Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jun 1.
Published in final edited form as: J Immunol. 2012 Apr 23;188(11):5365–5376. doi: 10.4049/jimmunol.1103553

Tumor-expressed iNOS controls induction of functional myeloid derived suppressor cells (MDSC) through modulation of VEGF release1

Padmini Jayaraman *, Falguni Parikh *, Esther Lopez-Rivera *, Yared Hailemichael ‡, Amelia Clark *, Ge Ma †, David Cannan *, Marcel Ramacher §, Masashi Kato , Willem W Overwijk ‡, Shu-Hsia Chen †, Viktor Y Umansky §, Andrew G Sikora *,†
PMCID: PMC3358566  NIHMSID: NIHMS366261  PMID: 22529296

Abstract

Inducible nitric oxide synthase (iNOS) is a hallmark of chronic inflammation which is also overexpressed in melanoma and other cancers. While iNOS is a known effector of myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, its pivotal position at the interface of inflammation and cancer also makes it an attractive candidate regulator of MDSC recruitment. We hypothesized that tumor-expressed iNOS controls MDSC accumulation and acquisition of suppressive activity in melanoma. CD11b+Gr1+ MDSC derived from mouse bone marrow cells cultured in the presence of MT-RET-1 mouse melanoma cells or conditioned supernatants expressed STAT3 and reactive oxygen species (ROS) and efficiently suppressed T cell proliferation. Inhibition of tumor-expressed iNOS with the small molecule inhibitor L-NIL blocked accumulation of STAT3/ROS-expressing MDSC, and abolished their suppressive function. Experiments with VEGF-depleting antibody and recombinant VEGF identified a key role for VEGF in the iNOS-dependent induction of MDSC. These findings were further validated in mice bearing transplantable MT-RET-1 melanoma, where L-NIL normalized elevated serum VEGF levels; downregulated activated STAT3 and ROS production in MDSC; and reversed tumor-mediated immunosuppression. These beneficial effects were not observed in iNOS “knockout” mice, suggesting L-NIL acts primarily on tumor-rather than host-expressed iNOS to regulate MDSC function. A significant decrease in tumor growth and a trend towards increased tumor-infiltrating CD8+ T cells was also observed in MT-RET transgenic mice bearing spontaneous tumors. These data suggest a critical role for tumor-expressed iNOS in the recruitment and induction of functional MDSC by modulation of tumor VEGF secretion and upregulation of STAT3 and ROS in MDSC.

INTRODUCTION

Tumor mediated immunosuppression is a major barrier to successful cancer immunotherapy. Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells originating in the bone marrow and recruited to peripheral sites by inflammation. While these cells are believed to have the potential to differentiate into mature macrophages, dendritic cells and other myeloid cells in the absence of inflammatory stress, cancer-associated inflammation can maintain MDSC in an immature and immunosuppressive state(1-3). Release of soluble mediators such as VEGF, GM-CSF, IL-1β, and other cytokines and growth factors induce T cell suppressive capacity of MDSC, and direct their trafficking into solid tumors where they mediate local immunosuppression. In addition to cancer, a variety of other chronic inflammatory conditions (such as infection, shock, trauma, and surgery) are associated with enhanced recruitment of MDSC (4-6).

MDSC inhibit T cell proliferation and activation through diverse mechanisms, including arginine depletion by expression of the enzyme arginase (ARG), production of reactive oxygen species (ROS)(7, 8), and expression of inducible nitric oxide synthase (iNOS) which leads to nitric oxide (NO) production.(9),(10, 11)iNOS is also overexpressed in many different solid tumors, and its expression is highly associated with diverse inflammatory processes in which iNOS can play a dual role as both an effector molecule and upstream mediator of cytokine release and other proinflammatory events (12). Thus, in addition to its well-described role as an effector mechanism of MDSC-mediated immunosuppression(7, 13), the cancer-associated aberrant expression of iNOS is an attractive candidate mediator of MDSC recruitment and activation. Since a number of strategies for pharmacologic inhibition of iNOS function and/or expression have been developed, including molecules which have entered clinical trials or clinical use, identification of iNOS as a key regulator of MDSC would have both biological and clinical significance.

In support of this hypothesis, there is some evidence that pharmacologic agents which modulate iNOS and NO can also affect MDSC accumulation in tumor-bearing animals. In mice bearing C26GM colon cancer, it was shown that treatment with phosphodiesterase-5 (PDE-5) inhibitor sildenafil, or the non-selective NOS inhibitor L-NAME decreased levels of GR1+ CD11b+MDSC in blood (14, 15). Another study demonstrated that the NO donor nitroaspirin modestly decreased tumor-infiltrating GR1+ CD11b+cells in C26GM model, which was associated with increased T cell function (16). However, as yet the potentially distinct roles of tumor- and host-expressed iNOS as mediators of MDSC recruitment and activation have not been systematically examined and potential mechanisms by which iNOS and NO may affect MDSC recruitment and differentiation are unknown.

In the present study, we use transplantable and spontaneous models of MT-RET syngeneic melanoma (17) to test the hypothesis that tumor-expressed iNOS directs MDSC recruitment, intratumoral trafficking, and acquisition of immunosuppressive function in the tumor-bearing state, and demonstrate a pivotal role for iNOS-dependent VEGF production in regulation of MDSC recruitment in vivo and in ex-vivo bone marrow culture. These data suggest that therapeutic strategies targeting NO production can potently reverse MDSC-mediated immunosuppression by interfering with inflammation-driven MDSC accumulation and acquisition of suppressor function.

MATERIALS AND METHODS

Mice and Tumor models

C57BL/6, iNOS-/-(B6.129P2-Nos2tm1Lau/J) and RAG-/-(B6.129S7-Rag1tm1Mom/J) mice were obtained from the Jackson Laboratory and housed in the Mount Sinai animal facility under pathogen-free conditions. All animal experiments were performed in accordance with the regulations of the local MSSM institutional animal care and use committee (IACUC). The B16 melanoma cell line was obtained from American Type Culture Collection (ATCC). The MT-RET-1 mouse melanoma tumor cell line (C57BL/6 background) is a transplantable tumor developed from a spontaneous melanoma growing in the MT-RET transgenic mouse (provided by Willem Overwijk, University of Texas MD Anderson cancer center, TX).

Transgenic Mice

C57BL/6 mice expressing human ret oncogene in melanocytes under the control of mouse metallothionein promoter-enhancer were kindly provided by Izumi Nakashima (Chubu University, Aichi, Japan). All mice were crossed and kept under specific pathogen free conditions in the animal facility of the German Cancer Research Center. Experiments were performed in accordance with government and institute guidelines and regulations. Spontaneous tumor development was assessed macroscopically and the survival of mice was monitored daily. Immunoblotting: Cells were lysed with NP40 lysis buffer in the presence of protease and phosphatase inhibitors. Protein lysates were subjected to 10% SDS-PAGE electrophoresis and transferred to nitrocellulose membranes. Membranes were probed with appropriate primary antibodies and incubated overnight at 4°C. Membranes were washed and incubated for 1 hour with secondary Ab conjugated with peroxidase. Results were visualized by chemiluminescence detection using a commercial kit (Millipore).

Ex-vivo generation of MDSC

Co-culture system

Bone marrow cells were derived aseptically by flushing the femur of naïve wild type (C57BL/6) mice. Single cell suspension was prepared in RPMI (Hyclone) complete medium. 106 BM cells were co-cultured with MT-RET tumor cells (transwell compartment) either in the presence or absence of 1mM of L-NIL, in the HTS Transwell-24 well plate with a treated polyester membrane of 0.4μm pore size (Corning). The plate was incubated at 37°C for a total of 6 days. Media and L-NIL were replaced on day 3. Cells from the lower compartment were harvested and stained for MDSC (GR1+CD11b+) or pSTAT3 or ROS (DCF-DA) on day 3 and day 6. All flow cytometry data was acquired using FACS Calibur and analyzed using flowjo7.6 software.

