Myeloid-Derived Suppressor Cells Modulate Immune Responses Independently of NADPH Oxidase in the Ovarian Tumor Microenvironment in Mice

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47phox−/−) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47phox−/− mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.


Introduction
Inflammatory cells that constitute the cancer microenvironment can limit or stimulate tumor growth. In cancers that are responsive to immune targeting, cytotoxic T lymphocytes are the major effector cells mediating antigen-driven anti-tumor immunity. However, factors produced by the tumor and its microenvironment can abrogate anti-tumor immunity and facilitate local spread and metastasis. The balance between immune responses that inhibit versus facilitate tumor growth can predict clinical outcome. Therapeutic targeting of immune pathways that facilitate tumor escape may extend periods of disease-free progression and, potentially, overcome barriers to durable anti-tumor immunity.
Epithelial ovarian cancer (EOC) is typically diagnosed at advanced stages. Even with optimal surgical debulking and chemotherapy, the vast majority of patients with advanced EOC will have progression of disease. However, there is considerable variability in progression-free survival and overall survival among patients with advanced EOC [15]. The role of immune surveillance in EOC was demonstrated by correlation of survival with tumor-infiltrating lymphocytes (TILs) [16]. Intraepithelial CD8 + TILs and a high CD8 + /Treg ratio were associated with favorable prognosis in patients with EOC [17,18]. Changes in the phenotype of tumor-infiltrating DCs influence EOC progression in mice [19]. MDSCs accumulate in the ascites of patients with advanced EOC and suppress T cell proliferation ex vivo [20]. These findings raise the potential for MDSCs in the EOC microenvironment as potential therapeutic targets. It is therefore important to understand mechanisms that lead to expansion and functional properties of MDSCs.
There is growing evidence that reactive oxidant intermediates (ROIs) and reactive nitrogen intermediates modulate the development of MDSCs. NADPH oxidase (NOX2) is the major source of ROIs in activated phagocytes. NADPH oxidase activation requires translocation of the cytoplasmic subunits p47 phox , p67 phox , and p40 phox and rac to the membrane-bound flavocytochrome consisting of gp91 phox and p22 phox (phox, phagocyte oxidase). Following activation, NADPH oxidase converts molecular oxygen to superoxide anion that can be subsequently converted to downstream ROI metabolites (e.g., hydrogen peroxide). NADPH oxidase is critical for antimicrobial host defense and also modulates inflammatory responses [21]. Prior studies in mice have shown that NOX2 enhances MDSC differentiation and function [22,23]. In the absence of NADPH oxidase, MDSCs lacked the ability to suppress T cell responses, and differentiated into mature macrophages and DCs [23]. In addition, ex vivo generation of human MDSCs from normal donor PBMCs by exposure to cytokines and tumor cell lines was associated with increased expression of NADPH oxidase constituent proteins [24,25].Taken together, these observations point to NADPH oxidase potentially favoring tumor progression by augmenting MDSC accumulation and immunosuppression in the tumor microenvironment.
EOC is characterized by peritoneal implants, ascites, and accumulation of tumor-associated macrophages and MDSCs [26,27]. The effect of NADPH oxidase in these myeloid cells on tumor progression and local immune responses is unclear. Our major goals were to delineate the role of NADPH oxidase in ovarian tumor progression and in modulating MDSC accumulation and function in murine EOC. We found that tumor progression was similar between WT and engineered NADPH oxidase-deficient mice. Granulocytic and monocytic MDSC accumulation in the peritoneum and spleen was similar between genotypes. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. We therefore conclude that in murine EOC, NADPH oxidase is dispensable for MDSC accumulation and immunosuppressive function. Understanding how the oxidative milieu modulates MDSC function, including NADPH oxidase-dependent and -independent pathways, may lead to novel ways to target these cells to enhance durable anti-tumor immunity.

Ethics statement
All mice were maintained under specific pathogen free conditions at the animal care facility at Roswell Park Cancer Institute and used in compliance with all relevant laws and institutional guidelines under a protocol approved by the Roswell Park Cancer Institute Institutional Animal Care and Use Committee.

