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Transforming growth factor-β (TGFβ) is an immunosuppressive cytokine produced by tumour cells and immune cells that can polarize many arms of the immune system. This Review covers the effects of TGFβ on NK cells, dendritic cells, macrophages, neutrophils, CD8+ and CD4+ effector and regulatory cells, and NKT cells in preclinical animal tumour models and in patients with cancer. Collectively, many recent studies favour the idea that blocking TGFβ signalling in the tumour microenvironment enhances antitumour immunity and may be beneficial for cancer therapy. An overview of the current drugs and reagents for inhibiting TGFβ signalling and their phase in clinical development is also provided.
As most tumours present self antigens, peripheral tolerance [G] has an important role in contributing to immune evasion by tumours. In addition, the overproduction of immunosuppressive cytokines, including transforming growth factor-β (TGFβ), by tumour cells and tumour-infiltrating lymphocytes also contributes to an immunosuppressive microenvironment. Many studies indicate that TGFβ can promote cancer metastasis through effects on the tumour microenvironment, by enhancing tumour cell invasion and by inhibiting the function of immune cells 1, 2. These findings have promoted interest in targeting TGFβ and its signalling pathway in patients with cancer. However, such targeting of TGFβ could result in adverse effects in normal tissues, as this pathway is also involved in multiple homeostatic processes (Figure 1). For example, TGFβ can function as a tumour suppressor to prevent tumorogenesis; however, overproduction of TGFβ is frequently associated with tumour metastasis and poor prognosis in patients with cancer (Figure 1). Although the molecular mechanisms behind this dichotomy of TGFβ functions are not fully elucidated, progress has been made in understanding the role of TGFβ in different stages of cancer. This topic has recently been reviewed and is not discussed here 1, 3–5.
This Review focuses on the tumour-promoting properties of TGFβ, which prevent effective antitumour immune responses once cancer has been established in the host. A successful immune response requires the proper activation and maturation of antigen-presenting cells (APCs) of the innate immune system that present antigen to adaptive immune cells. TGFβ can suppress or alter the activation, maturation and differentiation of both innate and adaptive immune cells, including natural killer (NK) cells, dendritic cells (DCs), macrophages, neutrophils, and CD4+ and CD8+ T cells 6. A dampened innate immune response leads to poor adaptive immunity, resulting in persistence of the tumour. In addition, TGFβ has an important role in the differentiation and induction of natural and induced regulatory T (TReg) cells, which also contribute to the tolerizing environment. Furthermore, in the presence of IL-6, TGFβ induces the differentiation of IL-17–producing CD4+ T helper 17 (TH17) cells and CD8+ cytotoxic T cells; although the role of IL-17-producing cells still remains controversial in tumour biology, given that these cells can exhibit both tumour-promoting and antitumour activities 7. As we discuss, many recent discoveries have been made towards understanding the biological effects of TGFβ on different immune cells, although multiple areas require further investigation. Finally, there is compelling evidence to support targeting TGFβ with inhibitors to enhance antitumour immunity in patients with cancer.
NK cells are innate lymphoid cells that have an important role in the antitumour response by recognizing and directly killing tumours and by rapidly producing chemokines and cytokines crucial for this function. For example, interferon-γ (IFNγ) production by NK cells is important for stimulating effector CD4+ TH1 cells that are required for clearing tumours. TGFβ attenuates IFNγ production by and the lytic activity of NK cells 8, 9. These might be direct effects of TGFβ or might result indirectly from cell-cell contact between NK cells and regulatory T cells producing this cytokine 10. In support of a direct effect, TGFβ signalling can suppress IFNγ production through SMAD3, a transcription factor downstream from this pathway, resulting in suppression of T-bet, a transcription factor required for IFNγ production 11.
Targeted killing by NK cells requires stimulating the NK activating receptors NKG2D, NKp46, NKp44 and NKp30 12. It has been shown that exogenously administered TGFβ inhibits NKp30 and NKG2D expression, leading to decreased ability of NK cells to kill target cells 13. TGFβ also decreases expression of NKG2D by NK cells and CD8+ T cells from glioma patients with a high tumour burden 14. In patients with lung and colorectal cancer, the downmodulation of NKG2D has been associated with increased serum levels of TGFβ 15. Furthermore, recent studies of isolated NK cells from healthy donors have shown that platelet-derived TGFβ results in downmodulation of NKG2D, causing a decrease in IFNγ production and degranulation functions essential for tumour destruction by these cells 16. Finally, surface-bound TGFβ on myeloid-derived suppressor cells [G] can inhibit NK cell cytolytic activity in an orthotopic liver cancer model 17. These observations indicate that TGFβ has immunosuppressive effects on NK cell killing functions in patients with cancer, and therefore might be a target for enhancing NK cell- mediated antitumour immune responses.
