RESUMO
Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. Herein, we describe a simplified intracellular staining workflow for classification of circulating human T and NK cells with cytolytic potential. We suggest reagents for measuring cytolytic proteins and identification of cell subsets within conventional and unconventional T cells and NK cells.
Assuntos
Células Matadoras Naturais , Linfócitos T Citotóxicos , Citometria de Fluxo , HumanosAssuntos
Hemoglobinúria Paroxística/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Anemia Aplástica , Animais , Doenças da Medula Óssea , Transtornos da Insuficiência da Medula Óssea , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/enzimologia , Hemoglobinúria Paroxística/patologia , Hemoglobinúria Paroxística/prevenção & controle , Humanos , CamundongosRESUMO
Tight regulation of hematopoietic stem cell (HSC) homeostasis ensures lifelong hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify Inositol-trisphosphate 3-kinase B (Itpkb) as an essential regulator of HSC homeostasis. Young Itpkb(-/-) mice accumulated phenotypic HSC, which were less quiescent and proliferated more than wild-type (WT) controls. Itpkb(-/-) HSC downregulated quiescence and stemness associated, but upregulated activation, oxidative metabolism, protein synthesis, and lineage associated messenger RNAs. Although they had normal-to-elevated viability and no significant homing defects, Itpkb(-/-) HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb(-/-) mice lost hematopoietic stem and progenitor cells and died with severe anemia. WT HSC normally repopulated Itpkb(-/-) hosts, indicating an HSC-intrinsic Itpkb requirement. Itpkb(-/-) HSC showed reduced colony-forming activity and increased stem-cell-factor activation of the phosphoinositide-3-kinase (PI3K) effectors Akt/mammalian/mechanistic target of rapamycin (mTOR). This was reversed by treatment with the Itpkb product and PI3K/Akt antagonist IP4. Transcriptome changes and biochemistry support mTOR hyperactivity in Itpkb(-/-) HSC. Treatment with the mTOR-inhibitor rapamycin reversed the excessive mTOR signaling and hyperproliferation of Itpkb(-/-) HSC without rescuing colony forming activity. Thus, we propose that Itpkb ensures HSC quiescence and function through limiting cytokine-induced PI3K/mTOR signaling and other mechanisms.
Assuntos
Anemia/genética , Anemia/mortalidade , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Homeostase/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Índice de Gravidade de DoençaRESUMO
Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αß-expressing 'single-positive' thymocytes from CD4(+)CD8αß(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.
Assuntos
Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Animais , Apoptose , Autoantígenos/imunologia , Sinalização do Cálcio , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Timócitos/imunologiaRESUMO
Natural killer (NK) cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections, tissue homeostasis, and reproduction. NK cell-based therapies are promising treatments for blood cancers. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize the loss of self-antigens or up-regulation of stress-induced ligands on pathogen-infected or tumor cells through invariant NK cell receptors (NKRs), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP(3) generation by PI3K and generation of diacylglycerol and IP(3) by phospholipase-Cγ (PLCγ). In the present study, we identify a novel role for the phosphorylated IP(3) metabolite inositol (1,3,4,5)tetrakisphosphate (IP(4)) in NK cells. IP(4) promotes NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP(4) limits NKR-induced IFNγ secretion, granule exocytosis, and target-cell killing, in part by inhibiting the PIP(3) effector-kinase Akt. This identifies IP(4) as an important novel regulator of NK cell development and function and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP(4) is a broadly important signaling paradigm.
Assuntos
Fosfatos de Inositol/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Transdução de Sinais/imunologia , Animais , Fosfatos de Inositol/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Citoplasmáticos e Nucleares/imunologia , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
Binding of the membrane phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) to the Pleckstrin Homology (PH) domain of the Tec family protein tyrosine kinase, Inducible T cell Kinase (ITK), is critical for the recruitment of the kinase to the plasma membrane and its co-localization with the TCR-CD3 molecular complex. Three aromatic residues, termed the FYF motif, located in the inner walls of the phospholipid-binding pocket of the ITK PH domain, are conserved in the PH domains of all Tec kinases, but not in other PH-domain containing proteins, suggesting an important function of the FYF motif in the Tec kinase family. However, the biological significance of the FYF amino acid motif in the ITK-PH domain is unknown. To elucidate it, we have tested the effects of a FYF triple mutant (F26S, Y90F, F92S), henceforth termed FYF-ITK mutant, on ITK function. We found that FYF triple mutation inhibits the TCR-induced production of IL-4 by impairing ITK binding to PIP(3), reducing ITK membrane recruitment, inducing conformational changes at the T cell-APC contact site, and compromising phosphorylation of ITK and subsequent phosphorylation of PLCγ(1). Interestingly, however, the FYF motif is dispensable for the interaction of ITK with two of its signaling partners, SLP-76 and LAT. Thus, the FYF mutation uncouples PIP(3)-mediated ITK membrane recruitment from the interactions of the kinase with key components of the TCR signalosome and abrogates ITK function in T cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência Conservada , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Interleucina-4/biossíntese , Células Jurkat , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosfolipase C gama/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Linfócitos T/metabolismo , TransfecçãoRESUMO
The present study focuses on a large family with an X-linked immunodeficiency in which there are variable clinical and laboratory phenotypes, including recurrent viral and bacterial infections, hypogammaglobulinemia, Epstein-Barr virus-driven lymphoproliferation, splenomegaly, colitis, and liver disease. Molecular and genetic analyses revealed that affected males were carriers of a hypomorphic hemizygous mutation in XIAP (XIAP(G466X)) that cosegregated with a rare polymorphism in CD40LG (CD40 ligand(G219R)). These genes are involved in the X-linked lymphoproliferative syndrome 2 and the X-linked hyper-IgM syndrome, respectively. Single expression of XIAP(G466X) or CD40L(G219R) had no or minimal effect in vivo, although in vitro, they lead to altered functional activities of their gene products, which suggests that the combination of XIAP and CD40LG mutations contributed to the expression of clinical manifestations observed in affected individuals. Our report of a primary X-linked immunodeficiency of oligogenic origin emphasizes that primary immunodeficiencies are not caused by a single defective gene, which leads to restricted manifestations, but are likely to be the result of an interplay between several genetic determinants, which leads to more variable clinical phenotypes.
