Introduction
The majority of tumor viruses are well controlled by the immune system and therefore cause only transient or no disease in their hosts after infection. It is apparent that the main role in this control can be ascribed to the presence of cytotoxic CD8
+ T cells, which are very effective in destroying virus infected or transformed cells. In recent years, the idea that CD4
+ T cells can also play a considerable role in protective anti-tumor responses has received growing attention. One important function of CD4
+ T cells is their help for CD8
+ T cell and antibody responses against virus or tumor antigens. However, studies using several tumor models have shown that CD4
+ T cells can efficiently eliminate major histocompatibility complex (MHC) class II expressing tumor cells [
1,
2] as well as tumors lacking MHC class II molecules [
3‐
5], demonstrating a direct role of CD4
+ T cells in tumor rejection. However, whether CD4
+ T cells can fully compensate for the effector functions of CD8
+ T cells in the control of virus-induced tumors remains unclear. In addition, the significance of regulatory T cells (Tregs) in inhibiting tumor-specific CD4
+ T-cell responses during tumor rejection in vivo has not been defined. It was previously reported that Tregs infiltrate tumors and draining lymph nodes, suggesting that they interfere with anti-tumor immune responses in general and thereby contribute to tumor growth and progression [
6]. Tregs inhibit the function of many adaptive and innate immune cells, including CD4
+ T cells, CD8
+ T cells, dendritic cells (DCs), macrophages, natural killer (NK) cells, and B cells through various molecular mechanisms [
7]. To define the role of Tregs in tumor immunity, most studies have used malignant tumor models, in which the immune system failed to prevent tumor progression. However, the task of effector CD4
+ T cells and Tregs should also be analyzed in effective anti-tumor immunity to fully understand their role in tumor biology.
To address these questions, we have used the Friend retrovirus (FV)-induced mouse tumor cell line of C57BL/6 origin, called FBL-3 cells [
8]. This cell line was generated by inoculation of FV complex into mice, which results in an EPO receptor-dependent proliferation signal in erythroid precursor cells. Integration of pro-virus can subsequently cause over-expression of the
Spi1 proto-oncogene and inactivation of the
p53 tumor suppressor gene resulting in host cell transformation. Thus, FV infection can induce fully malignant erythroleukemia in susceptible mouse strains. FBL-3 is a FV-transformed tumor cell line that does not produce infectious virus, but expresses highly immunogenic FV antigens [
9,
10]. After subcutaneous (s.c.) implantation of FBL-3 cells into mice, the tumor grows locally and subsequently regresses in a CD8
+ T-cell-dependent manner over a time period of 20 days [
11,
12]. Tumor-specific CD4
+ T cells seem to be less important for tumor rejection when functional CD8
+ T cells are present [
13]. However, if the pool of Tregs is expanded by a chronic infection, mice fail to reject transplants of FBL-3 tumors due to a Treg-mediated suppression of tumor-specific CD8
+ T-cell responses [
11]. In the present study, we used Foxp3 (forkhead box P3) transgenic mice expressing the diphtheria toxin (DT) receptor under the control of the Foxp3 promoter, which made it possible to selectively deplete Tregs in vivo and to determine the influence of Foxp3
+ Tregs on T-cell responses during tumor regression. We especially focused on the direct anti-tumor effect of CD4
+ T cells and found that these cells could fully compensate for the lack of cytotoxic CD8
+ T cells when their functional suppression by Tregs was interrupted.
Discussion
The CD4+-helper T-cell response represents a critical part of a functional immune system and is well characterized in many tumors but its effector role in the control of virus-induced tumors remains unclear. In the current research, we have used a highly immunogenic FV-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study the mechanisms of immunological control and escape during tumor formation. Our studies show that when CD8+ T cells were unable to control FBL-3 tumor development, cytotoxic effector CD4+ T cells were able to take over and eliminate the tumor. However, direct anti-tumor effects of CD4+ T cells were strictly regulated by Tregs, which thereby contributed toward tumor progression.
Here, we compared the kinetics of the CD4
+ T-cell response to FBL-3 tumor antigen in drLN with the CD8
+ T-cell response and found a more rapid development of the CD4
+ T-cell response after FBL-3 injection. Peak expansion of tumor-specific CD4
+ T cells was observed 2 days later as the CD8
+ T cell. In contrast, in a virus infection model with FV peak tetramer-positive CD8 T-cell, responses were far greater in magnitude [
18,
30] than peak tetramer-positive CD4 T-cell responses [
17]. In previous studies using the Moloney murine sarcoma and leukemia virus complex (MoMSV), which share the immunodominant CD4
+ and CD8
+ T-cell epitopes with FV, the burst size of the virus-specific CD8
+ T-cell response was considerably larger than that of virus-specific CD4
+ T cells in drLNs [
20].
