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1.
Nat Immunol ; 24(6): 991-1006, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37095377

RESUMO

Germinal center (GC) B cells undergo proliferation at very high rates in a hypoxic microenvironment but the cellular processes driving this are incompletely understood. Here we show that the mitochondria of GC B cells are highly dynamic, with significantly upregulated transcription and translation rates associated with the activity of transcription factor A, mitochondrial (TFAM). TFAM, while also necessary for normal B cell development, is required for entry of activated GC precursor B cells into the germinal center reaction; deletion of Tfam significantly impairs GC formation, function and output. Loss of TFAM in B cells compromises the actin cytoskeleton and impairs cellular motility of GC B cells in response to chemokine signaling, leading to their spatial disorganization. We show that B cell lymphoma substantially increases mitochondrial translation and that deletion of Tfam in B cells is protective against the development of lymphoma in a c-Myc transgenic mouse model. Finally, we show that pharmacological inhibition of mitochondrial transcription and translation inhibits growth of GC-derived human lymphoma cells and induces similar defects in the actin cytoskeleton.


Assuntos
Linfoma de Células B , Linfoma , Camundongos , Humanos , Animais , Linfócitos B/patologia , Centro Germinativo/patologia , Transcrição Gênica , Linfoma de Células B/genética , Linfoma de Células B/patologia , Camundongos Transgênicos , Microambiente Tumoral
2.
Nat Immunol ; 19(8): 838-848, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988091

RESUMO

Foxo transcription factors play an essential role in regulating specialized lymphocyte functions and in maintaining T cell quiescence. Here, we used a system in which Foxo1 transcription-factor activity, which is normally terminated upon cell activation, cannot be silenced, and we show that enforcing Foxo1 activity disrupts homeostasis of CD4 conventional and regulatory T cells. Despite limiting cell metabolism, continued Foxo1 activity is associated with increased activation of the kinase Akt and a cell-intrinsic proliferative advantage; however, survival and cell division are decreased in a competitive setting or growth-factor-limiting conditions. Via control of expression of the transcription factor Myc and the IL-2 receptor ß-chain, termination of Foxo1 signaling couples the increase in cellular cholesterol to biomass accumulation after activation, thereby facilitating immunological synapse formation and mTORC1 activity. These data reveal that Foxo1 regulates the integration of metabolic and mitogenic signals essential for T cell competitive fitness and the coordination of cell growth with cell division.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Proteína Forkhead Box O1/metabolismo , Linfócitos T Reguladores/fisiologia , Animais , Proliferação de Células , Células Cultivadas , Colesterol/metabolismo , Proteína Forkhead Box O1/genética , Perfilação da Expressão Gênica , Homeostase , Sinapses Imunológicas/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais
3.
PLoS Biol ; 20(7): e3001680, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35797414

RESUMO

Early career researchers (ECRs) are important stakeholders leading efforts to catalyze systemic change in research culture and practice. Here, we summarize the outputs from a virtual unconventional conference (unconference), which brought together 54 invited experts from 20 countries with extensive experience in ECR initiatives designed to improve the culture and practice of science. Together, we drafted 2 sets of recommendations for (1) ECRs directly involved in initiatives or activities to change research culture and practice; and (2) stakeholders who wish to support ECRs in these efforts. Importantly, these points apply to ECRs working to promote change on a systemic level, not only those improving aspects of their own work. In both sets of recommendations, we underline the importance of incentivizing and providing time and resources for systems-level science improvement activities, including ECRs in organizational decision-making processes, and working to dismantle structural barriers to participation for marginalized groups. We further highlight obstacles that ECRs face when working to promote reform, as well as proposed solutions and examples of current best practices. The abstract and recommendations for stakeholders are available in Dutch, German, Greek (abstract only), Italian, Japanese, Polish, Portuguese, Spanish, and Serbian.


