2. Immune cells action within tumor microenvironment
As surveillance cells in the tumor microenvironment, dendritic cells (DCs), natural killer (NK), and natural killer T cells (NKT) have been shown to infiltrate tumors and monitor the presence of novel antigen derived from tumors [
6]. DCs may be activated by danger signals released from stressed or necrotic tumor cells which may include cytokines, heat shock proteins, intracellular nucleotides, and intact double-stranded DNA [
6‐
8]. Activation and migration of DCs from the tumor site of antigen capture to secondary lymphoid organs is crucial in the initiation and amplification of immune response thereby, triggering a maturation program that includes expression of multiple costimulatory molecules and cytokines that result in efficient priming of effector T cell responses and beneficial antitumor immunity [
7]. Remarkably, the cytokines produced in the local microenvironment modulate the type of response that will be generated. Conversely, DCs at the tumor microenvironment, which possess an immature phenotype, is not necessarily conducive to the activation of antitumor immune responses due to tumor-derived local immunosuppressive cytokines milieu as transforming growth factor (TGF-β), and IL-10 and growth factors as vascular endothelial growth factor (VEGF) that may instead suppress or re-conditioning DCs function and/or myeloid-derived suppressor cells (MDSCs) [
7,
9]. The latter would result in secreting more of TGF-β to induce dampening of T cells and the augmented regulatory T (Treg) cell function, and ultimately inducing anergy thus favoring tumor evasion. Inevitably, efficient maturation of DCs at the tumor microenvironment is pivotal for priming T cells responses and mounting effective antitumor immunity [
7,
10‐
12].
MDSCs are heterogeneous population of myeloid derived cells of immature in nature. MDSCs have the ability to suppress T cell responses, cytokine production, and promote tumor angiogenesis and metastasis [
9,
11]. Normally these myeloid-derived cells are located in the bone marrow differentiate to mature granulocyte, monocytes/macrophages, or dendritic cells, and only 0.5% of peripheral blood mononuclear cells remains immature. In cancer condition, however, their differentiation step is blocked, proliferate and expand into MDSCs leading to a negative impact on the immune system as a whole and more specifically in the tumor microenvironment. MDSCs expand systemically in mice (CD11b+GR1+) with transplantable tumors or spontaneous tumors and in the peripheral blood of patients (CD11+CD14-CD33+) with different types of cancer. The induction and expansion of MDSCs is initiated by factors and cytokines produced by tumor stromal cells and activated T cells [
9,
11]. These factors and cytokines that induce MDSCs are usually driven by chronic inflammation. For instance, prostaglandin E2, cyclooxygenase 2, IL-6, colony stimulating factor and VEGF were found to induce the differentiation of CD11b+GR1+ MDSCs from bone marrow stem cells of mice, whereas COX-2 inhibitors or IL-1 deficient mice delayed tumor progression suggesting partial mediation of MDSCs by PGE2 and/or IL-1 [
9,
11,
13]. These studies suggest that inflammation promotes tumor progression through the induction of MDSCs that block immune surveillance and anti-tumor activity. MDSCs can directly affect T cell functions via cell-cell contact i.e. MHC-restricted and antigen-specific as well as indirectly through by suppressing CD4+ and CD8+ T cells, and inducing Treg cells [
9,
13]. However the latter two mechanisms depends on the subpopulation of MDSCs infiltrated in the tumor microenvironment with their produced cytokines (e.g. IL-10, TGF-β) or factors (e.g. nitric oxide, arginase, proxynitrate) [
9‐
13].
In the course of tumor mass, infiltration of NK cells into human neoplasm's appears to correlates with a better prognosis whereas low numbers of NK cells in advanced human neoplasms indicate that NK cells do not normally home efficiently to malignant tissues [
14]. The crosstalk between NK cells, DCs, and T cells initiates and sustains immune responses against tumors [
15]. In this process NK cells provide early and important sources of IFN-γ production as they might be the first to recognize developing tumors and thus producing the initial levels of IFN-γ [
16]. IFN-γ secreted at the tumor site by NK cells and NKT (type 1; invariant NKT) augments MHC expression on tumor cells increasing their immunogenicity which in turn can induce tumor cell death [
1]. NK cells not only operate in early stage of tumor development but also act as a helper in priming process of CD8
+ and Th1 cells by producing IFN-γ. Likewise, NK, NKT (type 1 NKT), and γδT cells are regarded as major sources of IFN-γ during the early phase of tumor development, whereas CD4
+ and CD8
+ T cells may become additional sources as adaptive immunity evolves [
17]. However, type II NKT cells were found to promote tumor progression by producing IL-13 which induces MDSCs accumulation and blocks their differentiation [
18].
