Abstract Body (Do not enter title and authors here): Introduction
Oxidized low density lipoproteins (OxLDL) present in atherosclerotic plaques. Epidemiological studies demonstrate that IgM antibody against malondialdehyde LDL (MDA-LDL), the major subtype of OxLDL, is inversely associated with severity of coronary artery disease (CAD), heart attack and death. In human, circulating marginal zone B cells (MZB) were recently identified as producers of these atheroprotective IgM to MDA-LDL. The frequency of circulating MZB cells correlates with CAD severity. We further discover that MZB cells from subjects with high CAD burden produce less atheroprotective IgM to MDA-LDL. This study explores phenotypic changes in MZB cells in relation to CAD severity and investigates underlying mechanisms

Hypothesis
The chemokine receptor CCR6 regulates the production of atheroprotective IgM against MDA-LDL by MZB cells and is downregulated in subjects with severe CAD.

Methods
Humanized mice were adoptively transferred with sort-purified human MZB cells from low and high CAD severity subjects. Single cell multi-omics (CITE-Seq) was performed on B cells from both low and high CAD groups. In-vitro migration study, imaging-flow cytometry, as well as CRISPR-mediated gene knock down were utilized to further investigate mechanisms.

Results
Humanized mice receiving MZB cells from low-CAD subjects (n = 7) produced higher levels of atheroprotective IgM than those receiving cells from high-CAD subjects (n = 6). Single-cell analysis of B cells from low (n = 30) and high (n = 30) CAD subjects revealed higher CCR6 expression in MZB cells from the low-CAD group. In vitro activation of MZB cells with MDA antigen led to downregulation of both CD24 and CCR6. Imaging flow cytometry and proximity ligation assays suggested co-localization of CD24 and CCR6. Blocking CD24 with a monoclonal antibody reduced surface CCR6 expression and impaired CCL20-induced migration. Finally, CCR6-knockdown MZB cells (n = 4) exhibited reduced splenic migration and IgM production in humanized mice compared to wild-type controls (n = 6).

Conclusion
MZB cells from individuals with severe CAD exhibit reduced CCR6 expression and impaired atheroprotective IgM production. CCR6 is critical for splenic migration of human MZB cells and subsequent IgM production.