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To understand the dynamic interplay between the human microbiome and host during health and disease, we analyzed the microbial composition, temporal dynamics, and associations with host multi-omics, immune, and clinical markers of microbiomes from four body sites in 86 participants over 6 years. We found that microbiome stability and individuality are body-site specific and heavily influenced by the host. The stool and oral microbiome are more stable than the skin and nasal microbiomes, possibly due to their interaction with the host and environment. We identify individual-specific and commonly shared bacterial taxa, with individualized taxa showing greater stability. Interestingly, microbiome dynamics correlate across body sites, suggesting systemic dynamics influenced by host-microbial-environment interactions. Notably, insulin-resistant individuals show altered microbial stability and associations among microbiome, molecular markers, and clinical features, suggesting their disrupted interaction in metabolic disease. Our study offers comprehensive views of multi-site microbial dynamics and their relationship with host health and disease.
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Estabilidad Central , Microbiota , Humanos , Piel/microbiología , Interacciones Microbiota-Huesped , BiomarcadoresRESUMEN
To understand dynamic interplay between the human microbiome and host during health and disease, we analyzed the microbial composition, temporal dynamics, and associations with host multi-omics, immune and clinical markers of microbiomes from four body sites in 86 participants over six years. We found that microbiome stability and individuality are body-site-specific and heavily influenced by the host. The stool and oral microbiome were more stable than the skin and nasal microbiomes, possibly due to their interaction with the host and environment. Also, we identified individual-specific and commonly shared bacterial taxa, with individualized taxa showing greater stability. Interestingly, microbiome dynamics correlated across body sites, suggesting systemic coordination influenced by host-microbial-environment interactions. Notably, insulin-resistant individuals showed altered microbial stability and associations between microbiome, molecular markers, and clinical features, suggesting their disrupted interaction in metabolic disease. Our study offers comprehensive views of multi-site microbial dynamics and their relationship with host health and disease. Study Highlights: The stability of the human microbiome varies among individuals and body sites.Highly individualized microbial genera are more stable over time.At each of the four body sites, systematic interactions between the environment, the host and bacteria can be detected.Individuals with insulin resistance have lower microbiome stability, a more diversified skin microbiome, and significantly altered host-microbiome interactions.
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Lipids can be of endogenous or exogenous origin and affect diverse biological functions, including cell membrane maintenance, energy management and cellular signalling. Here, we report >800 lipid species, many of which are associated with health-to-disease transitions in diabetes, ageing and inflammation, as well as cytokine-lipidome networks. We performed comprehensive longitudinal lipidomic profiling and analysed >1,500 plasma samples from 112 participants followed for up to 9 years (average 3.2 years) to define the distinct physiological roles of complex lipid subclasses, including large and small triacylglycerols, ester- and ether-linked phosphatidylethanolamines, lysophosphatidylcholines, lysophosphatidylethanolamines, cholesterol esters and ceramides. Our findings reveal dynamic changes in the plasma lipidome during respiratory viral infection, insulin resistance and ageing, suggesting that lipids may have roles in immune homoeostasis and inflammation regulation. Individuals with insulin resistance exhibit disturbed immune homoeostasis, altered associations between lipids and clinical markers, and accelerated changes in specific lipid subclasses during ageing. Our dataset based on longitudinal deep lipidome profiling offers insights into personalized ageing, metabolic health and inflammation, potentially guiding future monitoring and intervention strategies.
