Rationale: Monocytes are key effectors of the mononuclear phagocyte system, playing critical roles in regulating tissue homeostasis and coordinating inflammatory reactions, including those involved in chronic inflammatory diseases such as atherosclerosis. Monocytes have traditionally been divided into 2 major subsets termed conventional monocytes and patrolling monocytes (pMo) but recent systems immunology approaches have identified marked heterogeneity within these cells, and much of what regulates monocyte population homeostasis remains unknown. We and others have previously identified LYN tyrosine kinase as a key negative regulator of myeloid cell biology; however, LYN’s role in regulating specific monocyte subset homeostasis has not been investigated. Objective: We sought to comprehensively profile monocytes to elucidate the underlying heterogeneity within monocytes and dissect how Lyn deficiency affects monocyte subset composition, signaling, and gene expression. We further tested the biological significance of these findings in a model of atherosclerosis. Methods and Results: Mass cytometric analysis of monocyte subsets and signaling pathway activation patterns in conventional monocytes and pMos revealed distinct baseline signaling profiles and far greater heterogeneity than previously described. Lyn deficiency led to a selective expansion of pMos and alterations in specific signaling pathways within these cells, revealing a critical role for LYN in pMo physiology. LYN’s role in regulating pMos was cell-intrinsic and correlated with an increased circulating half-life of Lyn -deficient pMos. Furthermore, single-cell RNA sequencing revealed marked perturbations in the gene expression profiles of Lyn −/− monocytes with upregulation of genes involved in pMo development, survival, and function. Lyn deficiency also led to a significant increase in aorta-associated pMos and protected Ldlr −/− mice from high-fat diet–induced atherosclerosis. Conclusions: Together our data identify LYN as a key regulator of pMo development and a potential therapeutic target in inflammatory diseases regulated by pMos.

While immunotherapies such as immune checkpoint blockade and adoptive T cell therapy improve survival for a subset of human malignancies, many patients fail to respond. Phagocytes including dendritic cells (DCs), monocytes (Mos), and macrophages (MFs) orchestrate innate and adaptive immune responses against tumors. However, tumor-derived factors may limit immunotherapy effectiveness by altering phagocyte signal transduction, development, and activity. Using Cytometry by Time-of-Flight (CyTOF) we found that tumor-derived granulocyte-colony stimulating factor (G-CSF) altered myeloid cell distribution both locally and systemically. We distinguished a large number of G-CSF-induced immune cell subset and signal transduction pathway perturbations in tumor-bearing mice, including a prominent increase in immature neutrophil/myeloid-derived suppressor cell (Neut/MDSC) subsets and tumor-resident PD-L1+ Neut/MDSCs. G-CSF expression was also linked to distinct tumor-associated MF populations, decreased classic DCs, and splenomegaly characterized by increased splenic progenitors with diminished DC differentiation potential. G-CSF-dependent dysregulation of DC development was recapitulated in bone marrow (BM) cultures in vitro, using medium derived from G-CSF-expressing tumor cell cultures. Importantly, tumor-derived G-CSF impaired T cell adoptive cell therapy effectiveness and was associated with increased tumor volume and diminished survival of mice with mammary cancer. Treatment with neutralizing anti-G-CSF antibodies reduced colonic and circulatory Neut/MDSCs, normalized colonic immune cell composition and diminished tumor burden in a spontaneous model of mouse colon cancer. Analysis of human colorectal cancer (CRC) patient gene expression data revealed a significant correlation between survival and low G-CSF and Neut/MDSC gene expression. Our data suggest that normalizing G-CSF bioactivity may improve immunotherapy in cancers associated with G-CSF overexpression.

<p>Supplementary Figure S4 shows that G-CSF and G-CSFR signaling inhibit DC development and activation, in DCs derived in a OP9-DLL1 co-culture system.</p>

<p>Supplementary Figure S3 shows deep phenotyping of Neut/MDSCs and B cells in the bone marrow of mice bearing MT or MTG-CSF-/- tumors, using FlowSOM applied in tSNE maps.</p>

<p>Supplementary Figure S1 shows the characterization of MT and MTG-CSF-/- cell lines and tumors, including surface protein expression, tumor volume/weight, and immune cell subset content. Composition of immune cells in blood and spleen of MT and MTG-CSF-/- mice is also shown.</p>