mice per group and repeated 3 times. (D) Fold change in OT-I T-cell numbers compared with PBS control in the spleen of recipient mice adoptively transferred with OT-I cells, given 20 μg DEC-205:OVA intra footpad and challenged with 50 μg OVA protein in CFA subcutaneously. Cell detection was analyzed by FACS using CD45.1 and CD8 antibodies to detect OT-I CD45.1 cells gated on CD8. Values represent more than 4 mice per group, with mean values and SEMs.