Immune delineation of laryngeal papilloma reveals enhanced neutrophil associated gene profile.

Laryngeal papilloma (LP) is a rare benign disease, caused by recurrent multisite papillomas that are referred to as recurrent respiratory papillomatosis (RRP). RRP is caused primarily by two types of human papillomavirus (HPV): HPV6 and HPV11. The immune dysregulation within the microenvironment of the lesions has been shown to likely play a role in the development of RRP. The present study aimed at analyzing the transcriptional profile of immune response genes and cancer-related genes in the LP microenvironment. We used the NanoString® nCounter® analysis system to study expression of 730 genes among seven paired samples of LP and healthy laryngeal (HL) tissue. qRT-PCR and flow cytometric analysis was performed to confirm identified transcripts and follow-up scores of infiltrating immune cells, respectively. In total, 113 differentially expressed transcripts were detected of which 37 showed increased expression levels and 76 decreased expression levels in the LP samples compared to the HL samples (fold change ≥ 2). Transcripts with increased expression levels included S100As (A7, A8, and A12), CEACAM1, neutrophil activation associated cytokines (IL8), chemokines (CXCL6), and IL receptors, e.g., IL4R. Transcripts with decreased expression in LP were associated with innate and adaptive immunity. Overall, HPV6 and 11 were present in 67% and 33% of the patients, respectively. There was a significant increase in neutrophils and a significant decrease in CD8+ T cells in LP. LP samples display an immune profile characterized by enhanced expression of neutrophilic markers and significantly reduced T cell-associated markers.

Nasal administration of a probiotic assemblage in allergic rhinitis: A randomised placebo-controlled crossover trial.

BACKGROUND: Topical probiotics have been suggested as a treatment option for allergic rhinitis, as they may skew the immune response towards a beneficial type-1 non-allergic profile. So far observations in man have exclusively involved oral intake. The aim of this study was to examine whether a topical/nasal administration of a probiotic assemblage (PA) affects quality of life, symptoms and signs of allergic rhinitis in a nasal allergen challenge (NAC) model. METHODS: In a placebo-controlled and crossover design, 24 patients with seasonal allergic rhinitis were randomised to topical/nasal administration with a PA of Lactobacillus rhamnosus SP1, Lactobacillus paracasei 101/37 and Lactococcus lactis L1A or placebo for 3 weeks. Participants and investigators were blind to treatment allocation. The last week of each treatment period was combined with a NAC series. Efficacy variables were "Mini-Rhinoconjunctivitis Quality of Life Questionnaire" (Mini-RQLQ), "Total Nasal Symptom Score" (TNSS), "Peak Nasal Inspiratory Flow" (PNIF) and "Fractional Exhaled Nitric Oxide" (FeNO). In addition, to assess whether or not the PA produced any pro-inflammatory effect per se, soluble analytes were monitored in nasal lavage fluids. Finally, bacterial cultures, sampled using swabs from the middle nasal meatus, were assessed for the presence of the PA by MALDI-TOF analysis. RESULTS: Administration of the PA did not produce any nasal symptoms (cf. placebo). An innate immune response was discerned within the PA run (cf. baseline), but no change in nasal lavage fluid levels of cytokines/mediators was observed cf. placebo except for IL-17/IL-17A (a minor increase in the PA run). Administration of the PA did neither affect Mini-RQLQ, TNSS, PNIF nor FeNO. No evidence of persistent colonization was observed. CONCLUSIONS: Topical/nasal administration of a PA comprising Lactobacillus rhamnosus SP1, Lactobacillus paracasei 101/37 and Lactococcus lactis L1A, while likely evoking a minor innate immune response yet being safe, does not affect quality of life, symptoms or signs of allergic rhinitis. TRIAL REGISTRATION: not registered.

Changes in Intestinal Permeability Ex Vivo and Immune Cell Activation by Three Commonly Used Emulsifiers.