Immune escape of tumors has been studied in many models and has in part been attributed to active suppression mediated by CD4
+Foxp3
+ Tregs [
31]. The drLN is the site of the critical decision between immune activation and tolerance and has a major influence on host immunity. The current study shows that the Tregs from the drLNs appear to migrate into the tumor and influence the local microenvironment. The influence of Treg localization (sentinel lymph nodes and tumor) on cancer progression is still disputed [
32‐
35]. The assessment of circulating Tregs or Tregs infiltrating the tumor itself has been used as a prognostic indicator in human cancers but conflicting results were obtained in different tumor diseases. Curiel et al. [
32] reported that the presence of high numbers of CD4
+CD25
+Foxp3
+ cells in malignant ascites of ovarian carcinoma correlated with tumor staging and reduced survival. In colorectal cancer patients, it was demonstrated that Tregs in drLNs correlated better with disease progression and tumor stage [
33]. Another observation in breast carcinoma indicated that Treg localized within lymphoid aggregates, but not in the tumor area, had a negative impact on patients’ survival [
34]. The finding agrees with animal models of colon cancer [
36] and goes in line with our study. This suggests that Tregs in the drLNs can inhibit the development of effector cells such as cytotoxic CD4
+ and CD8
+ T cells, whereas Tregs in the tumor itself may have dual functions of controlling tumor-promoting inflammation on one hand but suppressing local effector T-cell responses on the other [
37]. Thus, in studies aimed at analyzing the impact of Tregs on tumor progression, it is critical to take the localization of Foxp3
+ Treg into account.
Although the perforin/granzyme pathway was previously known to be utilized by CD8
+ T cells and NK cells to lyse target cells, recent studies have suggested that Tregs may also use perforin/granzyme-mediated cytotoxicity as a mechanism to kill conventional T cells [
23,
24]. In our study, we showed that after FBL-3 challenge, Tregs in the drLNs started to produce GzmB. Moreover, the frequency of GzmB-producing Tregs correlated with the kinetics of the effector CD4
+ T-cell response. This observation and data from Treg depletion experiments strongly suggest that GzmB-producing Tregs suppress anti-tumor CD4
+ T-cell responses. Grossman et al. [
38] showed that activated human Tregs expressed granzyme A and/or B and could kill activated CD4
+ and CD8
+ T cells. This Treg-mediated killing was perforin-dependent [
38]. Similarly, Gondek et al. [
23] reported that murine Tregs stimulated with anti-CD3 mAb expressed GzmB and could suppress conventional CD4
+ T cells in a cell contact and GzmB-dependent manner. Using a system of tumor inoculation and adoptive transfer of Tregs, it was shown that GzmB was highly induced in tumor-associated Tregs by local factors in the tumor microenvironment [
39]. Thus, cytotoxic activity of Tregs might be a general mechanism of Treg-mediated suppression in tumor development. Interestingly, this is different to Treg activity in chronic infectious diseases in which most activated Tregs do not express granzymes [
16].
It was previously reported that CD8
+ T cells are essential in controlling FBL-3 progression [
11,
12]. After administration of anti-CD8 mAb, rejection of local tumors induced by FBL-3 challenge was blocked and mice died from systemic lymph node metastases [
12]. In agreement with these previous studies, we showed that the absence of CD4
+ T cells during FBL-3 formation did not significantly influence the tumor growth [
13]. Although we found a very potent and rapid CD4
+ T-cell response (production of cytokines and cytotoxins), these cells had only a minor effect on FBL-3 tumor rejection. Most likely, a direct anti-tumor effect of CD4
+ T cells is not required in a system in which the cytotoxic function of CD8
+ T cells is sufficient to mediate the successful rejection of the tumor. However, effector CD4
+ T cells could replace cytotoxic CD8
+ T cells but they were under tight regulation by Tregs. While CD8
+ T-cell ablation alone resulted in uncontrolled tumor growth [
12,
13], additional Treg depletion enabled the CD4
+ T cells to reject the tumor. Control experiments showed that this anti-tumor effect was exclusively mediated by effector CD4
+ T cells (Fig.
