Single-Cell Transcriptomics and Super-Resolution Confocal Imaging Reveal Differential Regulation and Subcellular Localization of Cathepsin L in Response to IFN-a in Primary CD14 Monocytes 9097

Abstract Description Interferons (IFNs) are essential for orchestrating immune responses, influencing autoimmune disorders, malignancies, and immune defense. Despite their importance to the immune response, a comprehensive single-cell transcriptomic dataset of the response to the three types of human IFN remains incomplete. To address this, we generated a single-cell RNA sequencing dataset profiling the responses of 13 immune cell types in human peripheral blood mononuclear cells (PBMCs) to IFN-α, IFN-β, IFN-γ, and IFN-λ1. Our results revealed that IFN-α stimulation exclusively upregulated cathepsin L (CTSL) mRNA expression in primary CD14 monocytes. Cathepsins are lysosomal proteases that play a key role in intracellular protein degradation and antigen processing. While CTSL mRNA levels increased in response to IFN-α, we did not observe a significant rise in CTSL protein levels measured by intracellular flow cytometry. Thus, we further investigated the subcellular localization change in CD14 monocytes upon IFN-α stimulation using super-resolution confocal microscopy and Aivia software for analysis. In steady-state CD14 monocytes, CTSL was randomly distributed in the cytoplasm. However, upon IFN-α stimulation, it colocalized with lysosomal markers. This suggests that IFN-α influences the immune function by modulating lysosomal protease activity. Our study provides a new scRNA-seq dataset for human immune cells and proposes a novel mechanism for the role of IFN-α in CD14 monocytes. Funding Sources Research reported in this publication was supported by The Allen Institute for Immunology Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)