RESUMO
Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.
Assuntos
Subpopulações de Linfócitos T , Transcriptoma , Criança , Humanos , Idoso , Envelhecimento/genética , Epitopos/metabolismo , Análise de Célula ÚnicaRESUMO
Type I and III interferons (IFNs) are robustly induced during infections and protect cells against viral infection. Both type I and III IFNs are also produced at low levels in the thymus at steady state; however, their role in T cell development and immune tolerance is unclear. Here, we found that both type I and III IFNs were constitutively produced by a very small number of AIRE+ murine thymic epithelial cells, independent of microbial stimulation. Antigen-presenting cells were highly responsive to thymic IFNs, and IFNs were required for the activation and maturation of thymic type 1 conventional dendritic cells, macrophages, and B cells. Loss of IFN sensing led to reduced regulatory T cell selection, reduced T cell receptor (TCR) repertoire diversity, and enhanced autoreactive T cell responses to self-antigens expressed during peripheral IFN signaling. Thus, constitutive exposure to IFNs in the thymus is required for generating a tolerant and diverse TCR repertoire.
Assuntos
Interferons , Camundongos Endogâmicos C57BL , Timo , Animais , Timo/imunologia , Camundongos , Interferons/imunologia , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most destructive bacterial infection in honeybees. Even antibiotic-sensitive strains of P. larvae can produce recurrent AFB months to weeks post-antibiotic treatment due to the survival of bacterial spores. Recently, phages that infect P. larvae have been shown to effectively combat AFB in the field. Here, we present evidence that phages not only bind to vegetative P. larvae but also bind to P. larvae spores. Spore binding was observed in the results of three specific experiments: (1) bacteria counted by flow cytometry generated quantitative data of FITC-labeled phages that were bound to vegetative bacteria as well as those bound to spores, (2) electron microscopy captured images of phages bound to the surface of spores in both horizontal and vertical positions, and (3) phages incubated with P. larvae spores bound to the spores and created plaques in vegetative bacteria under conditions not conducive to spore activation, indicating that binding to spores is reversible and that the phages are still active. Identification of phages with reversible spore-binding capability for use in phage therapy may improve treatment of sporulating bacterial infections.
RESUMO
Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021).
Assuntos
Citometria de Fluxo/métodos , Leucócitos Mononucleares , Monitorização Imunológica/métodos , Análise de Célula Única/métodos , Biologia Computacional , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/classificação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Análise de Sequência de RNARESUMO
SARS-CoV-2 has infected over 200 million and caused more than 4 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID-19 beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive immune activation that differed in character by age (young vs. old). We then characterized signals associated with recovery and convalescence to define and validate a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).