RESUMO
The antibody gene mutator activation-induced cytidine deaminase (AID) promiscuously damages oncogenes, leading to chromosomal translocations and tumorigenesis. Why nonimmunoglobulin loci are susceptible to AID activity is unknown. Here, we study AID-mediated lesions in the context of nuclear architecture and the B cell regulome. We show that AID targets are not randomly distributed across the genome but are predominantly grouped within super-enhancers and regulatory clusters. Unexpectedly, in these domains, AID deaminates active promoters and eRNA(+) enhancers interconnected in some instances over megabases of linear chromatin. Using genome editing, we demonstrate that 3D-linked targets cooperate to recruit AID-mediated breaks. Furthermore, a comparison of hypermutation in mouse B cells, AID-induced kataegis in human lymphomas, and translocations in MEFs reveals that AID damages different genes in different cell types. Yet, in all cases, the targets are predominantly associated with topological complex, highly transcribed super-enhancers, demonstrating that these compartments are key mediators of AID recruitment.
Assuntos
Linfócitos B/metabolismo , Carcinogênese , Citidina Desaminase/genética , Elementos Facilitadores Genéticos , Animais , Dano ao DNA , Humanos , Linfoma/metabolismo , CamundongosRESUMO
Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, â¼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.
Assuntos
Linfócitos B/metabolismo , Regulação da Expressão Gênica , Ativação Linfocitária , Linfócitos/metabolismo , Regiões Promotoras Genéticas , Animais , Linfócitos B/imunologia , Linhagem Celular Tumoral , DNA de Cadeia Simples/metabolismo , Elementos Facilitadores Genéticos , Estudo de Associação Genômica Ampla , Humanos , Linfócitos/citologia , Linfócitos/imunologia , Camundongos , Fator de Transcrição TFIIH/metabolismo , Transcrição GênicaRESUMO
Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αß T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αßTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αßTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αßTCR repertoire.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos T/citologia , Timócitos/metabolismo , Timo/metabolismo , Animais , Complexo Principal de Histocompatibilidade , Camundongos , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores Virais , Transdução de Sinais , Linfócitos T/metabolismo , Timo/imunologiaRESUMO
Regulatory T cells (Treg cells) can express the transcription factors T-bet and GATA-3, but the function of this expression and whether such cells represent stable subsets is still unknown. By using various reporter tools, we found that the expression of T-bet and GATA-3 in Treg cells was dynamically influenced by the cytokine environment. Treg cell-specific deletion of the gene encoding either T-bet (Tbx21) or GATA-3 (Gata3) alone did not result in loss of Treg cell function; however, mice with combined deficiency in both genes in Treg cells developed severe autoimmune-like diseases. Loss of Treg cell function correlated with upregulation of expression of the transcription factor RORγt and reduced expression of the transcription factor Foxp3. Thus, in the steady state, activated Treg cells transiently upregulated either T-bet or GATA-3 to maintain T cell homeostasis.
Assuntos
Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Tolerância Imunológica/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Autoimunidade , Células Cultivadas , Colite/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , CamundongosRESUMO
Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.
Assuntos
Citocinas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/metabolismo , Células T Matadoras Naturais/fisiologia , Timócitos/fisiologia , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Citotoxicidade Imunológica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , Ligação Proteica , Processamento Pós-Transcricional do RNA , Tretinoína/metabolismo , Regulação para Cima , Vitamina D/metabolismoRESUMO
Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4(+) lineage fate and Runx3 promoting the CD8(+) lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8(+) lineage fate, nor is it understood why ThPOK itself imposes the CD4(+) lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4(+) lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4(+) lineage fate in the thymus.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Linhagem da Célula , Subunidade alfa 3 de Fator de Ligação ao Core/fisiologia , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de Transcrição/fisiologia , Animais , Linfócitos T CD8-Positivos/fisiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4(+)CD8(lo) intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4(-)CD8(-) double-negative thymocytes positive for the T cell antigen receptor αß subtype (TCRαß) that 'preferentially' migrated to the intestine, where they re-expressed CD8α and were sequestered as CD8αα(+) intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8αα(+) IELs as the developmental fate of autoreactive thymocytes that survive negative selection.