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Resistencia a la Insulina , Humanos , Lipidómica , Envejecimiento , Ceramidas , InflamaciónRESUMEN
It is well established that intrauterine infections can pose a threat to pregnancy by gaining access to the placenta and fetus, and clinical studies have strongly linked bacterial infections with preterm labor. Although Chlamydia trachomatis (Ct) can infect the placenta and decidua, little is known about its effects on trophoblast cell immune function. We have demonstrated that Ct infects trophoblast cells to form inclusions and completes the life cycle within these cells by generating infectious elementary bodies. Moreover, infection with Ct leads to differential modulation of the trophoblast cell's production of cytokines and chemokines. Using two human first trimester trophoblast cell lines, Sw.71 and H8, the most striking feature we found was that Ct infection results in a strong induction of IL-1beta secretion and a concomitant reduction in MCP-1 (CCL2) production in both cell lines. In addition, we have found that Ct infection of the trophoblast results in the cleavage and degradation of NF-kappaB p65. These findings suggest that the effect of a Chlamydia infection on trophoblast secretion of chemokines and cytokines involves both activation of innate immune receptors expressed by the trophoblast and virulence factors secreted into the trophoblast by the bacteria. Such altered trophoblast innate immune responses may have a profound impact on the microenvironment of the maternal-fetal interface and this could influence pregnancy outcome.
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Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Citocinas/biosíntesis , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/microbiología , Trofoblastos/inmunología , Trofoblastos/microbiología , Línea Celular Transformada , Quimiocinas/biosíntesis , Infecciones por Chlamydia/metabolismo , Infecciones por Chlamydia/patología , Chlamydia trachomatis/crecimiento & desarrollo , Femenino , Células HeLa , Humanos , Inmunidad Innata , Intercambio Materno-Fetal/inmunología , Enfermedades Placentarias/inmunología , Enfermedades Placentarias/microbiología , Enfermedades Placentarias/patología , Embarazo , Complicaciones Infecciosas del Embarazo/patología , Resultado del Embarazo , Primer Trimestre del Embarazo/inmunología , Primer Trimestre del Embarazo/metabolismo , Trofoblastos/patologíaRESUMEN
Macrophage migration inhibitory factor (MIF) is a leaderless protein that is secreted from cells by a specialized, nonclassical export pathway. The release of MIF nevertheless is regulated and its production in response to different inflammatory, mitogenic, and hormonal stimuli plays an important role in diverse physiologic and pathologic processes. We report herein the identification of the Golgi complex-associated protein p115 as an intracellular binding partner for MIF. MIF interacts with p115 in the cytoplasm and the stimulated secretion of MIF results in the accumulation of both proteins in supernatants, which is consistent with MIF release from cells in conjunction with p115. The depletion of p115 from monocytes/macrophages decreases the release of MIF but not other cytokines following inflammatory stimulation or intracellular bacterial infection. Notably, the small molecule MIF inhibitor 4-iodo-6-phenylpyrimidine inhibits MIF secretion by targeting the interaction between MIF and p115. These data reveal p115 to be a critical intermediary component in the regulated secretion of MIF from monocytes/macrophages.
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Factores Inhibidores de la Migración de Macrófagos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Línea Celular , Citoplasma/metabolismo , Proteínas de la Matriz de Golgi , Humanos , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Macrófagos/metabolismo , Monocitos/metabolismo , Unión Proteica , Pirimidinas/farmacologíaRESUMEN
PURPOSE: To analyze the distribution, associated immune contexture, and clinical significance of human leukocyte antigen (HLA) class-I and HLA class-II subunits in non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using spatially resolved and quantitative multiplexed immunofluorescence we studied the tumor/stromal tissue distribution, cancer cell-specific defects, and clinicopathologic/survival associations of ß2 microglobulin (ß2M), HLA-A, and HLA-B,-C heavy chains, as well as HLA class-II ß chain in >700 immunotherapy-naïve NSCLCs from four independent cohorts. Genomic analysis of HLA genes in NSCLC was performed using two publicly available cohorts. RESULTS: Cancer cell-specific downregulation of HLA markers was identified in 30.4% of cases. ß2M was downregulated in 9.8% (70/714), HLA-A in 9% (65/722), HLA-B,-C in 12.1% (87/719), and HLA class-II in 17.7% (127/717) of evaluable samples. Concurrent downregulation of ß2M, HLA-B,-C, and HLA class-II was commonly identified. Deleterious mutations in HLA genes were detected in <5% of lung malignancies. Tumors with cancer cell-specific ß2M downregulation displayed reduced T cells and increased natural killer (NK)-cell infiltration. Samples with cancer cell HLA-A downregulation displayed modest increase in CD8+ T cells and NK-cell infiltration. Samples with cancer cell-selective HLA-B,-C or HLA class-II downregulation displayed reduced T cells and NK-cell infiltration. There was limited association of the markers with clinicopathologic variables and KRAS/EGFR mutations. Cancer cell-selective downregulation of the HLA subunits was associated with shorter overall survival. CONCLUSIONS: Our results reveal frequent and differential defects in HLA class-I and HLA class-II protein subunit expression in immunotherapy-naïve NSCLCs associated with distinct tumor microenvironment composition and patient survival.