DCs are APCs that have a major role in the initial activation and subsequent regulation of immune responses 18. In addition to activating adaptive immunity mediated by T cells, DCs can also activate NK cells 19. DCs can present antigen in an immunogenic or tolerogenic manner, and so they have an important role in determining the host response to tumours 18, 20. DC activation involves the upregulation of MHC and costimulatory molecule expression, alteration in motility and the formation of dendrites to increase the surface area for antigen presentation and interaction with lymphocytes 21. Non-activated or immature DCs can still present antigen, but in the absence of proper costimulation this results in T cell tolerance 22, 23. In the presence of immune-inhibitory signals such as IL-10 and/or corticosteroids, DCs can induce tolerance by T cell deletion and/or the activation and induction of TReg cells 24, 25. Hence, DCs can induce either immunity or peripheral tolerance and are an essential component of tumour immunity. TGFβ affects DC biology in several ways. TGFβ can immobilize DCs, thereby interfering with their migration and the transport of antigen to draining lymph nodes for presentation to adaptive immune cells, and might also directly induce DC apoptosis 26–28. Tumour-infiltrating DCs secrete TGFβ and respond to TGFβ, either in an autocrine or paracrine manner, by down-regulating expression of MHC class II molecules, the co-stimulatory molecules CD40, CD80 and CD86, and tumour necrosis factor (TNF), IFNα, IL-12 and CC-chemokine ligand 5 (CCL5) 6, 29. These immature or tolerogenic DCs promote the formation of TReg cells that potently inhibit the function of other T cells 30, 31. Activated DCs are also able to activate both natural and induced TReg cells 32–35; interestingly, the capacity of DCs to induce both types of TReg cell is greatly increased by TGFβ and IL-10 36, 37. So, in the context of these immunosuppressive cytokines in the tumour microenvironment, DCs take up tumour cells, become tolerogenic TGFβ-secreting cells, and promote the induction of tumour-specific TReg cells in both mice and humans 38–41. Therefore, a growing body of evidence points to the induction of TReg cells by TGFβ produced by DCs; as TReg cells are a major obstacle to tumour immunity, this indicates that the TGFβ pathway might be targeted to augment antitumour immunity in patients with cancer.
The mass of most solid tumours is made up of ~50% macrophages, and high levels of tumour- associated macrophages (TAMs) [G] are correlated with poor cancer prognosis 42, 43. Macrophages are a heterogeneous population and are typically defined as being of an M1 (classically activated) [G] or M2 (alternatively activated) [G] phenotype, similar to the CD4+ TH1 versus TH2 cell paradigm. M1 macrophages are induced by IFNγ and other proinflammatory stimuli and are efficient at presenting antigens, producing proinflammatory cytokines, activating TH1 responses, and in general mediating anti-tumor responses. In contrast, there are a variety of M2 macrophages induced by IL-10, immune complexes, glucocorticoids, IL-4 and IL-13 with various phenotypes that are involved in remodeling and repair of damaged tissue, parasite resistance, immune regulation and/or tumour promotion 44. Both TAMs and myeloid-derived suppressor cells (MDSCs), which are a heterogeneous population of immature DCs, macrophages, granulocytes, and other myeloid cells in early stages of their differentiation and have properties similar to those that have been described for M2 cells, are involved in cancer progression and metastasis. In skin cancer, TGFβ-mediated recruitment of macrophages into tumours has an important role in immune escape, as it converts a regressing tumour into a progressing tumour 45. It is suggested that TGFβ-recruited TAMs are highly phagocytic and can compete with DC function, thereby markedly decreasing the ability of DCs to present tumour antigens to the adaptive immune system 45. TAMs acquire their phenotype by expressing high levels of TGFβ, IL-10, macrophage galactose N-acetyl-galactosamine-specific lectin 1 (MGL1), Dectin-1, CXC-chemokine ligand 10 (CXCL10), CXCL9 and other IFN-responsive genes. By contrast, they produce low levels of IL-12, TNF and nitric oxide synthase 2 (NOS2). Interestingly, this cytokine phenotype is mediated by the inhibitory NF-κB subunit p50 and it is thought that the cytokine milieu of the tumour microenvironment is necessary to maintain the phenotype46–48. During tumour progression, TAMs can switch from an M1 to M2 phenotype, which is paralleled by a gradual inhibition of NF-κB activity 49.