Assuntos
Diferenciação Celular/imunologia , Deleção Clonal/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Antígenos CD28/imunologia , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Citometria de Fluxo , Tolerância Imunológica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de Sinais/imunologia , Timócitos/citologia , Timo/citologiaRESUMO
The common γ-chain (γc) plays a central role in signaling by IL-2 and other γc-dependent cytokines. Here we report that activated T cells produce an alternatively spliced form of γc mRNA that results in protein expression and secretion of the γc extracellular domain. The soluble form of γc (sγc) is present in serum and directly binds to IL-2Rß and IL-7Rα proteins on T cells to inhibit cytokine signaling and promote inflammation. sγc suppressed IL-7 signaling to impair naive T cell survival during homeostasis and exacerbated Th17-cell-mediated inflammation by inhibiting IL-2 signaling upon T cell activation. Reciprocally, the severity of Th17-cell-mediated inflammatory diseases was markedly diminished in mice lacking sγc. Thus, sγc expression is a naturally occurring immunomodulator that regulates γc cytokine signaling and controls T cell activation and differentiation.
Assuntos
Processamento Alternativo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Cadeias gama de Imunoglobulina/imunologia , Inflamação/imunologia , Células Th17/imunologia , Animais , Autoimunidade , Diferenciação Celular/imunologia , Proliferação de Células , Sobrevivência Celular/imunologia , Cadeias gama de Imunoglobulina/sangue , Cadeias gama de Imunoglobulina/genética , Imunomodulação , Subunidade beta de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-5/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transdução de Sinais/imunologiaRESUMO
Immature CD4(+)CD8(+) (double-positive (DP)) thymocytes are signaled via T cell antigen receptors (TCRs) to undergo positive selection and become responsive to intrathymic cytokines such as interleukin 7 (IL-7). We report here that cytokine signaling is required for positively selected thymocytes to express the transcription factor Runx3, specify CD8 lineage choice and differentiate into cytotoxic-lineage T cells. In DP thymocytes genetically engineered to be cytokine responsive, IL-7 signaling induced TCR-unsignaled DP thymocytes to express Runx3 and to differentiate into mature CD8(+) T cells, completely circumventing positive selection. We conclude that TCR-mediated positive selection converts DP cells into cytokine-responsive thymocytes, but it is subsequent signaling by intrathymic cytokines that specifies CD8 lineage choice and promotes differentiation into cytotoxic-lineage T cells.
Assuntos
Citocinas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Contagem de Células , Diferenciação Celular/imunologia , Linhagem da Célula , Subunidade alfa 3 de Fator de Ligação ao Core/imunologia , Citometria de Fluxo , Interleucina-7/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator de Transcrição STAT5/imunologia , Transdução de SinaisRESUMO
Immune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique proapoptotic protein signature (Pumaâºâºâºp-Bimâºâºp-JNKâºâºDUSP6â») and repressed expression of prosurvival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only â¼1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3âºCD25â» Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a proapoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Citocinas/imunologia , Fatores de Transcrição Forkhead/metabolismo , Subunidade gama Comum de Receptores de Interleucina/imunologia , Linfócitos T Reguladores/fisiologia , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Sobrevivência Celular , Células Cultivadas , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento , Linfopoese/genética , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
T-bet is a critical transcription factor for T helper 1 (Th1) cell differentiation. To study the regulation and functions of T-bet, we developed a T-bet-ZsGreen reporter mouse strain. We determined that interleukin-12 (IL-12) and interferon-γ (IFN-γ) were redundant in inducing T-bet in mice infected with Toxoplasma gondii and that T-bet did not contribute to its own expression when induced by IL-12 and IFN-γ. By contrast, T-bet and the transcription factor Stat4 were critical for IFN-γ production whereas IFN-γ signaling was dispensable for inducing IFN-γ. Loss of T-bet resulted in activation of an endogenous program driving Th2 cell differentiation in cells expressing T-bet-ZsGreen. Genome-wide analyses indicated that T-bet directly induced many Th1 cell-related genes but indirectly suppressed Th2 cell-related genes. Our study revealed redundancy and synergy among several Th1 cell-inducing pathways in regulating the expression of T-bet and IFN-γ, and a critical role of T-bet in suppressing an endogenous Th2 cell-associated program.
