Dr Pr Claude Leclerc

Patrick

Dr Dra Irmia

Juan Carlos

José Luis

Prof M François

Lemoine

Dr Géraldine Lescaille
email: geraldine.lescaille@gmail.com.

Keywords: Mucosal immunization, Head and Neck Cancer, therapeutic vaccine, orthotopic model

All my friends and the Magic PSA

List of tables and figures

Preface

Head and neck squamous cell carcinomas (HNSCCs) represent the sixth most frequent type of cancer in the world with more than 550 000 new cases per year. It is the fourth most prevalent cancer for males in France, after prostate, lung and colon cancers. These cancers are associated with multiple risk factors; among them the most important are alcohol and/or tobacco consumption. Nevertheless, some patients develop HNSCCs without exposure to these chemical carcinogens. Recently, human papillomavirus (HPV) has been associated with the development of some types of HNSCCs. Despite standard treatment strategies for HNSCCs -including surgery, radiation and chemotherapy-HNSCCs are very challenging to treat and present high relapse rates. The prognosis of HNSCCs remains poor, with a survival rate of 10-20% at 10 years. Due to this, there is an urgent need for innovative therapies that target specific features of HNSCCs.

Currently, immunotherapy is one of the most promising strategies for cancer treatment. Many human malignancies, including HNSCC are associated with quantitative and qualitative deficiencies in the immune system. Enhanced awareness of the immune alterations present in HNSCC, as well as better understanding the basic mechanisms of the immune system in carcinogenesis rationalizes the uses of immunotherapeutic strategies for treating HNSCCs. Moreover, recent clinical trials revealed the impressive efficacy of immunological checkpoint blockade in multiple types of metastatic cancers. In addition, preclinical studies provide evidence that some cytotoxic drugs have the ability to stimulate the immune system, resulting in anti-tumor immune responses that contribute to clinical efficacy of these agents. These observations raise the hypothesis that the next step for cancer treatment is the combination of conventional treatments and immunotherapies.

In the first part of this manuscript, I will present a review of the microenvironment's characteristics and immunoescape mechanisms of HNSCC, as well as the promising strategies of immunotherapy in this context. In the second part, I will present my work in the laboratory, whose main objective consisted in developing immunotherapeutic strategies in preclinical models of head and neck cancer. Results are discussed in the last part according to the literature, in order to present the perspectives of this contribution towards the improvement of the therapeutic care of HNSCC.

Chapter I. Head and Neck Cancers I.1 Generalities

I.1.1 Heterogeneity of Head and Neck Cancers

Head and neck cancers represent the sixth most frequent type of cancer in the world with annual global incidence and mortality rates estimated at 680 000 and 370 000 cases, respectively [START_REF] Ferlay | GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No[END_REF]. The vast majority (more than 90%) are head and neck squamous cell carcinomas (HNSCCs). HNSCCs are anatomically and clinically heterogeneous and arise from the mucosal surface of the oral cavity, oropharynx, hypopharynx, larynx, sinuses and other sites within the upper aerodigestive tract.

Tobacco and/or alcohol consumption are the predominant risk factors for the development of HNSCCs with a population attributable risk of 72%, of which 4% is due to alcohol alone, 33% is due to tobacco alone, and 35% is due to the combination of these factors [START_REF] Hashibe | Alcohol drinking in never users of tobacco, cigarette smoking in never drinkers, and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium[END_REF][START_REF] Hashibe | Interaction between tobacco and alcohol use and the risk of head and neck cancer: pooled analysis in the International Head and Neck Cancer Epidemiology Consortium. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. Otherwise, oral smokeless tobacco, especially when consumed in betel liquids, is a major cause of oral and oropharyngeal HNSCC in men (50%) and women (90%) in the Indian subcontinent [START_REF] Barnes | Pathology and Genetics of Head and Neck Tumours[END_REF]. However, in the last decades an increased incidence of HNSCCs associated with oral infection by the human type 16 papillomavirus (HPV-16) has been observed [START_REF] Chaturvedi | Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers[END_REF][START_REF] Chaturvedi | Human papillomavirus and rising oropharyngeal cancer incidence in the United States[END_REF], especially among young patients. Those HNSCCs developing in the oral cavity (OSCC) and in the oropharynx (OPSCC) are both associated with tobacco and alcohol abuse, but only OPSCC is associated with HPV-16. Previously published data support a dominant role for HPV-16 in economically developed (60 to 70%) versus developing countries (less than 10%).

Tobacco is responsible for more than 30% of all cancer deaths worldwide. Tobacco smoke contains more than 4000 chemicals, of which at least 60 have been shown to be carcinogenic, these carcinogens promote tumorigenesis by inducing genetic aberrations depending on carcinogen dose and host susceptibility to HNSCC [START_REF] Singh | Molecular pathogenesis of head and neck cancers[END_REF]. Chronic alcohol exposure also results in increased cancer incidence; the carcinogenic effect of alcohol involves both direct (increased p450 activity, resulting in an more activation of carcinogens) and indirect (acting as solvent, facilitating the entry of other carcinogens into cells, especially in the upper aerodigestive tract [START_REF] Seitz | Alcohol metabolism and cancer risk[END_REF].

Human papillomaviruses are small, non-encapsulated DNA viruses that can infect epithelial cells from many organisms, including humans. A fraction of people infected with high-risk subtypes of HPV are at risk for developing squamous cell carcinoma. HPV-16 is the most frequently detected subtype in squamous cell carcinoma and was found in up to 90% of HPV-positive tumors [START_REF] Gillison | Evidence for a causal association between human papillomavirus and a subset of head and neck cancers[END_REF][START_REF] Wiest | Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control[END_REF]. HPV DNA replicates to a high copy number in well-differentiated cells near the epithelial surface through the action of the E6 and E7 proteins and induces cell-cycle progression and viral DNA replication in differentiated keratinocytes. The human papillomavirus encodes up to 10 proteins, including the E6 protein, a 150 amino-acid (aa) protein containing two zinc-like fingers joined by an interdomain linker, which binds to and induces the degradation of the p53 tumor suppressor protein [START_REF] Werness | Association of human papillomavirus types 16 and 18 E6 proteins with p53[END_REF], and the E7 protein, a 98 aa zinc-binding phosphoprotein that binds and destabilizes the retinoblastoma (Rb) tumor suppressor protein [START_REF] Dyson | The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product[END_REF] leading to increased cell cycling and decreased apoptosis.

Even if the majority of head and neck cancers are squamous cell carcinomas, recent insight has revealed that this type of cancer is not homogeneous [START_REF] Leemans | The molecular biology of head and neck cancer[END_REF].

Various subclasses of HNSCCs can be distinguished at the histological [START_REF] Woolgar | Pitfalls and procedures in the histopathological diagnosis of oral and oropharyngeal squamous cell carcinoma and a review of the role of pathology in prognosis[END_REF], molecular [START_REF] Chung | Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression[END_REF] and genetic [START_REF] Smeets | Genetic classification of oral and oropharyngeal carcinomas identifies subgroups with a different prognosis[END_REF] level.

Genome-wide sequencing projects have identified a number of recurrently mutated genes in HNSCC, including TP53, CDKN2A, EGFR, PIK3CA, FAT1, NOTCH1, and chromatin related genes, among others [START_REF] Riaz | Unraveling the molecular genetics of head and neck cancer through genome-wide approaches[END_REF]. Furthermore, around 20% are HPV positive and 80% are HPV-negative. In addition, about 20% of HNSCC cases that are not caused by HPV seem to have only a few copy-number alterations, the rest presents high chromosome instability and TP53 mutations. These data underline head and neck cancers as a heterogeneous disease, and HPV-positive tumors as a specific subclass of HNSCCs due to the differences at the molecular level and clinical outcome [START_REF] Chung | Human papillomavirus in head and neck cancer: its role in pathogenesis and clinical implications[END_REF].

Feature HPV-negative HNSCC HPV-positive HNSCC

I.1.2 Management and prognosis of Head and Neck Cancers

The standard of care for HNSCC is the use of surgery in association with radiotherapy and/or chemotherapy [START_REF] Choong | Expanding role of the medical oncologist in the management of head and neck cancer[END_REF]. However, the choice of treatment for a patient must to be deliberated on a multidisciplinary board, taking into account individual parameters from the patient (general condition) and the tumor (localization, stage, etc.).

Despite current treatment, head and neck cancers have a survival rate of no more than 20% at ten years [START_REF] Chaturvedi | Human papillomavirus and rising oropharyngeal cancer incidence in the United States[END_REF]. The five year relative survival rates reported from 21 countries in the European Cancer Registry-based Study of Survival and Care of Cancer Patients (EUROCARE-4) were 44.4%, 48.5% and 45.42% for oral cavity cancer and 31.0%, 39.8% and 38.71% for oropharyngeal cancer, during the periods of 1990[START_REF] Kubota | Metastatic models of human cancer xenografted in the nude mouse: the importance of orthotopic transplantation[END_REF][START_REF] Pak | Mechanisms of immune suppression in patients with head and neck cancer: presence of CD34(+) cells which suppress immune functions within cancers that secrete granulocytemacrophage colony-stimulating factor[END_REF][START_REF] Bibby | Making the most of rodent tumour systems in cancer drug discovery[END_REF][START_REF] Karim-Kos | Recent trends of cancer in Europe: a combined approach of incidence, survival and mortality for 17 cancer sites since the 1990s[END_REF]), and 2000-2007[START_REF] De Angelis | Cancer survival in Europe 1999-2007 by country and age: results of EUROCARE--5-a population-based study[END_REF] respectively. The five year relative survival rate reported by the SEER program for the period 1996-2004 was 59.7% for oral cavity and pharyngeal cancers [START_REF] Ries | SEER Cancer Statistics Review, 1975-2004[END_REF].

Several characteristics of HNSCCs patients have been linked with favorable prognosis, including non-smoker, minimum exposure to alcohol, good performance status, and no comorbid disorders, all of which are related to HPV-positive tumor status [START_REF] Marur | HPV-associated head and neck cancer: a virus-related cancer epidemic[END_REF]. Furthermore, overall survival and free-disease survival of HPV-positive OPSCC patients is significantly better than that for HPV-negative OPSCCs patients as showed by several retrospective and prospective studies in the United States, Australia and Western

Europe [START_REF] Ang | Human papillomavirus and survival of patients with oropharyngeal cancer[END_REF][START_REF] Chaturvedi | Human papillomavirus and rising oropharyngeal cancer incidence in the United States[END_REF][START_REF] Posner | Survival and human papillomavirus in oropharynx cancer in TAX 324: a subset analysis from an international phase III trial[END_REF].

The improvement in survival may be a result of one or more of several reasons.

Augmented sensitivity to chemotherapy and radiotherapy has been attributed to absence of exposure to tobacco and presence of functional unmutated TP53 [START_REF] Bristow | The p53 gene as a modifier of intrinsic radiosensitivity: implications for radiotherapy[END_REF].

Increased survival of patients with HPV-positive cancer might also be attributable in part to absence of field cancerization related to tobacco and alcohol exposure [START_REF] Gillison | Evidence for a causal association between human papillomavirus and a subset of head and neck cancers[END_REF].

Nonetheless, HPV-positive OPSCCs patients have significantly better survival compared to HPV-negative patients even after adjustment for differences in favorable prognostic factors often observed among HPV-positive patients (younger age, better performance status, fewer co-morbidities, and less exposure to tobacco smoking) [START_REF] Ang | Human papillomavirus and survival of patients with oropharyngeal cancer[END_REF]. HPV-positive HNSCCs are in general, more sensitive to chemotherapy and radiation than HPV-negative tumors. Thus leading to some improvement in prognosis and therefore to longer survival.

I.2 Tumor microenvironment of HNSCC

Like other cancers, HNSCCs arise from the accumulation of genetic and epigenetic changes and abnormalities in cancer-associated signaling pathways, causing the acquisition of cancer-related phenotypes. However, the biology of a tumor can only be understood by studying tumor cells as well as the tumor microenvironment in which malignant cells subsist.

Cancer cells initiate tumors and drive tumor progression forward, carrying mutations that define cancer as a genetic disease. They have been portrayed as homogeneous cell populations until relatively late in the course of tumor progression. Many human tumors are histopathologically diverse, containing regions with various degrees of differentiation, proliferation, vascularity, inflammation, and invasiveness [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF].

The tumor microenvironment consists of extracellular matrix and diverse types of nonmalignant cells, including endothelial cells, cancer-associated fibroblasts (CAFs), pericytes and a variety of immune cells and their precursors [START_REF] Lee | Crosstalk between cancer cells and blood endothelial and lymphatic endothelial cells in tumour and organ microenvironment[END_REF].

I.2.1 Non-immune populations in the HNSCC microenvironment

HNSCC non-malignant stromal and parenchymal cells largely contribute to cancer progression through their crosstalk with cancer cells, extracellular matrix and other noncancer cells, using growth factors, proteases and other signaling molecules [START_REF] Koontongkaew | The tumor microenvironment contribution to development, growth, invasion and metastasis of head and neck squamous cell carcinomas[END_REF]. These cell-to-cell communications promote tumor growth [START_REF] Whiteside | The tumor microenvironment and its role in promoting tumor growth[END_REF], angiogenesis and metastatic invasion [START_REF] Watnick | The role of the tumor microenvironment in regulating angiogenesis[END_REF].

Journal of Cancer 2013, Vol. 4 70 tem to reject the tumor. However, this observation remained largely neglected for over a century until it was demonstrated that innate immune cells, in particular phagocytes, play an active role in promoting the tumorigenesis. In addition to leukocyte infiltra-tion, angiogenesis is now being recognized as another stromal reaction promoting cancer progression. Therefore, chronic inflammatory and neovascularization are critical, if not essential, for cancer progression [62][63][64].

The cellular microenvironment of H N SCC

Studies have demonstrated a higher incidence of malignancy in acquired or iatrogenic immunodeficient hosts, which suggest an important role for immunosurveillance in HNSCC [65][66][67][68]. In those with a competent immune system, this is an additional barrier to malignancy, which must be overcome and which shapes both the tumor and its microenvironment [69]. Although effective antitumor immune responses likely involve many components of the immune system, T-cells continue to be considered as the critical immune cells involved in antitumor immuni-Th2 cells and CD4+ T regulatory cells are capable of suppressing effective CD8+ antitumor responses. In fact, several investigators have found dysfunctional circulating and tumor-infiltrating T cells in HNSCC patients, with functional assays identifying multiple defects in T-cell activation and effector function, suggesting that the tumor has successfully suppressed an otherwise robust lymphocytic response [70][71][72].

Patterns of tumor-related leukocyte infiltration varies between primary tumors and metastatic lymph nodes in HNSCC with a local decrease in the number of CD8+ T-cells and increase in CD20+ B-cells being the most relevant findings. This indicated that suppression of local cellular immunity might be a mech-Journal of Cancer 2013, Vol. 4 70 tem to reject the tumor. However, this observation remained largely neglected for over a century until it was demonstrated that innate immune cells, in particular phagocytes, play an active role in promoting the tumorigenesis. In addition to leukocyte infiltra-

The cellular microenvironment of H N SCC

Studies have demonstrated a higher incidence of malignancy in acquired or iatrogenic immunodeficient hosts, which suggest an important role for immunosurveillance in HNSCC [65][66][67][68]. In those with a competent immune system, this is an additional barrier to malignancy, which must be overcome and which shapes both the tumor and its microenvironment [69]. Although effective antitumor immune responses likely involve many components of the immune system, T-cells continue to be considered as the critical immune cells involved in antitumor immunity. T lymphocytes are considered an essential component of antitumor immunity, with CD8+ T cells serving as cytotoxic effector cells and CD4+ Th1 cells Endothelial cells represent one of the more important non-immune components of the tumor stroma, forming the tumor-associated vasculature. They have a significant impact on the progression of HNSCC through secretion of factors involved in tumor proliferation and angiogenesis, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and IL-8 [START_REF] Campos | Endothelial derived factors inhibit anoikis of head and neck cancer stem cells[END_REF][START_REF] Li | Microvessel density and expression of vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived endothelial growth factor in oral squamous cell carcinomas[END_REF]; and through the activation of MAPK, Notch-1 and STAT3/Akt/ERK signaling [START_REF] Neiva | Cross talk initiated by endothelial cells enhances migration and inhibits anoikis of squamous cell carcinoma cells through STAT3/Akt/ERK signaling[END_REF][START_REF] Zeng | Crosstalk between tumor and endothelial cells promotes tumor angiogenesis by MAPK activation of Notch signaling[END_REF]. In addition to blood vessels, HNSCCs are typically infiltrated by lymphatic vessels, which are distributed throughout the tumor and the peritumoral regions [START_REF] Cao | Opinion: emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis[END_REF][START_REF] Zhao | Intratumoral lymphangiogenesis in oral squamous cell carcinoma and its clinicopathological significance[END_REF].

Increased tumor lymphatic vessel density correlates with metastasis to lymph nodes in HNSCC [START_REF] Frech | Lymphatic vessel density in correlation to lymph node metastasis in head and neck squamous cell carcinoma[END_REF]. Another component, the pericyte, is a specialized mesenchymal cell that wraps around the endothelial tubing of blood vessels. They provide paracrine signals to the endothelium and synthesize the vascular basement membrane that helps vessel walls to withstand the pressure of blood flow [START_REF] Dvorak | Tumor microenvironment and progression[END_REF]. Abnormal pericyte integration into tumor endothelium vessels destabilizes their integrity leading to cancer cell intravasation into the circulatory system; thus, enabling dissemination and metastasis [START_REF] Raza | Pericytes and vessel maturation during tumor angiogenesis and metastasis[END_REF]. In HNSCC, immunohistochemical analysis has shown high activity of new structurally abnormal vessel formation, indicated by non-homogeneous patterns of endothelial cells and loosely attached pericytes to the endothelium, together with precursor cells being incorporated into these structures [START_REF] Hollemann | New vessel formation in peritumoral area of squamous cell carcinoma of the head and neck[END_REF].

CAFs are the preponderant cell population of the tumor stroma with similarities to normal fibroblasts and myofibroblasts, but different biological roles and properties [START_REF] Xouri | Origin and function of tumor stroma fibroblasts[END_REF]. Two dominant patterns of distribution have been described in HNSCC: the 'network' pattern where CAFs occupy almost the entire tumor stroma and the 'spindle' pattern where CAFs are observed at the periphery of a tumor island [START_REF] Thode | Significance of myofibroblasts in oral squamous cell carcinoma[END_REF]. CAFs can enhance cancer cell proliferation, neoangiogenesis, and invasion and metastasis through the increased expression of various growth factors, cytokines and extracellular matrix proteins [START_REF] Shimoda | Carcinoma-associated fibroblasts are a rate-limiting determinant for tumour progression[END_REF]. In primary and metastatic HNSCC, they produce invasion-promoting signals that encourage production of TNF-α and IL-1α by tumor cells [START_REF] Koontongkaew | Tumor-stroma interactions influence cytokine expression and matrix metalloproteinase activities in paired primary and metastatic head and neck cancer cells[END_REF][START_REF] Leef | Molecular communication between tumor-associated fibroblasts and head and neck squamous cell carcinoma[END_REF]. These signals include a brain-derived neurotrophic factor, that promotes epithelial-mesenchymal transition (MET) facilitating metastasis in this type of cancers [START_REF] Dudas | Fibroblasts produce brain-derived neurotrophic factor and induce mesenchymal transition of oral tumor cells[END_REF], the hepatocyte growth factor, that binds to the MET receptor on HNSCC cells triggering invasion through the basement membrane [START_REF] Knowles | HGF and c-Met participate in paracrine tumorigenic pathways in head and neck squamous cell cancer[END_REF], and the insulin-like growth factor 2 and CCL7, that promotes HNSCC invasion [START_REF] Jung | Tumor-stromal crosstalk in invasion of oral squamous cell carcinoma: a pivotal role of CCL7[END_REF].

I.2.2 Immune populations in the HNSCC microenvironment

In addition to cancer cells and their surrounding stroma, the tumor microenvironment contains infiltrating cells of the innate and adaptive immune system. Currently, tumor immune-microenvironment is known to be crucial for understanding the tumor development and its response to treatment. Furthermore, tumors occurring in different anatomical sites differ in their immunecontexture and vary in their response to immunotherapy, suggesting that the tissue surrounding the tumor site can have a decisive role in determining its composition (Devaud et al., 2014). Indeed, the density and the composition of the immune microenvironment are heterogeneous between tumor types, and are very diverse from patient to patient [START_REF] Angell | From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer[END_REF].

These tumor infiltrating immune cells and their immune mediators and modulators can shape the tumor growth in two conflicting ways: tumor-promoting inflammation or anti-tumor immunity [START_REF] Grivennikov | Immunity, inflammation, and cancer[END_REF]. During tumor promoting inflammation, infiltrating immune cells supply direct and indirect mitogenic growth mediators, including the epidermal growth factor (EGF), transforming growth factor- (TGF-), tumor necrosis factor-a (TNF-), fibroblast growth factors (FGFs), and various chemokines, and cytokines [START_REF] Hanahan | Accessories to the crime: functions of cells recruited to the tumor microenvironment[END_REF]. In addition, these cells may produce proangiogenic and proinvasive molecules such as VEGF, PDGF, matrix-degrading enzymes, including MMP-9 and other matrix metalloproteinases, cysteine cathepsin proteases, and heparanase [START_REF] Kitamura | Immune cell promotion of metastasis[END_REF][START_REF] Qian | Macrophage diversity enhances tumor progression and metastasis[END_REF]. On the other hand, tumor-infiltrating immune cells support anti-tumor immunity by immunosurveillance and effector mechanism that leads, in the best case, to the elimination of cancer cells. This attribute raises the possibility that recruitment of certain immune cells may be a double-edge sword, by directly promoting angiogenesis and tumor progression while at the same time affording a means to tumor destruction.

Different leukocytes are present within the tumor-microenvironment granulocytes, mast cells, macrophages, and myeloid-derived suppressor cells (MDSC) are often present within the tumor mass, whereas natural killer (NK) cells are principally found in the stroma. CD8 + T cells congregate around the invasive margin and memory T cells are found in adjacent lymph tissue. Immature dendritic cells (DC) are more common within the core of the tumor as opposed to the stroma, whereas mature DCs congregate in tertiary lymphoid structures [START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF][START_REF] Gajewski | Innate and adaptive immune cells in the tumor microenvironment[END_REF].

T Lymphocytes

Immune cells recruited to the tumor include T cells (CD3 + TCR + ); which can be categorized according to their effector functions including CD8 + cytotoxic T lymphocytes (CTLs) and CD4 + T-helper (Th) cells. CTLs are thought to be the major effector immune cells directed against tumor cells, having the ability to recognize and kill malignant cells [START_REF] Boissonnas | In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor[END_REF]. CD4 + T-helper cells can be further subcategorized as Th1, Th2, Th17 and T regulatory (Treg) cells, they can promote or suppress anti-tumor immunity, as determined by their function [START_REF] Kim | CD4 T-cell subsets and tumor immunity: the helpful and the not-so-helpful[END_REF]. Th1 cells, determined by the T-bet transcription factor and the secretion of interferon gamma (IFN-), TNF-, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1); mediates antitumor immunity by enhancing priming and expansion of CD8 + T cells, and by recruiting NK and type I macrophages to tumor sites. Th2 cells, determined by the GATA-3 transcription factor and the secretion of IL-4, IL-5 and IL-13, may contribute to antitumor immunity by recruiting eosinophils and macrophages.

High densities of CTLs and Th1 cells, correlate with better survival in many different tumor types, including melanoma, HNSCCs, breast cancer, colorectal cancer, lung cancer, among others [START_REF] Fridman | The immune contexture in human tumours: impact on clinical outcome[END_REF][START_REF] Gooden | The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis[END_REF]. Indeed, increased amounts of intratumoral CD8 + Ki67 + cells have been linked with improved disease outcome in colorectal tumors [START_REF] Galon | Type, density, and location of immune cells within human colorectal tumors predict clinical outcome[END_REF]. Moreover, the presence of CD3 + CD8 + cells in HNSCCs, correlated with better clinical outcome and response to chemoradiotherapy (Balermpas et al., 2014a). However, recent findings in renal cell cancer, showed that the infiltration of CD8+ T cells was correlated with poor prognosis, modulated by the expression of immune checkpoints and the localization of DCs [START_REF] Giraldo | Orchestration and Prognostic Significance of Immune Checkpoints in the Microenvironment of Primary and Metastatic Renal Cell Cancer[END_REF]. Treg cells, characterized by the expression of the FoxP3 (forkhead box protein P3) transcription factor, are important for immunologic homeostasis. Intratumoral Tregs impede effective immunity against cancer and their presence correlates differently depending of the tumor [START_REF] Deleeuw | The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature[END_REF]. They were associated with poor prognosis in hepatocellular cancer, breast cancer, and melanoma; and good prognosis in colorectal and ovarian cancer. In HNSCC, Tregs positively correlates with loco-regional control, possibly through downregulation of the pro-tumoral inflammatory reaction [START_REF] Badoual | Prognostic value of tumorinfiltrating CD4+ T-cell subpopulations in head and neck cancers[END_REF]. However, the prognostic value of Treg may be different, depending on the tumor site [START_REF] Deleeuw | The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature[END_REF][START_REF] Weller | The role of tumour FoxP3 as prognostic marker in different subtypes of head and neck cancer[END_REF].

The Th17 subset depends of the expression of the STAT3 and RORt transcription factors and the production of IL-17A and IL-17F. They have been recovered from multiple human tumors including ovarian, gastric, prostate, colon, renal and pancreatic cancer where Th17 cells have shown both anti-tumorigenic and pro-tumorigenic functions [START_REF] Zou | T(H)17 cells in tumour immunity and immunotherapy[END_REF]. Furthermore, it have been shown that HNSCC and tumor draining lymph nodes are infiltrated with Th17 T cells in response to cytokines present in the HNSCC tumor microenvironment. Moreover, Th17 cells inhibit the proliferation and compromise the angiogenesis of HNSCC [START_REF] Kesselring | Human Th17 cells can be induced through head and neck cancer and have a functional impact on HNSCC development[END_REF].

Dendritic Cells

Dendritic cells are antigen-presenting cells responsible for the uptake, processing and cross-presentation of tumor antigens (TAs) to T cells; which are, in turn, activated to proliferate and secrete cytokines, forming a key part of the adaptive immune response [START_REF] Benencia | Dendritic cells the tumor microenvironment and the challenges for an effective antitumor vaccination[END_REF]. Multiple subsets of DCs have been described in human and mice.

They can be classified into two main categories: plasmacytoid DCs (pDCs) and conventional DCs (cDCs), cDCs can be further divided into lymphoid resident DCs and migratory DCs, which are present in peripheral tissues and non-lymphoid organs [START_REF] Segura | Inflammatory dendritic cells in mice and humans[END_REF]. Elevated intratumoral DCs have often, but not always, been associated with improved clinical outcome in HNSCC most likely due to the fact that DCs subsets cannot be appropriately discriminated [START_REF] Senovilla | Trial watch: Prognostic and predictive value of the immune infiltrate in cancer[END_REF]. However, the infiltration of Langerhans cells (LCs), a type of DC present in the epithelium of the mucosa, is associated with longer disease-free survival and decreased recurrence in HNSCC patients [START_REF] Yilmaz | MiR-221 as a pre-and postoperative plasma biomarker for larynx cancer patients[END_REF].

