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Experimental Therapeutics |
H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/ß TCR specific for MHC class II-restricted SFERFEIFPKE peptide, derived from influenza HA, were originally obtained from Harold von Boehmer (Basel Institute for Immunology, Basel, Switzerland) and then were crossed to a BALB/c background for >10 generations.
Reagents.
Pellets with ATRA and placebo were obtained from Innovative Research of America (Sarasota, FL). Control MHC class I (Kd)-restricted (IYSTVASSL) and MHC class II (I-Ad)-restricted (SFERFEIFPKE) HA-derived peptides, MHC class I-restricted mutant p53-derived peptide (KYICNSSCM), HPV-16-derived E7 peptide (H-2Kd-restricted, aa 49–57, RAHYNIVTF), as well as OVA control peptide (H-2Kb-restricted, aa 257–264, SIINFEKL) were purchased from SynPep Corporation (Dublin, CA). A recombinant vaccinia virus-encoding HA from the 1934 PR8 strain of influenza was a gift from Frank Guarneri (Johns Hopkins Institute, Baltimore, MD). A recombinant adenovirus encoding full open-reading frame of wild-type p53 gene was described elsewhere (21)
. The mAbs used for flow cytometry were CD45.2 (clone 104), Gr-1 (clone RB6–8C5), CD11b (clone M1/70), CD11c (clone HL3), I-Ab (clone AF6-120.1), CD86 (clone GL1) from BD PharMingen; CD4 (clone GK1.5) and anticlonotypic TCR (clone 6.5) are from Caltag (Burlingame, CA) and F4/80 from Serotec, Inc. (Raleigh, NC).
Tumor Models.
DA3, 7,12-dimethylbenz(a)anthracene-induced mammalian adenocarcinoma, was obtained from Dr. Diana Lopez (University of Miami, Miami, FL). DA3-HA tumor was generated by transfection of DA3 tumor cells with pIHA, which encodes the full open-reading frame of HA from the influenza virus A/PR/8/34. C3 fibrosarcoma was made by transformation of B6 mouse embryonic cells with HPV-16-induced (22)
and kindly provided by Dr. Martin Kast (Loyola University of Chicago, Maywood, IL). MHC class I- restricted HPV-16-derived peptide RAHYNIVTF expressed by this tumor was shown to elicit potent antitumor immune response (23)
. MethA cells were obtained from Dr. Lloyd J. Old. MethA tumor is methylcholantrene-induced sarcoma developed in BALB/c mice and passaged as an ascitic tumor. It is a relatively immunogenic tumor that carries a mutant endogenous p53 gene. The cell cultures were maintained at 37°C in a 5% CO2-humidified atmosphere. DA3-HA and C3 cell lines were maintained in a DMEM culture medium supplemented with OPI medium supplement (for DA3-HA), 10% FCS, 100 units/mg penicillin, 100 µg/ml streptomycin, 25 mM HEPES, and 2 mM glutamine (all from Life Technologies, Inc., Grand Island, NY). MethA cells were passaged in vivo as an ascitic tumor in BALB/c mice. To initiate the experiments, tumor cells in 0.1 ml of PBS were injected s.c. into the shaved right flank of each mouse. To establish tumor, mice were injected with 106 DA3-HA cells, 5 x 105 C3 cells, or 105 MethA cells.
ATRA Pellets Implantation and Treatment Protocols.
ATRA pellets (21-day release, 5 mg) or placebo pellets were implanted s.c. by trocar injection at the side contralateral to the tumor (into left flank). For in vivo assessment of ATRA effect on tumor-antigen specific CD4+ T-cell-mediated immune response, we used DA3-HA tumor model. ATRA (or placebo) pellets were implanted into BALB/c mice on day 4 after tumor inoculation. Seven days later, 2.5 x 106 double-positive CD4 and clonotypic TCR lymphocytes from HA-TCR-transgenic mice were injected i.v. Four days later (day 15 after tumor implantation), mice were in vivo primed by s.c. inoculation with 1 x 107 pfu of recombinant vaccinia encoding HA in 0.1 ml of PBS. Mice were sacrificed 1 week later, and antigen-specific response was evaluated.
