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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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Gr-1+ IMC can be found not only in spleens, but also in tumor tissues (12). Because Gr-1+ IMC could be differentiated into macrophages in tumor-free mice (12), it would be interesting and important to trace the fate of these cells in tumor tissues and compare immunosuppressive activity of macrophages localized in the vicinity of tumor and in lymphoid organs. No such direct study has been performed until now. These experiments may help to clarify the role of myeloid cells in cancer-associated nonresponsiveness. Despite the presence of large number of different immunosuppressive myeloid cells, tumor-bearing mice or cancer patients usually do not display significant systemic immunodeficiency (reviewed in Ref. 21). In a recent detailed study, Radoja et al. (22) investigated T cell function in multiple murine tumor models, including different transplantable tumors and also a transgenic model of spontaneous breast carcinoma. They demonstrated that in mice bearing sizable tumors, T cell functions (proliferation, cytokine production, induction of CD8+ alloreactive CTL, development of antikeyhole limpet hemocyanin CD4+ T cells, rejection of allogeneic or syngeneic regressor tumors) were not systemically reduced (22). However, significant number of studies have reported profound deficiency in T cell function in tumor tissues (reviewed in Refs. 23 and 24). Some authors have reported increased apoptosis of tumor-infiltrating lymphocytes (25), although contrary findings have also been shown (26). We hypothesized that myeloid cells localized in tumor tissues, and ones localized in peripheral lymphoid organs (spleen) may differ in their ability to suppress T cell responses. To test this hypothesis, we, for the first time, have performed a direct comparative analysis of myeloid cells freshly isolated from spleens and tumor tissues of tumor-bearing mice. We have identified the mechanisms, which are used by tumor-associated macrophages (TAM) to inhibit T cell function in vitro, and for the first time have determined that STAT1 transcription factor could be directly responsible for the observed TAM-mediated T cell apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Female BALB/c and C57BL/6 mice (6–8 wk of age) were obtained from the National Cancer Institute. H-2Kb congenic (CD45.1+) B6.SJL-PtrcaPep3b/BoyJ mice, TCR transgenic for OVA-derived peptide SIINFEKL on C57BL/6 background, and C.129S2-Stat6tm1Gru STAT6 knockout mice on BALB/c background were obtained from The Jackson Laboratory. STAT1 knockout mice were obtained from Y. Durbin (Children Research Institute, Columbus, OH) and were described in details elsewhere (27). TCR transgenic mice on BALB/c background expressing an 
TCR specific for MHC class II-restricted SFERFEIFPKE peptide, derived from influenza hemagglutinin (HA), were kindly provided by E. Sotomayor (H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL). The following tumor models were used in this study: CT-26 colon carcinoma (s.c.) on BALB/c background, C3 fibrosarcoma (s.c.) on C57BL/6 background, MethA sarcoma (s.c. and i.p.) on BALB/c background, and EL4 (s.c. and i.p.) lymphoma on C57BL/6 background. All tumor cells were maintained in vitro at 37°C in a 5% CO2 humidified atmosphere in complete culture medium. To establish s.c. tumors, mice were injected into shaved right flank with 105 C3 and CT-26 tumors or 2 x 105 MethA sarcoma or EL-4 cells. To establish i.p. tumors, mice were injected i.p. with 2 x 106 EL4 or MethA sarcoma cells.
Reagents
Arginase inhibitor NW-hydroxyl-nor-L-arginine (nor-NOHA) and inducible NO synthase (iNOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) were from Calbiochem. Anti-TGF--neutralizing Ab was obtained from R&D Systems. HA-derived peptide (I-Ad restricted, aa 110–120, SFERFEIFPKE) and OVA-derived peptide (H-2Kb restricted, aa 257–264, SIINFEKL) were purchased from SynPep. Anti-CD45.2, Gr-1, CD8, CD4, CD11b, and isotype control IgG2a, IgG2b Abs were obtained from BD Pharmingen, and anti-F4/80 Ab from Serotec. Abs against mouse arginase I were obtained from BD Pharmingen, and anti-iNOS from Upstate Biotechnology. Anti-STAT3, phospho-STAT3, STAT-1, and phospho-STAT1 Abs were purchased from Cell Signaling Technology.
