Abstract
Abstract Immune homeostasis is a delicate balance between the immune defense against foreign pathogens and suppression of the immune system to maintain self-tolerance and prevent autoimmune disease. Maintenance of this balance involves several crucial networks of cytokines and various cell types. Among these regulators, transforming growth factor-h (TGF-h) is a potent cytokine with diverse effects on hematopoietic cells. Its pivotal function within the immune system is to maintain tolerance via the regulation of lymphocyte proliferation, differentiation, and survival. In addition, TGF-h controls the initiation and resolution of inflammatory responses through the regulation of chemotaxis and activation of leukocytes in the periphery, including lymphocytes, natural killer cells, dendritic cells, macrophages, mast cells, and granulocytes.Through its pleiotropic effects on these immune cells, TGF-h prevents the development of autoimmune diseases without compromising immune responses to pathogens. However, overactivation of this pathway can lead to several immunopathologies under physiologic conditions including cancer progression, making it an attractive target for antitumor therapies. This review discusses the biological functions of TGF-h and its effects on the immune system and addresses how immunosuppression by this cytokine can promote tumorigenesis, providing the rationale for evaluating the immune-enhancing and antitumor effects of inhibitingTGF-h in cancer patients.

The pleiotropic cytokine, transforming growth factor-h (TGFh), plays a critical role in suppressing the immune response in the periphery to prevent an autoimmune response. TGF-hs are regulatory molecules with numerous effects on cell proliferation, differentiation, migration, and survival that affect multiple biological processes, including development, carcinogenesis, fibrosis, wound healing, and immune responses (1) . This system has evolved over the last billion years to regulate immune systems in multicellular organisms (2) . The effects of TGF-h on the immune system were first published in 1986 (3). Transgenic mice lacking TGF-h were established in the early 1990s and demonstrated the central role of this cytokine in inhibiting inflammation and autoimmune disease (4, 5) . The subsequent identification of TGF-h receptors (TGF-hR) and Smad factors as mediators of receptor signaling pathways revealed the molecular mechanisms behind TGF-h regulation of the immune response (6, 7) . At the turn of the 21st century, murine models containing cell type -specific activation of TGFh signaling have enhanced our understanding of the regulatory network of the TGF-h pathway in vivo (8 -12) and how attenuating its function may improve antitumor responses (13) . In this review, we discuss the regulatory functions of TGF-h in the immune system as well as how the immunosuppressive effects of this cytokine promotes cancer progression, providing the rationale for developing TGF-h inhibitors as antitumor agents.
TGF-B Expression and Signaling
The TGF-h superfamily consists of a large group of extracellular growth factors controlling multiple aspects of development. TGF-h superfamily members bind and activate transmembrane serine/threonine kinase receptors resulting in intracellular signaling (14) . This family contains not only the TGF-hs but also the bone morphogenetic proteins, activins, and growth differentiation factors (14) . Three TGF-h isoforms, TGF-h1, TGF-h2, and TGF-h3, have been identified in mammals that have similar biological function but are expressed in different tissues (15) . Among these three isoforms, TGF-h1 is predominantly expressed in the immune system. Unlike most cytokines, TGF-h is synthesized as an inactive large precursor molecule containing a self-inhibiting propeptide in addition to the active form of TGF-h. Proteolytic processing results in a heterodimer containing an active TGF-h in association with either a latency-associated protein or latent TGF-h -binding protein, which keeps TGF-h inactive (16) .
Under physiologic conditions, active TGF-h, either as a cell surface -bound or intracellular soluble form, is liberated from these proteins following additional stimuli from acidic pH, integrins, thrombospondin-1, and proteases, enabling it to exert its function by binding to its receptor (17 -21) .
