ORIGINAL RESEARCH published: 07 May 2021 doi: 10.3389/fimmu.2021.643298 Immunomodulation of T Helper Cells by Tumor Microenvironment in Oral Cancer Is Associated With CCR8 Expression and Rapid Membrane Edited by: Vitamin D Signaling Pathway Lewis Z. Shi, University of Alabama at Birmingham, 1 ´ 2 3,4,5 1 United States Marco Fraga , Milly Yañez , Macarena Sherman , Faryd Llerena , Mauricio Hernandez 6, Guillermo Nourdin 6, Francisco A´ lvarez 6, Joaqu´ın Urrizola 7, Reviewed by: Ce´ sar Rivera 8, Liliana Lamperti 1,9, Lorena Nova 10, Silvia Castro 1, Omar Zambrano 11, Hongru Zhang, 11 ´ 12 12 12 12 University of Pennsylvania, Alejandro Cifuentes , Leon Campos , Sergio Moya , Juan Pastor , Marcelo Nuñez , 12 12 ´ 1 ´ 13 14 United States Jorge Gatica , Jorge Figueroa , Felipe Zuñiga , Carlos Salomon , Gustavo Cerda , 5 1 15 16 Greg M. Delgoffe, Ricardo Puentes , Gonzalo Labarca , Mabel Vidal , Reuben McGregor 1 University of Pittsburgh, and Estefania Nova-Lamperti * United States 1 Molecular and Translational Immunology Laboratory, Clinical Biochemistry and Immunology Department, Pharmacy Faculty, *Correspondence: Universidad de Concepcio´ n, Concepcio´ n, Chile, 2 Anatomy Pathology Unit and Dental Service, Oral Pathology Department, Estefania Nova-Lamperti Hospital Las Higueras, Talcahuano, Chile, 3 Anatomy Pathology Unit, Hospital Guillermo Grant Benavente and Universidad [email protected]; de Concepcio´ n, Concepcio´ n, Chile, 4 Head and Neck Service, Hospital Guillermo Grant Benavente, Concepcio´ n, Chile, [email protected] 5 Dental Service, Hospital Guillermo Grant Benavente, Concepcio´ n, Chile, 6 MELISA Institute, San Pedro de la Paz, Chile, 7 Oral Maxillofacial Surgery Department, Dental Faculty, Universidad San Sebastia´ n, Concepcio´ n, Chile, 8 Department of Specialty section: Stomatology, Universidad de Talca, Talca, Chile, 9 PeveGen Laboratory, Concepcio´ n, Chile, 10 Centro de Salud Familiar This article was submitted to (CESFAM) Penco Lirque´ n, Penco, Chile, 11 Surgery Service, Hospital Las Higueras, Talcahuano, Chile, 12 Dental Service, Cancer Immunity and Maxillofacial Surgery Department, Hospital Las Higueras, Talcahuano, Chile, 13 Exosome Biology Laboratory, Centre for Immunotherapy, Clinical Diagnostics, UQ Centre for Clinical Research, Royal Brisbane and Women’s Hospital, Faculty of Medicine + a section of the journal Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia, 14 Advanced Microscopy Centre, Universidad Frontiers in Immunology de Concepcio´ n, Concepcio´ n, Chile, 15 Computer Science Department, Universidad de Concepcio´ n, Concepcio´ n, Chile, 16 Received: 17 December 2020 Department of Molecular Medicine and Pathology, School of Medical Sciences, The University of Auckland, Auckland, Accepted: 22 April 2021 New Zealand Published: 07 May 2021 Citation: The immune system plays a key role in the protective response against oral cancer; ´ Fraga M, Yañez M, Sherman M, however, the tumor microenvironment (TME) impairs this anti-cancer response by Llerena F, Hernandez M, Nourdin G, A´ lvarez F, Urrizola J, Rivera C, modulating T helper (Th) responses and promoting an anti-inflammatory environment. Lamperti L, Nova L, Castro S, Regulatory T cells (Tregs) and Th2 effector cells (Teff) are associated with poor prognosis Zambrano O, Cifuentes A, Campos L, Moya S, Pastor J, Nuñez M, Gatica J, in oral squamous cell carcinoma (OSCC). However, the main immunomodulatory Figueroa J, Zu´ ñiga F, Salomo´ nC, mechanisms associated with the enrichment of these subsets in OSCC remain Cerda G, Puentes R, Labarca G, unknown. We characterized Th-like lineages in Tregs and Teff and evaluated Vidal M, McGregor R and Nova-Lamperti E (2021) immunomodulatory changes induced by the TME in OSCC. Our phenotypic data Immunomodulation of T Helper Cells revealed a higher distribution of tumour-infiltrating CCR8+ and Th2-like Treg in OSCC by Tumor Microenvironment in Oral Cancer Is Associated With CCR8 compared with non-malignant samples, whereas the percentages of Th1 cells were Expression and Rapid Membrane reduced in cancer. We then analyzed the direct effect of the TME by exposing T cell Vitamin D Signaling Pathway. subsets to cancer secretomes and observed the OSCC secretome induced CCR8 Front. Immunol. 