Additionally, a low number of intratumoral S-100 + DCs predicts poor survival in patients with OSCCs [START_REF] Reichert | The number of intratumoral dendritic cells and zeta-chain expression in T cells as prognostic and survival biomarkers in patients with oral carcinoma[END_REF].

Natural Killers

NK cells are lymphocytes that mediate innate immunity and recognize and kill virally infected or malignant cells. They are capable of eliminating tumors with reduced or absent major histocompatibility complex (MHC) class I expression that evade CD8 + T cell-mediated control. In a variety of solid tumor such as gastric, renal, HNSCC and colorectal cancers the presence of high numbers of tumor-infiltrating NK cells correlates with improved prognosis of patients [START_REF] Moretta | Human NK cells: from surface receptors to the therapy of leukemias and solid tumors[END_REF]. Interestingly, the presence of NK cells were not correlated with the clinical outcome in lung cancer, suggesting that tumor microenvironment renders NK less tumoricidal by reducing NK receptor expression and IFN- secretion [START_REF] Platonova | Profound coordinated alterations of intratumoral NK cell phenotype and function in lung carcinoma[END_REF]. In HNSCC, it has been observed that NK cell numbers are diminished in peripheral blood of patients and did not correlate with the tumor site [START_REF] Accomando | Decreased NK cells in patients with head and neck cancer determined in archival DNA[END_REF][START_REF] Wulff | Decreased levels of circulating regulatory NK cells in patients with head and neck cancer throughout all tumor stages[END_REF].

However, aggressive infiltration in the peritumoral stroma by CD57 + inflammatory cells (30-60% of mature NKs), may contribute to an ineffective loco-regional anti-tumoral response [START_REF] Fraga | Infiltrating CD57+ inflammatory cells in head and neck squamous cell carcinoma: clinicopathological analysis and prognostic significance[END_REF].

Macrophages

Tumor-associated macrophages (TAMs) could exert phagocytic properties and can also present antigens to stimulate the adaptive pathway. In a classical and functional description, macrophages referred as 'M1' are activated with lipopolysaccharide (LPS) or IFN- to express pro-inflammatory cytokines (IL-12) and priming anti-tumor immune responses, whereas macrophages referred as 'M2' are IL-4-activated to express regulatory cytokines (IL-10) leading to anti-inflammatory responses and promoting tumor angiogenesis [START_REF] Ostuni | Macrophages and cancer: from mechanisms to therapeutic implications[END_REF]. In addition, it was showed that different tumor microenvironments could contain functionally distinct subsets of monocytes-derived TAMs that are poor antigen presenters and could suppress T-cell activation by using different mechanisms [START_REF] Movahedi | Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes[END_REF]. Increased numbers of TAMs correlates with poor prognosis in many cancers, including breast, bladder, prostate and colorectal cancer; but worse overall survival in patients with HNSCC, gastric, and urogenital cancer [START_REF] Biswas | Tumor-associated macrophages: functional diversity, clinical significance, and open questions[END_REF]. Indeed, in HNSCC, TAMs were associated with angiogenesis and high histopathological grade malignancy (El-Rouby, 2010), and the expression of the macrophage inflammatory protein-3a (MIP-3a) that was shown to promote oral cancer cell migration and invasion, and was correlated with poorer prognosis for patient survival [START_REF] Chang | Overexpression of macrophage inflammatory protein-3alpha in oral cavity squamous cell carcinoma is associated with nodal metastasis[END_REF].

Myeloid-derived suppressor cells

MDSCs are immature myeloid cells that are precursors of DC, macrophages, and granulocytes. Their accumulation has been documented in most patients and mice with cancer, where they were induced by various tumor-derived factors produced in the tumor microenvironment [START_REF] Ostrand-Rosenberg | Myeloid-derived suppressor cells: linking inflammation and cancer[END_REF]. MDSCs can suppress the effector functions of NK, and T cells by the production of reactive oxygen species, reactive nitrogen species and cytokines as well as interactions with other suppressor cells like Tregs (Ostrand- [START_REF] Ostrand-Rosenberg | Myeloid-derived suppressor cells: more mechanisms for inhibiting antitumor immunity[END_REF]. Low levels of circulating MDSC have been reported as a good prognosis factor in patients with B-cell lymphoma, lung cancer, melanoma, gastrointestinal neoplasms and bladder carcinoma [START_REF] Senovilla | Trial watch: Prognostic and predictive value of the immune infiltrate in cancer[END_REF]. In HNSCC, MDSCs can be defined as CD33 + IL-4 + CD14 + HLADR int/neg CD11b + cells. They are able to suppress T-cell proliferation and their intratumoral accumulation correlates with tumor recurrence. Moreover, the daily treatment with taladafil modifies the tumor microenvironment and reduces the number of MDSCs, increasing anti-tumor immunity in HNSCC patients [START_REF] Weed | Tadalafil reduces myeloid-derived suppressor cells and regulatory T cells and promotes tumor immunity in patients with head and neck squamous cell carcinoma[END_REF]. Also, it has been shown that HNSCC intratumoral CD34 + cells can suppress immune functions by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF) [START_REF] Pak | Mechanisms of immune suppression in patients with head and neck cancer: presence of CD34(+) cells which suppress immune functions within cancers that secrete granulocytemacrophage colony-stimulating factor[END_REF].

B Lymphocytes

B cells are lymphocytes with antigen-presenting properties and when activated differentiates into an antibody-secreting effector cell. They have also an immunoregulatory role in tumor microenvironment by the production of cytokines and chemokines to promote T-cell responses [START_REF] Nelson | CD20+ B cells: the other tumor-infiltrating lymphocytes[END_REF]. Infiltrating B cells are associated with good prognosis in other squamous cell cancers such as non-small cell lung cancer [START_REF] Germain | Tertiary Lymphoid Structure-Associated B Cells are Key Players in Anti-Tumor Immunity[END_REF]. In HNSCC, increased numbers of peritumoral B cells in lymph node metastasis were associated with favorable outcome [START_REF] Pretscher | Distribution of immune cells in head and neck cancer: CD8+ T-cells and CD20+ B-cells in metastatic lymph nodes are associated with favourable outcome in patients with oro-and hypopharyngeal carcinoma[END_REF].

Other immune cells

Polymorphonuclear (PMN) leukocytes like granulocytes, eosinophils and mast cells have also been found in tumor microenvironment. High levels of granulocytes have been associated with bad prognosis in tumors such as hepatocellular carcinoma [START_REF] Kuang | Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma[END_REF] and melanoma [START_REF] Jensen | Intratumoral neutrophils and plasmacytoid dendritic cells indicate poor prognosis and are associated with pSTAT3 expression in AJCC stage I/II melanoma[END_REF]. Analysis of HNSCC tumors, exhibited considerable infiltration by polymorphonuclear granulocytes, and strong infiltration was associated with poorer survival in advanced disease. Furthermore, the serum concentration of cytokines and chemokines that modulates PMN functions, such as IL-8, MIP-1 and RANTES, were significantly higher [START_REF] Trellakis | Polymorphonuclear granulocytes in human head and neck cancer: enhanced inflammatory activity, modulation by cancer cells and expansion in advanced disease[END_REF]. Tumor infiltration by eosinophils has been reported to be a positive prognostic indicator for gastric and lung cancer, still it gives no clear prognostic information in head and neck cancer [START_REF] Senovilla | Trial watch: Prognostic and predictive value of the immune infiltrate in cancer[END_REF]. Otherwise, robust tumor infiltration of mast cells has been linked to good prognosis in various cancers including HNSCC [START_REF] Khazaie | The significant role of mast cells in cancer[END_REF]. However, as HNSCC progresses, there is an increase in mast cell numbers, which is correlated with new vascular tube formation suggesting a role in angiogenesis [START_REF] Iamaroon | Increase of mast cells and tumor angiogenesis in oral squamous cell carcinoma[END_REF].

I.2.3 Immunosurveillance and immunoescape mechanisms in HNSCC

The interactions between the host and the tumor have been referred to as "immunosurveillance", where the immune system is able to spot, recognize and eliminate tumor cells. A new concept introduced in 2002, refers this interaction as "immunoediting", where the host immune system recognize and destroy sensitive tumor cells, but also can edits for survival of tumors that become resistant. This "immunoediting" process is composed of three phases: elimination, equilibrium and escape [START_REF] Schreiber | Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion[END_REF].

Immunosurveillance occurs during the elimination phase, in which the innate and adaptive system works together to detect a developing tumor and destroy it. In HNSCCs, tumors invoke a host immune response to the over expression of tumor-associated antigens (TAAs) and the secretion of cytokines and chemokines by the tumor, causing a leukocytic infiltrate into the tumor microenvironment [START_REF] Junker | Tumor associated antigen specific T-cell populations identified in ex vivo expanded TIL cultures[END_REF]. Among TAAs, p53 is the most expressed mutated gene in HNSCC (about 62%) [START_REF] Stransky | The mutational landscape of head and neck squamous cell carcinoma[END_REF]. Also we can find TAAs from the melanoma-associated antigens (MAGE) group like MAGE-A3 (51%) and MAGE-A4 (60%), and other antigens from the cancer-testis group like NY-ESO-1 [START_REF] Cuffel | Pattern and clinical significance of cancertestis gene expression in head and neck squamous cell carcinoma[END_REF]. Importantly, in HPV-positive HNSCC patients, CD8 + T cells against HPV oncogenic proteins like E7 can be found [START_REF] Heusinkveld | Systemic and local human papillomavirus 16-specific T-cell immunity in patients with head and neck cancer[END_REF].

DC activation due to TAAs and damage-association molecular patterns (DAMPs) is believed to be a crucial step in initiating immune responses against tumors in HNSCCs ( [START_REF] Kacani | Maturation of dendritic cells in the presence of living, apoptotic and necrotic tumour cells derived from squamous cell carcinoma of head and neck[END_REF]. An effective anti-tumor response would involve primed DCs migrating to regional lymph nodes, where they could present processed tumor antigens on HLA-I and II molecules to CD8 + and CD4 + T cells, and Th1 cells activation by cytokine secretion (IL-12 and TNF-) leading to secretion of IL-2 and IFN- to generate CTLs with specific cytotoxic activity against tumor cells bearing TAAs [START_REF] Allen | The clinical implications of antitumor immunity in head and neck cancer[END_REF].

However, tumor cell variants may show resistance to persistent CTLs and NKs attack and gradually survive and proliferate. Thus, entering the equilibrium phase in which the adaptive immune system sculpts tumor immunogenicity and prevents tumor cell growth [START_REF] Matsushita | Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting[END_REF]. Finally, in the escape phase, edited cancer cells efficiently overcome the immune recognition and destruction, so they expand and become a tumor clinically evident [START_REF] Schreiber | Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion[END_REF]. HNSCCs cells escape can occur through many different mechanisms at different levels, as described below.

Evasion of immune system detection

At the HNSCC tumor cell level, alterations leading to reduced immune recognition, such as loss of antigens, can be achieved through emergence during immunoediting of tumor cells that lack expression of immunodominant epitopes [START_REF] Badoual | Better understanding tumor-host interaction in head and neck cancer to improve the design and development of immunotherapeutic strategies[END_REF], loss or decreasing the expression of surface MHC class I molecules [START_REF] Ferris | Immune escape associated with functional defects in antigen-processing machinery in head and neck cancer[END_REF] or through loss of function of their antigen processing machinery [START_REF] Meissner | Defects in the human leukocyte antigen class I antigen processing machinery in head and neck squamous cell carcinoma: association with clinical outcome[END_REF]. The end result is the generation of poorly immunogenic tumor cell variants that become "invisible" to the immune system and thus acquire the capacity to grow progressively. However, even if 15% of primary and 40% of metastatic HNSCCs presents MHC class I molecules loss, this alteration is not correlated with clinical outcome [START_REF] Hasmim | Expression of EPHRIN-A1, SCINDERIN and MHC class I molecules in head and neck cancers and relationship with the prognostic value of intratumoral CD8+ T cells[END_REF]. Furthermore, patients TAA-specific CD8 + T cells that are expanded in vitro and exposed to autologous tumor cells can lyse the tumor cells when incubated with stimulatory factors like IFN- (Lopez-Albaitero et al., 2006).

Resistance to immune attack

HNSCC tumors may increase their resistance to cytotoxic effects of immune cells, through induction of anti-apoptotic mechanisms involving receptors and transcription factors. One receptor is toll-like receptor 4 (TLR-4), which normally binds to LPS; their overexpression is correlated with tumor grade and short survival [START_REF] Ren | Protein overexpression of CIRP and TLR4 in oral squamous cell carcinoma: an immunohistochemical and clinical correlation analysis[END_REF]. LPS binding to TLR-4 on HNSCC tumor cells enhanced proliferation and activated the nuclear factor-kappa B (NF-B) and PI3K/AKT anti-apoptotic ways [START_REF] Szczepanski | Triggering of Tolllike receptor 4 expressed on human head and neck squamous cell carcinoma promotes tumor development and protects the tumor from immune attack[END_REF].

Moreover, the expression in CD8 + T cells of another receptor implicated in the activation of PI3K/AKT anti-apoptotic way, the chemokine receptor 7 (CCR7), was associated with disease recurrence in HNSCCs patients [START_REF] Czystowska | The immune signature of CD8(+)CCR7(+) T cells in the peripheral circulation associates with disease recurrence in patients with HNSCC[END_REF].

Secretion or expression of inhibitory factors

Head and neck cancer cells can promote the development of an immunosuppressive microenvironment by producing regulatory cytokines and expressing negative costimulatory molecules. Among these cytokines IL-10, TGF- and prostaglandin E2 were described to interfere in immune reactivity to HNSCCs. Additionally, secretion by these cancer cells of GM-CSF and VEGF could obstruct the maturation of fully functional DCs [START_REF] Pries | Cytokines in head and neck cancer[END_REF]. A number of apoptosis-promoting factors have been identified in HNSCC including Galectin-1, Fas-L, TNF-related apoptosis inducing ligand (TRAIL), and programmed death-ligand 1 (PD-L1). Galectin-1 inhibits T-cell effector functioning by promoting T-cell apoptosis, blocking T-cell activation and inhibiting the secretion of proinflammatory cytokines [START_REF] Saussez | Galectins as modulators of tumor progression in head and neck squamous cell carcinomas[END_REF]. Tumor-derived cells and plasma microvesicles from HNSCCs patients express Fas-L and trigger apoptotic death of activated T cells expressing Fas [START_REF] Bergmann | Tumor-derived microvesicles in sera of patients with head and neck cancer and their role in tumor progression[END_REF]. Furthermore, OSCC may be capable to induce apoptosis in tumor-infiltrating lymphocytes (TILs) using the alternative TRAIL and TNF- pathways [START_REF] Kassouf | Oral cancer cell lines can use multiple ligands, including Fas-L, TRAIL and TNF-alpha, to induce apoptosis in Jurkat T cells: possible mechanisms for immune escape by head and neck cancers[END_REF]. In HPV-positive HNSCCs, PD-L1 is commonly expressed and it was showed that they could promote anergy, exhaustion or apoptosis in programmed cell death-1 (PD-1) expressing T cells [START_REF] Lyford-Pike | Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma[END_REF].

Recruitment of immune inhibitory cells

HNSCCs, also can promote immunosuppressive microenvironment by recruiting regulatory immune cells. Treg cells, MDSCs and TAMs are main leukocyte populations that play key roles in inhibiting host-protective anti-tumor responses [START_REF] Schreiber | Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion[END_REF].

Treg cells are CD4 + T cells that constitutively express CD25 and the transcription factor Foxp3. HNSCC intratumoral Tregs inhibit T-cell anti-tumor activity via a number of mechanisms including the production of immunosuppressive cytokines IL-10 and TGF- [START_REF] Strauss | A unique subset of CD4+CD25highFoxp3+ T cells secreting interleukin-10 and transforming growth factor-beta1 mediates suppression in the tumor microenvironment[END_REF], the expression of negative costimulatory molecules like cytotoxic Tlymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin mucin-3 (TIM-3) and PD-1 [START_REF] Jie | Intratumoral regulatory T cells upregulate immunosuppressive molecules in head and neck cancer patients[END_REF], and by consuming IL-2 a cytokine that is critical for maintaining CTL function. Importantly, CD4 + CD25 high FoxP3 + are increased in the peripheral blood and tumor tissue (Strauss et al., 2007a). As described above, Tregs positively correlates with locoregional control in HNSCC patients, possibly through downregulation of the pro-tumoral inflammatory reaction [START_REF] Badoual | Prognostic value of tumorinfiltrating CD4+ T-cell subpopulations in head and neck cancers[END_REF][START_REF] Weed | FOXP3 subcellular localization predicts recurrence in oral squamous cell carcinoma[END_REF]. However, while overall FoxP3 expression in tumor infiltrating CD4 + T cells does not correlate with tumor recurrence, nuclear FoxP3 localization is associated with recurrence of oral HNSCC within 3 years [START_REF] Weed | FOXP3 subcellular localization predicts recurrence in oral squamous cell carcinoma[END_REF]. Interestingly, PD-1 + T cells where positively correlated with a favorable clinical outcome in HPV-associated HNSCC (Badoual et al., 2013).

MDSCs and TAMs are intratumoral myeloid cells that can be converted into immunosuppressive cells by the tumor microenvironment [START_REF] Gabrilovich | Coordinated regulation of myeloid cells by tumours[END_REF]. In HNSCCs, MDSCs inhibit lymphocyte function by inducing Treg cells, producing TGF-, removing L-arginine required for T-cell function, or nitrating T-cell receptors [START_REF] Chikamatsu | Immunosuppressive activity of CD14+ HLA-DR-cells in squamous cell carcinoma of the head and neck[END_REF][START_REF] Pak | Mechanisms of immune suppression in patients with head and neck cancer: presence of CD34(+) cells which suppress immune functions within cancers that secrete granulocytemacrophage colony-stimulating factor[END_REF]. CD163 + TAMs are correlated with an unfavorable clinical outcome in HNSCC patients after definitive chemo-radiotherapy; and CD11b + TAMs correlates with early local recurrences (Balermpas et al., 2014b).

I.2.4 Immune-microenvironment according to HNSCC location

Head and neck cancers, in common with many other malignancies, are inflammatory foci by nature. It was previously shown that the presence of tumor infiltrating lymphocytes subsets in mixed populations of patients with HNSCCs of various sites were associated with a more favorable prognosis [START_REF] Badoual | Prognostic value of tumorinfiltrating CD4+ T-cell subpopulations in head and neck cancers[END_REF]Balermpas et al., 2014a), and infiltrates of TAMs were associated with nodal metastases [START_REF] Marcus | Prognostic factors in oral cavity and oropharyngeal squamous cell carcinoma[END_REF]. However, the degree of leukocyte infiltration appears to be specific depending on the site (OSCC or OPSCC) and is likely to be influenced by the differing microenvironments and the stage of the tumor (Green et al., 2013;Wansom et al., 2012;[START_REF] Wolf | Tumor infiltrating lymphocytes (TIL) and prognosis in oral cavity squamous carcinoma: a preliminary study[END_REF]. Better understanding of the immunologic characteristics of the microenvironment including numbers, location and function of tumor infiltrating lymphocytes and macrophages is necessary in order to explore and test strategies that might be beneficial to HNSCCs patients [START_REF] Badoual | Better understanding tumor-host interaction in head and neck cancer to improve the design and development of immunotherapeutic strategies[END_REF][START_REF] Wallis | Clinical relevance of immune parameters in the tumor microenvironment of head and neck cancers[END_REF].

Oral cavity

In oral cavity squamous cell carcinoma, the density of lymphocytic infiltrate at the tumor/host interface have been linked with good prognosis, while a lower overall density was associated with worse overall survival and local recurrence [START_REF] Brandwein-Gensler | Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival[END_REF].

Immunohistochemical analysis of resected specimens of OSCC showed that CD8 + T cells infiltrated the stroma and cancer nest, whereas most Tregs only occupied the stroma. Also, greater CD8 + cell counts and CD8 + /CCR4 + Treg ratio were associated with better survival [START_REF] Watanabe | Tumor-infiltrating lymphocytes, particularly the balance between CD8(+) T cells and CCR4(+) regulatory T cells, affect the survival of patients with oral squamous cell carcinoma. Oral surgery, oral medicine, oral pathology, oral radiology[END_REF]. This finding suggests that Tregs may be detrimental within the tumor microenvironment in OSCC. However, in a further study of Tregs investigating the relationship between CD4, CD25, CTLA-4, and FoxP3 staining and survival, they found that high CD4 + cell counts showed a correlation with decreased survival, whereas high CD25 + cell counts were associated with better overall survival in OSCC patients [START_REF] Moreira | T regulatory cell markers in oral squamous cell carcinoma: Relationship with survival and tumor aggressiveness[END_REF].

In addition to lymphocytic populations in the microenvironment, some other factors have demonstrated prognostic significance in OSCC. The expression of Granzyme B, a protein secreted by CTLs and NK cells, which induces apoptosis in abnormally proliferating cells, was associated with longer survival in OSCC [START_REF] Costa | The clinicopathological significance of the expression of Granzyme B in oral squamous cell carcinoma[END_REF]. There are cytokines and cell receptors, which also seem to be of prognostic significance, including IL-6 and TGF-1, both linked with worse overall survival [START_REF] Chen | Significance of the TGF-beta1/IL-6 axis in oral cancer[END_REF], and IL-10 linked with HPV-positive OSCC, and correlated with bad prognosis [START_REF] Chuang | Differential impact of IL-10 expression on survival and relapse between HPV16-positive and -negative oral squamous cell carcinomas[END_REF]. The ability of OSCC tumor cells to express PD-L1, a surface glycoprotein that causes T-lymphocyte dysfunction, has been linked to decreased numbers of TIL in the peritumoral region, but did not affect patients survival [START_REF] Cho | Relationship between the expressions of PD-L1 and tumor-infiltrating lymphocytes in oral squamous cell carcinoma[END_REF]. Besides, a low number of DCs was predictive for poor survival in OSCCs [START_REF] Reichert | The number of intratumoral dendritic cells and zeta-chain expression in T cells as prognostic and survival biomarkers in patients with oral carcinoma[END_REF].

Oropharynx

In OPSCCs, improved outcomes are associated with increased TILs independent of HPV status, suggesting that the local immune response may be more related with other factors such as tumor size or Karnofsky performance status (score from 100 = no evidence of disease to 0 = dead) (Wansom et al., 2012). High CD8 + T-cell infiltration and CD8 + T cells/Treg ratio were significantly positively correlated to a good clinical outcome in both HPV-positive and HPV-negative OPSCC. Moreover, HPV-positive status was associated with higher numbers of infiltrating CD8 + T cells and FoxP3 + T cells [START_REF] Nasman | Tumor infiltrating CD8+ and Foxp3+ lymphocytes correlate to clinical outcome and human papillomavirus (HPV) status in tonsillar cancer[END_REF]. CD20 + infiltration was associated with better survival rate in low risk OPSCC, but bad prognosis in high rate OPSCC patients, revealing that the impact of TILs on prognosis in these patients may be affected by type of treatment and the stage of disease [START_REF] Distel | Tumour infiltrating lymphocytes in squamous cell carcinoma of the oro-and hypopharynx: prognostic impact may depend on type of treatment and stage of disease[END_REF]. Additionally, increased numbers of intraepithelial CD8 + TILs in metastatic tumors and peritumoral B cells in lymph node metastasis were associated with favorable outcome [START_REF] Pretscher | Distribution of immune cells in head and neck cancer: CD8+ T-cells and CD20+ B-cells in metastatic lymph nodes are associated with favourable outcome in patients with oro-and hypopharyngeal carcinoma[END_REF].

Other sites

Higher density intratumoral and peritumoral TAMs predicted poorer survival in laryngeal SCC [START_REF] Lin | Clinical significance of tumorassociated macrophage infiltration in supraglottic laryngeal carcinoma[END_REF]. However, numbers of CD43 + and CD45 + RO T cells and DCs have been linked with improved survival in laryngeal SCC [START_REF] Esteban | Clinical significance of langerhans cells in squamous cell carcinoma of the larynx[END_REF][START_REF] Gabriel | Immunohistochemical analysis of lymphocytic infiltration in the tumor microenvironment in patients operated on for laryngeal cancer[END_REF]. In nasopharyngeal SCC, higher Treg/CD8 + ratios were seen in keratinizing type compared with non-keratinizing and undifferentiated carcinomas [START_REF] Yip | Increase in tumourinfiltrating lymphocytes with regulatory T cell immunophenotypes and reduced zeta-chain expression in nasopharyngeal carcinoma patients[END_REF]. In addition, CD8 + T cells expressing PD-1 were associated with poorer survival and locoregional control [START_REF] Hsu | Increase of programmed death-1-expressing intratumoral CD8 T cells predicts a poor prognosis for nasopharyngeal carcinoma[END_REF].

I.3 Pre-clinical models for HNSCC

To better understand the tumor and their microenvironment, and in order to develop and evaluate novel anticancer agents, appropriate animal pre-clinical models that can accurately recapitulate the disease process are required. The mice (Mus musculus) is the most frequently used animal species in laboratories because they are small in size, relatively inexpensive to maintain, reproduce rapidly, and can be genetically maintained. In addition, there are many available human and murine immortalized cell lines that have been tested for tumorigenicity in mice [START_REF] Schuh | Trials, tribulations, and trends in tumor modeling in mice[END_REF]. Several strains are used for the development of tumor preclinical models including immunocompetent strains such as BALB/c, C57Bl/6 and C3H, and immunodeficient strains like nude athymic (T-cell deficient), SCID or severe combined immunodeficiency (T and B-cell deficient), NOD-SCID or non-obese diabetic SCID (T and B-cell deficient, low NK), and NSG or NOD-SCID-IL2rg-/-(T, B and NK deficient) mice [START_REF] Zhou | Humanized NOD-SCID IL2rg-/-mice as a preclinical model for cancer research and its potential use for individualized cancer therapies[END_REF].

However, other animal species are also used in oncology research including the laboratory rat (Rattus norvegicus) which immunocompetent strains (Sprague-Dawley and Wistar) and some immunodeficient nude strains represent the most used among them [START_REF] Festing | Design and statistical methods in studies using animal models of development[END_REF], the syrian hamster (Mesocricetus auratus) and the Chinese hamster (Cricetulus griseus), used as preclinical models for pancreatic cancer and oral carcinoma [START_REF] Vairaktaris | The hamster model of sequential oral oncogenesis[END_REF], the rabbit (Oryctolagus cuniculus) used for more invasive strategies requiring bigger organisms [START_REF] Kreuter | Development of a rabbit pleural cancer model by using VX2 tumors[END_REF], the zebrafish (Danio rerio) an animal model used for embryogenesis and oncology research that allows a direct follow-up of tumor growth because of their ability to present transparent skin during their embryo stage [START_REF] Feitsma | Zebrafish as a cancer model[END_REF], and dogs and other bigger animals [START_REF] Khanna | The dog as a cancer model[END_REF].

Preclinical cancer models can be organized in different categories depending to the modality of tumor implantation and/or the nature and origin of tumor cells [START_REF] Mcconville | Preclinical Models of Tumor Growth and Response[END_REF]. Tumor models can be obtained by transplanting into the animal solid tumors or tumor cell lines in the original tumor site (orthotopic models) or a site that does not correspond to the original one (ectopic models). The nature of this transplant varies depending if it comes from the same animal species (syngeneic models) or another species, like human (xenogeneic models). In addition, tumors can be induced in situ by using different types of carcinogens (spontaneous and autochthonous models) or by the introduction of somatic mutations that are implicated in neoplastic transformation (transgenic models).