Signal transduction follows binding of TGF-h to a heterodimeric receptor complex consisting of type I and II transmembrane serine/threonine kinase subunits. Five type I (activin-like receptor kinase family) and seven type II receptors have been identified to form these TGF-hRs ( Fig. 1; ref. 14) . Intracellular signal transduction is mediated by phosphorylation of several transcription factors known as Smads as well as Smad-independent pathways involving kinase cascades, including Rho-Rac-cdc-42, Ras-extracellular signal-regulated kinase, Tak-MKK3/6-c-Jun NH 2 -terminal kinase, TAK-MKK4-p38, and phosphatidylinositol 3-kinase, and transcriptional regulators, such as TGF-hR interacting protein, protein phosphatase 2A, elongation initiation factors, and the immunophilin, FK506 binding protein 12 ( Fig. 1; refs. 22 -25) . Following TGF-h binding, the Smad-dependent pathway is activated when activin-like receptor kinase 5 phosphorylates Smad2 and Smad3, which translocate into the nucleus in a complex with Smad4 ( Fig. 1) . The resulting Smad complex binds to a target promoter and regulates gene expression through the recruitment of histone acetyl transferase or histone deacetylase (Figs. 1 and 2; refs. 26, 27) . Negative regulators of this pathway include Smad7, which competes with Smad2 and Smad3 for activinlike receptor kinase 5 binding and degrades activin-like receptor kinase 5 through the recruitment of Smurf-containing E3 ubiquitin-ligase complexes. The Smad-independent pathways activated following TGF-h binding to its receptor are poorly understood and their relevance to immune cell regulation is unknown.
TGF-B and the Key Effector Cells in the Antitumor
Response: Tcells and Natural Killer Cells TGF-b and T cells. CD8 + cytotoxic T cells (CTL) and natural killer (NK) cells play a critical role in the prevention, killing, and clearance of tumors and have been targeted in the development of anticancer immunotherapies (28, 29) . As a pleiotropic cytokine, TGF-h exerts its effects on multiple immune cell types (1) and its role in controlling T-cell functions has been extensively studied. TGF-h inhibition of T-cell proliferation in vitro was first documented using activated human T cells (3) . One mechanism behind TGF-h -mediated suppression of T-cell proliferation is through TGF-h blockade of the production of interleukin (IL)-2 ( Fig. 2) , a lymphokine known to activate T cells, NK cells, and other types of cells of the immune system (30, 31) . This effect can be partially reversed by the addition of exogenous IL-2 (3). Further work has shown that TGF-h suppression of IL-2 production may be through directly inhibiting IL-2 promoter activity through a cis-acting enhancer DNA element (32) . R-Smad3 has also been shown to be critical in mediating TGF-h inhibition of IL-2 production (33).
In addition, TGF-h inhibits T-cell proliferation through pathways not involving IL-2 suppression (3). For example, TGF-h also attenuates the expression of cell cycle regulators in T cells. Upon TGF-h treatment, cyclin-dependent kinase inhibitors, including p15, p21, and p27, are up-regulated, whereas cell cycle -promoting factors, including c-myc, cyclin D2, cyclin-dependent kinase 2, and cyclin E, are decreased (34 -39). The functional significance behind these effects of TGF-h as well as how TGF-h modulates these genes in T cells awaits further evaluation.
In addition to suppressing T-cell proliferation, TGF-h also controls T-cell effector functions. The expression of effector molecules by CTLs, such as IFN-g and perforin, is inhibited by TGF-h ( Fig. 2; refs. [40] [41] [42] [43] . Recent studies have shown that TGF-h is important for the inhibition of the exocytosis of granules and cytolytic function of CD8 + T cells (44) . For example, Mempel et al. (44) used multiphoton intravital microscopy in the lymph nodes of anesthetized mice to evaluate how CTLs interact with antigen-presenting target cells in the presence or absence of activated regulatory T cells (Tregs), a suppressive T-cell population identified by the constitutive expression of a forkhead family transcription factor, FoxP3 (44) . CTLs killed their targets at a 6.6-fold faster rate in the absence of Tregs, which can inhibit cytotoxic granule exocytosis by these effector cells (44) . This effect was dependent on TGF-h as CTLs refractory to signaling by this cytokine displayed normal killing in the presence of Tregs (44) .