12:643298. doi: 10.3389/fimmu.2021.643298 expression and reduced cytokine production from both subsets. Transcriptomic Frontiers in Immunology | www.frontiersin.org 1 May 2021 | Volume 12 | Article 643298 Fraga et al. T-Cell Immunomodulation in Oral Cancer analysis showed that the co-culture with OSCC secretome induced several gene changes associated with the vitamin D (VitD) signaling pathway in T cells. In addition, proteomic analysis identified the presence of several proteins associated with prostaglandin E2 (PGE2) production by rapid membrane VitD signaling and a reduced presence of the VitD binding protein. Thus, we analyzed the effect of VitD and PGE2 and observed that VitD promotes a regulatory Th2-like response with CCR8 expression whilst PGE2 also modulated CCR8 but inhibited cytokine production in combination with VitD. Finally, we evaluated the presence of CCR8 ligand in OSCC and observed increased chemokine CCL18, which was also able to upregulate CCR8 in activated Th cells. Overall, our data showed the immunomodulatory changes induced by the TME involving CCR8 expression and regulatory Th2 phenotypes, which are associated with PGE2 mediated VitD signaling pathway and CCL18 expression in OSCC. Keywords: oral cancer, immunomodulation, cancer immunology, Th-like Tregs, CCR8 INTRODUCTION higher in low-grade invasive OSCC cell lines than high-grade invasive OSCC cell lines (12, 13). Therefore, novel mechanisms Oral cancer is a malignant neoplasm developed in the oral cavity need to be addressed to understand how this cancer modulates with high mortality and morbidity due to late-stage diagnosis the immune system. In terms of metabolic changes, glycolysis- and high incidence of metastasis (1). Oral squamous cell related proteins and mitochondrial enzymes (14), are also carcinoma (OSCC) is the most common type of oral cancer, significantly increased in the carcinogenesis of OSCC making it representing more than 90% of the cases, and it has been linked is possible that the active glycolytic activity of cancer cells also with uncontrolled proliferation of squamous epithelial cells due affects the function of the immune cells. In terms of the immune to environmental-mediated genetic mutations. Risk factors such repertoire, it has been shown that OSCC includes cells with a as long-term use of tobacco, alcohol abuse, excessive sun pro-tumoral role such as tumor associated macrophages exposure, human papillomavirus (HPV) infection and a (TAMs), cancer-associated fibroblasts (CAF) and regulatory T weakened immune system have been associated with OSCC (2, cells (Tregs) (11). 3). In fact, it has been proposed that the origin of oral cancer is Previous data from our lab characterized the Th-like Tregs associated with DNA alteration mediated by environmental based on the expression of three chemokine receptors, immune carcinogens, since 3 to 6 mutations are required to transform a transcriptomic profiles and specific lineage cytokine production, healthy cell to a malignant cell (4). It is the impaired or defining Th1 as CXCR3+CCR6-CCR4+, Th2 as CXCR3-CCR6- overwhelmed anti-tumor immune response in the patient is CCR4+,Th17asCXCR3-CCR6+CCR4+ and Th1/17 the main factor that favors subsequent tumor progression (5). CXCR3+CCR6+CCR4+ (15). We found that Th2-like Tregs This altered response is not only associated with cancer expressed CCR8 and exhibited higher viability than other Th- cells escaping the immune control, but also to the like Tregs subsets, however suppression capacity was similar immunomodulatory effects of the tumor microenvironment by between subsets. However, Th2-like Tregs and Th2 Teff migrated contact dependent and soluble mechanisms, promoting a more than other Th-like subsets a phenomenon not mediated by regulatory immune repertoire and inducing an anti- CCR4 expression. Finally, we analyzed the presence of Th-like inflammatory environment. Tregs in blood, thymus, spleen, liver, skin, colon and tissues and The immunomodulatory mechanisms exerted by the tumor blood from patients with melanoma and colon cancer. We microenvironment include the contribution of cancer-associated observed a high presence of Th2-like Tregs and Th2 effector immune cells, the expression of inhibitory checkpoints (6) and cells (Teff) in melanoma and colorectal cancer at late-stage. Here the production of soluble factors such as proteins, metabolites, we progress these findings by investigating the distribution of chemical factors (7–9) and extracellular vesicles (10). In OSCC, these subsets in a cancer that has been traditionally associated the presence of cytokines such as IL-4, IL-5, IL-10, TGF-b, IL-17, with late-stage detection to evaluate if there is a specific subset IL-1a and immune-checkpoint inhibitors such as PD-L1 and enriched in well-established tumors and the main mechanism Indoleamine 2,3-Dioxygenase 1 (IDO1) have been associated associated with the enrichment of Th2 subsets in cancer areas. with poor prognosis (11). Several chemokines have also been In this study we analyzed the distribution of tissue resident associated with immunomodulation in OSCC such as CCL18, Th-like Tregs and Teff in OSCC compared to non-malignant CXCL13 and CCL4. It is however not clear whether the biopsies allowing us to investigate