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Alelos , Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase I/genética , Neoplasias Pulmonares/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Biología Computacional/métodos , Análisis Mutacional de ADN , Técnica del Anticuerpo Fluorescente/métodos , Técnica del Anticuerpo Fluorescente/normas , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidad , Mutación , PronósticoRESUMEN
Mutation in the EGFP domain of LDL receptor-related protein 6 (LRP6(R611C)) is associated with hypercholesterolemia and early-onset atherosclerosis, but the mechanism by which it causes disease is not known. Cholesterol uptake was examined in cells from LRP6(+/-) mice and LRP6(R611C) mutation carriers. Splenic B cells of LRP6(+/-) mice have significantly lower LRP6 expression and low-density lipoprotein (LDL) uptake than those of the wild-type littermates. Although similar levels of total LRP6 were found in lymphoblastoid cells (LCLs) of LRP6(R611C) mutation carriers and those of the unaffected family member, LDL uptake was significantly lower in the mutant cells. Mutant and wild-type receptors show similar affinities for apolipoprotein B at neutral pH. LRP6 colocalized with LDL and was coimmunoprecipitated with NPC1 (Niemann-Pick disease type C1), an endocytic regulator of LDL trafficking. However, the cellular localization of LRP6 in the mutant cells shifted from cell surface to late endosomes/lysosomes. Plasma membrane expression levels of LRP6(R611C) was lower compared to wild-type receptor and declined to a greater extent in LDL-rich medium. Further examinations revealed lower efficacy of apolipoprotein B dissociation from LRP6(R611C) compared to wild-type receptor at an acidic pH. These studies identify LRP6 as a receptor for LDL endocytosis and imply that R611C mutation results in reduced LRP6 membrane expression and decreased LDL clearance. Based on our findings, we conclude that the increased affinity of the mutant receptor for LDL in acidic pH leads to their impaired dissociation in late endosomes, which compromises their recycling to the plasma membrane.
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Proteínas Fluorescentes Verdes/genética , Proteínas Relacionadas con Receptor de LDL/genética , Lipoproteínas LDL/metabolismo , Mutación , Células 3T3 , Sustitución de Aminoácidos , Animales , Apolipoproteínas B/metabolismo , Linfocitos B/fisiología , Línea Celular , Membrana Celular/metabolismo , Endosomas/metabolismo , Regulación de la Expresión Génica , Tamización de Portadores Genéticos , Humanos , Inmunohistoquímica , Proteínas Relacionadas con Receptor de LDL/deficiencia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Lisosomas/metabolismo , Tasa de Depuración Metabólica , Ratones , Ratones Noqueados , Microscopía FluorescenteRESUMEN
INTRODUCTION: With expanding indications for programmed death 1 (PD-1) axis inhibitors in non-small cell lung cancer (NSCLC), acquired resistance (AR) to these therapies is increasingly being encountered. We sought to characterize clinical patterns of AR to PD-1 axis inhibitors in patients with advanced NSCLC, and evaluate subsequent outcome and management strategies for such patients. METHODS: Patients with NSCLC who developed AR to PD-1 axis inhibitor therapy initiated between December 2009 and February 2016 at one institution were identified and examined by clinical and radiographic features. AR was defined as progressive disease after initial response by either Response Evaluation Criteria in Solid Tumors v1.1 or immune-related response criteria. RESULTS: Twenty-six patients with AR to PD-1 axis inhibitor therapy were identified and evaluated. Median time to AR was 313 days; the 2-year survival rate from AR was 70% (95% confidence interval: 0.53-0.92). Twenty patients (77%) experienced AR in lymph nodes (LNs), including 11 patients with LN-only progression. Twenty-three (88%) patients had recurrence