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1.  CCL2 Blockade Augments Cancer Immunotherapy 
Cancer research  2009;70(1):109.
Since an immuno-inhibitory environment exists within tumors, successful vaccines will likely require additional approaches to alter the tumor microenvironment. Monocyte chemoattractant proteins (such as CCL2) are produced by many tumors and have both direct and indirect immuno-inhibitory effects. We hypothesized that CCL2 blockade would reduce immunosuppression and augment vaccine immunotherapy. Anti-murine-CCL2/CCL12 monoclonal antibodies were administered in three immunotherapy models: one aimed at the HPV-E7 antigen expressed by a non-small cell lung cancer line, one targeted to mesothelin expressed by a mesothelioma cell line, and one using an adenovirus expressing Interferon-α to treat a non-immunogenic, non-small cell lung cancer line. We evaluated the effect of the combination treatment on tumor growth and assessed the mechanism of these changes by evaluating cytotoxic T cells, immunosuppressive cells, and the tumor microenvironment. Administration of anti-CCL2/CCL12 antibodies along with the vaccines markedly augmented efficacy with enhanced reduction in tumor volume and cures of approximately half of the tumors. The combined treatment generated more total intra-tumoral CD8+ T-cells that were more activated and more anti-tumor antigen specific, as measured by tetramer evaluation. Another important potential mechanism was reduction in intratumoral T-regulatory (T-reg) cells. CCL2 appears to be a key proximal cytokine mediating immunosuppression in tumors. Its blockade augments CD8+ T cell immune response to tumors elicited by vaccines via multifactorial mechanisms. These observations suggest that combining CCL2 neutralization with vaccines should be considered in future immunotherapy trials.
doi:10.1158/0008-5472.CAN-09-2326
PMCID: PMC2821565  PMID: 20028856
CCL2; Cancer immunotherapy; Lung Cancer; Mesothelioma; T-lymphocytes
2.  T cell Surveillance of Oncogene-induced Prostate Cancer is Impeded by T Cell-derived TGF-β1 Cytokine 
Immunity  2011;35(1):123-134.
Summary
Tolerance induction in T cells takes place in most tumors and is thought to account for tumor evasion from immune eradication. Production of the cytokine TGF-β is implicated in immunosuppression, however the cellular mechanism by which TGF-β induces T cell dysfunction remains unclear. Using a transgenic model of prostate cancer, we showed that tumor development was not suppressed by the adaptive immune system, which was associated with heightened TGF-β signaling in T cells from the tumor-draining lymph nodes. Blockade of TGF-β signaling in T cells enhanced tumor antigen-specific T cell responses, and inhibited tumor development. Surprisingly, T cell- but not Treg cell-specific ablation of TGF-β1 was sufficient to augment T cell cytotoxic activity and blocked tumor growth and metastases. These findings reveal that T cell production of TGF-β1 is an essential requirement for tumors to evade immunosurveillance independent of TGF-β produced by tumors.
doi:10.1016/j.immuni.2011.04.019
PMCID: PMC3430371  PMID: 21757379
3.  Redirecting adaptive immunity against foreign antigens to tumors for cancer therapy 
Cancer biology & therapy  2007;6(11):1773-1779.
Immunotherapy for cancer is often limited by weak immunogenicity of tumor antigens. However, immune systems are usually strong and effective against foreign invading antigens. To test whether the destructive effect of adaptive immunity against foreign antigens can be redirected to tumors for cancer therapy, we immunized mice with adenovector expressing LacZ (Ad/CMV-LacZ). Subcutaneous syngeneic tumors were then established in the immunized animals or in naïve animals. The immune response against adenovirus or LacZ was redirected to tumors by intratumoral injection of Ad/CMV-LacZ. We found that immunization and treatment with the adenovector dramatically reduced the tumor growth rate compared with intratumoral administration of adenovector in naïve mice. Complete tumor regression was observed in about 50% of the immunized animals but not in the naïve animals. Similar effects were observed when oncolytic vaccinia virus was used to immunize and treat tumors. Lymphocyte infiltration in tumors was dramatically increased in the immunized group when compared with other groups. Moreover, immunity against parental tumor cells was induced in the animals cured with immunization and treatment with Ad/CMV-LacZ, as evidenced by the lack of tumor growth when the mice were challenged with parental tumor cells. Taken together, these results suggest that redirecting adaptive immunity against foreign antigens is a potential approach for anticancer therapy and that pre-existing immunity could enhance virotherapy against cancers.
PMCID: PMC2387205  PMID: 17986853
Gene therapy; cancer vaccine; virotherapy; immunotherapy
4.  The timing of TGF-β inhibition affects the generation of antigen-specific CD8+ T Cells 
BMC Immunology  2013;14:30.
Background
Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation.
Methods
BALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells.
Results
Inhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-βR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-βR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-βR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05).
Conclusions
These studies provide the first in vivo evidence that TGF-β may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-β in the immune system, and the future development of TGF-β inhibiting drugs.
doi:10.1186/1471-2172-14-30
PMCID: PMC3725164  PMID: 23865808
Malignant mesothelioma; Tumor immunology; Immune suppression; TGF-β; CD8+ Cytotoxic T cell
5.  Eradication of Metastatic Renal Cell Carcinoma after Adenovirus-Encoded TNF-Related Apoptosis-Inducing Ligand (TRAIL)/CpG Immunotherapy 
PLoS ONE  2012;7(2):e31085.
