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H. Lee Moffitt Cancer Center, University of South Florida, Tampa, FL 33612
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Abstract |
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production by T cells, but was mediated by integrins CD11b, CD18, and CD29. Blocking of these integrins with specific Abs abrogated ROS production and ImC-mediated suppression of CD8+ T cell responses. This study demonstrates a new mechanism of Ag-specific T cell inhibition mediated by ROS produced by ImCs in cancer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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However, immature myeloid cells are also an intrinsic part of the normal process of myeloid cell differentiation, and they are present in relatively small numbers in naive hosts. The number of immature myeloid cells significantly increases during bacterial infection and immunization with potent immunogens, such as vaccinia virus encoding IL-2 (11, 15) and superantigen (15). Neither naive nor immunized hosts suffer from Ag-specific T cell unresponsiveness. Understanding the mechanisms used by ImCs to suppress the T cell response is critically important for the development of effective methods to counter this phenomenon.
In this study we asked whether ImC from tumor-free and tumor-bearing hosts differ in their ability to suppress CD8+ T cells, and what could be a mechanism of Ag-specific inhibition of T cell response by ImC in cancer. We report, for the first time, that Ag-specific inhibition of CD8+ T cells by ImC could result from up-regulation of reactive oxygen species (ROS) production by these cells, and that this process is possibly mediated by integrins.
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Materials and Methods |
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Female BALB/c and C57BL/6 mice, aged 6–10 wk, were purchased from the National Cancer Institute (Frederick, MD). OT-1 TCR transgenic mice (C57BL/6-Tg(TCR
TCR
)1100Mjb) and IFN- receptor (IFN-R) knockout B6.129S7-Ifngrtm1Agt mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C3 fibrosarcoma was established in C57BL/6 mice by s.c. inoculation of 5 x 105 cells. Mice were immunized once s.c. with 0.3 mg of OVA protein emulsified in IFA at the base of tail. Isolation of Gr-1+ cells from immunized mice was performed on day 10 after vaccination.
Media and reagents
RPMI 1640 medium was supplemented with 10% FCS, 20 mM HEPES, 200 U/ml penicillin, 50 µg/ml streptomycin, 0.05 mM 2-ME, and 2-mM glutamine (all from Life Technologies, Grand Island, NY). OVA-derived peptide (H-2Kb-restricted, aa 257–264, SIINFEKL) and C3-derived peptide RAHYNIVTF were purchased from SynPep (Dublin, CA), IFA was obtained from Sigma-Aldrich (St. Louis, MO), and dichlorodihydrofluorescein diacetate (DCFDA) and dihydroethytium (DHE) were purchased from Molecular Probes (Eugene, OR). Superoxide dismutase (SOD), catalase, uric acid, and the arginase inhibitor, N-hydroxy-nor-L-arginine (nor-NOHA) were purchased from Calbiochem (La Jolla, CA). The following Abs were used for flow cytometry: Gr-1-allophycocyanin (clone RB6-8C5), CD11b-PE (clone M1/70), CD18-PE (clone C71/16), and CD29-PE (clone KM16; all from BD PharMingen, San Diego, CA).
Isolation of Gr-1+ splenocytes
Spleens were harvested under sterile conditions. Single-cell suspensions were prepared, and RBC were removed using ACK lysing buffer (Biosource International, Camarillo, CA). Splenocytes were resuspended in PBS, and 5–6 x 106 cells were incubated with 5 µg of biotinylated anti-Gr-1 mAbs (BD PharMingen) for 15 min on ice. Cells were washed with cold PBS to remove unbound Abs, then incubated with streptavidin microbeads for 15 min at 4°C. The Gr-1+ cell population was isolated on a MiniMACS column according to the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The purity of the Gr-1+ cell population was evaluated by flow cytometry and exceeded 90%.
Morphology and cytochemistry
For morphologic characterization, Gr-1+ cells were stained with H&E. Myeloperoxidase, -naphthyl acetate esterase, and naphthol AS-D chloroacetate esterase activities in isolated Gr-1+ cells were evaluated by cytochemical staining according to the manufacturer’s instructions using kits obtained from Sigma-Aldrich.
ELISPOT assay
ELISPOT assay was performed as described previously (12). Briefly, MultiScreen-HA plates (Millipore, Berford, MA) were precoated with anti-IFN- mAbs (clone R4-A2; BD PharMingen, San Diego, CA) by overnight incubation in PBS at 4°C. Splenocytes isolated from OVA-immunized mice (2 x 105 cells/well) were cultured for 24 h at 37°C in the presence of the specific SIINFEKL or control RAHYNIVTF peptides (10 µg/ml). Cells were then washed out with PBS containing 0.1% Tween, and plates were incubated overnight at 4°C with biotinylated anti-IFN- mAbs (clone XMG1.2, BD PharMingen). Results were visualized using avidin-alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Sigma-Aldrich). The number of spots cells in each well was scored in a blind fashion by two investigators and then recalculated per 106 cells.
Flow cytometry
One million cells were incubated for 30 min on ice in 100 µl of PBS with 1 µg of the relevant Abs and then washed twice with cold PBS. FACS data were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and were analyzed using CellQuest software (BD Biosciences). During the analysis of ROS in myeloid cells, several groups of cells with different levels of fluorescence were present. In this situation we used the geometric mean of fluorescence to measure fluorescence intensity.