Peritoneal macrophages from tumour-bearing hosts produce increased levels of TGFβ, are less differentiated and have deficiencies in inflammatory cytokine production owing to decreased expression of NF-κB and C/EBP transcription factors 50. In this model, tumour-derived factors TGFβ and prostaglandin E2, individually and additively downregulate NF-κB and C/EBP. There is also evidence that tumour-infiltrating MDSCs secrete high levels of TGFβ, which upregulates CD206 (a deactivation marker characteristic of M2 cells) expression in an autocrine manner 51. It is unclear how TGFβ production is induced in MDSCs and macrophages, but the mechanism might involve IL-13 and glucocorticoids 52, 53; the induced TGFβ might then contribute to the alternative activation of M2 macrophages by downregulating NF-κB expression.
Neutrophils are short-lived polymorphonuclear leukocytes with potent antimicrobial and inflammatory capacities. Despite their known function as professional phagocytes, the role of neutrophils in tumour progression has been controversial and has received little attention compared with that of macrophages. Initial studies characterizing the effect of TGFβ on the control of inflammatory responses showed that this factor was a potent chemotactic factor for neutrophils, promoting their migration but not degranulation or activation 6. Subsequent studies showed that neutrophil migration could also be indirectly affected by TGFβ through regulating the expression of adhesion molecules in the endothelium 54, 55, and that TGFβ could inhibit neutrophil cytotoxicity, suggesting that TGFβ might influence human neutrophil activity in vivo56. Recently, the contradictory role of neutrophils in both tumour suppression and tumour promotion either by directly or indirectly controlling tumour growth, angiogenesis and metastasis (reviewed in 57) was reevaluated in terms of the characterization of different types of tumour associated neutrophil (TAN) with polarized N1 or N2 phenotypes 58. These polarized populations are similar to those that have been described for macrophages; they are influenced by the microenvironment and seem to be controlled by TGFβ in the tumour proximity. N2 cells are characterized by an expression profile that promotes tumour angiogenesis and metastasis 59–61, and inhibits the antitumour immune response by the secretion of reactive oxygen species (ROS). ROS normally act as potent microbicidal agents but in the context of the tumour microenvironment, ROS could lead to oxidative damage and inhibition of T cell function 62. Depletion of this neutrophil subpopulation in untreated tumour-bearing mice was sufficient to inhibit tumour growth, even when CD8+ T cells were absent; highlighting the immunosuppressive potential of N2 cells 58–60. Under TGFβ-inhibiting conditions, as well as in response to certain activation signals, neutrophils acquire an antitumour N1 phenotype that promotes tumour death and inhibits tumour growth 57, 58, 63, 64. The lack of systemic effects on neutrophil polarization during TGFβ neutralization experiments indicated that the effect is mainly intratumoral, characterized by increased numbers of N1 TANS that express activating chemokines and cytokines as well as by changes in endothelial adhesion molecules expression 58. Interestingly N1 and N2 neutrophils were shown to control the activation status of CD8+ T cells. This interplay seemed to be reciprocal as activated CD8+ T cells were controlling the activation and migration of neutrophils to the tumour microenvironment as well65. Clearly a reevaluation of the role of neutrophils in tumour immunology as well as characterization of this polarized subpopulation by TGFβ may be required to design more effective immunotherapies.
CD8+ T cells are a crucial component of antitumour immunity, as tumour antigen-specific cytotoxic T lymphocytes (CTLs) have an important role in the cytolytic killing of tumour cells. Several studies have shown a direct correlation between the frequency of CTLs in tumour infiltrating lymphocytes (TILs) and the overall survival of cohorts of patients with cancer; in particular, the high ratio of intratumoral activated cytotoxic CD8+ T cells to Tregs leads to improved prognosis 66–69. TGFβ signalling in tumour-specific CTLs dampens their function and frequency in the tumour 70, and blocking TGFβ signalling on CD8+ T cells results in more rapid tumour surveillance and the presence of many more CTLs at the tumour site71. Several experimental protocols have been used to render CD8+ T cells unresponsive to TGFβ. In a model where a dominant-negative form of TGFβ receptor II (TGFβRII) is expressed by both CD4+ and CD8+ T cells, a strong antitumour immune response was associated with the proliferation and increased activity of tumour-specific CTLs 72, 73. Similarly, when tumour-specific CD8+ T cells are rendered unresponsive to TGFβ signalling by transduction with a similar TGFβRII dominant-negative construct before adoptive transfer, these cells infiltrate into tumours, secrete cytokines such as IFNγ and can successfully kill tumour cells 74, 75.