limited to one (54%) or two (35%) sites of disease. Fourteen patients (54%) continued PD-1 axis inhibitor therapy beyond progression. Three patients were re-challenged with the same PD-1 axis inhibitor after holiday from and progression off therapy, 2 again responded. Fifteen patients (58%) received local therapy to site(s) of AR, 11 continued respective PD-1 axis inhibitor after local therapy. The 2-year survival rate from AR among these 15 patients was 92% (95% confidence interval: 0.77-1). CONCLUSIONS: Acquired resistance to PD-1 axis inhibitors is often limited to one or two sites when local therapy and continuation of PD-1 axis inhibitor therapy can result in prolonged benefit. LN metastases appear to be particularly susceptible sites to AR. When progression of disease following response occurs after holiday from PD-1 axis inhibitor, re-challenge can again lead to tumor regression.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Inmunoterapia/métodos , Neoplasias Pulmonares/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana EdadRESUMEN
Mechanisms of acquired resistance to immune checkpoint inhibitors (ICI) are poorly understood. We leveraged a collection of 14 ICI-resistant lung cancer samples to investigate whether alterations in genes encoding HLA Class I antigen processing and presentation machinery (APM) components or interferon signaling play a role in acquired resistance to PD-1 or PD-L1 antagonistic antibodies. Recurrent mutations or copy-number changes were not detected in our cohort. In one case, we found acquired homozygous loss of B2M that caused lack of cell-surface HLA Class I expression in the tumor and a matched patient-derived xenograft (PDX). Downregulation of B2M was also found in two additional PDXs established from ICI-resistant tumors. CRISPR-mediated knockout of B2m in an immunocompetent lung cancer mouse model conferred resistance to PD-1 blockade in vivo, proving its role in resistance to ICIs. These results indicate that HLA Class I APM disruption can mediate escape from ICIs in lung cancer.Significance: As programmed death 1 axis inhibitors are becoming more established in standard treatment algorithms for diverse malignancies, acquired resistance to these therapies is increasingly being encountered. Here, we found that defective antigen processing and presentation can serve as a mechanism of such resistance in lung cancer. Cancer Discov; 7(12); 1420-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.
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Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias Pulmonares/genética , Humanos , Neoplasias Pulmonares/metabolismo , Transducción de SeñalRESUMEN
MHC class I tetramers are widely used, usually in combination with an antibody to CD8, to detect antigen specific T cells. Some anti-CD8alpha antibodies block the interaction of murine MHC class I tetramers with CD8 T cells, while others such as 53.6.7, enhance. To understand the molecular basis for this effect, we mapped the epitope for the enhancing antibody 53.6.7 and three other blocking antibodies using a panel of murine CD8alpha (Lyt-2) mutants expressed on COS-7 transfectants. Mutations in residues that contact MHC class I affected binding of the blocking antibodies. In contrast, antibody 53.6.7 was affected by a mutation in the residue T81A located on the D-E loop. In the cocrystal of CD8alphaalpha with MHC class I, two different complexes (A and B) were observed, indicating the existence of different CD8 conformations. The T81 residue does not make contact with MHC class I in either complex, however, neighboring residues in the D-E loop make very different contacts in the two different complexes. The most likely explanation for antibody enhancement of tetramer bindings is that binding of 53.6.7 to CD8alphabeta stabilizes a conformation with a higher affinity for interaction with MHC class I and suggests that the CD8 binding site is flexible.