Cell isolation
Tumor-bearing or naive mice were sacrificed, and their spleens and tumors were harvested under sterile conditions. Single cell suspensions were prepared, and red cells were removed from spleens using ammonium chloride lysing buffer. Solid tumors were dissected and chopped into small pieces using a scissors before incubation with a mixture of enzymes dissolved in RPMI 1640 (400 U/ml collagenase type IV, 0.05 mg/ml collagenase type I, 0.025 mg/ml hyaluronidase, all from Sigma-Aldrich; 0.01 mg/ml DNase I and 0.2 trypsin inhibitor unit/ml soybean trypsin inhibitor, both from Boehringer Mannheim) for 30 min at 37°C. Cells were recovered by centrifugation and resuspended in PBS, containing 1% of FBS. To harvest ascitic tumors or peritoneal cells from naive mice, mice were injected i.p. with 5 ml of ice-cold PBS and then aspirated. F4/80+ and Gr-1+ cells were isolated from tumors, spleen, or peritoneal cell suspension using magnetic microbeads and MiniMACS columns (Miltenyi Biotec). Briefly, cells were resuspended in MACS buffer, and 5–6 x 106 cells were incubated with 5 µg of biotinylated anti-Gr-1 or F4/80-PE mAbs for 10 min on ice. Cells were washed with cold buffer to remove unbound Abs, and then incubated with streptavidin or PE microbeads for 15 min at 4°C. Gr-1+ and F4/80+ cell populations were isolated on MiniMACS columns, according to the manufacturer‘s instructions. Purity of cell populations was evaluated by flow cytometry and exceeded 90%.
Flow cytometry
One million cells were incubated for 30 min on ice in 100 µl of PBS with 1 µg of relevant Abs, and then washed twice with cold PBS. Flow cytometry data were acquired using a FACSCalibur flow cytometer (BD Biosciences), and were analyzed with CellQuest software (BD Biosciences).
Ag-specific and CD3/CD28-induced T cell proliferation
Ag-specific T cell proliferation. Ag-specific proliferation of CD4 T cells was evaluated using HA-TCR transgenic mice, whereas CD8 T cell-mediated response was measured using OT-1 transgenic mice. Splenocytes from transgenic mice were depleted of RBC, washed with PBS, resuspended in complete culture medium, and placed in triplicates into U-bottom 96-well plate (2 x 105/well) in presence of cognate Ags (HA-derived peptide SFERFEIFPKE for TCR-HA transgenic mice or OVA-derived peptide SIINFEKL for OT-1 transgenic mice) and cultured for 4 days. Eighteen hours before harvesting, cells were pulsed with [3H]thymidine (1 µCi/well; Amersham Biosciences). [3H]Thymidine uptake was counted using a liquid scintillation counter and expressed as cpm.
CD3/CD28-induced T cell proliferation. Splenocytes were seeded in triplicates at concentration of 2 x 105 per well into U-bottom 96-well plates in presence of 1 µg/ml anti-CD3 Ab and 5 µg/ml anti-CD28 Ab for 72 h. Eighteen hours before harvesting, cells were pulsed with [3H]thymidine. Cell proliferation was evaluated, as described above.
Apoptosis
Apoptosis was evaluated by flow cytometry using annexin V-PE apoptosis detection kit (BD Pharmingen), according to manufacturer’s protocol.
Western blotting
Freshly isolated F4/80+ or Gr-1+ cells were lysed with radioimmunoprecipitation assay buffer in the presence of protease and phosphatase inhibitors. Samples (30 µg protein/lane) were subjected to electrophoresis in 10% SDS-polyacrylamide gels, and then blotted onto 0.45-µm nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with 5% dry skimmed milk in TBS (20 mM Tris-HCl, pH 7.6, 137 mM NaCl plus 0.1% (v/v) Tween 20) and then probed with rabbit Abs with appropriate specificity overnight at 4°C. Membranes were washed and incubated for 2 h at room temperature with secondary Ab (goat anti-rabbit Ab conjugated with HRP). Results were visualized by chemiluminescence detection using a commercial kit (Amersham Biosciences).
Arginase activity
Arginase activity was measured in cell lysates, as previously described by Corraliza et al. (28). Briefly, cells were lysed for 30 min with 100 µl of 0.1% Triton X-100. Subsequently, 100 µl of 25 mM Tris-HCl and 10 µl of 10 mM MnCl2 were added, and the enzyme was activated by heating for 10 min at 56°C. Arginine hydrolysis was conducted by incubating the lysate with 100 µl of 0.5 M L-arginine (pH 9.7) at 37°C for 15–120 min. The reaction was stopped with 900 µl of H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v). The urea concentration was measured at 540 nm after addition of 40 µl of -isonitrosopropiophenone (dissolved in 100% ethanol), followed by heating at 95°C for 30 min. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol urea per min.