Food additives such as emulsifiers are used in increasing quantities in the food industry. The aim of this study was to compare three different emulsifiers (polysorbate 80 (P80), carboxymethyl cellulose (CMC), and ß-lactoglobulin (ß-lac) with regards to their effect on the stimulation of immune cells and intestinal permeability. The immune stimulatory effects were studied in the myeloid cell line MUTZ-3-cells, while the change in intestinal permeability was studied in the Caco-2 cell line and ex vivo in the Ussing chamber system using small intestinal fragments from rats. The tested concentrations of the emulsifiers ranged from 0.02% up to 1%, which are concentrations commonly used in the food industry. The results showed that P80 affected both the myeloid cells and the intestinal permeability more than CMC (p < 0.05) and ß-lac (p < 0.05) at the highest concentration. CMC was found to neither affect the permeability in the intestine nor the MUTZ-3 cells, while ß-lac changed the permeability in the total part of the small intestine in rats. These findings indicate that P80 might be more cytotoxic compared to the other two emulsifiers.

The 21st Century movement within the area of skin sensitization assessment: From the animal context towards current human-relevant in vitro solutions.

In a regulatory context, skin sensitization hazard and risk evaluations of manufactured products and their ingredients (e.g. cosmetics) are mandatory in several regions. Great efforts have been made within the field of 21st Century Toxicology to provide non-animal testing approaches to assess the skin allergy potential of materials (e.g. chemicals, mixtures, nanomaterials, particles). Mechanistic understanding of skin sensitization process through the adverse outcome pathway (AOP) has promoted the development of in vitro methods, demonstrating accuracies superior to the traditional animal testing. These in vitro testing approaches are based on one of the four AOP key events (KE) of skin sensitization: formation of immunogenic hapten-protein complexes (KE-1 or the molecular initiating event, MIE), inflammatory keratinocyte responses (KE-2), dendritic cell activation (KE-3), and T-lymphocyte activation and proliferation (KE-4). This update provides an overview of the historically used in vivo methods as well as the current in chemico and in cell methods with and without OECD guideline designations to analyze the progress towards human-relevant in vitro test methods for safety assessment of the skin allergenicity potential of materials. Here our focus is to review 96 in vitro testing approaches directed to the KEs of the skin sensitization AOP.

Evaluation of in vitro testing strategies for hazard assessment of the skin sensitization potential of "real-life" mixtures: The case of henna-based hair-colouring products containing p-phenylenediamine.

BACKGROUND: Allergic contact dermatitis caused by henna-based hair-colouring products has been associated with adulteration of henna with p-phenylenediamine (PPD). OBJECTIVES: To develop a testing approach based on in vitro techniques that address key events within the skin sensitization adverse outcome pathway in order to evaluate the allergenic potential of hair-colouring products. METHODS: The following in vitro assays were used to test the sensitizing capacity of hair dye ingredients: the micro-direct peptide reactivity assay (mDPRA); the HaCaT keratinocyte-associated interleukin (IL)-18 assay; the U937 cell line activation test (U-SENS)/IL-8 levels; the blood monocyte-derived dendritic cell test; and genomic allergen rapid detection (GARD skin). Those techniques with better human concordance were selected to evaluate the allergenic potential of 10 hair-colouring products. RESULTS: In contrast to the information on the label, chromatographic analyses identified PPD in all products. The main henna biomarker, lawsone, was not detected in one of the 10 products. Among the techniques evaluated by testing hair dye ingredients, the mDPRA, the IL-18 assay, GARD skin and the U-SENS correlated better with human classification (concordances of 91.7%-100%) and were superior to the animal testing (concordance of 78.5%). Thus, these assays were used to evaluate hair-colouring products, which were classified as skin sensitizers by the use of different two-of-three approaches. CONCLUSIONS: Our findings highlight the toxicological consequences of, and risks associated with, the undisclosed use of PPD in henna-based "natural" "real-life" products.

Phl p 1-specific human monoclonal IgE and design of a hypoallergenic group 1 grass pollen allergen fragment.

