High expression of PD-L1 on conventional dendritic cells in tumour-draining lymph nodes is associated with poor prognosis in oral cancer.

INTRODUCTION: Oral squamous cell carcinoma (OSCC), while common and with a favorable prognosis in early stages, presents a marked reduction in survival rate upon metastasis to lymph nodes. Early detection of lymph node metastasis via biomarkers could enhance the therapeutic strategy for OSCC. Here, we explored dendritic cells (DCs) and cytotoxic T-cells in tumour-draining lymph nodes (TDLNs) as potential biomarkers. METHOD: Dendritic cells and cytotoxic T-cells in 33 lymph nodes were analyzed with multi-parameter flow cytometry in TDLNs, regional non-TDLNs surgically excised from 12 OSCC patients, and compared to 9 lymph nodes from patients with benign conditions. RESULTS: Our results displayed a higher proportion of conventional cDC1s with immunosuppressive features in TDLN. Further, high PD-L1 expression on cDC1 in TDLNs was associated with metastasis and/or recurrent disease risk. Also, elevated levels of memory CD8+ T-cells and terminally exhausted PD-1+TCF-1-CD8+ T-cells were observed in TDLNs and non-TDLNs compared to healthy lymph nodes. CONCLUSIONS: We conclude that TDLNs contain cells that could trigger an anti-tumor adaptive response, as evidenced by activated cDC1s and progenitor-like TCF-1+ T-cells. The detection of high PDL1 expression on cDC1s was indicative of TDLN metastasis and an adverse prognosis, proposing that PD-L1 on dendritic cells in TDLN could serve as a predictive biomarker of OSCC patients with a worse prognosis.

Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes.

The contribution of different immune cell subsets, especially T cells, in anti-tumor immune response is well established. In contrast to T cells, the anti-tumor contribution of B cells has been scarcely investigated. B-cells are often overlooked, even though they are important players in a fully integrated immune response and constitute a substantial fraction of tumor draining lymph nodes (TDLNs) known also as Sentinel Nodes. In this project, samples including TDLNs, non-TDLNs (nTDLNs) and metastatic lymph nodes from 21 patients with oral squamous cell carcinoma were analyzed by flow cytometry. TDLNs were characterized by a significantly higher proportion of B cells compared with nTDLNs (P = .0127). TDLNs-associated B cells contained high percentages of naïve B cells, in contrary to nTDLNs which contained significantly higher percentages of memory B cells. Patients having metastases in TDLNs showed a significantly higher presence of immunosuppressive B regulatory cells compared with metastasis-free patients (P = .0008). Elevated levels of regulatory B cells in TDLNs were associated with the advancement of the disease. B cells in TDLNs were characterized by significantly higher expression of an immunosuppressive cytokine-IL-10 compared with nTDLNs (P = .0077). Our data indicate that B cells in human TDLNs differ from B cells in nTDLNs and exhibit more naïve and immunosuppressive phenotypes. We identified a high accumulation of regulatory B cells within TDLNs which may be a potential obstacle in achieving response to novel cancer immunotherapies (ICIs) in head and neck cancer.

Acute odynophagia: A new symptom of COVID-19 during the SARS-CoV-2 Omicron variant wave in Sweden.

OBJECTIVE: The objective of this study is to present a novel clinical manifestation of infection with the Omicron variant of the SARS-CoV-2 virus affecting mainly young, vaccinated, and healthy adults. We describe a new group of COVID-19 patients seeking emergency care with symptoms similar to the life-threatening condition epiglottitis. Here, we present a case series and discuss management. METHODS: We performed a retrospective single-center case study of patients diagnosed with COVID-19 who were referred to the Ear, Nose, and Throat Emergency Department (ENT ED) between January 1 and January 23, 2022 with clinical symptoms such as acute odynophagia, severe sore throat, and fever. Ethical approval was obtained from the Swedish Ethical Review Authority (2020-02579). Informed consent was obtained from all patients included in the study. RESULTS: Twenty patients meeting inclusion criteria were identified. Fifteen patients were fully vaccinated against COVID-19. Four patients needed a short hospitalization for their symptoms. The most common diagnoses were COVID-19-associated acute viral laryngotracheitis and/or viral pharyngitis. Six patients presented with signs of secondary bacterial infection and were put on antibiotics. CONCLUSION: Previous variants of SARS-CoV-2 infection affected predominantly the lower respiratory tract and were associated with loss of smell and taste in many patients. The Omicron variant seems to affect predominantly the upper airways and cause acute laryngitis without olfactory dysfunction. In some patients, the clinical manifestation is similar to the symptoms of epiglottitis. In such a case, a prompt examination of the larynx is the gold standard to exclude inflammatory edema in the upper airways. None of the patients described in this study developed epiglottitis. In this study, we discuss the management of acute odynophagia in COVID-19 patients.