Confocal microscopy
Freshly purified F4/80+ or Gr-1+ cells were washed in cold PBS with 1% FBS, fixed in 1% paraformaldehyde, and then stained overnight with anti-phospho-STAT6 (Tyr641) or rabbit IgG, followed by FITC goat F(ab')2 anti-rabbit Ab (Southern Biotechnology Associates) in a solution containing PBS, 1% FBS, 0.2% saponin, and 3% cold fish gelatin (Sigma-Aldrich). After the final wash, the cells were resuspended in a minimal volume of Vectashield (Vector Laboratories), placed on a slide, and covered with a coverslip, and the edges were sealed with nail polish. Cells were observed on a Leica DM IRBC confocal microscope (Leica Microsystems).
NO production
Equal volumes of culture supernatants (100 µl) were mixed with Greiss reagent. After 10-min incubation at room temperature, the absorbance at 550 nm was measured using microplate plate reader (Bio-Rad). Nitrite concentrations were determined by comparing the absorbance values for the test samples to a standard curve generated by serial dilution of 0.25 mM sodium nitrite.
Statistical analysis
The statistical significance between values was determined by Student’s t test. All data were expressed as the mean ± SD. Probability values >0.05 were considered nonsignificant.
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Results |
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Previous studies have demonstrated that Gr-1+ cells accumulate in practically all tested mouse tumor models and contain immature myeloid cells at different stages of cell differentiation (10, 11). Adoptive transfer of these cells into naive tumor-free recipients or tumor-bearing mice results in their differentiation into mature myeloid cells, although in tumor-bearing mice significant proportion of donor cells retained their immature phenotype (Gr-1+CD11b+) (12). In this study, we investigated the fate and suppressive activity of myeloid cells localized in tumor tissues. Gr-1+ cells were isolated from spleens of C3 tumor-bearing C57BL/6 mice (CD45.2+). The phenotype of these cells is shown in Fig. 1A. Five million of these Gr-1+ cells were injected i.v. into each congenic CD45.1+ mouse-bearing C3 tumor (1–1.5 cm in diameter). Spleens and tumors were collected 3 days after the transfer, and the phenotype of donor’s CD45.2+ cells was evaluated using multicolor flow cytometry. Significant proportion of donor’s cells isolated from tumor site retained an immature phenotype (Gr-1+CD11b+). This population was substantially smaller in donors’ cells isolated from spleens (Fig. 1A). In spleen, equal proportion (20%) of donors’ cells had the phenotype of DCs (CD11c+IAb+) and macrophages (Gr-1–F4/80+) (Fig. 1A). In contrast, >70% of the donors’ cells isolated from tumors expressed F4/80 marker specific for macrophages. Practically all F4/80+ donors’ cells isolated from tumors were also Gr-1+ and CD11b+ (Fig. 1A and data not shown). The percentage of these cells was not changed if cells were collected 5 days after the transfer (data not shown). These results indicate that Gr-1+ myeloid cells are precursors of F4/80 TAM.
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What population of myeloid cells isolated from tumor tissue could be responsible for inhibition of T cells? Because F4/80+ macrophages are the major components of myeloid cells inside the tumors, we have investigated the ability of these cells to induce T cell suppression. F4/80+ cells were isolated from tumor or spleens of CT-26 tumor-bearing BALB/c mice using magnetic beads, and then added at different ratios to splenocytes from HA transgenic BALB/c mice stimulated with control or specific MHC class II-matched HA-derived peptide. Neither Gr-1+ nor F4/80+ cells isolated from spleens of tumor-bearing mice were able to significantly affect CD4-mediated Ag-specific T cell proliferation (Fig. 2A). Gr-1+ cells from tumor also failed to suppress T cell response. However, F4/80+ cells isolated from tumor significantly inhibited T cell proliferation at 1:4 ratio and almost completely blocked it at 1:2 ratio (Fig. 2B).