Tumor supernatant transfer

Tumor conditioned supernatants were derived from actively growing MT-RET cells in the presence or absence of L-NIL 1mM, 48 hours after initial culture. For neutralizing VEGF in TCCM obtained at 48 hours, VEGF neutralizing antibody (LEAF™ Purified Anti-mouse VEGF-A Antibody; Biolegend) was added at different concentrations (0.1μg/ml, 0.3μg/ml and 1μg/ml) for 4 hours at room temperature and spun to obtain supernatants. TCCM were then added at 30% v/v to 106 bone marrow cells (derived from naïve mice as mentioned above) in the presence or absence of exogenously added rVEGF (recombinant VEGF; Peprotech) at 0.1μg/ml, 0.3μg/ml, 1μg/ml and 10μg/ml for 5 days, with mediumreplaced on day 3. Cells were harvested for MDSC flow cytometry as previously described. For some experiments, the long-acting NO donor DiethylenetriamineNONOate (DETA-NONOate; Sigma) was added directly to bone marrow cultures.

Nitrotyrosine staining

Cells were stained with GR1FITC, CD11b APC and mouse anti-nitrotyrosine (Clone A8.2, Millipore) or isotype antibody for 30 minutes in the dark at 40C. Cells were then washed, spun and resuspended in staining buffer containing PE conjugated goat anti-mouse IgG and incubated further for 30 min in the dark at 40C. Cells were washed with staining buffer and re-suspended in 300 μl of staining buffer for FACS analysis using the FACScalibur.

VEGF-R staining

Cells were stained with GR1 FITC, CD11b APC and mouse anti-VEGF-R1(ebiosciences, clone Avas12a1) or VEGF-R2 (ebiosciences, clone AFL4) or isotype antibody for 30 minutes in the dark at 40C. Cells were then washed, spun and resuspended in staining buffer containing PE conjugated goat anti-mouse IgG and incubated further for 30 min in the dark at 40C. Cells were washed with staining buffer and re-suspended in 300 μl of staining buffer for FACS analysis using the FACScalibur.

Detection of ROS (Reactive Oxygen Species) levels in MDSC

Cells were stained with GR1 PE and CD11B APC for 20 minutes in the dark at 4oC, washed, spun and resuspended in 200μl of PBS++ (450 ml ddH2O, 125 μl 2M CaCl2, 250 μl 1M MgCl2, 50ml 10X PBS) containing 2μM DCFDA (Molecular probes/ Invitrogen) and incubated for 15minutes in dark at room temperature. Cells were washed with staining buffer and resuspended in 300 μl of staining buffer for FACS analysis using the FACS calibur. Data was analyzed using flowjo 7.6 software.

Detection of pSTAT3 levels in MDSC

Cells were stained with GR1 FITC and CD11b APC as mentioned above. They were fixed with BD fixation buffer and incubated for 10-30 minutes at 37°C. Cells were washed with staining buffer and permeablized by adding BD perm wash buffer III. The plate was incubated for 30 minutes on ice. Cells were again washed with staining bufferand stained with anti-pSTAT3 PE (BD Biosciences) 1 hour at room temperature. Cells were washed with staining buffer and resuspended in 300 μl of staining buffer for FACS analysis using the FACS calibur. Data was analyzed using Flowjo 7.6 software.

BioPlex Multiplex Assay for cytokine analysis

GM-CSF, G-CSF, M-CSF, IL-1, IL-6, MIP-1 α, MCP-1 and VEGF levels were measured in serum obtained from MT-RET or B16 tumor bearing mice from both control and L-NIL treated groups or tumor conditioned supernatants derived MT-RET cell lines cultured in the presence or absence of L-NIL using CTOT01 BioPlex Multiplex Assay kit (BD Biosciences) run on a Luminex (BD Biosciences) processor using the manufacturer’s instructions.

Transplantable tumor models

Mice were injected subcutaneously with 3×105 MT-RET or B16 tumor cells in suspension. Mice were manually restrained, and the tumors were measured twice a week with calipers. Tumor sizes were determined according to the bi-dimensional product of the longest measurement x its perpendicular. Once tumors became established (> 30 mm2, roughly 2 weeks), half of the mice received L-NIL (0.2%) in drinking water for 7 days, and the other half received plain drinking water. Water bottles were replaced promptly when the water level was low. After completing the course of L-NIL, all mice were sacrificed and different organs were collected.

Serum collection

Blood was collected by cardiac puncture and allowed to clot at room temperature prior to centrifugation to obtain serum.

Cell harvesting/purification

Spleens were mashed on a filter mesh cup (Fisher) that is placed on top of a 50 ml tube, using a syringe plunger. 10ml of RPMI containing 1% FBS was added on mesh cup and cells were centrifuged at 1400 rpm for 10 minutes at 4° C. The pellet was resuspended in 2ml of ACK lysing buffer (GIBCO) and incubated at room temperature for 3 minutes to remove red cells. Cells were washed with RPMI containing 1% FBS and the pellet was resuspended in 2ml of RPMI-CM. Femur was collected and the ends of the femur were cut to expose the cavity containing bone marrow. The bone marrow cells were flushed out with RPMI using a 27.5 G syringe. Tumors were collected and single cell suspensions were prepared as described above for spleen. Tumor infiltrating lymphocytes (TIL) were isolated using ficoll gradient centrifugation.

MDSC (GR1+CD11b+ cells) were stained in splenocytes, bone marrow cells, and TIL as described for ex-vivo experiments. T cells were identified by with CD4 APC and CD8 PERCP staining of single-cell suspensions from spleen and tumor. ROS detection from single-cell suspensions of splenocytes and TIL was done as described for ex-vivo experiments.

MDSC Suppression Assay

PurifiedMDSC (GR1+CD11b+ cells) were sorted from spleensor tumor of untreated MT-RET-bearing mice, L-NIL-treated or iNOS-KO mice using a MoFlo XDP cell sorter (Beckman-Coulter, Brea, CA). Further CD11b+ cells were sorted from Tumor infiltrating leukocytes from the tumors of untreated or L-NIL treated MT-RET-bearing mice.CFSE (Carboxyfluoresceindiacetatesuccinimidyl ester) 5mM stock solution was diluted to 20μM in PBS and added immediately to 2X cell concentrated cell suspension (5 × 107 cells/ml) of splenocytes of wild-type C57BL/6 mouse. The mixture was incubated for 10 minutes at 37° C. 5 volumes ice-cold RPMI/10% FBS were added and incubated for 5 minutes on ice. Cells were washed three times with RPMI CM. Sorted MDSC (GR1+CD11b+ from spleen or CD11b+ from TIL) cells from each condition were added to CFSE labeled wild type splenocytes at different ratios and activated with soluble anti-CD3 (0.3μg/ml) + anti-CD28 (0.5μg/ml) antibodies for 72 hours at 37° C.

Cells were harvested in 96 well plate and washed with staining buffer. 100μl of Master mix staining solution containing 1:400 anti CD8 -PERCP and 1:400 anti CD4 -APC antibodies was added to each well and incubated for atleast 20 minutes at 4° C. CFSE dilution was measured by flow cytometry (FL-1 channel) using FACS Calibur.

CTL Assay

Splenocytes or TIL harvested from tumor bearing mice were activated with gp100/trp2 peptides for 72 hours to enrich for tumor specific T cells. The activated cells were then cultured with varying ratios of gp100/trp2-pulsed EL-4 target cells and CTL assay was performed according to manufacturer’s instructions. (CytoTox 96® Non-Radioactive Cytotoxicity Assay from Promega).