Growing body of evidence indicates that tumor-specific CD4
+ T cells and there subtypes (Th1, Th2, Treg or Th17) are directly involved in mediating
in vivo tumor regression or evasion, as well as CD8+ T cells [
19] (Table
1). Each of the latter cell subtype is regulated by a different signal transducer and activator of transcription (STAT) and transcription factor and secretes unique repertoires of cytokines that mediate their responses as well as they possess a reciprocal relation between them in normal, physiological, and diseased conditions (Table
1). In addition, naïve T helper cells (Th0) also were found to be a major cell of tumor infiltrating lymphocytes (TIL) [
20]. These Th0 cells may produce IL-2, IFN-γ as well as IL-4 and may evolve in the tumor microenvironment depending on the danger signals provided in such environment [
20,
21]. The latter observation and the fact that cross regulation of Th1 and Th2 subsets occur through cytokine network has also been observed in TIL and peripheral blood of patients with other tumors [
22]. Proportions of Th1 cells identified on the basis of intracellular production of IFN-γ are markedly reduced, whereas proportions of Th2 cells producing IL-4 are significantly elevated [
23,
24]. Likewise, T cells infiltrating human cervical carcinomas exhibit enhanced Th2 cytokine profiles, particularly increased IL-4 and decreased IFN-γ production [
25]. Th2 polarization is dependent upon, and leads to, production of IL-4 which might have direct immunosuppressive effects on CD8
+ T cells at the tumor site. Furthermore, favoring Th2 development promotes tumor immune evasion leading to detrimental antitumor response [
26,
27]. Meanwhile, STAT1-deficient mice displayed an increase in Th2 polarization due to the block in IFN-γ signaling, and were more susceptible to tumor development [
28]. These results indicate the importance of polarizing TIL to Th1 cells to control and induce regression of solid tumor.
Table 1
CD4+ T lymphocytes and their functions in relation to transcription factors, priming and secreted cytokines in tumor progression or regression
Priming cytokines
| IL-12 IFN-γ | IL-4 | TGF-β and inflammatory cytokine (IL-1, IL-21, IL-6, TNF-α and IL-23) | TGF-β1 |
Transcription factors
| T-bet STAT1 | GATA3 STAT6 | RORγt RORα STAT3 | Foxp3 SMAD |
Major secreted effector cytokines
| IL-2 IFN-γ TNF-α | IL-4 IL-5 IL-13 | IL-17A (IL-17) IL-17F IL-21 IL-22 | TGF-β1 IL-10 |
Role in tumor microenvironment
| Help CD8+ T cells | Suppress CD8+ T cells | Recruit Th1 effector T cells/Suppress CD8+ T cells and promote early growth in the inflammatory environment? | Suppress CD4+ and CD8+ T cells |
Outcome
| Tumor regression | Tumor progression | Tumor regression/progression? | Tumor progression |
Currently, Th17 cells have been shown to play important roles in inflammation and autoimmune diseases, but their exact roles in tumor immunity are still meager and contradictory [
29,
30]. Some studies, however, detected low number of Th17 cells in several types of tumor such as ovarian cancer, [
31], non-Hodgkin's lymphoma [
32], and HER2 positive breast cancer patients [
33]. For instance, the levels of tumor-infiltrating Th17 cells were reduced in a group of ovarian cancer patients with more advanced disease and seemed to positively anticipate the outcome [
31]. Th17 cells were able to contribute to protective human tumor immunity through recruiting effector cells to the tumor microenvironment [
34]. Furthermore, tumor-specific Th17-polarized cells can eradicate large established melanoma in mice and were inversely correlated with Gleason score in prostate cancer patients [
34,
35]. A possible explanation lies in that Th17 CD4
+ T cells can be unstable and may evolve into IFN-γ-producing Th1-like cells capable of promoting tumor destruction. This effect also seems to be contingent on IFN-γ, as blocking antibodies prevented Th17 cell transfer from causing tumor regression [
34].