Despite evidence that antitumor immunity can be protective against renal cell carcinoma (RCC), few patients respond objectively to immunotherapy and the disease is fatal once metastases develop. We asked to what extent combinatorial immunotherapy with Adenovirus-encoded murine TNF-related apoptosis-inducing ligand (Ad5mTRAIL) plus CpG oligonucleotide, given at the primary tumor site, would prove efficacious against metastatic murine RCC. To quantitate primary renal and metastatic tumor growth in mice, we developed a luciferase-expressing Renca cell line, and monitored tumor burdens via bioluminescent imaging. Orthotopic tumor challenge gave rise to aggressive primary tumors and lung metastases that were detectable by day 7. Intra-renal administration of Ad5mTRAIL+CpG on day 7 led to an influx of effector phenotype CD4 and CD8 T cells into the kidney by day 12 and regression of established primary renal tumors. Intra-renal immunotherapy also led to systemic immune responses characterized by splenomegaly, elevated serum IgG levels, increased CD4 and CD8 T cell infiltration into the lungs, and elimination of metastatic lung tumors. Tumor regression was primarily dependent upon CD8 T cells and resulted in prolonged survival of treated mice. Thus, local administration of Ad5mTRAIL+CpG at the primary tumor site can initiate CD8-dependent systemic immunity that is sufficient to cause regression of metastatic lung tumors. A similar approach may prove beneficial for patients with metastatic RCC.
doi:10.1371/journal.pone.0031085
PMCID: PMC3270031  PMID: 22312440
6.  TGF-β Receptor Blockade Augments the Effectiveness of Adoptive T-Cell Therapy of Established Solid Cancers 
Purpose
Adoptive cellular immunotherapy has promise as an approach to eradicate established tumors. However, a significant hurdle in the success of cellular immunotherapy involves recently identified mechanisms of immune suppression on cytotoxic T-cells at the effector phase.
Transforming growth factor-β (TGF-β) is one of the most important of these immunosuppressive factors because it affects both T-cell and macrophage functions. We thus hypothesized that systemic blockade of TGF-β signaling combined with adoptive T-cell transfer would enhance the effectiveness of the therapy.
Experimental Design
Flank tumors were generated in mice using the OVA-albumin (OA) expressing thymoma cell line, EG7. Splenocytes from transgenic OT-1 mice (whose CD8 T-cells recognize an immunodominant peptide in OA) were activated in vitro and adoptively transferred into mice bearing large tumors in the presence or absence of an orally available TGF-β receptor-I kinase blocker (SM16).
Results
We observed markedly smaller tumors in the group receiving the combination of SM16 chow and adoptive transfer. Additional investigation revealed that TGF-β receptor blockade increased the persistence of adoptively transferred T-cells in the spleen and lymph nodes, increased numbers of adoptively transferred T-cells within tumors, increased activation of these infiltrating T-cells, and altered the tumor microenvironment with a significant increase in TNF-α and decrease in arginase mRNA expression
Conclusions
We found that systemic blockade of TGF-β receptor activity augmented the anti-tumor activity of adoptively transferred T-cells and may thus be a useful adjunct in future clinical trials.
doi:10.1158/1078-0432.CCR-08-0356
PMCID: PMC2491721  PMID: 18559619
tumor immunology; immunosuppression; TGFβ; Cytotoxic T-cells; cytokines; adoptive transfer
7.  The small molecule TGF-β signaling inhibitor SM16 synergizes with agonistic OX40 antibody to suppress established mammary tumors and reduce spontaneous metastasis 
Effective tumor immunotherapy may require not only activation of anti-tumor effector cells, but also abrogation of tumor-mediated immunosuppression. The cytokine, TGF-β, is frequently elevated in the tumor microenvironment and is a potent immunosuppressive agent and promoter of tumor metastasis. OX40 (CD134) is a member of the TNF-α receptor superfamily and ligation by agonistic antibody (anti-OX40) enhances effector function, expansion and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of a small molecule TGF-β signaling inhibitor, SM16, plus anti-OX40 in the poorly immunogenic, highly metastatic, TGF-β-secreting 4T1 mammary tumor model. Our data show that SM16 synergizes with anti-OX40 to elicit a potent anti-tumor effect against established primary tumors, with a 79% reduction in tumor size, a 95% reduction in the number of metastatic lung nodules and a cure rate of 38%. This positive treatment outcome was associated with a 3.2-fold increase of tumor-infiltrating, activated CD8+ T cells, an overall accumulation of CD4+ and CD8+ T cells, and an increased tumor-specific effector T cell response. Complete abrogation of the therapeutic effect in vivo following depletion of CD4+ and CD8+ T cells, suggests that the anti-tumor efficacy of SM16 + anti-OX40 therapy is T cell dependent. Mice that were cured of their tumors were able to reject tumor re-challenge and manifested a significant tumor-specific peripheral memory IFN-γ response. Taken together, these data suggest that combining a TGF-β signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.
doi:10.1007/s00262-011-1119-y
PMCID: PMC3595193  PMID: 21971588
Breast cancer; 4T1; OX40; TGF-β; TGFβRI/ALK5; small molecule kinase inhibitor; SM16
8.  TGF-β blockade controls ascites by preventing abnormalization of lymphatic vessels in orthotopic human ovarian carcinoma models 
Purpose
Ovarian cancer patients with malignant ascites have poor prognosis. The accumulation of ascites is caused by an imbalance between fluid extravasation from the blood vessels and reabsorption by lymphatic vessels. Whereas, the role of Transforming Growth Factor beta (TGF-β) in tumor progression has been well studied, the role of TGF-β in lymphatic vessel function is far from understood. Here, we sought to dissect the role of TGF-β blockade in the formation of ascites.