RESULTS

Inhibition of tumor-expressed iNOS decreases accumulation and suppressive function of GR1+ CD11B+MDSC in ex vivo bone marrow cultures

To assess the role of tumor-derived soluble factors in generation of MDSC, we used a modification of the ex-vivo bone marrow progenitor culture method described by Younet al(18), in which MDSC are differentiated from bone marrow cells in tumor/myeloid cell co-culture, or in the presence of tumor-conditioned supernatants. While the percentage of GR1+CD11b+ cells in 6 day culture remained stable-to-decreasing in medium alone, either co-culture with MT-RET-1 (Fig. S1A) cells across a permeable membrane or addition of tumor-conditioned supernatants was sufficient to induce ex-vivo accumulation of functional GR1+CD11b+ MDSC (Fig. 1A-B). Like in vivo-derived MDSC, these cells were capable of dose-dependent suppression of CD4 and CD8 T cell proliferation in suppression assay (Fig. 1A and 1C, and data not shown). MDSC accumulation from bone marrow cells co-cultured with MT-RET-1 tumor cells in a transwell chamber was significantly decreased in the presence of 1mM L-NIL (Fig. 1D), demonstrating that accumulation is iNOS-dependent.

Figure 1. Tumor -expressed iNOS is required for accumulation and functional activity of ex-vivo-derived CD11b+GR1+ MDSC.

Figure 1

Open in a new tab

A. Representative FACs plots of T cell (CD4 and CD8) proliferation as measured by CFSE dilution. B. Time course of accumulation of bone-marrow derived CD11b+GR1+ cells cultured in medium alone, in the presence of MT-RET conditioned supernatant, and in transwell co-culture with MT-RET cells. C. Dose response curve of ability of sorted ex-vivo MT-RET supernatant-derived CD11b+GR1+ MDSC(harvested on day 4 in culture)to inhibit CD4 and CD8 T cell proliferation. D. Relative accumulation (day 5-6) of CD11b+GR1+ MDSC in MT-RET transwell co-culture +/− 1mM L-NIL. E. Relative accumulation of MDSC(day 5)in the presence of MT-RET-conditioned supernatants alone, or MT-RET sups + 1mM L-NIL added to the tumor cells (L-NIL-tumor) and/or to the bone marrow cell culture (L-NIL-BM). Each graph summarizes data from at least 3 experiments.

To determine the relative importance of iNOS expression in the tumor and myeloid compartments, MT-RET-1 supernatant transfer experiments were performed (Fig. 1E). Incubation with the NO donor DETA-NONOate enhanced accumulation of MDSC (Fig. S2), and addition of L-NIL to bone marrow cultures partially suppressed tumor supernatant-induced MDSC accumulation, suggesting that there is some direct regulatory effect of NO on bone marrow cells. However, addition of L-NIL to the tumor cells during production of supernatants was more effective and almost completely reduced MDSC accumulation to baseline levels. The combination of L-NIL applied to both the tumor- and myeloid cultures was most effective of all, reducing MDSC accumulation to below baseline levels. The effect of L-NIL in tumor cell culture was not due to carry-over of L-NIL into the myeloid culture, since L-NIL is labile in aqueous solution, and equivalent concentrations of L-NIL incubated at 37°C in medium alone without tumor cells had no effect (data not shown). Thus, we conclude that inhibition of tumor-expressed iNOS is sufficient to suppress accumulation of functional MDSC induced by tumor-derived soluble mediators in ex-vivo culture.

L-NIL downregulates STAT3 activation and inhibits ROS production in MDSC

The transcription factor STAT3 is a major regulator of MDSC functional activity which is upregulated in both human- and murine-derived MDSC (19). VEGF and STAT3 can interact in a positive-feedback loop in which STAT3 activation drives VEGF expression and signaling through VEGF-R activates STAT3 in many cell types (20, 21). One consequence of STAT3 activation in MDSC is upregulation of NADPH oxidase subunits p47phox and gp91phox leading to production of reactive oxygen species (ROS) (22, 23). Thus we examined the effect of iNOS inhibition on STAT3 activation and ROS production by MDSC in ex-vivo bone marrow culture.

Bone-marrow-derived CD11b+GR1+ cells expressed low levels of resting ROS in the absence of tumor cells. However, ROS levels were dramatically upregulated in transwell culture with MT-RET-1 cells (Fig. 2A-B), and upregulation of ROS was suppressed to levels below baseline by addition of L-NIL to co-culture. Co-culture with MT-RET-1 cells also strongly upregulated levels of activated phospho-STAT3 in bone-marrow derived MDSC, and STAT3 activation was suppressed by addition of L-NIL (Fig. 2C). Parallel experiments examining the effect of L-NIL on T cell suppressive activity of MT-RET-1 supernatant-derived MDSC demonstrated that addition of L-NIL to the tumor cell culture was sufficient to completely abolish MDSC-mediated T cell suppression (Fig. 2D). Together, these data demonstrate that STAT3 activation and ROS production are induced in MDSC by soluble mediators, and downregulated by iNOS inhibition.

Figure 2. iNOS inhibition suppresses upregulation of ROS production and STAT3 activation by tumor-derived soluble mediators.

Figure 2

Open in a new tab

Representative histograms (A.) and quantitative summary from at least 3 experiments (B.) of effect of L-NIL on MT-RET-1-mediated ROS production by ex-vivo-derived MDSC (gated on CD11b+GR1+ cells)on day 6 after initial culture.C. Anti phospho-STAT3 antibody staining of permeablized cells is upregulated in CD11b+GR1+ MDSC in MT-RET / bone marrow cell transwell culture, and reversed by 1mM L-NIL (p≤0.05) on day 3 after initial cultures were set up. D. Ability of MT-RET supernatant to induce CD4 and CD8 T cell suppressive activity of ex-vivo-derived MDSC(harvested on day 4 in culture)is abolished by incubation of MT-RET cells with 1mM L-NIL. Each graph summarizes data from at least 3 experiments.

iNOS dependent VEGF secretion is required for tumor-induced MDSC accumulation

Tumor derived factors such as VEGF, GM-CSF, G-CSF, MCP-1 and inflammatory cytokines such as IL-1β and IL-6 play a pivotal role in eliciting MDSC from bone marrow as well as directing their accumulation in the spleen, tumor, and other peripheral sites (1).VEGF is a major angiogenic growth factor that is secreted by many murine cancer models, and has been shown to play an important role in the induction of MDSC by both mouse and human cancers (24). Release of VEGF from tumor cells has also been shown to be upregulated by iNOS expression. The potential for VEGF to be a direct modulator of MDSC differentiation and functional activity in our system is supported by the robust secretion of VEGF by MT-RET-1 tumor cells, and the expression of VEGF receptors VEGFR-1 and VEGFR3 on ex-vivo derived MDSC (Fig. 3A-B). VEGF release by MT-RET-1 cells was reduced >2-fold by L-NIL in vitro (Fig. 3A) without evidence of direct toxicity to tumor cells (Fig. S1B).

Figure 3. Tumor-secreted VEGF mediates iNOS-dependent cross-talk required for MDSC accumulation.

Figure 3

Open in a new tab

A. Relative VEGF levels in 48 hour supernatants derived from MT-RET-1 cells cultured in the presence or absence of 1mM L-NIL. B. Expression of VEGF-R-1 and 3 in CD11b+GR1+ MDSC derived ex-vivo from wild type bone marrow cultured with MT-RET-1 supernatants for 5 days. C. In vivo concentration of VEGF, IL-6, G-CSF and IL-1β in serum of untreated or L-NIL-treated wild-type mice on day 21 after MT-RET-1 tumor injection L-NIL treatment decreases VEGF in both serum from RET tumor bearing mice and RET tumor supernatants compared to their untreated counterparts (p≤0.05). VEGF concentration in serum was compiled from at least 3 experiments with n=5 mice per group. D.Ability of the indicated concentrations of anti-VEGF neutralizing antibody to block MT-RET supernatant-induced MDSC accumulation ex vivoat 5 days in culture. Neutralization of VEGF leads to significantly decreased accumulation of MDSC in BM cultures (p≤0.05). E. Ability of the indicated concentrations of recombinant murine VEGF to reverse L-NIL mediated suppression of MT-RET-1 supernatant-induced MDSC accumulation ex-vivoat 5 days in culture. Each graph summarizes data from at least 3 experiments.