Assuntos
Pesquisadores , Relatório de Pesquisa , Humanos , Poder Psicológico
4.
Cell Mol Life Sci ; 81(1): 350, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-39141086

RESUMO

Heparan sulfate (HS) proteoglycans are important regulators of cellular responses to soluble mediators such as chemokines, cytokines and growth factors. We profiled changes in expression of genes encoding HS core proteins, biosynthesis enzymes and modifiers during macrophage polarisation, and found that the most highly regulated gene was Sulf2, an extracellular HS 6-O-sulfatase that was markedly downregulated in response to pro-inflammatory stimuli. We then generated Sulf2+/- bone marrow chimeric mice and examined inflammatory responses in antigen-induced arthritis, as a model of rheumatoid arthritis. Resolution of inflammation was impaired in myeloid Sulf2+/- chimeras, with elevated joint swelling and increased abundance of pro-arthritic Th17 cells in synovial tissue. Transcriptomic and in vitro analyses indicated that Sulf2 deficiency increased type I interferon signaling in bone marrow-derived macrophages, leading to elevated expression of the Th17-inducing cytokine IL6. This establishes that dynamic remodeling of HS by Sulf2 limits type I interferon signaling in macrophages, and so protects against Th17-driven pathology.


Assuntos
Macrófagos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Células Th17 , Animais , Células Th17/imunologia , Células Th17/metabolismo , Camundongos , Macrófagos/metabolismo , Macrófagos/imunologia , Sulfatases/metabolismo , Sulfatases/genética , Sulfotransferases/metabolismo , Sulfotransferases/genética , Células Mieloides/metabolismo , Células Mieloides/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Experimental/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos Knockout , Interleucina-6/metabolismo , Interleucina-6/genética , Heparitina Sulfato/metabolismo
5.
Nat Methods ; 18(9): 1013-1026, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34446922

RESUMO

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.


Assuntos
Vesículas Extracelulares , Microscopia/métodos , Animais , Corantes/química , Epitopos , Vesículas Extracelulares/química , Vesículas Extracelulares/patologia , Vesículas Extracelulares/fisiologia , Corantes Fluorescentes/química , Humanos
6.
J Cell Sci ; 134(16)2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34423835

RESUMO

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here, we investigated the potential role of Bardet-Biedl syndrome 1 protein (BBS1), an essential core component of the BBS complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrated that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH1 (also known as WASHC1), a process that we demonstrated to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that functions upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling. This article has an associated First Person interview with the first author of the paper.


Assuntos
Síndrome de Bardet-Biedl , Cílios , Síndrome de Bardet-Biedl/genética , Polaridade Celular , Endossomos , Humanos , Proteínas Associadas aos Microtúbulos/genética , Sinapses , Linfócitos T
7.
PLoS Pathog ; 17(5): e1009584, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33970974

RESUMO

[This corrects the article DOI: 10.1371/journal.ppat.1008359.].

8.
PLoS Pathog ; 16(2): e1008359, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32084246

RESUMO

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.


Assuntos
HIV-1/metabolismo , Fusão de Membrana/fisiologia , Proteínas do Envelope Viral/metabolismo , Linfócitos T CD4-Positivos , Fusão Celular , Membrana Celular/metabolismo , Glicólise/fisiologia , HIV-1/fisiologia , Humanos , Fusão de Membrana/genética , Cultura Primária de Células , Análise de Célula Única , Vírion/metabolismo , Internalização do Vírus
9.
J Biol Chem ; 289(1): 520-8, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24235148

RESUMO

Mouse dendritic cells (DCs) can rapidly extend their Class II MHC-positive late endosomal compartments into tubular structures, induced by Toll-like receptor (TLR) triggering. Within antigen-presenting DCs, tubular endosomes polarize toward antigen-specific CD4(+) T cells, which are considered beneficial for their activation. Here we describe that also in human DCs, TLR triggering induces tubular late endosomes, labeled by fluorescent LDL. TLR triggering was insufficient for induced tubulation of transferrin-positive endosomal recycling compartments (ERCs) in human monocyte-derived DCs. We studied endosomal remodeling in human DCs in co-cultures of DCs with CD8(+) T cells. Tubulation of ERCs within human DCs requires antigen-specific CD8(+) T cell interaction. Tubular remodeling of endosomes occurs within 30 min of T cell contact and involves ligation of HLA-A2 and ICAM-1 by T cell-expressed T cell receptor and LFA-1, respectively. Disintegration of microtubules or inhibition of endosomal recycling abolished tubular ERCs, which coincided with reduced antigen-dependent CD8(+) T cell activation. Based on these data, we propose that remodeling of transferrin-positive ERCs in human DCs involves both innate and T cell-derived signals.