Experimental Design
We used soluble TGF-β Receptor II (sTβRII) to block TGF-β signaling in two orthotopic human ovarian carcinoma models: SKOV3ip1 and Hey-A8. We measured tumor proliferation, apoptosis, lymphangiogenesis and angiogenesis by immunohistochemical staining, and examined diaphragm lymphatic vessel network by intraperitoneal injection of a fluorescent dye. Diaphragm lymphatic vessel function was assessed by tracking fluorescent beads in the diaphragm and measuring their drainage rate.
Results
TGF-β blockade impaired tumor growth in both models, accompanied by a decreased tumor cell proliferation and angiogenesis. More strikingly, TGF-β blockade almost completely abolished ascites formation. TGF-β blockade significantly inhibited the expression of VEGF, which is the major contributor to ascites formation. At the same time, TGF-β blockade prevent ‘abnormalization’ of diaphragm lymphatic vessels and improved ascites drainage.
Conclusions
TGF-β blockade decreased ascites by both inhibiting ascites formation and improving ascites drainage. Based on our finding, it is reasonable to consider the use of TGF-β blockade as a palliative treatment for symptomatic ascites.
doi:10.1158/1078-0432.CCR-10-2429
PMCID: PMC3060297  PMID: 21278244
9.  Anti-transforming growth factor (TGF)-beta antibodies inhibit breast cancer cell tumorigenicity and increase mouse spleen natural killer cell activity. Implications for a possible role of tumor cell/host TGF-beta interactions in human breast cancer progression. 
Journal of Clinical Investigation  1993;92(6):2569-2576.
TGF-beta effects on angiogenesis, stroma formation, and immune function suggest its possible involvement in tumor progression. This hypothesis was tested using the 2G7 IgG2b, which neutralizes TGF-beta 1, -beta 2, and -beta 3, and the MDA-231 human breast cancer cell line. Inoculation of these cells in athymic mice decreases mouse spleen natural killer (NK) cell activity. Intraperitoneal injections of 2G7 starting 1 d after intraperitoneal inoculation of tumor cells suppressed intraabdominal tumor and lung metastases, whereas the nonneutralizing anti-TGF-beta 12H5 IgG2a had no effect. 2G7 transiently inhibited growth of established MDA-231 subcutaneous tumors. Histologically, both 2G7-treated and control tumors were identical. Intraperitoneal administration of 2G7 resulted in a marked increase in mouse spleen NK cell activity. 2G7 did not inhibit MDA-231 primary tumor or metastases formation, nor did it stimulate NK cell-mediated cytotoxicity in beige NK-deficient nude mice. Finally, serum-free conditioned medium from MDA-231 cells inhibited the NK cell activity of human blood lymphocytes. This inhibition was blocked by the neutralizing anti-TGF-beta 2G7 antibody but not by a nonspecific IgG2. These data support a possible role for tumor cell TGF-beta in the progression of mammary carcinomas by suppressing host immune surveillance.
Images
PMCID: PMC288452  PMID: 7504687
10.  Silencing CD36 gene expression results in the inhibition of latent-TGF-β1 activation and suppression of silica-induced lung fibrosis in the rat 
Respiratory Research  2009;10(1):36.
Background
The biologically active form of transforming growth factor-β1 (TGF-β1) plays a key role in the development of lung fibrosis. CD36 is involved in the transformation of latent TGF-β1 (L-TGF-β1) to active TGF-β1. To clarify the role of CD36 in the development of silica-induced lung fibrosis, a rat silicosis model was used to observe both the inhibition of L-TGF-β1 activation and the antifibrotic effect obtained by lentiviral vector silencing of CD36 expression.
Methods
The rat silicosis model was induced by intratracheal injection of 10 mg silica per rat and CD36 expression was silenced by administration of a lentiviral vector (Lv-shCD36). The inhibition of L-TGF-β1 activation was examined using a CCL-64 mink lung epithelial growth inhibition assay, while determination of hydroxyproline content along with pathological and immunohistochemical examinations were used for observation of the inhibition of silica-induced lung fibrosis.
Results
The lentiviral vector (Lv-shCD36) silenced expression of CD36 in alveolar macrophages (AMs) obtained from bronchoalveolar lavage fluid (BALF) and the activation of L-TGF-β1 in the BALF was inhibited by Lv-shCD36. The hydroxyproline content of silica+Lv-shCD36 treated groups was significantly lower than in other experimental groups. The degree of fibrosis in the silica+Lv-shCD36-treated groups was less than observed in other experimental groups. The expression of collagen I and III in the silica+Lv-shCD36-treated group was significantly lower than in the other experimental groups.
Conclusion
These results indicate that silencing expression of CD36 can result in the inhibition of L-TGF-β1 activation in a rat silicosis model, thus further preventing the development of silica-induced lung fibrosis.
doi:10.1186/1465-9921-10-36
PMCID: PMC2698900  PMID: 19439069
11.  Transforming growth factor beta is protective in host resistance against Listeria monocytogenes infection in mice. 
Infection and Immunity  1996;64(9):3901-3904.