Mice
Mice with a targeted disruption of the p47 phox gene (p47 phox2/2 ) have a defective phagocyte NADPH oxidase (NOX2), rendering phagocytes incapable of generating measurable superoxide [28]. p47 phox2/2 mice are backcrossed 14 generations in the C57BL/ 6Ncr. A p47 phox2/2 mouse breeding colony is established at Roswell Park Cancer Institute (Buffalo, NY) and gp91 phox2/2 female mice were purchased from Jackson Labs (Bar Harbor, ME). Female mice (age 6-8 weeks) were used in all experiments. All mice were maintained under specific pathogen free conditions at the animal care facility at Roswell Park Cancer Institute and used in compliance with all relevant laws and institutional guidelines under a protocol approved by the Roswell Park Cancer Institute Institutional Animal Care and Use Committee.

Tumor administration
Mice were administered intraperitoneal ID8 MOSEC cells (5610 6 cells in 200 mL PBS/mouse). For survival experiments, mice were monitored up to 150 days, and euthanized based on abdominal distention, ruffled fur, lethargy or inability to ambulate. In separate experiments, mice were sacrificed on day 42 or 90 after tumor administration for assessment of immunological endpoints. Day 42 corresponds to low tumor burden that models minimal residual disease in patients, and day 90 corresponds to advanced tumor burden.

Isolation and processing of mouse cells
Following sacrifice, peritoneal exudate cells (PECs) were collected by peritoneal lavage with PBS (5-8 ml, containing 1% FBS and 0.5 mM EDTA). PECs were subjected to red cell lysis with ACK buffer, followed by washing. Cell-free supernatants from peritoneal lavage were frozen for subsequent cytokine measurements. Lymph nodes and spleens were also harvested at sacrifice, and single cell suspensions were subjected to red cell lysis and washing. Isolated PECs, splenocytes and lymph node cells were either used within 24 h of harvest for flow cytometry and functional studies or frozen in liquid nitrogen in media containing 20% FBS and 5% DMSO.

Functional assays
MDSC function was evaluated based on suppression of stimulated T cell proliferation in co-culture experiments. Myeloid cells from tumor-bearing mice were purified with anti-CD11b magnetic beads using autoMACS according to the manufacturer's protocol (Miltenyi Biotec Inc., Auburn, CA). Granulocytic MDSCs were column-purified using anti-Ly6G magnetic beads. Following column separation, the purity of cell fractions was analyzed microscopically by Diff-Quick-stained cytospins (Fisher Scientific, Kalamazoo, MI,) and by flow cytometry.
Splenocytes from non-tumor-bearing C57BL/6 female mice were used as naïve T cell targets. Following red cell lysis and washing, splenocytes were incubated with 5 mM carboxyfluoresceindiacetate succinimidyl ester (CFSE; Invitrogen, Grand Island, NY) in PBS for 8 min as previously described [30]. Cells (2.5 or 5610 5 cells/well) were cultured in triplicate in 96-well plates coated with anti-CD3 (10 mg/ml) mAb (BD Biosciences) and B7.1 antigen (0.5 mg/ml) (R&D Systems Inc., Minneapolis, MN). Equal numbers of magnetically separated CD11b 2 , CD11b + , or Ly6G + cells isolated from tumor-bearing mice were added. After 72 h of co-culture, cells were collected, labeled with anti-CD4 and anti-CD8 mAb, and analyzed by flow cytometry. The proliferation of CFSE-labeled CD4 + and CD8 + T cells was evaluated by quantification of CFSE dilution staining as described [30]. The primary endpoint was the proportion of CFSE-loaded CD4 + and CD8 + T cells undergoing $1 replication.

Cytokine analysis
Cell-free supernatants from peritoneal ascites were collected from ID8 MOSEC-bearing mice at day 90. Cytokine and VEGF concentrations were measured by ELISA according to the manufacturer's instructions (BD Biosciences).

Reactive oxidant production
To measure the production of ROIs by granulocytic MDSCs, the oxidation sensitive dye H 2 DCFDA (Invitrogen) was used. PECs isolated from MOSEC-bearing WT and p47 phox2/2 mice at day 90, were stained with anti-CD11b and anti-Ly6G, and then incubated in 1% FBS-containing PBS at room temperature in dark with or without H 2 DCFDA (10 mM; Invitrogen) for 30 min. After washing, cells were incubated with or without PMA (50 nM) for 15 min, then transferred to ice, and immediately analyzed by flow cytometry.