Detailed understanding of how Abs of the IgE isotype interact with allergen at the onset of an allergic reaction is of great importance for deciphering mechanisms involved in the development of disease and may aid in the design of hypoallergenic variants. In this study, we have used a set of human monoclonal IgE Abs derived from the repertoires of allergic individuals, specific for the major timothy grass pollen allergen Phl p 1, to gain detailed information on the interaction between Abs and allergen. These allergen-specific IgE are to varying degrees cross-reactive toward both different allergen isoforms and various group 1 allergens originating from other grass species. The usage of human monoclonal IgE, as an alternative to polyclonal preparations or mouse Abs, allowed us to locate several important IgE-binding epitopes on the C-terminal domain of Phl p 1, all clustered to an IgE-binding "hot spot." By introducing three mutations in the IgE-binding area of the C-terminal domain we were able to significantly reduce its reactivity with serum IgE. In conclusion, our study shows the great potential of using human monoclonal IgE as a tool for studies of the molecular interactions taking place during allergic responses. Furthermore, we present a novel IgE-hyporeactive fragment with the potential to be used as a safer hypoallergenic alternative in specific immunotherapy than the pollen extracts used today.

Intradermal delivery of TLR agonists in a human explant skin model: preferential activation of migratory dendritic cells by polyribosinic-polyribocytidylic acid and peptidoglycans.

TLR agonists are attractive candidate adjuvants for therapeutic cancer vaccines as they can induce a balanced humoral and T cell-mediated immune response. With a dense network of dendritic cells (DCs) and draining lymphatics, the skin provides an ideal portal for vaccine delivery. Beside direct DC activation, TLR agonists may also induce DC activation through triggering the release of inflammatory mediators by accessory cells in the skin microenvironment. Therefore, a human skin explant model was used to explore the in vivo potential of intradermally delivered TLR agonists to stimulate Langerhans cells and dermal DCs in their natural complex tissue environment. The skin-emigrated DCs were phenotyped and analyzed for T cell stimulatory capacity. We report that, of six tested TLR-agonists, the TLR2 and -3 agonists peptidoglycan (PGN) and polyribosinic-polyribocytidylic acid (Poly I:C) were uniquely able to enhance the T cell-priming ability of skin-emigrated DCs, which, in the case of PGN, was accompanied by Th1 polarization. The enhanced priming capacity of Poly I:C-stimulated DCs was associated with a strong upregulation of appropriate costimulatory molecules, including CD70, whereas that of PGN-stimulated DCs was associated with the release of a broad array of proinflammatory cytokines. Transcriptional profiling further supported the notion that the PGN- and Poly I:C-induced effects were mediated through binding to TLR2/nucleotide-binding oligomerization domain 2 and TLR3/MDA5, respectively. These data warrant further exploration of PGN and Poly I:C, alone or in combination, as DC-targeted adjuvants for intradermal cancer vaccines.

Evaluation of high throughput gene expression platforms using a genomic biomarker signature for prediction of skin sensitization.

BACKGROUND: Allergic contact dermatitis (ACD) develops upon exposure to certain chemical compounds termed skin sensitizers. To reduce the occurrence of skin sensitizers, chemicals are regularly screened for their capacity to induce sensitization. The recently developed Genomic Allergen Rapid Detection (GARD) assay is an in vitro alternative to animal testing for identification of skin sensitizers, classifying chemicals by evaluating transcriptional levels of a genomic biomarker signature. During assay development and biomarker identification, genome-wide expression analysis was applied using microarrays covering approximately 30,000 transcripts. However, the microarray platform suffers from drawbacks in terms of low sample throughput, high cost per sample and time consuming protocols and is a limiting factor for adaption of GARD into a routine assay for screening of potential sensitizers. With the purpose to simplify assay procedures, improve technical parameters and increase sample throughput, we assessed the performance of three high throughput gene expression platforms--nCounter®, BioMark HD™ and OpenArray®--and correlated their performance metrics against our previously generated microarray data. We measured the levels of 30 transcripts from the GARD biomarker signature across 48 samples. Detection sensitivity, reproducibility, correlations and overall structure of gene expression measurements were compared across platforms. RESULTS: Gene expression data from all of the evaluated platforms could be used to classify most of the sensitizers from non-sensitizers in the GARD assay. Results also showed high data quality and acceptable reproducibility for all platforms but only medium to poor correlations of expression measurements across platforms. In addition, evaluated platforms were superior to the microarray platform in terms of cost efficiency, simplicity of protocols and sample throughput. CONCLUSIONS: We evaluated the performance of three non-array based platforms using a limited set of transcripts from the GARD biomarker signature. We demonstrated that it was possible to achieve acceptable discriminatory power in terms of separation between sensitizers and non-sensitizers in the GARD assay while reducing assay costs, simplify assay procedures and increase sample throughput by using an alternative platform, providing a first step towards the goal to prepare GARD for formal validation and adaption of the assay for industrial screening of potential sensitizers.

