Scott Lonning

Tumor Evasion of the Immune System by Converting CD4+CD25− T Cells into CD4+CD25+ T Regulatory Cells: Role of Tumor-Derived TGF-β

CD4+CD25+ T regulatory (Treg) cells were initially described for their ability to suppress autoimmune diseases in animal models. An emerging interest is the potential role of Treg cells in cancer development and progression because they have been shown to suppress antitumor immunity. In this study, CD4+CD25− T cells cultured in conditioned medium (CM) derived from tumor cells, RENCA or TRAMP-C2, possess similar characteristics as those of naturally occurring Treg cells, including expression of Foxp3, a crucial transcription factor of Treg cells, production of low levels of IL-2, high levels of IL-10 and TGF-β, and the ability to suppress CD4+CD25− T cell proliferation. Further investigation revealed a critical role of tumor-derived TGF-β in converting CD4+CD25− T cells into Treg cells because a neutralizing Ab against TGF-β, 1D11, completely abrogated the induction of Treg cells. CM from a nontumorigenic cell line, NRP-152, or irradiated tumor cells did not convert CD4+CD25− T cells to Treg cells because they produce low levels of TGF-β in CM. Finally, we observed a reduced tumor burden in animals receiving 1D11. The reduction in tumor burden correlated with a decrease in tumor-derived TGF-β. Treatment of 1D11 also reduced the conversion of CD4+ T cells into Treg cells and subsequent Treg cell-mediated suppression of antitumor immunity. In summary, we have demonstrated that tumor cells directly convert CD4+CD25− T cells to Treg cells through production of high levels of TGF-β, suggesting a possible mechanism through which tumor cells evade the immune system.

Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling

Imbalances in glucose and energy homeostasis are at the core of the worldwide epidemic of obesity and diabetes. Here, we illustrate an important role of the TGF-β/Smad3 signaling pathway in regulating glucose and energy homeostasis. Smad3-deficient mice are protected from diet-induced obesity and diabetes. Interestingly, the metabolic protection is accompanied by Smad3(-)(/-) white adipose tissue acquiring the bioenergetic and gene expression profile of brown fat/skeletal muscle. Smad3(-/-) adipocytes demonstrate a marked increase in mitochondrial biogenesis, with a corresponding increase in basal respiration, and Smad3 acts as a repressor of PGC-1α expression. We observe significant correlation between TGF-β1 levels and adiposity in rodents and humans. Further, systemic blockade of TGF-β signaling protects mice from obesity, diabetes, and hepatic steatosis. Together, these results demonstrate that TGF-β signaling regulates glucose tolerance and energy homeostasis and suggest that modulation of TGF-β activity might be an effective treatment strategy for obesity and diabetes.

Transforming growth factor beta subverts the immune system into directly promoting tumor growth through interleukin-17.

Overexpression of the immunosuppressive cytokine transforming growth factor beta (TGF-beta) is one strategy that tumors have developed to evade effective immunesurveillance. Using transplantable models of breast and colon cancer, we made the unexpected finding that CD8+ cells in tumor-bearing animals can directly promote tumorigenesis, by a mechanism that is dependent on TGF-beta. We showed that CD8+ splenocytes from tumor-bearing mice expressed elevated interleukin (IL)-17 when compared with naive mice, and that CD8+ T cells could be induced to make IL-17 on addition of TGF-beta and IL-6 in vitro. Treatment of mice with anti-TGF-beta antibodies in vivo reduced IL-17 expression both in the tumor and the locoregional lymph nodes. Although IL-17 has not previously been shown to act as a survival factor for epithelial cells, we found that IL-17 suppressed apoptosis of several tumor cell lines in vitro, suggesting that this altered T-cell polarization has the potential to promote tumorigenesis directly, rather than indirectly through inflammatory sequelae. Consistent with this hypothesis, knockdown of the IL-17 receptor in 4T1 mouse mammary cancer cells enhanced apoptosis and decreased tumor growth in vivo. Thus, in addition to suppressing immune surveillance, tumor-induced TGF-beta may actively subvert the CD8+ arm of the immune system into directly promoting tumor growth by an IL-17-dependent mechanism.