ROS production
The oxidation-sensitive dyes DCFDA and DHE were used for the measurement of ROS production by Gr-1+ cells. Cells were incubated at 37°C in DMEM in the presence of 2 µM DCFDA for 30 min or 2 µM DHE for 60 min, washed twice with cold PBS, and then labeled with allophycocyanin-conjugated anti-Gr-1 Ab and PE-conjugated anti-CD11b Abs. After incubation on ice for 20 min, cells were washed with cold PBS and analyzed by three-color flow cytometry using a FACSCalibur.
To block ROS production, Gr-1+ cells were incubated for 10 min at 37°C with different antioxidants, followed by 20-min incubation at 37°C with DCFDA. After that time cells were washed in cold PBS and kept on ice before analyzing by flow cytometry. The following reagents purchased from Calbiochem were used: SOD, 200 U/ml; SOD mimetic (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride), 20 µM; myeloperoxidase (MPO), 30 nM; catalase, 1000 U/ml; peroxynitrite scavenger (uric acid), 0.5 mM; and arginase inhibitor (nor-NOHA, diacetate salt), 2 µM.
Activation of ROS in vitro with immobilized Abs
Purified anti-CD11b, -CD18, -CD29, and -H-2Db Abs were immobilized by affinity binding of the Fc portion of the Abs to protein G-coated surfaces. Briefly, Reacti-Bind, protein G-coated, 96-well plates (Pierce, Rockford, IL) were washed with PBS. Fifty microliters of mAb at 10 µg/ml in blocking buffer (containing 5% of dry milk and 0.1% Tween 20 in PBS) was added to each well. After incubation for 2 h at room temperature, wells were gently washed three times with PBS. Freshly isolated Gr-1+ cells preloaded with DCFDA (3 x 105 cells in 50 µl of DMEM) were added to each well with immobilized Abs. Cells were incubated for 15 min at 37°C, and then the level of DCFDA oxidation was analyzed using a Wallac 1420 plate fluorometer (Wallac Oy, Turku, Finland). Readings were taken at 488/538 nm every 5 min for a total of 60 min.
MPO activity
MPO activity in Gr-1+ cells was determined as described by Grisham et al. (16). Freshly isolated Gr-1+ cells (1 x 106) were washed in 10 mM potassium phosphate buffer (pH 7.8) containing 30 mM KCl. Pellets were resuspended in a solution containing 0.5 ml of hexadecyltrimethyl ammonium bromide in 10 mM potassium buffer and centrifuged at 8000 x g for 15 min at 4°C. MPO activity was assessed in supernatants by measuring the H2O2-dependent oxidation of 3,3',5,5'-tetramethylbenzidine. The absorbance at 655 nm was measured using a microplate spectrophotometer (Bio-Rad, Hercules, CA), and the results were normalized to protein content.
Western blotting
Gr1+ cells were isolated from spleen of naive or tumor-bearing mice, washed twice with ice-cold PBS, and lysed in RIPA buffer. An equal amount of total protein was loaded on a 10% SDS-PAGE gel. After electrophoresis and transferring, membranes were blocked in 5% nonfat milk in TBS/Tween 20 for 1 h and then probed with primary goat anti-mouse MPO Ab (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by incubation with corresponding rabbit anti-goat secondary Ab conjugated with HRP. Membranes were developed using an ECL detection kit (Amersham Pharmacia Biotech, Arlington Heights, IL). Equal loading was assessed using anti--actin Ab (Santa Cruz Biotechnology).
Statistical analysis
The statistical significance between values was determined by Student’s t test. All data were expressed as the mean ± SD of triplicate determinations. Values of p > 0.05 were considered nonsignificant.
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Results |
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To compare the inhibitory potential of ImC from tumor-free and tumor-bearing mice, we isolated Gr-1+ cells from naive C57BL/6 mice, tumor-free C57BL/6 mice immunized with OVA, and C3 tumor-bearing C57BL/6 mice using the magnetic beads separation technique. As responders in an ELISPOT assay we used splenocytes from C57BL/6 mice immunized with the OVA-derived, MHC class I-restricted peptide SIINFEKL. Splenocytes were restimulated in vitro with control or specific MHC class I-bound peptides. As expected, specific peptide induced a significant increase in the number of IFN--producing T cells in the ELISPOT assay. Gr-1+ cells derived from tumor-bearing, but not from tumor-free, immunized or naive mice inhibited this response (Fig. 1). The experiments shown in Fig. 1 were performed at a 1/4 Gr-1+ cell/splenocyte ratio. Thus, ImC represented 20% of the total population of splenocytes. This proportion is substantially higher than observed in tumor-free naive mice (<5%) or immunized tumor-free mice (8–12%) (12, 17). The lack of suppressive activity in ImC from tumor-free mice at that concentration indicates that ImC isolated from tumor-bearing mice have a unique ability to suppress the CD8-mediated T cell response.