Assuntos
Apresentação de Antígeno/fisiologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Endossomos/imunologia , Monócitos/imunologia , Receptores Toll-Like/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Feminino , Antígeno HLA-A2/imunologia , Humanos , Imunidade Celular/fisiologia , Imunidade Inata/fisiologia , Molécula 1 de Adesão Intercelular/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Masculino , Camundongos , Monócitos/citologia , Transdução de Sinais/fisiologia
10.
J Biol Chem ; 289(8): 5000-12, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24356959

RESUMO

Most hereditary periodic fever syndromes are mediated by deregulated IL-1ß secretion. The generation of mature IL-1ß requires two signals: one that induces synthesis of inflammasome components and substrates and a second that activates inflammasomes. The mechanisms that mediate autoinflammation in mevalonate kinase deficiency, a periodic fever disease characterized by a block in isoprenoid biosynthesis, are poorly understood. In studying the effects of isoprenoid shortage on IL-1 ß generation, we identified a new inflammasome activation signal that originates from defects in autophagy. We find that hypersecretion of IL-1ß and IL-18 requires reactive oxygen species and is associated with an oxidized redox status of monocytes but not lymphocytes. IL-1ß hypersecretion by monocytes involves decreased mitochondrial stability, release of mitochondrial content into the cytosol and attenuated autophagosomal degradation. Defective autophagy, as established by ATG7 knockdown, results in prolonged cytosolic retention of damaged mitochondria and increased IL-1ß secretion. Finally, activation of autophagy in healthy but not mevalonate kinase deficiency patient cells reduces IL-1ß secretion. Together, these results indicate that defective autophagy can prime monocytes for mitochondria-mediated NLRP3 inflammasome activation, thereby contributing to hypersecretion of IL-1ß in mevalonate kinase deficiency.


Assuntos
Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/patologia , Interleucina-1beta/metabolismo , Mitocôndrias/metabolismo , Monócitos/metabolismo , Monócitos/patologia , Adolescente , Autofagia , Linhagem Celular , Criança , Pré-Escolar , Citosol/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Inflamassomos/metabolismo , Potencial da Membrana Mitocondrial , Deficiência de Mevalonato Quinase/metabolismo , Deficiência de Mevalonato Quinase/patologia , Modelos Biológicos , Oxirredução , Terpenos/metabolismo
11.
Blood ; 120(26): 5163-72, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23093620

RESUMO

The reactivation of human cytomegalovirus (HCMV) poses a serious health threat to immune compromised individuals. As a treatment strategy, dendritic cell (DC) vaccination trials are ongoing. Recent work suggests that BDCA-3(+) (CD141(+)) subset DCs may be particularly effective in DC vaccination trials. BDCA-3(+) DCs had however been mostly characterized for their ability to cross-present antigen from necrotic cells. We here describe our study of human BDCA-3(+) DCs in elicitation of HCMV-specific CD8(+) T-cell clones. We show that Fcgamma-receptor (FcγR) antigen targeting facilitates antigen cross-presentation in several DC subsets, including BDCA-3(+) DCs. FcγR antigen targeting stimulates antigen uptake by BDCA-1(+) rather than BDCA-3(+) DCs. Conversely, BDCA-3(+) DCs and not BDCA-1(+) DCs show improved cross-presentation by FcγR targeting, as measured by induced release of IFNγ and TNF by antigen-specific CD8(+) T cells. FcγR-facilitated cross-presentation requires antigen processing in both an acidic endosomal compartment and by the proteasome, and did not induce substantial DC maturation. FcγRII is the most abundantly expressed FcγR on both BDCA-1(+) and BDCA-3(+) DCs. Furthermore we show that BDCA-3(+) DCs express relatively more stimulatory FcγRIIa than inhibitory FcγRIIb in comparison with BDCA-1(+) DCs. These studies support the exploration of FcγR antigen targeting to BDCA-3(+) DCs for human vaccination purposes.


Assuntos
Antígenos Virais/farmacologia , Sangue/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Tecido Linfoide/imunologia , Receptores de IgG/imunologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/fisiologia , Antígenos de Superfície/metabolismo , Antígenos Virais/imunologia , Antígenos Virais/uso terapêutico , Sangue/metabolismo , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Apresentação Cruzada/efeitos dos fármacos , Apresentação Cruzada/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Sinergismo Farmacológico , Humanos , Imunoterapia Ativa/métodos , Imunoterapia Adotiva/métodos , Tecido Linfoide/metabolismo , Fosfoproteínas/imunologia , Fosfoproteínas/farmacologia , Receptores de IgG/antagonistas & inibidores , Receptores de IgG/fisiologia , Trombomodulina , Proteínas da Matriz Viral/imunologia , Proteínas da Matriz Viral/farmacologia
12.
Nat Commun ; 15(1): 4988, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862534

RESUMO

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA+ CAFs can form immunological synapses with Foxp3+ regulatory T cells (Tregs) in tumours. Notably, α-SMA+ CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA+ CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA+ CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA+ CAFs and Tregs in an autophagy-dependent manner.


