Deciphering the Role of Regulatory CD4 T Cells in Oral and Oropharyngeal Cancer: A Systematic Review

Background: Recruiting regulatory CD4 T cells (Tregs) into the tumor microenvironment is an important tumor escape mechanism. Diminishing these suppressive cells is therefore one of the targets of cancer immunotherapy. Selective depletion of Tregs has proven successful in enhancing anti-tumor immunity and therapeutic efficacy in multiple tumor types. However, the role of Tregs in oral/oropharyngeal cancers is unclear with conflicting evidence regarding the effect of these suppressive cells on tumor prognosis. In this study, we sought to review the role of Tregs in oral/oropharyngeal cancer with the aim of deciphering the controversy regarding their effect on tumor progression and prognosis. Methods: A systematic review of the literature pertaining to the role of Tregs in oral/oropharyngeal cancer was performed using Scopus, Embase, and PubMed. Forty-five records were deemed eligible and data describing methodology of Treg detection, tumor type, and association with prognosis were extracted. Results: Of the 45 eligible manuscripts accepted for this systematic review, thirty-nine studies reported data from human subjects while the remaining studies focused on animal models. Sixteen studies were carried out using peripheral blood samples, while samples from the tumor site were analyzed in 18 studies and 11 studies assessed both blood and tumor samples. The transcriptional factor, Foxp3, was the most commonly used marker for Treg identification (38/45). The findings of 25 studies suggested that an increase in Tregs in the tumor microenvironment and/or peripheral blood was associated with poorer prognosis. These conclusions were attributed to the suppression of immune responses and the consequent tumor progression. Conversely, nine studies showed an increase in Tregs in peripheral blood and/or tumor microenvironment was related to a favorable prognosis, particularly in the presence of human papilloma virus (HPV), the status of which was only assessed in 11 studies. Conclusions: This review underlines the importance of host immunity in the behavior of oral/oropharyngeal cancer. Furthermore, we report an apparent lack of clarity regarding the true role Tregs play in oral/oropharyngeal cancer progression which could be attributed to inconsistent detection techniques of Tregs. Our results therefore highlight the need for clearer methodologies and more robust phenotyping when defining Tregs.


INTRODUCTION
Head and neck cancer is the sixth most common malignancy with an estimated 686,000 new cases and 375,000 deaths reported annually (combined worldwide laryngeal, oral, and pharyngeal cancer incidence) (1). The majority of head and neck cancers are squamous cell carcinoma (SCC). Along with alcohol consumption, smoking and various forms of betel quid chewing [which have long been associated with the development of oral and oropharyngeal squamous cell carcinoma (OPSCC)], it is now recognized that human papilloma virus (HPV) infection plays an important role in the onset of HPV positive OPSCC (2).
Despite advances in diagnosis and treatment, OPSCC mortality rate has improved little over the years, with 5 year survival rates as low as 53% reported in England for cancers of the oral cavity (3). This is mainly attributed to late diagnosis and the absence of predictors of disease progression in oral premalignant lesions.
Recently a growing emphasis is being placed on the role of the immune system and its association with the occurrence and progression of cancer. Indeed, cancer immunotherapy is among the most important developments in cancer treatment. It was therefore not surprising that cancer immunotherapy was named the scientific breakthrough of the year in 2013 (4). Despite the impressive successes in cancer immunotherapy, the response in patients is sometimes short lived. This is due to factors that hamper the immune response against cancer such as the presence of the suppressive regulatory CD4 T cells (Tregs) in the tumor microenvironment (5).
Tregs are a subpopulation of CD4 + T lymphocytes which are capable of discerning self-antigens from non-self-antigens and suppressing the expansion of effector cells directed against self. The major subpopulations of Tregs include thymus-derived Tregs (tTregs), Tregs which have been induced peripherally by different cytokines (pTregs), and induced Tregs which are induced in vitro in the lab, (iTregs). All Treg types maintain regulatory functions, and their development and function are thought to be dependent on the expression of the transcription factor Forkhead box P3 (FoxP3), known as the "master regulator" of Treg regulatory functions (6,7).
It is therefore important to fully comprehend the causes of these contradictions to enable full understanding of the role that Tregs play in oral/oropharyngeal cancer. This will enable designing novel immune-therapeutics that optimize the anti-tumor immune response and ultimately clinical outcome.
In this study, we sought to review the role of Tregs in oral/oropharyngeal cancer with the aim of deciphering the controversy regarding their effect on tumor progression and patient prognosis.

METHODS
We conducted and reported this systematic review following the PRISMA statement (30).