In some tumour models, systemic blockade of TGFβ using a monoclonal antibody or kinase inhibitors to block downstream signalling prevents tumour recurrence by impacting the activity of various cell types, including an increase in the cytotoxic activity of CTLs76–79. However, inhibition of TGFβ by using the monoclonal antibody alone is not always sufficient to promote tumour rejections in all animal tumour models. In such models, the combination of a TGFβ-specific antibody with a vaccine resulted in a synergistic improvement in the inhibition of tumour growth that is mediated by increased number and activity of CD8+ T cells 80–82. It is speculated that in these models, the source of inhibitory TGFβ is the immune system itself and not the tumour, because the antibody-mediated blockade is effective at enhancing antitumour immune responses even when antibody is administered with a prophylactic vaccine before injection of tumour cells80. This effect of TGFβ is consistent with the recent finding where TGFβ was shown to be responsible for the apoptosis of short-lived effector T cells that comprise more than 90% of the effector pool after immunization with Listeria monocytogenes 83. TGFβ promotes the apoptosis of these effector T cells by downregulating the expression of BCL-2, which opposes the survival function of IL-15 on the short-lived effector population 83. It is possible that blocking TGFβ signalling with the neutralizing antibody during administration of the tumour vaccine inhibits the apoptosis of tumour-specific short-lived effector CD8+ T cells and therefore prevents the termination of expanding CTLs.
TGFβ-mediated inhibition of CTL function during tumour immunity might be through several mechanisms. TGFβ directly inhibits CTL function by suppressing the expression of several cytolytic genes, including the genes encoding granzyme A, granzyme B, IFNγ and Fas ligand 70. TGFβ also attenuates the effector function of antigen-specific memory CD8+ T cells by inhibiting T-bet expression resulting in inhibition of IFNγ production 84. TGFβ might also block T cell receptor (TCR) signalling of TILs by upregulating the expression of SPRED1 (sprouty-related, EVH1 domain containing 1), which is an inhibitor of the Ras/MAPK pathway 85. Interestingly, TGFβ can also influence CD8+ T cell-mediated tumour immunity by inducing IL-17 production by CD8+ T cells, although the effect of IL-17 on tumour growth versus immune surveillance remains controversial 86–89.
CD4+ T cells are central orchestrators of adaptive immunity; however, their role in antitumour immune responses has largely been overlooked, mainly owing to the lack of MHC class II expression by tumour cells. TGFβ has been shown to have effects on all subsets of CD4+ effector T cells controlling the expression of master transcriptional regulators in these cells. TGFβ inhibits T-bet and GATA-3 expression (which determine CTL, TH1 and TH2 cell differentiation) while it promotes FOXP3 and Rorγt expression (which determine TReg and TH17 cell differentiation) (reviewed in 90). The role of CD4+ T cells in tumour biology has been classically studied in the context of TReg cells, which are covered in a separate section of this Review. This section focuses on the specific role of TH cell subpopulations in the control of antitumour immune responses and how TGFβ within the tumour microenvironment could influence the polarization of each subset.
Early studies trying to understand the mechanism of tumor-induced immunosuppression 91 identified TGFβ as one of the major inhibitors of immune responses in the tumour microenvironment. Tumour-derived TGFβ was shown to inhibit TH1 responses by shifting infiltrating T cells towards a TH2 phenotype 92, and hence promoting a less efficient anti-tumor immune response. However, later studies comparing the efficacy of TH1 and TH2 effector cell subsets in mediating anticancer responses showed that both TH1 and TH2 cells increased the CTL-mediated antitumour response, although TH1 cells secreting IFNγ seemed to be more effective by promoting APC activation 93, 94. Studies using TCR-transgenic mice further support the requirement for CD4+ T cells to activate memory CTLs in vivo 95, and interestingly show tumour eradication by CD4+ T cells even in cases where tumours were resistant to CD8+ T cell- mediated rejection 96. These findings suggested a potential benefit of TH cells in cancer immunotherapy and started a search for the most effective anticancer CD4+ effector T cell population.