The role of transforming growth factor beta (TGF-beta) in host resistance against Listeria monocytogenes infection was studied with mice. The constitutive expression of TGF-beta 1 mRNA was observed in the spleens and livers of mice before and after infection. Injecting the mice with anti-TGF-beta 1 peptide serum resulted in diminished antilisterial resistance, whereas the administration of human platelet-derived TGF-beta 1 enhanced the resistance. Moreover, mice were protected against lethal infection when treated with TGF-beta 1. These results suggest the TGF-beta 1 might be involved in antilisterial resistance. On the other hand, injecting the mice with TGF-beta 1 resulted in a decrease in the titers of endogenous gamma interferon, tumor necrosis factor alpha, and interleukin-6, which are crucial in antilisterial resistance, in sera and in extracts of spleen and liver. Thus, a complicated mechanism might be involved in the role of TGF-beta 1 in host resistance against L. monocytogenes infection.
PMCID: PMC174310  PMID: 8751946
12.  Tumor Stroma Engraftment of Gene-Modified Mesenchymal Stem Cells as Anti-Tumor Therapy against Ovarian Cancer 
Cytotherapy  2013;15(1):20-32.
Many ovarian cancers originate from ovarian surface epithelium, where they develop from cysts intermixed with stroma. The stromal layer is critical to the progression and survival of the neoplasm and consequently is recruited into the tumor microenvironment. Using both syngenic mouse tumors (ID8-R), and human xenograft (OVCAR3, SKOV3) tumor models, we first confirmed intraperitoneally-circulating MSC could target, preferentially engraft and differentiate into α-SMA+ myofibroblasts, suggesting their role as “reactive stroma” in ovarian carcinoma development and confirming their potential as a targeted delivery vehicle for the intratumoral production of interferon-beta (IFNβ). Then, mice with ovarian carcinomas received weekly IP injections of IFNβ expressing MSC, resulting in complete eradication of tumors in 70% of treated OVCAR3 mice (P = 0.004) and an increased survival of treated SKOV3 mice compared with controls (P = 0.01). Similar tumor growth control was observed using murine IFNβ delivered by murine MSC in ID8-R ovarian carcinoma. As a potential mechanism of tumor killing, MSC produced IFNβ induced caspase-dependent tumor cell apoptosis. Our results demonstrate that ovarian carcinoma engraft MSC to participate in myofibrovascular networks and that IFNβ produced by MSC intratumorally modulates tumor kinetics, resulting in prolonged survival.
doi:10.1016/j.jcyt.2012.10.003
PMCID: PMC3709838  PMID: 23260083
mesenchymal stem cell; MSC; gene therapy; ovarian carcinoma; beta-interferon; ID8; non-invasive imaging
13.  Blockade of TGF-β enhances tumor vaccine efficacy mediated by CD8+ T cells 
Though TGF-β inhibition enhances anti-tumor immunity mediated by CD8+ T cells in several tumor models, it is not always sufficient for rejection of tumors. In the present study, to maximize the anti-tumor effect of TGF-β blockade, we tested the effect of anti-TGF-β combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significant delayed tumor growth, whereas anti-TGF-β antibodies alone did not show any anti-tumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-β antibodies compared to vaccinated mice without anti-TGF-β suggesting that anti-TGF-β synergistically enhanced irradiated tumor vaccine efficacy. CD8+ T cell-depletion completely abrogated the vaccine efficacy, so protection required CD8+ T cells. Depletion of CD25+ T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-β did not change the number of CD25+ T regulatory cells in un-vaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-β production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced anti-tumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-β enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-β levels found in cancer patients and that the effect is not dependent on TGF-β solely from CD4+CD25+ T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.
doi:10.1002/ijc.24961
PMCID: PMC2818422  PMID: 19830696
14.  In vivo antitumor activity of intratumoral fludarabine phosphate in refractory tumors expressing E. coli purine nucleoside phosphorylase 
Purpose
Systemically administered fludarabine phosphate (F-araAMP) slows growth of human tumor xenografts that express E. coli purine nucleoside phosphorylase (PNP). However, this treatment has been limited by the amount of F-araAMP that can be administered in vivo. The current study was designed to 1) determine whether efficacy of this overall strategy could be improved by intratumoral (IT) administration of F-araAMP, 2) test enhancement of the approach with external beam radiation, and 3) optimize recombinant adenovirus as a means to augment PNP delivery and bystander killing in vivo.
Methods
The effects of systemic or intratumoral F-araAMP in mice were investigated with human tumor xenografts (300 mg) in which 10% of the cells expressed E. coli PNP from a lentiviral promoter. Tumors injected with an adenoviral vector expressing E. coli PNP (Ad/PNP; 2 × 1011 viral particles, 2×/day × 3 days) and the impact of radiotherapy on tumors treated by this approach were also studied. Radiolabeled F-araAMP was used to monitor prodrug activation in vivo.
Results
Intratumoral administration of F-araAMP in human tumor xenografts expressing E. coli PNP resulted in complete regressions and/or prolonged tumor inhibition. External beam radiation significantly augmented this effect. Injection of large human tumor xenografts (human glioma, non-small cell lung cancer, or malignant prostate tumors) with Ad/PNP followed by intratumoral F-araAMP resulted in excellent antitumor activity superior to that observed following systemic administration of prodrug.