We used Luminex to profile cytokine expression levels in MT-RET 1 tumor bearing untreated and L-NIL-treated wild-type and iNOS-deficient mice (Fig. 3C). We observed non-significant trends towards decreased levels of IL-6, G-CSF, and IL-1β in serum from L-NIL-treated wild-type and iNOS knockout mice. However, elevated serum VEGF levels in tumor-bearing mice were significantly reduced by treatment with L-NIL (Fig. 3C, Fig. S4); the magnitude of this decrease was similar in L-NIL-treated mice bearing both early and late MT-RET tumors (data not shown). Although L-NIL also significantly decreased VEGF levels in iNOS-deficient mice, untreated iNOS knockout mice showed a paradoxical trend towards elevated serum VEGF levels with respect to wild-type mice, suggesting that it is tumor-rather than host-expressed iNOS that is responsible for enhanced VEGF production in tumor-bearing mice.

A functional role for VEGF in the accumulation of MDSC cultured with MT-RET-1 supernatants was confirmed by the ability of anti-VEGF neutralizing antibody to suppress accumulation of CD11b+GR1+ cells in a dose-dependent fashion (Fig. 3D). Conversely, addition of exogenous recombinant VEGF to L-NIL-treated MT-RET-1 supernatants was sufficient to restore the accumulation ofMDSC in ex-vivo culture, again in a dose-dependent fashion (Fig. 3E). These data suggest that upregulation of VEGF production is a key mechanism through which tumor-expressed iNOS regulates the induction of MDSC.

iNOS inhibition leads to decreased induction and accumulation of MDSC in vivo

It has been demonstrated in numerous tumor models that GR1+CD11b+ MDSC are produced in the bone marrow in response to tumor-derived soluble mediators which also direct their accumulation in spleen and tumor (25, 26). Since MDSC accumulate in increasing numbers as tumors grow in size (27), we studied accumulation and functional activity of MDSC in tumors treated beginning on day 14, at which point L-NIL had no effect on tumor growth (See Fig. 6A). We demonstrated efficacy of iNOS inhibition by staining for nitrotyrosine, a stable product formed by reaction of NO with ROS species. Nitrotyrosine levels in splenic MDSC from both iNOS deficient and L-NIL-treated mice were both significantly lower than in MDSC from untreated wild-type mice (Fig. 4C), demonstrating efficacy of pharmacologic iNOS inhibition with L-NIL, and suggesting that compensatory upregulation of other NOS isoforms does not occur in iNOSKO mice.

Figure 6. Pharmacologic iNOS inhibition reverses systemic T cell decline in tumor-bearing mice and enhances intratumoral accumulation of CD4 and CD8 T cells.

Figure 6

Open in a new tab

A. Growth of MT-RET untreated and “early” (day 4) L-NIL-treated melanoma in wild-type and RAG-KO mice demonstrates modest T/B cell-dependent anti-tumor effect. Treatment of established (day 14) tumors does not lead to decreases in tumor growth.B. Absolute and relative number of tumor-infiltrating CD4 and CD8 T cells in untreated and L-NIL-treated (WT or iNOS-KO) MT-RET-bearing mice on day 21. Each graph shown represents pooled data from at least 3 experiments with n=5 mice per group. C.Cytotoxicity assay against EL-4 target cells with effector cells from splenocytes or TIL of control or L-NIL treated mice (harvested on day 21 after MT-RET injection, enriched for gp100/trp2 specific T cells). CTL efficacy is expressed as percentage of target cell populations lysed. Graph is representative of 2 experiments with n=5 mice per group.

Figure 4. Pharmacologic iNOS inhibition or genetic ablation of host iNOS significantly reduce accumulation of MDSC in transplantable MT-RET-1 tumors.

Figure 4

Open in a new tab

A. Gating strategy and representative FACS plots of MDSC from tumor, spleen, and bone marrow of untreated and L-NIL-treated mice bearing transplantable MT-RET melanoma with percentages of GR1 Hi or Int populations. B. Relative percentage of CD11b+GR1+ MDSC populations in the tumor, spleen and bone marrow of untreated, L-NIL-treated, and iNOS-KO mice on day 21-23 after MT-RET-1 injection. Numbers are presented as percentage of total (high + intermediate) CD11b+ GR1+ double-positive cells. Tumors from L-NIL-treated mice have significantly decreased numbers ofCD11b+GR1+ cells *p≤0.01).C. Nitrotyrosine expressed by CD11b+GR1+ MDSC from spleens of untreated, L-NIL treated or iNOSknockoutMT-RET-1-bearing mice on day 22 normalized to isotypecontrol. L-NIL treated mice show decreased nitrotyrosine expression as compared to WT mice (*p≤0.01).Each graphrepresents pooled data from at least 3-5 experiments with n=5 mice per group.

Mice bearing transplantable MT-RET-1 (Fig.4A-B) or B16 melanoma (Fig. S4) had increased percentages of MDSC populations in bone marrow and substantial accumulation of intratumoral MDSC. Treatment with L-NIL decreased total MDSC in bone marrow and tumor by 2-3 fold in MT-RET-1-bearing mice, and significantly decreased total MDSC in tumor and spleen of B16-bearing mice (Fig. S4). Dolcettiet al(28) have recently classified GR1+ CD11b+ cells into 3 distinct populations of GR1hi, GR1int (intermediate), and GR1lo CD11b+ cells, each which differ in their expression of surface markers and mechanisms of immune suppression, but all of which have suppressive activity. However, the overall magnitude of MDSC decrease with L-NIL treatment was similar for GR1hi and GR1int MDSC (Fig. 4A and Table 1). The number of tumor-infiltrating MDSC was significantly decreased in MT-RET-1-bearing (Fig. 4B) and B16-bearing (Fig. S4) iNOS deficient mice; however, numbers of GR1+CD11b+cells in the bone marrow were notsignificantly reduced in iNOSKO mice.

Table 1.

Absolute numbers of CD11b+Gr-1+, Gr-1+Hi and Gr Int cells from TIL, Spleen and Bonemarrow.

CD11b+Gr-1+ Gr-1 Hi Gr-1 Int
TIL CONTROL 0.24±0.04 0.07±0.02 0.16±0.03
L-NIL 0.09±0.02* 0.02±0.01* 0.07±0.02*
NOS KO 0.05* 0.01* 0.04*
SPLEEN CONTROL 1.76±0.6 0.77±0.2 0.88±0.2
L-NIL 0.9±0.3 0.40±0.1 0.50±0.1
NOS KO 0.80±0.4 0.40±0.1 0.40±0.2
NO TUMOR 1.83±0.3 0.97±0.2 0.89±0.2
BONEMARROW CONTROL 3.31±0.9 2.31±0.6 0.82±0.2
L-NIL 1.7±0.3 1.19±0.2 0.52±0.1
NOS KO 2.07±0.7 1.61±0.5 0.45±0.1
NO TUMOR 1.10±0.1 0.29 0.42
Open in a new tab
*

p≤0.05

iNOS is required for acquisition of T cell suppressive function in MDSC

We utilized ex-vivo suppression assay to confirm that purified GR1+CD11B+cells derived from the spleens and tumorof MT-RET-1-bearing mice were competent to suppress T cell proliferation, and to assess the effect of L-NIL treatment on their suppressive function. Total GR1+CD11B+ cells consisting of both GR1hi and GR1int populations were sorted from control and L-NIL-treated splenocytes from MT-RET-1 tumor-bearing mice and cultured with CFSE labeled splenocytes activated with anti-CD3 and anti-CD28 antibodies (Fig 5A). While spleen- or tumor-derived MDSC from tumor-bearing untreated mice could efficiently suppress proliferation of both CD4 and CD8 T cells in a dose-dependent fashion, MDSC from L-NIL-treated mice failed to suppress proliferation even at a 1:1 ratio (Fig. 5A-B and Fig. S3). No suppression was observed when T cells were co-cultured with CD11b-GR1-cells from tumor-bearing mice, or GR1+CD11b+cells from tumor-free mice (Fig. 5B).

