Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.461
Filtrar
1.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445351

RESUMO

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.


Assuntos
Diagnóstico por Imagem/métodos , Proteínas/metabolismo , Análise de Célula Única/métodos , Anticorpos/metabolismo , Comunicação Celular , Imunofluorescência/métodos , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Formaldeído/química , Peroxidase do Rábano Silvestre/análise , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Técnicas Imunoenzimáticas/métodos , Tonsila Palatina/química , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Inclusão em Parafina , Proteínas/análise , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
2.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445493

RESUMO

Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163+CD14+ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-γ+ CD8 T cell response in C-strain vaccinated pigs, but not in pigs that received Alfort-187. Moreover, the frequency of CSFV-specific IFN-γ+ CD8 T cells was inversely correlated to the viral load in the tonsils of individual animals. Accordingly, we hypothesise that the activation of cDC1 is key in initiating local CSFV-specific CD8 T cell responses which curtail early virus replication and dissemination.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Tonsila Palatina/imunologia , Vacinas Virais/administração & dosagem , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Peste Suína Clássica/imunologia , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/fisiologia , Células Dendríticas/metabolismo , Interferon gama/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Células Mieloides/metabolismo , Tonsila Palatina/citologia , Tonsila Palatina/virologia , Receptores de Superfície Celular/metabolismo , Suínos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Carga Viral , Vacinas Virais/imunologia
3.
Immunity ; 54(8): 1788-1806.e7, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34166622

RESUMO

Lymphoid stromal cells (LSCs) are essential organizers of immune responses. We analyzed tonsillar tissue by combining flow cytometry, in situ imaging, RNA sequencing, and functional assays, defining three distinct human LSC subsets. The integrin CD49a designated perivascular stromal cells exhibiting features of local committed LSC precursors and segregated cytokine and chemokine-producing fibroblastic reticular cells (FRCs) supporting B and T cell survival. The follicular dendritic cell transcriptional profile reflected active responses to B cell and non-B cell stimuli. We therefore examined the effect of B cell stimuli on LSCs in follicular lymphoma (FL). FL B cells interacted primarily with CD49a+ FRCs. Transcriptional analyses revealed LSC reprogramming in situ downstream of the cytokines tumor necrosis factor (TNF) and transforming growth factor ß (TGF-ß), including increased expression of the chemokines CCL19 and CCL21. Our findings define human LSC populations in healthy tissue and reveal bidirectional crosstalk between LSCs and malignant B cells that may present a targetable axis in lymphoma.


Assuntos
Linfócitos B/imunologia , Células Dendríticas/imunologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Tonsila Palatina/imunologia , Células Estromais/imunologia , Células Cultivadas , Quimiocina CCL19/metabolismo , Quimiocina CCL21/metabolismo , Humanos , Integrina alfa1/metabolismo , Tonsila Palatina/citologia , Transdução de Sinais/imunologia , Células Estromais/citologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
4.
Nat Immunol ; 22(6): 757-768, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34031614

RESUMO

Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell-dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR-CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.


Assuntos
Linfócitos B/imunologia , Ativação do Complemento , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Centro Germinativo/imunologia , Animais , Animais Geneticamente Modificados , Linfócitos B/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Linhagem Celular Tumoral , Hematopoiese Clonal/imunologia , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Tonsila Palatina/citologia , Tonsila Palatina/patologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/metabolismo
5.
Methods Mol Biol ; 2285: 173-189, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928553

RESUMO

Antibody responses deeply rely on the interaction of antigen-primed B cells and CD4 helper T cells in the context of germinal center reactions, through signals provided by costimulatory molecules and cytokines. B-cell proliferation and differentiation in antibody-secreting plasma cells are processes that critically depend on the helper function of a specific CD4 T-cell subset, known as follicular helper T cells (Tfh). Here, we describe a method that mimics in vitro the cross talk between Tfh and B cells occurring in the germinal center. The procedure is based on setting up a coculture system with B cells and Tfh isolated from blood of healthy donors, or tonsils removed upon surgical intervention, in order to recapitulate in vitro the Tfh-dependent mechanisms leading to B cells' activation, proliferation, and differentiation.


