RESUMO
Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field. The protocol typically takes 8-10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells.
Assuntos
Sistema Imunitário/citologia , Dispositivos Lab-On-A-Chip , Fenótipo , Análise de Célula Única/instrumentação , Animais , Linfócitos B/citologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia de FluorescênciaRESUMO
Freezing of alum-based vaccines drastically alters their colloidal composition and leads to irreversible cluster formation. The loss of stability is well described, but the impact of frost damage on the functionality of the induced and secreted antibody repertoire has not been studied in detail. We therefore applied our single-cell measurement platform to extract the frequencies of Immunoglobulin G-secreting cells in combination with individual secretion rates and affinities. We showed that, frost-damaged or not, the tested vaccine was able to generate similar frequencies of total and antigen-affine IgG-secreting cells. Additionally, the frost-damaged vaccine stimulated a similar T-cell cytokine secretion pattern when compared to the regularly stored vaccine. However, frost-damaged vaccines induced no efficient affinity maturation and a complete collapse of the affinity distribution was observed. This study unveiled the impact of frost-damage to alum-based vaccines on the induced secreted antibody repertoire, and illustrated the power of functional single-antibody analysis.
Assuntos
Imunoglobulina G , CongelamentoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
T helper 17 (Th17) cells have crucial functions in mucosal immunity and the pathogenesis of several chronic inflammatory diseases. The lineage-specific transcription factor, RORγt, encoded by the RORC gene modulates Th17 polarization and function, as well as thymocyte development. Here we define several regulatory elements at the human RORC locus in thymocytes and peripheral CD4+ T lymphocytes, with CRISPR/Cas9-guided deletion of these genomic segments supporting their role in RORγt expression. Mechanistically, T cell receptor stimulation induces cyclosporine A-sensitive histone modifications and P300/CBP acetylase recruitment at these elements in activated CD4+ T cells. Meanwhile, NFAT proteins bind to these regulatory elements and activate RORγt transcription in cooperation with NF-kB. Our data thus demonstrate that NFAT specifically regulate RORγt expression by binding to the RORC locus and promoting its permissive conformation.
Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição NFATC/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Elementos Reguladores de Transcrição/genética , Células Th17/metabolismo , Timócitos/metabolismo , Ativação Transcricional , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem da Célula , Citometria de Fluxo , Células HEK293 , Código das Histonas , Humanos , Células Jurkat , Células Th17/citologia , Timócitos/citologia , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
OBJECTIVE: Recent genome-wide association studies have revealed numerous genetic associations between specific single-nucleotide polymorphisms (SNPs) and immune-mediated inflammatory diseases. The current challenge is to identify associations of the genetic variants with effector mechanisms implicated in pathogenesis. This study was undertaken to investigate the link between genetic variation at loci associated with spondyloarthritis (SpA) and the effector function of CD4+ T lymphocyte subsets involved in chronic inflammatory disease. METHODS: Expression of Th17 and Th1 cytokines and transcription factors was measured in CD4+ T cells isolated from patients with SpA. Correlation analyses were performed to assess potential associations of these expression levels with the patient's genotype at loci genetically linked to SpA. RESULTS: The effector functions of Th17 and Th1 cells in patients with SpA were found to be under combinatorial control by multiple SNPs at genes associated with the interleukin-23 (IL-23)/Th17 pathway. Patients with SpA carrying risk-associated alleles of genes in the IL-23/Th17 pathway expressed the highest levels of genes involved in the differentiation and function of Th17 and Th1 cells, whereas the presence of protective alleles was associated with low-level expression of these genes. In contrast, variation at loci that were genetically linked to SpA, but not associated with the IL-23 pathway, did not affect the expression of Th17- and Th1-specific genes, suggesting that these SNPs may contribute to the pathogenesis of SpA through distinct cellular mechanisms. CONCLUSION: These results show that genetic variations at genes associated with the IL-23 signaling pathway may influence the effector functions of Th17 and Th1 cells in patients with SpA. These findings provide a framework to delineate the mechanisms by which genetic variants contribute to pathology.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Interleucina-23/genética , Espondiloartropatias/metabolismo , Células Th1/metabolismo , Células Th17/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Variação Genética , Genótipo , Humanos , Interleucina-23/metabolismo , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Espondiloartropatias/genética , Adulto JovemRESUMO
Approximately 1 of every 250 newborns has some abnormality of genital and/or gonadal development. However, a specific molecular cause is identified in only 20% of these cases of disorder of sex development (DSD). We identified a family of French origin presenting with 46,XY DSD and congenital heart disease. Sequencing of the ORF of GATA4 identified a heterozygous missense mutation (p.Gly221Arg) in the conserved N-terminal zinc finger of GATA4. This mutation was not observed in 450 ancestry-matched control individuals. The mutation compromised the ability of the protein to bind to and transactivate the anti-Müllerian hormone (AMH) promoter. The mutation does not interfere with the direct protein-protein interaction, but it disrupts synergistic activation of the AMH promoter by GATA4 and NR5A1. The p.Gly221Arg mutant protein also failed to bind to a known protein partner FOG2 that is essential for gonad formation. Our data demonstrate the key role of GATA4 in human testicular development.