The critical physiologic role for TGF-h in regulating suppression of conventional CD4 + and CD8 + T cells has been illustrated by studies of genetically modified mice. For example, mice lacking TGF-h1 expression (TGF-h1 -/-mice) develop a multifocal inflammatory disease associated with increased inflammatory cytokine production (4, 5, 45) . However, because TGF-h1 is pleiotropic and acts on multiple immune cell types, it is unclear from these studies whether T cells were the direct targets of this cytokine. To answer this question, we generated mice expressing a dominant-negative form of TGFhRII from the CD4 promoter (CD4-dnThRII) in T cells, which resulted in the expression of the dominant-negative receptor (dnTGF-hRII) exclusively in both CD4 + and CD8 + lymphocytes (9) . These mice developed an autoimmune inflammatory phenotype associated with uncontrolled CD4 + T-cell differentiation into effector cells (9) . These mice were also resistant to tumor engraftment when challenged with the murine melanoma cell line B16-F10 and murine thymoma cell line EL-4, suggesting that blocking TGF-h signaling in T cells may enhance T-cell -mediated antitumor activity (13) . Adoptive transfer experiments revealed that the observed antitumor effects following tumor challenge were dependent on blockade of TGF-h signaling in CD8 + cells and T-cell ''help'' from either wild-type CD4 + or TGF-h dominant-negative receptor transgenic CD4 + cells (13) . In separate experiments conducted by Zhang et al. (46) , adoptive transfer of CTLs transfected with the dnTGF-bRII gene produced enhanced antitumor activity, thus confirming the immunosuppressive effects of TGF-h on effector cells and suggesting an approach for enhancing immune antitumor responses in cancer patients.
In addition to its inhibitory effects on the proliferation and the function of conventional T cells, TGF-h further contributes to immunosuppression by promoting the generation of Tregs. Tregs are frequently found at higher frequencies in the peripheral blood, lymph nodes, and tumor sites of cancer patients (47) . Although TGF-h can promote the generation of Tregs in vitro, it has been controversial whether TGF-h is involved in the generation or maintenance of Tregs under physiologic conditions. One recent study showed that Tregs lacking the TGFhRII developed normally in the thymus but were poorly maintained in the periphery (48) . That same study also showed that TGF-hRII -deficient Tregs were found to proliferate faster than the wild-type counterparts in the periphery, suggesting that TGF-h signaling is required to promote peripheral Treg survival independent of its proliferation potential (48) . These results contrasted with those in other animal models where Treg maintenance or development was unaffected in environments (49) . The discrepancies between these studies may be related to different experimental systems and mouse genetic backgrounds used in the experiments. The development of EGFP-FoxP3 and FoxP3-mRFP knock-in mice and FoxP3 intracellular staining will provide improved tools to identify Tregs based on FoxP3 expression (48, 50, 51) . These reagents will enable future studies evaluating the effects of TGF-h on Treg biology and how this cytokine and Tregs inhibit antitumor responses in mouse models and cancer patients.
TGF-b and NK cells. NK cells are lymphoid cells that participate in innate immunity and in early defense by recognizing and killing infected or tumorigenic cells and rapidly producing chemokines and cytokines. IFN-g production by NK cells is considered essential for stimulation of the Th1 response, and TGF-h attenuates this activity as well as the cytolytic activity of these cells (52, 53) . Although the mechanisms of TGF-h -mediated inhibition of NK production of IFN-g are unknown, TGF-h blocks the expression of receptors vital for the cytotoxic function of NK cells. Targeted killing by NK cells depends on the engagement of the NK receptors and coreceptors NKp46, NKp30, NKp44, and NKG2D (54, 55) . Exogenous TGF-h inhibits the expression of NKp30 and NKG2D receptors resulting in decreased killing function (56) . Our preliminary studies using transgenic mice containing NK cells resistant to TGF-h signaling (10) have revealed enhanced tumor rejection in these mice compared with wildtype littermates. 4 Furthermore, down-modulation of NKG2D has also been associated with elevated TGF-h levels in cancer patients (57) . This observation shows an immunosuppressive effect of TGF-h on NK cells under physiologic conditions in cancer patients and suggests a mechanism by which these cells are less effective at killing tumor cells. Further studies are planned to evaluate the antitumor effects of specifically blocking TGF-h signaling in NK cells and to determine the contribution of these cells in antitumor responses enhanced by approaches blocking TGF-h more broadly.