For evaluation of ATRA effect on CD8+ T-cell tumor antigen-specific response we used a C3 tumor model. On day 5 after tumor cell inoculation, C57Bl/6 mice were immunized with s.c. injection of 50 µg of C3 peptide (RAHYNIVTF) emulsified in CFA. ATRA (or placebo) pellets were implanted 7 days later, and the same day mice were given second immunization with peptide. On day 17 after tumor inoculation, mice were immunized with C3 peptide the third time, and 10 days later, ice was sacrificed.
To study antitumor effect of ATRA in combination with immunotherapy, we used the MethA tumor model. For immunization, DCs were generated from bone marrow of BALB/c mice and infected with Ad-p53 as described below. DCs were injected s.c. into mice (4 x 105 cells/mouse) on days 5, 10, and 15 after tumor cell inoculation. ATRA pellet (or placebo pellet) was implanted on day 7 after tumor cell injection.
Cell Isolation.
Mice were sacrificed by cervical dislocation, and their spleens, lymph nodes, tibias, and femurs were harvested under sterile conditions. Spleen cell suspensions were prepared, and red cells were depleted by incubation with ACK buffer. Cells were washed and resuspended in complete culture medium. Bone marrow cells were obtained by flushing the contents of the mouse femora and tibia with cold PBS. T lymphocytes were purified from spleens of naïve TCR-HA-transgenic mice using T-cell enrichment columns (MTCC-525; R&D Systems, Minneapolis, MN). Gr-1+ cells were isolated from spleens of C3 tumor-bearing mice using magnetic beads. Briefly, spleen cell suspension (107 splenocytes/ml) was incubated for 15 min at 4°C with biotinylated anti-Gr-1 mAbs (clone RB6-8C5; BD PharMingen). Cells were washed to remove unbound antibodies and then incubated with streptavidin microbeads for 15 min at 4°C. Positive Gr-1+ cell population was isolated on a MS+ MACS column according to the manufacturer’s instructions (Miltenyi Biotec GmbH, Auburn, CA). Purity of Gr-1+ cell population was evaluated by flow cytometry and exceeded 90%.
Generation of Bone Marrow-derived DC and Infection with p53-Adenovirus.
Generation of DCs and their infection with Ad-p53 was performed as described before (21)
. Briefly, DCs were generated from murine bone marrow using RPMI 1640 supplemented with 10% FCS, 20 ng/ml murine recombinant GM-CSF, and 10 ng/ml IL-4 (both from RDI, Flanders, NJ). The cultures were maintained at 37°C in 5% CO2-humidified atmosphere in 24-well plates. On day 3 of culture, floating cells were gently removed, and fresh medium with cytokines was replaced. On day 5, cells were collected, and DCs were enriched by centrifugation over a 13.5% metrizamide gradient (Accurate Chemicals, Westbury, NY). DCs were washed in serum-free medium and infected with Ad-p53 (20,000 viral particles/cell) for 2 h in 0.5 ml of serum-free medium supplemented with 20 ng/ml GM-CSF and 10 ng/ml IL-4 in 24-well plates followed by culturing in complete culture medium with the cytokines for an additional 24 h.
ELISPOT Assay.
ELISPOT assay was performed as described earlier (11)
. Briefly, MultiScreen-HA plates (Millipore Corporation, Berford, MA) were precoated with anti-IFN-
mAbs (cloneR4-A2; BD PharMingen, San Diego, CA) by overnight incubation in PBS at 4°C. Splenocytes from immunized mice were plated in wells (2 x 105 cells/well) in complete media and cultured for 24 h at 37°C in the presence of the specific (RAHYNIVTF) or control (SIINFEKL) peptide (10 µg/ml). Wells were gently rinsed with PBS containing 0.1% Tween 20 and after adding of biotinylated anti-IFN- mAbs (BD PharMingen), plates were incubated overnight at 4°C. Results were visualized using avidine-alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate (Sigma, St. Louis, MO). The number of spots in each well was scored by in blind fashion by two investigators and then recalculated per 106 cells.
ELISA.
ELISA was performed using antibodies and protocol developed by BD PharMingen. The sensitivity of the assay was 6 pg/ml.
Flow Cytometry.
Cells (106 in PBS containing 0.1% FCS) were incubated for 30 min with optimal concentration of antibodies on ice and then washed twice with cold PBS. Fluorescence-activated cell sorting data were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Fluorescence-activated cell sorting data were analyzed using CellQuest software (BD Biosciences).