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Anticuerpos Monoclonales/inmunología , Antígenos CD8/inmunología , Mapeo Epitopo , Antígenos de Histocompatibilidad Clase I/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos/genética , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/genética , Especificidad de Anticuerpos/inmunología , Biotinilación , Antígenos CD8/genética , Antígenos CD8/metabolismo , Células COS , Chlorocebus aethiops , Proteínas del Huevo/inmunología , Escherichia coli/genética , Citometría de Flujo , Antígenos H-2/genética , Antígenos H-2/inmunología , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oligopéptidos/inmunología , Ovalbúmina/inmunología , Fragmentos de Péptidos , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Conformación Proteica/efectos de los fármacos , TransfecciónRESUMEN
The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8ß splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8ß, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αß M-4, the frequency of MIP-1ß secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αß M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8ß M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.
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Antígenos CD8/química , Antígenos CD8/metabolismo , Citoplasma/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Western Blotting , Antígenos CD8/genética , Línea Celular , Células Cultivadas , Quimiocina CCL4/metabolismo , Citometría de Flujo , Humanos , Inmunoprecipitación , Mutagénesis Sitio-Dirigida , Estructura Terciaria de ProteínaRESUMEN
Women with antiphospholipid syndrome (APS) are at risk of recurrent pregnancy loss and obstetrical disorders, such as preeclampsia and intrauterine growth restriction (IUGR). Antiphospholipid antibodies (aPL) directly target the placenta by binding beta2-glycoprotein I (ß2GPI) expressed on the trophoblast. We recently demonstrated in human first trimester trophoblast cells that anti-ß2GPI antibodies (Abs) induce the secretion of IL-1ß in a Toll-like receptor 4 (TLR4)-dependent manner. IL-1ß secretion requires processing of pro-IL-1ß and this is mediated by the inflammasome, a complex of Nalp3, apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1. The objective of this study was to determine if aPL induce IL-1ß production in trophoblast via the inflammasome. Using a human first trimester trophoblast cell line, we demonstrated that a mouse anti-ß2GPI mAb and human polyclonal aPL-IgG induce IL-1ß processing and secretion, which was partially blocked upon caspase-1 inhibition. Nalp3 and ASC knockdown also attenuated anti-ß2GPI Ab-induced IL-1ß secretion. Furthermore, aPL stimulated the production of uric acid in a TLR4-dependent manner; and inhibition of uric acid prevented aPL-induced IL-1ß production by the trophoblast. These findings demonstrate that aPL, via TLR4 activation, induce a uric acid response in human trophoblast, which in turn activates the Nalp3/ASC inflammasome leading to IL-1ß processing and secretion. This novel mechanism may account for the inflammation at the maternal-fetal interface, which causes placental dysfunction and increases the risk of adverse pregnancy outcome in patients with APS.
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Anticuerpos Antifosfolípidos/farmacología , Proteínas Portadoras/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Trofoblastos/efectos de los fármacos , Ácido Úrico/inmunología , Animales , Síndrome Antifosfolípido/genética , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/patología , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Caspasa 1/genética , Caspasa 1/inmunología , Línea Celular , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/inmunología , Femenino , Regulación de la Expresión Génica , Humanos , Inflamasomas/genética , Interleucina-1beta/genética , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Embarazo , Primer Trimestre del Embarazo , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Transducción de Señal , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Trofoblastos/citología , Trofoblastos/inmunología , Ácido Úrico/metabolismo , beta 2 Glicoproteína I/genética , beta 2 Glicoproteína I/inmunologíaRESUMEN
PROBLEM: Preeclampsia is associated with hyperuricemia, which correlates with the disease severity. Levels of circulating uric acid increase before the clinical manifestations, suggesting that they may be causally related. Uric acid, or monosodium urate (MSU), activates the Nod-like receptor, Nalp3, leading to inflammasome activation and IL-1ß processing. Because preeclampsia is associated with placental immune/ inflammatory dysregulation, we sought to determine in the trophoblast, the presence of the Nalp3 inflammasome, and the effect of MSU on its activation. METHOD OF STUDY: Isolated first- and third-trimester trophoblasts were assessed for expression of the inflammasome components, Nalp1, Nalp3, and ASC. First-trimester trophoblast cells were incubated with or without MSU, and after which, IL-1ß secretion and processing and caspase-1 activation were determined. RESULTS: Trophoblast cells expressed Nalp1, Nalp3, and ASC under basal conditions. Following incubation with MSU, first-trimester trophoblast IL-1ß secretion was upregulated. This correlated with increased expression levels of active IL-1ß and active caspase-1. ASC knockdown reduced MSU-induced IL-1ß secretion. CONCLUSION: These findings demonstrate that uric acid activates the inflammasome in the trophoblast, leading to IL-1ß production. This may provide a novel mechanism for the induction of inflammation at the maternalfetal interface leading to placental dysfunction and adverse pregnancy outcome, including preeclampsia.