Statistical Analysis
Time to euthanasia was plotted using Kaplan-Meier curves and analyzed using the log-rank method. For immunologic endpoints, comparisons between two groups were assessed by student's t-test or Mann-Whitney if data were not normally distributed. Statistical analysis was performed using Graph Pad Prism 5 software.

NADPH oxidase does not affect overall survival in mice with ovarian cancer
To evaluate the role of NADPH oxidase in regulating ovarian tumor growth, we challenged WT and NADPH oxidase-deficient p47 phox2/2 mice with intraperitoneal MOSEC. Time to progres-sion requiring euthanasia was similar in WT and p47 phox2/2 mice ( Figure 1). Because phagocyte oxidase proteins could have NADPH oxidase-independent signaling, we evaluated survival following tumor challenge in another NADPH oxidase-deficient model (gp91 phox2/2 ) [31], and found no significant difference compared to WT mice (data not shown).
To assess tumor progression prior to overt disease requiring euthanasia, WT and p47 phox2/2 mice were sacrificed either at day 42 (modeling minimal residual disease) or day 90 (modeling advanced disease) after MOSEC administration. At day 42, there was no detectable ascites, but there was variable appearance of tumor nodules measuring ,1-5 mm in the peritoneal cavity of WT and p47 phox2/2 mice. At day 90, both genotypes had ascites with extensive tumor implants. There was no obvious effect of genotype at either time point based on visual inspection. These results show that NADPH oxidase does not have a major role in modulating the progression of ovarian tumor burden.
Effect of NADPH oxidase in modulating MDSCs in the ovarian tumor microenvironment and systemically NADPH oxidase could potentially have multiple effects on the tumor microenvironment that either promote or inhibit tumor progression, including modulation of the cytokine milieu, inflammatory cell recruitment, and antigen display and cross-presentation [32][33][34][35][36][37]. We undertook studies to evaluate the specific effects of NADPH oxidase on MDSC accumulation and function following MOSEC challenge. We evaluated MDSC accumulation in the peritoneal tumor microenvironment, draining lymph nodes (para-aortic and inguinal), and spleens at days 42 and 90 after MOSEC challenge in WT and NADPH oxidase-deficient mice. A well-defined granulocytic MDSC population (CD11b + Ly6-G + Ly6C low ) was observed in PECs and systemically in WT and p47 phox2/2 mice that increased in relation to tumor burden ( Figure 2). We did not observe a well-defined CD11b + Ly6G 2 Ly6-C high population that characterized monocytic MDSCs in other tumor-bearing models [2]; therefore the monocytic MDSC population was defined based on CD11b + Ly6G 2 Ly6C + expression (Figure 2A), realizing that this may contain a mixed population of monocytic cells.
In non-tumor-bearing mice, virtually all of the peritoneal myeloid cells were resident macrophages (CD11b + F4/80 + ) (data not shown). In MOSEC-bearing mice, tumor cells were the predominant cell population in peritoneal lavage fluid. Gating on peritoneal myeloid cells (CD11b + ), the proportion with granulocytic and monocytic MDSCs was greater in advanced (day 90)  In draining lymph nodes, there was a trend toward increased monocytic MDSC accumulation in p47 phox2/2 versus WT mice at day 42 but not at day 90. There was no effect of NADPH oxidase on versus early (day 42) stage tumor burden ( Figure 2B). In particular, the proportion of peritoneal granulocytic MDSCs was 8 to 9-fold greater at day 90 versus day 42. In draining lymph nodes, there was no consistent effect of tumor burden on the proportion of MDSCs ( Figure 2C). In spleens, there was an increased accumulation of MDSCs, particularly granulocytic MDSCs, in mice with advanced versus early disease ( Figure 2D). To our surprise, NADPH oxidase deficiency had no significant impact on the accumulation of granulocytic or monocytic MDSCs at early or advanced disease. Together, these data show that NADPH oxidase does not regulate MDSC accumulation in the local tumor microenvironment or systemically in murine EOC.
Both the tumor and inflammatory cells in the tumor microenvironment can modulate cytokine responses mediating MDSC accumulation and function. Since NADPH oxidase can have a key role in modulating cytokine responses to microbes and microbial products [36] and in angiogenesis [38], we evaluated whether NADPH oxidase regulates inflammatory mediators in the tumor microenvironment. We found that cytokine and VEGF concentrations in ascites (day 90) were similar between WT and p47 phox2/2 mice ( Figure 3). Thus, in the MOSEC tumor microenvironment, NADPH oxidase does not have a significant effect on modulation of mediators produced by MDSCs nor is it likely to influence MDSC recruitment. A limitation of this model is that we cannot distinguish tumor-derived from host-derived products in ascites.