Figure 5. CD11b+Gr1+ MDSC sorted from spleens of untreated MT-RET-1-bearing mice, but not L-NIL-treated mice, inhibit CD4 and CD8 T cell proliferation.

Figure 5

Open in a new tab

A. Relative proliferation of CD3/CD28-stimulated CD4 and CD8 T cells in the presence of different ratios of sorted CD11b+GR1+MDSC from spleens (left) or TIL (right) ofuntreated and L-NIL-treated miceon day 21 after MT-RET-1 injection orB. Relative proliferation of CD8 (top) and CD4 (bottom) T cells in the presence of 1:1 CD11b+Gr1+ MDSC or CD11b-GR1-cells sorted from splenocytes of iNOSKO mice, L-NIL-treated mice, or untreated mice in the presence or absence of 1mM L-NIL added to the sorted MDSC prior to the final wash step. Each graph shown above represents pooled data from at least 3 experiments with n=5 mice per group.L-NIL treated MDSC lose their ability to suppress T cell proliferation compared to WT (*p≤0.05)C.Effect of L-NIL or genetic ablation of host iNOS on ROS production in vivo by CD11b+GR1+ MDSC from spleenon day 21 after MT-RET injection. L-NIL treatment leads to decreased production of ROS from splenic GR1+Cd11b+ MDSC. D. L-NIL treatment in vivodownregulates activated (phosphorylated) STAT3 expression and pSTAT3/ total STAT3 ratio in MT-RET-1 tumors by Western blot. Each graph represents pooled data from at least 3 experiments.

Since iNOS is a known effector mechanism of MDSC-mediated T cell inhibtion, we performed additional experiments to determine whether suppression of ex-vivo MDSC function by L-NIL was caused by direct inhibition of iNOS expressed by MDSC. We found that the addition of even high doses (3mM) of L-NIL to purified MDSC prior to washing and plating does not reverse their ability to suppress T cell proliferation (Fig. 5B “L-NIL wash”), demonstrating that the effects of L-NIL are not due to carry-over of trace quantities of inhibitor into ex-vivo cultures. We also found that MDSC from iNOS deficient mice–which cannot utilize iNOS as an effector mechanism—retained greater than 50% CD8+ T cell suppressive capacity when compared to MDSC from L-NIL treated mice which had no suppressive capacity (Fig. 5B “iNOS KO”). Thus we conclude that the primary effect of L-NIL treatment is suppression of the tumor-directed acquisition of functional activity by MDSC in vivo.

iNOS inhibition in vivo also had an effect on MDSC ROS and activated p-STAT3 levels similar to that observed in the ex-vivo model. ROS levels in MDSC from spleen of L-NIL treated tumor-bearing mice were significantly reduced as compared to untreated mice (Fig. 5C). As was the case for VEGF levels, MDSC from tumor-bearing iNOS KO mice had ROS levels comparable to wild-type mice. Western blot analysis of total and phospho-STAT3 levels in MT-RET-1 tumors demonstrated robust phosphorylation of serine 727 and tyrosine 705 in tumors from untreated mice. STAT3 activation was downregulated by roughly two-fold in L-NIL-treated mice (Fig. 5D), suggesting that iNOS inhibition in vivodownregulates STAT3 activation in the tumor, in tumor-infiltrating myeloid cells, or both.However, we did not observe a similar downregulation of STAT3 phosphorylation in spleens of L-NIL-treated mice (data not shown).

Tumor-expressed iNOS drives cancer-associated immunosuppression and iNOS inhibition enhances immune-mediated tumor control

We studied the effect of L-NIL on growth of transplantable MT-RET-1 tumors in syngeneic mice, including: wild-type C57BL/6 mice, iNOS knockout mice, and RAG knockout mice which lack mature T cells and B cells. In immunocompetent wild-type mice, 7-10 days L-NIL treatment modestly inhibited growth of early (day 4) but not established (day 14) tumors (Fig. 6A andFig. S4A). While we (29) and others (30) have described direct anti-tumor activity of iNOS inhibition in several models of human xenograft growth in immunocompromised mice, inhibition of tumor growth by L-NIL in syngeneic melanoma models appears to depend on adaptive immune mechanisms, since L-NIL had no effect on tumor growth in RAG KO mice (Fig. 6A), and we observed no direct effect on growth of MT-RET-1 tumor cells in vitro (Fig.S1B).

Since the lack of effect in RAG-deficient mice strongly suggested that L-NIL acts through immune mechanisms, we examined the effect of L-NIL on CD4 and CD8 T cell accumulation in tumor-bearing wild-type and iNOS-deficient mice. Treatment of both 4-day (data not shown) and 14-day MT-RET-1 tumors with L-NIL increased numbers of tumor-infiltrating and splenic CD4 and CD8 T cells in wild-type mice (Fig 6B). While splenic T cell levels were reduced in the tumor-bearing state, L-NIL treatment restored numbers of splenic CD4 and CD8 T cells to numbers comparable to those seen in tumor-free mice (3×106 CD4 and 2.5×106 CD8 T cells) and reversed the splenomegaly observed in many tumor-bearing mice (data not shown). A similar trend towards recovery of splenic T cell numbers was observed in mice bearing transplantable B16 melanoma (Fig. S4). Increased T cell numbers in L-NIL-treated mice were also associated with enhanced per-cell cytolytic activity of CTL from spleen and TIL (Fig 6C) against EL4 cells pulsed with peptides for the gp100 and Trp2 melanoma antigens expressed by MT-RET-1 (Fig. S1C and data not shown). Thus, pharmacologic inhibition of INOS with L-NIL reverses both quantitative and qualitative tumor-mediated T cell dysfunction.

Unlike L-NIL treated mice, iNOSKO mice did not show enhanced numbers of splenic or tumor-infiltrating CD4 or CD8 T cells (Fig. 6B), or altered tumor growth (Fig. 6A). A similar lack of effect of host iNOS knockout on tumor growth and T cell numbers was observed in the B16 model (data not shown). L-NIL treatment of iNOS-deficient mice led to robust recovery of splenic CD4 and CD8 T cell numbers (Fig. 6B), demonstrating that that the failure of host iNOS ablation to restore T cell numbers in tumor-bearing mice is not due to upregulation of iNOS-independent compensatory mechanisms in iNOSKO mice. These data confirm a role for tumor-expressed iNOS in mediating tumor-mediated T cell dysfunction and suppression of anti-tumor immunity, most likely by driving accumulation and activation of MDSC.

iNOS inhibition enhances intratumoral CD8+ T cell infiltration and suppresses growth of spontaneous melanoma in MT-RET transgenic mice

To test the effect of iNOS inhibition on spontaneously-arising tumors, we compared melanoma growth in untreated and L-NIL-treated MT-RET transgenic mice. Mice were allowed to develop palpable tumors, and then randomized to 10 days treatment with L-NIL (0.2%) or control. Spontaneous melanomas tended to arise in the face, head, and neck (Fig. 7A), although they also involved other parts of the body. These tumors contained substantial numbers of GR1+CD11b+ MDSC (10-20% of all CD45+ leukocytes), and MDSC were also detected in the metastatic (tumor-infiltrated) lymph nodes, although at much lower levels (<1%; Fig. 7B). L-NIL treatment significantly suppressed tumor growth (Fig. 7C), as was shown previously with treatment of early MT-RET-1 transplantable tumors (Fig. 6A), and this was associated with a trend towards increased numbers of CD8+ T cells in the primary tumor (Fig. 7D). We also observed a modest but significant decrease in the number of NO-producing MDSC in both tumor and lymph node (7D).

Figure 7. iNOS inhibition suppresses melanoma growth and reduces number of MDSC in ret transgenic tumor bearing mice.

