Human monocytes in peripheral blood can be isolated either through elutriation, magnetic beads separation or gradient centrifugation. Monocytes are readily identifiable among the HLA-DR
+ CD11b
+ myeloid compartment, as they constitute 10–20% of all peripheral blood mononuclear cells (PBMC) obtained from standard density gradient centrifugation. Blood monocytes, however, still consist of a phenotypically and functionally heterogeneous populations of cells that are conventionally divided into 3 subsets based on the expression of CD14 and CD16 [
13]. The major CD14
highCD16
neg classical monocyte population is rapidly recruited to sites of inflammation or tissue damage, while the less-frequent CD14
lowCD16
high non-classical monocytes exhibit vascular surveillance functions during steady state. The CD14
highCD16
dim “intermediate” monocytes [
14] are the least abundant monocyte population, although their abundance can vary in pathological conditions [
15]. Recent studies of gene expression profiles of the monocyte subsets [
16‐
18] and of their kinetics in blood [
19] have shown that the monocyte population in blood is a developmental continuum. A minor fraction of classical monocytes differentiates in blood into intermediate monocytes that further transition into non-classical monocytes [
19]. In case a clear segregation of non-classical and intermediate monocytes is key, it is advised to use proper isotype controls in FACS sorting. Moreover, since natural killer (NK) cells may express CD16, it is crucial to include an NK-marker like CD56 (or CD335/NKp46) together with lymphoid lineage markers (CD3 and CD19) in the “dump channel”. Of note, a recent study compared the frequency and phenotypes of monocytes extracted from whole blood and gradient stratification, and found that the relative frequency of classical (CD14
highCD16
neg) versus non-classical (CD14
lowCD16
high) monocytes was significantly different [
13]. Thus it is important to disclose the source of blood monocytes for useful comparisons of data.
Human M-MDSC are present in the same density fraction as monocytes but differ from monocytes by low or the absence of HLA-DR expression. They are further characterized as lymphocyte lineage marker negative cells with the following phenotype CD11b
+HLA-DR
−CD14
+CD15
−. It is possible to use CD33 myeloid cell marker instead of CD11b. In this case, M-MDSC display high CD33 expression relative to PMN-MDSC [
20]. M-MDSC induction and expansion are mediated by a combination of soluble factors (e.g., VEGF, GM-CSF, M-CSF, S100A8/A9, IL-4, IL-6, IL-10, PGE2, MMP9, CXCL5, CXCL12 and C5a) produced by tumor and/or surrounding cells such as stromal cells, T cells or macrophages [
21]. These factors essentially trigger activation of members of the STAT family of proteins, such as STAT3, STAT6 and STAT1, ultimately leading to expression of genes involved in the blockade of myeloid differentiation and in genes with immune regulatory activity.
Expansion of immunosuppressive M-MDSC populations is observed in different cancer types including breast, colorectal cancer, melanoma, glioma and more, indicating that tumor derived factors can systemically activate this population in the blood of cancer patients [
22‐
25]. For a detailed critical review on MDSC in cancer we refer the reader to the companion review by Umansky et al. [
26] in this symposium-in-writing series.