Peritoneal granulocytic MDSCs from WT MOSEC-bearing mice have intact p47 phox -dependent NADPH oxidase function
We next assessed whether NADPH oxidase activity is intact in MDSCs from tumor-bearing mice. Peritoneal exudate cells harvested at day 90 after MOSEC challenge from WT and p47 phox2/2 mice were stimulated with PMA (50 nM for 15 min), and intracellular ROI production in granulocytic MDSCs (CD11b + Ly6G + ) was assessed by H 2 DCFDA fluorescence ( Figure 4A). WT granulocytic MDSCs had intact NADPH oxidase activity ( Figure 4B), whereas ROI generation was absent in p47 phox2/2 cells ( Figure 4C). We did not detect ROI generation in cultured MOSEC cells stimulated with PMA for 15 or 60 min (not shown), indicating that these cells are unlikely to be a source of ROIs in tumor-bearing mice. Together, these results point to p47 phox being required for NADPH oxidase activity in granulocytic MDSCs, but not significantly influencing MDSC accumulation in MOSEC-bearing mice.

T cell suppression by ovarian tumor induced-MDSCs is independent of NADPH oxidase
MDSCs are defined based on both surface marker expression and immunosuppressive function. Since NADPH oxidase did not significantly affect MDSC accumulation in MOSEC-bearing mice, we evaluated whether NADPH oxidase modulates MDSC function, focusing on granulocytic MDSCs. CD11b + cells and Ly6G + cells were isolated by magnetic separation from PECs of WT and p47 phox2/2 mice harvested at day 42 or 90 after MOSEC administration. Cytology and flow cytometry analysis of fractionated PECs showed concordant results, with similar findings between WT and p47 phox2/2 mice. Representative results from PECs harvested at day 90 are shown in Figure 5. The CD11bnegative fraction consisted predominantly of tumor cells, with sparse numbers of myeloid cells (,1%) ( Figure 5A). The CD11benriched fraction was comprised principally of macrophages (CD11b + F4/80 + ), but also contained granulocytic and monocytic MDSCs ( Figure 5B). The Ly6G-enriched fraction contained a mixed population of cells, with granulocytic MDSCs (CD11b + Ly6G + Ly6C low ) being the predominant myeloid cell population. Cytology confirmed the presence of cells with a distinct granulocytic morphology in the Ly6G + enriched fraction ( Figure 5C).
We next evaluated the effect of NADPH oxidase on the suppressive function of the granulocytic MDSC (Ly6G + )-enriched PEC population. Because of the limited number of this population, functional studies were restricted to granulocytic PECs from day 90 after MOSEC administration. Ly6G-enriched PECs from WT and p47 phox2/2 mice completely abrogated anti-CD3/B7.1stimulated proliferation of naïve CD4 + and CD8 + T cells ( Figure 7A). A limitation of these experiments is that Ly6G column purification led to enrichment of granulocytic cells, but not a pure population ( Figure 5C).
To determine the role of NADPH oxidase in the function of systemic granulocytic MDSCs, we evaluated the suppressive effect of Ly6G-enriched splenocytes from MOSEC-bearing WT and p47 phox2/2 mice harvested at day 90. Following column purification, the majority of cells in the Ly6G-enriched fraction had a granulocytic morphology, and 86% of CD11b+ cells expressed granulocytic MDSC (Ly6G + Ly6C low ) markers ( Figure 7B). Coculture experiments showed that the splenic Ly6G-enriched fraction from WT and p47 phox2/2 mice similarly suppressed anti-CD3/B7.1-stimulated proliferation of T cells ( Figure 7C). Taken together, these results show that functionally suppressive granulocytic MDSCs accumulate in the local peritoneal microenvironment and systemically in tumor-bearing mice independently of NADPH oxidase. . The p47 phox component is required for NADPH oxidase activity in granulocytic MDSCs. PECs from WT and p47 phox2/2 mice harvested at day 90 after MOSEC challenge were stimulated with PMA, and intracellular ROI production in CD11b + Ly6G + cells was assessed by H 2 DCFDA fluorescence. A) Gating on all non-aggregated cells (Gate 1), CD11b + Ly6G + cells (Gate 2) were defined using respective isotype controls. B and C) Stimulated ROI production was detectable in WT (B), but not in NADPH oxidase-deficient (C), granulocytic MDSCs. White plot = PMA stimulation; shaded plot = no-stimulation. Results are representative of two experiments. doi:10.1371/journal.pone.0069631.g004