Assuntos
Linfócitos B/imunologia , Comunicação Celular , Separação Celular , Citometria de Fluxo , Células T Auxiliares Foliculares/imunologia , Linfócitos B/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Citocinas/metabolismo , ELISPOT , Centro Germinativo/citologia , Humanos , Ativação Linfocitária , Tonsila Palatina/citologia , Fenótipo , Projetos de Pesquisa , Células T Auxiliares Foliculares/metabolismo , Fluxo de Trabalho
6.
Carbohydr Polym ; 264: 117992, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33910730

RESUMO

Biofunctional polymers have been widely used to enhance the proliferation and functionality of stem cells. Here, we report the development of a new biofunctional polymer, octanoyl glycol chitosan (OGC), and demonstrate its effects on the cell cycle and stem cell function using tonsil-derived mesenchymal stem cells (TMSCs). OGC treatment (100 µg/mL) significantly increased the proliferation of TMSCs, which could be attributed to cyclin D1 up-regulation in the G1 phase of the cell cycle. Additionally, OGC enhanced the ability of TMSCs to differentiate into adipocytes, chondrocytes, and osteoblasts. Taken together, this new biofunctional polymer, OGC, can promote stemness and osteogenesis, as well as induce stem cell proliferation by enhancing the intracellular metabolic rate and regulating the cell cycle. Thus, in the future, OGC could be a potential therapeutic additive for improving stem cell function.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quitosana/farmacologia , Células-Tronco Mesenquimais/metabolismo , Tonsila Palatina/citologia , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/química , Ciclina D1/metabolismo , Humanos , Osteogênese/efeitos dos fármacos , Consumo de Oxigênio , Tonsila Palatina/metabolismo , Polímeros/química , Polímeros/farmacologia , Engenharia Tecidual/métodos , Cicatrização/efeitos dos fármacos
7.
Front Immunol ; 12: 637832, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33859640

RESUMO

Human B-cell differentiation has been extensively investigated on genomic and transcriptomic grounds; however, no studies have accomplished so far detailed analysis of antigen-dependent maturation-associated human B-cell populations from a proteomic perspective. Here, we investigate for the first time the quantitative proteomic profiles of B-cells undergoing antigen-dependent maturation using a label-free LC-MS/MS approach applied on 5 purified B-cell subpopulations (naive, centroblasts, centrocytes, memory and plasma B-cells) from human tonsils (data are available via ProteomeXchange with identifier PXD006191). Our results revealed that the actual differences among these B-cell subpopulations are a combination of expression of a few maturation stage-specific proteins within each B-cell subset and maturation-associated changes in relative protein expression levels, which are related with metabolic regulation. The considerable overlap of the proteome of the 5 studied B-cell subsets strengthens the key role of the regulation of the stoichiometry of molecules associated with metabolic regulation and programming, among other signaling cascades (such as antigen recognition and presentation and cell survival) crucial for the transition between each B-cell maturation stage.


Assuntos
Antígenos/imunologia , Subpopulações de Linfócitos B/citologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Transdução de Sinais/imunologia , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Ativação Linfocitária/imunologia , Masculino , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Proteoma/genética , Transcriptoma/genética , Adulto Jovem
8.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925530

RESUMO

Regulatory B (Breg) cells are endowed with immune suppressive functions. Various human and murine Breg subtypes have been reported. While interleukin (IL)-10 intracellular staining remains the most reliable way to identify Breg cells, this technique hinders further essential functional studies. Recent findings suggest that CD9 is an effective surface marker of murine IL-10 competent Breg cells. However, the stability of CD9 and its relevance as a unique marker for human Breg cells, which have been widely characterized as CD24hiCD38hi, have not been investigated. Here, we demonstrate that CD9 expression is sensitive to in vitro B cell stimulations. CD9 expression could either be re-expressed or downregulated in purified CD9-negative B cells and CD9-positive B cells, respectively. We found no significant differences in the Breg differentiation capacity of the CD9-negative and CD9-positive B cells. Furthermore, CD9-positive B cells co-express CD40 and CD86, suggesting their nature as B cell activation or co-stimulatory molecules, rather than regulatory ones. Therefore, we report the relatively unstable CD9 as a distinct surface molecule, indicating the need for further research for a more reliable marker to purify human Breg cells.


