In addition to connecting high ARG levels with DNMT

In addition to connecting high ARG1 levels with DNMT3A/TET2 mutations in MDS/CMML patient biopsies, we also wanted to address the cell type demonstrating elevated ARG1. Following our mouse work, we hypothesized that overexpression of this protein would be found in myelomonocytic IOX4 including macrophages. Using IHC staining, we were able to find evidence to support this model. Aside from CD68-positive monocytes/macrophages, it appears that neutrophils (CD15-positive) and their precursors (CD33-positive) also overexpressed ARG1 in MDS/CMML cases. This observation is in keeping with one report that ARG1 was upregulated in peripheral blood neutrophils of 9/21 MDS patients sampled [35]. This group connected elevations in ARG1 with cell proliferation and extracellular matrix production in MDS patients [35]. Further experiments looking at the effects of ARG1 overexpression on proliferation, evasion of apoptosis and alteration of the local microenvironment should be pursued. Within the MDS cohort, patients with normal neutrophil counts were found to have elevated arginase activity whereas neutropenic individuals showed no elevations in activity. Importantly, age-matched controls (with normal neutrophil counts) did not have increased arginase. Although human neutrophils have been demonstrated to express ARG1, [25], [26] our results suggest a different character of ARG1-high neutrophils in MDS. We hypothesize that these cells may represent polymorphonuclear MDSCs, an ARG1-overexpressing cell type that cannot be readily distinguished from normal neutrophils by clinical hematology analyzers and is known to be elevated in MDS patients, although this will require confirmation [9]. Moving forward, approaches such as fluorescence activated cell sorting based on ARG1 expression, followed by gene expression analyses and ChIP-seq could add greatly to our understanding of ARG1 upregulation, especially in the context of DNMT3A/TET2 mutant MDS/CMML cases. In summary, our findings indicate that ARG1 overexpression in MDS/CMML patient BM is associated with earlier stages of MDS and CMML. Mutations in the epigenetic regulators DNMT3A and TET2 are most likely related to the increased expression of this protein through their effects on the BM immune environment. Further studies will need to be performed to establish a mechanism connecting these mutations with ARG1 expression. The novelty of this study lies in the observation that lower-grade MDS and CMML are linked to an increase in myelomonocytic cell ARG1 levels, as this has generally been found to be associated with poor survival in patients with solid cancers [17], [18], [19]. We hypothesize that the presence of ARG1 may be used as an indicator of inflammation and BM microenvironmental changes conducive to the formation of MDS and CMML. Future studies are required to explore the functional impacts of ARG1 expression on macrophages, neutrophils and the hematopoietic niche, as this will improve our understanding of myeloid cell function within the MDS/CMML BM microenvironment.