Discussion
Using genetically engineered NADPH oxidase-deficient mice, we found that NADPH oxidase did not affect tumor progression in murine EOC. The accumulation of MDSCs locally and systemically was similar in tumor-bearing WT and p47 phox2/2 mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo anti-CD3/B7.1-stimulated T cell proliferation was NADPH oxidase-independent. Together, our results show that MDSC accumulation and immunosuppression in murine EOC is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function. Since modulation of redox status is a potential therapeutic approach to limit or overcome MDSC-mediated immunosuppression in cancer, it is important to understand NADPH oxidant-dependent and -independent pathways that promote MDSC development.
Oxidative stress by activated monocytes, neutrophils, and MDSCs is considered to be one of the components of the chronic inflammatory environment that suppress T cell function. Consistent with this notion, CD8 + cells transduced with catalase (which depletes hydrogen peroxide) had enhanced viability and CTL function [39]. Prior studies have shown that ROI generation is one of the main characteristics of MDSCs from tumor-bearing mice [4,[40][41][42]. Additional studies point to a direct role for the phagocyte NADPH oxidase in promoting MDSC accumulation and function [22,23]. Corzo et al. [23] showed that splenic MDSCs from mice administered subcutaneous tumors had high levels of ROI generation and increased expression of NOX2 subunits, predominantly in gp91 phox and p47 phox compared with immature myeloid cells from naïve mice. In tumor-bearing mice, gp91 phox was required for MDSC-mediated suppression of ex-vivo antigen-stimulated T cell proliferation and IFN-c production [23]. These findings differ from our findings in murine EOC in which MDSC accumulation and function were NADPH oxidaseindependent. Thus, rather than NADPH oxidase being uniformly required for MDSC accumulation and function, these findings point to factors inherent to the tumor and/or inflammatory microenvironment determining whether NADPH oxidase is required or dispensable.
There are several potential differences in experimental design that might account for NOX2-dependent and -independent MDSC accumulation and function. One possibility relates to the use of p47 phox2/2 mice in our studies versus gp91 phox2/2 mice used by others [22,23]. Both p47 phox and gp91 phox are required components of NOX2 in both humans and in mice. Deficiency of either component leads to chronic granulomatous disease in humans [21] and to a consistent phenotype in engineered mice characterized by impaired host defense and excessive inflammatory responses to certain microbial products [28,31,35,43,44]. We found that p47 phox2/2 MDSCs, although completely deficient in stimulated NOX2 activity, had T cell suppressive properties similar to WT MDSCs. Still, we acknowledge the potential for these phox constituents to have NOX2-independent signaling that could affect MDSC accumulation and function. In tumor-bearing mice, MDSCs secrete and are induced by the myeloid-associated S100 proteins Figure 6. Myeloid peritoneal cells in MOSEC-bearing mice suppress T cell proliferation independently of NADPH oxidase. Purified myeloid (CD11b + ) PECs from MOSEC-bearing WT and p47 phox2/2 mice were co-cultured with CFSE-labeled splenocytes from naïve mice (E:T ratio: 1:1) in anti-CD3/B7.1-coated plates. After 72 h of culture, CD4 + and CD8 + T cell proliferation was assessed based on CFSE dilution as described in methods. A) Representation histograms showing that CD11b + , but not CD11b 2 , PECs from MOSEC-bearing WT and p47 phox2/2 mice (day 90) completely suppress anti-CD3/B7.1-stimulated CD4 + T cells proliferation. Anti-CD3/B7.1 stimulated and unstimulated CD4 + T cells are used as positive and negative controls, respectively. B) CD11b-enriched PECs from MOSEC-bearing WT and p47 phox2/2 mice at day 42 equally suppress both CD4 + and CD8 + T cell proliferation. In contrast, CD11b 2 PECs from the same mice incompletely suppressed