An Anti–Transforming Growth Factor β Antibody Suppresses Metastasis via Cooperative Effects on Multiple Cell Compartments

Overexpression of transforming growth factor beta (TGF-beta) is frequently associated with metastasis and poor prognosis, and TGF-beta antagonism has been shown to prevent metastasis in preclinical models with surprisingly little toxicity. Here, we have used the transplantable 4T1 model of metastatic breast cancer to address underlying mechanisms. We showed that efficacy of the anti-TGF-beta antibody 1D11 in suppressing metastasis was dependent on a synergistic combination of effects on both the tumor parenchyma and microenvironment. The main outcome was a highly significant enhancement of the CD8+ T-cell-mediated antitumor immune response, but effects on the innate immune response and on angiogenesis also contributed to efficacy. Treatment with 1D11 increased infiltration of natural killer cells and T cells at the metastatic site, and enhanced expression of coactivators (NKG2D) and cytotoxic effectors (perforin and granzyme B) on CD8+ T cells. On the tumor cells, increased expression of an NKG2D ligand (Rae1gamma) and of a death receptor (TNFRSF1A) contributed to enhanced immune cell-mediated recognition and lysis. The data suggest that elevated TGF-beta expression in the tumor microenvironment modulates a complex web of intercellular interactions that aggregately promote metastasis and progression. TGF-beta antibodies reverse this effect, and the absence of a major effect of TGF-beta antagonism on any one cell compartment may be critical for a good therapeutic window and the avoidance of autoimmune complications.

Resistance of Glioblastoma-Initiating Cells to Radiation Mediated by the Tumor Microenvironment Can Be Abolished by Inhibiting Transforming Growth Factor-β

The poor prognosis of glioblastoma (GBM) routinely treated with ionizing radiation (IR) has been attributed to the relative radioresistance of glioma-initiating cells (GIC). Other studies indicate that although GIC are sensitive, the response is mediated by undefined factors in the microenvironment. GBM produce abundant transforming growth factor-β (TGF-β), a pleotropic cytokine that promotes effective DNA damage response. Consistent with this, radiation sensitivity, as measured by clonogenic assay of cultured murine (GL261) and human (U251, U87MG) glioma cell lines, increased by approximately 25% when treated with LY364947, a small-molecule inhibitor of TGF-β type I receptor kinase, before irradiation. Mice bearing GL261 flank tumors treated with 1D11, a pan-isoform TGF-β neutralizing antibody, exhibited significantly increased tumor growth delay following IR. GL261 neurosphere cultures were used to evaluate GIC. LY364947 had no effect on the primary or secondary neurosphere-forming capacity. IR decreased primary neurosphere formation by 28%, but did not reduce secondary neurosphere formation. In contrast, LY364947 treatment before IR decreased primary neurosphere formation by 75% and secondary neurosphere formation by 68%. Notably, GL261 neurospheres produced 3.7-fold more TGF-β per cell compared with conventional culture, suggesting that TGF-β production by GIC promotes effective DNA damage response and self-renewal, which creates microenvironment-mediated resistance. Consistent with this, LY364947 treatment in irradiated GL261 neurosphere-derived cells decreased DNA damage responses, H2AX and p53 phosphorylation, and induction of self-renewal signals, Notch1 and CXCR4. These data motivate the use of TGF-β inhibitors with radiation to improve therapeutic response in patients with GBM.

Targeting the Transforming Growth Factor-β pathway inhibits human basal-like breast cancer metastasis

BackgroundTransforming Growth Factor β (TGF-β) plays an important role in tumor invasion and metastasis. We set out to investigate the possible clinical utility of TGF-β antagonists in a human metastatic basal-like breast cancer model. We examined the effects of two types of the TGF-β pathway antagonists (1D11, a mouse monoclonal pan-TGF-β neutralizing antibody and LY2109761, a chemical inhibitor of TGF-β type I and II receptor kinases) on sublines of basal cell-like MDA-MB-231 human breast carcinoma cells that preferentially metastasize to lungs (4175TR, 4173) or bones (SCP2TR, SCP25TR, 2860TR, 3847TR).ResultsBoth 1D11 and LY2109761 effectively blocked TGF-β-induced phosphorylation of receptor-associated Smads in all MDA-MB-231 subclones in vitro. Moreover, both antagonists inhibited TGF-β stimulated in vitro migration and invasiveness of MDA-MB-231 subclones, indicating that these processes are partly driven by TGF-β. In addition, both antagonists significantly reduced the metastatic burden to either lungs or bones in vivo, seemingly independently of intrinsic differences between the individual tumor cell clones. Besides inhibiting metastasis in a tumor cell autonomous manner, the TGF-β antagonists inhibited angiogenesis associated with lung metastases and osteoclast number and activity associated with lytic bone metastases. In aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.ConclusionsIn aggregate, these studies support the notion that TGF-β plays an important role in both bone-and lung metastases of basal-like breast cancer, and that inhibiting TGF-β signaling results in a therapeutic effect independently of the tissue-tropism of the metastatic cells. Targeting the TGF-β pathway holds promise as a novel therapeutic approach for metastatic basal-like breast cancer.