Assuntos
Autofagia , Fibroblastos Associados a Câncer , Sinapses Imunológicas , Linfócitos T Reguladores , Microambiente Tumoral , Linfócitos T Reguladores/imunologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/patologia , Humanos , Sinapses Imunológicas/metabolismo , Sinapses Imunológicas/imunologia , Animais , Microambiente Tumoral/imunologia , Camundongos , Autofagia/imunologia , Actinas/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Neoplasias/imunologia , Neoplasias/genética , Neoplasias/patologia , Camundongos Endogâmicos C57BL , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Feminino , Camundongos Knockout
13.
J Allergy Clin Immunol ; 129(3): 755-761.e7, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22130422

RESUMO

BACKGROUND: B cells of patients with common variable immunodeficiency (CVID) disorders display impairment in production of immunoglobulin class-switched antibodies, which is possibly contributed to by defects in early B-cell activation. On resting B cells, B-cell receptors (BCRs) are organized in oligomers that are signaling inactive. Their triggering by cognate antigen causes the lateral reorganization of BCRs and associated proteins into signalosomes, resulting in BCR-activated calcium entry. In resting cells the B-cell surface antigen CD20 is associated with the BCR but dissociates on signalosome formation. OBJECTIVE: We sought to determine whether CD20 dissociation from the BCR during early B-cell activation might contribute to the development of CVID disorders. METHODS: We evaluated BCR signalosome formation, internalization, and signaling in primary B cells of pediatric patients with CVID disorders and healthy control subjects. RESULTS: In many pediatric patients with CVID disorders, B cells exhibit significant deficits in BCR triggering-mediated calcium entry in the cytosol, which correlates with impaired plasmablast differentiation in vitro. These alterations did not originate from upregulation of CD22 or defects in calcium channels and did not involve gene mutations in phospholipase Cγ2 or Bruton tyrosine kinase. Instead, B cells from patients with CVID disorders exhibited reduced BCR dissociation from CD20. BCR or CD20 cross-linking induced less BCR internalization, and antibody-mediated CD20 triggering elicited less BCR downstream signaling, as measured based on secondary fluxes. CONCLUSIONS: We propose that CD20 dissociation from the BCR signalosome is pivotal to BCR-mediated calcium mobilization in the cytosol. Defects in CD20/BCR signalosome conformation might predispose to the spectrum of CVID disorders.


Assuntos
Antígenos CD20/metabolismo , Linfócitos B/metabolismo , Sinalização do Cálcio , Imunodeficiência de Variável Comum/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Adolescente , Antígenos CD20/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Diferenciação Celular , Separação Celular , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária , Masculino , Ligação Proteica , Agregação de Receptores , Receptores de Antígenos de Linfócitos B/imunologia
14.
J Exp Med ; 220(8)2023 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-37115585

RESUMO

The first immune-activating changes within joint resident cells that lead to pathogenic leukocyte recruitment during articular inflammation remain largely unknown. In this study, we employ state-of-the-art confocal microscopy and image analysis in a systemic, whole-organ, and quantitative way to present evidence that synovial inflammation begins with the activation of lining macrophages. We show that lining, but not sublining macrophages phagocytose immune complexes containing the model antigen. Using the antigen-induced arthritis (AIA) model, we demonstrate that on recognition of antigen-antibody complexes, lining macrophages undergo significant activation, which is dependent on interferon regulatory factor 5 (IRF5), and produce chemokines, most notably CXCL1. Consequently, at the onset of inflammation, neutrophils are preferentially recruited in the vicinity of antigen-laden macrophages in the synovial lining niche. As inflammation progresses, neutrophils disperse across the whole synovium and form swarms in synovial sublining during resolution. Our study alters the paradigm of lining macrophages as immunosuppressive cells to important instigators of synovial inflammation.


