Deubiquitination of NF-B members by
IntroductionCYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. 1 CYLD removes lysine-63 polyubiquitin chains from distinct members of the NF-B pathway 2-4 and mutations in CYLD dysregulate NF-B activity.Complete deletion of CYLD in mice (CYLD ko ) renders them susceptible to skin tumors, 5 but the CYLD ko mice do not display alterations of the immune system, as we could show for B-6 and T-cell development (A.W., N.H., S. Reissig, manuscript in preparation). Other knockout mouse models of CYLD point to a role for flCYLD in immunity, including hyperinduction of IFN␣ in virus-infected dendritic cells (DCs) 7 as well as protection from infections in its absence. 8 However, mice exclusively expressing the naturally occurring short splice variant of CYLD, sCYLD, are characterized by lymphomegaly, splenomegaly, and dramatic increases in B-cell numbers 6 caused by aberrant NF-B signaling. Because this pathway is important for the function of DCs, 9 which orchestrate innate and adaptive immune responses, we investigated the effect of sCYLD overexpression on DC function. We found CYLD ex7/8 DCs to be hyperresponsive to LPS, capable of inducing superior T-cell expansion on DC immunization in vivo, and able to suppress tolerance induced by ␣-DEC-205:OVA administration. As a molecular basis for this phenotype, we identified increased nuclear Bcl-3, p50, and p65 in resting and stimulated CYLD ex7/8 bone marrowderived DCs (BMDCs).
Methods
Mice and BMDC generationC57BL/6 wild-type (WT), CYLD ko , 5 and CYLD ex7/8 6 mice 6 to 8 weeks old were used as recipients and for BMDC generation as previously described. 10 St42 TCR Tg mice have been previously described. 11 OT-I mice 12 were obtained from Christian Kurts (Institute for Molecular Medicine & Experimental Immunology, Bonn, Germany). Approval for these studies was obtained from the review board of the Federal State of Rhineland-Palatinate, Germany. All animal experiments were in accordance with the guidelines of the Central Animal Facility Institution of the University of Mainz.
Flow cytometryFACSCanto cytometer and FlowJo software (Tree Star, Ashland, OR) using ␣-CD8 and ␣-CD90.1/CD45.1 (eBioscience, San Diego, CA) was used. ␣-IL-6, IL-10, and TNF-␣ antibodies from Cytometric Bead Array Flex system (Becton Dickinson, Franklin Lakes, NJ) were used and analyzed with FCAP Array Software (BD Biosciences, Mountain View, CA).
Immune toleranceRecipient mice were given 10 6 adoptively transferred OT-I cells, immunized with 20 g DEC-205:OVA, and boosted with 50 g OVA protein (Sigma-Aldrich, St Louis, MO) as previously described. 13Western blot analysis and electrophoretic mobility shift assay Western blot analysis was performed as previously described. 8 Nuclear and cytoplasmic fractions were prepared according to standard procedures. 14,15 NF-B Consensus Oligonucleotide was purchased from For personal use only. on May 7, 2018. by guest www.bloodjournal.org ...
