The adaptive immune system has evolved to mount robust responses to pathogens, and plays a critical role in inhibiting virus-induced tumors(Klein and Klein, 1977
). However, the functions of lymphocytes in control of non-viral tumors remain incompletely understood. In this report, we used an oncogene-induced prostate tumor model to show that despite substantial lymphocyte infiltration into tumors, the adaptive immune system did not protect mice from tumor development. This failure of tumor immune protection was mediated by TGF-β suppression of tumor antigen-specific T cell responses. TGF-β inhibited T cell priming in the tumor-draining lymph nodes, and impeded T cell proliferation and effector T cell differentiation. Furthermore, we showed that T cells themselves provide the essential source of TGF-β1 for the blockade of T cell responses to tumors to promote tumor growth and metastatic dissemination. These findings unveil a crucial immunosuppressive mechanism for tumor-associated T cell tolerance.
An important finding of this study is that the adaptive immune system did not naturally inhibit prostate tumor development in TRAMP mice. These observations, together with findings in a sporadic tumor model(Willimsky and Blankenstein, 2005
; Willimsky et al., 2008
), suggest that oncogene-induced spontaneous tumors do not induce protective adaptive immune responses. Yet, previous studies have provided strong evidence for T cell-dependent surveillance of carcinogen-induced tumors(Boesen et al., 2000
; Girardi et al., 2001
; Koebel et al., 2007
; Shankaran et al., 2001
; Smyth et al., 2000a
; Svane et al., 1996
). How tumors trigger differential T cell responses in these models remains to be determined. It is conceivable that extensive mutagenesis is induced during chemical carcinogenesis, which may generate high-affinity tumor-rejection antigens for T cells. In an SV40 Tag-induced tumor model, the immunogenicity of transformed cells causes systemic T cell unresponsiveness in tumor-bearing mice(Willimsky et al., 2008
). Therefore, it is plausible that tolerance to SV40 Tag-induced tumors may be associated with the transforming oncogene that itself is a potent T cell antigen. Future studies of tumors induced by non-antigenic oncogenes can be used to test this hypothesis. In contrast to TRAMP mice on Rag1-deficient background, deficiency of the activating receptor NKG2D leads to enhanced tumor development in TRAMP mice(Guerra et al., 2008
). NKG2D is expressed on effector T cells as well as on NK cells. Indeed, in addition to lymphocytes, high numbers of NK cells infiltrated the tumors in TRAMP mice (our unpublished observations). These observations suggest that NK cells may be responsible for NKG2D-dependent immunosurveillance in these mice. Thus, the effector functions of lymphocytes and innate immune cells are probably differentially regulated in response to spontaneous tumor development.
Multiple mechanisms have been proposed to explain the induction of T cell tolerance to tumors, such as the lack of high-affinity T cell antigens, weak co-stimulation and inflammation associated with tumor antigen presentation, and the immunosuppressive environment inside tumors(Blankenstein, 2007
; Drake et al., 2006
; Rabinovich et al., 2007
). Using Smad2 and Smad3 phosphorylation as a readout, we found that tumor development triggers increased TGF-β signaling in T cells from the tumor-draining lymph nodes, but not in the tumor-infiltrated T cells. Blockade of TGF-β signaling in T cells resulted in enhanced T cell proliferation and effector T cell differentiation in the tumor-draining lymph nodes. Intriguingly, the expression of the cytolytic enzyme GzmB in CD8+
T cells was further elevated in the tumor-infiltrated T cells. Although GzmB has been shown to be a direct target gene of Smad proteins(Thomas and Massague, 2005
), we cannot exclude the possibility that there are Smad-independent pathways by which TGF-β suppresses GzmB expression upon T cell tumor infiltration. An alternative explanation is that T cell activation and differentiation in the tumor-draining lymph node has a long-lasting effect on GzmB expression in tumor-infiltrated T cells. Altogether, these observations imply a novel TGF-β-dependent suppressive mechanism that inhibits anti-tumor T cell responses at the stage of T cell priming. In contrast to enhanced GzmB expression, expression of the inhibitory co-receptor PD-1 in tumor-infiltrating CD8+
T cells was diminished in mice with inhibited TGF-β signaling. TGF-β might directly promote PD-1 expression in tumor-infiltrating T cells. In addition, because PD-1 is expressed in exhausted T cells following chronic antigen stimulation, reduced PD-1 expression might be secondary to the tumor protection phenotype observed in these mice. The exact mechanism by which TGF-β regulates PD-1 expression is open for future investigation.
