Generation of human myeloid suppressor cells in the in vitro experimental model

Abstract

Myeloid suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that generally differentiate into macrophages, granulocytes, and dendritic cells. However, in pathology, these cells acquire a suppressor phenotype, blocking immune response. In particular, MDSC levels increase in many pathological conditions, including inflammation, sepsis, traumatic shock, autoimmune diseases, cancer, and pregnancy. Over the past 12 years, an interest in examining this cell population has been steadily increased [PUBMED: 2008 (65 articles); 2020 ( 650 entries)] that will expand our understanding of immune system functioning. In humans, MDSCs are characterized by HLA-DR-CD33+CD11b+ phenotype, in turn being subdivided into CD15+ or CD66+ granulocytic (G-MDSC), CD14+ monocytic (M-MDSC), and early (e-MDSC) MDSC bearing HLA-DR-CD11b+CD33+CD14-CD66b- phenotype. This work was aimed to develop an adequate experimental model allowing to evaluate cytokine-driven differentiation of human MDSCs from peripheral blood mononuclear cells in long-term in vitro culture system. For this, peripheral blood mononuclear cells were isolated from healthy donors induced to express MDSC phenotype with GM-CSF and IL-6 (40 or 20 ng/ml) cultured for 7, 14, 21 days. In several experiments, LPS (100 ng/ml) was added to the cultured cells 24 hours before immunophenotyping. The percentage of living Zombie Aquanegative cells in cultures (gated on cells according to FSC/SSC) ranged from 90.5-93.9%. No significant differences were observed between cultured cells. In our experimental conditions, the mean percentage of total MDSC subpopulation reached 2-2.3% of total living cells, exceeding that one by 9-10-fold found in freshly isolated mononuclear cells from healthy subjects. Based on the results of our experimental study, we found that induction of e-MDSC derived from human peripheral blood mononuclear cells requires two weeks of co-culture with 40 ng/ml IL-6 and 40 ng/ml GM-CSF. “Mature” MDSCs (M-MDSC + G-MDSC) yield was peaked in the following conditions: co-culture for 3 weeks with 20 ng/ml IL-6 and 20 ng/ml GM-CSF added with 100 ng/ml LPS 24 hours before completing protocol. Overall, further examining factors modulating MDSC differentiation will reveal conditions necessary for generating this suppressor cell subset potentially used in clinical practice.