γδT Cells: Unexpected Regulators of Cancer Development and Progression

Regulation via Secreted Cytokines

It was previously thought that a large fraction of mouse γδ T cells are pre-programmed in the embryonic thymus, prior to birth, to either secret IL-17 or IFN-γ (29–32). However, Bruno and Chien found that some cell populations of mouse γδ T cells can be induced in the periphery (31,33,34). Using breast cancer models, Coffelt et al showed that treatment with IL-1β neutralizing antibody decreases levels of IL-17 in the spleen, blood, LN, and lungs. His previous work confirmed that tumor-infiltrating γδT17 cells were the main producers of IL-17 within these tissues (6).

These findings are akin to those found by Carmi et al. in Lewis Lung Carcinoma metastatic models where they found IL-1β to be critical for γδT17 cell responses (35). Similarly, in vitro blockade of IL-6, TGF-β and IL-23 in tumor homogenate showed reduced numbers of γδT17 cells suggesting these factors could play a role in driving IL-17 production by γδ T cells. IL-6 was also found to be a critical recruiter and regulator of γδT17 cells in spontaneous pancreatic Kras models (36). These and many other studies found that γδT17 cells within the tumor microenvironment participate in a positive feedback loop in which γδT17 cells mainly recruit cells capable of further propagating their own immunosuppressive responses. For example, in an ovarian cancer study, the inflammatory macrophages induced by γδT17 cells could in fact produce TGF-β, IL-6 and IL-1β to increase further γδT17 cell responses by stimulating both proliferation of γδT17 cells as well as their IL-17 production (3). Another key example is the study performed by Ma et al. using hepatocellular carcinoma to show that γδT17 cells are induced by IL-1β and IL-23 from IL-17 recruited MDSCs in a positive feedback mechanism (8).

Regulation by microbiota and PAMP engagement

Since many γδ T cell populations locate at epithelial barriers, it is important to investigate how the microbiota influence γδ T cell function. In a spontaneous lung cancer model, Cheng et al. found that antibiotic treatment reduced IL-6 and IL-23 responses thereby suppressing γδT17 cells capabilities to secrete IL-17 and hamper anti-tumor responses (37). Using tissue samples from colorectal cancer patients, we showed that inflammatory DCs stimulated by bacterial products from a leaky colon epithelial barrier could produce IL-23 thus driving Vδ1⁺ γδT17 responses with the production of IL-17, GM-CSF and IL-8 (7).

In an alternative mechanism that enhances the non-specific, innate like capabilities of γδ cells, bacterial PAMP engagement can propagate a non-IL-17 dependent, γδ T pro-tumor effector function (11,24). Specifically, it was found that bacteria-activated MDSCs induce γδ T cells to produce galectin-1 through TLR5 (PAMP receptor for flagellin). Galectin-1 was shown to induce apoptosis on effector Th1 and CD8 T cells while enhancing the suppressive activity of Tregs. In the same study, Rutkowski et al. also found that increased expression of galectin-1in γδ T cells negatively correlates with 5-year survival rates within ovarian cancer patients without the TLR5 defective polymorphism (24).

More investigation on how microbiota regulate γδ T cells in cancer is necessary to determine to what extent the development of pro-tumorigenic γδ T cells is dependent on tumor infiltration of microbiota and other microbial interactions. Some bacterial products have already been shown to be important in promoting γδT17 responses in human colorectal and lung cancer (7,35,37). These studies are less surprising as it is well known that bacteria can induce the production of IL-1β and IL-23 which are key modulators of γδT17 IL-17 responses. However, it has yet to be elucidated as to whether bacteria can modulate γδT17 responses by other means than cytokine secretion and PAMP engagement. Understanding how γδT cells are regulated in the tumor microenvironment and determining whether they can be “re-educated,” as is possible with other pro-tumorigenic immune populations such as Tregs, MDSC and TAMs, is critical for improving the success of future immunotherapies targeting tumors where these cells are abundant and potent inducers of tumor growth and progression.