I.3.1 Ectopic pre-clinical models for HNSCC

Ectopic models are established when tumor grafts are performed on a site that does not match that of origin. Subcutaneous ectopic tumor grafts were used since the beginning of the 20th century as the standard for carcinogenesis research [START_REF] Levin | Tumor Inoculation into Organs and the Analogy between Human Cancer and the Tumors of White Mice and White Rats[END_REF]. HNSCC tumor models were developed in ectopic sites using human-derived cell lines injected in immunodeficient mice [START_REF] Langdon | Preclinical phase II studies in human tumor xenografts: a European multicenter follow-up study[END_REF][START_REF] Shimosato | Transplantation of human tumors in nude mice[END_REF], but also using syngeneic oral SCC murine-derived cell lines such as NR-S1 [START_REF] Tsushima | Predominant expression of B7-H1 and its immunoregulatory roles in oral squamous cell carcinoma[END_REF] and SCC-VII [START_REF] Strome | B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma[END_REF]. Interestingly, HPV-positive HNSCC and HPV-negative ectopic preclinical models where established using E6/E7 transduced primary mouse tonsil epithelial cells [START_REF] Williams | Preclinical models of HPV+ and HPV-HNSCC in mice: an immune clearance of HPV+ HNSCC[END_REF]. Another approach involves the direct implantation, serial implantation and propagation of freshly excised primary human tumors into immunodeficient mice to create a HNSCC primary tumorgraft that preserves genotypic and phenotypic features of the original tumor. Using nude and NSG mice this approach have shown to maintain molecular and histologic characteristics (Peng et al., 2013a) and to be useful for preclinical testing of therapeutic response of HNSCC tumorgrafts to radiation and chemotherapy [START_REF] Kimple | Development and characterization of HPV-positive and HPV-negative head and neck squamous cell carcinoma tumorgrafts[END_REF], and methylation alterations in this type of cancers [START_REF] Hennessey | Promoter methylation in head and neck squamous cell carcinoma cell lines is significantly different than methylation in primary tumors and xenografts[END_REF]. However, the main disadvantage of tumorgrafts is the unfeasibility for studying immune responses.

Ectopic tumor models are advantageous because of the ease of tumor establishment, measurement and reproducibility. However, ectopic site does not reproduce the primary tumor microenvironment as well as an orthotopic site does [START_REF] Killion | Orthotopic models are necessary to predict therapy of transplantable tumors in mice[END_REF]. Other disadvantages are the abundance of false-positive responses with drugs and the absence of metastasis, which can explain the differences in effectiveness of certain drugs in preclinical models and clinical trials [START_REF] Ruggeri | Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery[END_REF]. [START_REF] Killion | Orthotopic models are necessary to predict therapy of transplantable tumors in mice[END_REF]

Ectopic models Orthotopic models

Advantages

I.3.2 Orthotopic pre-clinical models for HNSCC

Orthotopic models are established when tumorgrafts are performed on a site that matches that of origin. They were described since 1970s as models of various tumor diseases [START_REF] Bibby | Making the most of rodent tumour systems in cancer drug discovery[END_REF][START_REF] Tan | Murine colon adenocarcinoma: syngeneic orthotopic transplantation and subsequent hepatic metastases[END_REF]. Although ectopic tumor models are often used in preclinical studies, these models lack the specific interactions that exist between the tumor cells and their native environment; the establishment of tumors at orthotopic sites may restore these distinct patterns of interactions and more closely simulates the natural tumor microenvironment with intact pathological, immunological responses and much higher metastatic rates, which recapitulates the human clinical course of tumor disease [START_REF] Ruggeri | Animal models of disease: pre-clinical animal models of cancer and their applications and utility in drug discovery[END_REF].

In HNSCCs, orthotopic models have been reported since late 1980s by injecting human cancer cells into the tongue or into the mouth's floor of nude mice [START_REF] Dinesman | Development of a new in vivo model for head and neck cancer[END_REF]) and more recently by injecting into immunocompetent mice, syngeneic murine cell lines like SCC-VII cells (Cui et al., 2005), HPV-16 E7 expressing TC-1 cells (Sandoval et al., 2013;[START_REF] Wu | Innovative DNA vaccine for human papillomavirus (HPV)-associated head and neck cancer[END_REF], and HPV-16 E7 expressing AT-84 oral SCC cells [START_REF] Paolini | Immunotherapy in new pre-clinical models of HPV-associated oral cancers[END_REF]). Mouth's floor or tongue implantation allows to reproduce some clinical signs like dysphagia and weight loss, however mice have to be euthanized in the first two weeks after tumor cell implantation because of tumor growth preventing correct feeding by the animals, resulting in unethical suffering and death (Bozec et al., 2009;[START_REF] Myers | An orthotopic nude mouse model of oral tongue squamous cell carcinoma[END_REF]. Orthotopic models are very useful to evaluate anti-tumor therapeutic approaches; in several studies these models can predict the clinical activity of certain molecules [START_REF] Bibby | Orthotopic models of cancer for preclinical drug evaluation: advantages and disadvantages[END_REF][START_REF] Bozec | Combined effects of bevacizumab with erlotinib and irradiation: a preclinical study on a head and neck cancer orthotopic model[END_REF]. In addition, there are many studies describing metastasis of orthotopic tumors to clinically relevant sites [START_REF] Cabanillas | Orthotopic murine model of head and neck squamous cell carcinoma[END_REF][START_REF] Kubota | Metastatic models of human cancer xenografted in the nude mouse: the importance of orthotopic transplantation[END_REF]. Although the improvements over ectopic tumors, orthotopic models have some limitations, these models are technically challenging to establish and to reproduce, and it can be difficult to evaluate tumor growth depending on the site [START_REF] Sano | Xenograft models of head and neck cancers[END_REF].

Few studies have been published in orthotopic sites other than the mouth or tongue.

One study reported an orthotopic model developed by implanting tumor fragments in the inner aspect of the mouse cheek. Importantly, this tumor model allowed a survival time of 30 days [START_REF] Atallah | Near-infrared fluorescence imaging-guided surgery improves recurrence-free survival rate in novel orthotopic animal model of head and neck squamous cell carcinoma[END_REF]. Additionally, in a model of sinonasal malignancy where tumor cells were injected in the right maxillary sinus or soft palate in mice, it was shown that this model recapitulates the malignant behavior of the tumor types seen in these patients [START_REF] Gelbard | An orthotopic murine model of sinonasal malignancy[END_REF]. Finally, two other studies established an orthotopic model of salivary cancer in the parotid glands suitable for anti-tumor strategies [START_REF] Choi | Vandetanib inhibits growth of adenoid cystic carcinoma in an orthotopic nude mouse model[END_REF][START_REF] Younes | Concomitant inhibition of epidermal growth factor and vascular endothelial growth factor receptor tyrosine kinases reduces growth and metastasis of human salivary adenoid cystic carcinoma in an orthotopic nude mouse model[END_REF].

I.3.4 In situ pre-clinical models for HNSCC

In situ tumor models including transgenic and genetically engineered mouse model (GEMM), and carcinogen induced tumor models, have been developed with increased complexity but also clinical relevance in certain cases.

Transgenic and GEMM models offer the possibility to study relationships between molecular changes and cancer development, as well as prevention and early interventions therapies. The most common way to generate this type of mouse models of cancers are to activate oncogenes or inactivate tumor-suppressor genes (or both) in vivo through the use of different techniques of genetic manipulations such as transgenic approaches created by microinjection of foreign DNA into the pronuclei of fertilized zygotes, gene-targeting approaches involving multiple steps that result in either deletions of the coding sequence of a gene (knockouts) or the introduction of exogenous sequences into the specific locus (knockins), and conditional and inducible systems that allow the induction of somatic mutations in a tissue-specific and time-controlled manner. Loss-of-function studies typically employ knockout or conditional knockout alleles, whereas gain-of-function studies use transgenic, conditional transgenic, and knockin approaches [START_REF] Cheon | Mouse models of cancer[END_REF]. The most common conditional system in the mouse is the Cre-loxP system, where Cre recombinase mediates site-directed DNA recombination at loxP sequences [START_REF] Lu | Mouse models for human head and neck squamous cell carcinomas[END_REF].

Using this approach two models have been described in HNSCCs that utilizes the keratin 5 or keratin 14 promoters to overexpress the oncogene K-ras G12D in oral epithelium of mice [START_REF] Caulin | Inducible activation of oncogenic K-ras results in tumor formation in the oral cavity[END_REF][START_REF] Vitale-Cross | Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis[END_REF]. Indeed, mice expressing CrePR1 (a fusion of Cre and progesterone receptor) under the control of the K5 or K14 promoter (that are expressed in the basal cells of stratified) were generated; additionally, the activation by Cre recombinase induces the excision of the Lox-Stop-Lox-K-ras G12D stop cassette and expression of K-ras G12D .

However, this approach targets a limited number of genes that is not reflective of the complex heterogeneity of human tumor cells and the development is costly and time consuming.

Other in situ approaches such as carcinogen-induced models are being used for HNSCC to recapitulate the time-dependent and multi-stage progression of tumor pathogenesis in response to environmental carcinogens and tumor-promoting agents. Nonetheless, the prolonged time frames and care in conducting these models is a factor of consideration. The most used chemicals for inducing oral cancer are 7,12-dimethylbenzaanthracene (DMBA) and 4-nitroquinoline-1 oxide (4-NQO), given mostly in the drinking water, leading to tumors on the tongue or mouth [START_REF] Kim | Animal models of cancer in the head and neck region[END_REF].

I.3.5 Monitoring of tumor growth in preclinical models

The basic method of tumor detection and quantitation is visual observation; this involves palpation followed by caliper measurements and calculation of tumor volume to generate an estimate of tumor burden. Moreover, increased interest in orthotopic and transgenic systems has created a demand for imaging-based methods for in vivo tumor monitoring including bioluminescence and fluorescence imaging, magnetic resonance imaging (MRI), X-ray computed tomography, and positron emission tomography (PET).

Bioluminescence imaging is a method that allows visualization of luciferase-driven light emitted from within an animal. In this approach, mice are inoculated with tumor cells that have been transfected with the luciferase gene, therefore the constitutive expression of luciferase allows assessment of tumor burden after systemic injection of substrate (luciferin) [START_REF] Edinger | Advancing animal models of neoplasia through in vivo bioluminescence imaging[END_REF]. Bioluminescence is suited to the detection of metastases and in situ tumor models, the image acquisition is generally rapid but low spatial resolution and two-dimensional images are the main limitations of this technique. In vivo fluorescence imaging can also be used to detect and monitor tumor growth; the fluorescent signal is emitted following excitation with monochromatic light of fluorescent or dye-labeled biological molecules [START_REF] Wessels | In vivo imaging in experimental preclinical tumor research--a review[END_REF].

Fluorescence imaging does not require the injection of exogenous substrate but signal attenuation in deep tissues and a high auto-fluorescence can be problematic. MRI combines outstanding soft tissue contrast with high spatial resolution. Therefore, tumors can usually be distinguished from normal tissue in rapid anatomical scans. Furthermore, differential uptake of contrast agent by the tumor compared with surrounding normal tissue, allows the tumor to be delineated in MR images [START_REF] Pautler | Mouse MRI: concepts and applications in physiology[END_REF]. MRI has been used in mouse models to detect tumors as small as 0.5mm in diameter in the brain, lung, liver, and pancreas, among others. Another technique, X-ray computed tomography has recently become feasible for preclinical cancer models, particularly for detection of lesions in bone, lung, and mammary glands [START_REF] Haines | A quantitative volumetric micro-computed tomography method to analyze lung tumors in genetically engineered mouse models[END_REF]. Three-dimensional images with high resolution can be produced and with the use of contrast agents and blood pool agents, soft tissues can also be imaged. Other approach, PET imaging, is increasingly used to study tumor biology, allowing higher image resolution and animal throughput. Tumor detection using micro-PET takes advantage of pathological changes in tumor cells that promote enhanced uptake of positron-emitting radiotracers [START_REF] Riemann | Small animal PET in preclinical studies: opportunities and challenges[END_REF]. An alternative method uses radioimmunotracers targeting tumor-specific antigens that provides distinction between normal and malignant tissues. As described before, CTLs are thought to be the major effector immune cells directed against tumor cells, having the ability to recognize and kill malignant cells [START_REF] Boissonnas | In vivo imaging of cytotoxic T cell infiltration and elimination of a solid tumor[END_REF]. To activate this potent anti-tumor immunity, antigen-presenting cells (APC), particularly DCs, must capture and process tumor protein antigens through the MHC class II pathway for presentation to CD4 + T cells, or translocated to the cytosol to enter the MHC class I pathway for "cross-presentation" to CD8 + T cells. Immature DC presenting the antigenic peptides are activated by pro-inflammatory cytokines to express costimulatory molecules and to increase the expression of MHC on their surface. Thus, tumor-specific naïve T cells differentiate into effector T cells as a result of the combination of signaling from TCR binding to the antigen peptide-loaded MHC on the DC and the binding of costimulatory molecules. Activated CTLs will then recognize cells expressing the specific antigens to which they were primed against and induce target cell lysis.

However, solid tumors continue to progress and metastasize in generally immunocompetent individuals due to different mechanism described above. The understanding of CTL effector mechanisms and the development of new immunotherapy strategies that enhances this and other anti-tumor responses are necessary.

Development and activation of CD8 + T cells

T cells are developed from bone marrow-derived prothymocytes in the thymus. The thymus contains three compartments: the subcapsular zone, the cortex and the medulla [START_REF] Chaplin | Overview of the immune response[END_REF]. In the subcapsular zone, double negative (CD4 -CD8 -) prothymocytes differentiate into double positive cells (CD4 + CD8 + ); and they proliferate and rearrange their TCR chains by somatic recombination. This process involves lymphoid-specific recombinase-activating gene (RAG1/RAG2) proteins and non-lymphoid-specific repair enzymes from the non-homologous end joining (NHEJ) system. RAG1/RAG2 proteins recognize, binds and cleaves DNA recombination signal sequences (RSS) flanking V (Variable), D (Diversity) and J (Joining) genes. Then, the resulting DNA ends are aligned and rejoined by NHEJ components including DNA-dependent protein kinase (DNA-PK), Ku 70/80, XRCC4, XRCC4-like factor (XLF), DNA ligase IV and Artemis nuclease. This process results in the V, D and J gene assembly producing a large diversity of TCR chains but also non-functional receptors. After leaving thymus, mature but naïve CD8 + T cells migrate to secondary lymphoid organs such as lymph nodes and the spleen, where they can spend an average of 24 hours until they are primed by APC like DCs, in this case this time is extended to 3-4 days. The establishment of cellular contact between naive CD8 + T cells and mature DCs provides the opportunity for antigen recognition through TCR-MHC interactions, other receptor-ligand interactions and delivery of soluble mediators that could result in clonal expansion and differentiation into effector and memory T cells [START_REF] Bousso | T-cell activation by dendritic cells in the lymph node: lessons from the movies[END_REF]. To achieve maximal expansion after activation, CD8 + T cells need to integrate multiple signals: the first signal is provided by TCR interaction with the complex MHC Class I-antigen expressed by DCs after cross-presentation, the second signal is provided by the interaction between costimulatory molecules expressed in mature DCs (CD80 and CD86) and their receptor in T cells (CD28), and the third signal is provided by cytokines such as IL-12 and type I IFN [START_REF] Curtsinger | Inflammatory cytokines as a third signal for T cell activation[END_REF]. CD4 + T cells play an important role in CD8 + activation process by helping in DCs activation after CD40-CD40L interaction and by secreting cytokines that are part of the third signal [START_REF] Wiesel | From crucial to negligible: functional CD8(+) T-cell responses and their dependence on CD4(+) T-cell help[END_REF].

Effector and memory CD8 + T cells

Once naïve CD8 + T cells (CD45RA + ) are activated by all three signals, they produce IL-2, undergo clonal expansion and acquire peripheral tissue-homing capabilities, effector cytokine release and cytolytic activity. When an effector CTLs (CD45RO + ) response has occurred and antigen is cleared from the system, these cells enter a death phase characterized by an apoptosis-induced contraction, and the remaining cells become longterm memory cells [START_REF] Gattinoni | Paths to stemness: building the ultimate antitumour T cell[END_REF]. Memory CD8 + T cells can persist for very long periods of time in the absence of antigen and remains quite stable overtime, largely through interactions with IL-15 and IL-7 that mediate survival and self-renewal [START_REF] Cui | Generation of effector CD8+ T cells and their conversion to memory T cells[END_REF]. In contrast to naïve T cells, memory T cells are capable of rapidly re-express critical genes and release cytokines such as TNF-, IFN- and IL-2 on restimulation [START_REF] Gattinoni | Paths to stemness: building the ultimate antitumour T cell[END_REF]. Based on the expression of CD62L and CCR7 surface markers, multiple sets of memory T cells have been characterized, most notably, the central memory (TCM) and effector memory (TEM) T cells [START_REF] Newell | Cytometry by time-of-flight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of CD8+ T cell phenotypes[END_REF]. TEM have an increased ability for immediate protection from a peripheral challenge, whereas TCM provide protection from systemic challenge and can generate a second wave of effector cells. Furthermore, in mice models, CD44 and CD62L surface markers are used to differentiate TCM from TEM and naïve T cells [START_REF] Chiu | Cutting edge: Central memory CD8 T cells in aged mice are virtual memory cells[END_REF].

CTL-mediated cytotoxicity

Cytolytic functions of CTLs are determined by multifaceted mechanisms including the interaction with pro-apoptotic receptors, degranulation of intracytoplasmic vesicles containing cytotoxic molecules and the secretion of cytokines like IFN- and TNF- [START_REF] Russell | Lymphocyte-mediated cytotoxicity[END_REF]. The Fas pathway and similar signaling pathways triggers an apoptotic response through Fas-associated death domain protein (FADD) and caspase-8 activation, this pathway also occurs for other death receptors such as TRAIL [START_REF] Barry | Cytotoxic T lymphocytes: all roads lead to death[END_REF]. The granule-dependent exocytosis pathway is established through intracellular signaling and microtubular mobilization that leads the preformed granules towards the point of contact with the target cell; releasing stored lytic molecules (Granzyme A, granzyme B and granulysin) and the pore-forming molecule, perforin. Together they induce apoptosis by caspases-independent or dependent pathways [START_REF] Chavez-Galan | Cell death mechanisms induced by cytotoxic lymphocytes[END_REF]. For example, once released into the cytoplasm, granzyme B initiate apoptotic cell death through direct cleavage of pro-caspase-3 or indirectly, through caspase-8. In addition, cleavage of BH3 interacting-domain death agonist (BID) results in its translocation to the mitochondria, leading to cytochrome c release and activation of caspase-9 through the interaction with the apoptotic protease-activating factor-1 (APAF-1).

Regulation of CD8 + T-cell activation

Two main mechanisms are involved in the contraction of the effector phase of the CD8 + T-cell response, the inhibition of T-cell expansion or the elimination of activated cells by apoptosis also referred as activation-induced cell death (AICD). The AICD process is induced by the same signals that lead to activation, these signals include re-stimulation of their TCR, binding of CD3 and the exposure to mitogens. AICD involves the engagement of death receptors like Fas, TRAIL and the TNF receptor. IL-2 is required for the sensitization towards AICD, through increased expression of the death receptor ligand Fas-L and decreased expression of the anti-apoptotic molecule c-FLIP [START_REF] Brenner | Concepts of activated T cell death[END_REF].

The mechanism involved in the inhibition of T-cell expansion includes co-inhibitory molecules such as CTLA-4 or PD-1, due to the presence of ITIM motifs in their structure [START_REF] Vigano | Positive and negative regulation of cellular immune responses in physiologic conditions and diseases[END_REF]. Indeed, the interaction of CTLA-4 (up-regulated on activated T cells) with CD80 or CD86 (expressed by DCs or other cells) competes with CD28 inducing an inhibitory signal that leads to the regulation of TCR and prevents T-cell activation.

Nowadays, different mechanisms have been proposed to be implicated in this inhibitory signal, including: altered phosphorylation of CD3 chains, disruption of the formation of ZAP-70 microclusters, interaction with phosphoinositide 3-kinase (PI3K) and association with the protein kinase isoform PKC-. However, no consensus have been established in this area [START_REF] Walker | Confusing signals: recent progress in CTLA-4 biology[END_REF]. Another molecule, PD-1 (expressed by CD4 + and CD8 + T cells, and other cells) is induced by TCR signaling and its interaction with PD-L1 (expressed by many cells) or PD-L2 (expressed by hematopoietic cell types) leads to the inhibition of CD28-mediated costimulation and thus lymphocyte proliferation and cytokine secretion. PD-1 signaling involves the inhibition of membrane proximal phosphorylation of CD3 chain and ZAP-70, and cell cycle arrest by the downstream Ras/Mek/Erk and AKT signaling pathways. PD-1 ligation inhibitory effects have been attributed to the recruitment of SHP-2 to the immunological synapse resulting in dephosphorylation of TCR signaling molecules [START_REF] Kulpa | PD-1 coinhibitory signals: the link between pathogenesis and protection[END_REF].

II.1.2 Immunotherapy and anti-tumor vaccination

Immunotherapy refers to a number of approaches intended to stimulate the immune system to induce responses in various diseases, particularly for cancer and degenerative diseases. Because current therapies for cancer rely on drugs that have severe effects on normal cells and causes morbidity and mortality, immunotherapy has the potential of being the most tumor-specific treatment [START_REF] Abbas | Cellular and Molecular Immunology[END_REF]. These strategies can be subdivided into passive immunotherapy, based on the transfer of ex vivo pre-activated immune cells or antibodies; and active immunotherapy, that aims at enhance anti-tumor immune response in the host. This strategy is rapid but does not lead to long-lived immunity.

Currently, there are some anti-tumor antibodies that are approved for the treatment of certain cancers and several others being considered. Many of these antibodies are specific for TAAs and others target the tumor microenvironment. Antibodies targeting epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2/Neu), and VEGF have shown therapeutic efficiency in non-leukemic cancers, whereas antibodies conjugated to radioactive isotopes, chemotherapeutic drugs, toxins or immunomodulators are used for the treatment of hematological malignancies [START_REF] Weiner | Monoclonal antibodies: versatile platforms for cancer immunotherapy[END_REF]. The anti-tumor effects of TAAstargeted mAb-based immunotherapy are mediated by inhibition of signaling pathways, antibody-dependent cell mediated cytotoxicity (ADCC) [START_REF] Ferris | Tumor antigen-targeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape[END_REF], and/or neutrophilmediated mechanisms [START_REF] Albanesi | Neutrophils mediate antibody-induced antitumor effects in mice[END_REF]. The overexpression of EGFR in HNSCC tumors provides a rationale for the use monoclonal antibodies against this antigen. Indeed, cetuximab, a chimeric mAb targeting the extracellular portion of EGFR, was found to enhance survival when combined with radiotherapy [START_REF] Bonner | Radiotherapy plus cetuximab for locoregionally advanced head and neck cancer: 5-year survival data from a phase 3 randomised trial, and relation between cetuximab-induced rash and survival[END_REF]. Additional anti-tumor mAb therapies for HNSCCs, including some that target HER2/Neu, VEGF or carcinoembryonic antigen (CEA)

were described [START_REF] Cohen | Erlotinib and bevacizumab in patients with recurrent or metastatic squamous-cell carcinoma of the head and neck: a phase I/II study[END_REF][START_REF] Rapidis | Immunotherapy of head and neck cancer: current and future considerations[END_REF].

Adoptive cellular immunotherapy (ACT) is based on the transfer of ex vivo cultured immune cells, which have anti-tumor reactivity into a tumor-bearing host. Moreover, immune ablation is an effective preconditioning regimen that can increase T-cell responses after ACT [START_REF] Rosenberg | Adoptive cell transfer: a clinical path to effective cancer immunotherapy[END_REF]. One of the most developed ACT strategies is the use of chimeric antigen receptors (CARs), where patients T cells are transfected with a construct encoding an antibody against a tumor surface antigen fused to T-cell signaling domains [START_REF] Kochenderfer | Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors[END_REF]. In HNSCCs, a popular approach is the collective ex vivo pretreatment of NK and T cells with high concentrations of interleukin-2, resulting in the so-called lymphokineactivated killer (LAK) cells, which have been re-infused into patients [START_REF] Saito | A combined lymphokine-activated killer (LAK) cell immunotherapy and adenovirus-p53 gene therapy for head and neck squamous cell carcinoma[END_REF].

Another approach is the injection into the tumor-feeding arteries of ex vivo generated NKT cells, in combination with nasal submucosal administration of Galcer-pulsed APC [START_REF] Yamasaki | Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy[END_REF]. In addition, ACT preliminary studies in oral HNSCCs have shown the ability of transferred cells to reactivate and expand HPV-16 E6/E7 specific T cells [START_REF] Ramos | Human papillomavirus type 16 E6/E7-specific cytotoxic T lymphocytes for adoptive immunotherapy of HPV-associated malignancies[END_REF].

b. NON-SPECIFIC ACTIVE IMMUNOTHERAPY

The earliest attempts to enhance anti-tumor responses in the host relied on non-specific immune stimulation. Nowadays, two potential approaches are to artificially provide costimulation for tumor-specific T cells and to provide cytokines that can enhance their activation. Indeed, host immunity against tumor can be shaped by administrating cytokines in a systemic or intratumor way. The largest clinical experience was obtained using IL-2, which stimulates activation and proliferation of T cells. However, Tregs cells can also be activated, leading to immunosuppression [START_REF] Lemoine | Massive expansion of regulatory T-cells following interleukin 2 treatment during a phase I-II dendritic cell-based immunotherapy of metastatic renal cancer[END_REF][START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCVinduced vasculitis[END_REF]. Cytokines such as IFN-, GM-CSF, IL-12, among others, are used as potential anti-tumor strategies. In HNSCCs, the use of recombinant IL-2 showed better survival rate in patients, when administered around the chin and neck lymph nodes [START_REF] De Stefani | Improved survival with perilymphatic interleukin 2 in patients with resectable squamous cell carcinoma of the oral cavity and oropharynx[END_REF]. IL-12 was also tested in HNSCC patients, where intratumoral administration of recombinant IL-12 elicited a switch from Th2 profile to Th1 profile in the loco-regional lymph nodes [START_REF] Van Herpen | Intratumoral administration of recombinant human interleukin 12 in head and neck squamous cell carcinoma patients elicits a T-helper 1 profile in the locoregional lymph nodes[END_REF].

Another strategy is based on the idea that tumor cells exploit various normal pathways of immune regulation or tolerance to evade host immune response. Therefore, blocking these inhibitory pathways will enhance anti-tumor response. Among the best studied of the immunological checkpoints is the inhibitory receptor of B7, the CTLA-4 which functions normally to shut off immune responses. Blockade of CTLA-4 using mAb has been used in preclinical studies showing rejection of several types of established transplantable tumors in mice and also in clinical trials of metastatic melanoma, prostatic, ovarian, breast and colonic cancers [START_REF] Peggs | Principles and use of anti-CTLA4 antibody in human cancer immunotherapy[END_REF]. Furthermore, CTLA-4 blockade reduced the frequency of granulocytic and monocytic MDSCs in the circulation of patients accompanied with reduction of arginase 1 (Arg1) + myeloid cells and progression-free survival (Pico de [START_REF] Pico De Coana | Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system[END_REF].