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We evaluated the phenotype of F4/80+ isolated from control and tumor-bearing mice. Typical results of these experiments are shown in Fig. 2E. F4/80+ macrophages isolated from spleens of naive tumor-free BALB/c and C57BL/6 mice contained equal proportion of Gr-1+ CD11b+ double-positive cells (36–39%). The proportion of these cells among F4/80+ macrophages isolated from spleens of tumor-bearing mice was slightly increased (46–51%). In contrast, >93% of F4/80+ cells isolated from tumor sites expressed both Gr-1 and CD11b markers (Fig. 2E). All tested tumor models demonstrated similar results (Fig. 2E and data not shown). Importantly, <30% of Gr-1+ cells isolated from tumor tissues expressed F4/80 marker (Fig. 2F). Among all cells in tumor tissues, the proportion of Gr-1–F4/80+ cells was 2–4%, and Gr-1+F4/80– cells 5–7%. These data indicate that substantial proportion of Gr-1+ cells in tumor tissues does not express F4/80 marker and probably represents myeloid cells at different stage of differentiation. This may explain different ability of F4/80+ and Gr-1+ cells to suppress T cell response.
Thus, these data indicate that Gr-1+ IMC in spleen or in the vicinity of tumors do not suppress CD4-mediated T cell response, which is consistent with our previous observations (19). In tumor-bearing mice, these cells differentiate into F4/80+ macrophages. However, in the vicinity of tumor and in spleens, the phenotype of these cells and their ability to suppress T cell response were profoundly different. If in spleens only one-half of F4/80+ cells were Gr-1+ and lacked the ability to suppress T cell response, practically all F4/80+ cells inside the tumors expressed Gr-1 marker and were highly immunosuppressive. These results were reproduced in at least four different tumor models on two different mouse strains.
Tumor-associated F4/80+ macrophages induce apoptosis of T cells, which is mediated by increased production of NO and arginase activity
Previous studies have demonstrated that macrophages isolated from tumor-bearing mice may induce apoptosis of T cells (33). To test this possibility, F4/80+ cells were isolated from spleens and tumors of C3 tumor-bearing mice and incubated with splenocytes from control syngeneic C57BL/6 mice stimulated with anti-CD3 and anti-CD28 Abs. Twenty-four and 48 h later, cells were collected and labeled with allophycocyanin-conjugated anti-CD4 Ab, FITC-conjugated anti-CD8 Ab, PE-conjugated annexin V, and 7-aminoactinomycin D (7-AAD). To maintain consistency with experiments described above, we used the same F4/80+ cells:splenocytes ratio, 1:4. The proportion of annexin V-positive, 7-AAD-negative apoptotic cells within populations of CD4+ or CD8+ T cells was calculated. Tumor-associated, but not spleen-derived F4/80+ cells induced significant level of apoptosis in both populations of T cells (Fig. 3A). The presence of F4/80+ TAM, however, did not induce apoptosis of nonstimulated T cells (data not shown). Similar effect was seen in a different tumor model in EL-4 tumor-bearing C57BL/6 mice. F4/80+ cells were isolated from spleens and peritoneal exudates of tumor-bearing mice and incubated with splenocytes from transgenic OT-1 C57BL/6 mice in the presence of specific OVA-derived peptide SIINFEKL. Twenty-four and 48 h later, cells were collected and labeled with anti-CD8 Ab, annexin V, and 7-AAD. F4/80+ macrophages isolated from the vicinity of tumor (peritoneum), but not from spleens of tumor-bearing mice induced apoptosis of CD8+ T cells (Fig. 3B). After 48 h in culture, this practically resulted in complete elimination of CD8+ cells (Fig. 3C). As in experiments described above, no induction of apoptosis was seen if T cells were not stimulated with specific peptide (data not shown). The presence of tumor-derived F4/80+ cells decreased the proportion of alive cells after 48 h of culture >5-fold (Fig. 3D). Previous studies implicated IFN-
and TNF- in macrophage-induced apoptosis of T cells. In our experimental model, neutralizing anti-IFN- Ab partially and anti-TNF- completely abrogated the effect of tumor-derived F4/80+ macrophages on T cell deletion (Fig. 3D). However, these Abs only partially reversed the inhibitory effect of F4/80+ macrophages on T cell proliferation (Fig. 3E). Overall, these data are consistent with previous publications (33) and indicate that IFN- and TNF- are important factors mediating the effect of tumor-derived F4/80+ macrophages.