RNase Protection Assay.
RNA was extracted from C3 and DA3-HA tumor cells using Trizol reagent (Life Technologies, Inc.). Twelve µg of each sample RNA was dried and resuspended in hybridization buffer. RNase protection assay was performed using BD PharMingen RiboQuant kit according to manufacturer’s protocol. Briefly, [32P]UTP-labeled multitemplate probe was synthesized in an in vitro transcription reaction using a template set that contained DNA templates for murine IL-3, IL-6, IL-10, IL-11, GM-CSF, M-CSF, G-CSF, VEGF, as well as L32 and GAPDH. The probe (2.8 x 105 cpm) was added to each sample. After hybridization, samples were treated with RNase, then hybridized probes were extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol. Samples were subjected to electrophoreses on 5% denaturing polyacrylamide gel. Gel was dried and exposed to phosphor screen. Quantitation was performed using ImageQuaNT software (Molecular Dynamics, Inc., Sunnyvale, CA). Each protected fragment was normalized against two housekeeping genes L32 and GAPDH.
Statistical Analysis.
Statistical analysis was performed using JMP statistical software (SAS Institute, Cary, NC).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment of Mice with ATRA Reduces the Number of ImCs.
In agreement with observations made by a number of research groups, growth of C3 fibrosarcoma or DA3-HA mammary adenocarcinoma was associated with substantial accumulation of Gr-1+/CD11b+ ImC in spleens (Fig. 1A)
. Three weeks after tumor cell injection, almost one-third of all splenocytes had the phenotype of ImCs. Absolute number of ImCs increased >15-fold (data not shown). Treatment with ATRA did not affect the total number of splenocytes in tumor-bearing mice, but it significantly decreased the proportion of ImCs. In C3 tumor-bearing mice treated with ATRA, the percentage of ImC was only 10.9 ± 2.4%, whereas ImCs comprised 27.2 ± 7.8% of the total splenocytes in mice treated with placebo (P < 0.01). In DA3-HA tumor-bearing mice, treatment with ATRA decreased the proportion of ImC from 34.1 ± 3.4 to 18.5 ± 3.9% (P < 0.05). In control tumor-free mice, Gr-1+CD11b+ ImCs represented >30% of bone marrow cells and <1.5% of lymph node cells (Fig. 1, B and C)
. In C3 tumor-bearing, mice the presence of ImCs increased 2-fold in both bone marrow and lymph nodes. Treatment of C3 tumor-bearing mice with ATRA significantly reduced the population of ImCs in both lymph nodes and bone marrow (Fig. 1, B and C)
. We investigated the time course of ATRA effect on the population of ImCs. Treatment of C3 tumor-bearing mice with ATRA or placebo was started 7 days after tumor inoculation. Mice were sacrificed 7, 14, and 21 days after the start of the treatment, and the proportion of Gr-1+CD11b+ ImCs was evaluated. The presence of ImCs in C3 tumor-bearing mice was significantly increased as early as 2 weeks after tumor inoculation (Fig. 1D)
. ATRA significantly reduced the presence of ImCs at all tested time points (Fig. 1D)
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. Tumor growth in vivo was also unchanged (Fig. 2B)
. This is consistent with previously reported observations that in therapeutic concentrations, ATRA does not directly inhibit growth of solid tumors (24)
. Thus, changes in tumor volume were not responsible for the effect of ATRA on ImCs.
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ATRA Induces Differentiation of ImCs.