Since Th17 cells are considered potent inducers of autoimmunity through the promotion of tissue inflammation and the mobilization of the innate immune system [
19], the resulting inflammatory mediators may contribute to tumor progression by upregulating immune suppressive cells of the adaptive and innate immune systems. Substantial evidence indicates that the inflammatory reaction at a tumor site can promote tumor growth and progression [
36,
37]. Tumor-associated inflammatory cytokines such as IL-6 and tumor necrosis factor (TNF)-α probably regulate Th17 cells in the tumor microenvironment of ovarian cancer mouse model [
38]. Also, tumor-activated monocytes (TAM) promote expansion of Th17 cells through secreting a set of key proinflammatory cytokines, such as IL-1, in the peritumoral stroma of hepatocellular carcinoma tissues [
39] and ovarian cancer patients [
31]. Notably, Kryczek
et al. reported the prevalence of Th17 cells in peripheral blood and tumor microenvironment in both human and mice [
40]. Elevated proportion of Th17 cells were also detected both in the peripheral blood and in the tumor-draining lymph nodes of patients with gastric cancer, which were associated with clinical stage [
41]. Th17 cells were also suggested as a prognostic marker in hepatocellular carcinoma [
42] suggesting that Th17 cells can also play an active role in tumor pathogenesis. Nonetheless, whether Th17 cells play the same roles in the different types and stages of cancers remains to be determined [
29,
30].
As a regulatory cell, CD4
+CD25
+ Treg cells have emerged as being particularly critical for the maintenance of immunologic tolerance [
43]. The cross-talk between Treg cells and targeted cells, such as APCs and T cells, is crucial for ensuring suppression by Treg cells in the appropriate microenvironment by soluble factors and direct cell-cell contact [
44]. For instance, Treg cells inhibit DCs function through binding of CTLA-4 to CD80/86 [
43‐
46] and IL-12-Th1 cell activation by producing TGF-β and IL-10 [
45,
46]. Notably, elevated proportions of CD4
+CD25
+ Treg in the total CD4
+ T cell populations are present in the tumor microenvironment of various types of cancer where they mediate immune suppression via down regulating the functions of CD4+, CD8+, NK and NKT cells [
47‐
49]. For instance, CD4
+CD25
+ Treg cells enhance susceptibility to 3-methylcholanthrene (MCA)-induced-tumorigenesis [
48] and depletion of CD4
+CD25
+ Treg cells reduced tumor growth of MCA-induced fibrosarcomas [
49].
Despite the disparate functions of Treg and Th17 subsets, both have been shown to be dependent on TGF-β for their differentiation [
50]. Therein, TGF-β induces the Treg specific transcription factor Foxp3. However, addition of IL-6 to TGF-β enhances differentiation of Treg cells into Treg/Th17 cells [
29,
50]. Thus, TGF-β in the absence of inflammatory cytokines will induce Foxp3
+ Treg cells differentiation [
50‐
52]. In addition, Treg/Th17 cells under the influence of IL-21 and IL-6 can differentiate into Th17 [
29]. Moreover, the cytokines that promote Th17 responses significantly counteract the activation and functionality of the Tregs [
29,
51,
52]. Thus, the cross-talk between immune cells within the tumor microenvironment involves interactions between expressed receptors on cells, types of cytokines and chemokines produced which are dependent on the stimulus or stimuli present in such environment. Such cross-talk leads to the outcome of tumor occurrence or no-tumor promotion. Therefore modulating such interactions and cytokines would be a target therapeutic tool to enhance the immune response against the tumor cells that results in eradicating them.