Conclusions
Activation of F-araAMP by E. coli PNP results in destruction of large tumor xenografts in vivo, augments radiotherapy, and promotes robust bystander killing. Our results indicate that intratumoral injection of F-araAMP leads to ablation of tumors in vivo with minimal toxicity.
doi:10.1007/s00280-012-1908-9
PMCID: PMC3423194  PMID: 22760227
tumor sensitization; low growth fraction malignancy; fludarabine; viral gene transfer; E. coli PNP
15.  Lymph Node-Targeted Immunotherapy Mediates Potent Immunity Resulting in Regression of Isolated or Metastatic HPV-Transformed Tumors 
Purpose
The goal of this study was to investigate the therapeutic potential of a novel immunotherapy strategy resulting in immunity to localized or metastatic HPV 16-transformed murine tumors.
Experimental design
Animals bearing E7-expressing tumors were co-immunized by lymph node injection with E7 49-57 antigen and TLR3-ligand (synthetic dsRNA). Immune responses were measured by flow cytometry and anti-tumor efficacy was evaluated by tumor size and survival. In situ cytotoxicity assays and identification of tumor-infiltrating lymphocytes and T regulatory cells were used to assess the mechanisms of treatment resistance in bulky disease. Chemotherapy with cyclophosphamide was explored to augment immunotherapy in late-stage disease.
Results
In therapeutic and prophylactic settings, immunization resulted in a considerable expansion of E7 49-57 antigen-specific T lymphocytes in the range of 1/10 CD8+ T cells. The resulting immunity was effective in suppressing disease progression and mortality in a pulmonary metastatic disease model. Therapeutic immunization resulted in control of isolated tumors up to a certain volume, and correlated with anti-tumor immune responses measured in blood. In situ analysis showed that within bulky tumors, T cell function was affected by negative regulatory mechanisms linked to an increase in T regulatory cells and could be overcome by cyclophosphamide treatment in conjunction with immunization.
Conclusions
This study highlights a novel cancer immunotherapy platform with potential for translatability to the clinic and suggests its potential usefulness for controlling metastatic disease, solid tumors of limited size, or larger tumors when combined with cytotoxic agents that reduce the number of tumor-infiltrating T regulatory cells.
doi:10.1158/1078-0432.CCR-09-0645
PMCID: PMC2756704  PMID: 19789304
T Cells; Peptides; Tumor Immunity; Vaccination; HPV
16.  Augmentation of Antitumor Immunity by Fusions of Ethanol-Treated Tumor Cells and Dendritic Cells Stimulated via Dual TLRs through TGF-β1 Blockade and IL-12p70 Production 
PLoS ONE  2013;8(5):e63498.
The therapeutic efficacy of fusion cell (FC)-based cancer vaccine generated with whole tumor cells and dendritic cells (DCs) requires the improved immunogenicity of both cells. Treatment of whole tumor cells with ethanol resulted in blockade of immune-suppressive soluble factors such as transforming growth factor (TGF)-β1, vascular endothelial growth factor, and IL-10 without decreased expression of major histocompatibility complex (MHC) class I and the MUC1 tumor-associated antigen. Moreover, the ethanol-treated tumor cells expressed “eat-me” signals such as calreticulin (CRT) on the cell surface and released immunostimulatory factors such as heat shock protein (HSP)90α and high-mobility group box 1 (HMGB1). A dual stimulation of protein-bound polysaccharides isolated from Coriolus versicolor (TLR2 agonist) and penicillin-inactivated Streptococcus pyogenes (TLR4 agonist) led human monocyte-derived DCs to produce HSP90α and multiple cytokines such as IL-12p70 and IL-10. Interestingly, incorporating ethanol-treated tumor cells and TLRs-stimulated DCs during the fusion process promoted fusion efficiency and up-regulated MHC class II molecules on a per fusion basis. Moreover, fusions of ethanol-treated tumor cells and dual TLRs-stimulated DCs (E-tumor/FCs) inhibited the production of multiple immune-suppressive soluble factors including TGF-β1 and up-regulated the production of IL-12p70 and HSP90α. Most importantly, E-tumor/FCs activated T cells capable of producing high levels of IFN-γ, resulting in augmented MUC1-specific CTL induction. Collectively, our results illustrate the synergy between ethanol-treated whole tumor cells and dual TLRs-stimulated DCs in inducing augmented CTL responses in vitro by FC preparations. The alternative system is simple and may provide a platform for adoptive immunotherapy.
doi:10.1371/journal.pone.0063498
PMCID: PMC3663747  PMID: 23717436
17.  A first or dominant immunization. II. Induced immunoglobulin carries transforming growth factor beta and suppresses cytolytic T cell responses to unrelated alloantigens 
Fresh sera from mice immunized by bearing an immunogenic tumor or by repeated injections of allogeneic spleen cells or xenogeneic erythrocytes powerfully suppress cytolytic T cell responses in one-way mixed lymphocyte cultures. Suppression is not antigen specific, though is mediated by immunoglobulin (Ig)G specific for the immunizing antigen. Suppression caused by IgG mimics that caused by active transforming growth factor beta (TGF-beta). IgG associates with or carries latent TGF-beta; however, suppression caused by the complex of IgG-TGF-beta requires macrophages (M phi), whereas active TGF-beta alone does not. Also, IgG dissociated from TGF-beta does not cause suppression, suggesting that M phi may take up Ig-TGF-beta, process the complex, and deliver active TGF-beta to lymphocytes. Indeed, suppression by immune serum was prevented by antibody to Fc receptors, by saturating Fc receptors with heterologous IgGs, and by antibodies against TGF-beta. The overall findings reveal a previously unrecognized regulatory circuit whereby IgG produced in response to one antigen nonspecifically downregulates cytolytic T lymphocyte responses to unrelated antigens. The findings introduce the intriguing possibility that TGF-beta delivered by IgG and processed by M phi may mediate important biological effects in processes such as wound healing, tumor growth, and some autoimmune diseases.
