RESUMO
BACKGROUND: Inborn errors of immunity (IEI) often lack specific disease models and personalized management. Signal transducer and activator of transcription (STAT)-1 gain of function (GoF) is such example of an IEI with diverse clinical phenotype with unclear pathomechanisms and unpredictable response to therapy. Limitations in obtaining fresh samples for functional testing and research further highlights the need for patient-specific ex vivo platforms. OBJECTIVE: Using STAT1-GoF as an example IEI, we investigated the potential of patient-derived expanded potential stem cells (EPSC) as an ex vivo platform for disease modeling and personalized treatment. METHODS: We generated EPSC derived from individual STAT1-GoF patients. STAT1 mutations were confirmed with Sanger sequencing. Functional testing including STAT1 phosphorylation/dephosphorylation and gene expression with or without Janus activating kinase inhibitors were performed. Functional tests were repeated on EPSC lines with GoF mutations repaired by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) editing. RESULTS: EPSC were successfully reprogrammed from STAT1-GoF patients and expressed the same pluripotent makers as controls, with distinct morphologic differences. Patient-derived EPSC recapitulated the functional abnormalities of index STAT1-GoF patients with STAT1 hyperphosphorylation and increased expression of STAT1 and its downstream genes (IRF1, APOL6, and OAS1) after IFN-γ stimulation. Addition of ruxolitinib and baricitinib inhibited STAT1 hyperactivation in STAT1-GoF EPSC in a dose-dependent manner, which was not observed with tofacitinib. Corrected STAT1 phosphorylation and downstream gene expression were observed among repaired STAT1-GoF EPSC cell lines. CONCLUSION: This proof-of-concept study demonstrates the potential of our patient-derived EPSC platform to model STAT1-GoF. We propose this platform when researching, recapitulating, and repairing other IEI in the future.
Assuntos
Mutação com Ganho de Função , Fator de Transcrição STAT1 , Células-Tronco , Humanos , Mutação , Fosforilação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Células-Tronco/imunologia , Células-Tronco/metabolismoRESUMO
During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN⺠cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN⺠blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV⺠serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1ß. Furthermore, circulating DC-SIGN⺠DC that were isolated directly from HIV-1⺠individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.
Assuntos
Apoptose/fisiologia , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Lectinas Tipo C/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Receptores de Superfície Celular/metabolismo , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD40/imunologia , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/patologia , Inativação Gênica , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Interações Hospedeiro-Patógeno , Humanos , Lectinas Tipo C/imunologia , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinase 5/imunologia , Ligação Proteica , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/imunologia , TransfecçãoRESUMO
Severe acute respiratory syndrome (SARS) is caused by infection of a previously undescribed coronavirus (CoV). L-SIGN, encoded by CLEC4M (also known as CD209L), is a SARS-CoV binding receptor that has polymorphism in its extracellular neck region encoded by the tandem repeat domain in exon 4. Our genetic risk association study shows that individuals homozygous for CLEC4M tandem repeats are less susceptible to SARS infection. L-SIGN is expressed in both non-SARS and SARS-CoV-infected lung. Compared with cells heterozygous for L-SIGN, cells homozygous for L-SIGN show higher binding capacity for SARS-CoV, higher proteasome-dependent viral degradation and a lower capacity for trans infection. Thus, homozygosity for L-SIGN plays a protective role during SARS infection.
