Single-Cell Transcriptomic Analysis Of Imaging-Sorted Circulating Immune Cell Doublets In Active Tuberculosis 2309416

Abstract Introduction Communication between immune cells through direct contact is a critical feature of immune responses. Our previous work has highlighted that a significant fraction of immune cell doublets detected in non-imaging flow cytometry are not technical artifacts, and instead hold biological relevance. In particular, using non-imaging FACS (fluorescence-activated cell sorting), we have recently shown that circulating T cell-monocyte complexes during infection hold transcriptomic signatures of active immune interactions. However, conventional non-imaging FACS lacks the spatial resolution necessary to characterize doublets accurately. Methods In this study, we employed the recently commercialized FACSDiscoverTM S8 spectral imaging cell sorter to characterize different phenotypes of circulating T cell-monocyte complexes in a cohort of patients with tuberculosis. Using various combinations of imaging parameters, we found that T cell-monocyte doublets can be classified as either synaptic or coincidental. Synaptic doublets were defined by high spatial overlap between the two cells forming complexes, representing true biological conjugates. In contrast, coincidental doublets were non-interacting cells but in close proximity during acquisition, thus representing technical artifacts rather than biological interactions. For each patient, synaptic and coincidental T cell-monocyte doublets were sorted, resulting in physical separation of the two cells forming the doublet, and processed for single-cell droplet sequencing. Results Sorting and single-cell sequencing analysis confirmed that synaptic doublets carried unique biological gene signatures associated with high metabolic activity and immune activation, while coincidental doublets resembled singlet T cells and monocytes. Conclusion Thus, image-based cell sorting provides unprecedented granularity in the study of immune doublets and enrichment for biological doublets over technical artifacts. Funding Source The Tullie and Rickey Families SPARK Awards for Innovations in Immunology, National Institute of Allergy and Infectious Diseases, Human Immunology Project Consortium, The Conrad Prebys Foundation Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)