Figure 7

Open in a new tab

A. H& E staining from axial and coronal sections of heads of tumor bearing ret transgenic mice.B.Representative plots showing percentage of MDSC populations gated on CD45+ cells in tumor and metastatic LN.C.Treatment with L-NIL (10 mice per group) induces a decrease in the weight of primary tumorD.CD8 T cells were quantified in both tumor and metastatic LN from both untreated and L-NIL treated mice and expressed as percentage of CD45+ leukocytes. NO was detected intracellularly in cells from treated (L-NIL) and non-treated (control) tumor bearing mice by flow cytometry, and results presented as percentage of NO+ cells within total Gr1+CD11b+ MDSC.

DISCUSSION

In the present study we demonstrate that tumor-expressed iNOS plays a key role in recruitment and activation of MDSC in the tumor-bearing state, and that iNOS inhibition reverses tumor-mediated immune suppression in mouse melanoma. The beneficial effect of iNOS inhibition on accumulation of functional MDSC, CD8 T cell numbers, and tumor growth is seen in both transplantable and spontaneous melanoma models. This role for iNOS as a mediator of recruitment of functional MDSC is distinct from its previously-described role as an effector mechanism of immunosuppression via the direct effects of high-output NO production (9) and formation of the reaction product peroxynitrite (11) on T cell activation, proliferation and survival. Rather, we found that iNOS inhibition suppressed VEGF release, STAT3 activation, and ROS upregulation required for induction of functional MDSC (see Fig. 8 for working model). These results are consistent with the known role of NO as a signal transduction mediator capable of controlling gene expression and cellular differentiation and development (31)

Figure 8.

Figure 8

Open in a new tab

Model of iNOS-mediated VEGF production in control of tumor-directed recruitment and functional maturation of MDSC.

Treatment with the selective iNOS inhibitor L-NIL reversed tumor-associated immunosuppression by decreasing numbers of tumor-infiltrating MDSC, abolishing the ability of MDSC to suppress T cell proliferation, restoring systemic and tumor-infiltrating CD4 and CD8 T cell numbers, and boosting antigen-specific cytotoxicity of CTL from spleen and TIL. Since MDSC are known to potently inhibit anti-tumor T cell responses (6, 9, 11) we focused on the effects of iNOS inhibition on MDSC activity and distribution in tumor-bearing mice as the likely primary mechanism of immune restoration in L-NIL treated mice. However, it is also possible that iNOS inhibition has direct or indirect effects on T cell migration and function independent of MDSC. In fact, a recent publication by Molon and coworkers describes a novel mechanism of chemokine nitration which prevents T cells from migrating intratumorally in several different tumor models (32). This could potentially account for the enhanced intratumoral CD4 and CD8 T cell numbers we observed in L-NIL treated mice, but not the increased number of T cells observed in the spleen. It also does not account for the higher per-cell cytotoxicity observed in splenocytes isolated from spleen and tumor of L-NIL treated mice. Rather, these effects are consistent with the nearly absolute loss of T cell suppressive capacity we observed in MDSC from L-NIL treated mice.

The loss of functional activity in MDSC from L-NIL treated mice suggests a failure of MDSC to upregulate or maintain mechanisms of T cell suppression that are normally induced by cancer. iNOSupregulation and production of NO is itself a direct effector mechanism of T cell suppression, particularly by monocytic MDSC (3, 18) However, it is unlikely that sufficient amounts of L-NIL, a reversible competitive antagonist of iNOS, were carried over into our ex-vivo suppressor assay to affect iNOS-dependent T cell suppression (see Fig. 5B). Also, suppressive activity was only partly impaired in MDSC derived from iNOSKO mice, suggesting that other suppressive mechanisms predominate in our system. These potential mechanisms include expression of the enzyme arginase (7, 10) and production of ROS by MDSC (33). We did not observe a significant change in arginase expression levels on MDSCafter in vivo L-NIL treatment (data not shown). However, we saw a significant decrease in ROS production after iNOS inhibition in both ex-vivo derived MDSC and MDSC isolated from tumor-bearing mice. Thus, rather than a direct effect on reactive nitrogen-mediated T cell inhibition, iNOS inhibition acts primarily by interfering with tumor-mediated upregulation of ROS production by MDSC.

ROS production in MDSC has been shown to be controlled by STAT3 activation and concomitant expression of ROS-producting NADPH oxidase subunits (30). STAT3 upregulation correlates with suppressive function in many studies of MDSC in both mice and humans, and has been shown to regulate the expression of NADPH oxidase subunits and production of high-output ROS (30). We observed that pharmacologic iNOS inhibition with L-NIL downregulates both STAT3 activation and ROS production by MDSC in ex-vivo culture and in RET-bearing mice. Since signaling through VEGFR has been shown to induce activation of suppressive activity in MDSC and STAT3 activation in myeloid cells (34, 35), VEGF is an attractive candidate soluble mediator of MDSC activation in our system. In fact, we found that iNOS-mediated release of VEGF is strongly implicated in accumulation of functional MDSC, since L-NIL suppresses VEGF levels in tumor-bearing mice in vivo and in RET culture, and since exogenous VEGF is sufficient to restore MDSC accumulation in L-NIL-treated ex vivo culture. Similarly, in ex vivo culture, anti-VEGF mAb alone was capable of mimicking the effect of iNOS inhibition by suppressing accumulation of GR1+CD11b+ MDSC, suggesting that iNOS-dependent VEGF production is required for MDSC induction. This mechanism is supported by the observation that VEGF levels are elevated in both tumor and serum of mice bearing spontaneous RET melanomas, and have been shown to correlate with tumor size and progressive immunosuppression (36). This is consistent with prior literature describing a key role for VEGF in MDSC induction and tumor-mediated immunosuppression in melanoma and other solid tumors (24, 37, 38). The ability of iNOS and NO expression in tumor cells to induce VEGF release has also been well-described (39, 40), and provides a logical mechanism through which modulation of iNOS could control the induction and activation of MDSC and – potentially - other immunosuppressive cells (41).

Since both tumor and host cells can express VEGF,(as well as other soluble inflammatory mediators, such as IL-6), the relative importance of these compartments in the iNOS-mediated induction of MDSC is as yet incompletely defined. Our ex-vivo supernatant transfer results, in which treatment of tumor cells with L-NIL causes robust suppression of MDSC accumulation, are consistent with a model in which tumor-expressed iNOS enhances VEGF release, and thus supports MDSC accumulation and activation. These results closely parallel the in vivo accumulation of GR1+CD11b+ MDSC in bone marrow of tumor-bearing mice, where only a slight trend towards decrease of MDSC is observed in iNOSKO mice, but inhibition of iNOS in both the tumor and host compartments with L-NIL significantlyreduces the number of MDSC to that seen in tumor-free mice.This is accompanied by a significant decrease (VEGF) or trend towards decrease (IL-6, IL-1β) of key inflammatory mediators in serum. Thus, the iNOS-dependent production of tumor-derived VEGF seems to play a pivotal role in MDSC induction from myeloid precursors. This is quite consistent with what is known about the RET gene driving oncogenesis in this model, a so called “inflammatory” oncogene which induces the expression of numerous proinflammatory mediators including VEGF, IL-6, and IL-1β.(36)(42)

The pivotal role of tumor-expressed iNOS in regulation of MDSC is supported by several points of evidence in our study. Despite efficient reduction of MDSC NO levels, as demonstrated by nitrotyrosine staining, ablation of host iNOS fails to decrease the number of MDSC infiltrating tumor and spleen, reverse immunosuppression, or suppress tumor growth. This is in stark contrast to the effect of pharmacologic iNOS inhibition, which affects iNOS in both tumor- and host-expressed compartments and has beneficial effects on MDSC function, T cell numbers, and tumor growth. In tumor-bearing iNOS KO mice we observed neither ROS downregulation (Fig 5) nor downregulation of activated STAT3 (data not shown), despite robust downregulation of these molecules with pharmacologic iNOS inhibition. These findings are supported by experiments with ex-vivo derived MDSC, where the suppression of MDSC accumulation and function is strongest with inhibition of tumor-expressed iNOS. Together, these data suggest a pivotal role for tumor-expressed iNOS in control of MDSC-mediated immunosuppression through modulation of VEGF release and VEGF-driven signaling events in the target MDSC.

