introduction |
introductionMyeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells that originate from immature myeloid cells (iMCs) (Obermajer et al. 2012). MDSCs contain the precursors to granulocytes, macrophages, and dendritic cells (DCs). MDSCs employ suppression of the lymphocyte function which poses a threat to anti-tumor immunity. MDSCs have not been widely studied and their functions are relatively enigmatic, but the role of MDSCs is being investigated to improve our understanding of their suppressive mechanisms (Poschke et al. 2012). A problem in MDSC research is the lack of ability to differentiate MDSCs due to their phenotypic heterogeneity and lack of research on functional differences between the two main types of MDSCs (Obermajer & Kalinski, 2012).
Some conditions of stress such as cancer affect myelopoiesis by inhibiting the normal process of iMC differentiation. Patients with cancer and tumor-bearing mice have been shown to have an abnormally high accumulation of MDSCs which increases the likelihood of tumor progression due to the higher amounts of immune suppressing cells (Mantovani, 2010). In a healthy individual, MDSCs serve as an immunosuppressant and are activated after pathogens are eradicated by lymphocytes in the body, but under chronic inflammatory conditions, MDSC differentiation is accelerated (Landskron et al., 2014). In addition, MDSCs are present in very high amounts in cancer patients and tumor-bearing mice, but are only found in trace amounts in healthy individuals (Obermajer & Kalinski, 2012). Cytokines, small proteins that regulate immune responses, control MDSC differentiation. Chronic inflammation triggers cellular events, such as the production of cytokines TGF- β, IL-1, IL-6, IL-10, promote cancer cell carcinogenesis and metastasis (Poschke et al. 2012). G-CSF, GM-CSF, TGF- β, IL-1, IL-6, IL-10, VEGF, PGE2, and MCP1 have all been reported to affect MDSC differentiation, however G-CSFs, IL-6, and MCP1s seem to be required in differentiation. In tumor-bearing patients, unregulated inflammation and cytokine production can induce malignant cell transformation and activation of MDSCs (Landskron et al. 2014; Obermajer & Kalinski, 2012; Poschke & Kiessling, 2012). Because of the suppressive nature of MDSCs, an increase in MDSC differentiation leads to cancer cell transformation and proliferation. MDSCs are widely studied because of their potential to be a target for a therapeutic drug against cancer; however, MDSCs have only been produced in vivo by inducing tumors within mice until recently (Liechtenstein et al. 2014; Dufain et al. 2015). Differentiation of MDSCs in vitro could be considered more ethical and humane as well as a cheaper method to produce MDSCs for future research. Because the cytokines secreted from 4T1 cells induce MDSC differentiation, if bone marrow derived stem cells are grown in 4T1 nutrient, then MDSC differentiation will occur. To determine that feasibility of producing large amounts of MDSCs in vitro, 4T1 medium will be used to differentiate MDSCs from bone marrow derived stem cells. Flow cytometry will be used to detect whether MDSCs are present in the population. The specific cellular interactions involving cytokines is still unknown, however G-CSFs, IL-6, and MCP1s seem to possess a higher affinity for MDSC differentiation. In order to test the significance of these cytokines, ELISA can be used to detect the presence of certain cytokines in 4T1 medium (suggesting they have some significance), and by neutralizing specific cytokines and testing for MDSC differentiation without those cytokines to determine whether cytokines are necessary for MDSC differentiation. 4T1 cells from culture are from the BALB/c lineage, which is separate from the black 6 species. Although the cytokines produced by BALB/c 4T1 cells are the same as those found in black 6, receptors are different between the two strains. If 4T1 media is added to black-6 stem cells, then MDSCs will still be differentiated despite the differences in receptors because the cytokines are the same between both species. |
review of literature MDSCs are defined as CD33+Lin-HLA-DR-/low cells that express CD34, CD33, CD11b, and IL4Ra and lack lineage (Lin) expression. MDSCs express high levels of immunosuppressive factors, namely inducible nitric oxide synthase (iNOS and NOS2) which suppresses T-cell responses. Reduced toxicity in natural killer cells depends on transforming growth factor b1 rather than arginase dependent. In addition, PD-L1/B7-H1, secreted by MDSCs, suppress antigen-specific immunity through regulatory T-cells (Treg). Prostaglandin E2 (PGE2) has been shown to be required to differentiate dendritic cells (DCs) to MDSCs in humans. PGE2 has also been shown to increase the amount of MDSCs in mice derived bone marrow in vitro. (Poschke et al. 2012)
