Head and neck squamous cell carcinomas (HNSCCs) belong to a group of diverse tumors, which can be induced by infection with human papillomavirus (HPV) or tobacco and alcohol consumption. The viral etiology of HNSCC relates to better clinical outcomes reflecting a different immune system response. Here, we retrospectively analyzed 97 tissue samples from oral and oropharyngeal carcinomas associated and non-associated with HPV infection using multispectral fluorescent immunohistochemistry. To evaluate the immune cell infiltration in tumor and stroma compartments, we designed four panels of four to five antibodies. We detected more T lymphocytes in the stroma, compared to the tumor parenchyma. In HPV positive (HPV+) in comparison to HPV negative (HPV-) tumors, higher counts of CD3+CD4+, CD3+CD8+, PD1+CD4+, PD1+CD8+ T cells, and ICOS- Treg cells were detected while more ICOS+ Treg cells and CTLA4+CD4+ T cells were observed in HPV- than in HPV+ tumors. The results of the univariate and multivariate analyses confirmed the predominant impact of HPV status on prognosis. More importantly, the number of CD8+PD-1+ T cells was identified as an independent factor, influencing the overall and/or disease-specific survival of patients with oral cavity or oropharyngeal carcinomas.

• Klíčová slova
• HPV, PD-1, head and neck cancer, survival,
• Publikační typ
• časopisecké články MeSH

Plasmacytoid dendritic cells (pDCs) are the most potent type I interferon-producing cells and play an important role in antiviral immunity. Tumor-infiltrating pDCs were shown to be predominantly pro-tumorigenic, with reduced ability to produce interferon alpha (IFNα) and confirmed capacity to prime regulatory T cells (Tregs) by the ICOS/ICOS-L pathway. Because a significant number of HNSCCs are induced by human papillomaviruses and show markedly different immune profiles than non-virally induced tumors, we compared the phenotype and functional capacity of HNSCC-infiltrating pDCs to the HPV status of the tumor. We observed a reduced capacity of pDCs to produce IFNα upon toll-like receptor activation in HPV-negative samples and a rather uncompromised functionality in HPV-associated tumors. Additionally, supernatants from non-virally induced but not HPV-associated tumor cell suspensions significantly inhibited IFNα production by peripheral blood-derived pDCs. We identified IL-10 and TNFα as the soluble pDC-suppressive factors with the highest variability between HPV-negative and HPV-positive tumor-derived supernatants. Additionally, we observed a positive correlation of tumor-infiltrating pDCs with Tregs in HPV-negative samples but not in virally induced tumors. Overall, our study indicates that the immunosuppressive cytokine milieu rich in IL-10 and TNFα in HPV-negative but not in HPV-positive HNSCC significantly affects the functional capacity of tumor-infiltrating pDCs, and such dysfunctional pDCs may further support the immunosuppressive tumor microenvironment by promoting the expansion of Tregs in the tumor tissue.

• Klíčová slova
• Head and neck cancer, Human papillomavirus, Interferon alpha, Plasmacytoid dendritic cells,
• MeSH
• biologické markery MeSH
• cytokiny metabolismus   MeSH
• dendritické buňky imunologie   metabolismus   patologie   MeSH
• dlaždicobuněčné karcinomy hlavy a krku etiologie   metabolismus   patologie   MeSH
• exprese genu MeSH
• infekce papilomavirem komplikace   virologie   MeSH
• interferon alfa imunologie   metabolismus   MeSH
• lidé MeSH
• náchylnost k nemoci MeSH
• nádorové mikroprostředí * imunologie   MeSH
• regulační T-lymfocyty imunologie   metabolismus   MeSH
• studie případů a kontrol MeSH
• T-lymfocyty - podskupiny imunologie   metabolismus   MeSH
• virová transformace buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• cytokiny MeSH
• interferon alfa MeSH

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV-) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV- tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers-cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)-was detected in HPV- patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

• Klíčová slova
• HPV, arginase 1, head and neck carcinoma, macrophages, prognosis,
• Publikační typ
• časopisecké články MeSH

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous disease that affects more than 800,000 patients worldwide each year. The variability of HNSCC is associated with differences in the carcinogenesis processes that are caused by two major etiological agents, namely, alcohol/tobacco, and human papillomavirus (HPV). Compared to non-virally induced carcinomas, the oropharyngeal tumors associated with HPV infection show markedly better clinical outcomes and are characterized by an immunologically "hot" landscape with high levels of tumor-infiltrating lymphocytes. However, the standard of care remains the same for both HPV-positive and HPV-negative HNSCC. Surprisingly, treatment de-escalation trials have not shown any clinical benefit in patients with HPV-positive tumors to date, most likely due to insufficient patient stratification. The in-depth analysis of the immune response, which places an emphasis on tumor-infiltrating immune cells, is a widely accepted prognostic tool that might significantly improve both the stratification of HNSCC patients in de-escalation trials and the development of novel immunotherapeutic approaches.