Another checkpoint target is the PD-1, an immunomodulatory molecule expressed after T-cell activation that when interacts with PD-L1 inhibits TCR-mediated proliferation and cytokine secretion. Blockade of PD-1/PD-L1 pathway is effective in enhancing T-cell killing of tumors in pre-clinical and clinical trials [START_REF] Nguyen | Clinical blockade of PD1 and LAG3--potential mechanisms of action[END_REF]. Tregs can compromise immune responses to tumor antigens; hence, depletion of these cells may also enhance anti-tumor immunity [START_REF] Waldmann | Effective cancer therapy through immunomodulation[END_REF]. Interestingly, PD-1 inhibitors have demonstrated a higher response rate in clinical trials in advanced melanoma patients compared to CTLA-4 blockers, and PD-L1 blocking antibodies are undergoing Phase I/II trials in melanoma, colorectal, renal, ovarian, pancreatic and head and neck cancers among others (Pico de [START_REF] Pico De Coana | Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system[END_REF].

Currently, there are no completed clinical trials of anti-CTLA-4 (ipilimumab) or anti-PD-1 (nivolumab) involving HNSCC patients. However, preliminary results of a phase 1b study evaluating the PD-1 inhibitor pembrolizumab in metastatic HNSCCs reported decreased tumor burden in 50% of evaluable patients [START_REF] Seiwert | A phase Ib study of MK-3475 in patients with human papillomavirus (HPV)-associated and non-HPVassociated head and neck (H/N) cancer[END_REF]. Importantly, it was showed in preclinical and clinical trials that combinatorial blockade of immune checkpoints, such as PD-1

and CTLA-4, resulted in significantly increased anti-tumor immunity when compared with blocking either checkpoint alone [START_REF] Drake | Breathing new life into immunotherapy: review of melanoma, lung and kidney cancer[END_REF]. This rationale can be used also in advanced head and neck cancers [START_REF] Swanson | Rationale for combined blockade of PD-1 and CTLA-4 in advanced head and neck squamous cell cancer-review of current data[END_REF].

Non-specific immune stimulation by intratumoral injection of inflammatory substances such as Bacillus Calmette-Guérin (BCG) mycobacteria, which may function as adjuvant and activates macrophages and stimulates T-cell responses, has been used for many years as immunotherapy for bladder cancers [START_REF] Gan | BCG immunotherapy for bladder cancer--the effects of substrain differences[END_REF]. Adjuvants are compounds that increase and/or modulate the intrinsic immunogenicity of an antigen; they are therefore required to assist vaccines to induce potent and persistent immune responses. We can distinguish between adjuvants that are carriers (liposomes and microspheres) and those that are immunostimulants (saponins and TLR agonists) or both (mineral salts and emulsions) [START_REF] Guy | The perfect mix: recent progress in adjuvant research[END_REF]. Since there is large range of adjuvants that can be combined, it is crucial to determine the optimal formulation that allow to optimally exploit the innate activation signals to ensure the development of safe and effective vaccines. Likewise, it has been shown that different formulations of the same vaccine components offer different protection [START_REF] O'hagan | New generation adjuvants--from empiricism to rational design[END_REF].

Moreover, some adjuvants such as TLR agonists alone or in combination with other approaches are being used as an immunotherapy strategy against cancer [START_REF] Vacchelli | Trial Watch: Toll-like receptor agonists for cancer therapy[END_REF]. For example TLR-9 agonist CpG oligodeoxynucleotide (CpG-ODN) was tested in a glioblastoma phase II clinical trial showing a 19% of 6-month progression free-survival and a good tolerance to treatment [START_REF] Carpentier | Intracerebral administration of CpG oligonucleotide for patients with recurrent glioblastoma: a phase II study[END_REF]. Furthermore, the right combination therapy of different TLR ligands such as Taxol (TLR-4 ligand) and Resiquimod (TLR-7/8 ligand) controls tumor growth in murine model of colorectal cancer [START_REF] Stier | Combinations of TLR ligands: a promising approach in cancer immunotherapy[END_REF]. Oral

HNSCCs have shown increased expression of various TLRs implicated in carcinogenesis, so the potential use of TLR agonists has been investigated [START_REF] Rich | Toll-like receptors and cancer, particularly oral squamous cell carcinoma[END_REF]. Among them, TLR9 agonist (CpG-ODN) and HNSCC cells influence the migration and the IFN- production of pDCs [START_REF] Thiel | Effect of head and neck cancer supernatant and CpG-oligonucleotides on migration and IFN-alpha production of plasmacytoid dendritic cells[END_REF], and TLR7 agonist (Imiquimod) inhibited the growth of oral HNSCC cells by inducing apoptosis and necrosis [START_REF] Ahn | Toll-like receptor 7 agonist, imiquimod, inhibits oral squamous carcinoma cells through apoptosis and necrosis[END_REF].

Peptide-based vaccines

Tumor vaccines make use of tumor antigens, which are derived from proteins that can be tumor specific or tumor associated (highly overexpressed in tumors but can also found in normal tissue). Five different classes of tumor-associated antigens have been defined

including cancer-testis antigen (i.e. melanoma antigen MAGE-A3), mutated protein, tumor virus (i.e. E6/E7 HPV-16 proteins), differentiation antigen and overexpressed protein (i.e.

MUC1) [START_REF] Stevanovic | Identification of tumour-associated T-cell epitopes for vaccine development[END_REF]. One approach developed for eliciting tumor-specific adaptive immune responses is the administration of purified or recombinant TAAs (or peptides) in the presence of adequate adjuvants. However, the antineoplastic activity of such responses is often limited in clinical trials (Pol et al., 2015a). MAGE-A3 and MUC1 peptide vaccines in association with adjuvants have been used in lung cancer and melanoma, showing more robust specific T cell and antibody responses, as well as objective tumor regressions [START_REF] Farkas | Vaccines based on abnormal self-antigens as tumorassociated antigens: immune regulation[END_REF][START_REF] Tartour | Lung cancer: potential targets for immunotherapy[END_REF]. In a HNSCCs phase I study using a Trojan system with HPV-16 derived peptides combined with MAGE-A3 and linked to a "penetrin" peptide sequence derived from human immunodeficiency virus (HIV) TAT protein, HPV specific T-cell responses against HLA-I restricted peptides were detected, but no clinical responses [START_REF] Voskens | Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma[END_REF]. Furthermore, in a phase II clinical trial using vaccination with multiple peptides derived from cancer testis antigens (LY6K, CDCA1 and IMP3), specific CTL responses correlated with better overall survival of patients with advanced HNSCCs were obtained [START_REF] Yoshitake | Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS[END_REF].

Cell-based vaccines

The main cell-based vaccination against cancer is based on DCs and includes: the use of untreated DCs, DCs exposed to antigens ex vivo, and strategies that allow loading of DCs in vivo. Therapeutic ex vivo cell-based vaccines with purified DCs from cancer patients have been used to immunize them against their own tumors. In this strategy, DCs are generated in culture from CD34+ hematopoietic progenitors or from peripheral bloodderived monocytes in the presence of GM-CSF and IL-4 or IL-13, incubated with tumor antigens or transfected with genes encoding these antigens and then re-injected into the patient [START_REF] Gilboa | DC-based cancer vaccines[END_REF]. Clinical studies analyzing different DC vaccine preparations, DC activators, antigen presentations and routes of injections concluded that DC-based vaccines are safe and can induce the expansion of CD4 + and CD8 + T cells specific to tumor antigens (Palucka and Banchereau, 2013a). Moreover, in HNSCC a phase I DCs p53 peptide vaccine demonstrated the safety and association with promising clinical outcome [START_REF] Schuler | Phase I dendritic cell p53 peptide vaccine for head and neck cancer[END_REF]). Yet, despite FDA approval of sipuleucel-T, a cellular product composed of enriched blood DCs pulsed with prostatic acid phosphatase and GM-CSF for treatment of prostate cancer [START_REF] Kantoff | Sipuleucel-T immunotherapy for castration-resistant prostate cancer[END_REF], clinically effective DC immunotherapy as monotherapy for solid tumors remains a distant goal. This objective can be achieved in combination with other anti-tumor therapies that augment DC function [START_REF] Datta | Rationale for a Multimodality Strategy to Enhance the Efficacy of Dendritic Cell-Based Cancer Immunotherapy[END_REF].

Nucleic acid-based vaccines

Therapeutic vaccines are also based on DNA or RNA nucleic acids. In DNA-based vaccines, purified DNA plasmids containing a TAA-encoding gene are administered (naked or using a vector) to cancer patients by intramuscular or intradermic injection in association or not with procedures that optimize DNA capture by cells (gene-gun, electroporation or transfection reagents such as PEI). Then, DNA will be processed and expressed by APCs or other transfected cells in order to develop specific CD8 + T cell or humoral immune responses (Cui et al., 2005). Despite the efficiency of naked DNA vaccines in generating anti-tumoral immune responses in animal models, the majority of clinical trials involving melanoma, prostate, B-cell lymphoma and colorectal cancers do not induce a sufficient immune response and the progression of disease is unaffected [START_REF] Bloy | Trial watch: Naked and vectored DNA-based anticancer vaccines[END_REF]. Therefore, combinatorial strategies that enhance and direct DNA vaccine immune response are required [START_REF] Lowe | Towards progress on DNA vaccines for cancer[END_REF]. Indeed, RNA-based vaccines are recently been used as an alternative to plasmid DNA giving some advantages: no need to be delivered into the nucleus but the cytoplasm, no integration into the host genome, and they can interact and trigger innate immune responses due to viral RNA recognition [START_REF] Geall | RNA: the new revolution in nucleic acid vaccines[END_REF]. These vaccines can be delivered naked, using viral vectors, using non-viral vectors such as lipids or polymers [START_REF] Midoux | Lipid-based mRNA vaccine delivery systems[END_REF], or by physical techniques including gene gun delivery and electroporation [START_REF] Johansson | Intradermal electroporation of naked replicon RNA elicits strong immune responses[END_REF].

Nowadays, RNA vaccination has been tested for cancer immunotherapy in preclinical and clinical trials of melanoma, prostate cancer, ovarian cancer and colon cancer eliciting antigenspecific cellular and humoral responses [START_REF] Sahin | mRNA-based therapeutics--developing a new class of drugs[END_REF].

Bacteria vector-based vaccines

Attenuated and genetically manipulated viruses and bacteria could be used as vectors for antigens. The bacterial vectors most used are salmonella, listeria, shigella, bordetella and BCG, whereas virus vectors are herpes virus, adenovirus, rhabdovirus, lentivirus and oncoretrovirus among others [START_REF] Leclerc | New technologies for vaccine development[END_REF]. Listeria monocytogenes (Lm)-based vaccines have

shown the ability to induce robust specific CTL responses against a wide-array of TAAs (including HPV-16 E6/E7, MAGE, HER2, Mesothelin, PSA, NY-ESO-1, etc.); therapeutic efficacy in preclinical models and clinical promise in HNSCC, pancreatic, breast, prostate and non-small lung cancer [START_REF] Souders | Listeria-based vaccines can overcome tolerance by expanding low avidity CD8+ T cells capable of eradicating a solid tumor in a transgenic mouse model of cancer[END_REF][START_REF] Wood | Attenuated Listeria monocytogenes: a powerful and versatile vector for the future of tumor immunotherapy[END_REF]. Furthermore, Salmonella-based vaccines carrying different antigens (IL-2, cytosine deaminase and VEGFR-2 DNA) are also being tested in clinical trials of hepatic metastasis, HNSCC, esophagus cancer, and pancreatic cancer [START_REF] Toussaint | Liveattenuated bacteria as a cancer vaccine vector[END_REF]. Otherwise, it was shown that the non-toxic B-subunit of Shiga toxin (STxB) from Shigella dysenteriae interacts with the glycolipid Gb3, expressed preferentially in DCs and B cells, and when STxB was coupled to antigens such as ovoalbumin (OVA) or HPV-16 E7 protein, they targeted the antigen in vivo to DCs and elicited strong and durable CTL response associated with tumor protection in mice [START_REF] Vingert | The Shiga toxin B-subunit targets antigen in vivo to dendritic cells and elicits anti-tumor immunity[END_REF]. Moreover, intranasal vaccination using this strategy, elicited mucosal CD8 + T-cell responses and control of HNSCC and lung cancer in mice (Sandoval et al 2013). Importantly, another strategy using a vaccine vector based on adenylate cyclase (CyaA) from Bordetella pertussis that target CD11b present in most DC in association with TLR4/TRIF activation, have shown strong specific CD4 + -independent CTL responses and are now been tested in cervical cancer and melanoma clinical trials as therapeutic vaccines [START_REF] Dadaglio | Antigen targeting to CD11b+ dendritic cells in association with TLR4/TRIF signaling promotes strong CD8+ T cell responses[END_REF]. [START_REF] Larocca | Viral vector-based therapeutic cancer vaccines[END_REF]. Importantly, the intratumoral injection of therapeutic vaccinia vaccine encoding HPV-16 E7 antigen (CRT-E7-VV) or recombinant vaccinia vaccine encoding HPV-16 E6/E7 antigens (TA-HPV) following Cisplatin treatment, led to increased E7-specific CD8+ T cells in blood and generated local and systemic antitumor responses in a HPV-induced tumor model [START_REF] Lee | Intratumoral injection of therapeutic HPV vaccinia vaccine following cisplatin enhances HPV-specific antitumor effects[END_REF]. Even more, adenoviral-based vaccines carrying mutated non-oncogenic HPV-16 E6/E7 induced specific cellular responses and in combination with chemotherapy/radiation controlled HPV-induced cancers in murine models [START_REF] Wieking | A non-oncogenic HPV 16 E6/E7 vaccine enhances treatment of HPV expressing tumors[END_REF]. In HNSCC, data with an adenoviral based p53 gene delivery product supports safety and clinical response in phase I and phase II trials after intratumoral injection [START_REF] Nemunaitis | Head and neck cancer: response to p53-based therapeutics[END_REF].

Viral vector-based vaccines

Non-viral vector-based vaccines

Because of their functional specialties, antigens can be delivered to DCs by targeting surface lectins (DC-SIGN, Clec9A and Langerin) resulting in humoral and/or cellular T-cell responses (Palucka and Banchereau, 2013b). This approach can be obtained using non-viral vectors such as lipid-based mRNA vaccines that achieve intracellular delivery using pHsensitive lipids or polymers [START_REF] Midoux | Lipid-based mRNA vaccine delivery systems[END_REF]. Moreover, efficacy of lipid-based vaccines has been proved in preclinical murine models, where they enhanced DCs transfection in vivo and vaccination against B16F10 melanoma tumor growth [START_REF] Perche | Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA[END_REF] and are being tested in clinical trials of ovarian, fallopian tube and peritoneal cancer (NCT01416038).

II.1.3 Virus-like particles (VLPs) and plasmo-retroVLPs (pVLPs)

Development of new therapeutic vaccines requires strategies capable of stimulating CTL responses and thus, to deliver antigen to MHC class I molecules expressed by DCs. To achieve this, delivery can exploit the 'classical' cytosolic MHC I pathway or take advantage of alternative pathways such as cross-presentation, where DCs acquire exogenous antigens and then present them bound to MHC I molecules [START_REF] Moron | New tools for antigen delivery to the MHC class I pathway[END_REF]. One potential strategy based on cross-presentation is the use of virus-like particles (VLPs). They consist of noninfectious viral structural proteins that assemble into particles structurally similar to infectious viruses but lacking viral nucleic acid. Currently, many VLP-based vaccines are developed and some approved for preventing diseases caused by HPV [START_REF] Schiller | An update of prophylactic human papillomavirus L1 virus-like particle vaccine clinical trial results[END_REF], hepatitis B Virus (HBV) [START_REF] Lacson | Antibody response to Engerix-B and Recombivax-HB hepatitis B vaccination in end-stage renal disease[END_REF], HIV [START_REF] Doan | Virus-like particles as HIV-1 vaccines[END_REF], Influenza virus [START_REF] Galarza | Virus-like particle (VLP) vaccine conferred complete protection against a lethal influenza virus challenge[END_REF], etc. In our laboratory a VLPs vaccine platform was developed specifically aimed at inducing such responses against multiple antigens displayed by recombinant retrovirus-based

VLPs made of Gag of Moloney murine leukemia virus (MuLV) and recently demonstrated their efficacy for vaccination against hepatitis C virus (HCV) [START_REF] Garrone | A prime-boost strategy using virus-like particles pseudotyped for HCV proteins triggers broadly neutralizing antibodies in macaques[END_REF].

VLPs are able to stimulate mainly the humoral response but also the cellular response of the immune system, because they can be taken up by APCs. For example when using HIV VLPs strong CD8 + T-cells immune responses can be obtained. This effect comes with the ability of Nef incorporated in VLPs to activate and mature primary human immature DCs [START_REF] Sistigu | Strong CD8+ T cell antigenicity and immunogenicity of large foreign proteins incorporated in HIV-1 VLPs able to induce a Nefdependent activation/maturation of dendritic cells[END_REF]. Moreover, it has been shown that VLPs induced DCs to secrete cytokines such as IL-12, IFN-, IL-6, among others [START_REF] Lenz | Papillomavirus virus-like particles induce cytokines characteristic of innate immune responses in plasmacytoid dendritic cells[END_REF][START_REF] Tegerstedt | Dendritic cells loaded with polyomavirus VP1/VP2Her2 virus-like particles efficiently prevent outgrowth of a Her2/neu expressing tumor[END_REF]. In the context of HPV vaccination, VLPs based on capsid L1/L2 proteins has shown to prevent disease by the induction of humoral responses. Furthermore, VLPs based on E7 oncoproteins retains the capacity to induce antibodies but also induce E7-specific CTL response [START_REF] Boisgerault | Virus-like particles: a new family of delivery systems[END_REF]. This rationale has been used to test VLP strategy as therapeutic vaccines for different cancers [START_REF] Ungaro | VLPs and particle strategies for cancer vaccines[END_REF]. In this line, HBV chimeric VLPs carrying MAGE-A3 have shown strong CTL activity and inhibition of established tumors in mice [START_REF] Zhang | Generation of chimeric HBc proteins with epitopes in E.coli: formation of virus-like particles and a potent inducer of antigen-specific cytotoxic immune response and anti-tumor effect in vivo[END_REF], and murine pneumotropic virus-VLP carrying HER2/neu together with CpG-ODN confers therapeutic effect in D2F2/E2 tumor-bearing mice [START_REF] Andreasson | Murine pneumotropic virus chimeric Her2/neu virus-like particles as prophylactic and therapeutic vaccines against Her2/neu expressing tumors[END_REF]. Additionally, in a clinical trial, HPV16 L1E7 chimeric VLP was used in patients with cervical intraepithelial neoplasia (CIN 2/3), showing humoral and cellular responses but no significant clinical efficacy [START_REF] Kaufmann | Vaccination trial with HPV16 L1E7 chimeric virus-like particles in women suffering from high grade cervical intraepithelial neoplasia (CIN 2/3)[END_REF].

II.2 Mucosal immunization II.2.1 Organization of the mucosal immune system

The mucosal immune system is a localized and specific immune organization protecting the inner surface of the human body, covering the mucosa of the oral-pharyngeal cavity, gastrointestinal (GI), respiratory and urogenital tract, as well as the exocrine glands [START_REF] Murphy | Janeway's immuniobiology, 8th edn[END_REF]. Structurally, the human mucosa includes a single-layer epithelium covered by mucus and antimicrobial products, fortified by both innate and adaptive immune cell components and natural microbiota [START_REF] Mcghee | Inside the mucosal immune system[END_REF]. However, the oral-pharyngeal mucosa possesses a stratified squamous epithelium instead of a single layer epithelium like in GI and respiratory tract; indeed, the oral-pharyngeal mucosa forms a mechanical barrier that is thicker and denser than GI mucosa, nonetheless permeable and fragile [START_REF] Novak | The immune privilege of the oral mucosa[END_REF].

The mucosal immune system can be separated into inductive and effector sites based upon their anatomical and functional properties. Inductive sites consists of secondary mucosaassociated lymphoid tissue (MALT) represented by the gut-associated lymphoid tissues (GALT), nasopharyngeal-associated lymphoid tissues (NALT), the bronchus-associated lymphoid tissue (BALT) and other less well characterized lymphoid sites. Effector sites includes the epithelium, lamina propria and salivary glands [START_REF] Kiyono | NALT-versus Peyer's-patch-mediated mucosal immunity[END_REF]. In MALTs, mucosally introduced antigens are taken in the epithelium by antigen-sampling cells and processed by DCs; these cells migrate to T-cell regions of the MALT and present the antigen to naïve T cells. In B-cell regions, germinal center formation and predominantly IgA class switching occurs. Finally, activated cells migrate to the mucosal effector tissue and form a cellular network including T helper cells, CTLs, B cells and epithelial cells, to provide the appropriate immune response [START_REF] Sato | The mucosal immune system of the respiratory tract[END_REF].

Oral mucosa presents effector sites but lacks inductive sites like MALTs, it has been hypothesized that DCs in the epithelium take up antigens, mature partially and migrate to the basal lamina, where they present antigens in the oral lymphoid foci to T cells to directly induce an effector response. Additionally, due to the large number of food antigens and antigens from the oral microflora in contact with the oral mucosa, tolerance induction represents an active process that include deletion and anergy of T cells, active inhibition by co-inhibitory receptors, and specific Tregs [START_REF] Novak | The immune privilege of the oral mucosa[END_REF]. Nonetheless, an inflammatory infiltrate composed of mainly lymphocytes and neutrophils can be regularly detected in the healthy gingiva.

II.2.2 Role of mucosal dendritic cells and T cells in oral immunity

Among the immune cells in the oral-pharyngeal mucosa, DCs are relatively better studied. In murine models, different subsets of CD11c + DCs, as well as pDCs reside in the epithelium of buccal, sublingual and gingival mucosa. In human, the frequencies of Langerhans cells (LCs) and other DCs are different depending on the mucosal site [START_REF] Hovav | Dendritic cells of the oral mucosa[END_REF].

DCs in the buccal mucosa (cheek)

In mice, at least four different DC subsets can be identified in the buccal tissue based on their expression of CD11c, CD103, langerin (Ln) and other markers: LCs located in the mucosal epithelium, interstitial DCs (iDCs) located in the lamina propria, langerin-expressing iDCs (Ln + iDCs) and CD103-expressing iDCs [START_REF] Hovav | Dendritic cells of the oral mucosa[END_REF]. Still in the mice, it has been shown that the buccal epithelium is an inductive site for priming CD8 + T cells (Desvignes et al., 1998). Moreover, buccal Ln + iDCs and iDCs mainly mediate presentation of the antigen to CD8 + T cells, while buccal LCs show limited function [START_REF] Nudel | Dendritic cells in distinct oral mucosal tissues engage different mechanisms to prime CD8+ T cells[END_REF]. In another murine study, it has been demonstrated that distinct subsets of buccal DCs have different migratory properties, buccal LCs expressed lower levels of costimulatory molecules and had slower rate compared with other buccal-DCs [START_REF] Aramaki | Differential expression of co-signal molecules and migratory properties in four distinct subsets of migratory dendritic cells from the oral mucosa[END_REF].

DCs in the sublingual mucosa

Oral mucosa has a relatively non-adsorptive nature. However, the epithelium of the sublingual mucosa is an exception, particularly thin it provides a useful route of entry for antigens, allergens, and drugs. In murine models, the frequency of LCs in the sublingual epithelium is much lower than in the buccal mucosa, iDCs are located in the lamina propria/submucosa interface, pDCs are also found, predominantly in the submucosa/muscular area [START_REF] Mascarell | Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells[END_REF]. Interestingly, a large population of CD11b + CD11c -cells that are F4/80 + also can be found in the lamina propria/submucosa interface [START_REF] Song | CCR7-CCL19/CCL21-regulated dendritic cells are responsible for effectiveness of sublingual vaccination[END_REF].

Studies in mice, point out that the sublingual and the vestibular mucosa are attractive immunological sites to induce tolerance. Furthermore, iDCs, pDCs and macrophage-like F4/80 + CD11b + CD11c -cells support the differentiation of IFN-/IL-10 producing regulatory T cells [START_REF] Mascarell | Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells[END_REF]Mascarell et al., 2011). In addition, sublingual iDCs were shown to transfer the antigen to the LNs, and present antigens to CD4 + T cells, migration was mediated by the CCR7-CCL19/CCL21 axis [START_REF] Song | CCR7-CCL19/CCL21-regulated dendritic cells are responsible for effectiveness of sublingual vaccination[END_REF].

Figure 13: Distribution and function of DCs in the murine oral mucosa [START_REF] Hovav | Dendritic cells of the oral mucosa[END_REF].

II.2.3 Strategies for mucosal immunization

The majority of current vaccination methods targets the systemic immune system and elicits only a weak or no mucosal immune response [START_REF] Lamichhane | The mucosal immune system for vaccine development[END_REF][START_REF] Neutra | Mucosal vaccines: the promise and the challenge[END_REF]. In contrast, mucosal immunization with appropriate vaccine delivery vehicle and route induces both protective mucosal and systemic immune responses, leading to a double layer of protection (Torrieri-Dramard et al., 2011).

As mentioned above, mucosal immune system uses a multifaceted regulatory system to maintain a balance between pathogen surveillance and tolerance to commensal microbes and dietary antigens. This approach is used for the induction of tolerance by immunotherapy for allergy mainly through the sublingual route [START_REF] Moingeon | Induction of tolerance via the sublingual route: mechanisms and applications[END_REF]. This propensity for tolerance to mucosal antigens means that mucosally delivered antigens are typically less immunogenic than antigens delivered by another route, and require potent adjuvants, vectors, or delivery platforms for effective mucosal vaccination [START_REF] Fujkuyama | Novel vaccine development strategies for inducing mucosal immunity[END_REF][START_REF] Rhee | Mucosal vaccine adjuvants update[END_REF]. Another challenge that must be faced is that vaccines are diluted in mucosal secretions when they are given orally or deposited directly on the surface. Thus relatively large doses of vaccine are required [START_REF] Neutra | Mucosal vaccines: the promise and the challenge[END_REF]. In addition, these strategies have to breach the epithelial barrier and "invade" organized mucosal lymphoid tissues like pathogens or live attenuated oral Salmonella typhi and poliovirus vaccines do.

Mucosal vaccine adjuvants

TLR agonists are generally used as adjuvants for non-mucosal and mucosal vaccination as well. Mucosal administration of CpG-ODN exhibits potent adjuvant activity and induce innate and Th1-type responses [START_REF] Mccluskie | Intranasal immunization of mice with CpG DNA induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution[END_REF]. Another TLR ligand mucosal adjuvant, AS04 incorporated in Cervarix HPV vaccine, is composed of alum and TLR4 ligand MPL and promotes a Th1-response towards co-administered antigens [START_REF] Didierlaurent | AS04, an aluminum salt-and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity[END_REF]. Other strategy uses bacterial toxins are derivatives as mucosal adjuvants; this includes Vibrio cholerae toxin (CT) and Escherichia coli heat labile enterotoxin (LT) and their mutants and subunits. It has been claimed that CT primarily induces Th2 type immune responses, while LT activates both Th1 and Th2 responses [START_REF] Rhee | Mucosal vaccine adjuvants update[END_REF]. Moreover, adding cytokines and chemokines, to directly obtain the adjuvant effect and avoid the use of toxins, was used in different studies.

For example, the combination IL-1, IL-12, IL-18 and GM-CSF after nasal immunization, can bring a strong mucosal and systemic Th1 response as CT [START_REF] Staats | Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization[END_REF].

