Abstract Description Marginal zone (MZ) B cells in the spleen are innate-like lymphocytes crucial for producing antibodies against blood-borne pathogens. Despite evidence of heterogeneity, the overall composition of MZ B cells remains insufficiently defined. Given their diverse immune roles, MZ B cells are likely composed of distinct subpopulations. In previous research, we identified two subtypes of MZ B cells, CD80high and CD80low, which differ genetically and functionally. These differences include variations in B cell receptor repertoires, antibody and cytokine secretion, and resistance to radiation. In this study, we utilized single-cell RNA sequencing (scRNA-seq) was employed to explore MZ B cell heterogeneity. Analysis of 5,734 single cells revealed five clusters. Gene ontology analysis highlighted populations such as Interferon Stimulated Gene (ISG)+, Cycling, and regulatory B cells. A key challenge in scRNA-seq is the identification of rare or novel cell types due to limitations in reference marker gene sets. To overcome this, bulk RNA sequencing data was integrated to refine the classification of CD80high and CD80low MZ B cell clusters. Ultimately, six distinct MZ B cell clusters were identified: CD80high, CD80low, ISG+, regulatory, cycling, and an unknown subpopulation. Further characterization of these clusters could reveal new diagnostic and therapeutic opportunities targeting MZ B cells to enhance immune-related disease outcomes. Funding Sources Supported by RS-2024-00341200; 2023R1A2C2004510; RS-2024-00405650 Topic Categories Technological Innovations in Immunology (TECH)