Next, we evaluated the effect of ATRA on ImC differentiation in vivo in adoptive transfer experiments. Gr-1+ cells were isolated from spleens of C3 tumor-bearing C57BL/6 (CD45.2+) mice using a magnetic bead separation technique. The purity of Gr-1+CD11b+ ImCs was >90% (data not shown). ImCs (3 x 106) were transferred i.v. into B6.SJL-PtrcaPep3b/BoyJ congenic (CD45.1+) mice. Pellets containing ATRA or vehicle alone (placebo) were implanted into recipient mice 1 week before the transfer of ImCs. The proportion of donors’ CD45.2+ cells in the total population of splenocytes, and the presence of immature and mature myeloid cells within the population of CD45.2+ cells was evaluated 3 and 5 days after the transfer using multicolor flow cytometry. Three days after adoptive transfer into mice treated with placebo donors’ CD45.2+ cells represented 1.1 ± 0.3% of nucleated cells in recipients’ spleens. Five days after the transfer, their presence slightly reduced to 0.8 ± 0.3% (P > 0.1). On day 3 and 5 after the transfer into ATRA-treated mice, the proportion of the donors’ CD45.2+ cells in the spleens was similar to that in placebo treated mice (1.2 ± 0.3 and 0.9 ± 0.3%, respectively). After transfer into control mice, a significant proportion of donors’ cells (25%) retained the phenotype of ImCs (Gr-1+CD11b+; Fig. 3
). The rest of the cells consisted of CD11c+IAb+ DCs, F4/80+ macrophages, and a small proportion of Gr-1+CD11b- granulocytes (Fig. 3)
. In the presence of ATRA, the proportion of ImCs decreased rapidly (16.3% on day 3, P < 0.05), and by day 5, it reached the level observed in naïve tumor-free mice (3.3%). The proportions of DCs, macrophages, and granulocytes among donors’ CD45.2+ cells were significantly higher in ATRA-treated mice than in mice treated with placebo (Fig. 3)
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. No changes in the populations of CD11c+B220+Gr-1+ plasmocytoid DCs or F4/80+CD11b- mature macrophages were found. This was associated with significant increase in the number of granulocytes (Fig. 4A)
. Seven days of ATRA treatment resulted in a dramatic increase in the number of DCs. It was restored to the control level (Fig. 4A)
. Because ATRA did not significantly affect the total number of splenocytes (data not shown), all changes in the absolute numbers of DCs were caused by an increased proportion of these cells. ATRA did not change the number of mature macrophages or granulocytes in spleens (Fig. 4A)
. The same effect of ATRA was seen in spleens isolated after 14 days of treatment (24 days after tumor inoculation; data not shown). Lymph nodes isolated from C3 tumor-bearing mice 17 days after tumor inoculation did not show significant changes in the populations of myeloid cells (data not shown). This is consistent with previously reported data (25)
and reflects the fact that DC turnover in spleens is significantly higher than in lymph nodes (26
, 27)
. However, by day 24 after tumor inoculation, a decrease in the population of DCs became evident in lymph nodes as well (Fig. 4B)
. Treatment of mice with ATRA significantly increased the number of DCs and granulocytes (Fig. 4B)
. Thus, it appears that ATRA induces differentiation of ImCs into mature cells, preferentially DCs.
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As expected, immunization of control mice resulted in significant expansion of the clonotype-positive CD4+ T cells (Fig. 5A)
. These T cells produced large amounts of IL-2 and IFN- in response to restimulation with HA-specific peptide (Fig. 5, B and C)
. There was no expansion of antigen-specific T lymphocytes obtained from tumor-bearing mice treated with placebo pellets. These cells did not respond to stimulation with specific peptide (Fig. 5, A and B)
. These data are consistent with previously reported characteristics of this model of tumor-induced immune tolerance. Treatment of tumor-bearing mice with ATRA resulted in significant expansion of antigen-specific CD4+ T cells. Vaccination with vaccinia-HA increased the proportion of these cells in spleen even further (Fig. 5A)
. Production of IL-2 in response to specific peptide was restored to the control level both in spleen and in lymph nodes of these mice. (Fig. 5B)
. IFN- production was increased at a lesser extent and did not reach control level (Fig. 5C)
. These results indicate that ATRA administration can substantially reduce tumor-induced CD4+ T-cell tolerance.