PMCID: PMC2191145  PMID: 8350058
18.  Systemic Inhibition of Transforming Growth Factor-β in Glioma-Bearing Mice Improves the Therapeutic Efficacy of Glioma-Associated Antigen Peptide Vaccines 
Purpose
A variety of cancers, including malignant gliomas, overexpress transforming growth factor-β (TGF-β), which helps tumors evade effective immune surveillance through a variety of mechanisms, including inhibition of CD8+ cytotoxic T lymphocytes (CTL) and enhancing the generation of regulatory T (Treg) cells. We hypothesized that inhibition of TGF-β would improve the efficacy of vaccines targeting glioma-associated antigen (GAA)-derived CTL epitopes by reversal of immunosuppression.
Experimental Design
Mice bearing orthotopic GL261 gliomas were treated systemically with a TGF-β neutralizing monoclonal antibody, 1D11, with or without subcutaneous (s.c.) vaccinations of synthetic peptides for GAA-derived CTL epitopes, GARC-1 (77-85) and EphA2 (671-679) emulsified in incomplete Freund's adjuvant.
Results
Mice receiving the combination regimen exhibited significantly prolonged survival compared with mice receiving either 1D11 alone, GAA-vaccines alone or mock-treatments alone. TGF-β neutralization enhanced the systemic induction of antigen-specific CTLs in glioma-bearing mice. Flow cytometric analyses of brain infiltrating lymphocytes revealed that 1D11 treatment suppressed phosphorylation of Smad2, increased GAA-reactive/interferon (IFN)-γ-producing CD8+ T cells, and reduced CD4+/FoxP3+ Treg cells in the glioma microenvironment. Neutralization of TGF-β also up-regulated plasma levels of interleukin (IL)-12, macrophage inflammatory protein-1α and IFN-inducible protein-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment up-regulated plasma IL-15 levels and promoted the persistence of GAA-reactive CD8+ T cells in glioma-bearing mice.
Conclusions
These data suggest that systemic inhibition of TGF-β by 1D11 can reverse the suppressive immunological environment of orthotopic tumor-bearing mice both systemically and locally, thereby enhancing the therapeutic efficacy of GAA-vaccines.
doi:10.1158/1078-0432.CCR-09-1067
PMCID: PMC2783346  PMID: 19861464
Transforming growth factor β; glioma vaccines; type-1 immune response; regulatory T cells
19.  Suppression of lung cancer in murine model: treated by combination of recombinant human endostsatin adenovirus with low-dose cisplatin 
Background
The sustained growth of tumors necessitates neovascularization. As one of the potent endogenous vascular inhibitors, endostatin has been widely used in antiangiogenesis therapy for tumor. Cisplatin is normally administered in chemotherapy for lung cancer but accompanied with serious side effects. In the current study, we investigated a novel chemo-antiangiogenesis therapeutic strategy to both improve toxic effects on lung cancer cells and reduce damages to normal cells in the anti-tumor therapy.
Methods
In vitro, we transduced LLC cells with Ad-hEndo and collected supernatants. Western blotting analysis of the supernatants revealed expression of endostatin. In vivo, to fully investigate the suppression effect on murine lung cancer of the combination therapy, we injected recombinant human endostatin adenovirus intratumorally plus a low dose of cisplatin intraperitoneally routinely. The tumor volume and survival time were observed. Angiogenesis was apparently inhibited within the tumor tissues and on the alginate beads. Assessment of apoptotic cells by the TUNEL assay was conducted in the tumor tissues.
Results
The combination treatment significantly suppressed the tumor growth and prolonged survival time of the murine LLC tumor model. This anti-tumor activity was associated with decreased microvessel density and increased apoptotic index of tumor cells.
Conclusion
According to the results in this study, recombinant human endostatin adenovirus in combination with a low dose of cisplatin demonstrated apparent synergistic anti-tumor activity without marked toxicity. Thus, these observations may provide a rational alternative for lung cancer treatment.
doi:10.1186/1756-9966-28-31
PMCID: PMC2657125  PMID: 19265510
20.  TGF-Beta Suppresses VEGFA-Mediated Angiogenesis in Colon Cancer Metastasis 
PLoS ONE  2013;8(3):e59918.