With the clinical application of regulation of MDSC in cancer patients, within the past few years, more and more functional studies on MDSCs have been published. (Dufain et al., 2015; Mantovani, 2010; Obermajer & Kalinski, 2012) Recently, the molecular pathways that are involved in MDSCs differentiation and mobilization have been studied in the presence of tumors. According to Mantovani (2010), c/EBPb transcription factor is important in the differentiation of bone marrow derived MDSCS in vitro. In addition, STAT3 (signal transducer and activator of transcription 3) promotes MDSC expansion and mobilization, and IFR8 has been shown to regulate MDSC-inducing signals. Important cytokines that have implications in MDSC differentiation include G-CSF, GM-CSF, IL-6, IL-10, VEGF, PGE2, and IL-1. G-CSF, GM-CSF, and IL-6 namely are associated with in vitro differentiation of MDSCs that are functionally the same as MDSCs produced in vivo (Mantovani, 2010). In vitro differentiation of MDSCs have shown to either have a low yield or production of functionally different MDSCs. In order to produce large amounts of MDSCs, Dufain et al. (2015) developed an in vitro system of differentiating MDSCs from bone marrow cells using medium from tumor cells. Arg-1 and iNOS inhibitors have been shown to reduce MDSC immunosuppressive functions. Dufain et al.’s (2015) study demonstrated that large amounts of GM-CSF are required to differentiate MDSCs from bone marrow derived stem cells, that MDSCs found in the spleen are both phenotypically and functionally different from those found in or near tumors, and that their in vitro generated MDSCs are phenotypically and functionally similar to MDSCs produced naturally with the presence of a tumor. (Dufain et al., 2015) A study by Obermajer and Kalinski (2012) concluded that MDSCs can be generated from bone marrow with successful functional differentiation of the subpopulations CD115+Ly-6C+ and GR-1+CD115Ly-6C-, or monocytic and granulocyte progenitors respectively. In mice, use of cytokine/growth factors and inducing bone marrow derived stem cells by lipopolysaccharide induced expansion of MDSC populations with normal functionality. In humans, one viable method of in vitro generation of MDSCs is to use myelomonocytic precursor cells in peripheral blood in a media with the presence of IL-4 + GM-CSD and tumor derived cytokines. Obermajer and Kalinski (2012) concluded that using a single determining factor, PGE2, larger numbers of monocytic MDSCs could be generated. In addition, the expansion of iMCs into MDSCs can be induced by vascular endothelial growth factor (GM-CSF and IL-6) and “Toll-like receptor ligands” (such as IL-1b, IFNg, and PGE2). All of these cytokines have the ability to induce COX2 expression and PGE2 production showing that these two factors are important in MDSC development. Using PGE2, Obermaier and Kalinski (2012) demonstrated the ability of cytokine rich medium to induce development of MDSCs to produce a large amount of MDSCs in vitro. (Obermajer & Kalinski, 2012) The upregulation of various cytokines in cancerous cells causes MDSC differentiation. This differentiation leads to the suppression of immune functions due to deactivation of lymphocytes (namely T cells). T cells are required to activate adaptive immune response, so the decrease of T cells leaves the body unable to detect and destroy cancerous target cells. Because MDSCs are so heavily involved in cancer cell growth and metastasis of a tumor, research into MDSCs could lead to new discoveries in cancer cell development and function as well as the development of an anti-tumor drug. Unfortunately, the acquisition of sufficient MDSCs required for studying their morphology and function cannot be obtained in vivo. The purpose of this research project is to streamline the process of in vitro MDSC differentiation. In addition, we will study characteristics of MDSCs such as the ability of Black-6 (C57BL/6) to be differentiated by BALB/c produced cytokines, the cell’s response to interleukin-6 (IL-6), and the rate of replication of MDSCs in a healthy vs tumor-bearing host. |