TGF-B and Dendritic Cells Involved in the Antitumor Response
Dendritic cells are professional antigen-presenting cells essential for immunity and tolerance (58) . They are extremely potent antigen-presenting cells that can initiate antigen-specific immune responses through presentation to T cells as well as activate B cells and NK cells. Because they can activate both the adaptive and innate arms of the immune system, these cells have been targeted for developing cancer vaccines. Cancer vaccines based on administration of antigen-loaded dendritic cells have been used in several clinical trials involving advanced melanoma, renal cell carcinoma, breast cancer, non-Hodgkin's lymphoma, multiple myeloma, prostate cancer, lung cancer, and colorectal cancer patients (reviewed in refs. 29 and 58). However, to date, the majority of cancer patients have not derived clinical benefit (reviewed in refs. 29 and 58) .
Improvements in dendritic cell function may be possible through inhibition of TGF-h. TGF-h regulates the antigen presentation function of differentiated dendritic cells. On stimulation with bacterial components, inflammatory cytokines or costimulatory receptors, dendritic cells up-regulate MHC class II and costimulatory molecules and cytokines, including IL-12, which induce T-cell activation and differentiation. Exogenous TGF-h administration to lipopolysaccharidestimulated dendritic cells inhibits the expression of MHC class II and costimulatory molecules (59) . In addition, exogenous administration of this cytokine prevents the production of IL-12 induced by the inflammatory cytokines IL-1 and tumor necrosis factor-a (59). These effects may ultimately result in the inhibition of T-cell activation and differentiation required for an effective antitumor response, thus providing the rationale for evaluation of blocking TGF-h signaling in dendritic cells as well as T cells and NK cells.
TGF-B, the Immune System, and Cancer
High levels of TGF-h are produced by many types of tumors, including melanomas and cancers of the breast, colon, esophagus, stomach, liver, lung, pancreas, and prostate, as well as hematologic malignancies (60, 61) . Also, both the TGF-hRs type I and II as well as Smad proteins are mutated in several cancers (reviewed in ref. 62 ). In addition, tumors can promote TGF-h production by the surrounding cells in the tumor microenvironment (63) . In early stages of tumorigenesis, TGF-h seems to act as a tumor suppressor. Gastrointestinal tumors with microsatellite instabilities and mutations in the TGF-hRII and pancreatic cancers containing Smad4 mutations do not respond appropriately to ligand-receptor interactions and thus become resistant to the signaling and antitumor activity by this cytokine (64, 65) . During later stages of tumorigenesis, TGF-h can foster tumor growth, progression, and metastasis by enhancing the epithelial to mesenchymal transition and promoting tumor angiogenesis (66, 67) . Interestingly, higher levels of TGF-h expression are observed in metastatic breast and colon metastases compared with the primary tumors (68, 69) , reinforcing its role in promoting tumor progression in later stages of the disease. Elevated TGF-h levels correspond with poor clinical outcome of cancer patients. High serum TGF-h levels in gastric carcinoma patients have been correlated with earlier recurrence rates for this cohort (70) . Retrospective analyses of archival tumors obtained from breast, colon, and lung cancer patients have suggested a relationship between high levels of TGF-h with increased disease progression, metastasis, and death in breast and colon cancer patients and a worse 5-year survival in lung cancer patients (71 -73) .
In addition to its direct effects on tumor cells and angiogenesis, TGF-h enables tumors to evade immune surveillance (74) and killing through the mechanisms reviewed above and summarized in Fig. 3 . TGF-h promotes Treg generation on TCR stimulation and it is conceivable that tumors promote immune privilege by generating Tregs through TGF-h production. This may be clinically relevant as high levels of Tregs portend a poor prognosis in ovarian cancer patients (75) and these cells infiltrate tumors in patients with various cancers as previously discussed above.