Assuntos
ADP-Ribosil Ciclase 1/imunologia , Linfócitos B Reguladores/imunologia , Antígeno CD24/imunologia , Glicoproteínas de Membrana/imunologia , Tetraspanina 29/imunologia , Tecido Adiposo/citologia , Biomarcadores/análise , Diferenciação Celular/imunologia , Criança , Humanos , Interleucina-10/imunologia , Ativação Linfocitária , Células-Tronco Mesenquimais/imunologia , Tonsila Palatina/citologia , Regulação para Cima
9.
Nature ; 592(7852): 133-137, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33597749

RESUMO

Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (TFH) cells and, consequently, receive more contact-dependent T cell help1. As these GC B cells and TFH cells do not maintain long-lasting contacts in the chaotic GC environment2-4, it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on TFH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of TFH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from TFH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.


Assuntos
Quimiocina CCL22/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Células Cultivadas , Quimiocina CCL17/deficiência , Quimiocina CCL17/genética , Quimiocina CCL22/deficiência , Quimiocina CCL22/genética , Feminino , Humanos , Masculino , Camundongos , Tonsila Palatina/citologia , Receptores CCR4/deficiência , Receptores CCR4/genética , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Regulação para Cima
10.
BMC Vet Res ; 17(1): 88, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33618723

RESUMO

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5. RESULTS: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level. CONCLUSIONS: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition.


Assuntos
Tonsila Palatina/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Animais , Genótipo , Imunidade Inata/genética , Tonsila Palatina/citologia , Tonsila Palatina/metabolismo , Tonsila Palatina/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/isolamento & purificação , Sus scrofa , Suínos , Transcriptoma , Carga Viral/veterinária , Viremia/veterinária , Viremia/virologia
11.
J Immunol Methods ; 490: 112953, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33359172

RESUMO

The sphingosine 1-phosphate receptor type 1 (S1PR1) has several important functions, including stabilizing endothelial barrier and maintaining lymphocyte circulation. These functions are critically dependent on the regulation of S1PR1 cell surface expression. Currently available antibodies against human S1PR1 are not able to pick up cell surface expression on living cells by flow cytometry due to intracellular epitopes or unspecific binding. Here we describe the generation of a mouse monoclonal antibody specific for the N-terminal region of human S1PR1. It has an immunoglobulin M (IgM) kappa isotype and detects cell surface expression of recombinant human S1PR1 on overexpressing cells. Due to unspecific intracellular cell staining, it cannot be used for staining of dead cells and tissue slides or in microscopic analyses. It is also not suitable for Western blot analysis and immunoprecipitation. However, the antibody can stain for endogenous S1PR1 on human endothelial cell lines and primary human umbilical vein endothelial cells (HUVEC). Incubation of these cells with various S1PR1 agonists revealed potent S1PR1 internalization, which was not the case with the specific antagonist W146. Surprisingly, human T and B cells isolated from blood and palatine tonsils did not show specific staining, demonstrating significantly lower endogenous S1PR1 surface expression on lymphocytes than on endothelial cells.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/metabolismo , Linfoma de Burkitt/metabolismo , Células Endoteliais/metabolismo , Imunoglobulina M/isolamento & purificação , Receptores de Esfingosina-1-Fosfato/metabolismo , Linfócitos T/metabolismo , Anilidas/farmacologia , Animais , Linfoma de Burkitt/patologia , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Especificidade de Órgãos , Organofosfonatos/farmacologia , Tonsila Palatina/citologia , Receptores de Esfingosina-1-Fosfato/imunologia
12.
Front Immunol ; 11: 565458, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33133077