Human blood dendritic cell subsets exhibit discriminative pattern recognition receptor profiles.

Dendritic cells (DCs) operate as the link between innate and adaptive immunity. Their expression of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), enables antigen recognition and mediates appropriate immune responses. Distinct subsets of human DCs have been identified; however their expression of PRRs is not fully clarified. Expressions of CLRs by DC subpopulations, in particular, remain elusive. This study aimed to identify and compare PRR expressions on human blood DC subsets, including CD1c(+) , CD141(+) and CD16(+) myeloid DCs and CD123(+) plasmacytoid DCs, in order to understand their capacity to recognize different antigens as well as their responsiveness to PRR-directed targeting. Whole blood was obtained from 13 allergic and six non-allergic individuals. Mononuclear cells were purified and multi-colour flow cytometry was used to assess the expression of 10 CLRs and two TLRs on distinct DC subsets. PRR expression levels were shown to differ between DC subsets for each PRR assessed. Furthermore, principal component analysis and random forest test demonstrated that the PRR profiles were discriminative between DC subsets. Interestingly, CLEC9A was expressed at lower levels by CD141(+) DCs from allergic compared with non-allergic donors. The subset-specific PRR expression profiles suggests individual responsiveness to PRR-targeting and supports functional specialization.

Circulating CD1c(+) DCs are superior at activating Th2 responses upon Phl p stimulation compared with basophils and pDCs.

The contributing role of circulating human dendritic cell (DC) populations and basophils in the presentation and augmentation of Th2 responses remains to be determined. The present study aimed at elucidating the functional role of CD1c(+) myeloid DCs (mDCs), CD123(+) plasmacytoid DCs (pDCs), monocyte-derived DCs and basophils in allergen presentation and Th2 activation. By coculturing Phleum pratense (Phl p)-pulsed CD1c(+) mDCs, CD123(+) pDCs, monocyte-derived DCs and basophils with autologous CD4(+) effector memory T cells, we assessed T-cell proliferation as well as the frequency of interleukin-4- and interferon-γ-producing T cells. Interestingly, a Th2-stimulating ability was observed for Phl p-challenged CD1c(+) mDCs and monocyte-derived DCs, while CD123(+) pDCs and basophils did not affect the Th-balance. In addition, both Phl p-pulsed CD1c(+) mDCs and monocyte-derived DCs stimulated increased T-cell proliferation compared to basophils and CD123(+) pDCs. Together, these results point to a prominent role for circulating CD1c(+) mDCs in allergen presentation and augmentation of Th2 responses, making them promising therapeutic targets for Type I hypersensitivity reactions.

Fine tuning of the innate and adaptive immune responses by Interleukin-2.

Novel immunotherapies for cancer and other diseases aim to trigger the immune system to produce durable responses, while overcoming the immunosuppression that may contribute to disease severity, and in parallel considering immunosafety aspects. Interleukin-2 (IL-2) was one of the first cytokines that the FDA approved as a cancer-targeting immunotherapy. However, in the past years, IL-2 immunotherapy is not actively offered to patients, due to limited efficacy, when compared to other novel immunotherapies, and the associated severe adverse events. In order to design improved in vitro and in vivo models, able to predict the efficacy and safety of novel IL-2 alternatives, it is important to delineate the mechanistic immunological events triggered by IL-2. Particularly, in this review we will discuss the effects IL-2 has with the bridging cell type of the innate and adaptive immune responses, dendritic cells. The pathways involved in the regulation of IL-2 by dendritic cells and T-cells in cancer and autoimmune disease will also be explored.