Search Strategy
A systematic search of PubMed, Embase, and Scopus (from their commencements to May 2017 when the search was performed), for studies in the English language with no species restrictions and for studies related to the role of Tregs in oral and oropharyngeal cancer. The following keywords were used in searching: ("head and neck cancer" or "head and neck malignancy" or "oropharyngeal") and ("epithelial dysplasia" or "oropharyngeal premalignancy") and ("tumor microenvironment" or "cancer immunology" or "tumor infiltrating lymphocytes" or "TILs" or "circulating immune cells" or "peripheral immune cells") and ("regulatory T cells" or "regulatory T lymphocyte" or "regulatory CD4 T lymphocyte" or "regulatory CD4 T cells" or "Treg" or "Tregs" or "Foxp3+ or CD4+Foxp3+" or "CD25+" or "CD4+CD25+" or "suppressive immune cells" or "suppressive lymphocytes").
We scrutinized the reference lists of the identified reports, reviews, meta-analyses, and other relevant publications to find additional pertinent studies. The "related articles" function was also used to broaden the search.
Our inclusion criteria were: 1-Studies must have been published as original articles 2-Studies must have been published in English 3-Studies assessing the role of Tregs in oral and oropharyngeal cancer.
Our exclusion criteria were: 1-Letters to the editor, conference abstracts, review, and systemic review articles 2-Studies that focused on thyroid, laryngeal, esophageal, and salivary gland tumors.

Data Extraction
The studies which met the inclusion criteria were summarized and data extraction was performed using a pre-defined form by one of the authors (CO) and accuracy checks were performed on over 75% of the manuscripts by (RA). Data extracted included: author, journal, year of publication, sample size, tumor type, tumor site, species, whether blood or tumor sample were used, method of sample analysis, markers used to detect Tregs, role of Tregs in tumor progression/prognosis, HPV status, correlations between HPV status and Tregs, tTregs vs. pTregs, and any data related to oral epithelial dysplasia. Due to the huge variation in the study designs, the number of samples, the tumor site, and the method for detecting Tregs within tumor or blood samples, meta analyses of the results were not possible.

Manuscripts Included in the Systematic Review
Of 715 identified citations, we identified 54 articles which met the inclusion criteria. Following full text screening, 45 articles were deemed to be eligible for inclusion in this study. Reasons for exclusion included irrelevant manuscripts which did not tackle the role of Tregs in oral and oropharyngeal cancer (n = 478), manuscripts that focused on tumors other than oral or oropharyngeal; laryngeal/esophageal (n = 82), salivary gland (n = 44), thyroid gland (n = 32) or gastric tumors (n = 18), review articles (n = 13), one study looked at the role of Tregs in periodontal disease and two articles were excluded because they assessed the expression of Foxp3 in tumor cells rather than assessing Tregs. Figure 1 shows the flow diagram of the studies retrieved for this systematic review.

Data Summary
The full characteristics of the study populations are displayed in Table 1.

HPV Status
Only 11 manuscripts looked at the HPV status of the tumors (24-29, 32, 36, 38, 43, 47), and out of those, only 10 included HPV positive cases in their studies (24-29, 32, 36, 38, 47). Half these studies reported no difference in Treg levels between HPV positive and negative tumors (24,27,29,32,36). Two manuscripts reported a decrease in Treg proportion in HPV positive (28,38) [one associated with an increase in TIL (28)], and three reported an increase in Treg associated with an overall increase in TIL (24,25,47). Four studies associated HPV positive tumors with better survival compared to HPV negative (24,27,28,47). One study found no correlation between HPV status and survival (29). Three studies correlated an increase in Tregs in HPV positive tumors with better prognosis (24,26,28), however, one of the studies suggested that it was associated with the overall increase in TIL (28).
No apparent correlation was found between the site of the tumor and the outcome. Only one study reported higher numbers of Tregs in Oral SCC lesions in comparison to oropharyngeal tumors (24).
As for the markers used, remarkably, Foxp3 was the only marker used to identify Tregs in five out of the nine studies that suggested a positive clinical outcome with an increase of Tregs (22,25,(27)(28)(29). Five out of the seven studies that used CTLA-4 as a marker of Treg correlated the presence of Treg with a poor clinical outcome (18,42,54,55,58). The remaining two did not conclude a definitive role for Tregs (36,50). On the other hand, out of the four studies that assessed GITR, two associated Tregs with poor clinical outcome and disease progression (54,55), while the other two did not conclude a role (50,60). All four studies that assessed TGF-β as a marker of Treg function found a negative clinical outcome associated with Treg (18,42,48,55).