Mucosal delivery systems and vectors

Depending of the mucosal site targeted by immunization, antigens can be degraded and epitopes destroyed by pH and enzymes of the mucosa tissue. However, antigen protection can be afforded by a range of methods including encapsulation in lipid vesicles, use of polymeric materials, enteric coatings and plant-based systems [START_REF] Mann | Delivery systems: a vaccine strategy for overcoming mucosal tolerance?[END_REF]. Antigen delivery by liposome formulations is used for oral vaccination due to their stability in acidic solutions and bile. For example, chitosan-coated liposomes loaded with calcitonin, were found to penetrate intestinal mucosa after oral administration [START_REF] Takeuchi | Effectiveness of submicron-sized, chitosan-coated liposomes in oral administration of peptide drugs[END_REF].

Furthermore, intranasal mRNA delivery using lipid polymer nanoparticles in mice showed a good expression of reporter proteins, when mRNA alone does not [START_REF] Sharma | Polymer nanotechnology based approaches in mucosal vaccine delivery: challenges and opportunities[END_REF].

Another strategy, using QuilA into a mixture of cholesterol and phospholipids (ISCOMATRIX) associated with inactivated influenza vaccine, induced humoral responses after intranasal administration [START_REF] Coulter | Intranasal vaccination with ISCOMATRIX adjuvanted influenza vaccine[END_REF]. Otherwise, recent studies have provided direct evidence that oral MucoRice-cholera toxin B-subunit induced Ag-specific antibodies that played a critical role against CT-induced diarrhea [START_REF] Ranasinghe | New advances in mucosal vaccination[END_REF].

The use of bacteria and virus vectors offers tremendous potential for the development of mucosal vaccines. Lactic acid bacteria, such as Lactobacillus casei, represent an alternative platform for mucosal delivery of therapeutic molecules or vaccines .

These non-pathogenic bacteria have been shown to be effective in delivering HPV-16 E7

antigen to the mucosa and induced mucosal cytotoxic response after oral immunization [START_REF] Adachi | Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cytotoxic lymphocytes against HPV16 E7[END_REF]. Using another strategy such as virus-like particles (VLPs), it has been shown that intranasal delivery of recombinant VLPs in mice induces both systemic and mucosal immune responses, whereas parenteral delivery induces systemic but poor mucosal immune responses [START_REF] Jackson | Intranasal vaccination with murabutide enhances humoral and mucosal immune responses to a virus-like particle vaccine[END_REF][START_REF] Sedlik | Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses[END_REF]. Nonetheless, larger doses of VLPs are required to induce the equivalent systemic immune response seen with co-delivery with a mucosal adjuvant [START_REF] Hjelm | TLR7 and 9 agonists are highly effective mucosal adjuvants for norovirus virus-like particle vaccines[END_REF]. Furthermore, the association of this strategy with an aloe-derived mucoadhesive dry powder formulation stabilizes the VLP and promotes the antigen depot effect, eliminating the need for inclusion of a mucosal adjuvant [START_REF] Velasquez | Intranasal delivery of Norwalk virus-like particles formulated in an in situ gelling, dry powder vaccine[END_REF]. Finally, intranasal vaccination in mice of HIV-specific retrovirus-based VLPs (pVLPs) displaying at their surface HIV-GP140 antigens, have shown better mucosal and systemic immune responses when compared with standard DNA vaccination [START_REF] Pitoiset | Enveloped virus-like particle platforms: vaccines of the future?[END_REF].

Mucosal immunization routes

Traditional routes of mucosal immunization include the oral, nasal and pulmonary routes. However, it has been demonstrated that rectal, vagina, buccal and sublingual mucosal immunizations are also effective strategies for protective immunity against infections, to treat allergy and autoimmune diseases.

Despite the effectiveness of oral vaccination using live attenuated vaccines to provide systemic immunity; these responses cannot be obtained with all vaccine strategies, due mainly to the small quantity of antigens that survive degradation in the intestine and cross the intestinal

wall. Yet, repeated large dose administration of vaccine antigens, vaccination using attenuated pathogenic bacteria, detoxified toxins and mucosal binding proteins as carriers ameliorates this strategy [START_REF] Russell-Jones | Oral vaccine delivery[END_REF]. Because of the low bioavailability observed in oral vaccination, intranasal route represents a good alternative for mucosal delivery. Indeed, FluMist and Fluenz, two approved intranasal vaccines composed of live attenuated multivalent influenza virus vaccine, have shown to increase the level of secreted nasal IgA and generate cell-mediated IFN-

. Yet, even when intranasal vaccination could lead to strong mucosal CTL responses against tumors (Sandoval et al., 2013), this route prevents sufficient antigen delivery and subsequent APC presentation due to anatomic and physiologic characteristics of the nasal cavity [START_REF] Riese | Intranasal formulations: promising strategy to deliver vaccines[END_REF]. Otherwise, for pulmonary vaccination, inhalation devices and powder or liquid formulation strategies can be used [START_REF] Tonnis | Devices and formulations for pulmonary vaccination[END_REF]. Moreover, other routes like vaginal and rectal mucosa are being used for the prevention of HIV transmission [START_REF] Yu | Mucosal HIV transmission and vaccination strategies through oral compared with vaginal and rectal routes[END_REF].

Mucosal vaccine delivery of the mouth can be subdivided into sublingual and buccal delivery. Sublingual delivery occurs through the mucosa of the ventral surface of the tongue and the floor of the mouth under the tongue, whereas buccal delivery occurs through the inner mucosa located in the cheeks, the gums and upper and lower inner lips [START_REF] Kraan | Buccal and sublingual vaccine delivery[END_REF].

Sublingual route have been used mostly for tolerance induction, it was shown that ovalbumin antigen crosses the epithelial barrier within 15 to 30 min and the uptake by sublingual DCs occurs within 30 and 60 min after sublingual administration in mice [START_REF] Mascarell | Oral dendritic cells mediate antigen-specific tolerance by stimulating TH1 and regulatory CD4+ T cells[END_REF]. A recent work demonstrates that antigen-bearing DCs that have captured the antigen in the sublingual mucosa are encountered in distant lymph nodes and spleen, suggesting that sublingual DCs are capable to enter the blood circulation to seed distant lymphoid organs (Hervouet et al., 2014). Furthermore, sublingual route enhanced humoral and cellular immune responses after HPV-16 L1 protein vaccination with adjuvants [START_REF] Cho | Enhanced humoral and cellular immune responses after sublingual immunization against human papillomavirus 16 L1 protein with adjuvants[END_REF].

As described above, the tolerogenic trend of the oral mucosa is due principally to the difference in their populations according to the mouth location. Sublingual mucosa has shown the presence of tolerogenic DC, whereas the buccal mucosa presents DC populations implicated in T-cell prime [START_REF] Hovav | Dendritic cells of the oral mucosa[END_REF]. Based on this rationale, buccal mucosa represents a promising route and offers several advantages for vaccination, including the permeability of the epithelium, the accessibility of Langerhans cells, and the induction of T-cell responses. Indeed, topical or transepithelial buccal immunization with measles virus nucleoprotein, induced recruitment of DCs in the buccal mucosa and priming of CD8 + CTL responses (Etchart et al., 2001). Moreover, electroporation mediated DNA vaccination with influenza virus nucleoprotein using the buccal mucosa route, resulted in robust and sustainable humoral and cellular immune responses, seen in both in the mucosa and the blood (Kichaev et al., 2013).

Thesis objectives

Despite current therapy HNSCCs have a bad prognosis, therefore the development of innovative vaccine strategies and adequate pre-clinical tumor models will be required to implement vaccine-based therapies for HNSCCs. The aim of my PhD project was to develop therapeutic strategies of vaccination in preclinical models for head and neck cancer.

The first objective was to validate the plasmo-retroVLP (pVLP) strategy, an approach that combines DNA-based and VLP-based vaccination, using an HPV-induced murine tumor model because HPV-16 is one of the etiologies of HNSCC. Thus, we developed plasmids that express the HPV-16 E7 oncoprotein (pVLP-E7) able to form VLPs E7 by self-assembly in vitro and when injected intradermally in the mice in combination with electroporation. Moreover, we studied the ability of pVLP-E7 to generate specific immune responses and anti-tumor effects in vivo using a preclinical model developed by subcutaneously injecting a murine tumoral cell line (TC-1) that express oncogenic proteins of HPV-16 (E6 and E7) in the flank of animals.

Because subcutaneous models do not provide specific interactions between HNSCC cells and their native environment, the second objective was to validate an orthotopic model of HNSCC to study our strategy. For this purpose, we studied the immune microenvironment of the gingiva from OSCC patients and healthy gingiva. Then in mice, we injected TC-1 cells and a murine cell line of oral cancer (NR-S1) in the cheek of animals to mimic OSCC environment.

The third objective was to validate a new route for mucosal vaccination. To achieve this, we studied the ability of pVLP-E7 to generate specific immune responses when injected in the intra-cheek or the intranasal mucosal routes of vaccination in comparison to the nonmucosal intradermic route. Furthermore, we tested this ability using an E7 polypeptide-based strategy in comparison to the pVLP-E7 strategy.

The fourth objective was to validate the intra-cheek route as a therapeutic vaccination in an orthotopic murine model of HNSCC. Indeed, in a context of strong loco-regional risk of HNSCC recurrence, the orthotopic model allows us to study mucosal routes in the same territory and the loco-regional responses to our pVLP vaccination strategy. Thus, using the TC-1 orthotopic model, we studied the tumor-specific immune responses and anti-tumoral responses of intra-cheek pVLP-E7 vaccination in comparison to intradermic route.

Results

Efficacy of DNA vaccines forming E7 recombinant retroviral virus-like particles

for the treatment of HPV-induced cancers.

We developed a strategy of DNA vaccination able to create non-infectious virus-like particles, which express HPV-16 E7 antigen (pVLP-E7). VLPs are more immunogenic than recombinant proteins and able to stimulate, both the humoral and the cellular arms of the immune system. One interest of the strategy is to combine the straight forwardness, the large scale and low cost of DNA vaccines with the immunostimulatory properties of VLP vaccines.

Here, we have shown that pVLP-E7 strategy was able to induce E7 specific immune response in vivo and in vitro in a HLA-A2 model. Indeed, we demonstrated that the combination of DC from HLA-A2 donors with VLPs expressing E7, and co-cultured in the presence of autologous T cells could induce E7-specific CD8 T responses. Then, we have shown that preventive vaccination using this strategy, fully protected mice from HPV-induced tumors. Furthermore, in the curative model, we showed a statistically significant tumor regression in mice vaccinated at early times. Importantly, we have shown the ability of pVLP-E7 to induce anti-tumor responses in later terms, when combined with TLR-agonist adjuvants.

My contribution for this publication as the second author (two first authors with equal contribution), included the production of pVLP-E7 for the preventive and therapeutic approaches, and the partial involvement in the acquisition, analysis and interpretation of data from the evaluation of preventive vaccinations with pVLP-E7 in the TC-1 induced tumors, the efficacy of therapeutic vaccinations in mice bearing TC-1 tumors, and the improvement of the efficacy of pVLP-E7 therapeutic vaccinations by combining the strategy with adjuvants.

2.

Intra-cheek immunization as a novel vaccination route for therapeutic vaccines of head and neck squamous cell carcinomas using an orthotopic pre-clinical model.

We have previously described the efficiency of therapeutic intradermic (ID) DNA vaccinations using plasmo-virus like particles carrying the HPV-E7 oncoprotein from HPV-16 (pVLP-E7) to control the growth of ectopic tumors models of HPV-induced head and neck cancers. We therefore wondered whether mucosal intra-cheek (IC) vaccinations could elicit better immune and anti-tumoral responses using an orthotopic murine model of head and neck squamous cell carcinoma (HNSCC).

We showed that human gingiva derived from oral squamous cell carcinoma (OSCC) patients, showed higher tumor-infiltrating lymphocytes in comparison to healthy gingiva. In the same line, murine mucosal orthotopic tumors, developed by injecting TC-1 cells (expressing E7) or NR-S1 cells (oral SCC) in the mice cheek, elicited stronger inflammatory responses and more tumor-induced weight loss when compared to subcutaneous ectopic models. Otherwise, we found that IC vaccination with pVLP-E7 could elicit better cervico-facial anti-E7 cellular immune responses than ID route while intranasal route was unable to induce any immune responses. Furthermore, IC route also increased the infiltration of E7-specific CD8+ T cells in non-draining lymph nodes.

When vaccinations were performed in mice bearing well-established TC-1 tumors, a better E7-specific CD8+ T-cell response was obtained using the IC route. Moreover, combination therapy of IC vaccination with TLR agonists, such as Imiquimod and CpG-ODN, led to rejection of established tumors and long-term protection from tumor rechallenge.

This therapeutic effect was associated with the infiltration of E7-specific CD8+ T cells in tumor and tumor-draining lymph nodes. Our findings demonstrate that IC mucosal vaccination with pVLP-E7 associated with adjuvants is efficient against mucosal orthotopic tumors; together, they provide a new valuable therapeutic strategy for HNSCCs.

For note, this manuscript has been submitted in "Cancer Research" journal and is under revision.

Intra-cheek immunization as a novel vaccination route for therapeutic vaccines of head and neck squamous cell carcinomas using an orthotopic pre-clinical model

Rodney Macedo1 , Juliette Rochefort

Introduction

Head and neck squamous cell cancers (HNSCCs) represent the sixth most frequent type of cancer in the world with global incidence and mortality rates annually estimated at 540 000 and 270 000 cases, respectively [START_REF] Islami | Alcohol drinking and esophageal squamous cell carcinoma with focus on light-drinkers and never-smokers: a systematic review and meta-analysis[END_REF]. HNSCCs are anatomically and clinically heterogeneous and arise from the mucosal surface of the oral cavity (OSCC), oropharynx (OPSCC), hypopharynx, larynx, sinuses and other sites within the upper aero-digestive tract.

Traditionally, HNSCCs are associated with alcohol and tobacco abuse [START_REF] Tramacere | A metaanalysis of alcohol drinking and oral and pharyngeal cancers. Part 1: overall results and dose-risk relation[END_REF]. However, there is an increased incidence of HNSCCs occurring in younger population without exposure to these chemical carcinogens [START_REF] Majchrzak | Oral cavity and oropharyngeal squamous cell carcinoma in young adults: a review of the literature[END_REF], especially in OSCCs and OPSCCs. Moreover, OPSCCs are frequently associated to human papillomavirus type-16 (HPV-16) ( 4). Standard treatments for HNSCCs combine surgery, radiation and chemotherapy depending upon the site of the disease and the degree of invasion and metastases. However, HNSCCs are very challenging to treat, and 35% to 55% of patients develop loco-regional or metastatic recurrence within two years. Thus, the prognosis of these patients remains poor, with a survival rate of less than 10-20% at 10 years [START_REF] Ho | Decision making in the management of recurrent head and neck cancer[END_REF]. Thus, there is an urgent need to develop innovative therapies for HNSCCs.

In a previous published report [START_REF] Lescaille | Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers[END_REF], we have developed an experimental strategy of therapeutic vaccines based on the use of plasmid DNA encoding retrovirus-like particles (pVLPs), an approach that combines DNA vaccination and VLP formation, to treat TC-1 tumor-bearing mice. Indeed, TC-1 cells, which over-express E6 and E7 oncoproteins from HPV-16, were subcutaneously (SC) implanted into the flank of animals. When tumors were well established, mice were intra-dermally (ID) vaccinated with pVLP harboring a non-oncogenic mutated E7 protein (pVLP-E7). Injection of pVLPs was associated with local electroporation in order to improve the immunization efficiency. We first showed that the pVLP strategy was more efficient than DNA vaccination or VLP alone to induce antigen-specific immune responses and anti-tumor effects. Thus, therapeutic vaccinations with pVLP-E7, when combined with TLR agonists such as CpG-ODN and Imiquimod, were able to control the growth of advanced tumors and to cure 50% of the mice resulting in a long-term disease free survival.

Although these data are encouraging, the ectopic model used is not adequate as a pre-clinical model for HNSCCs, making it difficult to extrapolate the efficacy of therapeutic vaccines using pVLPs. Thus, an orthotopic tumor model that recapitulates HNSCC characteristics must be developed. Furthermore, considering the mucosal origin of these cancers and the necessity to generate better loco-regional responses, it might be of interest to test different mucosal vaccination routes. Indeed, it have been shown that mucosal immunizations are more efficient to selectively elicit anti-tumor specific T-cell responses against mucosal tumors [START_REF] Nizard | Mucosal vaccines: novel strategies and applications for the control of pathogens and tumors at mucosal sites[END_REF]. In order to address these questions, we first validated a new orthotopic tumor model consisting of infusing tumor cells into the cheek of animals, and then we evaluated different mucosal immunizations routes using pVLPs. Our findings showed that intra-cheek (IC) vaccinations using pVLP-E7, as compared to ID vaccination, gave rise to a higher mobilization of CD8 + specific T cells in tumor draining lymph nodes (TdLNs) and in the tumor environment resulting in better anti-tumor effects and in a long-term protection.

In vivo tumor monitoring

C57BL/6 mice were injected with 5.10 4 TC-1-Luc cells either subcutaneously (SC) in the flank, intra-cheek (IC) or intra-lingual (IL). C3H/HeNRj mice were injected with 5.10 6 NR-S1 using the SC or IC route. All mice were anesthetized before tumor graft as previously described [START_REF] Lescaille | Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers[END_REF]. Mice were monitored every 2-3 days for tumor progression and individual weight. Tumor growth was determined using a caliper and according to the formula:

(length×width 2 )/2. For monitoring luciferase activity, mice were intraperitoneally injected with D-luciferin (150mg/kg) (Promega, Madison, WI, USA), bioluminescence images were acquired using IVIS Spectrum (Caliper Life Sciences, Tremblay, France) and luciferase expression was analyzed with the Living Image 4.2 software (Caliper Life Sciences). Mice were sacrificed when tumors reached volumes of 700-900 mm 3 (IC) or 1400-1600 mm 3 (SC), or when body weight loss was more than 15% (IL).

Tissue analysis by flow cytometry

Human cell suspensions were obtained from tumor and gingival samples after non-enzymatic digestion using Cell Recovery Solution (Corning, Avon, France) at 4°C for 1 hour. After filtering, washing and counting, cells were stained with Fixable Viability Dye eFluor780 (eBioscience, Paris, France) at 2-8°C for 30 minutes. Murine cell suspensions were obtained from tumors, lymph nodes or spleen by enzymatic dissociation using 1 mg/mL of collagenase IV (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and 0.2 mg/mL of DNase (Roche, Boulogne-Billancourt, France). After counting cells, they were stained using Fixable Viability Dye eFluor780 at 2-8°C during 10 minutes. Human or murine cell suspensions were incubated with monoclonal antibodies (mAbs) (Supplementary Table I) at 4°C during 20 minutes, and permeabilized with Foxp3/TFs Staining Buffer Set (eBioscience) for intracellular staining, according to manufacturer's instructions. Acquisition and data analyses were performed using LSRII flow cytometer (Becton Dickinson, Pont de Claix, France) and FlowJo v8.8.7 software (TreeStar, Ashland, OR, USA).

Immunization of mice

C57BL/6JRj mice were immunized using the intradermic (ID), intra-cheek (IC) or intranasal (IN) routes, three times at 2-day intervals. Two plasmids were used for pVLP-E7 vaccination:

pGag-E7 containing the mutated non-oncogenic E7 protein and pVSV-G coding for the vesicular stomatitis virus-G envelope protein [START_REF] Lescaille | Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers[END_REF]. For ID and IC immunization, 10µg pVLP-E7 (7.5μg pGag-E7 + 2.5μg pVSV-G) in 40µL of 0.5%NaCl were injected using the ID route (lower back) or the IC route (submucosally into the cheek inner face) and immediately electroporated in both injection sites using a BTX ECM830 generator (Harvard Apparatus, Les Ulis, France) and CUY650 P3 electrodes (Sonidel limited, Dublin, Ireland) as previously described [START_REF] Lescaille | Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers[END_REF]. For IN immunization, 10μg pVLP-E7 in 50µL of 5% glucose with PEIformulated (Ozyme, Montigny-le-Bretonneux, France) were administrated slowly into one nostril [START_REF] Torrieri-Dramard | Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses[END_REF]. As control groups, mice were ID or IC injected with 20 µg of E743-57 (GQAEPDRAHYNIVTF) polypeptide (Polypeptide Laboratories, Strasbourg, France) mixed with 50μg of CpG oligodeoxynucleotides (CpG-ODN, Li28-Litenimod, kindly provided by AF. Carpentier). In some groups, pVLP-E7 vaccination was combined with Imiquimod (5mg/mice, Aldara 5%, MedaPharma, Solna, Sweden) used as a topical treatment and CpG-ODN directly injected (50μg CpG-ODN in 50μl of 0.9%NaCl) into the tumor. All mice were anesthetized before immunizations.

ELISpot assays

E7-specific IFN- production by splenocytes and lymph node cells was determined as follows: briefly, cells (5 × 10 5 cells/well) were stimulated at 37°C in 5% CO2 for 24 hours with 5µg/mL of H-2D b -restricted immunodominant HPV-16 E749-57 peptide (RAHYNIVTF) (Anaspec, Fremont, USA). After revelation, spots were counted using the AID ELISpot reader (ELR03, AID Autoimmun Diagnostika, Strassberg, Germany). Results are presented as the mean of triplicate wells, and numbers of spots are expressed for 10 6 cells.

Tetramer Staining

For the detection of infiltrating E7-specific CD8 + T cells, tumors and TdLNs cells were dissociated as described above, and CD8 + cells were purified by MACS using anti-CD8 microbeads (Miltenyi Biotec, Paris, France). Cells were stained with CD45, CD3e, CD8a, CD49a mAbs (all from Biosciences) and E749-57/Db Tetramers (Beckman Coulter, Villepinte, France). Then, tubes were incubated 20-30 min in the dark at room temperature, and analyzed by flow cytometry.

In vivo CD8 + T-cell depletion

To evaluate the role of CD8 + T cells in the anti-tumor effect, CD8 + T cells were in vivo depleted as follows: 100 μg of anti-CD8 mAbs (rat IgG2b mAb, clone YTS 169.4 from Proteogenix) per mice or isotype control mAbs were intraperitoneally injected one week before therapeutic vaccination and then once a week as previously described [START_REF] Sandoval | Mucosal imprinting of vaccine-induced CD8(+) T cells is crucial to inhibit the growth of mucosal tumors[END_REF].

Anti-tumoral long-term protection

IC or ID vaccinated mice showing complete tumor regression at day 200, were orthotopically rechallenged with 5.10 4 TC-1-Luc cells. Naive mice were used as controls. Mice were monitored as described above for tumor progression up to day 400. At day 250, blood (300-350µL) was collected by retro-orbital puncture into heparinized tubes. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using LSA 1077 (PAA, Pasching, Austria). PBMC were stained with CD3e, CD8a, CD49a mAbs (all from Biosciences), CD4, CD44, CD62L mAbs (all from Biolegend, Saint-Quentin-Yvelines, France) and E749-57/Db Tetramers as described above, and then analyzed by flow cytometry.

Statistical analyses

Student t-test or one-way ANOVA with Tukey's correction was used for normally distributed data. Mann-Whitney or Kruskal-Wallis with Dunn's correction were used for non parametric data. Kaplan-Meier log-rank analysis was used to evaluate the survival differences between groups. Statistical analysis was conducted using Prism 6.0 software (GraphPad, La Jolla, CA, USA). Only p values < 0.05 were considered as significant. Results are presented as mean ± SEM of n separate experiments.

Results

Validation of an orthotopic tumor model for oral squamous cell carcinomas

In order to develop an orthotopic tumor model that shares anatomical and cellular features of HNSCC, we first evaluated by multiparametric flow-cytometry (Figure 1A) the inflammatory cellular components of OSCC microenvironment comparatively to healthy gingiva. Analysis of tumor microenvironment (Figure 1B) showed significant increases of total CD45 + cells, granulocytes, macrophages, myeloid (mDC) and plasmacytoid dendritic cells (pDC), and T lymphocyte subsets, albeit not B lymphocytes. Thus, these data underlined the inflammatory features of OSCCs and the presence of adaptive immune cells within the tumor microenvironment.

Secondly, we designed two orthotopic murine models using TC-1-Luc cells where cells were 2C). In the spleen, no significant differences concerning all innate and adaptive cell subpopulations were seen between the two tumor models (Supplementary Figure S2). To better validate the orthotopic tumor model and eliminate a bias due to the fact that TC-1-Luc cells are genetically modified lung epithelial cells, we have also injected NR-S1 cells, which do not express HPV-16 oncoproteins or luciferase, either subcutaneously or into the cheek of mice. As observed for TC-1-Luc tumors, a significant global infiltration of adaptive and innate cells was found in the orthotopic IC NR-S1 model, as compared to the ectopic SC NR-S1 model (Figure 2D and2E). These findings indicate that intra-cheek infusions of either TC-1-Luc cells or NR-S1 cells gave rise to more inflammatory tumor microenvironments that may be related to the peculiar anatomic localization and mucosal development of these tumors, as observed for OSCCs in humans. Thus, the orthotopic IC model appears as a suitable model for mimicking OSCCs. have been proposed to treat HPV-related cancers, we compared this strategy versus pVLP-E7 alone using the IC or the ID route. Results showed that IC immunizations significantly elicited higher numbers of E7-specific CD8 + T cells in CLN whatever the type of the vaccine as compared to ID immunizations (Figure 3B). Furthermore, pVLP-E7 alone gave rise to higher anti-E7 CD8 + T-cell responses than E7 polypeptide combined with CpG-ODN, particularly using the IC route (p < 0.001) (Figure 3B).

We further addressed whether the vaccination route may induce or not a different anti-tumor effect in mice bearing TC-1-Luc orthotopic tumors. Thus, mice were vaccinated either ID or IC with pVLP-E7 at days 7-9-11 following tumor cell infusion. IC vaccinations were performed on the contro-lateral side of the IC orthotopic tumors. Analysis of tumor cell suspensions obtained 7 days after vaccinations revealed higher percentages of H-2D b /E7 tetramer + CD8 + T cells within tumor microenvironment of mice vaccinated by IC route comparatively to ID route (Figure 3C). Moreover, analysis of anti-E7 CD8 + responses by IFN-γ ELISpot assay in TdLNs showed that IC vaccinations gave rise to significantly higher specific CD8 + T-cell responses (p < 0.0001) than ID vaccinations (Figure 3D). Whether IC vaccinations may have a better therapeutic effect than ID vaccinations was further studied.

Mice grafted with TC-1-Luc cells using the IC orthotopic model were IC or ID vaccinated with pVLP-E7 at days 7-9-11 after tumor cell infusion. A decrease of the tumor growth was observed comparatively to untreated mice after pVLP-E7 vaccinations using both routes, resulting in a significant prolonged survival (Figure 3E and 3F). However, no complete tumor regression was observed after either IC or ID vaccinations, and all mice were euthanized.