Next-generation CD40 agonists for cancer immunotherapy.

INTRODUCTION: There is a need for new therapies that can enhance response rates and broaden the number of cancer indications where immunotherapies provide clinical benefit. CD40 targeting therapies provide an opportunity to meet this need by promoting priming of tumor-specific T cells and reverting the suppressive tumor microenvironment. This is supported by emerging clinical evidence demonstrating the benefits of immunotherapy with CD40 antibodies in combination with standard of care chemotherapy. AREAS COVERED: This review is focused on the coming wave of next-generation CD40 agonists aiming to improve efficacy and safety, using new approaches and formats beyond monospecific antibodies. Further, the current understanding of the role of different CD40 expressing immune cell populations in the tumor microenvironment is reviewed. EXPERT OPINION: There are multiple promising next-generation approaches beyond monospecific antibodies targeting CD40 in immuno-oncology. Enhancing efficacy is the most important driver for this development, and approaches that maximize the ability of CD40 to both remodel the tumor microenvironment and boost the anti-tumor T cell response provide great opportunities to benefit cancer patients. Enhanced understanding of the role of different CD40 expressing immune cells in the tumor microenvironment may facilitate more efficient clinical development of these compounds.

IL-2-mediated hepatotoxicity: knowledge gap identification based on the irAOP concept.

Drug-induced hepatotoxicity constitutes a major reason for non-approval and post-marketing withdrawal of pharmaceuticals. In many cases, preclinical models lack predictive capacity for hepatic damage in humans. A vital concern is the integration of immune system effects in preclinical safety assessment. The immune-related Adverse Outcome Pathway (irAOP) approach, which is applied within the Immune Safety Avatar (imSAVAR) consortium, presents a novel method to understand and predict immune-mediated adverse events elicited by pharmaceuticals and thus targets this issue. It aims to dissect the molecular mechanisms involved and identify key players in drug-induced side effects. As irAOPs are still in their infancy, there is a need for a model irAOP to validate the suitability of this tool. For this purpose, we developed a hepatotoxicity-based model irAOP for recombinant human IL-2 (aldesleukin). Besides producing durable therapeutic responses against renal cell carcinoma and metastatic melanoma, the boosted immune activation upon IL-2 treatment elicits liver damage. The availability of extensive data regarding IL-2 allows both the generation of a comprehensive putative irAOP and to validate the predictability of the irAOP with clinical data. Moreover, IL-2, as one of the first cancer immunotherapeutics on the market, is a blueprint for various biological and novel treatment regimens that are under investigation today. This review provides a guideline for further irAOP-directed research in immune-mediated hepatotoxicity.

In vivo dendritic cell reprogramming for cancer immunotherapy.

Immunotherapy can lead to long-term survival for some cancer patients, yet generalized success has been hampered by insufficient antigen presentation and exclusion of immunogenic cells from the tumor microenvironment. Here, we developed an approach to reprogram tumor cells in vivo by adenoviral delivery of the transcription factors PU.1, IRF8, and BATF3, which enabled them to present antigens as type 1 conventional dendritic cells. Reprogrammed tumor cells remodeled their tumor microenvironment, recruited, and expanded polyclonal cytotoxic T cells; induced tumor regressions; and established long-term systemic immunity in multiple mouse melanoma models. In human tumor spheroids and xenografts, reprogramming to immunogenic dendritic-like cells progressed independently of immunosuppression, which usually limits immunotherapy. Our study paves the way for human clinical trials of in vivo immune cell reprogramming for cancer immunotherapy.

A proteomics dataset capturing myeloid cell responses upon cellular exposure to fungicides, adjuvants and fungicide formulations.

Dendritic cells are the sentinels of the immune system, linking the innate and adaptive immune response. Myeloid and dendritic cell models have been successfully used in in vitro approaches to predict adverse outcomes such as skin sensitization. We here exposed a well-characterized human dendritic cell-like cell line to agricultural chemicals, including fungicide formulations, active ingredients, adjuvants and defined mixtures for 24 h to profile induced changes on protein levels. Cell pellets were harvested and prepared for bottom-up label-free analysis with peptide separation on an EASY-nano LC system 1200 coupled online with a QExactive HF-X mass spectrometer with data-dependent acquisition (DDA). The raw data files and processed quantitative data have been deposited to ProteomeXchange with the data identification number PXD034624 and are described here. The data in this article may serve as a resource for researchers interested in e.g. human toxicology, immunology, cell biology and pharmacology.