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. This was consistent with many previous observations that immunotherapy of established tumors is often ineffective. In contrast, in the presence of ATRA, the decrease in tumor growth was much more sustained. By day 35, there was more than a 3-fold difference in tumor size between the group of mice treated with combination of ATRA and immunization and all other groups (P < 0.01). In a separate set of experiments, we evaluated the immune response to vaccination. On day 27, mice were sacrificed, and the presence of antigen-specific CTL precursors was evaluated by IFN- production in ELISPOT assay. T cells derived from spleens of placebo-treated, placebo-treated and immunized with peptide, or ATRA-treated mice did not respond to in vitro restimulation with the specific peptide (Fig. 6B)
. In contrast, a significant number of antigen-specific IFN--producing T cells was found in the spleens of ATRA-treated tumor-bearing mice that had been immunized with C3-derived peptide (Fig. 6B)
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5-fold smaller than in mice from any other group (P < 0.001; Fig. 6C
). In a separate group of experiments, we evaluated the immune response to vaccination. Four weeks after tumor inoculation, mice were sacrificed, and the presence of antigen-specific CTL precursors was evaluated by IFN- production in ELISPOT assay. As specific peptide, we used H2Kd-restricted mutant p53-derived peptide (KYICNSSCM). This peptide is specific for MethA sarcoma. Previous study demonstrated that treatment of MethA sarcoma with DCs in combination with -irradiation induced potent immune response against this particular epitope, probably via mechanism of epitope spreading (28)
. In control, H2Kd-restricted HA-derived peptide (IYSTVASSL) was used. Only T cells derived from spleens of immunized mice treated with ATRA demonstrated significant level of antigen-specific immune response 4 weeks after tumor inoculation (Fig. 6D)
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DISCUSSION |
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, RARß, RAR) and the RXRs (RXR, RXRß, RXR). Each receptor subtype is encoded by a separate gene (29)
. In most cases, an efficient transduction of the ATRA signal requires heterodimerization between RAR and RXR. Our previous experiments showed that ImCs expressed both RAR and RXR types of ATRA receptors (V. Gorelov, unpublished observation). In this study, we first investigated whether in vitro observations can be confirmed in vivo. We selected pellets containing 5 mg of ATRA because this dose did not result in toxicity but still provided a clear effect on ImC. This dose resulted in <0.1 µM serum concentration of ATRA. In all three tested tumor models, ATRA administration substantially reduced the proportion and absolute number of ImCs in spleens. There could be several possible explanations of this fact: (a) ATRA has an antitumor effect in our experimental tumor models, and decreased tumor mass produces less factors able to stimulate ImC production; (b) ATRA does not affect tumor growth but inhibits production by tumors of ImC growth factors; (c) ATRA induces differentiation of ImCs; and (d) ATRA induces apoptosis of ImCs.
ATRA at concentration as high as 2 µM did not affect the growth of selected tumor cells in vitro. It was possible that ATRA could affect tumor growth not directly but via endothelial cells, which could interfere with tumor vascular supply. To test this possibility, we evaluated tumor growth in vivo in mice treated with 5 mg of ATRA pellets. No differences from the control group were found in any of the used animal tumor models. It has been reported that at higher concentrations, ATRA was able to suppress growth of some solid tumors. However, clinical trials performed in the past several years yielded a very limited effect. This could be partly explained by decreased expression of the specific receptors (24) . Hyperproduction of myeloid cell growth factors such as M-CSF, G-CSF, GM-CSF, or factors able to affect myelopoiesis such as VEGF by tumor cells could be responsible for an accumulation of ImCs in cancer. Therefore, we asked whether ATRA effects on ImCs in tumor-bearing mice could be explained by the decreased production of some of these growth factors. In our experiments, ATRA did not inhibit expression of any of the growth factors. Moreover, it increased expression of G-CSF in DA3-HA tumor cells. These findings were consistent with previous observations that ATRA might increase the production of some myeloid cell growth factors by bone marrow stromal cells (30) and in acute promyelocytic leukemia (31) . However, the fact that ATRA did not suppress the expression of some myeloid growth factors and VEGF, by itself, was not sufficient to rule out this mechanism because there are number of other factors that potentially could be involved in that process. Therefore, we directly evaluated ATRA effect on differentiation of ImCs in vivo using adoptive transfer of ImCs into congenic mice. Our experiments demonstrated that ATRA induces rapid differentiation of ImCs into mature DCs, macrophages, and granulocytes. Five days after the transfer, the presence of donors’ ImCs in ATRA-treated mice was reduced to the normal level, whereas in placebo-treated mice, one-fourth of the donors’ cells still expressed the phenotype of ImCs. This was consistent with our previous observations in vitro (11) and the fact that ATRA treatment did not affect the total number of splenocytes in vitro or in vivo. These data also argue against the possibility that ATRA-induced apoptosis of ImCs may be responsible for the elimination of these cells from spleens. Tumor-bearing mice had a reduced number of DCs in spleens and lymph nodes. This was in agreement with previously reported observations (25 , 32) . ATRA treatment did not significantly affect the total number of splenocytes or lymph node cells but increased the number of DCs in these mice. Because this increase was associated with a decreased presence of ImCs in these mice, the results of these experiments demonstrated that ATRA might reduce presence of ImCs by differentiating them into mature myeloid cells.