Assuntos
Moléculas de Adesão Celular/genética , Lectinas Tipo C/genética , Receptores de Superfície Celular/genética , Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Animais , Células CHO/virologia , Moléculas de Adesão Celular/metabolismo , Chlorocebus aethiops , Estudos de Coortes , Cricetinae , Cricetulus , Predisposição Genética para Doença , Homozigoto , Hong Kong/epidemiologia , Humanos , Intestino Delgado/fisiologia , Lectinas Tipo C/metabolismo , Pulmão/fisiologia , Pulmão/virologia , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Superfície Celular/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/epidemiologia , Sequências de Repetição em Tandem , Células Vero/virologiaRESUMO
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with multiple etiological factors. The SLE susceptibility locus on chromosome 16p13 encodes a novel gene CLEC16A and its functional relationship with SLE is unclear. This study aimed to investigate the expression correlation of the two major CLEC16A spliced transcripts with SLE development. Expressions of the long (V1) and short (V2) CLEC16A isoforms in the peripheral blood mononuclear cells (PBMCs) were assayed by quantitative real time PCR and compared between healthy individuals and SLE patients. Correlation of CLEC16A isoform expression levels with SLE susceptibility, disease severity and twelve clinical parameters were also evaluated. Full length transcripts of CLEC16A V1 and V2 isoforms were readily amplified from PBMCs of healthy controls and patients at varying abundance. Compared with healthy controls (n = 86), expression levels of V1 and V2 were significantly reduced by ~two- and four-fold respectively in SLE patients (n = 181). The relative V2/V1 ratio was also significantly reduced by approximately two-fold. With regard to SLE disease parameters, only a weak positive correlation was found between CLEC16A V1 expression levels and SLE disease activity index (SLEDAI) score. Taken together, CLEC16A was found to be a susceptibility factor for SLE, with possible contribution to the development of the disease.
Assuntos
Lectinas Tipo C/metabolismo , Leucócitos/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Lectinas Tipo C/genética , Lúpus Eritematoso Sistêmico/diagnóstico , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte de Monossacarídeos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11blo/- pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.
Assuntos
Reações Cruzadas/imunologia , Epitopos/imunologia , Galactose/imunologia , Imunogenicidade da Vacina , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/imunologia , Vacinas contra Influenza/genética , Células Matadoras Naturais/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologiaRESUMO
Two closely related trans-membrane C-type lectins dendritic cell-specific intracellular adhesion molecules (ICAM)-3 grabbing non-integrin (DC-SIGN or CD209) and liver/lymph node-specific ICAM-3 grabbing non-integrin (L-SIGN also known as DC-SIGNR, CD209L or CLEC4M) directly recognize a wide range of micro-organisms of major impact on public health. Both genes have long been considered to share similar overall structure and ligand-binding characteristics. This review presents more recent biochemical and structural studies, which show that they have distinct ligand-binding properties and different physiological functions. Of importance in both these genes is the presence of an extra-cellular domain consisting of an extended neck region encoded by tandem repeats that support the carbohydrate-recognition domain, which plays a crucial role in influencing the pathogen-binding properties of these receptors. The notable difference between these two genes is in this extra-cellular domain. Whilst the tandem-neck-repeat region remains relatively constant size for DC-SIGN, there is considerable polymorphism for L-SIGN. Homo-oligomerization of the neck region of L-SIGN has been shown to be important for high-affinity ligand binding, and heterozygous expression of the polymorphic variants of L-SIGN in which neck lengths differ could thus affect ligand-binding affinity. Functional studies on the effect of this tandem-neck-repeat region on pathogen-binding, as well as genetic association studies for various infectious diseases and among different populations, are discussed. Worldwide demographic data of the tandem-neck-repeat region showing distinct differences in the neck-region allele and genotype distribution among different ethnic groups are presented. These findings support the neck region as an excellent candidate acting as a functional target for selective pressures exerted by pathogens.
Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Infecções/genética , Lectinas Tipo C/genética , Lectinas Tipo C/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Células Dendríticas/imunologia , Suscetibilidade a Doenças/imunologia , Humanos , Lectinas Tipo C/química , Ligantes , Modelos Biológicos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/químicaRESUMO
A major challenge in the field of transplantation is to prevent graft rejection and prolong graft survival. Tolerance induction is a promising way to achieve long-term graft survival without the need for potent immunosuppression and its associated side effects. The recent success of co-stimulatory blockade by the chimeric protein CTLA4Ig in the modulation of the recipient's immune system and the prolongation of graft survival in animal models suggests a possible application of CTLA4Ig in clinical transplantation. To produce sufficient amounts of CTLA4Ig for future clinical application, we sought to use the mammary gland as a bioreactor and produce CTLA4Ig in the milk of transgenic farm animals. Prior to the generation of transgenic farm animals, we tested our strategy in mice. Using the promoter of the sheep beta-lactoglobulin gene, we expressed our CTLA4Ig chimeric gene in the mammary gland of transgenic mice. The yield of CTLA4Ig was fivefold higher in transgenic milk than that from transfected cells. Purified milk-derived CTLA4Ig is biologically active and suppresses T cell activation. We showed that the production of CTLA4Ig in the milk has no adverse immunosuppression effect on the transgenic animals and the offsprings that were fed with the transgenic milk. The findings suggest that the approach to produce CTLA4Ig in milk by transgenesis is feasible; further studies involving farm animals are warranted.
