[PMC free article] [PubMed] [CrossRef] [Google Scholar] 3. is associated with a better prognosis for advanced melanoma, breast and lung cancer (1C3). This and emerging data suggest targeting the host immune system can enhance cancer therapies(4). While some patients have a positive response to immunotherapy, the majority remain refractory (5). A better understanding of the entire immunosuppressive landscape – both the adaptive and the innate systems – is necessary to improve this promising treatment modality. The overall melanoma response rate of the CTLA-4 antagonist, ipilimumab, is ~10% and the PD-1 antagonist, pembrolizumab, exceeds 35% (6,7). A lack of functional or tumor-infiltrating T cells are often cited as reasons for therapeutic failure (8,9). Several additional T cell-centric therapies are currently being investigated in clinical trials (10C14). While many acknowledge the importance of the myeloid compartment in the tumor microenvironment (TME), including tumor-associated macrophages (15,16) MDSCs (17,18) and tolerizing or inactive antigen presenting cells (APCs) (19,20), cancer therapeutics based on the innate immune system are less common (e.g. IDO and PI3K- inhibitors (21C23)). To increase the efficacy and durability of immunotherapy, additional methods reversing the immunosuppressive TME and a better understanding of immunotherapy resistance mechanisms are needed. TYRO3, AXL, MERTK transmembrane receptor tyrosine kinases (TAM RTKs) regulate the innate immune system, dampening inflammatory responses to prevent chronic inflammation and auto-immunity (24,25). The activity of these receptors is stimulated by protein ligands e.g. GAS6 and PROTEIN S (26,27). The protein ligands have a higher affinity and are most effective when they bridge the cell Mouse monoclonal antibody to Rab4 surface TAM receptors to a source of lipid phosphatidylserine (PtdSer) exposed on apoptotic cells, aggregating platelets, exosomes and certain viruses that expose PtdSer EMD-1214063 on their surface (apoptotic mimicry) (24). GAS6 and PROTEIN EMD-1214063 S are expressed in multiple cell types including epithelial and immune cells (28) and can be secreted by tumor cells dampening the immune response (29). Genetic deletion of MERTK in mice leads to heightened inflammatory responses and mild auto-immunity (30). When tumors are implanted in Animal Studies: C57BL/6J and OT-I (C57BL/6-Tg(TcraTcrb)1100Mjb/J) mice were purchased from Jackson Labs. Mm00444547_m1), (Mm00437221_m1), (Mm00434920_m1), (Mm00490378_m1), (Mm01343426_m1)) (ThermoFisher) or validated primers (below) and PowerUp SYBR Green Master Mix (ThermoFisher, A25742) were used for conventional qPCR. GAPDH was used as a reference gene for normalization and gene expression was determined using the 2 2?CT method. Forward: 5 EMD-1214063 CTCCAAGCCAAAGTCCTTAGAG 3 Reverse: 5 AGGAGCTGTCATTAGGGACATC 3 Forward: 5 AGGAAGTGGGCCGAAGGAT EMD-1214063 3 Reverse: 5 GAAACTATGGAGCACAGCCACAT 3 Forward: 5 GTCTACATGTTCCAGTATGACTCC 3 Reverse: 5 AGTGAGTTGTCATATTTCTCGTCGT 3 Forward: 5 CAGGCCAGAGCAGCATCTTC 3 Reverse: 5 GCCAGCCTCGTGTTTTATTCC 3 Forward: 5 CTCCCGTGGCTTCTAGTGC 3 Reverse: 5 GCCTTAGTTTGGACAGGATCTG 3 ROS Production Assay: 10,000 MDSCs were plated in a black 96-well plate (Costar, 3916) and incubated overnight at 37C with 5% CO2. ROS Production was measured by DCFDA cellular ROS detection assay kit (Abcam, ab113851) according to manufacturers directions. Fluorescence was measured at 535nm with an EnSpire plate reader (PerkinElmer). Peripheral Blood Cell Isolation from Murine Blood: Murine blood was obtained by terminal cardiac puncture and contained in heparin-coated tubes. Cells were isolated by density gradient centrifugation using Lympholyte-M (Cederlane Labs). Peripheral Blood Cell Isolation from Human Blood: 20ml blood was collected from 25 healthy donors or 18 metastatic melanoma patients under protocol LCCC 1715 after written consent was obtained. Inclusion criteria for healthy cohort: 1. Healthy donors were older than 18 years of age. 2. Male or female. 3. No documented diagnosis of cancer. 3. Donors consented to providing a one-time blood sample of up to 20ml. 4. Consented to abstraction of their medical records as evidence by signed HIPPA form. 5. Signed institutional review board (IRB)-approved informed consent document. Inclusion criteria for melanoma cohort: 1. 18 years of age or older. 2. Male or Female. 3. English-speaking. 4. Appointment at the NC Cancer Hospital (NCCH) for treatment of EMD-1214063 a pathologically or diagnostically confirmed diagnosis of metastatic melanoma. 5. Patient consented to providing a baseline blood sample of up to 20ml. 6. Consents to abstraction of their medical records as evidence by signed HIPPA form for this protocol. 7. Signed an institutional review board (IRB)-approved informed consent document for this protocol. Exclusion criteria for both cohorts: 1. Younger than 18 years of age. 2. Dementia, altered mental status, or any psychiatric condition that would prohibit the understanding or rendering of informed consent. 3. Diagnosis of autoimmune diseases, or chronic.