anti-CD3/B7.1-stimulated T cell proliferation. C) CD11b-enriched PECs from MOSEC-bearing WT and p47 phox2/2 mice at day 90 completely suppressed T cell proliferation while the CD11b-negative fraction had no significant effect on T cell proliferation. Myeloid PECs collected on day 42 were pooled because of limited cell number, while nonpooled PECs from individual mice were analyzed from day 90 harvests. Individual experiments were performed using PECs from 3 mice per genotype per time point, and each experiment was repeated at least once using PECs from different mice, with similar results. Comparison between genotypes: p = NS. doi:10.1371/journal.pone.0069631.g006 [7,22,45], which can prime NOX2 activity. Cheng et al. [22] reported that S100A9 expressed by hematopoietic progenitor cells inhibited their differentiation to DCs and macrophages and induced generation of MDSCs via a gp91 phox -dependent pathway. The lack of effect of NOX2 in MDSC accumulation in our studies indicates that, at least in the MOSEC model, MDSC generation in vivo is NOX2-independent. Other factors that could influence the requirement for NOX2 in MDSC differentiation relate to differences in tumor-derived products (e.g., secreted growth factors, cytokines, chemokines, and angiogenic products) and the local tumor microenvironment (e.g. peritoneal fluid versus subcutaneous). We believe that a strength of our approach is that we focused on MDSCs in the ascites of MOSEC-bearing mice -which are likely to be the most relevant in modulating anti-tumor immunity in the local tumor microenvironment.
Both tumor-derived factors and products produced by non-tumor cells in the microenvironment, including G-CSF, GM-CSF, cytokines (e.g., IL-1b, IL-4, IL-6, interferon-c), S100A8/A9, cyclooxygenase-2 and prostaglandin E2, indoleamine 2,3-dioxygenase, and ROIs can promote MDSC development and/or immunosuppressive activity [22,24,[45][46][47][48][49]. Because MDSCs can be induced by multiple factors, it is possible that no single molecule is essential for generating MDSCs [1]. In addition, the lack of effect of NOX2 on MDSC accumulation and function does not rule out an effect of other sources of ROIs. There are NOX2-independent sources of ROI generation, including xanthine oxidase [50] and mitochondrial ROIs [51] that have antimicrobial host defense capacity and, conceivably, could contribute to MDSC generation and/or function in the absence of NOX2. These NOX2-independent pathways could potentially react with nitrogen intermediates to generate peroxynitrite, which is required for nitration of TCR/CD8 and induction of T cell tolerance [4]. As a precedent for this concept, the interaction of xanthine oxidase and reactive nitrogen intermediates overcame the requirement for NOX2 as a mediator of ROImediated acute lung injury [52]. We speculate that NOX2 and other ROI-generating enzymes represent alternative pathways for ROI generation that can prime the development of MDSCs. Further work using small molecule inhibitors and genetically engineered mice with deficiencies in specific ROI-generating pathways will be required to test this concept.  . Peritoneal and splenic granulocytic MDSCs from tumor-bearing mice suppress T cell proliferation independently of NADPH oxidase. Ly6G-enriched PECs and splenocytes from MOSEC-bearing WT and p47 phox2/2 mice (day 90) were co-cultured with splenocytes from non-tumor-bearing WT mice (E:T ratio: 1:1). A) Ly6G-enriched PECs completely suppressed anti-CD3/B7.1-stimulated CD4 + and CD8 + T cell proliferation. PECs from 3 mice per genotype were evaluated. B) In Ly6G-enriched splenocytes, the majority of cells had a granulocytic morphology (arrows), and 86% of CD11b + cells expressed granulocytic MDSC markers (Ly6G + Ly6C low ). C) Ly6G-enriched splenocytes from WT and p47 phox2/2 mice modestly suppressed anti-CD3/B7.1-stimulated CD4 + and CD8 + T cell proliferation. N = 3 mice per genotype were used in this experiment, and results are representative of 3 experiments. Comparison between genotypes: p = NS. doi:10.1371/journal.pone.0069631.g007