Altogether, our results show that IC vaccinations are superior over ID vaccinations for eliciting local and loco-regional immune responses in tumor-free mice as well as in tumorbearing mice. Nonetheless, no curative effect was observed with pVLP-E7 alone using either the IC or ID route of vaccination. For ELISpot assays, cells were loaded with E7 49-57 peptide and background obtained with cells not pulsed with the E7 49-57 peptide was subtracted. Data have been obtained using 4-6 mice per group per experiment, except for Figure

89

3A where some data were pooled from three separate experiments. NS, non-statistical difference = p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Therapeutic advantage of intra-cheek vaccinations when combined with adjuvants

Although IC immunizations gave rise to better specific immune responses than ID vaccinations, no major therapeutic effect was observed. We have previously described that pVLP-E7 vaccine administrated by ID route in combination with adjuvants such as Imiquimod and CpG-ODN, which act as TLR7 and TLR9 agonists respectively, enhanced the anti-tumor response and cured mice with established ectopic SC TC-1 tumors ( 6). Therefore, using the orthotopic IC model, we compared the anti-tumor effects of IC and ID pVLP-E7 vaccinations in combination with these adjuvants at days 7-9-11. Interestingly, when combined with adjuvants, IC and ID vaccinations resulted in a significant therapeutic effect on the tumor growth, as compared to non-treated mice, whereas adjuvants alone only had a slight, albeit non-significant, effect (Figure 4A). Furthermore, IC vaccinations combined with adjuvants gave rise to a significant (p < 0.05) and better long-term tumor-free survival (58%), as compared to ID vaccinations combined with adjuvants (25%), for at least 200 days (figure 4B). These data confirmed the therapeutic advantage of IC vaccinations over ID route.

Intra-cheek therapeutic effects correlates with better specific CD8 + T-cell responses

Whether stronger anti-tumoral responses obtained after IC vaccinations + adjuvants, as compared to ID vaccinations + adjuvants, could be explained by higher CD8 + specific immune responses was then studied. For this purpose, tumor-bearing mice were pVLP-E7 vaccinated in combination with adjuvants at days 7-9-11, and then CD8 + T cell responses were analyzed in cell suspensions obtained from tumors and TdLNs at day 18. Figure 5A (left top panel) shows that ID and IC vaccinations in association with adjuvants induce a significant increase (p < 0.05 and p < 0.0001, respectively) of the absolute number of total CD8 + T cells in TdLNs, comparatively to non-vaccinated mice. Furthermore, only IC vaccinations significantly enhanced the effect of adjuvants (p < 0.01). In tumors (Figure 5A, left bottom panel), only IC vaccination + adjuvants increased the CD8 + T-cell density, as compared to other groups of mice.

The presence of CD8 + specific T cells was further examined using H 2 -D b E7 tetramers.

Significant higher numbers of E7-specific CD8 + T cells were found in TdLNs (Figure 5A, middle top panel), and in tumors (Figure 5A middle bottom panel) when mice were vaccinated using the IC route and adjuvants. The fact that IC vaccinations induced a higher mobilization of E7-specific cells was confirmed by IFN-γ ELISpot Assay (Figure 5B). To explain this preferential recruitment of CD8 + T cells at the mucosal site after IC vaccination, we analyzed the expression of the CD49a integrin, known to be particularly expressed by mucosal T cells. IC vaccinations demonstrated their superiority comparatively to ID vaccinations to induce E7-specific mucosal CD8 + T cells in TdLNs (Figure 5A, right top panel). Even if adjuvants alone, through their inflammatory effects, can mobilize CD8 + T cells, no significant increase of E7-specific CD8 + T cells could be observed. Furthermore, analysis of tumor microenvironment also revealed a higher density of tetramer E7 + CD8 + T cells and tetramer E7 + CD49a + CD8 + T cells (Figure 5A, middle and right bottom panels), when mice were vaccinated by using the IC route, as compared to the ID route.

Overall, our findings indicate that the better anti-tumor efficiency observed after IC vaccinations correlates with higher specific immune responses in TdLNs and tumor microenvironment. Because the presence of Tregs in these sites may be a major hurdle for the efficacy of effector specific T cells to eradicate tumors, we wondered whether our vaccine strategy may or not diminish the density of Tregs and/or the balance between Tregs and effector T cells (see Figure 2B and2C). In both routes of vaccinations, the Treg density did not changed (Figure 5C) strongly suggesting that the vaccine efficiency was due to the stimulation of specific effector cells. This was further demonstrated by treating mice with anti-CD8 depleting mAbs prior and during IC vaccinations. Indeed, in CD8-depleted mice the anti-tumor efficiency of IC vaccinations completely disappeared (Figure 5D), confirming the crucial role of CD8 + T cells in the therapeutic effect of IC vaccinations.

Long-term protection effects of intra-cheek vaccinations against tumor relapses

Because of the high relapse rate of HNSCC, that more often occur locally or locoregionally, it will be of importance that therapeutic vaccines induce long-term protections.

For this purpose, mice showing a total regression of TC-1-Luc tumors after vaccination using ID (n=3/12 mice) or IC route (n=7/12 mice) were rechallenged at day 200 with 5.10 4

TC-1-Luc cells injected into the contralateral cheek with regard to the initial tumor development. A group of naive mice receiving TC-1-Luc cells was used as tumor growth control. All cured mice were protected from TC-1-Luc tumor cell growth and could survived at least for 400 days (Figure 6A). Moreover, to study memory responses, specific E7 CD8 + T cells were evaluated by using H2-D b -E7 tetramers in the blood of mice 6 weeks after rechallenge. A representative analysis of circulating E7-tetramer + CD8 + T cells shows that IC vaccinated mice have a much higher percentage (Mean for 7 mice: 2.09 ± 0.08%, Max = 4.96 %) than the ID group (Mean for 3 mice: 0.51 ± 0.38%, Max = 0.93 %) or naïve mice (Mean for 7 mice: 0.11 ± 0.01%, Max=0.17%) (Figure 6B). Furthermore, central memory and effector memory CD8 + T cells were distinguished using CD44 and CD62L markers. In the vaccinated groups, CD8 + CD44 + CD62L high (central memory) and CD8 + CD44 + CD62L low (effector memory) E7-specific CD8 + T cells could be detected (Figure 6C). Interestingly, IC vaccinations as compared to ID vaccinations induced higher percentage of central memory (1.49 ± 0.62 %, n=7 vs 0.18 ± 0.09%, n=3) and of effector memory (0.51 ± 0.20%, n=7 vs 0.15 ± 0.06%, n=3) E7-specific CD8 + T cells. Noteworthy, the percentage of E7-specific mucosal (CD49a + ) CD8 + T cells still remained much higher in rechallenged mice previously vaccinated by the IC route (1.76 ± 0.75%, n=7), as compared to those vaccinated by the ID route (0.33 ± 0.18%, n=3) (Figure 6C, right panel). Overall, our findings indicate that IC vaccination gave rise to a long-term anti-tumor response, to better central memory and effector memory specific responses, and to a better recruitment of mucosal effector CD8 + T cells, as compared to ID vaccinations.

Discussion

Because prognosis of HNSCC remains poor with a high risk of local recurrence, it is very important to develop therapeutic strategies that aim at eliciting both systemic and local immune responses in order to induce tumor regression and to avoid tumor relapse, thanks to long-term protection. Deciphering tumor microenvironment is crucial to understand the tumor development and to develop immune-based therapies. Indeed, tumors occurring at different anatomical sites differ in their microenvironmental content and may vary in their response to immunotherapy, suggesting that normal tissue surrounding the tumor site can have a decisive role in determining its composition [START_REF] Devaud | Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy[END_REF]. Moreover, in HNSCC the degree of leukocyte infiltration appears to be dependent upon the tumor site (OSCC or OPSCC) and is likely to be influenced by the differing microenvironments and the stage of the tumor [START_REF] Green | Increased prevalence of tumour infiltrating immune cells in oropharyngeal tumours in comparison to other subsites: relationship to peripheral immunity[END_REF][START_REF] Wansom | Infiltrating lymphocytes and human papillomavirus-16--associated oropharyngeal cancer[END_REF][START_REF] Wolf | Tumor infiltrating lymphocytes (TIL) and prognosis in oral cavity squamous carcinoma: a preliminary study[END_REF]. Our data showing an increased numbers of both innate and adaptive cells in gingiva-carcinoma cancers, comparatively to human normal gingiva are in line with other reports showing the inflammatory nature of these cancers [START_REF] Wallis | Clinical relevance of immune parameters in the tumor microenvironment of head and neck cancers[END_REF]. In order to evaluate immunotherapy strategies, such as therapeutic vaccines, development of orthotopic models which provide specific interactions between cancer cells and their native microenvironment ( 16) is crucial. Because inducible oral-specific tumor models imply labor-intensive processes [START_REF] Tang | Oral cavity and esophageal carcinogenesis modeled in carcinogen-treated mice[END_REF][START_REF] Raimondi | Rapamycin prevents early onset of tumorigenesis in an oral-specific K-ras and p53 two-hit carcinogenesis model[END_REF], we chose to develop orthotopic tumor models using cell lines. Although we tested the injection of cells in the basis of the tongue as described [START_REF] Sandoval | Mucosal imprinting of vaccine-induced CD8(+) T cells is crucial to inhibit the growth of mucosal tumors[END_REF], considering the risk of early death and the difficulty of monitoring intra-lingual tumor in mice [START_REF] Bozec | Combination of sunitinib, cetuximab and irradiation in an orthotopic head and neck cancer model[END_REF], we decided to inject TC-1-Luc cells into the mucosa of the cheek, as published by others [START_REF] Yang | The drug efficacy and adverse reactions in a mouse model of oral squamous cell carcinoma treated with oxaliplatin at different time points during a day[END_REF]. We observed significant increases of both innate and adaptive immune cells in the tumor microenvironment as compared to ectopic TC-1-Luc tumors. This observation was reproduced using the NR-S1 cell line that originates from a spontaneous murine oral carcinoma.

While very few orthotopic models have been established using syngeneic murine cell line derived from oral cancer [START_REF] Cui | Effect of YM529 on a model of mandibular invasion by oral squamous cell carcinoma in mice[END_REF][START_REF] Vahle | Multimodal imaging analysis of an orthotopic head and neck cancer mouse model and application of anti-CD137 tumor immune therapy[END_REF], our data clearly indicate that intra-cheek model display inflammatory features close to those observed in patients with OSCCs, and represents an adequate pre-clinical model for oral HNSCCs.

In a context of strong loco-regional risks of recurrence, the orthotopic model allows to study in the same territory, mucosal routes of vaccination and their loco-regional responses. Indeed, mucosal routes possess the advantage over the parenteral route of eliciting local and systemic T-cell responses as well as humoral responses [START_REF] Torrieri-Dramard | Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses[END_REF]. Most of mucosal routes that have been studied are vaginal, rectal or sublingual routes. Sublingual immunotherapy has been widely used for therapeutic allergy vaccines [START_REF] Mascarell | Induction of allergen-specific tolerance via mucosal routes[END_REF]. Although several studies have showed the efficiency of the sublingual route to induce tolerance, others have observed cell-mediated immune responses against pathogens [START_REF] Appledorn | Sublingual administration of an adenovirus serotype 5 (Ad5)-based vaccine confirms Toll-like receptor agonist activity in the oral cavity and elicits improved mucosal and systemic 102 cell-mediated responses against HIV antigens despite preexisting Ad5 immunity[END_REF][START_REF] Choi | A single sublingual dose of an adenovirus-based vaccine protects against lethal Ebola challenge in mice and guinea pigs[END_REF][START_REF] Domm | Robust antigenspecific humoral immune responses to sublingually delivered adenoviral vectors encoding HIV-1 Env: association with mucoadhesion and efficient penetration of the sublingual barrier[END_REF][START_REF] Park | Sublingual immunization with a live attenuated influenza a virus lacking the nonstructural protein 1 induces broad protective immunity in mice[END_REF], or tumors [START_REF] Singh | Sublingual vaccination induces mucosal and systemic adaptive immunity for protection against lung tumor challenge[END_REF]. Similarly, Sandoval et al. have reported that the intranasal route was able to induce good anti-tumoral responses in a model of oral cancer and lung [START_REF] Sandoval | Mucosal imprinting of vaccine-induced CD8(+) T cells is crucial to inhibit the growth of mucosal tumors[END_REF]. However, IN immunizations with pVLPs, which require PEI for IN immunization [START_REF] Torrieri-Dramard | Intranasal DNA vaccination induces potent mucosal and systemic immune responses and cross-protective immunity against influenza viruses[END_REF], did not promote in our hands detectable immune responses against HPV-16 E7 oncoprotein. More interestingly, we have shown that the IC vaccinations can induce better immune and anti-tumoral responses particularly in TdLNs, as compared to ID vaccinations. The advantage of IC immunizations, comparatively to ID immunizations, was also confirmed by using long peptides and adjuvants (see Figure 3B). Although no studies have used buccal route for anti-tumoral vaccination, good immune responses have already been observed against different pathogens [START_REF] Etchart | Dendritic cells recruitment and in vivo priming of CD8+ CTL induced by a single topical or transepithelial immunization via the buccal mucosa with measles virus nucleoprotein[END_REF][START_REF] Kichaev | Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses[END_REF]. Indeed, humoral and cellular responses against the influenza virus nucleoprotein (NP) of influenza H1N1 have been observed using DNA vaccination associated with electroporation [START_REF] Kichaev | Electroporation mediated DNA vaccination directly to a mucosal surface results in improved immune responses[END_REF]. Oral mucosa appears to be an attractive site for vaccine delivery due to their accesibility, anatomy and physiology. In addition, several studies revealed the buccal epithelium as an inductive site for efficient priming of CD8 + T lymphocytes [START_REF] Etchart | Dendritic cells recruitment and in vivo priming of CD8+ CTL induced by a single topical or transepithelial immunization via the buccal mucosa with measles virus nucleoprotein[END_REF][START_REF] Desvignes | The murine buccal mucosa is an inductive site for priming class I-restricted CD8+ effector T cells in vivo[END_REF]. Cheek localization, contrary to sublingual and intranasal routes, easily allows the use of electroporation, necessary when using a DNA strategy. Furthermore, the relatively high frequency of DCs, in particular of Langerhans cells and the low numbers of mast cells in human [START_REF] Allam | Distribution of Langerhans cells and mast cells within the human oral mucosa: new application sites of allergens in sublingual immunotherapy?[END_REF] in the buccal region makes the cheek mucosa an attractive site for vaccine delivery.

Previously, we have shown that the combination of intradermic pVLP vaccinations with adjuvants, such as Imiquimod and CpG-ODN, improved tumor growth inhibition [START_REF] Lescaille | Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers[END_REF]. Here, we observed that IC vaccinations using the same strategy were even better than ID vaccinations for inducing tumor regression in an orthotopic model of oral cancer. Interestingly, IC route was able to induce a dramatic local and loco-regionally increase of the E7-specific CD8 + T cells/Tregs ratio, independently of the presence of adjuvants. Moreover, we demonstrated that IC vaccinations favored a preferential recruitment of antigen-specific CD8 + T cells expressing the mucosal integrin CD49a in tumor microenvironment and TdLNs (Figure 5A). Interestingly, in a previous report Sandoval et al also observed an increase of specific CD49a + CD8 + T cells in intra-lingual tumors after IN mucosal vaccination [START_REF] Sandoval | Mucosal imprinting of vaccine-induced CD8(+) T cells is crucial to inhibit the growth of mucosal tumors[END_REF]. Then, these data and ours suggest a link between IN or IC vaccination and the induction of a mucosal homing program on CD8 + T cells that controlled their trafficking. This hypothesis has been also suggested by others [START_REF] Hervouet | Antigen-bearing dendritic cells from the sublingual mucosa recirculate to distant systemic lymphoid organs to prime mucosal CD8 T cells[END_REF]. Thus, such mucosal vaccination routes will be of great importance for therapeutic vaccines designed to treat mucosal cancers.

Overall, our findings have shown the advantage of an oral mucosal route of vaccination to induce long-term anti-tumoral responses by using plasmo-retroVLPs as vaccine vectors for antigen delivery and by using a new pre-clinical orthotopic model of oral cancer. In this report, E7 oncoprotein was used as an antigen model applicable for HPV-related HNSCCs.

Then, it will be worthwhile to validate our vaccine strategy using pVLPs carrying other tumor-associated antigens particularly involved in non HPV-related HNSCCs [START_REF] Laban | Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer[END_REF]. Our data are encouraging to rapidly envision a clinical trial for HNSCC because pVLPs, like DNA vaccine, are easy to produce under good manufacturing procedures and the adjuvants used, i.e CpG-ODN and Imiquimod, are already available as clinical grade reagents. Such vaccinebased clinical trials would be proposed after tumor mass reduction using standard chemo/radiotherapy, known to induce immunological cell death [START_REF] Kroemer | Immunogenic cell death in cancer therapy[END_REF], in combination with immune checkpoint inhibitors like PD-1/PD-L1 and/or anti-CTL4 mAbs [START_REF] Perez-Gracia | Orchestrating immune check-point blockade for cancer immunotherapy in combinations[END_REF][START_REF] Badoual | PD-1expressing tumor-infiltrating T cells are a favorable prognostic biomarker in HPVassociated head and neck cancer[END_REF]. Considering that cancers of the oral cavity are known for their high risk of loco-regional recurrence, it is important to develop new strategies that can stimulate better loco-regional responses. Mucosal vaccination was reported as an interesting approach to generate good responses of this type. Hence, we tested intranasal and intra-cheek mucosal routes of vaccination. The sublingual route could not be used because of the limitation for the use of electroporation or PEI injection for DNA transfection. We observed that the intra-cheek route was better than the intradermic and intranasal routes of vaccination, inducing good locoregional T-cell responses as well as systemic and remote responses. Furthermore, in orthotopic tumor-bearing mice, we observed a long-term survival of 58% when using the intra-cheek route against 25% with intradermal vaccination (p <0.05), both in combination with adjuvants.

Different curative immunization strategies have been tested in preclinical models of cancers including, vectors (bacterial, viral, non-viral), proteins, peptides, nucleic acids (RNA, DNA) and cell-based vaccination. In HNSCCs, many strategies focused on driving a specific immune response against HPV antigens (E6 and E7) were tested in preclinical and some clinical trials including other HPV-induced cancers. Indeed, specific CD8 + and Th1 CD4 + responses have been observed in 47% of patients with vulvar dysplastic lesions caused by HPV, using long E6/E7 overlapping peptides [START_REF] Kenter | Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia[END_REF]. However, it has been shown that peptide-based vaccines, exhibited various drawbacks, including low immunogenicity. Otherwise, VLP-based vaccines have shown to be more immunogenic than protein strategies. We decided to work with pVLPs because of their ability to generate VLPs that induce good humoral and more importantly T-cell responses. Furthermore, the use of this pVLP strategy is particularly interesting because of its safety compared to the use of other strategies such as viral or bacterial vectors.

Although different preclinical studies showed interesting results with therapeutic antitumor mucosal vaccination, at the moment no clinical trial is in progress in HNSCC. Moreover, even if the advantage of the mucosal intra-cheek pVLPs strategy was demonstrated here, using an HPV-induced preclinical tumor model, it is still necessary to carry out studies focused to improve the anti-tumoral response and more importantly to assess their effectiveness in the treatment of HPV-negative HNSCC. Nonetheless, our data are encouraging to rapidly envision a clinical trial for HNSCC. Such vaccine-based clinical trials could be proposed after tumor mass reduction using standard chemo/radiotherapy, in combination with immune checkpoint inhibitors (Perez-Gracia et al., 2014) and other strategies discussed below.

Improvement of vaccination strategies for HNSCC

Vaccination against HPV-negative HNSCC TAAs

Vaccines may target two forms of TA: (1) tumor specific antigens (TSA), or [START_REF] Tramacere | A metaanalysis of alcohol drinking and oral and pharyngeal cancers. Part 1: overall results and dose-risk relation[END_REF] tumor associated antigens (TAA). TSA are oncoproteins that are unique for the tumor; Cancer Testis antigens, such as those of the melanoma associated antigen (MAGE) family, are antigens expressed in many types of cancer, including HNSCCs [START_REF] Li | Evaluation of melanoma antigen (MAGE) gene expression in human cancers using The Cancer Genome Atlas[END_REF]. In oral squamous cell carcinoma (OSCC), MAGE-A3 is present in 44% of these cancers and MAGE-A4 in 53% of cases [START_REF] Ries | Expression of melanoma-associated antigens in oral squamous cell carcinoma[END_REF], and it is considered that 75% of these cancers express at least one or the other of these two antigens. Using this rationale, it would be interesting to test an immunotherapy strategy that specifically target MAGE-A3 and MAGE-A4

antigens in these cancers. Immunotherapeutic strategies, including using antigenic peptides directed against MAGE-A3 were studied in metastatic melanoma in the past with disappointing results [START_REF] Kruit | Phase 1/2 study of subcutaneous and intradermal immunization with a recombinant MAGE-3 protein in patients with detectable metastatic melanoma[END_REF]. Since then, strategies have evolved using different vectors, especially in combination with other therapeutic alternatives [START_REF] Kruit | Selection of immunostimulant AS15 for active immunization with MAGE-A3 protein: results of a randomized phase II study of the European Organisation for Research and Treatment of Cancer Melanoma Group in Metastatic Melanoma[END_REF]. Moreover, MAGE-A3

Trojan vaccines induced humoral and cellular immune responses in patients with advanced HNSCC [START_REF] Voskens | Induction of MAGE-A3 and HPV-16 immunity by Trojan vaccines in patients with head and neck carcinoma[END_REF]. Regarding MAGE-A4, few strategies have been developed, but a study in animals showed that it is possible to obtain CTL immune responses against MAGEA4 and NY-ESO-1 [START_REF] Muraoka | Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8(+) T cells[END_REF]. These immunotherapy approaches against MAGE-A3/MAGE-A4, could be developed using the pVLP platform. Furthermore, specific immune responses and anti-tumoral responses after intra-cheek mucosal vaccination will be tested in an orthotopic model obtained after injection of oral OSCC NR-S1 tumor cells transfected to express MAGE-A3/MAGE-A4. Finally, these strategies could be combined to adjuvants or immune-modulators to induce better specific anti-tumor immune responses.

Improvement in the vaccination schedule

Following anti-tumoral vaccination, high numbers of antigen-specific memory CD8 + T cells are usually desired, since this number correlates with host protection. One approach to generate this response is to utilize a system of prime-boost vaccination. Indeed, repeated administration of the same vaccine (homologous booster) have shown to be successful in boosting the humoral response to antigen, but is far less effective at generating increased numbers increased numbers of CD8 + T cells due to rapid clearance of the homologous boosting agent by the primed immune system [START_REF] Woodland | Jump-starting the immune system: prime-boosting comes of age[END_REF]. In contrast, heterologous prime-boost vaccination, where an antigen is primed in CD8+ T cells with one vector and then the same antigen is delivered in another vector at a later time, is much more effective at generating increase numbers of memory CD8+ T cells [START_REF] Lu | Heterologous prime-boost vaccination[END_REF]. Such continuous antigen stimulation in a microenvironment that is often immunosuppressive may favor T-cell exhaustion, especially using DC-based immunotherapy, where booster vaccinations induced a long-lasting CTL response but did not sustain central memory CTLs and was detrimental for overall survival in mice [START_REF] Ricupito | Booster vaccinations against cancer are critical in prophylactic but detrimental in therapeutic settings[END_REF]. Moreover, partial CD4+ depletion reduces Tregs after multiple vaccinations and restores therapeutic efficacy, suggesting that these cells are limiting the efficacy of these strategy [START_REF] Lacelle | Partial CD4 depletion reduces regulatory T cells induced by multiple vaccinations and restores therapeutic efficacy[END_REF].

Nevertheless, it also has been shown that in some cases, there is evidence that boosting augmented the antigen-specific immune response against tumors. For example, booster vaccination of patients with non-small cell lung cancer using MAGE-A3 protein revealed long-term immunologically memory when combined with adjuvants, but antigen tolerance in the absence of adjuvant, compromising further immunization attempts [START_REF] Atanackovic | Booster vaccination of cancer patients with MAGE-A3 protein reveals long-term immunological memory or tolerance depending on priming[END_REF]. In a phase 2 study, PROSTVAC, an heterologous prime-boost regimen with two different poxvirus-based vectors carrying the prostate specific antigen (PSA), showed improved overall survival in patients with metastatic castration-resistant prostate cancer and is now being validated in a phase 3 study (PROSPECT). Importantly, in a prostate murine model, PROSTVAC resulted in significant anti-tumor efficacy and depletion of CD4+ and CD8+ revealed that both subsets contribute to this effect [START_REF] Mandl | Elucidating immunologic mechanisms of PROSTVAC cancer immunotherapy[END_REF]. Additionally, another murine study showed that a prime-boost strategy using homologous or heterologous DNA/fowlpox virus expressing the HPV-16 E6 protein resulted in a higher number of TC-1 tumor-free mice [START_REF] Bissa | A prime/boost strategy using DNA/fowlpox recombinants expressing the genetically attenuated E6 protein as a putative vaccine against HPV-16-associated cancers[END_REF]. These data suggest that the prime-boost strategy could be a good improvement for our strategy, when combined with adjuvants or immune-modulators that counteract the immunosuppressive tumor microenvironment.

pVLP Engineering

Several strategies have been developed to increase the immunogenicity of DNA-based vaccines, including the use of DC stimulatory molecules such as Fms-like tyrosine kinase-3 ligand (Flt3L), CD40L and other factors. Co-injection of Flt3L, IL-18 or GM-CSF plasmids with Her-2/neu DNA vaccination has shown to enhance cellular and antitumor immunity induced in preventive and therapeutic murine models [START_REF] Chang | Enhanced efficacy of DNA vaccination against Her-2/neu tumor antigen by genetic adjuvants[END_REF]. Furthermore, the engineering of E6 and E7 DNA vaccines to generate an optimal vaccine by fusion of E6 and E7, addition of a tissue plasminogen activator signal sequence and addition of CD40L or Flt3L; showed 25 times stronger E6 and E7-specific CD8 + T-cell responses than the initial E6/E7 fusion construct [START_REF] Seo | Optimal induction of HPV DNA vaccine-induced CD8+ T cell responses and therapeutic antitumor effect by antigen engineering and electroporation[END_REF]. Using this rationale, we could include genes of Flt3L or CD40L in the pVLP platform to enhance their action as therapeutic vaccine.

Improvement in vaccine delivery

To acquire an adequate in vivo immune response against tumor antigens, it is essential that pVLPs (as other DNA vaccines) are efficiently delivered and transferred into the nuclei of mammalian cells. To achieve this, a large number of viral (retrovirus, adenovirus, adenoassociated virus, lentivirus, etc.), physical (electroporation, DNA particle bombarding by gene gun, hydrodynamic, ultrasound, etc.), and non-viral (cationic liposomes, cationic polymers and lipid-polymer systems) delivery systems are being employed [START_REF] Nayerossadat | Viral and nonviral delivery systems for gene delivery[END_REF].

Among them, electroporation is the predominant strategy used to deliver DNA vaccines. And it is due to the immune responses observed that are comparable or superior to other well studied vaccine platforms including viral vectors [START_REF] Sardesai | Electroporation delivery of DNA vaccines: prospects for success[END_REF]. Moreover, DNA delivery by electroporation using the mucosal route has shown both humoral and cellular immune responses without tissue damage (Kichaev et al., 2013). Otherwise, non-viral vectors have shown also crucial advantages over viral vectors to improve their toxicity and targeting problems; these approaches include cationic liposomes, biomaterials (chitosan, cyclodextran), cationic polymers (polypeptides, polyethilenimine) and dendrimers [START_REF] Yang | Design, preparation and application of nucleic acid delivery carriers[END_REF]. Furthermore, DNA formulation with certain types of polymers and cationic liposomes has shown better gene transfer efficiency when compared to electroporation of naked DNA in different tumor models [START_REF] Anwer | Formulations for DNA delivery via electroporation in vivo[END_REF]. Overall, other non-viral delivery systems could be tested in comparison or in combination with electroporation to improve pVLP vaccination.

