These cells arise from myeloid precursors in response to tumor-derived growth factors and pro-inflammatory cytokines (6, 7)

These cells arise from myeloid precursors in response to tumor-derived growth factors and pro-inflammatory cytokines (6, 7). as well as NK cells. In vitro co culture experiments revealed that MDSC inhibited the IFN responsiveness of splenocytes from normal mice. Treatment of C26 bearing mice with gemcitabine or an anti-GR1 antibody led LY2119620 to depletion of MDSC and restored splenocyte IFN responsiveness. Spleens from C26 bearing animals displayed elevated levels of iNOS protein and nitric oxide (NO). In vitro treatment of splenocytes with a nitric oxide donor led to a decreased STAT1 IFN response. The elevation in NO in C26 bearing mice was associated with increased levels of nitration on STAT1. Finally, splenocytes from iNOS knockout mice bearing C26 tumors exhibited a significantly elevated IFN-response as compared to control C26 tumor bearing mice. These data suggest that NO produced by MDSC can lead to reduced interferon responsiveness in immune cells. have also demonstrated the unique importance of IFN-/ sensitivity in cells of the hematopoietic lineage for immunoediting and anti-tumor immunity (3). Interferons are now accepted as crucial mediators of immunosurveillance and are important in both innate and adaptive anti-tumor immune responses. A functional immune system in patients with malignancy is also critical for the success of immune-based therapies, such as exogenously administered cytokines, vaccines and targeted antibodies via the induction of type I interferons (e.g. IFN-, LY2119620 IFN-) and type II IFNs (IFN-). Our group as well as others have determined that immune effector cells from patients with advanced cancers exhibit reduced activation of IFN-induced signaling pathways (4, 5). One potential mechanism for the immune inhibition seen in tumor bearing hosts is the presence of increased numbers of immune suppressor cells. Myeloid-derived suppressor cells (MDSC) are a heterogenous populace of early myeloid cells that accumulate in the blood and tumors of patients with malignancy. Their figures correlate with tumor burden (6). These cells arise from myeloid precursors in response to tumor-derived growth factors and pro-inflammatory cytokines (6, 7). MDSC are explained phenotypically in murine models as GR1+CD11b+, with subsets expressing IL-4R (7, 8). MDSC have been shown to reside in the peripheral blood, lymphoid tissue, and tumor tissue of mice in a number of experimental models (6, 9C13). Prior studies have exhibited that MDSC can inhibit the effector function of NK and T cells in tumor-bearing animals through multiple mechanisms, including the release of immune-suppressive cytokines, the generation of nitric oxide and reactive oxygen species, and the depletion of arginine or cystine from your tumor microenvironment (6, 14, 15). Studies in murine models show that disruption of MDSC function can reverse immune tolerance to tumor antigens, stimulate anti-tumor immune responses and markedly inhibit tumor growth (6, 7). We hypothesized that elevated numbers of MDSC present in the setting of advanced malignancy would inhibit the response of immune cells to LY2119620 type I and II interferons. The results of our experiments exhibited that mice bearing C26 adenocarcinoma tumors exhibit elevated numbers of MDSC which led to increased nitration on STAT1 and impaired responsiveness of immune effector cells to interferons. Methods Cytokines and Reagents Recombinant murine IL-6 and IFN- were Rabbit polyclonal to HMGB4 purchased from R & D Systems, Inc. (Minneapolis, MN). Recombinant Universal Type I Interferon (IFN-A/D) was purchased from PBL Biomedical Sciences (Piscataway, NJ). S-nitroso-stimulation with PBS, IFN-A/D or IFN- as explained previously (4). Data were expressed as specific fluorescence (Fsp = Ft C Fb), where Ft represents the median value of total staining and Fb represents the median value of background staining with an isotype control antibody (4, 17, 18). CD4, CD8, and CD49b antibodies were used for surface staining of immune subsets (BD Biosciences). For nitration circulation cytometry, splenocytes were co-labeled with anti-STAT1-PE and anti-nitrotyrosine-alexafluor-488 antibody (BD Biosciences and Millipore). Real Time PCR Following TRIzol extraction (Invitrogen) and RNeasy purification (Qiagen), total.