DISCUSSION
Cancer immunotherapy to reactivate anti-tumor immunity is one of the most important recent developments in cancer treatment. For some patients, targeting the immune system to boost its anti-tumor activity can generate enduring disease remission, but despite the impressive successes in cancer immunotherapy, the response in patients is sometimes transient. This is attributed to multiple factors including the exhaustion of tumor-specific CD8 T cells in addition to induced suppression of the immune response against cancer. One of the major immune escape mechanisms in cancer patients is the conversion and dominance of suppressive immune cells within the tumor microenvironment that hamper the function of anti-tumor effector T cells. Regulatory CD4 T cells (Tregs) are among the most studied suppressor cells in the tumor microenvironment and their role in mediating tumor progression has been reported in many types of cancer. Indeed, reducing the number of Tregs has been reported to enhance anti-tumor immunity and promote tumor regression (9)(10)(11)(12)(13)(14).
However, in head and neck cancer and particularly in OPSCC, the role of Tregs in mediating tumor progression and affecting the overall clinical outcome is not clear. In fact, there are conflicting reports in the literature; while a considerable number of studies reported a similar role for Tregs in mediating tumor escape mechanisms and facilitating tumor progression (15)(16)(17)(18)(19)(20), other studies reported an opposite role and associated Tregs with a positive clinical outcome (21)(22)(23)(24)(25)(26)(27)(28)(29).
In this systematic review, we attempted to assess the body of knowledge available about the role that Tregs play in head and neck cancer with the aim of understanding the reasons for this contradiction in describing the role that Tregs play in disease progression and the clinical outcome.
One of the potential reasons for the controversy in the literature, is different reports from different species. We therefore included all the manuscripts from all species to assess whether the species under study affected the reported outcome. The only study that assessed both human and murine samples reported a role for Tregs in promoting tumor progression (32). One of the two studies that assessed murine sample did not reach a conclusion about the role of Tregs (34), while the second murine study reported a role for Tregs in enhancing tumor progression (19). Similar results about the role of Tregs in promoting disease progression were reported in the only study that assessed rat samples (41) and the two canine samples (52,53). These findings ruled out any role for inter-species variability in the controversy in the literature.
In recent years, the incidence of HPV positive oropharyngeal cancers has increased and is on the rise. Surprisingly, we report that HPV status was assessed in only 11 studies out of the 45 included in this systematic review. HPV-associated tumors are a distinct subtype with different intra-tumoral immune cell infiltration and better prognosis (24,27,28,47). Therefore, phenotyping tumors according to their HPV positivity is essential when assessing the role of different immune cells in anti-tumor immunity.
Despite the advances in head and neck cancer diagnosis and treatment, the mortality rate is still high. This is mainly attributed to late diagnosis and the lack of predictors of disease progression. Premalignant lesions are altered tissues that carry a higher risk of developing into malignancy, but unfortunately markers to predict malignant transformation into malignancy in these lesions are lacking. Surprisingly, among all the reviewed articles in this study, only three studies assessed premalignant lesions in animal models (32,34,41), and only one of these studies assessed samples from human patients (32). All three studies reported an increase in suppressive Tregs with disease progression from normal through dysplastic to neoplastic lesions (32,34,41). In their study, Ma et al. reported a correlation between disease stage and Tregs and in particular in A2AR expression. They reported that blocking A2AR reduced Tregs in the tumor bearing mice and enhanced anti-tumor immune response (32). Understanding the immune response within premalignant lesions is crucial to predict their progression into malignancy and to design treatments to modulate the immune response to eliminate these lesions before transforming into cancerous lesions.
Interestingly, distinction between thymic vs. peripheral Treg within the tumor microenvironment was not made nor assessed in any of the identified manuscripts in this systematic review. This is not a surprise given the lack of markers that accurately determine the origin of Tregs, but certainly measuring intratumoral numbers of converted Treg, defined by markers such as CD103 or S1PR may yield more precision for the role that Treg play within the tumor microenvironment.
In this systematic review, we found that Foxp3 is the most commonly used marker for Treg identification. In fact, it was the only marker used in 13 studies (16, 20, 22, 25, 27-29, 34, 35, 40, 47, 51, 56). Many of the studies used immunohistochemistry and a single stain to detect Foxp3. This causes a potential problem as Foxp3 is highly expressed in other activated T cell subsets including effector T cells. In fact, it has been proposed that under certain inflammatory condition, Foxp3+ Tregs might become unstable adopting a phenotype that is more characteristic of effector CD4+ T cells (61). Foxp3 might be a marker of activation rather than a marker of regulation and a key identifier of Tregs. Therefore, using dual staining and colocalization of markers such as CD4 and Foxp3 could clarify the role of Treg TIL subset in the tumor microenvironment more accurately.
Furthermore, Foxp3 is expressed in tumor cells. In fact, it has been reported that tongue SCC tumor cells express Foxp3 and its expression significantly associated with disease progression and poor patient outcome (62). The same group found that Foxp3 expressed in tumor cells has distinct biological functions compared with that in Tregs (63). On the other hand, the expression of Foxp3 in tumor cells is associated with an increase in the secretion of sCTLA-4, which was recently reported to be a favorable predictor of clinical outcome in advanced cancers (64). This could explain the reported association between Tregs, as identified by Foxp3 expression, and a favorable clinical outcome.
This adds to the controversy regarding the role of Tregs and emphasizes the need to use more than one method and different markers to detect Tregs within the tumor microenvironment. Remarkably, immunohistochemistry was the only used method to assess Tregs in 21 studies (16, 20, 22, 24-29, 31-36, 40, 47, 48, 51, 56, 57). A study further suggests that the overall expression of Foxp3 in Tregs by itself is not an important predictor of clinical outcome, but rather the localization of Foxp3 is the important predictor of outcome. Weed et al. reported that in oral SCC, nuclear Foxp3 is associated with a higher probability of early disease recurrence in comparison to cytoplasmic Foxp3, which is associated with a lower probability of recurrence (43). Immune profiling and the pattern of TIL within the tumors is of great importance as reported by Feng et al. after we conducted our search. They reported that the distance between the suppressive Foxp3+ Tregs and the effector CD8+ T cells are predictive of patient overall survival (65). Surprisingly, despite the great advances in different assays that added to the confidence in defining Treg markers, our study did not find any major changes in the assays or markers used to detect Tregs over the 12 year period (2005-2017) that the manuscripts included in this review covered. These reports emphasize the importance of using different markers, assays and analyses in the study of immune cells in cancer patients.
Checkpoint inhibitor antibodies represent a novel type of cancer immunotherapy that has seen notable success in the treatment of different cancers (66). One of the major targets of checkpoint inhibitors is CTLA-4, which is highly expressed on Tregs. Only seven studies out of the 45 studies that we assessed looked at the expression of CTLA-4 in Treg or used it as an identifier for these suppressor cells (18,36,42,50,54,55,58), five of which correlated the presence of Treg with a poor clinical outcome (18,42,54,55,58). One of these studies reported a higher frequency of CTLA-4+ Tregs (identified as CD4+CD25 hi Foxp3+) in non-responder patients to Cetuximab (18), suggesting a potential use of this immune checkpoint as a biomarker for response to therapy. GITR, another immune checkpoint was assessed in four studies as a marker of Treg function (50,54,55,60), two of which associated Tregs with poor clinical outcome and disease progression (54,55). TGF-β was assessed in four studies as by the membrane bound form (LAP), or using RT-PCR (18,42,48,55), all of which correlated Tregs to poor clinical outcome.