Combination of vaccination with conventional and innovative approaches

In recent years it has been shown that conventional cancer treatments, for example chemotherapy and radiation therapy may have immunogenic effects. Furthermore, it has recently been demonstrated that certain molecules of chemotherapy treatments (like doxorubicin, bleomycin, cyclophosphamide, oxaliplatin, etc.) could participate in the activity of the immune system and contribute to a better overall clinical efficacy [START_REF] Pol | Trial Watch: Immunogenic cell death inducers for anticancer chemotherapy[END_REF].

This restricted panel of chemotherapeutics can induce a combination of tumor cell stress and death that is immunogenic (immunogenic cell death: ICD), characterized by the recruitment of T cells, increased ratio of CTLs/Treg cells, innate immunity cells, and the production of pro-inflammatory cytokines which in some cases can control cancer cells (Kroemer et al., 2013). Moreover, the clinical profile of anticancer chemotherapy based on ICD inducers may be ameliorated by the concomitant administration of various immunostimulatory interventions [START_REF] Pol | Trial Watch: Immunogenic cell death inducers for anticancer chemotherapy[END_REF].

Combination with chemotherapy

Using this approach, different immune strategies were described in combination with chemotherapeutic agents. For example, adoptive transfer of autologous T cells genetically modified to express anti-CD19 or anti-CD20 CARs have shown safety and efficacy in the treatment of patients with B-cell malignancies upon a course of conventional chemotherapy (Vacchelli et al., 2013a). Besides, the combination of Oxaliplatin with IL-7 inhibited the growth of tumors in lung and abdomen metastasis model of colon cancer in mice, this effect correlated with increase of tumor-infiltrating CD8 + T cells and decrease in Treg cells [START_REF] Gou | Chemo-immunotherapy with oxaliplatin and interleukin-7 inhibits colon cancer metastasis in mice[END_REF].

As described before, some HNSCCs are associated with HPV infection. Thus, various immune strategies are using HPV-16 E6 or E7 as target antigens for developing therapeutic vaccines. It has been shown that the combination of low-dose cyclophosphamide administered as daily or single dose with the pNGVL4a-CRT/E7 (detox) DNA vaccine had a synergistic antitumor effect in TC-1 tumor-bearing mice, this effect is correlated with the reduction of Treg and increased tumor-infiltration of specific CD8 + T cells (Peng et al., 2013b). Interestingly, decreased expression of E6/E7 oncoproteins increased the sensitivity of HPV-positive HNSCC cell lines to irradiation  Cisplatin [START_REF] Ziemann | Increased sensitivity of HPV-positive head and neck cancer cell lines to x-irradiation +/-Cisplatin due to decreased expression of E6 and E7 oncoproteins and enhanced apoptosis[END_REF].

Combination with DNA-repair inhibitors

Because HNSCCs are very challenging to treat, they have a high relapse and bad prognosis. Even when treated with conventional approaches, there is an urgent need for innovative strategies that will target specific features of HNSCCs [START_REF] Da Silva | Recurrent oral cancer: current and emerging therapeutic approaches[END_REF]. causing subsequent cell death [START_REF] Quanz | Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response[END_REF].

Dbait molecules have already demonstrated efficacy in combination with radiotherapy and chemotherapy in preclinical models of colorectal cancer, melanoma and glioblastoma [START_REF] Biau | A preclinical study combining the DNA repair inhibitor Dbait with radiotherapy for the treatment of melanoma[END_REF][START_REF] Devun | Preclinical study of the DNA repair inhibitor Dbait in combination with chemotherapy in colorectal cancer[END_REF]Quanz et al., 2009a). Moreover, a first phase I/II clinical trial is currently underway, which combines Dbait and radiotherapy in the treatment of metastatic melanoma (DRIIM assay). In collaboration with Marie Dutreix team and DNA Therapeutics®, our laboratory's team is testing different protocols associating Dbait with radiotherapy or chemotherapy on HNSCCs and characterizing prediction biomarkers by using different pre-clinical models. Indeed, using HNSCCs immunocompetent murine models developed by injecting ectopically or orthotopically TC-1 tumor cells, we are going to evaluate the therapeutic effect of pVLP vaccines in combination with Dbait strategy. We will also analyze the possible effect of Dbait in stimulating the immune response by causing ICD in tumor cells. Furthermore, using oral HNSCC patient-derived tumor xenograft (PDX) models, we will study the therapeutic effect of Dbait in a model that recapitulates the geneexpression and histology patterns of HNSCCs (Peng et al., 2013a).

As preliminary experiments, we have tested the effect of Dbait alone or in association with cis-platinium and 5-FU (a conventional regimen for HNSCCs), on the tumor growth of

Combination with targeting cancer therapies

The most active area of immunotherapy in HNSCC in the last decade has been the development of monoclonal antibodies (mAbs) targeting TA on the cell surface. The majority of inquiry has focused on EGFR. EGFR is over-expressed in HNSCC; this high expression correlates with increased stage at presentation and poor prognosis [START_REF] Grandis | Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer[END_REF]. Cetuximab, the first FDA-approved molecularly targeted drug in HNSCC, improves response and survival when added to radiation in locally advanced HNSCC, or to chemotherapy in recurrent/ metastatic disease [START_REF] Bonner | Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck[END_REF][START_REF] Vermorken | Platinum-based chemotherapy plus cetuximab in head and neck cancer[END_REF].

However, the advantage of the use of this molecule is not clear. Two other anti-EGFR mAbs have been clinically evaluated in HNSCC (Panitumumab, and nimotuzumab); the clinical activity of panitumumab appears inferior to cetuximab [START_REF] Giralt | Panitumumab plus radiotherapy versus chemoradiotherapy in patients with unresected, locally advanced squamous-cell carcinoma of the head and neck (CONCERT-2): a randomised, controlled, open-label phase 2 trial[END_REF][START_REF] Vermorken | Cisplatin and fluorouracil with or without panitumumab in patients with recurrent or metastatic squamouscell carcinoma of the head and neck (SPECTRUM): an open-label phase 3 randomised trial[END_REF]. Interestingly, it has been shown in two studies that the addition of nimotuzumab conferred a significant survival advantage in advanced-stage HNSCC.

Because is known that VEGF is overexpressed in HNSCC, several studies have examined the use of bevacizumab in HNSCC. Bevacizumab is a recombinant humanized IgG1 monoclonal antibody that binds VEGF-A and was the first agent against this protein approved by the Food and Drug Administration. Despite the decrease in VEGF levels in patients treated with bevacizumab, the post-treatment VEGF levels have not been shown to be consistently associated with efficacy. Nonetheless, some anti-angiogenic molecules inhibit the development of immunosuppressive mechanisms developed by the tumors to escape the immune system (such as regulatory T cells, myeloid-derived suppressor cells, and immunosuppressive cytokines). It seems that bevacizumab as well as sunitinib treatment reduces the number of immature myeloid cells and consequently MDSCs that can arise from them [START_REF] Fricke | Vascular endothelial growth factor-trap overcomes defects in dendritic cell differentiation but does not improve antigen-specific immune responses[END_REF][START_REF] Ko | Sunitinib mediates reversal of myeloid-derived suppressor cell accumulation in renal cell carcinoma patients[END_REF][START_REF] Osada | The effect of anti-VEGF therapy on immature myeloid cell and dendritic cells in cancer patients[END_REF]. Furthermore, polarization of TAMs with an M2-skewed phenotype into an immune-supportive M1-like phenotype treatment could be induced by low dose anti-angiogenic treatment [START_REF] Huang | Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy[END_REF].

Other proposed mechanisms by which anti-angiogenic drugs can promote an immune-supportive tumor microenvironment include the increase of cell adhesion molecules (CAM), which promote leukocyte endothelium interactions [START_REF] Dings | Enhancement of Tcell-mediated antitumor response: angiostatic adjuvant to immunotherapy against cancer[END_REF][START_REF] Dirkx | Antiangiogenesis therapy can overcome endothelial cell anergy and promote leukocyteendothelium interactions and infiltration in tumors[END_REF][START_REF] Dirkx | Tumor angiogenesis modulates leukocyte-vessel wall interactions in vivo by reducing endothelial adhesion molecule expression[END_REF] and the reduction of regulatory T-cell numbers (Li et al., 2005). In a phase II study, sunitinib demonstrated activity in recurrent or metastatic HNSCC [START_REF] Machiels | Phase II study of sunitinib in recurrent or metastatic squamous cell carcinoma of the head and neck: GORTEC 2006-01[END_REF]. Moreover these anti-angiogenic treatments potentiate some immunotherapy strategies in preclinical models [START_REF] Adotevi | Targeting human telomerase reverse transcriptase with recombinant lentivector is highly effective to stimulate antitumor CD8 T-cell immunity in vivo[END_REF][START_REF] Huang | Combining antiangiogenic therapy with immunotherapy exerts better therapeutical effects on large tumors in a woodchuck hepatoma model[END_REF].

Again, in a HNSCC orthotopic model, combination of sunitinib, cetuximab and irradiation resulted in completely abolished tumor growth (Bozec et al., 2009). These findings on the impact of anti-angiogenic drug on immunosuppression favors the potential benefit of the combination with immunotherapeutic strategies.

Combination of vaccination with modulation of the tumor microenvironment

Early failures of immunotherapy protocols and improved knowledge of the tumor microenvironment suggested that HNSCC, as other cancers, escape immunosurveillance by different mechanisms described above; thus, this tumor-associated immunosuppression could explain the resistance of tumors to immunotherapy in certain cases. Recent improvements have allowed the design of immune modulators, defined as molecular adjuvants that in combination with vaccination rationally improve the immune response. Immune modulators used in vaccines are of two types, ones that amplify the response, and ones that overcome negative regulatory mechanisms or checkpoint inhibitors [START_REF] Berzofsky | Strategies to use immune modulators in therapeutic vaccines against cancer[END_REF].

Some cytokines, or certain adjuvants such as TLR ligands, have been described as capable to lift the state of anergy of T cells infiltrating the tumors and make them refractory to immunosuppressive mechanisms [START_REF] Derre | BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination[END_REF]. Cytokine-based therapy has been used both loco-regionally and systematically with limited success in HNSCC, albeit with significant toxicities in some patients. Several responses have been observed with IL-2 therapy in HNSCC patients [START_REF] De Stefani | Improved survival with perilymphatic interleukin 2 in patients with resectable squamous cell carcinoma of the oral cavity and oropharynx[END_REF][START_REF] Whiteside | Evidence for local and systemic activation of immune cells by peritumoral injections of interleukin 2 in patients with advanced squamous cell carcinoma of the head and neck[END_REF]. Some TLR ligands have already shown increased adjuvanticity and improved potential of anticancer vaccination in clinical trials [START_REF] Vacchelli | Trial Watch: Toll-like receptor agonists for cancer therapy[END_REF]; and more TLR agonists are under development in pre-clinical trials [START_REF] Baxevanis | Toll-like receptor agonists: current status and future perspective on their utility as adjuvants in improving anticancer vaccination strategies[END_REF]. Here, we have shown that the use of Imiquimod and CpG in combination with our DNA vaccination plasmoVLP in our HNSCC models allowed us to obtain stronger specific CD8 + T-cell responses and better anti-tumor responses.

We chose to use Imiquimod, a TLR-7 agonist, because this adjuvant induced clearance of HPV and regression of lesions in HPV-induced vulvar intraepithelial neoplasia [START_REF] Terlou | Imiquimod-induced clearance of HPV is associated with normalization of immune cell counts in usual type vulvar intraepithelial neoplasia[END_REF]. In addition, it has been shown that Imiquimod inhibits the growth of oral HNSCC cells by inducing apoptosis and necrosis [START_REF] Ahn | Toll-like receptor 7 agonist, imiquimod, inhibits oral squamous carcinoma cells through apoptosis and necrosis[END_REF]. Moreover, in a murine model similar to the one we used, the combination of vaccination DNA encoding a fusion protein calreticulin to E7 (CRT / E7) with the topical use of Imiquimod, increased the anti-E7 CD8 response and reduced MDSC in the tumor microenvironment [START_REF] Chuang | Treatment with imiquimod enhances antitumor immunity induced by therapeutic HPV DNA vaccination[END_REF].

Furthermore, we decided to use CpG, a TLR-9 agonist, in combination with Imiquimod to optimize anti-tumor responses in our model. Indeed, the use of CpG has been shown to increase the efficacy of different anti-cancer vaccines [START_REF] Carpentier | Cancer immunotherapy with CpG-ODN[END_REF]. It has been shown

that CpG antagonize the tumor-promoting effect of HNSCC cells by inhibiting the secretion of IL-1 and IL-10 (Brocks et al., 2007). Another approach that fuses E7 DNA with a gene encoding CpG and together with electroporation could increase anti-tumor effects of vaccination [START_REF] Ohlschlager | The combination of TLR-9 adjuvantation and electroporation-mediated delivery enhances in vivo antitumor responses after vaccination with HPV-16 E7 encoding DNA[END_REF]. In addition, a recent study showed that treatment of OSCC cells with CpG-ODN resulted in increased IL-6 secretion that promotes T-cell immune responses [START_REF] Ruan | The secretion of IL-6 by CpG-ODN-treated cancer cells promotes T-cell immune responses partly through the TLR-9/AP-1 pathway in oral squamous cell carcinoma[END_REF].

The second group of immune modulators overcomes the immunosuppressive tumor microenvironment by blocking negative regulatory cells and factors. In line with this notion, three checkpoint blockers have already been approved for use in the treatment of advanced, unresectable or metastatic melanoma: ipilimumab (anti-CTLA-4 mAb), pembrolizumab (anti-PD-1 mAb) and nivolumab (another anti-PD-1 mAb). Furthermore, this and other strategies are being testes in various cancers preclinical and clinical trials [START_REF] Buque | Trial Watch: Immunomodulatory monoclonal antibodies for oncological indications[END_REF].

There are similarities and distinctions of immune modulation between the PD-1/PD-L1

and the CD28/CTLA-4 systems, which could determine the action of PD-1 or CTLA-4 mAbs [START_REF] Topalian | Immune checkpoint blockade: a common denominator approach to cancer therapy[END_REF]. For example, blockade of PD-1/PD-L1 could have greater specificity for tumor antigen-specific T cells and less effect on autorreactive T cells, suggesting a higher antitumor activity and lower adverse effects compared to CTLA-4 blockade. However, combination therapy with both checkpoint inhibitors might further improve the strength of the therapeutic effect [START_REF] Homet Moreno | Anti-PD-1 therapy in melanoma[END_REF]. Moreover, it have been shown that PD-1 and CTLA-4 blockade had synergistic effects when combined to vaccination, expanding infiltrating T cells and reducing Treg within melanoma tumors in mice [START_REF] Curran | PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors[END_REF].

At this day, the efficacy of association between vaccination and the use of immune checkpoint inhibitors has not been proved in human. A phase I trial combining ipilimumab with a vaccine containing transgenes for prostate specific antigen (PSA) and for a triad of costimulatory molecules (PROSTVAC) is promising [START_REF] Jochems | A combination trial of vaccine plus ipilimumab in metastatic castration-resistant prostate cancer patients: immune correlates[END_REF]. Patients in the phase I study had a median overall survival of about 31 months for all dose levels of ipilimumab. Those who received the highest dose of ipilimumab (10 mg/kg) had the longest median overall survival: 37 months. In addition, about 20% of patients on the highest dose of ipilimumab were still alive at 80 months. Furthermore, in melanoma, a phase I/II trial for evaluating a peptide vaccine (mixture of six melanoma-associated helper peptides) plus ipilimumab (NCT02385669) and a phase I trial for evaluating NY-ESO-1 vaccine (protein or overlapping peptides) in combination with ipilimumab (NCT01810016) are currently recruiting patients.

Conclusions

Despite conventional strategies (surgery, chemotherapy, radiation), the prognosis of head and neck cancers is poor and do not exceed 20% at 10 years. Moreover, when the treatment is successful, the quality of life of patients is very altered. Thus, is important that the evolvement of new therapeutic strategies continue in order to develop safe, effective, and easy-to-use treatments presenting few side effects as possible. Immunotherapy is one of the most promising strategies for the cancer treatment. In HNSCC, different immunotherapy strategies have been developed in particular in HPV-induced HNSCC showing interesting results in preclinical studies.

In this work, we developed a DNA vaccination strategy able to induce non-infectious virus-like particles (VLPs) expressing the HPV-16 E7 antigen. This vaccine strategy has the advantage of requiring a simple, fast, inexpensive and widely production. After showing the in vitro ability of this approach to induce responses in human DCs, we have shown that in using both the ectopic and the intra-cheek orthotopic murine preclinical models of HPVinduced HNSCC, this plasmoVLP strategy allowed to generate tumor-specific CD8 + T-cell responses as well as good anti-tumor response. We also showed, in the orthotopic murine model of HNSCC, that when using an intra-cheek mucosal route of vaccination, this pVLP strategy is able to induce better tumor-specific CD8 + T cell and anti-tumoral responses in comparison to the intradermal route of vaccination.

The combination of conventional treatments and immunotherapy strategies appears to be a promising approach. Hence, it will be interesting to associate plasmoVLP vaccination using the intra-cheek route and conventional treatment like radiotherapy/chemotherapy and/or surgery. More importantly, while the E7 oncoprotein seems to be a good antigen for HPVinduced HNSCC therapeutic vaccines, p53 or antigens from the MAGE family appears to be interesting to test for the development of HPV-negative HNSCC strategies. Together, our results and the perspectives discussed above reveals that this strategy in combination with other conventional and innovative approaches constitute a feasible and promising therapy for HNSCCs, encouraging us to envision a clinical trial for this type of cancers.

Development of therapeutic vaccine strategies and pre-clinical animal tumor models for head and neck cancers

Abstract:

Head and neck squamous cell cancer (HNSCC) associated with alcohol and tobacco consumption, and recently with human papillomavirus-16 (HPV-16), have bad prognosis despite current therapies. Development of innovative vaccine strategies and adequate pre-clinical tumor models are required to better evaluate HNSCCs. We developed a DNA vaccination that creates non-infectious virus-like particles, which express HPV-16 E7 oncoprotein (pVLP-E7). Results showed that pVLP-E7 induced an E7-specific immune response in vivo and in vitro. Moreover, using an ectopic model of HNSCC that expresses E6/E7 (TC-1), we found that pVLP-E7 intradermic (ID) immunizations induced antitumoral responses at early stages. For larger established tumors, pVLP-E7 vaccines were only efficient when administered with TLR-7 and TLR-9 agonists. In an orthotopic model that shares anatomical and inflammatory features with human HNSCC we observed that intra-cheek (IC) infusion of either TC-1 or NR-S1 cells into mice elicited higher numbers of inflammatory infiltrates in the tumor compared to ectopic models. Using this orthotopic IC model, we found that mucosal IC pVLP-E7 vaccination elicited better vaccine-specific CD8+ T-cell responses than ID administration in naive and tumor-bearing mice. Furthermore, pVLP-E7 IC immunizations in combination with TLR agonists led to rejection of established tumors and long-term protection, both of which were associated with E7specific CD8+ T cell infiltration in tumors and lymph nodes. Our findings demonstrate that pVLP-E7 IC vaccination with adjuvants is efficient against these tumor models and together provides a valuable therapeutic strategy for HNSCCs.

Développement de stratégies vaccinales thérapeutiques et des modèles animaux de tumeurs pré-cliniques pour les cancers des voies aéro-digestives supérieures

Résumé:

Les cancers des voies aéro-digestives supérieures, liés à la consommation d'alcool et de tabac mais également à l'HPV-16, ont un pronostic médiocre malgré les traitements actuels. Le développement de nouvelles stratégies innovantes dans des modèles précliniques adaptés est ainsi nécessaire. Nous avons préalablement développé une stratégie vaccinale ADN permettant l'auto-assemblage in vivo de pseudoparticules virales non infectieuses exprimant l'oncoprotéine E7 de l'HPV-16 (pVLP-E7). Nous avons notamment montré que l'injection de pVLP-E7 en intradermique (ID) était capable d'induire de bonnes réponses anti-tumorales dans un modèle murin de cancer obtenu en injectant dans le flanc des cellules d'une lignée exprimant les antigènes E6 et E7 de l'HPV-16, mais qu'il était nécessaire d'ajouter des adjuvants de types agoniste de TLR 7 et 9 dans des tumeurs avancées. Afin de tester de nouvelles voies vaccinales dans un modèle pertinent, nous avons développé un modèle orthotopique intrabuccal présentant des caractéristiques anatomiques et inflammatoires plus proches des cancers observés chez l'homme que le modèle ectopique. Dans ce modèle, nous avons testé une voie vaccinale muqueuse intrajugale qui a montré de meilleures réponses T CD8+ spécifiques en comparaison à la voie ID. Nous avons montré que ce type de vaccination en association à des adjuvants, était efficace dans des tumeurs établies, en lien avec une infiltration intratumorale et ganglionnaire de lymphocytes T CD8+ spécifique, permettant également une protection lors de rechallenge tumoral. Cette stratégie apparaît donc prometteuse dans le traitement de ces cancers fréquemment récidivants.

Table 1 :

 1 Different clinical and biological characteristics of HNSCC ..................................... Table 2: Features of the ectopic and orthotopic models ..........................................................

Figure 1 :

 1 Figure 1: The tumor microenvironment ..................................................................................

Figure 2 :

 2 Figure 2: Immunosurveillance and immunoescape mechanisms ............................................

Figure 3 :

 3 Figure 3: Immune parameters linked with clinical outcome in OSCC (A) and OPSCC (B) ..

Figure 4 :

 4 Figure 4: Representation of the categories of preclinical cancer models in use .....................

Figure 5 :

 5 Figure 5: Strategies for the generation of genetically engineered mouse models ...................

Figure 6 :

 6 Figure 6: Differentiation and maturation of T cells in the thymus ...........................................

Figure 7 :

 7 Figure 7: T-cell activation ........................................................................................................

Figure 8 :

 8 Figure 8: Immunotherapy strategies and their action in the cancer-immunity cycle ...............

Figure 9 :

 9 Figure 9: Activation of innate and adaptive immunity by TLR agonists ................................

Figure 10 :

 10 Figure 10: Mechanisms of action of DNA vaccines ................................................................

Figure 11 :

 11 Figure 11: VLP and pVLP immunization ................................................................................

Figure 12 :

 12 Figure 12: Comparison of mucosal immune system in oral mucosa (a) and gut mucosa (b) ..

Figure 13 :

 13 Figure 13: Distribution and function of DCs in the murine oral mucosa. ................................

Figure 14 :

 14 Figure 14: Antigen delivery and presentation following sublingual or buccal vaccination ....

Figure 15 :

 15 Figure 15: Effects of Dbait ± chemotherapy on the growth of TC-1 tumors .........................

Figure 1 :

 1 Figure 1: The tumor microenvironment[START_REF] Koontongkaew | The tumor microenvironment contribution to development, growth, invasion and metastasis of head and neck squamous cell carcinomas[END_REF]

Figure 2 .

 2 Figure 2. The tumor microenvironment (TME). TME comprises different stromal cells in addition to tumor cells. These include vascular or lymphatic endothelial cells, supporting pericytes, fibroblasts, and both innate and adaptive infiltrating immune cells. Moreover, TME contains non-cellular components, including extracellular matrixes, growth factors, proteases, protease inhibitors and other signaling molecules that play important roles in stromal reactions in TME.

Figure 2 .

 2 Figure 2. The tumor microenvironment (TME). TME comprises different stromal cells in addition to tumor cells. These include vascular or lymphatic endothelial cells, supporting pericytes, fibroblasts, and both innate and adaptive infiltrating immune cells. Moreover, TME contains non-cellular components, including extracellular matrixes, growth factors, proteases, protease inhibitors and other signaling molecules that play important roles in stromal reactions in TME.

  Th2 cells and CD4+ T regulatory cells are capable of suppressing effective CD8+ antitumor responses. In fact, several investigators have found dysfunctional circulating and tumor-infiltrating T cells in HNSCC patients, with functional assays identifying multiple defects in T-cell activation and effector function, suggesting that the tumor has successfully suppressed an otherwise robust lymphocytic response [70-72]. Patterns of tumor-related leukocyte infiltration varies between primary tumors and metastatic lymph nodes in HNSCC with a local decrease in the number of CD8+ T-cells and increase in CD20+ B-cells being the most relevant findings. This indicated that suppression of local cellular immunity might be a mechanism by which tumor cells evade host immunity [73]. Patients with tumors expressing HPV16 had an increased frequency of CD8+ T cells specific for pep-

Figure 2 :

 2 Figure 2: Immunosurveillance and immunoescape mechanisms[START_REF] Schreiber | Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion[END_REF]

Figure 3 :

 3 Figure 3: Immune parameters linked with clinical outcome in OSCC (A) and OPSCC (B)[START_REF] Wallis | Clinical relevance of immune parameters in the tumor microenvironment of head and neck cancers[END_REF]

Figure 4 :

 4 Figure4: Representation of the categories of preclinical cancer models in use[START_REF] Mcconville | Preclinical Models of Tumor Growth and Response[END_REF]

Figure 5 :

 5 Figure5: Strategies for the generation of genetically engineered mouse models[START_REF] Cheon | Mouse models of cancer[END_REF]

  These cells then move to the thymic cortex, where TCR chains are also rearranged to form a mature  TCR, being later tested for positive selection. Here, functional TCRs that recognize MHC class I expressed on cortical epithelial cells become CD8 + single positive (CD4 -CD8 + ); those that are selected on MHC class II become CD4 + single positive (CD4 + CD8 -), and those that fails in recognizing MHC undergoes apoptosis and are cleared by thymic cortical macrophages. Thymocytes that survive positive selection move to the thymic medulla for negative selection. Here, cells that recognize with high affinity selfpeptides expressed by thymic medullary epithelial cells are removed by apoptosis, because of the potential risk of autoreactivity. Finally, cells that survive negative selection are exported to the peripheral circulation.

Figure 6 :

 6 Figure 6: Differentiation and maturation of T cells in the thymus[START_REF] Chaplin | Overview of the immune response[END_REF].

Figure 7 :

 7 Figure7: T-cell activation[START_REF] Kershaw | Gene-engineered T cells for cancer therapy[END_REF]

Figure 8 :

 8 Figure8: Immunotherapy strategies and their action in the cancer-immunity cycle[START_REF] Chen | Oncology meets immunology: the cancer-immunity cycle[END_REF]

Figure 9 :

 9 Figure 9: Activation of innate and adaptive immunity by TLR agonists (Thomas and Hassan, 2012).

  innovative DNA vaccine encoding an invariant chain-Pan-DR-Epitope (Li-PADRE) enhances specific CD8 + T-cell immune responses in HNSCCs models[START_REF] Wu | Innovative DNA vaccine for human papillomavirus (HPV)-associated head and neck cancer[END_REF]. Currently, naked DNA constructs encoding proteins from HPV-associated HNSCC are being tested in phase I (HPV-6) and phase I/II (HPV16/18) clinical trials[START_REF] Bloy | Trial watch: Naked and vectored DNA-based anticancer vaccines[END_REF].

Figure 10 :

 10 Figure 10: Mechanisms of action of DNA vaccines[START_REF] Xu | Intranasal DNA Vaccine for Protection against Respiratory Infectious Diseases: The Delivery Perspectives[END_REF].

