Multiplexed enrichment and genomic profiling of peripheral blood cells reveal subset-specific immune signatures

Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system’s technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.National Institute of Allergy and Infectious Diseases (U.S.) (Grant U24 AI118668

An immune-cell signature of bacterial sepsis

Dysregulation of the immune response to bacterial infection can lead to sepsis, a condition with high mortality. Multiple whole-blood gene-expression studies have defined sepsis-associated molecular signatures, but have not resolved changes in transcriptional states of specific cell types. Here, we used single-cell RNA-sequencing to profile the blood of people with sepsis (n = 29) across three clinical cohorts with corresponding controls (n = 36). We profiled total peripheral blood mononuclear cells (PBMCs, 106,545 cells) and dendritic cells (19,806 cells) across all subjects and, on the basis of clustering of their gene-expression profiles, defined 16 immune-cell states. We identified a unique CD14+ monocyte state that is expanded in people with sepsis and validated its power in distinguishing these individuals from controls using public transcriptomic data from subjects with different disease etiologies and from multiple geographic locations (18 cohorts, n = 1,467 subjects). We identified a panel of surface markers for isolation and quantification of the monocyte state and characterized its epigenomic and functional phenotypes, and propose a model for its induction from human bone marrow. This study demonstrates the utility of single-cell genomics in discovering disease-associated cytologic signatures and provides insight into the cellular basis of immune dysregulation in bacterial sepsis

Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro

Bacterial sepsis and severe COVID-19 share similar clinical manifestations and are both associated with dysregulation of the myeloid cell compartment. We previously reported an expanded CD14⁺ monocyte state, MS1, in patients with bacterial sepsis and validated expansion of this cell subpopulation in publicly available transcriptomics data. Here, using published datasets, we show that the gene expression program associated with MS1 correlated with sepsis severity and was up-regulated in monocytes from patients with severe COVID-19. To examine the ontogeny and function of MS1 cells, we developed a cellular model for inducing CD14⁺ MS1 monocytes from healthy bone marrow hematopoietic stem and progenitor cells (HSPCs). We found that plasma from patients with bacterial sepsis or COVID-19 induced myelopoiesis in HSPCs in vitro and expression of the MS1 gene program in monocytes and neutrophils that differentiated from these HSPCs. Furthermore, we found that plasma concentrations of IL-6, and to a lesser extent IL-10, correlated with increased myeloid cell output from HSPCs in vitro and enhanced expression of the MS1 gene program. We validated the requirement for these two cytokines to induce the MS1 gene program through CRISPR-Cas9 editing of their receptors in HSPCs. Using this cellular model system, we demonstrated that induced MS1 cells were broadly immunosuppressive and showed decreased responsiveness to stimulation with a synthetic RNA analog. Our in vitro study suggests a potential role for systemic cytokines in inducing myelopoiesis during severe bacterial or SARS-CoV-2 infection