RESUMO

Complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) of myeloid cells are known for long to participate in actin linked functions like phagocytosis, adhesion, and migration. The expression and role of these two ß2-integrins however, in human B lymphocytes have only scarcely been studied so far, although it has been shown recently that CD11c+ B cells are mainly memory cells. In our systematic study we investigated B cells isolated from tonsils and peripheral blood of healthy donors. We found, that while only 5% of resting tonsillar B cells expressed CD11c, their number increased up to 26% after 3 days of BCR stimulation. Lower, but still remarkable percentage of B lymphocytes were positive for CD11c after stimulation via TLR9 alone or via TLR9 and BCR simultaneously. At the same time, we detected no significant expression of CD11b on resting or activated tonsillar B cells. Blood B lymphocytes showed a similar expression pattern of both ß2-integrins. We demonstrated that CD11c molecules appearing on the surface of B cells are newly synthesized, reaching the number of 9,500 per activated B cell. We found that CR4 expressing B cells belong to the memory pool and the increase of CD11c expression on tonsillar B cells upon BCR mediated activation occurs parallel with class switching. Analysis of the function of CD11c revealed, that this ß2-integrin contributes to the adhesion and migration of activated B lymphocytes. We also demonstrated that the CR4 mediated adhesion promotes the proliferation of the BCR activated cells. Our studies are the first to demonstrate that CD11c expressed on BCR-activated human B cells are not only passive markers but functional drivers of memory B cell responses.


Assuntos
Linfócitos B/imunologia , Antígeno CD11c/metabolismo , Antígenos CD18/metabolismo , Adesão Celular/imunologia , Movimento Celular/imunologia , Proliferação de Células/fisiologia , Memória Imunológica , Ativação Linfocitária , Doadores de Sangue , Células Cultivadas , Humanos , Tonsila Palatina/citologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptor Toll-Like 9/metabolismo
13.
J Immunol ; 205(10): 2679-2693, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-33020148

RESUMO

Human NK cells develop in tonsils through discrete NK cell developmental intermediates (NKDIs), yet the mechanistic regulation of this process is unclear. We demonstrate that Notch activation in human tonsil-derived stage 3 (CD34-CD117+CD94-NKp80-) and 4A (CD34-CD117+/-CD94+NKp80-) NKDIs promoted non-NK innate lymphoid cell differentiation at the expense of NK cell differentiation. In contrast, stage 4B (CD34-CD117+/-CD94+NKp80+) NKDIs were NK cell lineage committed despite Notch activation. Interestingly, whereas NK cell functional maturation from stage 3 and 4A NKDIs was independent of Notch activation, the latter was required for high NKp80 expression and a stage 4B-like phenotype by the NKDI-derived NK cells. The Notch-dependent effects required simultaneous engagement with OP9 stromal cells and were also stage-specific, with NOTCH1 and NOTCH2 receptors regulating stage 3 NKDIs and NOTCH1 primarily regulating stage 4A NKDIs. These data establish stage-specific and stromal-dependent roles for Notch in regulating human NK cell developmental plasticity and maturation.


Assuntos
Diferenciação Celular/imunologia , Células Matadoras Naturais/fisiologia , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Plasticidade Celular/imunologia , Células Cultivadas , Humanos , Imunidade Inata , Lectinas Tipo C/metabolismo , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Cultura Primária de Células , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais/imunologia
15.
Vet Microbiol ; 246: 108732, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32605752

RESUMO

Campylobacter jejuni colonizes the chicken gut at a high density without causing disease. However, consumption of poultry products contaminated with this bacterium causes gastroenteritis in humans. Therefore, it is critically important to reduce the Campylobacter burden in poultry products to prevent transmission to humans. Evidence indicates that enhancing intestinal mucosal immune responses is of paramount importance for preventing or reducing Campylobacter colonization in chickens. In view of this, the present study was undertaken to evaluate host responses to different C. jejuni-derived ligands, including lipooligosaccharide (LOS), outer membrane proteins (OMPs), and genomic DNA, with the ultimate goal of identifying a ligand with potent immunostimulatory capacity to serve as a mucosal vaccine adjuvant against enteric infections in chickens. The results revealed that C. jejuni pathogen-associated molecular patterns (PAMPs) varied in their ability to induce the expression of cytokines and chemokines in chicken macrophages and cecal tonsil mononuclear cells and nitric oxide production in macrophages. In addition, C. jejuni OMPs demonstrated superior activity over LOS and DNA ligands in eliciting cytokine expression associated with T helper (Th)1 and Th2 responses (interferon [IFN]-γ and interleukin [IL]-13, respectively), in addition to expression of pro-inflammatory cytokines (IL-1ß), chemokine (CXCLi2), and regulatory cytokines (IL-10 and TGFß1/4) in cecal tonsil cells. Importantly, in addition to their ability to induce innate responses, OMPs could also function as antigens to elicit C. jejuni-specific antibody responses and thereby confer dual protection against C. jejuni infection. Further studies are required to assess the protective efficacy of C. jejuni OMPs against C. jejuni infection in chickens.