The epidemiologic and biological studies implicating TGF-h in tumor promotion and progression provide a strong rationale for developing TGF-h inhibitors as therapeutic agents for advanced cancer. Various inhibitors of TGF-h signaling are being evaluated in preclinical models and early clinical trials including soluble protein receptors, TGF-h antibodies, small-molecule kinase inhibitors, oligonucleotides, peptide aptamers, and tumor vaccines, which are summarized in Table 1 . Currently, the majority of the TGF-h signaling inhibitors are in preclinical studies. However, a blocking oligonucleotide (AP12009), an antibody to all three isoforms of human TGF-h (GC-1008), a small-molecule inhibitor of the TGF-hRI kinase (LY573636), and a vaccine using allogeneic tumor cells have entered clinical cancer trials (76 -78). 5, 6 The most advanced studies to date involve approaches inhibiting TGF-h locally, including the synthetic antisense oligodeoxynucleotide, AP12009, and allogeneic tumor vaccines (76 -78) . The antisense AP12009 can bind to TGF-h2 mRNA in tumor cells, ultimately leading to decreased expression of TGF-h2. In preclinical studies, AP12009 reduced proliferation in glioma, pancreatic cancer, and malignant melanoma cell lines (76) . Animal studies further showed that intrathecal bolus injections and intracerebral infusions of AP12009 were well tolerated (76) . Subsequent phase I/II open-label dose escalation studies showed that this compound was safe and well tolerated when administered as intratumoral injections to patients with high-grade gliomas (76) . Following treatment with AP12009, patient-derived peripheral blood mononuclear cells showed enhanced immune cell -mediated cytotoxicity against tumor cells in vitro after stimulation with IL-2 and coincubation with autologous glioma cells (76) . These results suggest that this oligodeoxynucleotide may enhance antitumor responses in glioma patients and warrant further evaluation in larger clinical trials. To this end, an international phase II trial in adult patients with recurrent high-grade glioma has recently completed enrollment (76) . Given that AP12009 also inhibits pancreatic cancer and malignant melanoma cell lines, phase I/II studies evaluating this compound in pancreatic carcinoma and malignant melanoma patients are also under way (76) .
Clinical studies have also been initiated with an allogeneic whole-cell tumor vaccine that has been stably transfected with a TGF-h2 antisense plasmid vector. Expression of this vector leads to reduced TGF-h2 expression by the vaccine cells (77) , which could be an advantage considering that the endogenous TGF-h generated by the whole-cell vaccine might inhibit its efficacy. Gliosarcoma (9L) cells were stereotactically implanted in the forebrains of Fisher 344 rats and the animals were immunized with irradiated 9L cells stably transfected with a TGF-h2 antisense vector (pCEP-4 TGF-h) 5 days later (79) . All (11 of 11) of the vaccinated rats survived 12 weeks compared with 2 of 15 and 3 of 10 rats vaccinated with gliosarcoma cells transfected with an empty vector or the IL-2 gene, respectively (79) . Furthermore, a cohort of surviving rats rechallenged with 9L cells 12 weeks following vaccination survived for 6 months compared with a 5-week survival for unvaccinated control rats (79), suggesting that the vaccine induced a sustained antitumor response with immunologic memory. At necropsy, histopathology of the tumor implantation sites revealed no microscopic evidence of the originally implanted tumor cells (79) . These results are surprising given that TGF-h was only inhibited in the vaccine tumor cells and not in the tumor bed and surrounding stroma where the cytokine could be readily produced and exert its immunosuppressive effects to promote tumor progression in the rats. Nevertheless, these preclinical studies led to early clinical trials showing that s.c. injections of these vaccines were well tolerated in phase I and II trials enrolling glioma patients and stage II, III, and IV lung cancer patients (77, 78) . Elevated serum levels of either IFN-g or immunoglobulins against vaccine cells were noted in some patients on these trials (77, 78) . However, it was unclear whether any immune response was elicited against relevant tumor antigens. The authors reported clinical responses in vaccinated patients (77, 78) , although assessment of response and clinical benefit is particularly difficult in this patient population. Therefore, larger clinical trials will be necessary to further characterize the activity of this approach.