4g). To define the suppressive effects of Tregs on conventional T cells, one has to take their functions on CD4 and CD8 populations into account. Our current study shows that Treg depletion had no biological effect on CD8
+ T-cell-mediated tumor rejection. Although CD8
+ T-cell functions were augmented after Treg depletion (data not shown), this did not result in faster tumor rejection, most likely because the antitumor CTL were very efficient even under the suppressing influence of Tregs. In contrast, in FV infection, the functional suppression by Tregs mainly targeted CD8
+ T cells [
40], resulting in the development of functional exhausted CD8
+ T cells and in high FV loads in lymphatic organs [
16]. Earlier Iwashiro et al. [
11] demonstrated that mice persistently infected with FV have approximately twice the normal percentage of splenic CD4
+CD25
+ Tregs and lose their ability to reject the implantation of FBL-3 cells. In a mouse model of spontaneous mammary carcinoma, the depletion of Tregs resulted in CD4
+ T-cell activation and subsequent development of efficient CD8
+ T-cell activity [
41]. In two other models of antitumor immunity, it was proposed that Tregs diminished CD8
+ T-cell function by consuming IL-2 [
42] or by preventing cytotoxic granule release [
43]. In a model of autoimmunity, other authors proposed that DCs were central to Treg inhibition in vivo and attenuated priming of CD4-helper cells [
44]. Hence, it seems to depend on the environment and the type of immunity whether Tregs preferentially regulate CD4
+ or CD8
+ T-cell populations. In addition, the specificity of Treg depletion might also influence the results in certain experiments. The use of non-specific Treg targeting agents such as CD25-depleting antibodies, which in addition to CD25
+ Tregs also deplete recently activated CD25
+ effector T cells, can complicate the interpretation of the data. In our study, we used transgenic DEREG mice, which express a diphtheria toxin receptor under the control of the
Foxp3 promoter, allowing highly selective depletion of Foxp3
+Tregs even during ongoing immune responses [
45]. Our finding that Tregs preferentially influence the anti-tumor CD4
+ T-cell immune response revealed several aspects that had not been reported previously. The CD4
+ T cells residing in the drLNs of mice depleted of Tregs and CD8
+ T cells showed major changes to those of non-depleted mice. We demonstrated that in vivo depletion of Tregs and CD8
+ T cells in FBL-3-bearing DEREG transgenic mice augments GzmB production by CD4
+ T cells and increases FV-specific CD4
+ T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the Foxp3
+ Tregs control the proliferation and function of effector CD4
+ T cells and prevent the induction of efficient CD4
+ T cells with cytotoxic potential. It was previously shown that in the murine mammary carcinoma model, deletion of Tregs resulted in increasing numbers of IFN-γ and IL-2-producing CD4
+ T cells at tumor sites [
41]. However, the CD4
+ T cells alone, even in the absence of Treg suppression, were not sufficient to abrogate tumor progression [
41] but the Treg removal was performed by using anti-CD25 antibodies and depletion of Tregs was incomplete [
41]. Another group has also used DEREG mice for selective depletion of Tregs [
46]. However, the regression of the melanoma growth, which was induced by depletion of Foxp3
+ Tregs, was critically dependent on the presence of CD8
+ T cells in this model and additional elimination of those cells resulted in ongoing tumor progression [
46].
We show here that the immune system may still be capable of controlling tumor development in the absence of the main cytotoxic population (CD8
+ T cells). However, underlying mechanisms that influence the activity of effector CD4
+ T cells in the absence of CD8
+ T cells are poorly defined. CD8
+Foxp3
+ T cells were reported to mediate immunosuppression in cancer patients [
47]. Depletion of these CD8
+Foxp3
+ Tregs in addition to CD4
+Foxp3
+ Tregs might allow for antitumor cytotoxic function of CD4
+ T cells and tumor rejection. Moreover, some of the molecules that are produced by CD8
+ T cells, such as granzymes, may have immunomodulatory effects on other T cells [
48].