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Enzymatic activity and ROS production by ImC
Gr-1+ cells were isolated from spleens of naive, immunized, and tumor-bearing mice using the magnetic bead separation technique. We evaluated the activity of three enzymes specific for myeloid cells: MPO, -naphthyl acetate esterase, and naphthol AS-D chloroacetate esterase. In three experiments performed, 42–45% of Gr-1+ cells were positive for -naphthyl acetate esterase, and the same 42–45% cells were positive for naphthol AS-D chloroacetate esterase. These proportions remained the same in all tested groups of mice (Fig. 2A). Gr-1+ cells derived from mice with tumors showed a profound deficit of MPO activity, a phenomenon not found in tumor-free immunized and naive mice (Fig. 2A). Only 7.0 ± 2.2.% of the total Gr-1+ splenocytes from tumor-bearing mice were positive for MPO, whereas 43.5 ± 2.7% of Gr-1+ cells from immunized and 33.0 ± 3.0% of Gr-1+ cells from naive mice showed activity of this enzyme (p < 0.05; Fig. 2, A and B). MPO activity was also measured in the total cell lysates using a spectrophotometer. Gr-1+ cells from tumor-bearing mice demonstrated substantially lower MPO activity than Gr-1+ cells isolated from tumor-free control mice (Fig. 2C). We asked whether decreased activity of MPO was due to a lack of that protein. Gr-1+ cells were isolated from spleens of naive and tumor-bearing mice, whole cell lysates were prepared, and the presence of MPO was evaluated using Western blotting. Gr-1+ cells isolated from tumor-bearing mice had the same level of MPO as Gr-1+ cells from tumor-free animals (Fig. 2C).
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The pool of ROS may include different types of molecules, ranging from singlet oxygen to hydrogen peroxide. We examined what type of ROS are produced by ImC. Gr-1+ cells were isolated from tumor-bearing mice, and different oxygen species were neutralized using specific inhibitors or scavengers. The effects of these compounds were evaluated by flow cytometry using DCFDA. Catalase reduced ROS levels in ImCs >4-fold, indicating that H2O2 contributed greatly to the overall level of ROS in these cells (Fig. 4A). Uric acid had similar effects, suggesting that peroxynitrite could be a substantial part of the ROS pool. However, the most noticeable differences were found in the effect of the arginase inhibitor nor-NOHA. It decreased ROS levels in ImCs >10-fold (Fig. 4A). This strongly suggests that arginine metabolites play a critical role in the generation of ROS in tumor-bearing mouse-derived ImCs. SOD did not significantly affect the levels of ROS (Fig. 4A), suggesting a rather minor contribution of superoxide to the total ROS pool, results consistent with the lack of change in DHE oxidation.
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-producing cells, but completely abrogated the Ag-specific inhibitory effect of ImC (Fig. 4B). Catalase increased the background level of IFN- production, but also abrogated the effect of ImC (Fig. 4B). Ag-specific activation of ROS production in ImC
These data demonstrate a critical role for ROS in ImC-mediated suppression of T cells. We and others have shown that this suppression requires direct cell-cell contact and is Ag specific. The necessity of direct cell-cell contact is evident from the fact that ROS are short-lived substances operating through very short distances. However, the Ag-specific nature of the suppression may have two potential mechanisms. First, Ag-specific interaction between ImC and T cells may simply provide prolonged contact between these cells, which may be necessary for ROS to exert their effects. Second, Ag-specific interaction between T cells and ImC may affect the level of ROS produced by ImC. To test these possibilities, Gr-1+ cells isolated from C3 tumor-bearing or tumor-free naive C57BL/6 mice were loaded with either the C3-derived peptide RAHYNIVTF or the OVA-derived peptide SIINFEKL, then incubated for different times, either alone or with T cells isolated from transgenic OT-1 mice bearing TCR specific for SIINFEKL. The Gr-1+ cell/T cell ratio was 1/1. To distinguish Gr-1+ cells during the analysis, cells were labeled with anti-Gr-1 Ab conjugated with allophycocyanin. Only Gr-1+ cells were analyzed. A typical example of such analysis is shown in Fig. 5A. Neither of the two peptides alone affected the levels of ROS in ImCs from control or tumor-bearing mice (data not shown). After a 1-h incubation with OT-1 T cells, ImC isolated from tumor-bearing mice and loaded with specific peptide (SIINFEKL) had >2-fold higher levels of ROS than ImCs loaded with control C3-derived peptide. No such effect was observed in ImC isolated from control mice (Fig. 5A). These data suggest that interaction of Gr-1+ cells with Ag-specific T cells in the presence of the specific Ag may stimulate ROS production by ImCs from tumor-bearing, but not control, mice.
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We also investigated the type of ROS produced after ImC incubation with T cells and specific peptide. ImC were incubated with OT-1 T cells and specific peptide as described above in the presence of different inhibitors of ROS. The level of ROS in Gr-1+ ImC was evaluated using DCFDA. The results showed that the same molecules contributed to the ROS pool in ImC after their contact with T cells as in nonstimulated ImC (Fig. 5B).
