TRANSCRIPTOMIC ANALYSIS OF HUMAN CIRCULATING MONOCYTES: FOCUS ON MEMBRANE-SPANNING 4A GENE FAMILY MEMBERS.

Within the mononuclear phagocyte system (MPS), monocytes represent the unique population able to operate as both effector and precursor cells. These incredibly plastic cells, mainly present in peripheral blood, are essential components of the innate immune system and play central roles both in homeostatic and pathological conditions. Monocytes are key determinants for the surveillance of endothelial integrity and repair, regulation of wound healing and replenishment of tissue resident macrophages (TRMs). Moreover, they serve as first line of defence against infections and hold a key position in several human diseases. Despite the fact that current classification distinguishes three major subsets (classical, intermediate and non-classical), monocytes represent an extremely heterogeneous population in terms of phenotype and specialized functions. To overcome the remarkable lack of consensus on the identity and interrelationship of monocyte subsets, we have performed a single cell RNA sequencing analysis on circulating mononuclear cells of healthy donors. We identified 8 cluster of monocytes. C0 and c2 resembled neutrophil-like monocytes (NeuMo) and, together with c1, displayed distinct inflammatory programs and activation states. Of the two other clusters of classical monocytes, c7 significantly expressed high levels of antiviral genes, including IFN-related genes, while c12 corresponded to circulating monocyte-platelet aggregates (MPA). C6 and c3 resembled the intermediate and non-classical monocyte populations, respectively. Finally, a small cluster of CD16+ cells (c13) was characterized by the specific expression of genes of the complement system. We then moved to the analyses of a public transcriptomic dataset obtained from peripheral blood mononuclear cells (PBMCs) of gastrointestinal cancer patients at 3 time points of treatment. We were able to identify all previously defined monocyte subsets. Preliminary data showed the specific expansion of the IFN-related cluster c7 exclusively in responder patients after treatment with immunotherapy, suggesting a potential role of this population in response to therapy. Analyses of the Membrane-Spanning 4-domain subfamily A (MS4A) protein family, representing a set of proteins whose roles in regulating myeloid cell function are now emerging, identified MS4A4A as potential markers for the commitment to circulating non-classical monocytes in humans. The expression of MS4A4A on a fraction of CD16+ monocytes was further confirmed at the protein level by flow cytometry. Given the selective expression of this protein on CD16+ cells and based on solid preliminary data from our laboratory, we 3 hypothesized that MS4A4A may play a role in the biology of this subset of cells. Interestingly, bulk RNA sequencing of CD16-MS4A4A-, CD16+MS4A4A- and CD16+MS4A4A+ monocytes revealed enrichment of the Fc gamma receptor (FcγR) and Fc epsilon receptor (FcεR) pathways in MS4A4Apos cells. Implementation of these findings with functional assays is in program and will be essential to deeper investigate a possible role of MS4A4A as modulator of FcRs function in monocytes.