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1.
Proc Natl Acad Sci U S A ; 117(29): 17156-17165, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611812

RESUMO

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels of Nr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77tg) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearranged Vα14-Jα18 TCR-α chain gene into the Nur77tg (Nur77tg;Vα14tg) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4-producing NKT2 cell subset but not IFN-γ-producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions.


Assuntos
Diferenciação Celular , Tolerância Imunológica , Células T Matadoras Naturais , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Antígenos de Linfócitos T , Timócitos
2.
Sci Rep ; 7(1): 15594, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29142275

RESUMO

Semi-invariant natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. NKT cell survival during development requires signal processing by activated RelA/NF-κB. Nonetheless, the upstream signal(s) integrated by NF-κB in developing NKT cells remains incompletely defined. We show that the introgression of Bcl-xL-coding Bcl2l1 transgene into NF-κB signalling-deficient IκBΔN transgenic mouse rescues NKT cell development and differentiation in this mouse model. We reasoned that NF-κB activation was protecting developing NKT cells from death signals emanating either from high affinity agonist recognition by the T cell receptor (TCR) or from a death receptor, such as tumor necrosis factor receptor 1 (TNFR1) or Fas. Surprisingly, the single and combined deficiency in PKC-θ or CARMA-1-the two signal transducers at the NKT TCR proximal signalling node-only partially recapitulated the NKT cell deficiency observed in IκBΔN tg mouse. Accordingly, introgression of the Bcl2l1 transgene into PKC-θ null mouse failed to rescue NKT cell development. Instead, TNFR1-deficiency, but not the Fas-deficiency, rescued NKT cell development in IκBΔN tg mice. Consistent with this finding, treatment of thymocytes with an antagonist of the inhibitor of κB kinase -which blocks downstream NF-κB activation- sensitized NKT cells to TNF-α-induced cell death in vitro. Hence, we conclude that signal integration by NF-κB protects developing NKT cells from death signals emanating from TNFR1, but not from the NKT TCR or Fas.


Assuntos
NF-kappa B/genética , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Animais , Apoptose/genética , Diferenciação Celular/genética , Humanos , Ativação Linfocitária/genética , Linfócitos/imunologia , Camundongos , Camundongos Transgênicos , NF-kappa B/imunologia , Proteína Quinase C-theta/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Transdução de Sinais/genética , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Fator de Transcrição RelA/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteína bcl-X/genética , Receptor fas/genética
3.
Proteomics ; 17(12)2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28508465

RESUMO

Adjuvants enhance immunity elicited by vaccines through mechanisms that are poorly understood. Using a systems biology approach, we investigated temporal protein expression changes in five primary human immune cell populations: neutrophils, monocytes, natural killer cells, T cells, and B cells after administration of either an Adjuvant System 03 adjuvanted or unadjuvanted split-virus H5N1 influenza vaccine. Monocytes demonstrated the strongest differential signal between vaccine groups. On day 3 post-vaccination, several antigen presentation-related pathways, including MHC class I-mediated antigen processing and presentation, were enriched in monocytes and neutrophils and expression of HLA class I proteins was increased in the Adjuvant System 03 group. We identified several protein families whose proteomic responses predicted seroprotective antibody responses (>1:40 hemagglutination inhibition titer), including inflammation and oxidative stress proteins at day 1 as well as immunoproteasome subunit (PSME1 and PSME2) and HLA class I proteins at day 3 in monocytes. While comparison between temporal proteomic and transcriptomic results showed little overlap overall, enrichment of the MHC class I antigen processing and presentation pathway in monocytes and neutrophils was confirmed by both approaches.


Assuntos
Apresentação de Antígeno , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/uso terapêutico , Proteoma/metabolismo , Adjuvantes Imunológicos , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Humanos , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Mapas de Interação de Proteínas , Proteômica , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo
4.
PLoS One ; 12(1): e0167488, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28099485