The FET cell line, derived from an early stage colon carcinoma, is non-tumorigenic in athymic nude mice. Engineered FET cells that express TGF-α (FETα) display constitutively active EGFR/ErbB signaling. These cells readily formed xenograft tumors in athymic nude mice. Importantly, FETα cells retained their response to TGF-beta-mediated growth inhibition, and, like the parental FET cells, expression of a dominant negative TGF-beta type II receptor (DNRII) in FETα cells (FETα/DNRII) abrogated responsiveness to TGF-beta-induced growth inhibition and apoptosis under stress conditions in vitro and increased metastatic potential in an orthotopic model in vivo, which indicates metastasis suppressor activity of TGF-beta signaling in this model. Cancer angiogenesis is widely regarded as a key attribute for tumor formation and progression. Here we show that TGF-beta signaling inhibits expression of vascular endothelial growth factor A (VEGFA) and that loss of autocrine TGF-beta in FETα/DNRII cells resulted in increased expression of VEGFA. Regulation of VEGFA expression by TGF-beta is not at the transcriptional level but at the post-transcriptional level. Our results indicate that TGF-beta decreases VEGFA protein stability through ubiquitination and degradation in a PKA- and Smad3-dependent and Smad2-independent pathway. Immunohistochemical (IHC) analyses of orthotopic tumors showed significantly reduced TGF-beta signaling, increased CD31 and VEGFA staining in tumors of FETα/DNRII cells as compared to those of vector control cells. These results indicate that inhibition of TGF-beta signaling increases VEGFA expression and angiogenesis, which could potentially contribute to enhanced metastasis of those cells in vivo. IHC studies performed on human colon adenocarcinoma specimens showed that TGF-beta signaling is inversely correlated with VEGFA expression, indicating that TGF-beta-mediated suppression of VEGFA expression exists in colon cancer patients.
doi:10.1371/journal.pone.0059918
PMCID: PMC3607554  PMID: 23536895
21.  Potent systemic therapy of Multiple Myeloma utilizing Oncolytic Vesicular stomatitis virus coding for Interferon-beta 
Cancer Gene Therapy  2012;19(7):443-450.
Multiple myeloma (MM) is an incurable malignancy of plasma secreting B-cells disseminated in the bone marrow. Successful utilization of oncolytic virotherapy for myeloma treatment requires a systemically administered virus that selectively destroys disseminated myeloma cells in an immune-competent host. Vesicular stomatitis virus (VSV) expressing Interferon-β (IFNβ) is a promising new oncolytic agent that exploits tumor-associated defects in innate immune signaling pathways to specifically destroy cancer cells. We demonstrate here that a single, intravenous dose of VSV-IFNβ specifically destroys subcutaneous and disseminated 5TGM1 myeloma in an immune competent myeloma model. VSV-IFN treatment significantly prolonged survival in mice bearing orthotopic myeloma. Viral murine IFNβ expression further delayed myeloma progression and significantly enhanced survival compared to VSV expressing human IFNβ. Evaluation of VSV-IFNβ oncolytic activity in human myeloma cell lines and primary patient samples confirmed myeloma specific oncolytic activity but revealed variable susceptibility to VSV-IFNβ oncolysis. The results indicate that VSV-IFNβ is a potent, safe oncolytic agent that can be systemically administered to effectively target and destroy disseminated myeloma in immune competent mice. IFNβ expression improves cancer specificity and enhances VSV therapeutic efficacy against disseminated myeloma. These data show VSV-IFNβ to be a promising vector for further development as a potential therapy for treatment of Multiple myeloma.
doi:10.1038/cgt.2012.14
PMCID: PMC3380174  PMID: 22522623
Oncolytic; virotherapy; myeloma; Vesicular stomatitis virus; systemic
22.  Mechanisms of Decreased Expression of Transforming Growth Factor-Beta Receptor Type I at Late Stages of HPV16-Mediated Transformation 
Cancer letters  2009;282(2):177-186.
Transforming growth factor-beta (TGF-β) signaling is disrupted in many cancers, including cervical cancer, leading to TGF-β resistance. Although initially sensitive, human papillomavirus type 16 (HPV16) immortalized human keratinocytes (HKc/HPV16) become increasingly resistant to the growth inhibitory effects of TGF-β during in vitro progression to a differentiation resistant phenotype (HKc/DR). We have previously shown that loss of TGF-β sensitivity in HKc/DR is attributed to decreased expression of TGF-β receptor type I (TGF-β RI), while the levels of TGF-β receptor type II (TGF-β RII) remain unchanged. The present study explored molecular mechanisms leading to reduced TGF-β RI expression in HKc/DR. Using TGF-β RI and TGF-β RII promoter reporter constructs, we determined that acute expression of the HPV16 oncogenes E6 and E7 decreased the promoter activity of TGF-β RI and TGF-β RII by about 50%. However, promoter activity of TGF-β RI is decreased to a greater extent than TGF-β RII as HKc/HPV16 progress to HKc/DR. Reduced TGF-β RI expression in HKc/DR was found not to be linked to mutations within the TGF-β RI promoter or to promoter methylation. Electrophoretic mobility shift and supershift assays using probes encompassing Sp1 binding sites in the TGF-β RI promoter found no changes between HKc/HPV16 and HKc/DR in binding of the transcription factors Sp1 or Sp3 to the probes. Also, Western blots determined that protein levels of Sp1 and Sp3 remain relatively unchanged between HKc/HPV16 and HKc/DR. Overall, these results demonstrate that mutations in or hypermethylation of the TGF-β RI promoter, along with altered levels of Sp1 or Sp3, are not responsible for the reduced expression of TGF-β RI we observe in HKc/DR. Rather the HPV16 oncogenes E6 and E7 themselves exhibit an inhibitory effect on TGF-β receptor promoter activity.
doi:10.1016/j.canlet.2009.03.014
PMCID: PMC2710415  PMID: 19344999
TGF-beta receptors; HPV-mediated transformation; Human keratinocytes
23.  Overexpression and Immunosuppressive Functions of Transforming Growth Factor 1, Vascular Endothelial Growth Factor and Interleukin-10 in Epithelial Ovarian Cancer 
Objective
Transforming growth factor-1 (TGF-β1), vascular endothelial growth factor (VEGF), and interleukin-10 (IL-10) may be critical cytokines in the microenvironment of a tumor, playing roles in immune suppression. This study was conducted to elucidate the roles and immunosuppressive functions of these cytokines in epithelial ovarian cancer (EOC).