Assuntos
Campylobacter/imunologia , Quimiocinas/genética , Citocinas/genética , Imunidade nas Mucosas , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Adjuvantes Imunológicos/análise , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Campylobacter/genética , Galinhas/imunologia , DNA Bacteriano/imunologia , Interações Hospedeiro-Patógeno/imunologia , Leucócitos Mononucleares/microbiologia , Ligantes , Lipopolissacarídeos/imunologia , Macrófagos/microbiologia , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Tonsila Palatina/microbiologia
16.
Nat Commun ; 11(1): 3677, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32699279

RESUMO

Through the formation of concentration gradients, morphogens drive graded responses to extracellular signals, thereby fine-tuning cell behaviors in complex tissues. Here we show that the chemokine CXCL13 forms both soluble and immobilized gradients. Specifically, CXCL13+ follicular reticular cells form a small-world network of guidance structures, with computer simulations and optimization analysis predicting that immobilized gradients created by this network promote B cell trafficking. Consistent with this prediction, imaging analysis show that CXCL13 binds to extracellular matrix components in situ, constraining its diffusion. CXCL13 solubilization requires the protease cathepsin B that cleaves CXCL13 into a stable product. Mice lacking cathepsin B display aberrant follicular architecture, a phenotype associated with effective B cell homing to but not within lymph nodes. Our data thus suggest that reticular cells of the B cell zone generate microenvironments that shape both immobilized and soluble CXCL13 gradients.


Assuntos
Linfócitos B/imunologia , Microambiente Celular/imunologia , Quimiocina CXCL13/metabolismo , Células Dendríticas Foliculares/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Linhagem Celular , Quimiocina CXCL13/imunologia , Simulação por Computador , Células Dendríticas Foliculares/citologia , Células Dendríticas Foliculares/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Biológicos , Tonsila Palatina/citologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Estromais/imunologia , Células Estromais/metabolismo
17.
Int J Mol Med ; 46(3): 1166-1174, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32582998

RESUMO

Bone marrow (BM) transplantation (BMT) represents a curative treatment for various hematological disorders. Prior to BMT, a large amount of the relevant anticancer drug needed to be administered to eliminate cancer cells. However, during this pre­BMT cytotoxic conditioning regimen, hematopoietic stem cells in the BM and thymic epithelial cells were also destroyed. The T cell receptor (TCR) recognizes diverse pathogen, tumor and environmental antigens, and confers immunological memory and self­tolerance. Delayed thymus reconstitution following pre­BMT cytotoxic conditioning impedes de novo thymopoiesis and limits T cell­mediated immunity. Several cytokines, such as RANK ligand, interleukin (IL)­7, IL­22 and stem cell factor, were recently reported to improve thymopoiesis and immune function following BMT. In the present study, it was found that the co­transplantation of tonsil­derived mesenchymal stromal cells (T­MSCs) with BM­derived cells (BMCs) accelerated the recovery of involuted thymuses in mice following partial pre­BMT conditioning with busulfan­cyclophosphamide treatment, possibly by inducing FMS­like tyrosine kinase 3 ligand (FLT3L) and fibroblast growth factor 7 (FGF7) production in T­MSCs. The co­transplantation of T­MSCs with BMCs also replenished the CD3+ cell population by inhibiting thymocyte apoptosis following pre­BMT cytotoxic conditioning. Furthermore, T­MSC co­transplantation improved the recovery of the TCR repertoire and led to increased thymus­generated T cell diversity.


Assuntos
Transplante de Medula Óssea/métodos , Células-Tronco Mesenquimais/citologia , Tonsila Palatina/citologia , Linfócitos T/citologia , Timo/citologia , Animais , Complexo CD3 , Feminino , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tonsila Palatina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo
18.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597837

RESUMO

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.