Assuntos
Transdução de Sinais , Proteínas com Domínio T/imunologia , Células Th2/imunologia , Animais , Diferenciação Celular , Fator de Transcrição GATA3/imunologia , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Knockout , Fator de Transcrição STAT4/deficiência , Fator de Transcrição STAT4/imunologia , Proteínas com Domínio T/deficiência , Células Th1/imunologia , Células Th2/citologia , Toxoplasma/imunologia , Toxoplasmose/imunologiaRESUMO
Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1ß but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1ß-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Células-Tronco Hematopoéticas/metabolismo , Inflamassomos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Dermatite/imunologia , Dermatite/metabolismo , Fluoruracila/farmacologia , Hematopoese/efeitos dos fármacos , Hematopoese/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/virologia , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Camundongos , Camundongos Knockout , Mutação , Pancitopenia/imunologia , Pancitopenia/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Mice that express a single transgenic T cell receptor have a low incidence of T cell lymphoma development. We investigated whether this tumor development is restricted by surveillance mechanisms that are exerted by IL-15-dependent cells. Lymphoma incidence was increased to between 30 and 60% when TCR transgenes were expressed in IL-15-deficient mice. Mice in which NK cells had been depleted genetically or with neutralizing antibodies allowed lymphoma growth while the absence of CD8 T cells was without consequence. Half of the emerged T cell lymphomas carried Notch1 mutations. The distinct phenotype of the lymphomas involved expression of PD1, CD30, CD24, the stress receptor ligand Mult1 and MHC class I down-regulation. NK cells were able to directly lyse lymphoma cells, and neutralizations of Mult1 and class I expression prevented NK cell degranulation. Together these data support an involvement of NK cells in tumor surveillance of nascent T cell lymphomas.
Assuntos
Células Matadoras Naturais/metabolismo , Linfoma de Células T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Feminino , Humanos , Interleucina-15/imunologia , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Linfoma/patologia , Linfoma de Células T/genética , Linfoma de Células T/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
The transcription factors GATA-3 and ThPOK are required for intrathymic differentiation of CD4(+) T cells, but their precise functions in this process remain unclear. Here we show that, contrary to previous findings, Gata3 disruption blocked differentiation into the CD4(+) T cell lineage before commitment to the CD4(+) lineage and in some contexts permitted the 'redirection' of major histocompatibility complex class II-restricted thymocytes into the CD8(+) lineage. GATA-3 promoted ThPOK expression and bound to a region of the locus encoding ThPOK established as being critical for ThPOK expression. Finally, ThPOK promoted differentiation into the CD4(+) lineage in a way dependent on GATA-3 but inhibited differentiation into the CD8(+) lineage independently of GATA-3. We propose that GATA-3 acts as a specification factor for the CD4(+) lineage 'upstream' of the ThPOK-controlled CD4(+) commitment checkpoint.
Assuntos
Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica/imunologia , Fatores de Transcrição/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citometria de Fluxo , Fator de Transcrição GATA3/metabolismo , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timo/citologia , Timo/imunologia , Fatores de Transcrição/metabolismoRESUMO
Missense mutations in the C-terminal B30.2 domain of pyrin cause familial Mediterranean fever (FMF), the most common Mendelian autoinflammatory disease. However, it remains controversial as to whether FMF is due to the loss of an inhibitor of inflammation or to the activity of a proinflammatory molecule. We generated both pyrin-deficient mice and "knockin" mice harboring mutant human B30.2 domains. Homozygous knockin, but not pyrin-deficient, mice exhibited spontaneous bone marrow-dependent inflammation similar to but more severe than human FMF. Caspase-1 was constitutively activated in knockin macrophages and active IL-1ß was secreted when stimulated with lipopolysaccharide alone, which is also observed in FMF patients. The inflammatory phenotype of knockin mice was completely ablated by crossing with IL-1 receptor-deficient or adaptor molecule ASC-deficient mice, but not NLRP3-deficient mice. Thus, our data provide evidence for an ASC-dependent NLRP3-independent inflammasome in which gain-of-function pyrin mutations cause autoinflammatory disease.
Assuntos
Doenças Autoimunes/imunologia , Proteínas de Transporte/imunologia , Proteínas do Citoesqueleto/genética , Mutação , Imunidade Adaptativa , Animais , Doenças Autoimunes/patologia , Células Cultivadas , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/imunologia , Macrófagos/imunologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Pirina , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/imunologiaRESUMO
Th17 cells have been described as short lived, but this view is at odds with their capacity to trigger protracted damage to normal and transformed tissues. We report that Th17 cells, despite displaying low expression of CD27 and other phenotypic markers of terminal differentiation, efficiently eradicated tumors and caused autoimmunity, were long lived, and maintained a core molecular signature resembling early memory CD8(+) cells with stem cell-like properties. In addition, we found that Th17 cells had high expression of Tcf7, a direct target of the Wnt and ß-catenin signaling axis, and accumulated ß-catenin, a feature observed in stem cells. In vivo, Th17 cells gave rise to Th1-like effector cell progeny and also self-renewed and persisted as IL-17A-secreting cells. Multipotency was required for Th17 cell-mediated tumor eradication because effector cells deficient in IFN-γ or IL-17A had impaired activity. Thus, Th17 cells are not always short lived and are a less-differentiated subset capable of superior persistence and functionality.