Contradictory reports regarding the role of IL-17 in cancer have made it difficult to conclude whether or not T cells expressing this cytokine would be beneficial against tumours 97, 98. Accumulation of TH17 cells in the tumour microenvironment has been reported in several types of cancer, as well as the expression of IL-6, IL-1β and TGFβ by tumour cells, which are key cytokines controlling TH17 cell differentiation and proliferation 99, 100. TH17 cell-polarized tumour specific CD4+ T cells were shown to be more efficient than TH1 -polarized cells in tumour rejection after adoptive transfer and this efficiency was probably dependent on IFNγ rather than IL-17 production 88. Similar observations, transferring CD8+IL-17+ cells which then become IFNγ+ producing cells were reported; however, some discrepancies have been found regarding the role of IFNγ in these models as the use of lymphopenic hosts promotes loss of a TH17 cell phenotype and acquisition of a TH1 cell phenotype in the transferred cells, potentially masking the real effects of IL-17 in controlling antitumour immunity 87, 89, 101.
Recent findings indicate that the differentiation state of T cells, naïve versus effector/memory, might also be important for mounting more efficient antitumour responses as single transfers of naïve CD4+ T cells were able to eradicate established tumours independent of CD8+ T cells, NK cells and NKT cells 102, 103.
Growing evidence suggests that control of the cytokines expressed in the tumour microenvironment can promote tumour eradication by controlling TReg and TH17 cell polarization in the tumour. Exogenous administration of IL-2 in tumour-bearing mice increased TReg cell and decreased TH17 cell frequencies in the tumour 104, whereas antagonizing the effects of TGFβ by administering IL-7 has been shown to be useful in the promotion of TH17 cells 105. A complete understanding of the dynamic cytokine network, including the role of TGFβ in controlling T cell polarization in the tumour, as well as characterization of the molecular signals mediating TH cell differentiation will be crucial for dissecting the beneficial use of TH cells in future immunotherapies against cancer.
TReg cells are an immunosuppressive T cell population that express the forkhead family transcription factor, FoxP3, and can suppress antitumour immune responses 106. These cells are a heterogeneous population containing at least two distinct subsets known as natural TReg (nTReg) cells and adaptive or induced TReg (iTReg) cells 106. nTReg cells develop in the thymus, express the IL-2 receptor α chain (CD25) and maintain self tolerance in an antigen-independent manner. iTReg cells, by contrast, develop in the periphery in response to self- or tumour- antigens and express variable levels of CD25 106. Although nTReg cells and iTReg cells have been identified as separate subsets of regulatory T cells, their phenotype and function have not been fully established in preclinical tumour-bearing animal models and patients with cancer. TGFβ could be involved in generating TReg cells in vivo and this cytokine may assist subsets of TReg cells in suppressing effector cell function in the tumour microenvironment (reviewed in 2 and covered in more detail in the section detailing effects of TGFβ on effector cells). High levels of TReg cells in patients with cancer can be inversely correlated with survival 107, 108. Although the precise mechanism(s) behind increased TReg cells in malignancies are unknown, TGFβ, as well as other tumour-produced chemical mediators working in concert with this cytokine, PGE2 and H-Ferritin have been implicated in inducing TReg cells 109, 110. In addition, the production of CCL22 by TAMs surrounding tumours might mediate TReg cell trafficking to the tumour bed through CCR4 111. Recently, IL-23 production in the tumour microenvironment has been implicated in promoting the proliferation of intratumoural TReg cells as these cells express the IL-23 receptor, have evidence of STAT3 signalling (downstream of IL-23 receptor) and are decreased in number in tumour-bearing animals treated with a blocking antibody specific for the IL-23 receptor 25. This cytokine may complement the effects of TGFβ, which also seems to increase the number of intratumoural TReg cells.
Recently a new regulatory T cell subtype has been identified that can be induced and expanded in mice bearing orthotopic liver, lung and melanoma tumours 112. Unlike the conventional TReg cells described above, this regulatory subtype lacks expression of CD25 and FOXP3. Instead, the cells express the IL-2 receptor β chain (CD122), IL-10, TGFβ1 and the early activation marker, CD69 112. Activation of CD69 by the agonistic Ab against CD69 (H1.2F3) results in high levels of membrane-bound TGFβ expression by these cells through the activation of ERK and this might contribute to the ability of this regulatory T cell subset to suppress CD4+ T cell proliferation and promote the growth of established tumours 112. Although these results suggest that another subset of regulatory T cells associated with TGFβ production can suppress the antitumour response, the role of these cells in patients with cancer remains to be determined. TGFβ, in combination with IL-2, is required for the conversion of naïve T cells to iTReg cells in vitro 113, 114. Furthermore, the induction of TReg cells by TGFβ might be a mechanism by which tumours escape the antitumour immune response as several tumours can produce TGFβ2. Blockade of TGFβ with antibodies or genetic manipulation leads to decreased numbers of induced TReg cells in some models of tumour-bearing animals 39, 115. Therefore, targeting TGFβ signalling in the tumour microenvironment could attenuate the immunosuppressive effects of iTReg cells, resulting in increased antitumour immunity.