Assuntos
Imunoconjugados/metabolismo , Imunossupressores/metabolismo , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Abatacepte , Animais , Antígeno B7-1/imunologia , Células CHO , Cromatografia de Afinidade , Cricetinae , Feminino , Citometria de Fluxo , Humanos , Imunoconjugados/genética , Imunoconjugados/imunologia , Imunossupressores/imunologia , Imunossupressores/isolamento & purificação , Lactação , Teste de Cultura Mista de Linfócitos , Glândulas Mamárias Animais/imunologia , Camundongos , Camundongos Transgênicos , Leite/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Suínos , TransfecçãoRESUMO
Dendritic cells (DCs) are the most potent antigen-presenting cells, and are regarded as "natural adjuvants" for the induction of primary T or T-dependent immunity. DCs in the peripheral sites capture and process antigens. Encounter of exogenous or endogenous stimuli mature the function of DCs, and they thus acquire T-cell stimulatory capacity and distinct chemotactic behavior which enables them to migrate to lymphoid tissue. In the secondary lymphoid organs, they present antigens to T- and B-cells and stimulate their proliferation. Dendritic cells are also involved in tolerance induction, in particular, to self antigens. DCs also play a key role in the transmission of many pathogens, and therefore may become targets for designing new therapies. DCs have been manipulated in vitro and in vivo for cancer immunotherapy. In this article, we provide a concise overview of DC biology and its current and future role in clinical settings.
Assuntos
Células Dendríticas/fisiologia , Animais , Apresentação de Antígeno , Divisão Celular , Movimento Celular , Humanos , Tolerância Imunológica , Imunoterapia , Modelos Biológicos , Neoplasias/terapia , Linfócitos T/metabolismoRESUMO
Genetic polymorphisms have been demonstrated to be associated with vulnerability to human infection. ICAM3, an intercellular adhesion molecule important for T cell activation, and FCER2 (CD23), an immune response gene, both located on chromosome 19p13.3, were investigated for host genetic susceptibility and association with clinical outcome. A case-control study based on 817 patients with confirmed severe acute respiratory syndrome (SARS), 307 health care worker control subjects, 290 outpatient control subjects, and 309 household control subjects unaffected by SARS from Hong Kong was conducted to test for genetic association. No significant association to susceptibility to SARS infection caused by the novel coronavirus (SARS-CoV) was found for the FCER2 and the ICAM3 single nucleotide polymorphisms. However, patients with SARS homozygous for ICAM3 Gly143 showed significant association with higher lactate dehydrogenase levels (P=.0067; odds ratio [OR], 4.31 [95% confidence interval {CI}, 1.37-13.56]) and lower total white blood cell counts (P=.022; OR, 0.30 [95% CI, 0.10-0.89]) on admission. These findings support the role of ICAM3 in the immunopathogenesis of SARS.
Assuntos
Antígenos CD/genética , Moléculas de Adesão Celular/genética , Predisposição Genética para Doença , L-Lactato Desidrogenase/sangue , Polimorfismo de Nucleotídeo Único/genética , Síndrome Respiratória Aguda Grave/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Frequência do Gene , Genótipo , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Síndrome Respiratória Aguda Grave/fisiopatologiaRESUMO
Sonic hedgehog (Shh) is a crucial morphogen in the development of numerous tissues and organs, including the nervous system, gastrointestinal tract and lung. Recent findings suggest that Shh plays an important role in thymocyte development and peripheral T cell function. Here we report that the Shh receptors, patched and smoothened, are expressed in resting and activated T cells and their expression is regulated upon T cell activation. Shh protein is also detected on the surface of freshly isolated T cells. Although exogenous Shh alone does not activate resting T cells, it exhibits co-stimulatory activity which is reflected in its ability to potentiate CD3-mediated proliferation and cytokine production by CD4(+) T cells. The co-stimulatory effect is most prominent at sub-optimal TCR stimulation level. Gene expression analysis reveals that Shh signaling in CD4(+) T cells modulates a different set of transcriptional targets from that in neuronal cells. Furthermore, Shh co-stimulation modulates the expression of a subset of CD28-responsive genes, including cyclin A and B cell translocation gene 2.