Mechanism of Ag-specific up-regulation of ROS by Gr-1+ ImC
Thus, it appears that ROS production significantly increased after ImC incubation with T cells in the presence of the specific peptide. What could cause this up-regulation? IFN- is a major cytokine produced by activated T cells, and it has been implicated in the up-regulation of ROS production in hepatocytes, fibroblasts, and macrophages (21, 22, 23, 24). We suggested that the release of IFN- by T cells could stimulate ROS production by ImC. To test this hypothesis we incubated Gr-1+ ImC isolated from tumor-bearing mice for 1 h with different concentrations of IFN- (1–300 ng/ml). IFN- did not affect the level of ROS at any tested concentration (data not shown). It was possible that the addition of exogenous IFN- did not have the same effect on ImC as endogenous IFN-. To test this possibility we used Gr-1+ ImC isolated from tumor-bearing mice lacking IFN-R. These ImC were incubated with T cells from OT-1 mice at a 1/1 ratio in the presence of control (RAHYNIVTF) or specific (SIINFEKL) peptides, and the level of ROS production was evaluated in Gr-1+ cells as described above. In parallel, ImC from wild-type C57BL/6 tumor-bearing mice were used. Peptides alone did not affect ROS production in Gr-1+ cells (data not shown). The presence of the specific peptide during incubation of IFN-R+/+ ImC with T cells resulted in a 2-fold increase in ROS production compared with control peptide. Almost 50% of all ImCs expressed high levels of ROS (Fig. 6A). IFN-R-/- ImC incubated with T cells and control peptide had substantially lower levels of ROS than their wild-type counterparts. However, specific peptide induced a similar 2-fold increase in ROS production in ImC from IFN-R-/- mice as in those from wild-type mice. We also evaluated the possible immunosuppressive effect of ImC from IFN-R-/- mice on T cells. ImC isolated from IFN-R-/- tumor-bearing mice had the same ability to suppress an Ag-specific T cell response as ImC from IFN-R+/+ mice (Fig. 6B). Thus, taken together, these data suggested that IFN- production by Ag-specific T was not a factor affecting ROS production in ImC and was probably not involved in ImC-mediated T cell suppression.
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production was evaluated 24 h later in an ELISPOT assay. Preincubation of Gr-1+ ImC with each adhesion molecule abrogated the suppressive effect of these cells on CD8+ T cells (Fig. 7B).
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Discussion |
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These data raise questions about the biological role of ImCs. Indeed, these cells are an intrinsic part of myeloid cell differentiation and are present in healthy individuals. Their production is significantly increased in response to vaccination or bacterial infection. However, neither healthy nor vaccinated individuals suffer from systemic immunosuppression. Moreover, tumor-bearing mice and cancer patients do not display signs of systemic immunosuppression until the tumor becomes bulky or in the late stages of metastatic disease.
As we demonstrated previously, highly purified ImCs from tumor-bearing mice suppress CD8+ T cell response. This suppression was NO independent and Ag specific, required direct cell-cell contact, and was dependent on MHC class I (12). However, ImC obtained from neither naive nor vaccinated mice were able to suppress CD8+ T cells. Our experiments revealed striking differences between Gr-1+ ImC isolated from tumor-bearing and tumor-free mice. Tumor-bearing mouse-derived ImC had 5-fold fewer MPO-positive cells than the population of ImC obtained from tumor-free mice. This was associated with a >3-fold higher level of ROS. These differences were not restricted to only one type of tumor or one strain of mouse. We observed this in CT-26 tumor-bearing BALB/c mice as well as in C3 tumor-bearing C57BL/6 mice. MPO catalyzes a reaction between hydrogen peroxide and chloride to generate hypochlorous acid, a potent oxidant with strong microbicidal activity (30). In addition, a new important function for MPO has recently been demonstrated to play a buffering role to protect the granule proteases from oxidative degradation by high levels of H2O2 (31). We hypothesized that the lack of MPO activity might affect the levels of ROS in these cells because MPO is a main consumer of hydrogen peroxide (32).
It appears that H2O2 is important component of ROS in ImCs. This was confirmed by the fact that catalase, which converts H2O2 to oxygen and water, decreased ROS levels in these cells >4-fold. However, it appears that the largest contribution to the ROS pool is arginase activity. The arginase inhibitor nor-NOHA decreased the total level of ROS in ImC derived from tumor-bearing mice by >10-fold. Arginase catalyzes the hydrolysis of L-arginine to urea and L-ornithine. L-arginine is used by NO synthase as a substrate for the generation of NO (33). However, low concentrations of L-arginine result in low NO formation and high generation of superoxide (O2·-) (reviewed in Ref. 34). Thus, high arginase activity in tumor-bearing mouse-derived ImC may have lowered the level of L-arginine and resulted in increased production of O2·- instead of NO. Superoxide itself is very unstable and is converted to H2O2 and oxygen. This is consistent with our data showing that in ImC, ROS accumulates primarily in the form of H2O2, not O2·-. There are several reports showing increased arginase activity in squamous cell and basal cell carcinomas of the skin as well as in prostate cancer (35, 36). Consistent with our findings, overexpression of arginase in macrophages promoted tumor growth (37). Several known tumor-derived factors, such as TGF- and IL-10, are able to increase arginase activity in macrophages (38, 39), and a number of other cytokines and growth factors produced by tumor can induce ROS production, including IL-6, IL-3, platelet-derived growth factor, GM-CSF, and fibroblast growth factor (reviewed in Ref. 40). Constant production of these factors in tumor-bearing mice could lead to the different levels of ROS observed in ImC from tumor-bearing and tumor-free mice.