Tumors and other host cells secrete TGF-β, and it has been unclear which specific cell type-produced TGF-β mediates the suppression of T cell responses to tumors. Earlier, our work demonstrated an essential role for T cell-produced TGF-β1 in the control of Th1 and Th17 cell differentiation, and the inflammatory diseases inflicted by the effector CD4+
T cells(Li et al., 2007
). Here in this report, we found that T cell-produced TGF-β1 is also crucial for the inhibition of CD8+
T cell differentiation to cytolytic T cells and for the control of tumor development. The absence of TGF-β1 in T cells resulted in diminished T cell TGF-β signaling in the tumor-draining lymph node, providing a cellular mechanism for TGF-β suppression of T cell priming. Tumor growth likely releases high amounts of tumor-associated antigens. Thus, it is conceivable that increased T cell TGF-β1 secretion is triggered by chronic antigen stimulation of T cells in the tumor-draining lymph nodes. It is important to note that TGF-β1 is secreted as an inactive form that needs to be liberated from the constraints of the latency-associated protein(Annes et al., 2003
). Recent studies have revealed that dendritic cell-expressed αvβ8 integrin is required for the activation of latent TGF-β1 and for the regulation of T cell responses(Lacy-Hulbert et al., 2007
; Travis et al., 2007
). Therefore, the selectively enhanced TGF-β signaling in tumor-draining lymph node T cells may also be due to the specific requirement of dendritic cells to prime naïve T cells. However, the exact TGF-β1-producing T cell subset required for the control of tumor immune tolerance remains to be determined. Abrogation of TGF-β1 from CD4+
regulatory T cells was insufficient to inhibit tumor growth, suggesting that autocrine TGF-β1 might be involved in inhibiting effector T cell responses to tumors. It is also possible that Treg cell- and effector T cell-produced TGF-β1 might be redundant in promoting tumor T cell tolerance. Future studies using T cell subset-specific TGF-β1-deficient mouse models can be used to differentiate these possibilities.
Blockade of TGF-β signaling in T cells or T cell-specific deletion of Tgfb1
gene inhibits tumor development in TRAMP mice. These findings provide strong evidence that in the absence of self-directed TGF-β signaling in T cells, a spontaneous non-virus-induced tumor can induce and sustain tumor antigen-specific T cell responses to protect the host from tumor development. Increased TGF-β1 has been detected in another sporadic tumor model(Willimsky and Blankenstein, 2005
; Willimsky et al., 2008
). Importantly, high TGF-β1 levels are specifically associated with the induction of general cytotoxic T lymphocyte unresponsiveness in tumor-bearing mice(Willimsky et al., 2008
). Although the precise function of increased TGF-β1 remains to be determined in that model, these observations suggest that TGF-β-dependent T cell suppression may provide a general mechanism for defective tumor immunity in mice. Tumor-specific T cell responses have also been detected in cancer patients(Boon et al., 2006
; Lee et al., 1999
); yet, similar to experimental models, tumors grow progressively as a possible consequence of failed T cell surveillance. It will therefore be of great interest to determine whether the self-directed TGF-β pathway also controls T cell tolerance to primary tumors in humans.
Disseminated metastases are the primary cause of mortality in cancer patients. Cancer has very complex manifestations; tumor suppressor mechanisms can affect primary tumor development without any significant impact on tumor metastatic growth and vice versa. This dichotomy indicates that mechanisms controlling primary tumor growth and metastasis could be independent. We have shown here that, besides fostering primary tumor development, T-cell production of TGF-β1 facilitates tumor metastatic growth as deletion of the Tgfb1 gene from T cells also inhibited the colonization of lungs by the aggressive B16 tumors as well as the establishment of the peritoneal cavity by EL-4 tumors. These findings uncovered surprisingly comprehensive effects of T cell TGF-β1 production on tumor development and dissemination, and singularly revealed that absence of TGF-β1 from T cells is sufficient to break tolerance to tumors.
Our findings showed enhancement of anti-tumor immune responses in the absence of T cell-produced TGF-β1 independent of tumor-produced TGF-β1. In contrast, work published by several groups has shown that tumor-produced TGF-β promotes tumor escape from immune surveillance(Friese et al., 2004
; Kao et al., 2003
; Liu et al., 2007
; Torre-Amione et al., 1990
). One explanation for these different findings is that most of these studies based their conclusions on observations of specific cell lines or transplanted tumors. It is plausible that mechanisms of TGF-β-mediated T cell tolerance differ between primary and transplanted models. The TGF-β receptors are widely expressed in tumor cells and multiple lineages of tumor stromal cells(Massague, 2008
). Tumor cells and other cells in the immediate vicinity of the tumor will most likely consume TGF-β secreted by tumors before it reaches the draining lymph nodes to regulate T cell differentiation. Thus, tumor-derived TGF-β1 probably makes only a minor contribution to the observed TGF-β-mediated T cell tolerance, which we demonstrated is initiated at the stage of T cell priming. Nevertheless, the definitive function of tumor cell-produced TGF-β1 in control of tumor immunosurveillance awaits the generation of tumor cell-specific TGF-β1-deficient mice.
In conclusion, we have revealed in this report a TGF-β1-dependent mechanism that subverts T cell anti-tumor responses resulting in ineffective control of tumor growth and metastasis. These findings may have profound implications for targeting TGF-β for cancer immunotherapy. A systemic TGF-β blocking strategy will probably neutralize all the TGF-β, and hosts lose the benefits of the cytostatic effects of TGF-β on tumor cells(Pu et al., 2009
). Targeted blockade of the self-directed TGF-β signaling pathway in T cells will likely provide a better strategy to specifically awaken anti-tumor immunity and eradicate cancer.