  Recombinant retrovirus-based VLPs can be produced either ex vivo after cell transfection with plasmid DNA encoding wild type or chimeric Gag proteins and envelope glycoproteins or in vivo after injection of the same plasmid DNA named plasmo-retroVLPs (pVLPs). This vaccine strategy combines the efficiency of VLP-based vaccines with the simplicity and versatility of DNA-based vaccines, giving the advantage of a simple, fast, inexpensive and large-scale production. Not to mention, this technique keeps the particulate antigen presentation, and the production processes ensure reproducibility and quality in vaccine preparations. It has been previously demonstrated in our laboratory that pVLPs induce higher cellular[START_REF] Bellier | DNA vaccines encoding retrovirus-based virus-like particles induce efficient immune responses without adjuvant[END_REF] and humoral[START_REF] Bellier | DNA vaccines expressing retrovirus-like particles are efficient immunogens to induce neutralizing antibodies[END_REF] immune responses against vaccine antigens in comparison to standard DNA vaccines that do not form VLPs. Likewise, pVLP pseudotyped with HCV E1 and E2 glycoproteins, used as boost in a mice primed with serotype 5 recombinant adenovirus vectors (rAD5) containing E1/E2, induced significantly increased E1 and E2 specific IFN- CD4 + and CD8 + T-cell responses and E2-specific antibody generation[START_REF] Desjardins | Recombinant retrovirus-like particle forming DNA vaccines in prime-boost immunization and their use for hepatitis C virus vaccine development[END_REF]. This same VLP strategy induced strong specific T-cell responses and neutralizing antibodies, in the absence of recombinant virus vector priming[START_REF] Huret | Recombinant retrovirusderived virus-like particle-based vaccines induce hepatitis C virus-specific cellular and neutralizing immune responses in mice[END_REF].

Figure 11 :

 11 Figure 11: VLP and pVLP immunization

Figure 12 :

 12 Figure 12: Comparison of mucosal immune system in oral mucosa (a) and gut mucosa (b)[START_REF] Novak | The immune privilege of the oral mucosa[END_REF]

Figure 14 :

 14 Figure14: Antigen delivery and presentation following sublingual or buccal vaccination[START_REF] Kraan | Buccal and sublingual vaccine delivery[END_REF]

Figure 1 .

 1 Figure 1. Oral Squamous Cell Cancers are inflammatory neoplasms. Single cell suspensions were obtained from human OSCC samples (n=7) and healthy gingiva (n=7), and analyzed by flow cytometry. A, Gating strategy: after dead cells and doublets exclusion, nine CD45 + gated subpopulations were identified: (a) CD15 + CD11b + (granulocytes), (b) CD14 + CD11b + (macrophages), (c) CD19 + CD3 -(B cells), (d) CD56 + CD3 - (Natural Killer cells), (e) CD3 + CD4 + (CD4 + T cells), (f) CD3 + CD8 + (CD8 + T cells), (g) CD3 + CD4 + CD25 + FoxP3 + CD127 -(Tregs), (h) Lin-1 neg (CD3 -CD19 -CD56 -) HLA-DR + CD11c -(pDC) and (i) Lin-1 neg CD11c + HLA-DR + CD14 -(mDC). B, Total number of CD45 + cells and of different CD45 + gated subsets are presented per gram (g) of tumor. NS, non-statistical difference = p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2 .

 2 Figure 2. Orthotopic intra-cheek tumors: models for OSCCs. C57BL/6 mice (4-5 mice per group) were injected with TC-1-Luc cells using subcutaneous (SC), intra-cheek (IC) or intra-lingual (IL) routes. A, A representative bioluminescence imaging is shown one-week after TC-1-Luc injection (left). Kaplan-Meier curves show tumor-specific survival rates (right). B and C, Flow cytometry analysis of cell suspensions from tumors and tumor-draining lymph nodes (TdLNs), two weeks after TC-1-Luc cell injection. For gating strategy, see supplementary Figure S1. Axillar (ALN) and cervical (CLN) lymph nodes from non-immunized mice were used as controls for SC and IC TdLNs, respectively. Numbers of CD45 + cells and of different CD45 + gated cell

Figure 3 .

 3 Figure 3. Advantage of IC vaccinations for inducing high loco-regional specific CD8 + T-cell responses. A, IFN- ELISpot assay performed at day 10 in LNs and spleen cells from C57BL/6 mice immunized at days 0-2-4

Figure 4 .

 4 Figure 4. Adjuvants enhanced the anti-tumor effects of IC vaccinations. C57BL/6 mice (8-12 mice per group) bearing intra-cheek TC-1-Luc tumors were ID or IC immunized at days 7-9-11 (arrow) with pVLP-E7 in the presence of adjuvants: CpG-ODN + Imiquimod (CpG/IMQ). As controls, one group of mice received CpG/IMQ alone and another received PBS. A, Monitoring of tumor volume measured every 2-4 days; tumor-free rates are indicated in cursive. B, Kaplan-Meier curves showing tumor-free survival rates. Data presented were pooled from three separate experiments. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

Figure 5 .

 5 Figure 5. The therapeutic effect of IC vaccinations correlates with better specific CD8 + T-cell responses.C57BL/6 mice (4-6 mice per group) bearing intra-cheek TC-1-Luc tumors were ID or IC immunized at days 7-9-11 (arrow) with pVLP-E7 in the presence of adjuvants: CpG-ODN + Imiquimod (CpG/IMQ). As controls, one group of mice received CpG/IMQ alone and another received PBS. A, Detection by flow cytometry of E7-tetramer + cells in single cell suspensions obtained from tumors (pooled) and TdLNs at day 18. Numbers of E7 49-57 -tetramer + , CD8 + and CD49a + expressing cells/mm 3 (tumors) and per x10 6 cells (TdLNs) are presented. B, E7-specific IFN- ELISpot assay: cells from TdLNs were loaded with E7 49-57 peptide and spot numbers are expressed by 10 6 cells. The background obtained with cells not pulsed with the E7 49-57 peptide has been subtracted C, Number of Treg cells/mm 3 of tumors. D, Effect of the in vivo CD8 depletion in C57BL/6 mice bearing intra-cheek TC-1-Luc tumors when IC immunized with pVLP-E7 combined with CpG/IMQ at days7-9-11 (arrow). One week before the

Figure 6 .

 6 Figure 6. Long-term protection effects of IC vaccinations. Immunized C57BL/6 mice (3-7 mice per group) showing complete tumor regression, were rechallenged with TC-1-Luc cells at day 200. A, Monitoring of tumor volume measured every 2-4 days. B, Blood detection by flow cytometry of E7 49-57 -tetramer expressing cells in CD8 + T cells at day 250. A representative analysis is shown C, Percentages of CD62L, CD44 and CD49a cells in CD8 + T cells. Data presented have been obtained from 3-7 mice. NS, non-statistical difference = p > 0.05; *, p < 0.05; **, p < 0.01.

  Supplementary Figure S1. Gating strategy for multiparametric flow cytometry analysis of cell suspensions from tumors, TdLNs and spleen in mice. After dead cells and doublets exclusion, nine CD45 + gated subpopulations were identified: (a) Ly6G + CD11b + (granulocytes), (b) NKp46 + I-A/I-E -(NK cells), (c) CD11c + Ly6C + CD11b -(pDC), (d) CD11c + excluding pDCs (mDC), (e) I-A/I-E + CD11b + CD11c -(macrophages), (f) CD19 + CD3 -(B cells), (g) CD3 + CD4 + (CD4 + T cells), (h) CD3 + CD8 + (CD8 + T cells) and (icytometry analysis of spleen cells from mice injected with TC-1-Luc cells. C57BL/6 mice (4-5 mice per group) were injected with TC-1-Luc cells using two routes: subcutaneous (SC) and intra-cheek (IC). Two weeks after, single cell suspensions were obtained from the spleens were analyzed by multiparametric flow cytometry. Spleens from non-immunized mice were used as controls. Total numbers of CD45 + cells and of the different innate and adaptive subsets are presented per x10 6 cells. Results are expressed as mean ± SEM. NS: non-statistical difference = p > 0.05.

  therefore, they are not expressed in normal host cells (e.g. mutated p53 protein or the E6/E7 HPV oncoproteins). HPV is an ideal vaccine target, due to the expression of their viral oncoproteins in HPV-induced tumors to maintain the transformed state. In HNSCC, most of the current studies include the development of vaccination strategies in HPV-induced cancers[START_REF] Gildener-Leapman | Tailored immunotherapy for HPV positive head and neck squamous cell cancer[END_REF], which represent 20-93% of cancers of the oropharynx HNSCCs (tonsil and base of tongue). These strategies, as well as the vaccine strategy we used, are based on the expression of the HPV-16 E7 or E6 oncoproteins that are over-expressed in these cancers. However, the main risk factors of HNSCC cancers of the oral cavity are alcohol and tobacco, and therefore they are not mainly associated with HPV infections. At present day, no biomarker of HPV-negative HNSCC is considered valid; thus, it is necessary to develop new therapeutic targets to improve the prognosis of these patients.In HPV-negative HNSCC, overexpressed wild type (WT) TAAs, such as p53, are potential vaccine targets. Although TP53 mutation is the most commonly identified mutation in HPV-negative HNSCC, most mutations result in the accumulation of p53; nonmutated portions of the protein are susceptible to degradation into WT peptide sequences appropriate for immune presentation. A phase I trial examining p53 multipleepitope/dendritic cell vaccine in HNSCC patients was reported. Following definitive therapy, patients with locally advanced HNSCC were vaccinated with WT p53 sequences pre-loaded onto autologous DCs. Results shown a two-year disease-free survival of 88% and increased post-vaccination p53-specific T-cell frequencies in 11/16 patients (69%) with IFN- secretion detected in 4/16 patients[START_REF] Schuler | Phase I dendritic cell p53 peptide vaccine for head and neck cancer[END_REF]. Nonetheless, one of the inconveniences of DC vaccination lies in the fact that is really hard to produce them.Otherwise, strategies of immunotherapy targeting the in vivo DCs to obtain a T specific stimulation were studied showing promising results. Thus, it would be interesting to use other types of vectors in order to obtain anti-p53 responses in HNSCC tumors.

  Neoplastic cells escape the lethal effects of chemotherapy and radiotherapy and thus, acquire resistance; through different mechanism including enhanced intracellular detoxification, increased ability to repair DNA and enhanced tolerance to DNA damage. Therefore, inhibiting DNA repair is a promising strategy for increasing the efficacy of conventional DNA-damaging agents such as chemotherapy and radiotherapy. Recently, the group of Marie Dutreix in collaboration with DNA Therapeutics® has developed a new class of drugs for sensitizing resistant cancers to conventional therapies by inhibiting DNA repair. "DNA repair bait" (Dbait) molecules inhibit DNA repair pathways by trapping proteins involved in DNA damage sensing and signaling. Dbait trap key damage signal transducers (the DNA-dependent protein kinase (DNA-PK) and the Poly-ADP-Ribo-Polymerase (PARP)), and trigger their activation, which amplifies "false" DNA damage signaling. These "false" signals impair the recruitment of downstream DNA repair enzymes at the damage sites on chromosomes leading to repair inhibition and accumulation of unrepaired damage

TC- 1

 1 injected into the flank of immunocompetent C57BL/6 mice. Interestingly, the data (figure 1) clearly show that Dbait + chemotherapy significantly (p < 0.05) diminished the growth of TC-1 tumors comparatively to the control group (non-treated animals) or Dbait alone or chemotherapy alone.

Figure 15 :

 15 Figure 15: Effects of Dbait  chemotherapy on the growth of TC-1 tumors

Table 1 :

 1 Different clinical and biological characteristics of HNSCC[START_REF] Leemans | The molecular biology of head and neck cancer[END_REF]

	Incidence	Decreasing	Increasing
	Etiology	Smoking, excessive alcohol use Oral sex
	Age	Above 60 years	Under 60 years
	Field cancerization Yes	Unknown
	TP53 mutations	Frequent	Infrequent
	Predilection site	None	Oropharynx
	Prognosis	Poor	Favorable

Table 2 :

 2 Features of the ectopic and orthotopic models

  Many viral vectors are used for the development of vaccines against HIV, tuberculosis or malaria among others. Several clinical trials for cancer immunotherapy were conducted in metastatic renal cell carcinoma, colon cancer, prostate cancer and non-small cell lung cancer and other cancers using viral vectors such as vaccinia virus, modified virus Ankara, Fowlpox virus, alphavirus, adenovirus, measles virus, herpes simplex virus and vesicular stomatitis virus. The intrinsic properties of each virus carry advantages and disadvantages (immunogenicity, manipulation, no mutagenesis, tropism, tumor specificity, etc.). Nonetheless, some vectors induce the development of host-induced neutralizing antibodies to the vector itself, limiting its continued use. Many of this viral vector-based vaccines, specially vaccinia virus Ankara (MVA), vaccinia or Avipox and alphavirus vectors have been tested in clinical trials of melanoma, renal, colon, prostate, and non -small lung cancer showing humoral and cellular immune responses and better survival in some of them

  1,2 , Maude Guillot-Delost 1 , Kae Tanaka 1 , Aline Le Moignic 1 , Clara Noizat 1 , Claude Baillou 1 , Véronique Mateo 1 , Antoine F. Carpentier 3 , Eric Tartour4 , Chloé Bertolus 1,5 , Bertrand Bellier 6 , Géraldine Lescaille 1,2* and François M.

	Lemoine 1,7*
	Authors' Affiliations:

Sorbonne Universités, UPMC/Paris 06, UMR-S INSERM U1135, CNRS ERL 8255, Centre d'Immunologie et Maladies Infectieuses (CIMI-Paris); Paris, France.

Paris Diderot/Paris 07, Sorbonne Paris Cité; Assistance Publique-Hôpitaux de Paris (AP-HP), Groupe hospitalier Pitié-Salpêtrière, Department of Odontology; Paris, France.

[START_REF] Majchrzak | Oral cavity and oropharyngeal squamous cell carcinoma in young adults: a review of the literature[END_REF] Université Paris 13; AP-HP, Hôpital Avicenne, Department of Neurology; Bobigny, France

.[START_REF] Fakhry | HPV and head and neck cancers: state-of-thescience[END_REF] Paris Descartes/Paris 05, Sorbonne Paris Cité; INSERM U970, Paris-Cardiovascular Research Center (PARCC); AP-HP, Hôpital Européen Georges Pompidou, Service d'Immunologie Biologique; Paris,

France.[START_REF] Ho | Decision making in the management of recurrent head and neck cancer[END_REF] Sorbonne Universités, UPMC Univ-Paris 06; AP-HP, Groupe hospitalier Pitié-Salpêtrière, Department of Maxillo-facial Surgery; Paris

, France.[START_REF] Lescaille | Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers[END_REF] Sorbonne Universités, UPMC/Paris 06, UMR-S INSERM U959, CNRS, FRE 3632, Immunology-Immunopathology-Immunotherapy (I3); Paris, France.

Acknowledgements

This work was financially supported by grants from Fondation ARC pour la Recherche sur le Cancer and La Ligue Nationale contre le Cancer. RM received a fellowship from the Association pour la Recherche sur la Moelle Osseuse (ARMO). JR received a one-year fellowship from INSERM. JR and KT were partly supported by the Fondation "Les Gueules Cassées".

DCs in the gingival mucosa

The gingiva does not contain submucosa and the lamina propria is bound directly to the membrane that lines the alveolar bone. In mice, three DC subsets are distinguished: iDCs represents the largest DC population, low percentages of LCs are located in the gingival epithelium in comparison to buccal and skin tissues, and a minor population of CD103 + iDCs [START_REF] Arizon | Langerhans cells down-regulate inflammation-driven alveolar bone loss[END_REF]. In humans, a low frequency of LCs with higher expression of the IgE Fc receptor (FcRI) was detected in vestibular mucosa in comparison to skin (Allam et al., 2008).

Moreover, LCs and CD68 + macrophages contribute to antigen/allergen uptake in human gingiva [START_REF] Mascarell | Characterization of oral immune cells in birch pollenallergic patients: impact of the oral allergy syndrome and sublingual allergen immunotherapy on antigen-presenting cells[END_REF]. In gingiva, LCs were not directly involved in T-cell priming while iDCs presents peptides to both CD4 + and CD8 + T cells [START_REF] Arizon | Langerhans cells down-regulate inflammation-driven alveolar bone loss[END_REF].

Role of T cells in oral immunity

Mucosal T cells are divided into two major subsets based on TCR and co-receptor type they express: conventional (type a) mucosal T cells that express an TCR with CD4 or CD8  as co-receptor, comprising antigen-induced effector/memory T cells; and non conventional (type b) mucosal T cells that contain TCR or TCR conventional TCR co-receptors, these cells reside permanently at the mucosal effector sites [START_REF] Cheroutre | Mucosal T lymphocytes--peacekeepers and warriors[END_REF]. Type a mucosal T cells reside within the lamina propria, whereas most type b lymphocytes are associated with the epithelium. T-cells populations such as intraepithelial lymphocytes (IEL) in the oral-pharyngeal mucosa are less studied. Nonetheless, intraepithelial  and  T cells have been described in the oral mucosa [START_REF] Patinen | Intraepithelial lymphocytes bearing the gamma/delta receptor in the oral and jejunal mucosa in patients with dermatitis herpetiformis[END_REF]. Furthermore, CD8 + IELs in the oralpharyngeal mucosa have been identified recently [START_REF] Wu | The mucosal immune system in the oral cavity-an orchestra of T cell diversity[END_REF].

IELs and conventional lymphocytes undertake the elimination of foreign antigen; conventional CD8 + T cells raise their numbers in the oral mucosa in response to OSCC tumors [START_REF] Zancope | Differential infiltration of CD8+ and NK cells in lip and oral cavity squamous cell carcinoma[END_REF]. These cells showed a higher expression of perforin and Granzyme B proteins [START_REF] Costa | Distinct expression of perforin and granzyme B in lip and oral cavity squamous cell carcinoma[END_REF]. Otherwise, T cells are also implicated in oral mucosal tolerance, CD4 + T cells seems to be more important for the perpetuation of tolerance than CD8 + T cells, additionally  T cells plays an important role in mucosal homeostasis [START_REF] Novak | The immune privilege of the oral mucosa[END_REF].

Furthermore, the expression of the mucosal integrin subunit  CD49a, by effectors specific CD8 + T cells, was correlated to the induction of a mucosal homing in this cells and their effective anti-tumor response in human mucosal lung cancer (Sandoval et al 2013).

Disclosure of Potential Conflicts of Interest:

No potential conflicts of interest were disclosed. Head and neck squamous cell cancers (HNSCCs) arise from various mucosal sites of the upper aero-digestive tract. Despite current therapy, HNSCCs remain with a poor prognosis raising the need for the development of innovative therapies. We have previously described the efficacy of therapeutic intra-dermic (ID) plasmid DNA vaccines that encode retroviral virus-like particles carrying the E7 oncoprotein from HPV-16 (pVLP-E7) as a tumorassociated antigen model, to control the growth of ectopic HPV-derived TC-1 tumors. In order to establish a better pre-clinical model for HNSCCs, we first validated a new orthotopic model consisting of infusing TC-1 cells or NR-S1 cells, that derived from a spontaneous oral cavity murine tumor, into the cheek of animals. Interestingly, TC-1 and NR-S1 tumor microenvironments resemble to those of human HNSSCs. In order to identify a better immunization route for therapeutic vaccines that would correlate with efficient anti-tumor effects and a long-term protection for such mucosal neoplasms, we then evaluated different mucosal immunizations with pVLP-E7. Our findings showed that intra-cheek (IC) vaccinations using pVLP-E7 in combination with TLR agonists (Imiquimod and CpG-ODN), as compared to ID vaccination, gave rise to a higher mobilization of CD8 + specific effector T cells in both tumor draining lymph nodes and tumor microenvironment resulting to better anti-tumor effects and to a long-term protection against tumor rechallenge. Thus, using this new intra-cheek tumor model and an innovative IC mucosal vaccination route, we provide valuable pre-clinical data to envision the use of pVLPs in combination with adjuvants, as therapeutic vaccines, for HNSCCs.

Word

Material and Methods

Human samples

Tumors samples were obtained during surgical resection of primary OSCCs (Maxillo-Facial Surgery department, Pitié-Salpêtrière Hospital; Paris, France). Gingival tissues were collected from healthy subjects undergoing preventive wisdom tooth extraction (Odontology department, Pitié-Salpêtrière Hospital; Paris, France). Samples were obtained after informed written consent according to local ethic committee authorization.

Mice

Seven-to 8-week-old female C57BL/6JRj (H-2 b ) or C3H/HeNRj (H-2 k ) mice were purchased from Janvier (Le Genest Saint Isle, France) and kept under specific pathogen-free conditions at the UMS28 animal facility (UFR 969, Pitié-Salpêtrière). Experiments were performed according to the European Economic Community guidelines and approved by local ethics committee.

Cell lines

TC-1 cells (CRL-2785; American Type Culture Collection [ATCC], Manassas, VA, USA) have been previously described [START_REF] Lescaille | Efficacy of DNA vaccines forming e7 recombinant retroviral virus-like particles for the treatment of human papillomavirus-induced cancers[END_REF]. TC-1-Luc cells (a generous gift from T.C. Wu, John Hopkins University, MD, USA) were genetically engineered to express the luciferase protein. NR-S1 cells (kindly provided by Dr. K. Ando, National Institute of Radiological Science, Tokyo, Japan), derived from a spontaneous oral carcinoma in C3H mice [START_REF] Usui | Effect of PS-K, a protein polysaccharide, on pulmonary metastases of a C3H mouse squamous cell carcinoma[END_REF].

Discussion and perspectives

The aim of my work was to develop a therapeutic DNA strategy able to create viruslike particles (VLPs) for the immunotherapy of HNSCC using an adequate preclinical model.

While VLPs are well studied in various models and are known to raise antibody responses, especially in the preventive vaccination for HPV infections [START_REF] Koutsky | A controlled trial of a human papillomavirus type 16 vaccine[END_REF], little is known about anti-tumoral efficacy. However it has been shown that VLPs may induce T-cell responses when an appropriate antigen is introduced into the VLPs platform. Given the difficulty of VLP production and the possibility of generating the particles in vivo, we chose to use a DNA strategy called plasmo-retroVLP (pVLP), which involves injecting the DNA required to produce the constituent proteins of VLPs. This strategy has many advantages, firstly an easy manufacture and inexpensive large scale, and secondly a reproducibility and a satisfactory quality of the vaccine preparations.

To validate our strategy, we have chosen to use a TC-1 tumor murine model expressing the oncogenic proteins E6 and E7 of HPV-16, known to be expressed in HPVinduced HNSCC. Thus, in order to induce anti-tumor T-cell responses against E7, we have inserted the gene for this oncoprotein in the Gag gene of MuLV, for production of VLP-E7;

for added security, we chose to use a deleted protein receptor site on the Rb protein, to make the resultant protein non-oncogenic (ΔE7). Furthermore, we have shown that our strategy of pVLP-E7 can produce IFN-γ responses in human DCs and also in the mice, where in addition they allowed the generation of anti-tumor responses using a subcutaneous TC-1 tumor model.

Since the microenvironment of cancer is dependent on their location, we have developed various orthotopic models of oral cavity HNSCC. Thus, we have used an intralingual tumor model as described before (Sandoval et al., 2013), as well as an intra-cheek tumor model. Given the very limited survival in the intra-lingual group and the difficulty for monitoring tumor growth, we decided to test our strategy in the intra-cheek tumor model.

Using the ectopic subcutaneous model, we have shown that intradermal vaccination of pVLP-E7 in combination with adjuvants (TLR agonists 7 and 9) made it possible to induce antitumor responses with long-term survival of 50%. Nonetheless, this response was only 25%

when using the orthotopic tumor model.

Combination with radiotherapy

Concerning the radiotherapy, it has been shown that it can stimulate the immune system, and have a synergic anti-tumor effect when combined with immunotherapy strategies or immune-modulators [START_REF] Lee | Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment[END_REF]. Radiation results in tumor cell death but also in increased expression of tumor associated antigens and MHC class I molecules by the tumor enhancement of antigen presentation and cross-presentation by DCs, potentially through local release of cytokines such as IFN- or IFN- [START_REF] Hiniker | Immunotherapy and radiation[END_REF].

Interest in combining radiation and immune-based therapies for the treatment of cancer is growing; indeed, a great deal of preclinical research into combining radiation and therapeutic vaccination has been translated into clinical studies especially for prostate cancer [START_REF] Hodge | The tipping point for combination therapy: cancer vaccines with radiation, chemotherapy, or targeted small molecule inhibitors[END_REF]. This strategy was used in an orthotopic murine model of HNSCC, where tumor-bearing mice were vaccinated with tumor cells previously irradiated and infected with vaccinia virus expressing IL-2; this approach resulted in larger number of specific CD4 + and CD8 + T cells infiltrating the spleen and tumor-draining lymph nodes [START_REF] Dasgupta | Recombinant vaccinia virus expressing interleukin-2 invokes anti-tumor cellular immunity in an orthotopic murine model of head and neck squamous cell carcinoma[END_REF]. More recently, a retrospective clinical study in HNSCC patients that were treated with IMRT (Intensity-Modulated Radiation Therapy), a new type of radiotherapy, and concurrent cetuximab showed the feasibility of this strategy. However, further investigations are necessary [START_REF] Zwicker | IMRT reirradiation with concurrent cetuximab immunotherapy in recurrent head and neck cancer[END_REF]. Furthermore, low dose radiation associated with HPV E7 DNA strategy made TC-1 tumor cells more susceptible to lysis of anti-E7 CTL and significantly increased therapeutic antitumor effects in mice [START_REF] Tseng | Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts[END_REF]. Importantly, a recent study combining irradiation with Shiga Toxin B (StxB)-based HPV-16 E7 vaccination induced specific CD8 + T-cell antitumor responses and CD8+ T cell memory [START_REF] Mondini | Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer[END_REF].

These important findings pave the way for therapeutically relevant associations between certain chemotherapy and immunotherapy strategies, and between radiation and immunostimulatory therapies, to increase the anti-tumor immunity.

Preclinical results showing an evidence of the PD-1/PD-L1 pathway in immune resistance of HPV-associated HNSCC [START_REF] Lyford-Pike | Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma[END_REF], the strong cytoplasmic expression of PD-L1 in patients with locally advanced OSCCs (Oliveira- [START_REF] Oliveira-Costa | Gene expression patterns through oral squamous cell carcinoma development: PD-L1 expression in primary tumor and circulating tumor cells[END_REF], as well as the promising preliminary results of clinical trials evaluating pembrolizumab (KEYNOTE-012 study), tremelimumab (MEDI4736 study) and ipilimumab in combination with other therapies in patients with HPV and non-HPV associated HNSCCs, bring us the rationale to combine this therapy in HNSCCs [START_REF] Swanson | Rationale for combined blockade of PD-1 and CTLA-4 in advanced head and neck squamous cell cancer-review of current data[END_REF].

Since TGF- induces immunosuppressive Tregs and other mechanisms that can support tumor growth, it constitutes another interesting target for cancer therapy. Using this approach, monotherapy with 1D11, a mAb that neutralizes TGF-, does not show any impact on tumor growth in a subcutaneous TC-1 tumor model; nonetheless combination of this strategy with an E7 peptide vaccine significantly improved the vaccine efficacy to reduce tumor size [START_REF] Terabe | Synergistic enhancement of CD8+ T cell-mediated tumor vaccine efficacy by an anti-transforming growth factor-beta monoclonal antibody[END_REF].