CD4+ and CD8+ T cells in sentinel nodes exhibit distinct pattern of PD-1, CD69, and HLA-DR expression compared to tumor tissue in oral squamous cell carcinoma.

Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti-programmed cell death protein 1 (PD-1) treatment is predominantly studied in tumor-infiltrating lymphocytes (TILs). Its impact on PD-1 expressing cells in tumor-draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD-1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD-1, CD69, and HLA-DR on CD4+ and CD8+ T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD-1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD-1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD-1 negative TILs expressed high levels of human leukocyte antigen - DR isotype and CD69. In contrast, PD-1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3+ PD-1+ cells in tumor-draining lymph nodes had significantly lower disease-free and overall survival rates (log-rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD-1 levels in TDLNs correspond with survival and potentially with response to anti-PD-1 therapy. Such knowledge may ultimately help guide anti-PD-1 treatment.

Human Blood and Tonsil Plasmacytoid Dendritic Cells Display Similar Gene Expression Profiles but Exhibit Differential Type I IFN Responses to Influenza A Virus Infection.

Influenza A virus (IAV) infection constitutes an annual health burden across the globe. Plasmacytoid dendritic cells (PDCs) are central in antiviral defense because of their superior capacity to produce type I IFNs in response to viruses. Dendritic cells (DCs) differ depending on their anatomical location. However, only limited host-pathogen data are available from the initial site of infection in humans. In this study, we investigated how human tonsil PDCs, likely exposed to virus because of their location, responded to IAV infection compared with peripheral blood PDCs. In tonsils, unlike in blood, PDCs are the most frequent DC subset. Both tonsil and blood PDCs expressed several genes necessary for pathogen recognition and immune response, generally in a similar pattern. MxA, a protein that renders cells resistant to IAV infection, was detected in both tonsil and blood PDCs. However, despite steady-state MxA expression and contrary to previous reports, at high IAV concentrations (typically cytopathic to other immune cells), both tonsil and blood PDCs supported IAV infection. IAV exposure resulted in PDC maturation by upregulation of CD86 expression and IFN-α secretion. Interestingly, blood PDCs secreted 10-fold more IFN-α in response to IAV compared with tonsil PDCs. Tonsil PDCs also had a dampened cytokine response to purified TLR ligands compared with blood PDCs. Our findings suggest that tonsil PDCs may be less responsive to IAV than blood PDCs, highlighting the importance of studying immune cells at their proposed site of function.

The TLR7 agonist imiquimod induces bronchodilation via a nonneuronal TLR7-independent mechanism: a possible role for quinoline in airway dilation.

Toll-like receptor (TLR) 7 agonists are known to reduce allergic airway inflammation. Their recently reported ability to rapidly relax airways has further increased their interest in the treatment of pulmonary disease. However, the mechanisms behind this effect are not fully understood. The present study, therefore, aimed to determine whether airway smooth muscle (ASM)-dependent mechanisms could be identified. TLR7 agonists were added to guinea pig airways following precontraction with carbachol in vitro or histamine in vivo. Pharmacological inhibitors were used to dissect conventional pathways of bronchodilation; tetrodotoxin was used or bilateral vagotomy was performed to assess neuronal involvement. Human ASM cells (HASMCs) were employed to determine the effect of TLR7 agonists on intracellular Ca(2+) ([Ca(2+)]i) mobilization. The well-established TLR7 agonist imiquimod rapidly relaxed precontracted airways in vitro and in vivo. This relaxation was demonstrated to be independent of nitric oxide, carbon monoxide, and cAMP signaling, as well as neuronal activity. A limited role for prostanoids could be detected. Imiquimod induced [Ca(2+)]i release from endoplasmic reticulum stores in HASMCs, inhibiting histamine-induced [Ca(2+)]i The TLR7 antagonist IRS661 failed to inhibit relaxation, and the structurally dissimilar agonist CL264 did not relax airways or inhibit [Ca(2+)]i This study shows that imiquimod acts directly on ASM to induce bronchorelaxation, via a TLR7-independent release of [Ca(2+)]i The effect is paralleled by other bronchorelaxant compounds, like chloroquine, which, like imiquimod, but unlike CL264, contains the chemical structure quinoline. Compounds with quinoline moieties may be of interest in the development of multifunctional drugs to treat pulmonary disease.