To demonstrate that the increased expression of GzmB by effector CD4
+ T cells resulted in antigen-specific cytotoxicity in vivo after depletion of Foxp3
+ Tregs, we performed an in vivo CTL assay. CD4
+ T cells showed potent killing activity of peptide-loaded targets in the drLN of FBL-3-bearing mice. The acquisition of cytotoxic activity by tumor-reactive CD4
+ T cells is particularly striking since it only emerged when the suppressive function of Tregs was blocked. From previous work, we knew that CD4
+ T cells can help rejection of tumors through indirect effects on NK cells [
49] and tumor-infiltrating macrophages [
1,
50,
51] and Tregs can negatively influence this help by inhibiting IFN-γ synthesis [
52]. Cytotoxic CD4
+ targeting viral antigens [
53‐
55] and alloantigens [
56‐
58] have been described previously, but in these models, the influence of Tregs on effector CD4
+ T-cell activity was not investigated but the tumor or the pathogen was eliminated by adoptive transfer of effector CD4
+ T cells [
57,
59]. Quezada et al. [
57] showed effector CD4
+ T-cell-dependent tumor rejection when radiotherapy and adoptive transfer of tumor-reactive CD4
+ T cells were combined with the blockade of the CTLA-4 molecule that is expressed on Tregs [
60,
61], suggesting that Tregs influenced cytotoxic CD4
+ T cells in this model as well.
As published previously, CD4
+ T cells cannot directly recognize FBL-3, since they only express MHC class I but not MHC class II molecules in vitro [
27] (Supplementary figure S3a,). However, our in vivo experiments strongly suggest cytotoxic activity of CD4
+ T cells against FBL-3 tumor cells. In the current study (Supplementary figure S3b), we showed that after being in the host environment, a proportion of the inoculated MHC II-deficient FBL-3 cells became I-A
b-positive. In addition, CD4
+ T cells can support killing of tumor cells by indirect mechanisms including recruitment of antigen-presenting cells [
27,
62] or killing stroma cells [
63] that support tumor growth. A combination of such mechanisms can elucidate the antigen-specific elimination of FBL-3 tumors by cytotoxic CD4
+ T cells.
It is well established that solid tumors are often infiltrated by macrophages. As previously published, these cells play an important role in the FBL-3 tumor model [
27]. In our study, we illustrate significant expansion of activated CD11b
+F4/80
+ macrophages that produce TRAIL in drLNs after depletion of Tregs and CD8
+ T cells. Moreover, infiltration of CD11b
+ cells into the tumor mass was shown, which suggests an antitumor effector function of macrophages. Activated macrophages have been reported to secrete a number of molecules with tumorocidal effects, like nitric oxide and reactive oxygen intermediates, and thus can partly mediate antitumor activity [
64]. In addition, antitumor function of macrophages can be stimulated by IFN-γ-producing CD4
+ T cells. However, macrophages alone could not control FBL-3 cell growth in the absence of CD4
+ and CD8
+ T cells (Fig.
4g). Thus, we believe that tumor-specific CD4
+ T cells can facilitate direct anti-tumor activity, which can be supported by TRAIL
+ macrophages inducing the classical macrophage activation pathway that contributes to inhibition of tumor cell growth [
65].
The direct antitumor role of CD4
+ T cells has been controversially discussed since most tumors do not express MHC II and thus cannot be directly recognized by CD4
+ T cells. Nonetheless, there are a number of HLA-DR-expressing tumors in patients [
66]. Moreover, HLA-DR expression is associated with better prognosis in colorectal cancers [
67]. The ability of MHC II-negative tumors to start to express class II molecules in the tumor microenvironment can be applied in cancer immunotherapy. Recent investigations in melanoma patients showed effectiveness of adoptive CD4
+ T-cell therapy [
68]. Thus, analyzing for HLA-DR expression of the primary tumor and metastasis and subsequent adoptive CD4
+ T-cell transfer might be a promising approach for future immunotherapy.
Cytotoxic CD4
+ T-cell activity against MHC II-negative tumors can also be used in cancer immunotherapy. Genetic modification of cytotoxic T cells with chimeric antigen receptors (CARs) specific for tumor antigen allows MHC-independent antigen recognition that nonetheless retains the T-cell effector mechanisms that are needed to eliminate tumor cells [
69].
In conclusion, our studies have established a critical role for cytotoxic CD4+ T cells in the context of oncoviral diseases. We propose that effector CD4+ T cells, which are largely regulated by Foxp3+ Tregs during tumor formation, are capable of maintaining immune control against FBL-3 tumor via direct killing and can functionally replace CD8+ T cells. We suggest that cytotoxic CD4+ T-cell immune responses may be induced therapeutically to enhance resistance against oncovirus-associated tumors.