Reversal of immune dysfunction by L-NIL was associated with modest immune-dependent suppression of growth of early transplantable MT-RET-1 tumors; a more profound decrease in spontaneous tumor growth was also observed in L-NIL treated MT-RET transgenic mice, possibly related to the much slower initial growth rate of spontaneous tumors. Thus iNOS inhibition is sufficient to restore endogenous immunity in melanoma-bearing mice, but not to levels sufficient to mediate tumor regression or eradication. Combining iNOS inhibition with other immunomodulatory approaches, such as anti-tumor vaccination or adoptive transfer of anti-tumor T cells, is a logical next step since this would simultaneously increase the number of high-affinity tumor-specific CTL while disarming mechanisms which lead to their death or inactivation upon entry into the tumor. Thus, iNOS inhibition may function as relatively simple and non-toxic strategy for conditioning the imunosuppressive tumor microenvironment, and could potentially be combined in a modular fashion with any number of other immunotherapeutic approaches.

The aberrant expression of iNOS and other inflammatory molecules has been recently identified as a critical tipping point in progression of many different cancers (43)(44), and a logical focus for targeted molecular therapy (29).iNOS and NO have already been shown to promote tumor growth, survival, and treatment resistance in melanoma and other cancers. Understanding the regulation of MDSC recruitment and function by tumor-expressed iNOS will provide important insights into the link between inflammatory signaling and development of the immunosuppressive tumor microenvironment, and new opportunities for therapeutic intervention.

Supplementary Material

1
2
3
4
5

ACKNOWLEDGEMENTS

We thank Lloyd Mayer and Huabao Xiong of the Mount Sinai School of Medicine Immunology Institute for reviewing our manuscript and offering helpful comments.

Footnotes

1

Grant Support: The National Cancer Institute of the NIH, grant number K08CA154963.

Dr. Mildred Scheel Foundation for Cancer Research Grant 108992 (to V.U.), and the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer (V.U.).