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Inflamasomas/efectos de los fármacos , Interleucina-1beta/metabolismo , Preeclampsia/inmunología , Trofoblastos/efectos de los fármacos , Ácido Úrico/farmacología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Caspasa 1/inmunología , Caspasa 1/metabolismo , Línea Celular , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/metabolismo , Progresión de la Enfermedad , Activación Enzimática/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Hiperuricemia , Inflamasomas/inmunología , Inflamasomas/metabolismo , Relaciones Materno-Fetales , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas NLR , Preeclampsia/sangre , Preeclampsia/fisiopatología , Embarazo , Trofoblastos/metabolismo , Trofoblastos/patología , Ácido Úrico/sangreRESUMEN
The CD8alphabeta heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8(+) T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8(+) T cells. In total CD8(+) T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8(+) T cells. Upon stimulation of CD8(+) T cells, the M-2 variant mRNA levels were elevated 10-20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8beta fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8(+) T cell responses.
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Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Empalme Alternativo , Animales , Complejo CD3/metabolismo , Antígenos CD8/genética , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Humanos , Memoria Inmunológica , Activación de Linfocitos , Lisosomas/inmunología , Lisosomas/metabolismo , Ratones , Persona de Mediana Edad , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/metabolismo , UbiquitinaciónRESUMEN
The intracellular survival of the bacterial pathogen Chlamydia trachomatis depends on protein synthesis by the microbe soon after internalization. Pharmacologic inhibition of bacterial translation inhibits early trafficking of the parasitophorous vacuole (inclusion) to the microtubule-organizing center (MTOC) and promotes its fusion with lysosomes, which is normally blocked by Chlamydia. Depletion of cellular tryptophan pools by gamma interferon-inducible indoleamine-2,3-dioxygenase (IDO) is believed to be the major innate immune mechanism controlling C. trachomatis infection in human cells, an action to which the bacteria can respond by converting into a nonreplicating but highly reactivatable persistent state. However, whether severe IDO-mediated tryptophan starvation can be sufficient to fully arrest the chlamydial life cycle and thereby counteract the onset of persistence is unknown. Here we demonstrate that at low exogenous tryptophan concentrations a substantial fraction of C. trachomatis bacteria fail to traffic to the MTOC or to switch into the conventional persistent state in gamma interferon-induced human cells. The organisms stay scattered in the cell periphery, do not retain infectivity, and display only low transcriptional activity. Importantly, the rate at which these aberrant Chlamydia bacteria become reactivated upon replenishment of cellular tryptophan pools is substantially lower. Thus, severe tryptophan depletion in cells with high IDO activity affects chlamydial development more rigorously than previously described.