Key Roles for Transforming Growth Factor β in Melanocyte Stem Cell Maintenance

Melanocyte stem cells in the bulge area of hair follicles are responsible for hair pigmentation, and defects in them cause hair graying. Here we describe the process of melanocyte stem cell entry into the quiescent state and show that niche-derived transforming growth factor beta (TGF-beta) signaling plays important roles in this process. In vitro, TGF-beta not only induces reversible cell cycle arrest, but also promotes melanocyte immaturity by downregulating MITF, the master transcriptional regulator of melanocyte differentiation, and its downstream melanogenic genes. In vivo, TGF-beta signaling is activated in melanocyte stem cells when they reenter the quiescent noncycling state during the hair cycle and this process requires Bcl2 for cell survival. Furthermore, targeted TGF-beta type II receptor (TGFbRII) deficiency in the melanocyte lineage causes incomplete maintenance of melanocyte stem cell immaturity and results in mild hair graying. These data demonstrate that the TGF-beta signaling pathway is one of the key niche factors that regulate melanocyte stem cell immaturity and quiescence.

Synergistic Enhancement of CD8+ T Cell–Mediated Tumor Vaccine Efficacy by an Anti–Transforming Growth Factor-β Monoclonal Antibody

Purpose: Transforming growth factor-β (TGF-β) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4+ and CD8+T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti–TGF-β (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy. Experimental Design: The effect of combination treatment with a peptide vaccine and anti–TGF-β was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment. Results: Anti–TGF-β significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti–TGF-β alone had no impact. The number of tumor antigen–specific CTL with high functional avidity as measured by IFN-γ production and lytic activity was significantly increased in vaccinated mice by TGF-β neutralization. Although TGF-β is known to play a critical role in CD4+Foxp3+ Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti–TGF-β did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-β neutralization had no effect on interleukin-17–producing T cells, which are induced by TGF-β and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-β, was not sufficient to eliminate all sources of suppressive TGF-β. Finally, the synergistic protection induced by anti–TGF-β vaccine augmentation was mediated by CD8+ T cells since anti-CD8 treatment completely abrogated the effect. Conclusions: These results suggest that TGF-β blockade may be useful for enhancing cancer vaccine efficacy. (Clin Cancer Res 2009;15(21):6560–9)

Transforming Growth Factor-β Regulates House Dust Mite–induced Allergic Airway Inflammation but Not Airway Remodeling

RATIONALE It is now believed that both chronic airway inflammation and remodeling contribute significantly to airway dysfunction and clinical symptoms in allergic asthma. Transforming growth factor (TGF)-beta is a powerful regulator of both the tissue repair and inflammatory responses, and numerous experimental and clinical studies suggest that it may play an integral role in the pathogenesis of asthma. OBJECTIVES We investigated the role of TGF-beta in the regulation of allergic airway inflammation and remodeling using a mouse model of house dust mite (HDM)-induced chronic allergic airway disease. METHODS We have previously shown that intranasal administration of an HDM extract (5 d/wk for 5 wk) elicits robust Th2-polarized airway inflammation and remodeling that is associated with increased airway hyperreactivity. Here, Balb/c mice were similarly exposed to HDM and concurrently treated with a pan-specific TGF-beta neutralizing antibody. MEASUREMENTS AND MAIN RESULTS We observed that anti-TGF-beta treatment in the context of either continuous or intermittent HDM exposure had no effect on the development of HDM-induced airway remodeling. To further confirm these findings, we also subjected SMAD3 knockout mice to 5 weeks of HDM and observed that knockout mice developed airway remodeling to the same extent as HDM-exposed littermate controls. Notably, TGF-beta neutralization exacerbated the eosinophilic infiltrate and led to increased airway hyperreactivity. CONCLUSIONS Collectively, these data suggest that TGF-beta regulates HDM-induced chronic airway inflammation but not remodeling, and furthermore, caution against the use of therapeutic strategies aimed at interfering with TGF-beta activity in the treatment of this disease.

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+ CTLs 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 s.c. vaccinations of synthetic peptides for GAA-derived CTL epitopes, GARC-1 (77-85) and EphA2 (671-679), emulsified in incomplete Freunds 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/IFN-γ–producing CD8+ T cells, and reduced CD4+/FoxP3+ Treg cells in the glioma microenvironment. Neutralization of TGF-β also upregulated plasma levels of interleukin-12, macrophage inflammatory protein-1α, and IFN-inducible protein-10, suggesting a systemic promotion of type-1 cytokine/chemokine production. Furthermore, 1D11 treatment upregulated plasma interleukin-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 immunologic environment of orthotopic tumor-bearing mice both systemically and locally, thereby enhancing the therapeutic efficacy of GAA vaccines. (Clin Cancer Res 2009;15(21):6551–9)