REFERENCES

  • 1.Bronte V. Myeloid-derived suppressor cells in inflammation: uncovering cell subsets with enhanced immunosuppressive functions. Eur J Immunol. 2009;39:2670–2672. doi: 10.1002/eji.200939892. [DOI] [PubMed] [Google Scholar]
  • 2.Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: linking inflammation and cancer. J Immunol. 2009;182:4499–4506. doi: 10.4049/jimmunol.0802740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 2009;9:162–174. doi: 10.1038/nri2506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Goni O, Alcaide P, Fresno M. Immunosuppression during acute Trypanosoma cruzi infection: involvement of Ly6G (Gr1(+))CD11b(+ )immature myeloid suppressor cells. Int Immunol. 2002;14:1125–1134. doi: 10.1093/intimm/dxf076. [DOI] [PubMed] [Google Scholar]
  • 5.Giordanengo L, Guinazu N, Stempin C, Fretes R, Cerban F, Gea S. Cruzipain, a major Trypanosoma cruzi antigen, conditions the host immune response in favor of parasite. Eur J Immunol. 2002;32:1003–1011. doi: 10.1002/1521-4141(200204)32:4<1003::AID-IMMU1003>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  • 6.Makarenkova VP, Bansal V, Matta BM, Perez LA, Ochoa JB. CD11b+/Gr-1+ myeloid suppressor cells cause T cell dysfunction after traumatic stress. J Immunol. 2006;176:2085–2094. doi: 10.4049/jimmunol.176.4.2085. [DOI] [PubMed] [Google Scholar]
  • 7.Bronte V, Serafini P, Mazzoni A, Segal DM, Zanovello P. L-arginine metabolism in myeloid cells controls T-lymphocyte functions. Trends Immunol. 2003;24:302–306. doi: 10.1016/s1471-4906(03)00132-7. [DOI] [PubMed] [Google Scholar]
  • 8.Adeegbe D, Serafini P, Bronte V, Zoso A, Ricordi C, Inverardi L. In vivo induction of myeloid suppressor cells and CD4+Foxp3+ T regulatory cells prolongs skin allograft survival in mice. Cell Transplant. 2010 doi: 10.3727/096368910X540621. [DOI] [PubMed] [Google Scholar]
  • 9.Mazzoni A, Bronte V, Visintin A, Spitzer JH, Apolloni E, Serafini P, Zanovello P, Segal DM. Myeloid suppressor lines inhibit T cell responses by an NO-dependent mechanism. J Immunol. 2002;168:689–695. doi: 10.4049/jimmunol.168.2.689. [DOI] [PubMed] [Google Scholar]
  • 10.Bronte V, Serafini P, De Santo C, Marigo I, Tosello V, Mazzoni A, Segal DM, Staib C, Lowel M, Sutter G, Colombo MP, Zanovello P. IL-4-induced arginase 1 suppresses alloreactive T cells in tumor-bearing mice. J Immunol. 2003;170:270–278. doi: 10.4049/jimmunol.170.1.270. [DOI] [PubMed] [Google Scholar]
  • 11.Nagaraj S, Gupta K, Pisarev V, Kinarsky L, Sherman S, Kang L, Herber DL, Schneck J, Gabrilovich DI. Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med. 2007;13:828–835. doi: 10.1038/nm1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Seago ND, Clark DA, Miller MJ. Role of inducible nitric oxide synthase (iNOS) and peroxynitrite in gut inflammation. Inflamm Res. 1995;44(Suppl 2):S153–154. doi: 10.1007/BF01778308. [DOI] [PubMed] [Google Scholar]
  • 13.Ochoa AC, Zea AH, Hernandez C, Rodriguez PC. Arginase, prostaglandins, and myeloid-derived suppressor cells in renal cell carcinoma. Clin Cancer Res. 2007;13:721s–726s. doi: 10.1158/1078-0432.CCR-06-2197. [DOI] [PubMed] [Google Scholar]
  • 14.Serafini P, Meckel K, Kelso M, Noonan K, Califano J, Koch W, Dolcetti L, Bronte V, Borrello I. Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function. J Exp Med. 2006;203:2691–2702. doi: 10.1084/jem.20061104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Capuano G, Rigamonti N, Grioni M, Freschi M, Bellone M. Modulators of arginine metabolism support cancer immunosurveillance. BMC Immunol. 2009;10:1. doi: 10.1186/1471-2172-10-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.De Santo C, Serafini P, Marigo I, Dolcetti L, Bolla M, Del Soldato P, Melani C, Guiducci C, Colombo MP, Iezzi M, Musiani P, Zanovello P, Bronte V. Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination. Proc Natl Acad Sci U S A. 2005;102:4185–4190. doi: 10.1073/pnas.0409783102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Iwamoto T, Takahashi M, Ohbayashi M, Nakashima I. The Ret Oncogene Can Induce Melanogenesis and Melanocyte Development in Wv/Wv Mice. Exp Cell Res. 1992;200:410–415. doi: 10.1016/0014-4827(92)90189-f. [DOI] [PubMed] [Google Scholar]
  • 18.Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol. 2008;181:5791–5802. doi: 10.4049/jimmunol.181.8.5791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Cheng P, Corzo CA, Luetteke N, Yu B, Nagaraj S, Bui MM, Ortiz M, Nacken W, Sorg C, Vogl T, Roth J, Gabrilovich DI. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein. J Exp Med. 2008;205:2235–2249. doi: 10.1084/jem.20080132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Bartoli M, Platt D, Lemtalsi T, Gu X, Brooks SE, Marrero MB, Caldwell RB. VEGF differentially activates STAT3 in microvascular endothelial cells. FASEB J. 2003;17:1562–1564. doi: 10.1096/fj.02-1084fje. [DOI] [PubMed] [Google Scholar]
  • 21.Niu G, Wright KL, Huang M, Song L, Haura E, Turkson J, Zhang S, Wang T, Sinibaldi D, Coppola D, Heller R, Ellis LM, Karras J, Bromberg J, Pardoll D, Jove R, Yu H. Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene. 2002;21:2000–2008. doi: 10.1038/sj.onc.1205260. [DOI] [PubMed] [Google Scholar]
  • 22.Kusmartsev S, Gabrilovich DI. Inhibition of myeloid cell differentiation in cancer: the role of reactive oxygen species. J Leukoc Biol. 2003;74:186–196. doi: 10.1189/jlb.0103010. [DOI] [PubMed] [Google Scholar]
  • 23.Corzo CA, Cotter MJ, Cheng P, Cheng F, Kusmartsev S, Sotomayor E, Padhya T, McCaffrey TV, McCaffrey JC, Gabrilovich DI. Mechanism regulating reactive oxygen species in tumor-induced myeloid-derived suppressor cells. J Immunol. 2009;182:5693–5701. doi: 10.4049/jimmunol.0900092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Gabrilovich D, Ishida T, Oyama T, Ran S, Kravtsov V, Nadaf S, Carbone DP. Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo. Blood. 1998;92:4150–4166. [PubMed] [Google Scholar]
  • 25.Dolcetti L, Marigo I, Mantelli B, Peranzoni E, Zanovello P, Bronte V. Myeloid-derived suppressor cell role in tumor-related inflammation. Cancer Lett. 2008;267:216–225. doi: 10.1016/j.canlet.2008.03.012. [DOI] [PubMed] [Google Scholar]
  • 26.Serafini P, Borrello I, Bronte V. Myeloid suppressor cells in cancer: recruitment, phenotype, properties, and mechanisms of immune suppression. Semin Cancer Biol. 2006;16:53–65. doi: 10.1016/j.semcancer.2005.07.005. [DOI] [PubMed] [Google Scholar]
  • 27.Diaz-Montero CM, S. M, Nishimura MI, Garrett-Mayer E, Cole DJ, Montero AJ. Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin-cyclophosphamide chemotherapy. Cancer Immunol Immunother. 2009 doi: 10.1007/s00262-008-0523-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Dolcetti L, Peranzoni E, Ugel S, Marigo I, Gomez A. Fernandez, Mesa C, Geilich M, Winkels G, Traggiai E, Casati A, Grassi F, Bronte V. Hierarchy of immunosuppressive strength among myeloid-derived suppressor cell subsets is determined by GM-CSF. Eur J Immunol. 2010;40:22–35. doi: 10.1002/eji.200939903. [DOI] [PubMed] [Google Scholar]
  • 29.Sikora AG, Gelbard A, Davies MA, Sano D, Ekmekcioglu S, Kwon J, Hailemichael Y, Jayaraman P, Myers JN, Grimm EA, Overwijk WW. Targeted inhibition of inducible nitric oxide synthase inhibits growth of human melanoma in vivo and synergizes with chemotherapy. Clin Cancer Res. 2010;16:1834–1844. doi: 10.1158/1078-0432.CCR-09-3123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ohtsu N, Takaoka K, Segawa E, Hashitani S, Noguchi K, Kishimoto H, Urade M. Antitumor effects of inhibitors of nitric oxide synthase or cyclooxygenase-2 on human KB carcinoma cells overexpressing COX-2. Oncol Rep. 2010;24:31–36. doi: 10.3892/or_00000825. [DOI] [PubMed] [Google Scholar]
  • 31.Yamanaka N, O’Connor MB. Nitric oxide directly regulates gene expression during Drosophila development: need some gas to drive into metamorphosis? Genes Dev. 2011;25:1459–1463. doi: 10.1101/gad.2080411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Molon B, Ugel S, Del Pozzo F, Soldani C, Zilio S, Avella D, De Palma A, Mauri P, Monegal A, Rescigno M, Savino B, Colombo P, Jonjic N, Pecanic S, Lazzarato L, Fruttero R, Gasco A, Bronte V, Viola A. Chemokine nitration prevents intratumoral infiltration of antigen-specific T cells. J Exp Med. 2011;208:1949–1962. doi: 10.1084/jem.20101956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kusmartsev S, Nefedova Y, Yoder D, Gabrilovich DI. Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species. J Immunol. 2004;172:989–999. doi: 10.4049/jimmunol.172.2.989. [DOI] [PubMed] [Google Scholar]
  • 34.Nefedova Y, Huang M, Kusmartsev S, Bhattacharya R, Cheng P, Salup R, Jove R, Gabrilovich D. Hyperactivation of STAT3 is involved in abnormal differentiation of dendritic cells in cancer. J Immunol. 2004;172:464–474. doi: 10.4049/jimmunol.172.1.464. [DOI] [PubMed] [Google Scholar]
  • 35.Nefedova Y, Nagaraj S, Rosenbauer A, Muro-Cacho C, Sebti SM, Gabrilovich DI. Regulation of dendritic cell differentiation and antitumor immune response in cancer by pharmacologic-selective inhibition of the janus-activated kinase 2/signal transducers and activators of transcription 3 pathway. Cancer Res. 2005;65:9525–9535. doi: 10.1158/0008-5472.CAN-05-0529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zhao F, Falk C, Osen W, Kato M, Schadendorf D, Umansky V. Activation of p38 mitogen-activated protein kinase drives dendritic cells to become tolerogenic in ret transgenic mice spontaneously developing melanoma. Clin Cancer Res. 2009;15:4382–4390. doi: 10.1158/1078-0432.CCR-09-0399. [DOI] [PubMed] [Google Scholar]
  • 37.Kusmartsev S, Gabrilovich DI. Effect of tumor-derived cytokines and growth factors on differentiation and immune suppressive features of myeloid cells in cancer. Cancer Metastasis Rev. 2006;25:323–331. doi: 10.1007/s10555-006-9002-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kusmartsev SA, Kusmartseva IN, Afanasyev SG, Cherdyntseva NV. Immunosuppressive cells in bone marrow of patients with stomach cancer. Adv Exp Med Biol. 1998;451:189–194. doi: 10.1007/978-1-4615-5357-1_30. [DOI] [PubMed] [Google Scholar]
  • 39.Jozkowicz A, Cooke JP, Guevara I, Huk I, Funovics P, Pachinger O, Weidinger F, Dulak J. Genetic augmentation of nitric oxide synthase increases the vascular generation of VEGF. Cardiovasc Res. 2001;51:773–783. doi: 10.1016/s0008-6363(01)00344-3. [DOI] [PubMed] [Google Scholar]
  • 40.Tao J, Tu YT, Li JW, Feng AP, Huang CZ, Wu Y, Shen GX. Endogenous production of nitric oxide contributes to proliferation effect of vascular endothelial growth factor-induced malignant melanoma cell. Clin Exp Dermatol. 2006;31:94–99. doi: 10.1111/j.1365-2230.2005.01922.x. [DOI] [PubMed] [Google Scholar]
  • 41.Niedbala W, Cai B, Liu H, Pitman N, Chang L, Liew FY. Nitric oxide induces CD4+CD25+ Foxp3 regulatory T cells from CD4+CD25 T cells via p53, IL-2, and OX40. Proc Natl Acad Sci U S A. 2007;104:15478–15483. doi: 10.1073/pnas.0703725104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Borrello MG, Alberti L, Fischer A, Degl’innocenti D, Ferrario C, Gariboldi M, Marchesi F, Allavena P, Greco A, Collini P, Pilotti S, Cassinelli G, Bressan P, Fugazzola L, Mantovani A, Pierotti MA. Induction of a proinflammatory program in normal human thyrocytes by the RET/PTC1 oncogene. Proc Natl Acad Sci U S A. 2005;102:14825–14830. doi: 10.1073/pnas.0503039102. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS366261-supplement-1.docx (12.6KB, docx)
2
NIHMS366261-supplement-2.tif (2.2MB, tif)
3
NIHMS366261-supplement-3.tif (1.5MB, tif)
4
NIHMS366261-supplement-4.tif (5.7MB, tif)
5
NIHMS366261-supplement-5.tif (6MB, tif)

RESOURCES