It appears that the main target for arginase and oxygen species on T cells is CD3
. L-arginine starvation results in a decrease in CD3 expression in Jurkat cells, due in part to decreased mRNA stability (41). Otsuji et al. (10) found that oxidative stress, caused by tumor-derived macrophages (Mac-1+ splenocytes that coexpressed Gr-1) suppressed -chain expression in T cells. Granulocyte-derived H2O2 has been shown to be involved in the inhibition of IFN- production and the suppression of CD3-chain expression by T cells in advanced cancer patients (18).
Thus, increased ROS production by ImC derived from tumor-bearing mice seems to be a major factor responsible for inhibition of the CD8+ T cell response. ROS are short-lived substances, exerting their effect over a very short distance, which could explain the finding that ImC need direct contact with T cells to suppress their response and also explain the Ag-specific nature of the inhibition. Previous studies in vitro and in vivo have indicated that the Ag-specific interaction between T cells and APCs is much more stable and lasts much longer than interaction in the absence of the Ag (42, 43). Gr-1+ ImC express MHC class I molecules, but a low or undetectable level of MHC class II (12). This lack of MHC class II molecules may preclude them from the formation of Ag-specific interactions with CD4+ cells, which may explain the lack of suppression of CD4+ T cells (12).
Ag-specific interactions between ImC and T cells resulted in a significant increase in ROS production by ImC, possibly further contributing to inhibition of the T cell response. This effect was observed only in ImC obtained from tumor-bearing, not tumor-free, mice. Our initial hypothesis was that T cells activate ROS production via IFN-, because activation of ROS production by IFN- has been described previously (21, 23). To test this hypothesis we used ImC isolated from tumor-bearing IFN-R knockout mice, which should not react to IFN- released by T cells. Despite the fact that the background level of ROS in IFN-R-/- ImC was lower than that in wild-type cells, they up-regulated ROS after interaction with Ag-specific T cells to the same degree as their wild-type counterparts. In addition, IFN-R-/- ImC inhibited the CD8+ T cell response in a similar manner as IFN-R+/+ ImC. This indicates that IFN- probably does not play a major role in that process. Adhesion molecules, and integrins in particular, have been recently implicated in ROS production by macrophages and fibroblasts (44, 45, 46). Adhesion molecules also play an important role in the interaction between T cells and APCs. All ImC isolated from tumor cells express high level of integrin M-chain (CD11b). Our experiments have shown that CD11b as well as integrin 2-chain (CD18) and integrin 1-chain (CD29) are actively involved in up-regulation of ROS production in ImC. Moreover, neutralization of these molecules resulted in abrogation of the immunosuppressive effect of ImC. Therefore, the lack of up-regulation of ROS production and immunosuppression in ImC obtained from tumor-free mice could be explained by the significantly lower level of integrins compared with ImCs from tumor-bearing mice.
In conclusion, our data suggest a new mechanism for Ag-specific, ImC-mediated suppression of CD8+ T cells in cancer. Tumor-derived factors constantly stimulate the production of activated ImC. These cells have high level of arginase activity, which results in accumulation of ROS, mainly in the form of H2O2. Inhibition of T cell activity by ImC requires the presence of Ag and direct contact with Ag-specific T cells. Specific Ag stabilizes cell contact between ImC and Ag-specific T cells, which results in increased ROS production via signaling through integrins. Freshly isolated ImC do not express MHC class II, but are positive for MHC class I, which may explain the inhibition of CD8+, but not CD4+, T cells by these ImCs. The requirement for direct contact with T cells in the presence of specific Ags also explains the lack of systemic immunosuppression in tumor-bearing hosts despite the abundant presence of immature myeloid cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dmitry Gabrilovich, H. Lee Moffitt Cancer Center, University of South Florida, MRC-2E, Room 2067, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: dgabril{at}moffitt.usf.edu
3 Abbreviations used in this paper: ImC, immature myeloid cell; DCFDA, dichlorodihydrofluorescein diacetate; DHE, dihydroethytium; IFN-R, IFN- receptor; MPO, myeloperoxidase; nor-HOHA, N-hydroxy-nor-L-arginine; ROS, reactive oxygen species; SOD, superoxide dismutase.
Received for publication July 2, 2003. Accepted for publication November 6, 2003.