4. The paradigm role of TGF-β, IL-17 and IFN-γ within tumor microenvironment
Since the circular nature of the relationship between inflammation and cancer has proven to be evident, it is important to emphasize the following generalizations; inflammation can cause cancer; inflammation can cause mutation; mutation can cause inflammation; mutation can cause cancer; cancer can cause inflammation; and inflammation can suppress cancer [
98]. In addition, inflammation can modulate the immune response to enhance tumor growth [
17,
21,
26,
40]. Since TGF-β is conventionally regarded as an anti-inflammatory cytokine whereas IFN-γ and IL-17 are considered proinflammatory cytokines, the paradigm role of these cytokines is complex in cancer immunology and tumor microenvironment. TGF-β demonstrates both tumor suppressor and oncogenic activities [
92]. In the current paradigm, the suppressor activities dominate in normal tissue. On the other hand, changes in TGF-β expression and cellular responses during tumorigenesis tip the balance in favor of its oncogenic activities to drive malignant progression, invasion and metastasis both
in vitro and
in vivo[
92,
99]. In fact, understanding the roles of TGF-β produced from T cells, Treg, MDSCs and even B regulatory cells within the tumor microenvironment requires insight into the changing response patterns of many interacting cell types [
11,
92,
100,
101]. For instance, mutations in type II TGF-β receptor gene,
TGFBR2, are frequent in human colon cancer with microsatellite instability [
102], and down regulation of TGF-β-SMAD signaling pathway is associated with poor prognosis in breast cancer patients [
103]. Furthermore, deletion of
TGFBR2, in mammary epithelial cells results in increasing chemokines that recruit further MDSCs [
94]. These MDSCs secretes high levels of TGF-β and metalloproteinases [
94]. In a tumor microenvironment, the latter two enhances tumor growth and metastasis. In addition, loss of
TGFBR2 in fibroblasts or T cells leads to prostate intraepithelial neoplasia and colon carcinoma [
104]. These data suggest controlling inflammation reduces tumorigenesis and inflammation in an established tumor enhances it growth and metastasis. On the other hand, TGF-β, as a tumor promoter and considered to be pre-oncogenic, is produced from many tumor types. For instance, TGF-β-SMAD activity is usually high in aggressive gliomas [
105]. Several studies showed the blocking TGF-β reduced tumor progression through several parameters including high infiltration of CD8+ T cells and suppression of Treg and MDSCs [
93,
86,
106].
Th17 cells differentiation requires TGF-β in humans and mice as well. However, TGF-β alone is not sufficient for the induction of Th17 in mice as it requires IL-6 plus TGF-β [
77,
83,
107,
108]. Actually, the discovery that TGF-β is a crucial cytokine for Th17 cell development suggested that Th17 and Treg cell subsets share reciprocal developmental pathways from uncommitted CD4
+ precursors [
50]. Moreover, there is a direct lineage relationship of Th17 cells with Treg cells. Indeed, these two subsets require TGF-β for lineage commitment and it is further observed that there are direct interactions between the lineage specific transcription factor Foxp3 of Treg and RORγt of Th17. Further work showed that TGF-β signaled in a concentration dependent manner to promote the expression of both Foxp3 and RORγt. Foxp3 directly bound to RORγt preventing Th17 differentiation; an effect relieved by IL-6, IL-21 and IL-23, therefore confirming the suppressive function of Foxp3 on RORγt [
52]. Practically, such interactions can't be disregarded within local tumor microenvironment but even still need to be fully further investigated. A study has revealed that in parallel to high levels of CD4
+Foxp3
+ Treg cells, IL-17
+ T cells including CD4
+ T cells and CD8
+ T cells were kinetically induced in the tumor microenvironment in multiple mouse and human tumors. Furthermore, IL-17
+ T cells demonstrated a dynamic differentiation; Th17/Treg, Th17, and Th17/Th1 in the tumor microenvironment thus providing new insight of IL-17
+ T cells potential role in tumor immune pathology and therapy [
40].