Asunto(s)
Chlamydia trachomatis/inmunología , Triptófano/metabolismo , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidad , Citoplasma/microbiología , Expresión Génica/inmunología , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/inmunología , Centro Organizador de los Microtúbulos/microbiología , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Transcripción Genética/inmunología , VirulenciaRESUMEN
In an effective immune response, CD8+ T cell recognition of virally derived Ag, bound to MHC class I, results in killing of infected cells. The CD8alphabeta heterodimer acts as a coreceptor with the TCR, to enhance sensitivity of the T cells to peptide/MHC class I, and is two orders of magnitude more efficient as a coreceptor than the CD8alphaalpha. To understand the important interaction between CD8alphabeta and MHC class I, we created a panel of CD8beta mutants and identified mutations in the CDR1, CDR2, and CDR3 loops that decreased binding to MHC class I tetramers as well as mutations that enhanced binding. We tested the coreceptor function of a subset of reducing and enhancing mutants using a T cell hybridoma and found similar reducing and enhancing effects. CD8beta-enhancing mutants could be useful for immunotherapy by transduction into T cells to enhance T cell responses against weak Ags such as those expressed by tumors. We also addressed the question of the orientation of CD8alphabeta with MHC class I using CD8alpha mutants expressed as a heterodimer with wild-type CD8alpha or CD8beta. The partial rescuing of binding with wild-type CD8beta compared with wild-type CD8alpha is consistent with models in which either the topology of CD8alphaalpha and CD8alphabeta binding to MHC class I is different or CD8alphabeta is capable of binding in both the T cell membrane proximal and distal positions.
Asunto(s)
Antígenos CD8/química , Antígenos CD8/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Mutagénesis , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Antígenos CD8/genética , Células COS , Chlorocebus aethiops , Humanos , Ratones , Datos de Secuencia MolecularRESUMEN
A well functioning lab is a productive lab. Here the guiding principles of good lab management are discussed.
Asunto(s)
Alergia e Inmunología , Laboratorios/organización & administración , Laboratorios/normas , InvestigadoresRESUMEN
The mouse thymic leukemia (TL) Ag is a nonclassical MHC class I molecule that binds with higher affinity to CD8alphaalpha than CD8alphabeta. The interaction of CD8alphaalpha with TL is important for lymphocyte regulation in the intestine. Therefore, we studied the molecular basis for TL Ag binding to CD8alphaalpha. The stronger affinity of the TL Ag for CD8alphaalpha is largely mediated by three amino acids on exposed loops of the conserved alpha3 domain. Mutant classical class I molecules substituted with TL Ag amino acids at these positions mimic the ability to interact with CD8alphaalpha and modulate lymphocyte function. These data indicate that small changes in the alpha3 domain of class I molecules potentially can have profound physiologic consequences.
Asunto(s)
Antígenos CD8/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Antígenos CD8/química , Antígenos CD8/genética , Secuencia Conservada , ADN/genética , Humanos , Hibridomas , Técnicas In Vitro , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Linfocitos T/inmunología , TransfecciónRESUMEN
The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8alphaalpha homodimers with a 10-fold higher affinity than H-2K(b) class I molecules. To understand the molecular basis for this difference, we created a panel of CD8alpha mutants and tested the ability of the CD8alphaalpha homodimers to bind to H-2K(b) tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the alpha3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2K(b) class I sequences are highly conserved in the alpha3 domain of MHC class I, this suggests that CD8 contacts the alpha3 domain of TL and H-2K(b) in a similar manner. In contrast, mutations in residues on the A and B beta strands of CD8 that are involved in contact with beta(2)-microglobulin affected interaction with the H-2K(b) tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2K(b). The unique high affinity binding of TL with CD8alphaalpha is most likely a result of amino acid differences in the alpha3 domain between TL and H-2K(b), particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8alphaalpha-H-2K(b) cocrystal.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antígenos CD8/metabolismo , Antígenos H-2/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglobulina beta-2/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Antígenos CD8/genética , Antígenos CD8/inmunología , Células COS , Regiones Determinantes de Complementariedad/metabolismo , Dimerización , Sueros Inmunes/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Timo/inmunología , Timo/metabolismo , TransfecciónRESUMEN
To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.