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J.-I. Youn, S. Nagaraj, M. Collazo, and D. I. Gabrilovich Subsets of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice J. Immunol., October 15, 2008; 181(8): 5791 - 5802. [Abstract] [Full Text] [PDF]
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P. Sinha, C. Okoro, D. Foell, H. H. Freeze, S. Ostrand-Rosenberg, and G. Srikrishna Proinflammatory S100 Proteins Regulate the Accumulation of Myeloid-Derived Suppressor Cells J. Immunol., October 1, 2008; 181(7): 4666 - 4675. [Abstract] [Full Text] [PDF]
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 P. Cheng, C. A. Corzo, N. Luetteke, B. Yu, S. Nagaraj, M. M. Bui, M. Ortiz, W. Nacken, C. Sorg, T. Vogl, et al. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein J. Exp. Med., September 29, 2008; 205(10): 2235 - 2249. [Abstract] [Full Text] [PDF]
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D.-M. Kuang, Q. Zhao, J. Xu, J.-P. Yun, C. Wu, and L. Zheng Tumor-Educated Tolerogenic Dendritic Cells Induce CD3{epsilon} Down-Regulation and Apoptosis of T Cells through Oxygen-Dependent Pathways J. Immunol., September 1, 2008; 181(5): 3089 - 3098. [Abstract] [Full Text] [PDF]
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S. Watanabe, K. Deguchi, R. Zheng, H. Tamai, L.-x. Wang, P. A. Cohen, and S. Shu Tumor-Induced CD11b+Gr-1+ Myeloid Cells Suppress T Cell Sensitization in Tumor-Draining Lymph Nodes J. Immunol., September 1, 2008; 181(5): 3291 - 3300. [Abstract] [Full Text] [PDF]
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P. A. Cohen, G. K. Koski, B. J. Czerniecki, K. D. Bunting, X.-Y. Fu, Z. Wang, W.-J. Zhang, C. S. Carter, M. Awad, C. A. Distel, et al. STAT3- and STAT5-dependent pathways competitively regulate the pan-differentiation of CD34pos cells into tumor-competent dendritic cells Blood, September 1, 2008; 112(5): 1832 - 1843. [Abstract] [Full Text] [PDF]
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G. Willimsky, M. Czeh, C. Loddenkemper, J. Gellermann, K. Schmidt, P. Wust, H. Stein, and T. Blankenstein Immunogenicity of premalignant lesions is the primary cause of general cytotoxic T lymphocyte unresponsiveness J. Exp. Med., July 7, 2008; 205(7): 1687 - 1700. [Abstract] [Full Text] [PDF]
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S. Kusmartsev, E. Eruslanov, H. Kubler, T. Tseng, Y. Sakai, Z. Su, S. Kaliberov, A. Heiser, C. Rosser, P. Dahm, et al. Oxidative Stress Regulates Expression of VEGFR1 in Myeloid Cells: Link to Tumor-Induced Immune Suppression in Renal Cell Carcinoma J. Immunol., July 1, 2008; 181(1): 346 - 353. [Abstract] [Full Text] [PDF]
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 K. Shah, S. Hingtgen, R. Kasmieh, J. L. Figueiredo, E. Garcia-Garcia, A. Martinez-Serrano, X. Breakefield, and R. Weissleder Bimodal Viral Vectors and In Vivo Imaging Reveal the Fate of Human Neural Stem Cells in Experimental Glioma Model J. Neurosci., April 23, 2008; 28(17): 4406 - 4413. [Abstract] [Full Text] [PDF]
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S. Nagaraj and D. I. Gabrilovich Tumor Escape Mechanism Governed by Myeloid-Derived Suppressor Cells Cancer Res., April 15, 2008; 68(8): 2561 - 2563. [Abstract] [Full Text] [PDF]
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K. Movahedi, M. Guilliams, J. Van den Bossche, R. Van den Bergh, C. Gysemans, A. Beschin, P. De Baetselier, and J. A. Van Ginderachter Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity Blood, April 15, 2008; 111(8): 4233 - 4244. [Abstract] [Full Text] [PDF]
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 J. Wels, R. N. Kaplan, S. Rafii, and D. Lyden Migratory neighbors and distant invaders: tumor-associated niche cells Genes & Dev., March 1, 2008; 22(5): 559 - 574. [Abstract] [Full Text] [PDF]
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I. Vaknin, L. Blinder, L. Wang, R. Gazit, E. Shapira, O. Genina, M. Pines, E. Pikarsky, and M. Baniyash A common pathway mediated through Toll-like receptors leads to T- and natural killer-cell immunosuppression Blood, February 1, 2008; 111(3): 1437 - 1447. [Abstract] [Full Text] [PDF]
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P.-Y. Pan, G. X. Wang, B. Yin, J. Ozao, T. Ku, C. M. Divino, and S.-H. Chen Reversion of immune tolerance in advanced malignancy: modulation of myeloid-derived suppressor cell development by blockade of stem-cell factor function Blood, January 1, 2008; 111(1): 219 - 228. [Abstract] [Full Text] [PDF]
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C. Melani, S. Sangaletti, F. M. Barazzetta, Z. Werb, and M. P. Colombo Amino-Biphosphonate Mediated MMP-9 Inhibition Breaks the Tumor-Bone Marrow Axis Responsible for Myeloid-Derived Suppressor Cell Expansion and Macrophage Infiltration in Tumor Stroma Cancer Res., December 1, 2007; 67(23): 11438 - 11446. [Abstract] [Full Text] [PDF]