Concerning tumor development, some human tumor cells could express IL-17 that represents an early event in the development of the inflammatory reaction within the tumor microenvironment which may successively influence tumor phenotype and growth [
29]. Ostensibly, IL-17 seems to be a pleiotropic cytokine, as other inflammatory cytokines, with possible protumor or antitumor effects which often depends on the immunogenicity and degree of inflammation in the tumor itself [
29,
82]. Meth-A cells transfected with the human IL-17 gene can induce tumor-specific antitumor immunity by augmenting the expression of major histocompatibility complex (MHC) class I and II antigens; this antitumor immunity may be mediated by CD4
+ and CD8
+ T cells [
76]. On the other hand, using IL-17 knockout mice, Wang et al. showed that disruption of IL-17 reduced tumorigenesis and it was associated with less STAT3 activation, STAT3-associated proliferative and antiapoptotic gene expression, hyperplasia, and MDSCs tumor infiltration within the tumor microenvironment [
83]. The latter results indicate the role of IL-17-STAT3 pathway in cancer-associated inflammation in the tumor microenvironment. Furthermore, IL-17 up-regulated elaboration of a variety of proangiogenic factors by fibroblasts as well as tumor cells revealing a novel role for IL-17 as a CD4 or γδT cell-derived mediator as a tumor promoter by inducing angiogenesis and tumor growth [
80,
83]. A recent work showed that G-197A allele of the
IL-17A gene promoter was significantly associated with an increased risk of subsequent development of intestinal-type gastric cancer upon its association with the progression of gastric mucosal inflammation and the development of gastric mucosal atrophy [
109]. In addition, a very recent report by Kryczek et al. [
110] showed for the first time an IL-17+ regulatory T cells expressing FoxP3 (IL-17+Foxp3+CD4+ T cells) in the tumor microenvironment of inflammatory tumors such as colon cancer as well as chronic inflammation tissue of the colon but not in renal cell carcinoma, melanoma or ovarian carcinomas. The latter is supported by the two stage skin carcinogenesis model using 7,12-dimethyl benz(a) anthracene/12-O-tetradecanoylphorbel-13-acetate (TPA)-induced papilloma where the latter upregalated IL-17 expression in the skin papilloma and TH17 in the draining lymph nodes. Depletion or neutralization of IL-17 led reduced keratinocyte proliferation and delayed papilloma development [
55]. However, the latter observations were also correlated with IFNγ R deficiency or neutralization of IFN-γ and IFN-γ response to induce the expression of TNF-α, IL-6, TGF-β during the papilloma promoting stage. This stresses that the differential behavior of IL-17 within inflammatory versus non-inflammatory solid tumors and opens a new strategy for suppressing these cells in inflammation-induced solid tumors via down regulating SMAD3/4 and STAT3 signaling pathways. Similarly, it was found that
in vivo inactivation of genes that govern MDSCs accumulation, such as STAT3 and STAT6 restores T cell function and promotes tumor regression [
13].
The mouse prostate cancer cell line TRAMP-C2 secretes TGF-β1 and show low MHC-I expression. Treatment with IFN-γ increased MHC-I expression and antagonized the immunosuppressant activity of TGF-β [
66]. Since IFN-γ and TGF-β show reciprocal antagonistic effects, treatment of TRAMP-C2 with IFN-γ not only restored MHC-I expression but also improved the establishment of an effective antitumor memory immune response and thus the control of ongoing tumor growth [
66].
In vitro, studies with hepatic stellate cells demonstrate that TGF-β-dependent activation of SMAD3/4 responsive reporter construct was significantly decreased by IFN-γ due to the fact that IFN-γ induced the activity of the SMAD7 promoter and SMAD7 protein expression via STAT-1 signaling providing a novel approach for opposing profibrogenic activities of TGF-β in liver cells and indicating a TGF-β antagonizing function by IFN-γ [
96].
Treatment of cancer is critically dependent on IFN-γ and it is ability to activate macrophages, T cytotoxic, NK cells, and regulate MDSCS, Treg cells. However, IFN-γ relation to IL-17 and TH17 cells depending on the tumor environment context and the stability of TH17 [
55,
110]. Nonetheless, the finding that IFN-γ receptor knockout mice exhibit severely impaired antitumor capability illustrated the importance of IFN-γ in tumor immunity and the induction of IFN-γ via STAT1 activation and inhibition of STAT6 and 3. All in all, IFN-γ is correlated with several direct and indirect antitumor properties and tumor responsiveness to IFN-γ is necessary for IFN-γ-dependent inhibition of tumor angiogenesis by CD4
+ T cells.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HA did the literature search and drafted the manuscript. KM conceived the idea and did literature search on specific points and finalized the manuscript. Both authors approved the final version.