γδ T cells promote tumor progression via IL-17-a-mediated immunosuppression

The promotion of tumor progression by IL-17 is multi-faceted. In many types of tumors, including pancreatic adenocarcinoma; hepatocellular carcinoma; and non-small cell lung, colorectal, and breast cancers, increased levels of IL-17 are associated with metastasis and poor prognosis (38) (8) (7). The consensus among the literature points to two main mechanisms by which IL-17 can influence tumor progression and metastasis: recruitment of immunosuppressive cell types like MDSCs and tumor infiltrating neutrophils, and increased angiogenesis (39). Wu et al. show that IL-17 γδ T cell infiltration is indicative of tumor progression and metastasis in human colorectal cancer patients (7). They found that inflammatory DCs stimulated by bacterial products produce IL-23 driving Vδ1⁺ γδT17 responses with the production of IL-17, GM-CSF and IL-8. This IL-17, GM-CSF and IL-8 production led to increased infiltration of PMN-MDSCs correlating with further cancer progression. He and colleagues showed that IL-17 is necessary for both the recruitment and development of immunosuppressive MDSCs as IL-17-deficient mice showed a decrease in the ability of MDSCs to suppress CD8⁺ T cell proliferation (40). More recently, Coffelt and colleagues identified tumor-infiltrating neutrophils within a breast cancer model that were regulated by IL-17 and capable of suppressing CD8⁺ T cell activation (41). IL-17 depletion in these mice showed a decreased number of circulating cKIT⁺ neutrophils and decreased metastatic tumor burden.

Another main way IL-17 has been shown to perpetuate tumor progression is via angiogenesis. In both a MCA induced fibrosarcoma model and ID8 ovarian tumor model, it has been shown that IL-17 markedly increases the transcription of pro-angiogenic factors, Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) within the tumor (3,39). Similarly, a deficiency in IL-17 resulted in decreased vascular density and arrest of tumor growth. The second mechanism of angiogenesis was proposed by Numasaki and colleagues who showed increased secretion of angiogenic CXC chemokines CXCL1, CXCL5, CXCL6, and CXCL8 in three different non-small cell lung carcinoma cell lines in the presence of IL-17a (42,43).

γδ T cell suppression through mimicry of Treg and Th-2-like activity

Treg and Th2 cells have distinct immunoregulatory capabilities, mediated in particular by the characteristic cytokines these cell types secrete. γδ T cells portray a Treg like phenotype by having the capability to produce more conventional suppressive cytokines including IL-10 and TGF-β in order to inhibit αβ T cell immune responses (10,28,44–46). In mice, an accumulation of IL-10 and TGF-β producing γδ T cells resulted in much lower adaptive and NK anti-tumor responses (46). Using rejection models, including the transplantation of tumors derived from one strain onto another background, it has been shown that the suppressive capabilities of IL-10⁺ γδ T cells can in fact inhibit rejection of the tumor by the adaptive and anti-tumor γδ T cell immunosurveillance (45).

Similarly, through their ability to secrete IL-4, γδ T cells have been show to adopt a Th2-like phenotype. For example, within the context of a melanoma mouse model, Hao and colleagues found a Vγ1⁺ population capable of suppressing anti-tumor Vγ4⁺ γδ T cells through production of IL-4 (4). This Th2-like phenotype was further confirmed by demonstrating that these particular cells expressed high levels of GATA-3, a transcription factor driving Th2 immunity known to be particular powerful in promoting tumor progression and suppressing anti-tumor immune responses. In humans, elevated levels of IL-4 have been found in the microenvironment of a variety of different cancers including renal cell cancer, non-small cell lung cancer, prostate, colon, and breast cancers (44). Unsure of how this affects γδ T cells, Mao and colleagues challenged human peripheral blood samples with IL-4. Interestingly, the results showed that IL-4 stimulation led to an increased absolute number and ratio of Vδ1⁺ to Vδ2⁺ cells. Furthermore, following IL-4 stimulation, the Vδ1⁺ cells increased their IL-10 secretion resulting in inhibited in vitro expansion of the Vδ2⁺ γδ T cells, cells known for their potent anti-tumor killing capabilities (44).