Although TGF-h secretion from tumor cells may be important in inhibiting antitumor responses by the immune system, localized blockade of TGF-h in tumors may not be the most effective approach as several other cell types in the tumor microenvironment can produce this immunosuppressive cytokine, including Tregs and stromal cells. Systemic blockade of TGF-h via systemic administration of a small-molecule or antibody inhibitor may be more effective at enhancing antitumor responses that would be inhibited by this cytokine. To this end, monoclonal antibodies blocking TGF-h and smallmolecule inhibitors of TGF-h signaling are also entering clinical trials. 5, 6 These approaches have shown acceptable safety profiles as well as improved antitumor activity in the preclinical studies summarized in Table 1 . The results from these animal studies have led to the development of early clinical trials evaluating systemic blockade of TGF-h in cancer patients with refractory disease. A multicenter, open label, dose-escalation phase I study evaluating a monoclonal antibody, GC1008, in patients with advanced renal cell carcinoma or malignant melanoma opened last year and is accruing patients. 5 Patients eligible for this study will receive up to four doses of GC1008. In addition to the antibody approach, LY573636, a small-molecule inhibitor blocking TGF-h kinase activity is currently being evaluated in clinical trials. Recently, a phase I clinical trial evaluating this small-molecule inhibitor in refractory ovarian cancer, non -small cell lung cancer, soft tissue sarcoma, and thymoma patients reported that this compound was well tolerated as a 2-h i.v. infusion administered every 21 days (80). The main dose-limiting toxicity was bone marrow suppression manifested as grade 3/4 thrombocytopenia and neutropenia (80) . Subsequent phase II studies of metastatic melanoma patients 6 and non -small cell lung cancer patients 7 evaluating this approach are in progress. Although systemic blockade of TGF-h has been well-tolerated in animal models, it is possible that significant toxicities including autoimmune disease will be observed in clinical trials, particularly if used in combination with other immune activators. Murine models in which TGF-h signaling is blocked in CD4 + and CD8 + lymphocytes develop autoimmune colitis as they age, suggesting that autoimmunity may be observed By understanding the roles of TGF-h in promoting tumorigenesis and developing inhibitors to selectively inhibit TGF-h during the specific time points when it promotes progression of later stages of disease, we may be able to design more effective immunotherapies augmenting the antitumor response in cancer patients. The clinical benefits from initial trials evaluating blockade of TGF-h are unclear and larger studies are necessary to determine the efficacy of these approaches. Monoclonal antibodies and small-molecule inhibitors have been evaluated in preclinical models and early clinical trials assessing systemic blockade of TGF-h signaling are in progress.
Although the preclinical murine studies discussed in this review have suggested that TGF-h blockade can enhance antitumor activity, the effect can be overcome by increased tumor burden, 8 indicating that this approach will likely need to be combined with chemotherapies or other immunotherapies to effectively eradicate tumors in cancer patients. Other such strategies of this nature are being developed and are the subject of papers in this issue of Clinical Cancer Research Focus (82 -85) . However, the toxicities of systemic blockade of this cytokine remain unknown and may worsen with additional chemotherapies or immunotherapies. Preclinical studies evaluating combinatorial immunotherapies and chemotherapies with TGF-h blockade are under way. We anticipate that the results from these animal studies will guide future trials evaluating combinations of TGF-h blockade, immunotherapies, and chemotherapies in cancer patients.
Conclusions
Regulation of the immune response is vital to prevent the development of autoimmune diseases. As a critical immune suppressive cytokine, TGF-h has pleiotropic functions in regulating multiple immune cells in a context-dependent manner. However, dysregulation of the immunosuppressive function of TGF-h promotes tumorigenesis, particularly in later stages of cancer development. Further studies of the mechanisms and signaling pathways mediating TGF-h will lead to an improved understanding of how our immune system achieves self-tolerance and mounts effective immune responses against unsolicited foreign antigens and tumors. This knowledge may ultimately lead to the development of innovative therapeutic strategies to manipulate the effects of TGF-h on the immune system to treat cancer patients.

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