Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment.

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.

Cross-platform immunophenotyping of human peripheral blood mononuclear cells with four high-dimensional flow cytometry panels.

Immunophenotyping using high dimensional flow cytometry is a central component of human immune system multi-omic studies. We present four high parameter flow cytometry panels for deep immunophenotyping of human peripheral blood mononuclear cells (PBMC). This set of four 25+ color panels include 64 cell surface markers to resolve broad immune compartment populations, as well as activation and memory of specific T, B, natural killer (NK), and myeloid lineages. Common lineage bridging markers are integrated into each panel to allow for inter-panel quality control through major lineage frequency verification. These panels were developed using a five laser BD Symphony A5 conventional cytometer and successfully transferred to a five laser Cytek Aurora spectral cytometer capable of acquiring the panels. Nine representative PBMC samples were stained with the four phenotyping panels and acquired on both instruments to evaluate population frequency and visual staining patterns for gating between the systems. Both instruments produced comparable high quality flow cytometry data and supported our decision to acquire samples on the spectral cytometer moving forward. This modular set of panels and instrument performance metrics provide guidelines for designing flow cytometry experiments suitable for longitudinal or cross-sectional immune profiling.

Sterile production of interferons in the thymus affects T cell repertoire selection.

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.

Longitudinal Multi-omic Immune Profiling Reveals Age-Related Immune Cell Dynamics in Healthy Adults.

The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examined human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults' memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age.

Phages Bind to Vegetative and Spore Forms of Paenibacillus larvae and to Vegetative Brevibacillus laterosporus.

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.

Optimized workflow for human PBMC multiomic immunosurveillance studies.

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).

Longitudinal immune dynamics of mild COVID-19 define signatures of recovery and persistence.

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).