Determining the downstream roles of pro-tumor γδ T cells that are immunosuppressive is critical considering they can affect the use of immunotherapies. Traxlmayr et al. found during clinical trial studies that DC production of IL-12 and pyrophosphate activation can in fact induce regulatory γδ T cell responses capable of hampering the proliferative capabilities of tumor-specific CD4 and CD8 T cells (47). A recent study demonstrated that tumor-infiltrating CD39⁺γδ T cells have potent immunosuppressive activity via secretion of adenosine in human colorectal cancer (48). More investigation is needed to better characterize which γδ T cell populations are capable of becoming regulatory and how these suppressive populations operate in situ.

Interface between γδ T cells and DC results in Senescence

Dating back to 1989, Patel et al. were the first to describe a suppressive effector function for γδ T cells in vitro (49). Since then much work has focused on studying the duality of immunoregulatory versus pro-inflammatory γδ T cells. In 2007, Peng et al. were first to find an immunoregulatory Vδ1⁺ γδ T cell population in human breast cancer (50). Co-culture of BTIL31 γδ T cells with DCs resulted in their decreased expression of maturation markers like CD86, CD80, and CD83. Additionally, co-cultured DCs were incapable of stimulating naïve CD4⁺ T cells following LPS stimulation. In a later paper, Peng and colleagues expanded upon this finding by showing γδ T cell induced senescence in DCS as measured by increased SA-β-galactosidase (senescence associated β-galactosidase) expression (51). These DCs again showed decreased markers of maturation, decreased cytokine secretion capabilities, and decreased antigen presentation capacity. Additionally, CD4⁺ and CD8⁺ T cells co-cultured with tumor derived γδ T cells also showed upregulation of SA-β-galactosidase expression as well as increased negative regulatory functioning as measured by IL-10/TGF-β secretion and inhibition of responding CD4⁺ cells. These findings suggest that tumor-programmed γδ T cells use senescence as one mechanism to control anti-tumor immune responses within the tumor microenvironment.

Emerging Regulatory Role of γδ T cells via Modulation of PD-1/PDL-1

The immune checkpoint molecules, PD-1 and its ligand PDL-1/2, are one of the main regulatory mechanisms of pro-tumor immunity with the ability to inhibit the effector function of T cells and to perpetuate the development of Tregs. Since the response rate for PD-1/PDL-1 therapy still hovers around 40% with a large proportion of partial responders, further investigation into the mechanisms modulating the PD-1/PDL-1 axis is warranted (52,53). Recent studies using both a breast and colorectal cancer model have shown that a saturated IL-17a tumor microenvironment can cause upregulation of PDL-1 expression on the tumor cells themselves and tumor infiltrating inflammatory DCs (54) (7). However, little is known about the expression profile of PDL-1/PD-1 on tumor-associated γδT17 cells and whether these cells themselves can increase expression of immune checkpoint molecules thereby directly modulating the tumor microenvironment.

Daley et al began to probe for an answer to this question using a murine model of PDA and human PDA tissues (28). PDA proves to be a useful model for studying tumor associated effects of γδ T cells because up to 75% of all CD3⁺ cells within human PDA tissue are γδ T cells. Further phenotyping of these cells demonstrated a unique T effector memory profile expressing higher FasL, NK1.1, CD39, CD44, JAML, OX40 and Vγ4⁺ as compared to the spleen control tissue. Depletion of these cells resulted in decreased ductal dysplasia and fibrosis along with a massive influx of CD4⁺ and CD8⁺ cells with restored functionality as assessed by increased TNF-α and IFN-γ production. In vitro studies suggested that tumor-infiltrating γδ T cells inhibited αβT cell activation via PD-1/PD-L1 ligation. Addition of α-PD-L1 antibody to the WT model system restored CD4⁺ and CD8⁺ T cell functionality in a manner similar to γδ T cell ablation. However, PD-L1 blockade failed to induce tumor protection in TCR δ-deficient mice with no enhanced CD4⁺ and CD8⁺ T cell infiltration. Therefore, via cell-to-cell contact by PD-L1, tumor infiltrating γδ T cells are capable of restraining αβ T cell activation thus promoting tumor progression.