Single Cell Profiling Identifies IgM To MDA-LDL Producing Human B Cells And Atheroprotective Role Of CD24

Murine B-1 cells produce IgMs that inactivate oxidation-specific epitopes and protect against atherosclerosis. Despite compelling data that IgM to MDA-LDL (IgM MDA-LDL ) is inversely associated with coronary artery disease (CAD) and cardiovascular (CV) events in human, the human B cell subtype that produces IgM MDA-LDL is unknown. Here, we utilized mass cytometry to identify the 11 circulating human B cell subtypes and discovered that only the frequency of CD20 + CD27 + IgM + cells was associated with IgM MDA-LDL levels. Notably, high expression of CD24, a GPI-anchored sialoglycoprotein, was associated with IgM MDA-LDL (r = 0.57, p = 0.002) and inversely associated with CAD severity (r = -0.58, p=0.001). Adoptive transfer (AT) of sorted CD20 + CD27 + IgM + CD24hi (B 27+IgM+CD24hi ) and CD24lo/- (B 27+IgM+CD24lo/- ) human B cells into humanized mice demonstrated that CD24 is critical for trafficking to the spleen and IgM production. Bulk RNAseq coupled with pathway analysis of B 27+IgM+CD24hi and B 27+IgM+CD24lo/- cells revealed enhanced BCR and CCR6 signaling in B 27+IgM+CD24hi . Imaging flow cytometry indicated colocalization between CD24 and CCR6 which was abrogated by CD24 blocking antibody (CD24mAb). Trans-well migration and internalization assays demonstrated that CD24 blockade increased CCL20-induced CCR6 internalization (p < 0.05, n =4), and reduced CCL20-induced migration of B 27+IgM+CD24hi cells (p < 0.05, n =4). CD24mAb treatment of B 27+IgM+CD24hi cells prior to AT into humanized mice also reduced the amount of surface CCR6 on B 27+IgM+CD24hi cells in vivo (p < 0.05, n = 4). Both CCR6 knockout and CD24 inhibition of B 27+IgM+CD24hi cells reduced migration to the spleen (p < 0.05, n = 4), and plasma level of human IgM (p = 0.057, n = 4). Lastly, single cell multi-omics sequencing of PBMCs from 60 CAD subjects revealed elevated CCR6 and IgM signaling in B 27+IgM+CD24hi cells in subjects with less severe CAD. Results provide the first evidence for a role for CD24 in regulating CCR6-mediated B cell homing to the spleen, IgM production and CAD in humans. Findings may have important implications for the new CD24 immunotherapy entering clinical trials and raise the possibility that augmenting CD24 and/or CCR6 on B 27+IgM+ cells may be an effective atheroprotective strategy.