RESUMO

BACKGROUND: Vaccine development for influenza A/H5N1 is an important public health priority, but H5N1 vaccines are less immunogenic than seasonal influenza vaccines. Adjuvant System 03 (AS03) markedly enhances immune responses to H5N1 vaccine antigens, but the underlying molecular mechanisms are incompletely understood. OBJECTIVE AND METHODS: We compared the safety (primary endpoint), immunogenicity (secondary), gene expression (tertiary) and cytokine responses (exploratory) between AS03-adjuvanted and unadjuvanted inactivated split-virus H5N1 influenza vaccines. In a double-blinded clinical trial, we randomized twenty adults aged 18-49 to receive two doses of either AS03-adjuvanted (n = 10) or unadjuvanted (n = 10) H5N1 vaccine 28 days apart. We used a systems biology approach to characterize and correlate changes in serum cytokines, antibody titers, and gene expression levels in six immune cell types at 1, 3, 7, and 28 days after the first vaccination. RESULTS: Both vaccines were well-tolerated. Nine of 10 subjects in the adjuvanted group and 0/10 in the unadjuvanted group exhibited seroprotection (hemagglutination inhibition antibody titer > 1:40) at day 56. Within 24 hours of AS03-adjuvanted vaccination, increased serum levels of IL-6 and IP-10 were noted. Interferon signaling and antigen processing and presentation-related gene responses were induced in dendritic cells, monocytes, and neutrophils. Upregulation of MHC class II antigen presentation-related genes was seen in neutrophils. Three days after AS03-adjuvanted vaccine, upregulation of genes involved in cell cycle and division was detected in NK cells and correlated with serum levels of IP-10. Early upregulation of interferon signaling-related genes was also found to predict seroprotection 56 days after first vaccination. CONCLUSIONS: Using this cell-based systems approach, novel mechanisms of action for AS03-adjuvanted pandemic influenza vaccination were observed. TRIAL REGISTRATION: ClinicalTrials.gov NCT01573312.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Biologia de Sistemas/métodos , Adolescente , Adulto , Anticorpos Antivirais/sangue , Formação de Anticorpos/imunologia , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Quimiocina CXCL10/sangue , Células Dendríticas/imunologia , Método Duplo-Cego , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Influenza Humana/imunologia , Interleucina-6/sangue , Células Matadoras Naturais/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Neutrófilos/imunologia , Vacinação , Adulto Jovem
5.
Mol Cell Biol ; 35(22): 3854-65, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26324326

RESUMO

Hdac3 is a key target for Hdac inhibitors that are efficacious in cutaneous T cell lymphoma. Moreover, the regulation of chromatin structure is critical as thymocytes transition from an immature cell with open chromatin to a mature T cell with tightly condensed chromatin. To define the phenotypes controlled by Hdac3 during T cell development, we conditionally deleted Hdac3 using the Lck-Cre transgene. This strategy inactivated Hdac3 in the double-negative stages of thymocyte development and caused a significant impairment at the CD8 immature single-positive (ISP) stage and the CD4/CD8 double-positive stage, with few mature CD4(+) or CD8(+) single-positive cells being produced. When Hdac3(-/-) mice were crossed with Bcl-xL-, Bcl2-, or TCRß-expressing transgenic mice, a modest level of complementation was found. However, when the null mice were crossed with mice expressing a fully rearranged T cell receptor αß transgene, normal levels of CD4 single-positive cells were produced. Thus, Hdac3 is required for the efficient transit from double-negative stage 4 through positive selection.


Assuntos
Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/genética , Linfócitos T/citologia , Animais , Antígenos CD4/análise , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/análise , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/metabolismo , Proteína bcl-X/genética
6.
J Immunol ; 187(12): 6335-45, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-22084435

RESUMO

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-x(L) expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-x(L), and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.


Assuntos
Diferenciação Celular/imunologia , Homeostase/imunologia , Interleucina-15/fisiologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Homeostase/genética , Interleucina-15/deficiência , Interleucina-15/genética , Fígado/citologia , Fígado/imunologia , Fígado/metabolismo , Contagem de Linfócitos , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células T Matadoras Naturais/citologia , Baço/citologia , Baço/imunologia , Baço/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismo , Proteína bcl-X/biossíntese , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
7.
PLoS One ; 6(4): e18534, 2011 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-21494556

RESUMO

BACKGROUND: Tumor growth is intimately linked with stromal interactions. Myeloid derived suppressor cells (MDSCs) are dramatically elevated in cancer patients and tumor bearing mice. MDSCs modulate the tumor microenvironment through attenuating host immune response and increasing vascularization. METHODOLOGY/PRINCIPAL FINDINGS: In searching for molecular mediators responsible for pro-tumor functions, we found that regulator of G protein signaling-2 (Rgs2) is highly increased in tumor-derived MDSCs compared to control MDSCs. We further demonstrate that hypoxia, a common feature associated with solid tumors, upregulates the gene expression. Genetic deletion of Rgs2 in mice resulted in a significant retardation of tumor growth, and the tumors exhibit decreased vascular density and increased cell death. Interestingly, deletion of Rgs2 in MDSCs completely abolished their tumor promoting function, suggesting that Rgs2 signaling in MDSCs is responsible for the tumor promoting function. Cytokine array profiling identified that Rgs2-/- tumor MDSCs produce less MCP-1, leading to decreased angiogenesis, which could be restored with addition of recombinant MCP-1. CONCLUSION: Our data reveal Rgs2 as a critical regulator of the pro-angiogenic function of MDSCs in the tumor microenvironment, through regulating MCP-1 production.