• Klíčová slova
• antitumor immune response, head and neck squamous cell carcinoma, human papillomavirus (HPV), immune infiltrate, treatment de-escalation, tumor microenvironment,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: Standard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with high morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients. Therefore, a better stratification of patients and the development of novel therapeutic protocols are crucially needed. The importance of tumor-infiltrating B cells (TIL-Bs) in shaping antitumor immunity remains unclear; therefore, we analyzed frequency, phenotype, prognostic value and possible roles of TIL-Bs in OPSCC. METHODS: We utilized transcriptomic analysis of immune response-related genes in 18 OPSCC samples with respect to human papillomavirus (HPV) status. The density and localization of CD20+, CD8+ and DC-LAMP+ cells were subsequently analyzed in 72 tissue sections of primary OPSCC samples in relation to patients' prognosis. The immunohistochemical approach was supplemented by flow cytometry-based analysis of phenotype and functionality of TIL-Bs in freshly resected primary OPSCC tissues. RESULTS: We observed significantly higher expression of B cell-related genes and higher densities of CD20+ B cells in HPV-associated OPSCC samples. Interestingly, CD20+ TIL-Bs and CD8+ T cells formed non-organized aggregates with interacting cells within the tumor tissue. The densities of both intraepithelial CD20+ B cells and B cell/CD8+ T cell interactions showed prognostic significance, which surpassed HPV positivity and CD8+ TIL density in stratification of OPSCC patients. High density of TIL-Bs was associated with an activated B cell phenotype, high CXCL9 production and high levels of tumor-infiltrating CD8+ T cells. Importantly, the abundance of direct B cell/CD8+ T cell interactions positively correlated with the frequency of HPV16-specific CD8+ T cells, whereas the absence of B cells in tumor-derived cell cultures markedly reduced CD8+ T cell survival. CONCLUSIONS: Our results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8+ T cell interactions can predict patients with excellent prognosis, who would benefit from less invasive treatment. We propose that in extensively infiltrated tumors, TIL-Bs might recruit CD8+ T cells via CXCL9 and due to a highly activated phenotype contribute by secondary costimulation to the maintenance of CD8+ T cells in the tumor microenvironment.

• Klíčová slova
• HNSCC, HPV, Tumor-infiltrating B lymphocytes,
• MeSH
• adjuvantní chemoradioterapie MeSH
• aktivace lymfocytů MeSH
• B-lymfocyty imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• dlaždicobuněčné karcinomy hlavy a krku imunologie   mortalita   terapie   virologie   MeSH
• dospělí MeSH
• infekce papilomavirem imunologie   mortalita   terapie   virologie   MeSH
• Kaplanův-Meierův odhad MeSH
• kohortové studie MeSH
• krční disekce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mezibuněčná komunikace imunologie   MeSH
• nádorové mikroprostředí imunologie   MeSH
• nádory orofaryngu imunologie   mortalita   terapie   virologie   MeSH
• orofarynx patologie   chirurgie   MeSH
• Papillomaviridae imunologie   izolace a purifikace   MeSH
• prognóza MeSH
• progrese nemoci MeSH
• senioři MeSH
• tumor infiltrující lymfocyty imunologie   MeSH
• výběr pacientů MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Similarly to other types of malignant tumours, the incidence of head and neck cancer is increasing globally. It is frequently associated with smoking and alcohol abuse, and in a broader sense also with prolonged exposure to these factors during ageing. A higher incidence of tumours observed in younger populations without a history of alcohol and tobacco abuse may be due to HPV infection. Malignant tumours form an intricate ecosystem of cancer cells, fibroblasts, blood/lymphatic capillaries and infiltrating immune cells. This dynamic system, the tumour microenvironment, has a significant impact on the biological properties of cancer cells. The microenvironment participates in the control of local aggressiveness of cancer cells, their growth, and their consequent migration to lymph nodes and distant organs during metastatic spread. In cancers originating from squamous epithelium, a similarity was demonstrated between the cancer microenvironment and healing wounds. In this review, we focus on the specificity of the microenvironment of head and neck cancer with emphasis on the mechanism of intercellular crosstalk manipulation for potential therapeutic application.

• Klíčová slova
• IL-6, cancer, cancer ecosystem, cancer microenvironment, cancer therapy, cancer-associated fibroblast, cytokine, extracellular matrix, tumour-associated macrophages,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Monitoring immune responses to solid cancers may be a better prognostic tool than conventional staging criteria, and it can also serve as an important criterion for the selection of individualized therapy. Multiparametric phenotyping by mass cytometry extended possibilities for immunoprofiling. However, careful optimization of each step of such method is necessary for obtaining reliable results. Also, with respect to procedure length and costs, sample preparation, staining, and storage should be optimized. Here, we designed a panel of 31 antibodies which allows for identification of several subpopulations of lymphoid and myeloid cells in a solid tumor and peripheral blood simultaneously. For sample preparation, disaggregation of tumor tissue with two different collagenases combined with DNase I was compared, and removal of dead or tumor cells by magnetic separation was evaluated. Two possible procedures of barcoding for single-tube staining of several samples were examined. While the palladium-based barcoding affected the stability of several antigens, the staining with two differently labeled CD45 antibodies was suitable for cells isolated from a patient's blood and tumor. The storage of samples in the intercalation solution for up to two weeks did not influence results of the analysis, which allowed the measurement of samples collected within this interval on the same day. This procedure optimized on samples from patients with head and neck squamous cell carcinoma enabled identification of various immune cells including rare subpopulations.

• MeSH
• analýza jednotlivých buněk MeSH
• antigeny CD45 imunologie   MeSH
• deoxyribonukleasa I metabolismus   MeSH
• imunofenotypizace metody   MeSH
• kolagenasy metabolismus   MeSH
• lidé MeSH
• lymfocyty fyziologie   MeSH
• monoklonální protilátky metabolismus   MeSH
• myeloidní buňky fyziologie   MeSH
• nádory diagnóza   imunologie   MeSH
• palladium metabolismus   MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• taxonomické DNA čárové kódování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD45 MeSH
• deoxyribonukleasa I MeSH
• kolagenasy MeSH
• monoklonální protilátky MeSH
• palladium MeSH