Assuntos
Ligante de CD40/genética , Imunodeficiência de Variável Comum/genética , Transtornos Linfoproliferativos/genética , Polimorfismo de Nucleotídeo Único , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Adolescente , Adulto , Arginina/genética , Criança , Epistasia Genética/fisiologia , Família , Feminino , Genes Ligados ao Cromossomo X , Glutamina/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Polimorfismo de Nucleotídeo Único/fisiologia , Adulto JovemRESUMO
X-linked lymphoproliferative syndromes (XLP) are primary immunodeficiencies characterized by a particular vulnerability toward Epstein-Barr virus infection, frequently resulting in hemophagocytic lymphohistiocytosis (HLH). XLP type 1 (XLP-1) is caused by mutations in the gene SH2D1A (also named SAP), whereas mutations in the gene XIAP underlie XLP type 2 (XLP-2). Here, a comparison of the clinical phenotypes associated with XLP-1 and XLP-2 was performed in cohorts of 33 and 30 patients, respectively. HLH (XLP-1, 55%; XLP-2, 76%) and hypogammaglobulinemia (XLP-1, 67%; XLP-2, 33%) occurred in both groups. Epstein-Barr virus infection in XLP-1 and XLP-2 was the common trigger of HLH (XLP-1, 92%; XLP-2, 83%). Survival rates and mean ages at the first HLH episode did not differ for both groups, but HLH was more severe with lethal outcome in XLP-1 (XLP-1, 61%; XLP-2, 23%). Although only XLP-1 patients developed lymphomas (30%), XLP-2 patients (17%) had chronic hemorrhagic colitis as documented by histopathology. Recurrent splenomegaly often associated with cytopenia and fever was preferentially observed in XLP-2 (XLP-1, 7%; XLP-2, 87%) and probably represents minimal forms of HLH as documented by histopathology. This first phenotypic comparison of XLP subtypes should help to improve the diagnosis and the care of patients with XLP conditions.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Transtornos Linfoproliferativos/diagnóstico , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Técnicas Imunoenzimáticas , Lactente , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/terapia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Fenótipo , Estudos Retrospectivos , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Taxa de Sobrevida , Adulto JovemAssuntos
Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Transtornos Linfoproliferativos/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Agamaglobulinemia/genética , Apoptose/fisiologia , Infecções por Vírus Epstein-Barr/etiologia , Infecções por Vírus Epstein-Barr/imunologia , Predisposição Genética para Doença , Humanos , Células Matadoras Naturais/patologia , Subpopulações de Linfócitos/patologia , Linfo-Histiocitose Hemofagocítica/genética , Masculino , Modelos Biológicos , Fenótipo , Estrutura Terciária de Proteína , Esplenomegalia/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/deficiência , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologiaRESUMO
The homeostasis of the immune response requires tight regulation of the proliferation and apoptosis of activated lymphocytes. In humans, defects in immune homeostasis result in lymphoproliferation disorders including autoimmunity, haemophagocytic lymphohystiocytosis and lymphomas. The X-linked lymphoproliferative syndrome (XLP) is a rare, inherited immunodeficiency that is characterized by lymphohystiocytosis, hypogammaglobulinaemia and lymphomas, and that usually develops in response to infection with Epstein-Barr virus (EBV). Mutations in the signalling lymphocyte activation molecule (SLAM)-associated protein SAP, a signalling adaptor molecule, underlie 60% of cases of familial XLP. Here, we identify mutations in the gene that encodes the X-linked inhibitor-of-apoptosis XIAP (also termed BIRC4) in patients with XLP from three families without mutations in SAP. These mutations lead to defective expression of XIAP. We show that apoptosis of lymphocytes from XIAP-deficient patients is enhanced in response to various stimuli including the T-cell antigen receptor (TCR)-CD3 complex, the death receptor CD95 (also termed Fas or Apo-1) and the TNF-associated apoptosis-inducing ligand receptor (TRAIL-R). We also found that XIAP-deficient patients, like SAP-deficient patients, have low numbers of natural killer T-lymphocytes (NKT cells), indicating that XIAP is required for the survival and/or differentiation of NKT cells. The observation that XIAP-deficiency and SAP-deficiency are both associated with a defect in NKT cells strengthens the hypothesis that NKT cells have a key role in the immune response to EBV. Furthermore, by identifying an XLP immunodeficiency that is caused by mutations in XIAP, we show that XIAP is a potent regulator of lymphocyte homeostasis in vivo.