Assuntos
Quimiocina CCL2/genética , Células Mieloides/metabolismo , Células Mieloides/patologia , Neovascularização Patológica/metabolismo , Proteínas RGS/metabolismo , Microambiente Tumoral , Regulação para Cima/genética , Animais , Morte Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Quimiocina CCL2/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/patologia , Proteínas RGS/deficiência , Transdução de Sinais
8.
Immunity ; 33(2): 254-65, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20691614

RESUMO

Follicular (FO) and marginal zone (MZ) B cells are maintained in distinct locations within the spleen, but the genetic basis for this separation is still enigmatic. We now report that B cell sequestration requires lineage-specific regulation of migratory receptors by the transcription factor Klf2. Moreover, using gene-targeted mice we show that altered splenic B cell migration confers a significant in vivo gain-of-function phenotype to FO B cells, including the ability to quickly respond to MZ-associated antigens and pathogens in a T cell-dependent manner. This work demonstrates that in wild-type animals, naive FO B cells are actively removed from the MZ, thus restricting their capacity to respond to blood-borne pathogens.


Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Movimento Celular , Imunidade Humoral , Baço/citologia , Baço/imunologia , Animais , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos T-Independentes/genética , Antígenos T-Independentes/imunologia , Medula Óssea/imunologia , Diferenciação Celular , Células Cultivadas , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/imunologia , Camundongos , Camundongos Knockout , Receptores CCR/imunologia
11.
EMBO J ; 28(22): 3579-90, 2009 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-19816402

RESUMO

The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.


Assuntos
Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Adaptação Biológica/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos CD1d/química , Antígenos Glicosídicos Associados a Tumores/imunologia , Células Cultivadas , Galactosilceramidas/química , Galactosilceramidas/imunologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Relação Estrutura-Atividade , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia
12.
Cell Host Microbe ; 1(2): 109-19, 2007 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-18005689

RESUMO

Staphylococcus aureus, a bacterium responsible for tremendous morbidity and mortality, exists as a harmless commensal in approximately 25% of humans. Identifying the molecular machinery activated upon infection is central to understanding staphylococcal pathogenesis. We describe the heme sensor system (HssRS) that responds to heme exposure and activates expression of the heme-regulated transporter (HrtAB). Inactivation of the Hss or Hrt systems leads to increased virulence in a vertebrate infection model, a phenotype that is associated with an inhibited innate immune response. We suggest that the coordinated activity of Hss and Hrt allows S. aureus to sense internal host tissues, resulting in tempered virulence to avoid excessive host tissue damage. Further, genomic analyses have identified orthologous Hss and Hrt systems in Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis, suggesting a conserved regulatory system by which Gram-positive pathogens sense heme as a molecular marker of internal host tissue and modulate virulence.


Assuntos
Meio Ambiente , Heme/fisiologia , Heme/toxicidade , Staphylococcus aureus/patogenicidade , Aclimatação , Ferro/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Virulência
13.
Immunity ; 25(3): 487-97, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16949316

RESUMO

Invariant natural killer T (iNKT) cell-derived cytokines have important functions in inflammation, host defense, and immunoregulation. Yet, when and how iNKT cells undergo effector differentiation, which endows them with the capacity to rapidly secrete cytokines upon activation, remains unknown. We discovered that granulocyte-macrophage colony-stimulating factor (Csf-2)-deficient mice developed iNKT cells that failed to respond to the model antigen alpha-galactosylceramide because of an intrinsic defect in the fusion of secretory vesicles with the plasma membrane. Exogenous Csf-2 corrected the functional defect only when supplied during the development of thymic, but not mature, splenic Csf-2-deficient iNKT cells. Thus, we ascribe a unique function to Csf-2, which regulates iNKT cell effector differentiation during development by a mechanism that renders them competent for cytokine secretion.


Assuntos
Diferenciação Celular/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/deficiência , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/fisiologia , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Timo/metabolismo
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