COP9 signalosome subunit 8 is essential for peripheral T cell homeostasis and antigen receptor-induced entry into the cell cycle from quiescenceRESULTSDeletion of Csn8Csn8 deficiency impairs peripheral T cellsCsn8 promotes T cell proliferation and survival in vivoCsn8 regulates TCR-dependent responsesCsn8 is required for cell cycle entry from quiescenceCsn8 in proximal TCR signalingCsn8 controls Rb phosphorylation and gene expressionDISCUSSIONMETHODST cell culture and flow cytometryT cell proliferation and IL-2 productionMixed bone marrow chimerasAdoptive transfer of T cellsRetroviral transductionImmunoblotQuantitative RT-PCRAdditional methodsStatistical analysisFigure 1 Csn8 is essential for mammalian embryonic development.Figure 2 Loss of Csn8 impairs peripheral T cell homeostasis.Figure 3 Defective activation of Csn8-deficient T cells.Figure 4 Csn8 is required for TCR-induced proliferation.Figure 5 Quiescent Csn8-deficient MEFs fail to reenter the cell cycle.Figure 6 Proximal TCR signaling events.Figure 7 Expression of cell cycle regulators after TCR activation.Figure 8 Defects in transcriptional regulation of cell cycle-related genes.ACKNOWLEDGMENTSAUTHOR CONTRIBUTIONSReferencesCOP9 signalosome subunit 8 is essential for peripheralT cell homeostasis and antigen receptor–induced entryinto the cell cycle from quiescenceSuchithra Menon1,4, Hongbo Chi2–4, Huiyong Zhang1, Xing Wang Deng1, Richard A Flavell2& Ning Wei1Engagement of antigen receptors triggers the proliferation and functional activation of lymphocytes. Here we report that T cellhomeostasis and antigen-induced responses require the COP9 signalosome (CSN), a regulator of the ubiquitin-proteasomesystem. Conditional deletion of the CSN subunit Csn8 in peripheral T lymphocytes disrupted formation of the CSN complex,reduced T cell survival and proliferation in vivo and impaired antigen-induced production of interleukin 2. Moreover, Csn8-deficient T cells showed defective entry into the cell cycle from the G0 quiescent state. This phenotype was associated witha lack of signal-induced expression of cell cycle–related genes, including G1 cyclins and cyclin-dependent kinases, and withexcessive induction of p21Cip1. Our data define a CSN-dependent pathway of transcriptional control that is essential for antigen-induced initiation of T cell proliferation.T lymphocytes are pivotal in adaptive immunity. Mature naive T cellscirculate through the blood and peripheral lymphoid organs in aquiescent state (G0) until they encounter cognate antigen. Engagementof T cell antigen receptors (TCRs) and costimulatory molecules triggersa signaling cascade culminating in the transcriptional induction ofgenes crucial for the entry of quiescent T cells into the cell cycle1,2,alterations in the expression of surface proteins, secretion of cytokines,and differentiation into effector and memory T lymphocytes3.After TCR stimulation, the transition from the G0 phase throughthe G1 phase to the S phase of the cell cycle takes 25–30 h, andsubsequent cell divisions occur fairly rapidly, each requiring approxi-mately 5–6 h (ref. 3). A key regulator of the G0-G1 transition is theretinoblastoma protein (Rb); inactivation of Rb is sufficient for cellsarrested at the G0 phase to reenter the cell cycle4. Rb and the relatedpocket proteins inhibit the E2F family of transcription factors andthereby prevent entry into S phase5. This inhibition is relieved by G1cyclin and cyclin-dependent kinase (CDK) complexes, which phos-phorylate Rb during the G1 phase6–8. These cyclins and CDKs areexpressed in small amounts in naive T cells and are strongly inducedin response to TCR stimulation3. The G0-G1 transition also involvesan increase in cell size, which is a prerequisite for the entry of cells intothe cell cycle but is dispensable for the induction of effector functionsin T cells9,10.The ubiquitin-proteasome system is critical in immunity and cellcycle regulation8,11. The COP9 signalosome (CSN) is an evolutionarilyconserved protein complex of eight subunits (Csn1–Csn8) that inter-acts with deubiquitinating enzymes and kinases12–14. Arguably themost characterized activity of CSN is its ability to cleave and removethe ubiquitin-like protein Nedd8 (also called Rub1) from cullinubiquitin ligase subunits (‘deneddylation’)15,16. Cullin ‘neddylation’facilitates the assembly and activity of cullin-RING ubiquitin ligasessuch as SCF (Skp1–cullin 1–F-box protein) complexes17. CSN dened-dylation activity requires the metalloprotease motif located in Csn5(also called Jab1), as well as the integrity of the CSN complex18.Consequently, deletion of any of the CSN subunits abolishes CSN-mediated cullin deneddylation12. Whereas deneddylation represents achief activity of CSN, it alone cannot explain all of the phenotypicdefects associated with the loss of CSN. For the fission yeast Schizo-saccharomyces pombe, only those mutants lacking genes encodingCsn1 and Csn2 show a slow-growth phenotype, although all mutantslacking genes encoding Csn are defective in deneddylation14,19,20.ForArabidopsis thaliana, deletion of the Csn1 amino-terminal domaindoes not affect deneddylation of Cul1 but causes gene expressiondefects and growth arrest21. These observations indicate that physio-logically important functions of CSN in addition to the deneddylaseenzymatic activity remain to be defined.Cops8 (called Csn8 here) encodes the smallest and the leastconserved but first identified subunit of CSN12,22.Csn8,originallycalled ‘COP9’ (constitutively photomorphogenic 9), was initiallyidentified in arabidopsis during a screen for mutants that demonstratelight-grown seedling characteristics when grown in darkness23. Herewe generated mice allowing germline and conditional deletion of Csn8to investigate the function of Csn8 in T cell activation and prolifera-tion. We report that deletion of Csn8 specifically in peripheral T cells© 2007 Nature Publishing Group http://www.nature.com/natureimmunologyReceived 17 May; accepted 24 August; published online 30 September 2007; doi:10.1038/ni15141Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06511, USA.2Howard Hughes Medical Institute, Department ofImmunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.3Department of Immunology, St. Jude Children’s Research Hospital, Memphis,Tennessee 38105, USA.4These authors contributed equally to this work.