Methods
The expression levels of TGF-β1, VEGF and IL-10 in malignant tissue were evaluated by immune- histochemistry and compared with corresponding borderline, benign, and tumor-free tissues. Moreover, relationships among the levels of these cytokines and correlations between expression and the prognosis of EOC were analyzed by Pearson rank correlations and multi-factor Logistic regression. The roles of TGF-β1, VEGF, and IL-10 in the immunosuppressive microenvironment of ovarian cancer were studied through dendritic cell (DC) maturation and CD4+CD25+FoxP3+ Treg generation in vitro experiments.
Results
TGF-β1, VEGF, and IL-10 were expressed in 100%, 74.69%, and 54.96% of EOC patients, respectively. TGF-β1 was an independent prognostic factor for EOC. IL-10 was significantly co-expressed with VEGF. In vitro, VEGF and TGF-β1 strongly interfered with DC maturation and consequently led to immature DCs, which secreted high levels of IL-10 that accumulated around the tumor site. TGF-β1 and IL-10 induced Treg generation without antigen presentation in DCs.
Conclusions
TGF-β1, VEGF and IL-10 play important roles in EOC and can lead to frequent immune evasion events.
doi:10.1007/s11670-012-0130-y
PMCID: PMC3555263  PMID: 23359769
Epithelial ovarian cancer; Tumor microenvironment; Immunosuppression; Cytokines
24.  Elucidating the Mechanism of Regulation of Transforming Growth Factor β Type II Receptor Expression in Human Lung Cancer Cell Lines12 
Neoplasia (New York, N.Y.)  2011;13(10):912-922.
Lung carcinogenesis in humans involves an accumulation of genetic and epigenetic changes that lead to alterations in normal lung epithelium, to in situ carcinoma, and finally to invasive and metastatic cancers. The loss of transforming growth factor β (TGF-β)-induced tumor suppressor function in tumors plays a pivotal role in this process, and our previous studies have shown that resistance to TGF-β in lung cancers occurs mostly through the loss of TGF-β type II receptor expression (TβRII). However, little is known about the mechanism of down-regulation of TβRII and how histone deacetylase (HDAC) inhibitors (HDIs) can restore TGF-β-induced tumor suppressor function. Here we show that HDIs restore TβRII expression and that DNA hypermethylation has no effect on TβRII promoter activity in lung cancer cell lines. TGF-β-induced tumor suppressor function is restored by HDIs in lung cancer cell lines that lack TβRII expression. Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by either activated Ras or epidermal growth factor signaling is involved in the down-regulation of TβRII through histone deacetylation. We have immunoprecipitated the protein complexes by biotinylated oligonucleotides corresponding to the HDI-responsive element in the TβRII promoter (-127/-75) and identified the proteins/factors using proteomics studies. The transcriptional repressor Meis1/2 is involved in repressing the TβRII promoter activity, possibly through its recruitment by Sp1 and NF-YA to the promoter. These results suggest a mechanism for the downregulation of TβRII in lung cancer and that TGF-β tumor suppressor functions may be restored by HDIs in lung cancer patients with the loss of TβRII expression.
PMCID: PMC3201568  PMID: 22028617
25.  Antitumor Activity of EBV-specific T Lymphocytes Transduced With a Dominant Negative TGF-β Receptor 
Summary
Transforming growth factor (TGF)-β is produced in most human tumors and markedly inhibits tumor antigen-specific cellular immunity, representing a major obstacle to the success of tumor immunotherapy. TGF-β is produced in Epstein-Barr virus (EBV)-positive Hodgkin disease and non-Hodgkin lymphoma both by the tumor cells and by infiltrating T-regulatory cells and may contribute the escape of these tumors from infused EBV-specific T cells. To determine whether tumor antigen-specific cytotoxic T lymphocytes (CTLs) can be shielded from the inhibitory effects of tumor-derived TGF-β, we previously used a hemagglutinin-tagged dominant negative TGF-βRII expressed from a retrovirus vector to provide CTLs with resistance to the inhibitory effects of TGF-β in vitro. We now show that human tumor antigen-specific CTLs can be engineered to resist the inhibitory effects of tumor-derived TGF-β both in vitro and in vivo using a clinical grade retrovirus vector in which the dominant negative TGF-β type II receptor (DNRII) was modified to remove the immunogenic hemagglutinin tag. TGF-β–resistant CTL had a functional advantage over unmodified CTL in the presence of TGF-β–secreting EBV-positive lymphoma, and had enhanced antitumor activity, supporting the potential value of this countermeasure.
doi:10.1097/CJI.0b013e318177092b
PMCID: PMC2745436  PMID: 18463534
non-Hodgkin lymphoma; Hodgkin disease; TGF-β; cytotoxic T lymphocyte

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