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Thus, these results indicate that T cell inhibition induced by TAM is mediated by the combined effect of two major factors: increased production of NO and high arginase activity.
Role of STAT transcription factors in TAM-mediated T cell inhibition
We asked which signaling pathway in myeloid cells could be responsible for the observed phenomena. Recent study implicated STAT6 as one of the possible factors mediating immunosuppressive function of myeloid cells (14). Therefore, we first compared the level of STAT6 expression in TAM and F4/80+ macrophages isolated from naive mice. F4/80+ TAM had substantially higher level of phospho-STAT6 than their control counterparts (Fig. 5A). To clarify the role of STAT6 in F4/80+ cell-mediated T cell suppression, EL-4 tumors were established in wild-type STAT6+/+ and STAT6–/– knockout mice. F4/80+ cells were isolated from tumor tissues and analyzed. No differences in T cell-suppressive activity between TAM isolated from STAT6+/+ and STAT6–/– mice were found (Fig. 5B). TAM isolated from STAT6+/+ and STAT6–/– tumor-bearing mice had also the same level of arginase activity (Fig. 5C). These data indicate that although TAM had increased level of STAT6 activity, it was not directly responsible for the increased level of arginase activity and T cell immunosuppression.
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Most of previous studies that described immunosuppressive myeloid cells in spleen of tumor-bearing mice used not freshly isolated splenocytes, but cells cultured for several days in vitro. To clarify the effect of culture on immunosuppressive activity of myeloid cells, we have isolated Gr-1+ cells from spleens of CT-26 tumor-bearing BALB/c mice and cultured them for 5 days in the presence of GM-CSF. The presence of GM-CSF was necessary to preserve viability of IMC. Resulting population of cells was represented mostly by F4/80+ macrophages (data not shown). In contrast to Gr-1+ IMC or F4/80+ macrophages freshly isolated from spleens of tumor-bearing mice, cultured cells had a profound inhibitory effect on T cell proliferation (Fig. 6A). This effect was associated with increased arginase activity and NO production (Fig. 6, B and C). STAT1 was not detected in freshly isolated Gr-1+ cells, but was clearly seen in cells after 5 days in culture (Fig. 6D). These data indicate that during in vitro culture, IMC differentiate into immunosuppressive macrophages and that process is associated with up-regulation of STAT1. We asked what condition could convert splenic F4/80+ macrophage into immunosuppressive cells? F4/80+ cells were isolated from spleens of EL-4 tumor-bearing mice and cultured either in complete medium alone or with EL-4 tumor cells. Because F4/80+ cells are mature macrophages, we used short incubation period without presence of growth factors. Two days later, F4/80+ cells were reisolated from cultures using magnetic beads separation technique. Neither freshly isolated F4/80+ cells, nor cells cultured in medium alone were able to suppress Ag-specific T cell proliferation or produce NO (Fig. 6, E and F). In contrast, macrophages cultured for 48 h with tumor cells acquired the ability to inhibit T cell and produce large amount of NO (Fig. 6, E and F). These data indicate that tumor environment may support conversion of mature macrophages to immunosuppressive cells, and this process is associated with increased NO production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Because F4/80+ cells represented a great majority of myeloid cells in tumor tissues, we focused further analysis on these cells. F4/80+ macrophages isolated from spleens of tumor-bearing mice failed to suppress T cell responses, whereas F4/80+ cells isolated from tumor tissues demonstrated profound inhibition of T cell proliferation in response to different stimuli. These findings were reproduced in two different mouse strains and four different tumor models. It is known that TAM may inhibit T cell responses by inducing apoptosis of activated T cells via up-regulation of NO production and arginase activity. Previous studies have implicated NO, PGs, and TNF- in macrophage-mediated T cell suppression (33, 34, 35). L-Arginine plays a central role in the normal function of the immune system. It is metabolized in macrophages by inducible NO synthase to produce NO, important in the cytotoxic mechanisms, and by arginase I and arginase II to synthesize L-ornithine and urea. Previous studies from Ochoa and colleagues (36, 37) have demonstrated that activated macrophages can modulate extracellular levels of L-arginine and alter T cell function. Human T cells stimulated and cultured in the absence of L-arginine lose the expression of the TCR 
-chain and have an impaired proliferation and a decreased cytokine production. Very recently, they have shown up-regulation of arginase I in tumor-associated myeloid cells (38). Consistent with our observations, they have found very little arginase I activity in Gr-1+CD11b+ cells, whereas the bulk of activity was associated with Gr-1–CD11b+ macrophages (38). Bronte and colleagues (20, 30) have recently demonstrated that induction of arginase and iNOS together causes activated T lymphocytes to undergo apoptosis. In this study, we for the first time demonstrated that this process of simultaneous activation of iNOS and arginase I indeed takes place in TAM. Arginase I and iNOS are both competing for the substrate. Previous studies demonstrated that overexpression of arginase in murine macrophage cell line J774A.1 enhanced L-ornithine and putrescine production and attenuated NO production by the LPS-activated macrophages (39). This suggests that there should be some powerful signaling to simultaneously activate both these enzymes. We suggested that it is likely to be mediated by STAT family of transcription factors.