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Y. Nefedova, M. Fishman, S. Sherman, X. Wang, A. A. Beg, and D. I. Gabrilovich Mechanism of All-Trans Retinoic Acid Effect on Tumor-Associated Myeloid-Derived Suppressor Cells Cancer Res., November 15, 2007; 67(22): 11021 - 11028. [Abstract] [Full Text] [PDF]
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S. K. Bunt, L. Yang, P. Sinha, V. K. Clements, J. Leips, and S. Ostrand-Rosenberg Reduced Inflammation in the Tumor Microenvironment Delays the Accumulation of Myeloid-Derived Suppressor Cells and Limits Tumor Progression Cancer Res., October 15, 2007; 67(20): 10019 - 10026. [Abstract] [Full Text] [PDF]
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R. Marhaba, M. Vitacolonna, D. Hildebrand, M. Baniyash, P. Freyschmidt-Paul, and M. Zoller The Importance of Myeloid-Derived Suppressor Cells in the Regulation of Autoimmune Effector Cells by a Chronic Contact Eczema J. Immunol., October 15, 2007; 179(8): 5071 - 5081. [Abstract] [Full Text] [PDF]
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E. Ambrosino, M. Terabe, R. C. Halder, J. Peng, S. Takaku, S. Miyake, T. Yamamura, V. Kumar, and J. A. Berzofsky Cross-Regulation between Type I and Type II NKT Cells in Regulating Tumor Immunity: A New Immunoregulatory Axis J. Immunol., October 15, 2007; 179(8): 5126 - 5136. [Abstract] [Full Text] [PDF]
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B. Zhu, Y. Bando, S. Xiao, K. Yang, A. C. Anderson, V. K. Kuchroo, and S. J. Khoury CD11b+Ly-6Chi Suppressive Monocytes in Experimental Autoimmune Encephalomyelitis J. Immunol., October 15, 2007; 179(8): 5228 - 5237. [Abstract] [Full Text] [PDF]
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P. Sinha, V. K. Clements, S. K. Bunt, S. M. Albelda, and S. Ostrand-Rosenberg Cross-Talk between Myeloid-Derived Suppressor Cells and Macrophages Subverts Tumor Immunity toward a Type 2 Response J. Immunol., July 15, 2007; 179(2): 977 - 983. [Abstract] [Full Text] [PDF]
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 P. Filipazzi, R. Valenti, V. Huber, L. Pilla, P. Canese, M. Iero, C. Castelli, L. Mariani, G. Parmiani, and L. Rivoltini Identification of a New Subset of Myeloid Suppressor Cells in Peripheral Blood of Melanoma Patients With Modulation by a Granulocyte-Macrophage Colony-Stimulation Factor-Based Antitumor Vaccine J. Clin. Oncol., June 20, 2007; 25(18): 2546 - 2553. [Abstract] [Full Text] [PDF]
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M. J. Delano, P. O. Scumpia, J. S. Weinstein, D. Coco, S. Nagaraj, K. M. Kelly-Scumpia, K. A. O'Malley, J. L. Wynn, S. Antonenko, S. Z. Al-Quran, et al. MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis J. Exp. Med., June 11, 2007; 204(6): 1463 - 1474. [Abstract] [Full Text] [PDF]
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T. A. Dietlin, F. M. Hofman, B. T. Lund, W. Gilmore, S. A. Stohlman, and R. C. van der Veen Mycobacteria-induced Gr-1+ subsets from distinct myeloid lineages have opposite effects on T cell expansion J. Leukoc. Biol., May 1, 2007; 81(5): 1205 - 1212. [Abstract] [Full Text] [PDF]
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K. H. T. Paraiso, T. Ghansah, A. Costello, R. W. Engelman, and W. G. Kerr Induced SHIP Deficiency Expands Myeloid Regulatory Cells and Abrogates Graft-versus-Host Disease J. Immunol., March 1, 2007; 178(5): 2893 - 2900. [Abstract] [Full Text] [PDF]
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J. Vieweg, Z. Su, P. Dahm, and S. Kusmartsev Reversal of Tumor-Mediated Immunosuppression Clin. Cancer Res., January 15, 2007; 13(2): 727s - 732s. [Abstract] [Full Text] [PDF]
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G. Varga, S. Balkow, M. K. Wild, A. Stadtbaeumer, M. Krummen, T. Rothoeft, T. Higuchi, S. Beissert, K. Wethmar, K. Scharffetter-Kochanek, et al. Active MAC-1 (CD11b/CD18) on DCs inhibits full T-cell activation Blood, January 15, 2007; 109(2): 661 - 669. [Abstract] [Full Text] [PDF]
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P. Serafini, K. Meckel, M. Kelso, K. Noonan, J. Califano, W. Koch, L. Dolcetti, V. Bronte, and I. Borrello Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function J. Exp. Med., November 27, 2006; 203(12): 2691 - 2702. [Abstract] [Full Text] [PDF]
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M. Munder, H. Schneider, C. Luckner, T. Giese, C.-D. Langhans, J. M. Fuentes, P. Kropf, I. Mueller, A. Kolb, M. Modolell, et al. Suppression of T-cell functions by human granulocyte arginase Blood, September 1, 2006; 108(5): 1627 - 1634. [Abstract] [Full Text] [PDF]
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G. Lizee, L. G. Radvanyi, W. W. Overwijk, and P. Hwu Improving Antitumor Immune Responses by Circumventing Immunoregulatory Cells and Mechanisms. Clin. Cancer Res., August 15, 2006; 12(16): 4794 - 4803. [Abstract] [Full Text] [PDF]
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J. A. Van Ginderachter, S. Meerschaut, Y. Liu, L. Brys, K. De Groeve, G. Hassanzadeh Ghassabeh, G. Raes, and P. De Baetselier Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) ligands reverse CTL suppression by alternatively activated (M2) macrophages in cancer Blood, July 15, 2006; 108(2): 525 - 535. [Abstract] [Full Text] [PDF]