CONCLUSIONS
In conclusion, our systematic review emphasized the existing controversy regarding the role of Tregs in head and neck cancer, and in particular in OPSCC. We conclude that similar to most cancer types, Tregs contribute to tumor escape mechanisms and are therefore associated with poor clinical outcome. The inconsistent results reported in the literature could be due to the use of different markers to identify Tregs, variation in patient recruitment criteria or a heterogeneous cancer population. Indeed, we observed major differences in the reported outcomes between studies that assessed tumor samples, and those that assessed blood samples, suggesting the need to assess both to reach a more definitive understanding of the role of different immune cells in disease progression. HPV status, an important prognostic marker in OPSCC, was not assessed in majority of the studies, which could explain some of the discrepancy in the findings.
Our findings therefore suggest the need to define a better and more robust method to detect Tregs in the tumor microenvironment and in the periphery using a combination of methodologies, markers and analyses. We suggest using a combination of markers to define Tregs in the periphery and within TIL, including CD4, CD3, CD25, CD127lo, FoxP3, and CTLA-4. We also propose incorporating the regulatory properties of tumor cells as well as TIL for a complete picture of the tumor microenvironment.

AUTHOR CONTRIBUTIONS
CO performed the search, screening and data extraction. FW contributed to results interpretation and writing the manuscript.
RA designed the study, checked the accuracy of the data extraction, and contributed to results interpretation and writing the manuscript.