STAT is a critical component of diverse signal transduction pathway that is actively involved in cellular survival, proliferation, and differentiation. Following ligand binding to the receptor, the activated Janus family of tyrosine kinases (JAK) phosphorylates receptors on target tyrosine sites, which generates docking sites for STATs. Subsequently, recruited STATs are phosphorylated and dimerized, followed by their translocation into the nucleus, where they modulate expression of target genes. The STAT family of transcription factors consists of seven members (reviewed in Ref. 40). Previous studies implicated STAT6 as possible factor involved in immune suppression associated with myeloid cells (14, 41, 42). We have demonstrated important role of STAT3 in accumulation of Gr-1+ IMC in cancer (13), and a number of groups demonstrated important role of STAT1 in regulation of iNOS activity (43, 44). Present study demonstrated that increased STAT6 activity in TAM was not responsible for their immunosuppressive activity. In line with our previous observation, myeloid cells in spleen of tumor-bearing mice had increased level of STAT3 activity and undetectable STAT1 activity. However, surprisingly opposite results were obtained in TAM. These cells had no detectable STAT3 activity, but increased STAT1. These data are consistent with recent observation that STAT3 inhibits transcription of the iNOS by interacting with NF-
B (45). These data are also consistent with recent observation that pharmacological inhibition of STAT3 activity in macrophages with AG490 did not affect the level of NO production (E. Sotomayor, unpublished observation). TAM isolated from STAT1 knockout mice failed to suppress T cell responses. Importantly, these cells lacked arginase I activity and had very low iNOS expression and NO production. Taken together, these data indicate that STAT1 activation in TAM may be responsible for up-regulation of iNOS and arginase I activity in these cells that in turn result in T cell suppression. At this time, it is not clear what factor or factors of the tumor microenvironment are responsible for up-regulation of STAT1 in TAM. It is possible that interaction between TAM and tumor cells or tumor-associated fibroblasts or endothelial cells may contribute to this effect. In our experiments, tumor cells were able to convert nonsuppressive splenic F4/80+ macrophage to highly immunosuppressive cells.
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants CA84488 and CA100062 to D.I.G. 
2 Address correspondence and reprint requests to Dr. Dmitry I. Gabrilovich, H. Lee Moffitt Cancer Center, University of South Florida, MRC-2, Room 2067, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: dgabril{at}moffitt.usf.edu; or Dr. Sergei Kusmartsev, H. Lee Moffitt Cancer Center, University of South Florida, MRC-2, Room 2068, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: kusmarsa{at}moffitt.usf.edu
3 Abbreviations used in this paper: IMC, immature myeloid cell; 7-AAD, 7-aminoactinomycin D; DC, dendritic cell; HA, hemagglutinin; iNOS, inducible NO synthase; L-NMMA, NG-monomethyl-L-arginine; nor-NOHA, NW-hydroxyl-nor-L-arginine; TAM, tumor-associated macrophage.