Upper airway microbiome transplantation for patients with chronic rhinosinusitis.

BACKGROUND: Chronic or recurrent rhinosinusitis without polyps (CRSsNP) is characterized by a persistent inflammation of the sinonasal mucosa. The underlying cause is unclear but increasing interest has been directed toward changes in the sinonasal microbiome as a potential driver. METHODS: Twenty-two patients diagnosed with CRSsNP were treated with antibiotics for 13 days, followed by 5 consecutive days of nasal microbiome transplants from healthy donors. Outcome measures were 22-item Sino-Nasal Outcome Test (SNOT-22) questionnaire, total nasal symptom score (TNSS), endoscopic grading, 16S ribosomal RNA (rRNA) next generation sequencing (microbiome analysis), and nasal lavage fluid analysis of inflammatory cytokines. Patients were examined at the start of the study and after antibiotic treatment as well as 10 days and 3 months after the transplant series. RESULTS: At the end of the study, patients reported significantly reduced SNOT-22 scores and microbiome analysis showed significantly increased abundance and diversity. No significant change was observed for TNSS or endoscopic scoring. CONCLUSION: Nasal microbiome transplants obtained from healthy individuals and administered as nasal lavages to patients with CRSsNP are feasible. The patients reported significant and lasting reduction of symptoms and these findings were associated with a lasting increase in abundance and diversity of the local bacterial flora. The observations, which need to be confirmed by randomized controlled trials, may constitute a new treatment avenue for these difficult to treat patients where antibiotics only provide short lasting symptom control.

Exploring Spatial Heterogeneity of Immune Cells in Nasopharyngeal Cancer.

Nasopharyngeal cancer (NPC) is a malignant tumor. In a recent publication, we described the presence and distribution of CD8+ T cells in NPC and used the information to identify 'inflamed', 'immune-excluded', and 'desert' immune phenotypes, where 'inflamed' and 'immune-excluded' NPCs were correlated with CD8 T cell infiltration and survival. Arguably, more detailed and, in particular, spatially resolved data are required for patient stratification and for the identification of new treatment targets. In this study, we investigate the phenotype of CD45+ leukocytes in the previously analyzed NPC samples by applying multiplexed tissue analysis to assess the spatial distribution of cell types and to quantify selected biomarkers. A total of 47 specified regions-of-interest (ROIs) were generated based on CD45, CD8, and PanCK morphological staining. Using the GeoMx® Digital Spatial Profiler (DSP), 49 target proteins were digitally quantified from the selected ROIs of a tissue microarray consisting of 30 unique NPC biopsies. Protein targets associated with B cells (CD20), NK cells (CD56), macrophages (CD68), and regulatory T cells (PD-1, FOXP3) were most differentially expressed in CD45+ segments within 'immune-rich cancer cell islet' regions of the tumor (cf. 'surrounding stromal leukocyte' regions). In contrast, markers associated with suppressive populations of myeloid cells (CD163, B7-H3, VISTA) and T cells (CD4, LAG3, Tim-3) were expressed at a higher level in CD45+ segments in the 'surrounding stromal leukocyte' regions (cf. 'immune-rich cancer cell islet' regions). When comparing the three phenotypes, the 'inflamed' profile (cf. 'immune-excluded' and 'desert') exhibited higher expression of markers associated with B cells, NK cells, macrophages, and myeloid cells. Myeloid markers were highly expressed in the 'immune-excluded' phenotype. Granulocyte markers and immune-regulatory markers were higher in the 'desert' profile (cf. 'inflamed' and 'immune-excluded'). In conclusion, this study describes the spatial heterogeneity of the immune microenvironment in NPC and highlights immune-related biomarkers in immune phenotypes, which may aid in the stratification of patients for therapeutic purposes.

