The mechanisms that allow the maintenance of immunological memory remain incompletely defined. Here we report that tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 1, a protein recruited in response to several costimulatory TNFR family members, is required for maximal CD8 T cell responses to influenza virus in mice. Decreased recovery of CD8 T cells in vivo occurred under conditions where cell division was unimpaired. In vitro, TRAF1-deficient, antigen-activated T cells accumulated higher levels of the proapoptotic BH3-only family member Bim, particularly the most toxic isoform, Bim S. In the presence of excess IL-15, memory phenotype T cells with similar surface phenotype and comparable levels of Bcl-2 family members could be generated from WT or TRAF1-deficient T cell receptor transgenic OT-I T cells. However, when the memory CD8 T cells were allowed to compete for survival signals in the absence of antigen in vivo, the TRAF1-deficient T cells showed decreased recovery compared with TRAF1-sufficient T cells. This defect in T cell recovery in vivo was alleviated by introduction of siRNA to down-modulate Bim in TRAF1-deficient memory T cells. These studies identify the TRAF1 signaling axis and Bim down-regulation as critical for CD8 memory T cell survival in vivo.Bcl-2 family ͉ influenza virus ͉ knockout/transgenic mice
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 ...
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