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Establishment of the Major Compatibility Complex-Dependent Development

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Establishment of the Major Compatibility Complex-Dependent Development of CD4+ and CD8+ T Cells by the Cbl Family ProteinsIntroductionResultsGeneration of c-Cbl and Cbl-b Double-Deficient MiceThe Cbl Double Deficiency Enhances Thymic Negative SelectionAltered Ratio of CD8+ to CD4+ Thymocytes in Cbl Double-Deficient MiceThe Cbl Double Deficiency Leads to MHC-Independent CD4+ and CD8+ TnbspCellnbspDevelopmentMost TCR-Proximal Signaling Pathways Were Not Spontaneously Activated in Cbl Double-Deficient ThymocytesConstitutive Hyperactivation of NF-kappaB in Cbl-/-, Cblb-/- ThymocytesImpaired Pre-TCR Downmodulation and Constitutive Pre-TCR Signaling in Cbl-/-, Cblb-/- DP ThymocytesDiscussionExperimental ProceduresMiceFlow Cytometric AnalysisCell Transfer and Bone Marrow Chimera StudiesBiochemical StudiesThymocyte Proliferation and IL-2 Production AssayElectrophoresis Mobility Gel Shift AssaySupplemental DataAcknowledgmentsReferencesImmunity 25, 571–581, October 2006 ª2006 Elsevier Inc. DOI 10.1016/j.immuni.2006.08.021Establishment of the Major CompatibilityComplex-Dependent Developmentof CD4+and CD8+T Cells by the Cbl Family ProteinsFang Huang,1,4,5Yasuyuki Kitaura,1,4,6IhnKyung Jang,1,4Mayumi Naramura,2,7Hemanta H. Kole,2Liping Liu,1,8Haiyan Qin,1Mark S. Schlissel,3and Hua Gu1,*1Department of MicrobiologyColumbia University College of Physiciansand Surgeons701 West 168thStreetNew York, New York 100322Laboratory of ImmunogeneticsNational Institute of Allergy and Infectious DiseasesNational Institutes of HealthRockville, Maryland 208523Department of Molecular and Cell BiologyUniversity of California, BerkeleyBerkeley, California 94720SummaryCasitas B cell lymphoma (Cbl) proteins are negativeregulators for T cell antigen receptor (TCR) signaling.Their role in thymocyte development remains unclear.Here we show that simultaneous inactivation of c-Cbland Cbl-b in thymocytes enhanced thymic negativeselection and altered the ratio of CD4+and CD8+Tcells. Strikingly, the mutant thymocytes developedinto CD4+- and CD8+-lineage T cells independent ofthe major histocompatibility complex (MHC), indicat-ing that the CD4+- and CD8+-lineage developmentprograms are constitutively active in the absence ofc-Cbl and Cbl-b. The mutant double-positive (DP)thymocytes exhibited spontaneous hyperactivationof nuclear factor-kappa B (NF-kB). Additionally, theyfailed to downregulate the pre-TCR and pre-TCR sig-naling. Thus, our data indicate that Cbl proteins playa critical role in establishing the MHC-dependentCD4+and CD8+T cell development programs. Theylikely do so by suppressing MHC-independent NF-kBactivation, possibly through downmodulating pre-TCR signaling in DP thymocytes.IntroductionT lymphopoiesis is a sequential developmental processregulated by thymic environmental signals (Fowlkes andPardoll, 1989; Love and Chan, 2003; von Boehmer,1988). The first step of T cell development involves suc-cessive rearrangements of the V-(D)-J genes in CD4 andCD8 double-negative (DN) T precursors, and only thosecells with a successfully rearranged TCRb gene may de-velop further into CD4 and CD8 DP stage of thymocytes.This step in T cell development is controlled by signalingof growth factor receptors such as interleukine-7 recep-tor (IL-7R) and pre-TCR and occurs independently fromthe MHC because pre-TCR signaling is constitutively ac-tive in the absence of the MHC (Hayday et al., 1999; vonBoehmer and Fehling, 1997). At the DP stage, thymo-cytes may decide to become either CD4+helper-T (Th)or CD8+cytotoxic-T (Tc) cells and are positively andnegatively selected based on the interaction betweenthe TCR and the MHC expressed by thymic accessorycells (Kaye et al., 1989; Sha et al., 1988; Teh et al.,1988). While thymocytes expressing a TCR that recog-nize MHC-I choose CD8+T cell fate and develop intoTc cells, cells that express a TCR recognizing MHC-IIcommit to CD4+lineage and become Th cells (Cosgroveet al., 1991; Grusby et al., 1991; Zijlstra et al., 1990).Development of CD4+and CD8+T cells is regulated bysignals delivered by the TCR; however, it remains un-clear how the incoming TCR signals are connected tothe CD4+and CD8+lineage-development program. Cur-rent opinion indicates that engagement of the TCR withMHC initiates a TCR-downstream signaling cascade,starting with tyrosine phosphorylation of the immunore-ceptor tyrosine-based activation motifs (ITAMs) of theCD3 complex by the Src-family tyrosine kinase Lck (lym-phocyte-specific protein tyrosine kinase) (Weiss andLittman, 1994). The phosphorylated ITAMs then recruittyrosine kinase Zap70 (zeta-chain associated protein70 kDa) to the engaged TCR complexes, leading toZap70 activation. Upon activation, Zap70 phosphory-lates membrane protein LAT (linker for activation of Tcells), which then assembles a complex of multisignal-ing molecules, including Grb2 (growth-factor receptorbinding protein 2), Gads (Grb2-related adaptor protein),SLP-76 (the SH2-domain-containing leukocyte phos-phoprotein of 76 kDa), Vav, PLCg-1 (phospholipaseCgamma-1), and Itk (interleukine-2 tyrosine kinase)(Koretzky and Myung, 2001; Lucas et al., 2003). Coor-dination between these signaling components leadsto multiple downstream cellular responses, includingCa2+mobilization, cytoskeleton reorganization, and ac-tivation of various nuclear factors that eventually deter-mine the various T cell development programs.The Cbl family of proteins are signaling adaptors thatassociate with many different receptors and intracellularsignaling components (Rao et al., 2002; Thien et al.,2001). c-Cbl and Cbl-b, two highly homologous mem-bers of the Cbl family, are coexpressed in hematopoi-etic-lineage cells and function as E3-ubiquitin ligases(Joazeiro et al., 1999). Biochemical analyses revealthat Cbl proteins promote the ubiquitination of severalCbl-associated proteins, thereby regulating the degra-dation or intracellular association of these molecules(Liu, 2004). In the immune system, protein ubiquitinationhas been implicated in the regulation of T-lymphocyte*Correspondence: [email protected] authors contributed equally to this work.5Present address: Fu Dan University Medical School, Shanghai200032, P. R. China.6Present address: RIKEN BioSource Center, Tsukuba-Shi, Ibaraki305-0074, Japan.7Present address: Department of Medicine, Evanston NorthwesternHealthcare Research Institute, Northwestern University, Illinois60201.8Present address: Neuroinflammation Research Center, Depart-ment of Neurosciences,


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