Received for publication August 27, 2004. Accepted for publication February 1, 2005.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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P. Serafini, S. Mgebroff, K. Noonan, and I. Borrello Myeloid-Derived Suppressor Cells Promote Cross-Tolerance in B-Cell Lymphoma by Expanding Regulatory T Cells Cancer Res., July 1, 2008; 68(13): 5439 - 5449. [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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Y. Sawanobori, S. Ueha, M. Kurachi, T. Shimaoka, J. E. Talmadge, J. Abe, Y. Shono, M. Kitabatake, K. Kakimi, N. Mukaida, et al. Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice Blood, June 15, 2008; 111(12): 5457 - 5466. [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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B. Zhang, Y. Zhang, N. A. Bowerman, A. Schietinger, Y.-X. Fu, D. M. Kranz, D. A. Rowley, and H. Schreiber Equilibrium between Host and Cancer Caused by Effector T Cells Killing Tumor Stroma Cancer Res., March 1, 2008; 68(5): 1563 - 1571. [Abstract] [Full Text] [PDF]
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S. K. Biswas, A. Sica, and C. E. Lewis Plasticity of Macrophage Function during Tumor Progression: Regulation by Distinct Molecular Mechanisms J. Immunol., February 15, 2008; 180(4): 2011 - 2017. [Abstract] [Full Text] [PDF]
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 S. R. Perri, B. Annabi, and J. Galipeau Angiostatin inhibits monocyte/macrophage migration via disruption of actin cytoskeleton FASEB J, December 1, 2007; 21(14): 3928 - 3936. [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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W. Badn, E. Visse, A. Darabi, K. E. Smith, L. G. Salford, and P. Siesjo Postimmunization with IFN-{gamma}-Secreting Glioma Cells Combined with the Inducible Nitric Oxide Synthase Inhibitor Mercaptoethylguanidine Prolongs Survival of Rats with Intracerebral Tumors J. Immunol., September 15, 2007; 179(6): 4231 - 4238. [Abstract] [Full Text] [PDF]
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J. E. Talmadge Pathways Mediating the Expansion and Immunosuppressive Activity of Myeloid-Derived Suppressor Cells and Their Relevance to Cancer Therapy Clin. Cancer Res., September 15, 2007; 13(18): 5243 - 5248. [Abstract] [Full Text] [PDF]
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S. Yu, C. Liu, K. Su, J. Wang, Y. Liu, L. Zhang, C. Li, Y. Cong, R. Kimberly, W. E. Grizzle, et al. Tumor Exosomes Inhibit Differentiation of Bone Marrow Dendritic Cells J. Immunol., June 1, 2007; 178(11): 6867 - 6875. [Abstract] [Full Text] [PDF]
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C. Liu, S. Yu, J. Kappes, J. Wang, W. E. Grizzle, K. R. Zinn, and H.-G. Zhang Expansion of spleen myeloid suppressor cells represses NK cell cytotoxicity in tumor-bearing host Blood, May 15, 2007; 109(10): 4336 - 4342. [Abstract] [Full Text] [PDF]
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M.-H. Tsai, J. A. Cook, G. V.R. Chandramouli, W. DeGraff, H. Yan, S. Zhao, C. N. Coleman, J. B. Mitchell, and E. Y. Chuang Gene Expression Profiling of Breast, Prostate, and Glioma Cells following Single versus Fractionated Doses of Radiation Cancer Res., April 15, 2007; 67(8): 3845 - 3852. [Abstract] [Full Text] [PDF]
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 M. Stempelj, M. Kedinger, L. Augenlicht, and L. Klampfer Essential Role of the JAK/STAT1 Signaling Pathway in the Expression of Inducible Nitric-oxide Synthase in Intestinal Epithelial Cells and Its Regulation by Butyrate J. Biol. Chem., March 30, 2007; 282(13): 9797 - 9804. [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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 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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K. C. McKenna and J. A. Kapp Accumulation of Immunosuppressive CD11b+ Myeloid Cells Correlates with the Failure to Prevent Tumor Growth in the Anterior Chamber of the Eye J. Immunol., August 1, 2006; 177(3): 1599 - 1608. [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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A. Sanchez-Aguilera, C. Montalban, P. de la Cueva, L. Sanchez-Verde, M. M. Morente, M. Garcia-Cosio, J. Garcia-Larana, C. Bellas, M. Provencio, V. Romagosa, et al. Tumor microenvironment and mitotic checkpoint are key factors in the outcome of classic Hodgkin lymphoma Blood, July 15, 2006; 108(2): 662 - 668. [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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P. C. Rodriguez, C. P. Hernandez, D. Quiceno, S. M. Dubinett, J. Zabaleta, J. B. Ochoa, J. Gilbert, and A. C. Ochoa Arginase I in myeloid suppressor cells is induced by COX-2 in lung carcinoma J. Exp. Med., October 3, 2005; 202(7): 931 - 939. [Abstract] [Full Text] [PDF]
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