Assuntos
Células-Tronco/metabolismo , Células Th17/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/genética , Perfilação da Expressão Gênica , Interleucina-17/biossíntese , Camundongos , Camundongos Transgênicos , Neoplasias/imunologia , Células-Tronco/citologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/citologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.
Assuntos
Síndrome de Birt-Hogg-Dubé/genética , Doenças Renais Císticas/genética , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Síndrome de Birt-Hogg-Dubé/patologia , Cardiomegalia/genética , Cardiomegalia/patologia , Proliferação de Células/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Humanos , Rim/patologia , Doenças Renais Císticas/patologia , Neoplasias Renais/patologia , Camundongos , Camundongos Knockout , Mutação de Sentido IncorretoRESUMO
The lineage fate of developing thymocytes is determined by the persistence or cessation of T cell receptor (TCR) signaling during positive selection, with persistent TCR signaling required for CD4 lineage choice. We show here that transcriptional upregulation of CD4 expression is essential for error-free lineage choice during major histocompatibility complex class II (MHC II)-specific positive selection and is critical for error-free lineage choice in TCR-transgenic mice whose thymocytes compete for the identical selecting ligand. CD4 upregulation occurred for endogenously encoded CD4 coreceptors, but CD4 transgenes were downregulated during positive selection, disrupting MHC II-specific TCR signaling and causing lineage errors regardless of the absolute number or signaling strength of transgenic CD4 proteins. Thus, the kinetics of CD4 coreceptor expression during MHC II-specific positive selection determines the integrity of CD4 lineage choice, revealing an elegant symmetry between coreceptor kinetics and lineage choice.
Assuntos
Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linhagem da Célula , Antígenos de Histocompatibilidade Classe II/imunologia , Regulação para Cima , Animais , Antígenos CD4/genética , Linfócitos T CD4-Positivos/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
How CD4-CD8 differentiation is maintained in mature T cells is largely unknown. The present study has examined the role in this process of the zinc finger protein Zbtb7b, a critical factor for the commitment of MHC II-restricted thymocytes to the CD4+ lineage. We showed that Zbtb7b acted in peripheral CD4+ T cells to suppress CD8-lineage gene expression, including that of CD8 and cytotoxic effector genes perforin and Granzyme B, and was important for the proper repression of interferon-gamma (IFN-gamma) during effector differentiation. The inappropriate expression of IFN-gamma by Zbtb7b-deficient CD4+ T cells required the activities of Eomesodermin and Runx transcription factors. Runx activity was needed for Granzyme B expression, indicating that Runx proteins control expression of the cytotoxic program. We conclude that a key function of Zbtb7b in the mature CD4+ T cell compartment is to repress CD8-lineage gene expression.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição/deficiência , Fatores de Transcrição/imunologiaRESUMO
Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays a central role in induction of allergic inflammatory responses. Its principal targets have been reported to be dendritic cells and/or CD4 T cells; epithelial cells are a principal source. We report in this study the development of a reporter mouse (TSLP-ZsG) in which a ZsGreen (ZsG)-encoding construct has been inserted by recombineering into a bacterial artificial chromosome immediately at the translation initiating ATG of TSLP. The expression of ZsG by mice transgenic for the recombinant BAC appears to be a faithful surrogate for TSLP expression, particularly in keratinocytes and medullary thymic epithelial cells. Limited ZsG and TSLP mRNA was observed in bone marrow-derived mast cells, basophils, and dendritic cells. Using the TSLP-ZsG reporter mouse, we show that TNF-α and IL-4/IL-13 are potent inducers of TSLP expression by keratinocytes and that local activation of Th2 and Th1 cells induces keratinocyte TSLP expression. We suggest that the capacity of TSLP to both induce Th2 differentiation and to be induced by activated Th2 cells raises the possibility that TSLP may be involved in a positive feedback loop to enhance allergic inflammatory conditions.