CD8+ T cells can become suppressor cells similar to CD4+ TReg cells, and TGFβ can induce CD8+ T cells to express FOXP3 116, 117. CD8+ regulatory T cells are induced under immunosuppressive conditions such as the tumour microenvironment 118–121. The role of tumour-infiltrating CD8+ regulatory T cells is less well understood than that of CD4+ TReg cells. It was recently shown that infiltration of CD8+ T cells into the immunosuppressive microenvironment of prostate tumours can convert tumour-specific CD8+ effector T cells into regulatory cells, and that this regulatory activity could be blocked by a TGFβ-specific antibody119. In another recent study, CD8+CD25+FOXP3+ regulatory T cells were isolated from colorectal cancer tissue and shown to have suppressive activity ex vivo. In this study, TGFβ and IL-6 induced the generation of CD8+ regulatory T cells in a synergistic manner 120. However, as these CD8+ regulatory T cells represent only a small number of CD8+ T cells in vivo, more investigation is needed to fully understand how they are induced and what is their clinical relevance in patients with cancer.
NKT cells are a heterogenous subset of T cells that also have properties of NK cells, and thus bridge the innate and adaptive immune responses. Unlike other T cells that recognize MHC class I-presented peptides, NKT cells recognize self and foreign glycolipids presented by the nonclassical MHC class I molecule CD1d 122. There are two main subtypes of NKT cells that have opposing roles in the antitumour immune response: Type I NKT cells (invariant NKT or iNKT cells) and Type II NKT cells.
iNKT cells are defined by use of a semi-invariant TCR involving Vα14Jα18 in mice and Vα24Jα18 in humans, and respond to α-galactosylceramide, resulting in increased antitumour responses through IFNγ production that activates CD8+ T cells and NK cells 123, 122. Defects in iNKT cells have been identified in patients with cancer in later stages of the disease and increased numbers of circulating and intratumoural iNKT cells have been associated with improved prognosis 124. Targeting iNKT cells with activating agents is being evaluated in clinical trials (reviewed in 123). TGFβ has been implicated in suppressing this cell subset in patients with cancer and a recent evaluation of iNKT cells from patients with metastatic melanoma and renal cell carcinoma suggested that blocking TGFβ in vitro could enhance iNKT cell activation ex vivo 125.
Type II NKT cells have diverse repertoires of TCRs and, in contrast to iNKT cells, suppress the antitumour response through several mechanisms, including TGFβ production 122. In a murine fibroblast tumor model this subset of NKT cells can express high levels of IL-13, leading to the production of TGFβ by MDSCs, which in turn results in attenuated antitumour responses by CD8+ effector T cells 76. However, in a different tumour model, evaluating antibody-mediated blockade of TGFβ combined with a peptide vaccine against a lung cancer tumor line (TC1) suggested that the IL-13 pathway augmented by type II NKT cells might not be the mechanism behind the enhanced antitumour activity observed in vaccinated mice 81. These results suggest a more complex interplay, yet to be determined, between TGFβ, type II NKT cells and effector immune cells responsible for the antitumour response.
Successful cancer immunotherapy depends on overcoming the immunosuppressive milieu in the tumour microenvironment in patients with cancer. TGFβ has a crucial immunosuppressive role in both the innate and the adaptive arms of the immune response (Figures 2 and and3).3). In terms of the innate immune response, TGFβ inhibits IFNγ production by NK cells causing dampened CD4+ TH1 cell responses. It downregulates expression of the activating receptor NKG2D on NK cells resulting in decreased cytolytic activity and overall poor antitumour responses. TGFβ also influences tumour antigen presentation by decreasing DC migration and promoting DC apoptosis in some tumour models. In general, TGFβ inhibits DC maturation and cytokine production, thereby promoting a tolerogenic environment. In addition, TGFβ produced by tolerogenic DCs contributes to TReg cell differentiation. TGFβ can also favour the differentiation of M2 versus M1 macrophages by inhibiting NF-κB activation. TAMs are a subtype of M2 cells that are recruited to the tumour by TGFβ and also produce high levels of TGFβ. TAMs in the tumour microenvironment compete with DCs for antigen uptake but cannot properly present antigen. TGFβ also promotes the differentiation of N1 to N2 neutrophils, which similar to M2 macrophages, are less cytotoxic. So, blocking TGFβ can induce an expression profile in the tumour microenvironment that promotes better antigen uptake and presentation, resulting in more robust priming and activation of the adaptive anti-tumour immune response.