Assuntos
Separação Celular/métodos , Imunidade Inata/imunologia , Imunidade nas Mucosas/imunologia , Leucócitos Mononucleares/citologia , Tecido Linfoide/imunologia , Tonsila Palatina/citologia , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/imunologia , Tecido Linfoide/citologia , Tonsila Palatina/imunologia
19.
FASEB J ; 34(7): 9269-9284, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32413173

RESUMO

Monocytic cells perform crucial homeostatic and defensive functions. However, their fate and characterization at the transcriptomic level in human tissues are partially understood, often as a consequence of the lack of specific markers allowing their unequivocal identification. The 6-sulfo LacNAc (slan) antigen identifies a subset of non-classical (NC) monocytes in the bloodstream, namely the slan+ -monocytes. In recent studies, we and other groups have reported that, in tonsils, slan marks dendritic cell (DC)-like cells, as defined by morphological, phenotypical, and functional criteria. However, subsequent investigations in lymphomas have uncovered a significant heterogeneity of tumor-infiltrating slan+ -cells, including a macrophage-like state. Based on their emerging role in tissue inflammation and cancer, herein we investigated slan+ -cell fate in tonsils by using a molecular-based approach. Hence, RNA from tonsil slan+ -cells, conventional CD1c+ DCs (cDC2) and CD11b+ CD14+ -macrophages was subjected to gene expression analysis. For comparison, transcriptomes were also obtained from blood cDC2, classical (CL), intermediate (INT), NC, and slan+ -monocytes. Data demonstrate that the main trajectory of human slan+ -monocytes infiltrating the tonsil tissue is toward a macrophage-like population, displaying molecular features distinct from those of tonsil CD11b+ CD14+ -macrophages and cDC2. These findings provide a novel view on the terminal differentiation path of slan+ -monocytes, which is relevant for inflammatory diseases and lymphomas.


Assuntos
Amino Açúcares/metabolismo , Células Dendríticas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Tonsila Palatina/metabolismo , Tonsilite/genética , Estudos de Casos e Controles , Células Cultivadas , Células Dendríticas/citologia , Perfilação da Expressão Gênica , Humanos , Macrófagos/citologia , Monócitos/citologia , Tonsila Palatina/citologia , Tonsilite/metabolismo , Tonsilite/patologia
20.
Mediators Inflamm ; 2020: 6982438, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32322164

RESUMO

Methods: We isolated T-MSCs from human palatine tonsil and evaluated the ingredients of T-MSCs-CM. The effect of T-MSCs-CM was evaluated in the AR mouse model that was randomly divided into five groups (negative control, positive control, and T-MSCs-CM treated (0.1 mg, 1 mg, and 10 mg)). To investigate the therapeutic effect, we analyzed rhinitis symptoms, serum immunoglobulin (Ig), inflammatory cells, and cytokine expression. We also assessed T cell receptor signal, including MAP kinase (ERK/JNK), p65, and NFAT1. Results: We identified the increment of TGF-ß1, PGE2, and HGF in the T-MSCs-CM. In an animal study, the T-MSCs-CM-treated group showed significantly reduced allergic symptoms and infiltration of eosinophils and neutrophils in the nasal mucosa, whereas there was no significant difference in total IgE and the OVA-specific IgE level. Additionally, we found that the 10 mg T-MSCs-CM-treated group showed a significantly decreased IL-4 mRNA expression, compared to the (+) Con group. In the analysis of T cell receptor signal, the phosphorylation of MAP kinases, translocation of p65, and activation of NFAT1 were inhibited after T-MSCs-CM. Conclusions: Our findings suggest that T-MSCs-CM showed a partial immunomodulatory effect on the AR mouse model by the inhibition of T cell activation via MAP kinase, p65, and NFAT1.


Assuntos
Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mucosa Nasal/citologia , Tonsila Palatina/citologia , Rinite Alérgica/metabolismo , Rinite Alérgica/terapia , Animais , Western Blotting , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina E/metabolismo , Camundongos Endogâmicos BALB C , Mucosa Nasal/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...