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R. Kim, M. Emi, K. Tanabe, and K. Arihiro Tumor-Driven Evolution of Immunosuppressive Networks during Malignant Progression Cancer Res., June 1, 2006; 66(11): 5527 - 5536. [Abstract] [Full Text] [PDF]
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A. Pina, P. H. N. Saldiva, L. E. C. Restrepo, and V. L. G. Calich Neutrophil role in pulmonary paracoccidioidomycosis depends on the resistance pattern of hosts J. Leukoc. Biol., June 1, 2006; 79(6): 1202 - 1213. [Abstract] [Full Text] [PDF]
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I. Kryczek, L. Zou, P. Rodriguez, G. Zhu, S. Wei, P. Mottram, M. Brumlik, P. Cheng, T. Curiel, L. Myers, et al. B7-H4 expression identifies a novel suppressive macrophage population in human ovarian carcinoma J. Exp. Med., April 17, 2006; 203(4): 871 - 881. [Abstract] [Full Text] [PDF]
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Z. F. M. Vasconcelos, B. M. dos Santos, J. Farache, T. S. S. Palmeira, R. B. Areal, J. M. T. Cunha, M. A. Barcinski, and A. Bonomo G-CSF-treated granulocytes inhibit acute graft-versus-host disease Blood, March 1, 2006; 107(5): 2192 - 2199. [Abstract] [Full Text] [PDF]
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B. Huang, P.-Y. Pan, Q. Li, A. I. Sato, D. E. Levy, J. Bromberg, C. M. Divino, and S.-H. Chen Gr-1+CD115+ Immature Myeloid Suppressor Cells Mediate the Development of Tumor-Induced T Regulatory Cells and T-Cell Anergy in Tumor-Bearing Host Cancer Res., January 15, 2006; 66(2): 1123 - 1131. [Abstract] [Full Text] [PDF]
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S. K. Bunt, P. Sinha, V. K. Clements, J. Leips, and S. Ostrand-Rosenberg Inflammation Induces Myeloid-Derived Suppressor Cells that Facilitate Tumor Progression J. Immunol., January 1, 2006; 176(1): 284 - 290. [Abstract] [Full Text] [PDF]
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P. Sinha, V. K. Clements, and S. Ostrand-Rosenberg Interleukin-13-regulated M2 Macrophages in Combination with Myeloid Suppressor Cells Block Immune Surveillance against Metastasis Cancer Res., December 15, 2005; 65(24): 11743 - 11751. [Abstract] [Full Text] [PDF]
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X. Song, Y. Krelin, T. Dvorkin, O. Bjorkdahl, S. Segal, C. A. Dinarello, E. Voronov, and R. N. Apte CD11b+/Gr-1+ Immature Myeloid Cells Mediate Suppression of T Cells in Mice Bearing Tumors of IL-1{beta}-Secreting Cells J. Immunol., December 15, 2005; 175(12): 8200 - 8208. [Abstract] [Full Text] [PDF]
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S. Kusmartsev, S. Nagaraj, and D. I. Gabrilovich Tumor-Associated CD8+ T Cell Tolerance Induced by Bone Marrow-Derived Immature Myeloid Cells J. Immunol., October 1, 2005; 175(7): 4583 - 4592. [Abstract] [Full Text] [PDF]
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E. Suzuki, V. Kapoor, A. S. Jassar, L. R. Kaiser, and S. M. Albelda Gemcitabine Selectively Eliminates Splenic Gr-1+/CD11b+ Myeloid Suppressor Cells in Tumor-Bearing Animals and Enhances Antitumor Immune Activity Clin. Cancer Res., September 15, 2005; 11(18): 6713 - 6721. [Abstract] [Full Text] [PDF]
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L. Brys, A. Beschin, G. Raes, G. H. Ghassabeh, W. Noel, J. Brandt, F. Brombacher, and P. D. Baetselier Reactive Oxygen Species and 12/15-Lipoxygenase Contribute to the Antiproliferative Capacity of Alternatively Activated Myeloid Cells Elicited during Helminth Infection J. Immunol., May 15, 2005; 174(10): 6095 - 6104. [Abstract] [Full Text] [PDF]
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S. Kusmartsev and D. I. Gabrilovich STAT1 Signaling Regulates Tumor-Associated Macrophage-Mediated T Cell Deletion J. Immunol., April 15, 2005; 174(8): 4880 - 4891. [Abstract] [Full Text] [PDF]
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 C. De Santo, P. Serafini, I. Marigo, L. Dolcetti, M. Bolla, P. Del Soldato, C. Melani, C. Guiducci, M. P. Colombo, M. Iezzi, et al. Nitroaspirin corrects immune dysfunction in tumor-bearing hosts and promotes tumor eradication by cancer vaccination PNAS, March 15, 2005; 102(11): 4185 - 4190. [Abstract] [Full Text] [PDF]
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M. Munder, F. Mollinedo, J. Calafat, J. Canchado, C. Gil-Lamaignere, J. M. Fuentes, C. Luckner, G. Doschko, G. Soler, K. Eichmann, et al. Arginase I is constitutively expressed in human granulocytes and participates in fungicidal activity Blood, March 15, 2005; 105(6): 2549 - 2556. [Abstract] [Full Text] [PDF]
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P. Sinha, V. K. Clements, and S. Ostrand-Rosenberg Reduction of Myeloid-Derived Suppressor Cells and Induction of M1 Macrophages Facilitate the Rejection of Established Metastatic Disease J. Immunol., January 15, 2005; 174(2): 636 - 645. [Abstract] [Full Text] [PDF]
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