Assuntos
Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica , Proteínas Hedgehog/fisiologia , Ativação Linfocitária , Animais , Ciclina A/genética , Ciclina A/metabolismo , Feminino , Genes Supressores de Tumor , Proteínas Hedgehog/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Proteínas Supressoras de TumorRESUMO
Calcineurin has been demonstrated as one of the key enzymes in TCR-mediated signaling cascades that lead to the transcription of a variety of cytokines including IL-2. In this study, we addressed the role of calcineurin in lymphocyte development and peripheral T cell responses using the lymphocytic choriomeningitis virus glycoprotein peptide p33-specific, TCR (P14)-transgenic T cells that were deficient in calcineurin subunit A alpha-isoform (CNAalpha(-/-)). Fetal thymic organ culture of P14/CNAalpha(-/-) lobes showed no defect in positive or negative selection of thymocytes. In addition, peptide-induced peripheral T cell deletion was also normal in CNAalpha-deficient T cells. In terms of mature T cell function, a reduction in proliferation, and IL-2 and IFN-gamma production was observed upon stimulation of P14/CNAalpha(-/-) T cells with the antigenic peptide. Impaired NF-AT nuclear localization was also observed. These results suggest that CNAalphais important for mature T cell function, but has a limited role in thymocyte development.
Assuntos
Calcineurina/metabolismo , Proteínas Nucleares , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Linfócitos T/imunologia , Animais , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Calcineurina/deficiência , Calcineurina/genética , Diferenciação Celular , Divisão Celular , Deleção Clonal , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas/imunologia , Técnicas In Vitro , Interferon gama/biossíntese , Interleucina-2/biossíntese , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fatores de Transcrição NFATC , Fragmentos de Peptídeos/imunologia , Transdução de Sinais , Linfócitos T/citologia , Fatores de Transcrição/metabolismo , Proteínas Virais/imunologiaAssuntos
Neoplasias Nasofaríngeas/metabolismo , Receptores CXCR4/biossíntese , Receptor 3 Toll-Like/biossíntese , Receptor 6 Toll-Like/biossíntese , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Metástase Neoplásica , Receptores CXCR4/genéticaRESUMO
The costimulatory protein ICOS is inducibly expressed on activated T cells. Previous results have shown that ICOS(-/-) mice are defective in germinal center formation, antibody (Ab) production and class switch as well as Th1 and Th2 cytokine production in response to protein or parasite antigens. However, ICOS-Ig failed to block antiviral Ab responses. To date the immune response to viruses has not been examined in ICOS(-/-) mice. In this report we compared antiviral Ab responses to LCMV, VSV and influenza virus in ICOS(-/-) versus wild-type mice. Our results show that ICOS is important in the Ab response to all three viruses, with greater effects on primary as compared to secondary responses. Although ICOS(-/-) mice are impaired in some immune responses following influenza infection, the effects were less severe than for CD28(-/-) mice. There was no defect in initial influenza-specific CD8 T cell expansion in ICOS(-/-) mice or in cytotoxic effector function. However, ICOS was important in maintaining CD4 cytokine production and CD8 T cell numbers late in the primary response. Upon secondary infection, ICOS(-/-) mice show wild-type levels of influenza-specific CD8 T cells, whereas CD28(-/-) mice show greatly impaired secondary CD8 T cell expansion. Overall, our results show that ICOS plays a clear role in the primary response to viruses at the level of Ab production, germinal center formation and Th cytokine production, but has diminished effects following secondary viral challenge.