Targeted spatial proteomic analysis of CD8+ T- and myeloid cells in tonsillar cancer.

Background: Tonsillar cancer is caused by high-risk human papillomavirus (HPV), tobacco smoking, and alcohol abuse. Aspects of the patient's immune response to this disease have arisen as prognostic factors and treatment targets, reflecting differences in the type and protein expression profile of immune cells. Because tonsillar cancers are heterogenous lesions such data need to be spatially resolved. Methods: In this study, we aim to explore inter-patient and intra-tumoral sources of variation in tonsillar cancer using immunofluorescence and targeted spatial proteomics to interrogate a cohort of 105 patients. Furthermore, we assess prognostic factors and elucidate molecular targets. We have used CD8, CD11c, and Pan-cytokeratin (PanCK) to quantify and locate immune cells driving antigen-specific cellular immunity. Guided by immunofluorescence information, we selected 355 CD8+, CD11c+, or PanCK+ areas inside and outside (i.e., stroma) cancer-cell islets, to quantify 43 immune-related proteins using digital spatial profiling. Results: Quantitative analysis of immunofluorescence in combination with clinical data revealed that the abundance of total CD8+ cells and CD8+ cells infiltrating cancer-cell islets, respectively, were associated with higher 5-year disease-free survival and overall survival, independently of HPV-status and clinical stage. Comparison of CD8+ cells inside and outside cancer-cell islets revealed an upregulation of effector CD8+ T-cell and immune checkpoint molecules in the former. Among these, the expression of PD-L1 by CD8+ T-cells was associated with lower all-cause mortality in a univariate proportional hazards model. Similarly, a comparison of tumor boundary and stroma CD11c+ cells showed upregulation of both co-stimulatory and immune checkpoint molecules with proximity to tumor cell islets. Conclusion: Our findings highlight the relevance of analyzing aspects of tumor micro-architecture in the search of prognostic markers and molecular targets for tonsillar cancer. The abundance of intra-tumoral CD8+ T-cells can be considered a positive predictive marker for tonsillar cancer, while the significance of PD-L1 expression by intra-tumoral CD8+ T-cells warrants further evaluation. Location-based differences in CD8+ and CD11c+ cells suggest an immune cell-altering effect on the tumor microenvironment, and grant new insight into which cells that can be targeted by novel therapeutic agents.

Human papillomavirus-associated head and neck squamous cell carcinoma cells rely on glycolysis and display reduced oxidative phosphorylation.

Introduction: Head and neck squamous cell carcinoma (HNSCC) constitutes a heterogeneous group of cancers. Human papilloma virus (HPV) is associated with a subtype of HNSCC with a better response to treatment and more favorable prognosis. Mitochondrial function and metabolism vary depending on cancer type and can be related to tumor aggressiveness. This study aims to characterize the metabolism of HPV-positive and HPV-negative HNSCC cell lines. Methods: Oxidative phosphorylation (OXPHOS) and glycolysis were assessed in intact cells, in four HNSCC cell lines using Seahorse XF Analyzer. OXPHOS was further studied in permeabilized cells using high-resolution respirometry in an Oroboros O2K. Metabolomic analysis was performed using mass spectroscopy. Results: The HPV-negative cell lines were found to display a higher OXPHOS capacity and were also able to upregulate glycolysis when needed. The HPV-positive cell line had a higher basal glycolytic rate but lower spare OXPHOS capacity. These cells were also unable to increase respiration in response to succinate, unlike the HPV-negative cells. In the metabolomic analysis, the HPV-positive cells showed a higher kynurenine/tryptophan ratio. Discussion: HPV-positive HNSCC preferred glycolysis to compensate for lower OXPHOS reserves, while the HPV-negative HNSCC displayed a more versatile metabolism, which might be related to increased tumor aggressiveness. The higher kynurenine/tryptophan ratio of HPV-positive HNSCC might be related to increased indoleamine 2,3-dioxygenase activity due to the carcinoma's viral origin. This study highlights important metabolic differences between HPV-positive and HPV-negative cancers and suggests that future metabolic targets for cancer treatment should be individualized based on specific tumor metabolism.