Assuntos
Artrite , Humanos , Infiltração de Neutrófilos , Artrite/patologia , Macrófagos , Membrana Sinovial/patologia , Inflamação/patologia , Antígenos
15.
Crit Rev Immunol ; 31(2): 85-98, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21542788

RESUMO

Common variable immunodeficiency (CVID) is the most frequently diagnosed symptomatic primary immunodeficiency. CVID develops as a consequence of absence or malfunction of proteins involved with immunoglobulin production by plasma and memory B-cells. The last decade has brought us clarification of several genetic predispositions to the development of CVID. Despite considerable effort, however, for eighty-five percent of CVID patients, disease etiology remains undefined. We propose that in subsets of patients, CVID may involve defective assembly of protein complexes, which is crucial for example for B cell activation upon antigen triggering of the B cell receptor/co-receptor complex. Such defective protein-protein interactions may not be uncovered by standard gene sequencing methods, and may involve epigenetic or post-transcriptional regulation. In this review, we summarize recent developments in CVID research and propose additional approaches to the clarification of etiology of CVID patient groups, necessary for development of tailored treatment options.


Assuntos
Linfócitos B/metabolismo , Imunodeficiência de Variável Comum/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Epigênese Genética , Humanos , Memória Imunológica , Mutação/genética , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais
16.
Oxf Open Immunol ; 2(1): iqab016, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35593707

RESUMO

Destabilization of balanced immune cell numbers and frequencies is a common feature of viral infections. This occurs due to, and further enhances, viral immune evasion and survival. Since the discovery of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which manifests in coronavirus disease 2019 (COVID-19), a great number of studies have described the association between this virus and pathologically increased or decreased immune cell counts. In this review, we consider the absolute and relative changes to innate and adaptive immune cell numbers, in COVID-19. In severe disease particularly, neutrophils are increased, which can lead to inflammation and tissue damage. Dysregulation of other granulocytes, basophils and eosinophils represents an unusual COVID-19 phenomenon. Contrastingly, the impact on the different types of monocytes leans more strongly to an altered phenotype, e.g. HLA-DR expression, rather than numerical changes. However, it is the adaptive immune response that bears the most profound impact of SARS-CoV-2 infection. T cell lymphopenia correlates with increased risk of intensive care unit admission and death; therefore, this parameter is particularly important for clinical decision-making. Mild and severe diseases differ in the rate of immune cell counts returning to normal levels post disease. Tracking the recovery trajectories of various immune cell counts may also have implications for long-term COVID-19 monitoring. This review represents a snapshot of our current knowledge, showing that much has been achieved in a short period of time. Alterations in counts of distinct immune cells represent an accessible metric to inform patient care decisions or predict disease outcomes.

17.
Front Cell Dev Biol ; 9: 673446, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34368126

RESUMO

The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a- and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1 high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1 high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for fine-scale imaging studies.

18.
Oxf Open Immunol ; 2(1): iqaa007, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33575657

RESUMO

COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients' long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation.

19.
Elife ; 82019 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-31469364

RESUMO

Planar supported lipid bilayers (PSLB) presenting T cell receptor (TCR) ligands and ICAM-1 induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but incorporation of other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. We demonstrate selective enrichment of CD40L and ICOS in SE in response to addition of CD40 and ICOSL, respectively, to SLB presenting TCR ligands and ICAM-1. SE are enriched in tetraspanins, BST-2, TCR signaling and ESCRT proteins. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2. CD40L+ SE retain the capacity to induce dendritic cell maturation and cytokine production.


Assuntos
Ligante de CD40/análise , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Receptores de Antígenos/análise , Linfócitos T Auxiliares-Indutores/metabolismo , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Proteoma/análise
20.
Nat Commun ; 9(1): 1597, 2018 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-29686427

RESUMO

Endocytosis of surface receptors and their polarized recycling back to the plasma membrane are central to many cellular processes, such as cell migration, cytokinesis, basolateral polarity of epithelial cells and T cell activation. Little is known about the mechanisms that control the organization of recycling endosomes and how they connect to receptor endocytosis. Here, we follow the endocytic journey of the T cell receptor (TCR), from internalization at the plasma membrane to recycling back to the immunological synapse. We show that TCR triggering leads to its rapid uptake through a clathrin-independent pathway. Immediately after internalization, TCR is incorporated into a mobile and long-lived endocytic network demarked by the membrane-organizing proteins flotillins. Although flotillins are not required for TCR internalization, they are necessary for its recycling to the immunological synapse. We further show that flotillins are essential for T cell activation, supporting TCR nanoscale organization and signaling.


Assuntos
Endocitose/fisiologia , Ativação Linfocitária/fisiologia , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Transdução de Sinais/imunologia
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