In terms of the adaptive immune response, TGFβ can also directly dampen the function of CD8+ and CD4+ T cells while promoting the recruitment and differentiation of regulatory T cells at the tumour bed (Figure 3). This cytokine inhibits the cytotoxic function of tumour specific CTLs and promotes apoptosis of the short-lived effector CD8+ T cells. TGFβ also controls the differentiation of several key CD4+ T cell subsets in tumour immunology, including TH1, TH17 and TReg cell subpopulations. Importantly, the effect of TGFβ on the differentiation of CD4+ T cells is influenced by the cytokine milieu in the tumour microenvironment, suggesting that modulating the relative abundance of such factors could probably promote antitumour responses in vivo. It is well documented that both TGFβ and regulatory T cells have key roles in suppressing the antitumour immune response; however, the precise contributions of TGFβ and different regulatory T cell subsets in suppressing effector cell function are still being evaluated (Figure 4). For example, although TGFβ can induce CD8+ T cells to become regulatory cells expressing FOXP3, the precise role of CD8+FOXP3+ T cells in tumour immunity remains unclear. TGFβ is also implicated in suppressing antitumour iNKT cell function; however, the interplay between TGFβ and immunosuppressive Type II NKT cells is less clear. Given that TGFβ can actively modulate inflammation and tolerance induction in the many ways described above, TGFβ blockade might enhance antitumour immunity through effects on numerous components of the immune response.
The immunosuppressive effects of TGFβ on immune cell subsets leading to dampened antitumour immune responses as described above strongly support the development of TGFβ inhibitors to treat patients with cancer. Several inhibitors of TGFβ signalling, summarized in Table 1, are in various stages of development, targeting several steps in the TGFβ signalling cascade (Figure 5). Although most of these approaches are in preclinical studies, several clinical trials have evaluated TGFβ inhibition in patients with cancer using an antibody (GC1008), blocking oligonucleotides (AP12009), small molecule inhibitors (LY373636 and LY2157299) and a vaccine approach 126–133. The results from these trials evaluating TGFβ blockade alone indicate that there is limited clinical benefit; however, trials evaluating the small molecule inhibitors and antibody-mediated blockade are ongoing (Table 1).
Although systemic blockade of TGFβ has been well-tolerated in preclinical studies, given the pleiotropic effects of this cytokine, one potential concern of this type of therapy is the development of significant autoimmune toxicities in humans. This could be particularly problematic if TGFβ blockade is used in combination with other immune-activating agents, such as CTLA-4- or PD1-specific antibodies (which are also being evaluated as single agents in clinical trials and have shown several autoimmune toxicities) 134. Other potential toxicities of blocking TGFβ might result from the cytokine’s homeostatic functions in other tissues outside of the immune system, including angiogenesis and the development of musculoskeletal tissues.
The manipulation of local TGFβ sources in the tumour should be considered in the future as a strategy to inhibit the dominant immunosuppressive intratumoural environment while promoting antitumour immunity. Challenges to this approach will include being able to target the tumour microenvironment with TGFβ inhibitors without affecting TGFβ function in the rest of the host to maintain systemic homeostatic processes. This might require novel delivery systems, as well as effective TGFβ-specific drugs that have minimum systemic toxicities to the recipient.
Given that TGFβ affects the activity and differentiation of numerous immune cell types, it is unclear which of the effects of TGFβ is most important in the tumour microenvironment. This is, in part, due to the pleiotropic nature of TGFβ and the contextual and combinatorial effects that this cytokine can have at a range of biological concentrations on different cell types at various stages of their development. Therefore, there remain several questions regarding the basic biology of this cytokine as well as regarding the best strategy to modulate this pathway alone and in combination with other pathways to enhance antitumour immunity in patients with cancer.