Next, we asked whether this effect had any consequences for antitumor immune response. Freshly isolated ImCs expressed MHC class I but not MHC class II molecules and suppressed only CD8-mediated but not CD4-mediated responses (11)
. However, within several days after adoptive transfer into control or tumor-bearing mice, these cells up-regulated MHC class II expression (33)
. In addition, after several days in culture, ImCs differentiate into immunosuppressive macrophages that produce high levels of NO and suppress both CD4+ and CD8+ T cells (8
, 34)
. Therefore, we suggested that elimination of ImCs in vivo might improve not only CD8-mediated but also CD4-mediated immune responses. To test this hypothesis, two experimental tumor models were used. HA-expressing tumor abrogated the response of antigen-specific CD4+T cells to vaccination. It manifested in lack of expansion of antigen-specific CD4+T cells and diminished production of IL-2 and IFN- in response to the stimulation with MHC class II-matched specific antigen. This phenomenon has been previously described in this type of experimental system (12
, 35)
. ATRA treatment increased the proportion of antigen-specific T cells and IL-2 production to the control level. IFN- production by T cells was significantly increased. However, it did not reach the control level. A similar effect was observed in MHC class I-restricted T-cell response to vaccination. Tumor-bearing mice immunized with MHC class I-bound specific peptide failed to maintain antigen-specific response 10 days after the last immunization, whereas T cells from immunized mice treated with ATRA showed significant response. These data indicate that ATRA considerably improves antitumor immune response.
In two experimental systems, using two different methods of immunization, ATRA dramatically enhanced the antitumor immune effect of vaccination. It appears that this treatment increases not the strength of the responses because it did not increase the number of mice rejecting tumors but the longevity of the responses. One of the well-described problems of immunotherapy of established tumors is the limited duration of its effect. Tumor growth resumes soon after the finish of the therapy. That has seriously weakened the overall efficacy of the treatment. This is associated with inhibition of tumor-specific immune response. In a previous study, we directly confirmed that by implanting tumor to mice previously immunized with irrelevant antigen, preexisted strong CTL response diminished within 10 days after tumor implantation (36) . In tumor models, used in this study, tumor growth resumed in about a week after the last immunization. However, in both models, in mice treated with ATRA, tumor growth remained suppressed a long time after that. It is consistent with the mechanism of ATRA effects on ImC. Decreased presence of ImC in tumor-bearing mice reduces their immunosuppressive effect on T cells, which allow CTLs persist longer and keep tumor growth under control. It appears that the treatment with ATRA alone will not be sufficient to provide sustained long-lasting effect of immunization. Repeated vaccination will be required to maintain clonal outgrowth of antigen-specific T cells. However, ATRA may be a critical factor supporting the persistence of these CTLs and, thus, dramatically improving the effect of vaccination.
In summary, here we demonstrated that decreased presence of ImCs might dramatically improve antitumor immune response and enhance the effect of vaccination. ATRA provides such an effect and may be a valuable addition to different cancer vaccines.
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FOOTNOTES |
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1 These authors equally contributed to this work. 
2 To whom requests for reprints should be addressed, at H. Lee Moffitt Cancer Center, University of South Florida, MRC-2E, Room 2067, 12902 Magnolia Drive, Tampa, FL 33612. Phone: (813) 903-6863; Fax: (813) 632-1328; E-mail: dgabril{at}moffitt.usf.edu
3 The abbreviations used are: ImC, immature myeloid cell; DC, dendritic cell; TCR, T-cell receptor; HA, hemagglutinin; ATRA, all-trans-retinoic acid; HPV, human papillomavirus; mAb, monoclonal antibody; CFA, complete Freund adjuvant; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; VEGF, vascular endothelial growth factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ad-p53, adenovirus containing wild-type p53 gene; RAR, retinoic acid receptor; RXR, retinoid X receptor.
