Unraveling the heterogeneity of immune cell doublets in human blood using high-throughput spectral imaging cell sorting 3361

Abstract Description We have previously demonstrated that 1) many immune cell doublets fail to be categorized as such by conventional flow cytometry analyzers and sorters, 2) imaging parameters are the best features to distinguish doublets from singlets, 3) a significant fraction of doublets in human blood show signatures of immune synapses, indicating biological relevance. Here, we took advantage of the high-throughput spectral imaging cell sorter BD FACSDiscover S8 to further characterize immune cell doublet heterogeneity. We continued our work on biological T cell-monocyte doublets in tuberculosis (TB) by further classifying them into synaptic or coincidentally interacting doublets using various imaging parameters. Sorting and single-cell sequencing confirmed that synaptic doublets carried unique biological gene signatures, while coincidental doublets resembled singlet T cells and monocytes. Based on CD3 imaging parameters, we also identified additional subsets within synaptic doublets with distinct transcriptomic signatures and interaction properties. Second, we leveraged the spectral component of the BD FACSDiscover S8 and designed a 23-color panel composed of lineage markers representing major immune cell types. We applied this panel to healthy and TB cohorts and built an atlas of all subtypes of immune cell doublets present in human blood, along with their abundance at steady-state and during infection. Thus, imaging cell sorting is a powerful technology to study immune cell doublets. Topic Categories Technological Innovations in Immunology (TECH)