Although it is thought that tumour cells are an important source of TGFβ in the tumour microenvironment, immune cells themselves might in fact be a larger source of this cytokine, which is produced by effector T cells, regulatory T cells, APCs and MDSCs. Identifying the most relevant source of TGFβ will be important, as localized immunotherapy in the tumour microenvironment might be safer than systemic therapies that could interfere with the systemic homeostatic functions of TGFβ. In addition, TGFβ is expressed and synthesized as an inactive latent form unable to bind to its receptor. TGFβ becomes activated by interacting with molecules such as plasmin, matrix metalloproteinase, reactive oxygen species, thrombospondin-1, or integrins αvβ6 or αvβ8 6, 90. Notably, the cells that can activate TGFβ may be different than those that produce this potent cytokine, and thus this activation step provides a means for TGFβ to integrate signals from multiple cell types 90. We know little about how TGFβ is activated in a tumour-bearing environment and whether tolerogenic DCs, TAMs or MDSCs have a greater capacity to activate TGFβ than their immunogenic counterparts. Therefore, a precise understanding of the mechanisms by which immunosuppressive cell subsets work alone and together, and their specific involvement in producing and/or activating TGFβ, may improve cancer immunotherapies.
Further studies are also warranted to evaluate the effect of increasing innate and adaptive immune responses in a tumour-bearing host. For example, inhibition of TGFβ offers a means to manipulate T cell polarization in vivo that can change an immunosuppressive environment into a more antitumour environment once a tumour has established in the host, as is the case in cancer patients when these types of therapies are generally being considered. Once the exact role of IL-17 and TH17 cells in antitumour immunity has been defined, modulation of TGFβ levels might also be used to alter the ratio between TReg cells and TH17 cells in the tumour microenvironment. In addition, blocking TGFβ signalling in combination with tumour vaccines promotes antitumour immunity that is mediated, in part, by CD8+ T cells80–82, 135, which could lead to a long-term response with immunological memory.
Additional areas for future research related to the development of agents that efficiently block TGFβ and its activity include pharmacodynamic profiling of tissue TGFβ concentrations and the optimization of strategies to block the most appropriate TGFβ-dependent signalling pathways. For example, the blockade of SMAD-independent pathways of TGFβ-dependent signalling, including MAPKs, Rho GTPases and PI3K that are involved in tumour progression and metastasis, could lead to new strategies to enhance antitumour immunity (Figure 5). Mutations in SMAD2, SMAD3 and SMAD4 lead to cancer progression 136, 137, indicating that the tumour-suppressor properties of TGFβ involve SMAD-dependent signalling and therefore SMAD-dependent pathways may not be ideal therapeutic targets. Understanding the intricate signalling pathways controlled by TGFβ, as well as the mechanism(s) leading to its opposing effects in tumour biology, could lead to new strategies against cancer 138.
Furthermore, identifying the ideal timing of TGFβ blockade in the host, if used in combination with vaccines, cytokine therapies (IL-2 and IL-15) or other immune-activating antibodies (such as CTLA4- or PD1/PD1L-specific antibodies), would be very informative 139. Finally, designing optimal methods to deliver the most effective TGFβ inhibitor to the tumour microenvironment and the evaluation of exposing expanded T cell subsets to these drugs ex vivo to enhance adoptive T-cell based immunotherapies are all areas requiring further investigation.
So far, the clinical trials evaluating blockade of TGFβ in patients with cancer do not show a clear clinical benefit. Therefore, larger studies are warranted to clarify the toxicity and efficacy of these strategies. In addition, the optimal dose and timing of TGFβ blockade, as well as the ideal combination of this approach with other immunotherapies, remain unknown. These questions are currently being addressed in ongoing preclinical studies and are likely to be the focus of future clinical trials.
In addition to the effects of TGFβ on various immune cell subsets to promote tumour progression, this pleiotropic cytokine enhances tumourigenesis in a number of pathways not directly involving the antitumor response. For example, TGFβ promotes autocrine mitogen production to enhance tumor cell proliferation. In addition, TGFβ augments a number of biologic process supporting metastasis formation, including: tumor cell motility, cancer cell priming for metastasis development, extravasation of tumor cells from the primary site, osteoclast mobilization which can support osseus metastasis deposits and angiogenesis to nourish primary tumor and secondary metastases. These effects are beyond the scope of this review and are discussed in detail in References 1 and 2.
R.A.F is an investigator of the Howard Hughes Medical Institute. This work is supported by a post-doctoral fellowship grant from the Cancer Research Institute (to S.S.), the Yale Skin SPORE through a Yale Skin SPORE Career Development Award (to S.H.W.) and a post-doctoral fellowship from PEW Charitable Trust: PEW Latin American Fellow Program in Biomedical Sciences (to P.L.L.). Additional support from NIH grants CA121974 and DK051665 (to R.A.F.) and JDRF grant 32-2008-352 (to R.A.F.).
The authors declare that they have no competing financial interests.