4 S. Kusmartsev et al. submitted for publication.
Received 9/30/02. Accepted 5/23/03.
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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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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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C. E. Clark, S. R. Hingorani, R. Mick, C. Combs, D. A. Tuveson, and R. H. Vonderheide Dynamics of the Immune Reaction to Pancreatic Cancer from Inception to Invasion Cancer Res., October 1, 2007; 67(19): 9518 - 9527. [Abstract] [Full Text] [PDF]
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S. Darmanin, J. Chen, S. Zhao, H. Cui, R. Shirkoohi, N. Kubo, Y. Kuge, N. Tamaki, K. Nakagawa, J.-i. Hamada, et al. All-trans Retinoic Acid Enhances Murine Dendritic Cell Migration to Draining Lymph Nodes via the Balance of Matrix Metalloproteinases and Their Inhibitors J. Immunol., October 1, 2007; 179(7): 4616 - 4625. [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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I. Fricke, N. Mirza, J. Dupont, C. Lockhart, A. Jackson, J.-H. Lee, J. A. Sosman, and D. I. Gabrilovich Vascular Endothelial Growth Factor-Trap Overcomes Defects in Dendritic Cell Differentiation but Does Not Improve Antigen-Specific Immune Responses Clin. Cancer Res., August 15, 2007; 13(16): 4840 - 4848. [Abstract] [Full Text] [PDF]
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H.-J. Ko, Y.-J. Kim, Y.-S. Kim, W.-S. Chang, S.-Y. Ko, S.-Y. Chang, S. Sakaguchi, and C.-Y. Kang A Combination of Chemoimmunotherapies Can Efficiently Break Self-Tolerance and Induce Antitumor Immunity in a Tolerogenic Murine Tumor Model Cancer Res., August 1, 2007; 67(15): 7477 - 7486. [Abstract] [Full Text] [PDF]
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Y. Huang, X. Chen, M. M. Dikov, S. V. Novitskiy, C. A. Mosse, L. Yang, and D. P. Carbone Distinct roles of VEGFR-1 and VEGFR-2 in the aberrant hematopoiesis associated with elevated levels of VEGF Blood, July 15, 2007; 110(2): 624 - 631. [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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K. Yanagisawa, M. A. Exley, X. Jiang, N. Ohkochi, M. Taniguchi, and K.-i. Seino Hyporesponsiveness to Natural Killer T-Cell Ligand {alpha}-Galactosylceramide in Cancer-Bearing State Mediated by CD11b+ Gr-1+ Cells Producing Nitric Oxide Cancer Res., December 1, 2006; 66(23): 11441 - 11446. [Abstract] [Full Text] [PDF]
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N. Mirza, M. Fishman, I. Fricke, M. Dunn, A. M. Neuger, T. J. Frost, R. M. Lush, S. Antonia, and D. I. Gabrilovich All-trans-Retinoic Acid Improves Differentiation of Myeloid Cells and Immune Response in Cancer Patients. Cancer Res., September 15, 2006; 66(18): 9299 - 9307. [Abstract] [Full Text] [PDF]
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Y. Nefedova, S. Nagaraj, A. Rosenbauer, C. Muro-Cacho, S. M. Sebti, and D. I. Gabrilovich 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., October 15, 2005; 65(20): 9525 - 9535. [Abstract] [Full Text] [PDF]
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Y. Nefedova, P. Cheng, D. Gilkes, M. Blaskovich, A. A. Beg, S. M. Sebti, and D. I. Gabrilovich Activation of Dendritic Cells via Inhibition of Jak2/STAT3 Signaling J. Immunol., October 1, 2005; 175(7): 4338 - 4346. [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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 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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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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 L. M. Hengesbach and K. A. Hoag Physiological Concentrations of Retinoic Acid Favor Myeloid Dendritic Cell Development over Granulocyte Development in Cultures of Bone Marrow Cells from Mice J. Nutr., October 1, 2004; 134(10): 2653 - 2659. [Abstract] [Full Text] [PDF]
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 D. E. Spaner Amplifying cancer vaccine responses by modifying pathogenic gene programs in tumor cells J. Leukoc. Biol., August 1, 2004; 76(2): 338 - 351. [Abstract] [Full Text] [PDF]
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