TNFα-blockade stabilizes local airway hyperresponsiveness during TLR-induced exacerbations in murine model of asthma.

Viral infections are a common cause of asthma exacerbation. These maladies are sometimes complicated by bacterial infections. Toll-like receptors (TLRs) are in the forefront of our microbial defence, with TLR3 responding to viral and TLR4 to bacterial stimulation. The present study was designed to evaluate the effect of concomitant TLR3 and TLR4 stimulation in a murine model of allergic asthma.BALB/c mice were stimulated intranasally with a combination of poly(I:C) and LPS activating TLR3 and TLR4, respectively. This resulted in the development of airway hyperresponsiveness (AHR) in the proximal part of the lung, along with signs of neutrophilic inflammation. Analysis of the bronchioalveolar lavage fluid (BALF) revealed a marked increase in TNFα. In contrast, the allergic airway inflammation induced by ovalbumin administration to sensitized mice caused AHR in the whole lung along with an increase in eosinophils and lymphocytes in the BALF and lung.When poly(I:C) + LPS were given to mice with an ongoing allergic airway inflammation induced by ovalbumin, the AHR was further increased in the peripheral lung and neutrophils appeared together with eosinophils and lymphocytes in the BALF and lung. Treatment with the TNFα-blocking antibody infliximab blunted the AHR increase, without affecting the cells influx in BALF.To conclude; a combined TLR3- and TLR4-stimulation, representing a concomitant viral and bacterial infection, causes an AHR that is further exaggerated during an ongoing allergic inflammation. The airway stabilizing effect of infliximab indicates the possible future use of TNFα blockade in treatment of microbial induced exacerbations of allergic asthma.

Tumour-draining lymph nodes in head and neck cancer are characterized by accumulation of CTLA-4 and PD-1 expressing Treg cells.

INTRODUCTION: High Tregs infiltration within the tumour microenvironment (TME) of various cancers shows a positive correlation with poor prognosis. Despite the fact that tumour draining lymph nodes (TDLNs) are recognized as key organs playing a crucial role in response to immunotherapy and modulating anti-cancer immunity, the distribution of Tregs and their role in TDLNs remain uncertain thus far. The purpose of this project is to investigate the density of Tregs in TDLNs and non-TDLNs and their expression of immune checkpoint molecules - PD-1 and CTLA-4. METHODS: Samples including TDLNs, non-TDLNs and metastatic lymph nodes (LNs) from 23 patients with oral squamous cell carcinoma (OSCC) were analyzed by multicolour flow cytometry with a focus on Tregs population and expression of CTLA-4 and PD-1. RESULTS: TDLNs and metastatic LNs were characterized by a significantly higher infiltration of Tregs defined as CD4+FoxP3+CD25highCD127low cells and significantly higher expression of CTLA-4 and PD-1 on Tregs compared with non-TDLNs. Tregs in TDLNs and metastatic LNs co-expressed CTLA-4 and PD-1 abundantly. High expression of these immune check-point molecules correlated with positive N-stage but not with T-stage. CONCLUSION: TDLNs and metastatic LNs are characterized by a high accumulation of Tregs expressing high levels of CTLA-4 and PD-1. High infiltration of Tregs can be a potential driver of an immunosuppressive milieu in TDLNs that can, in turn, favour cancer progression. High accumulation of Tregs expressing CTLA-4 and PD-1 in TDLNs is associated with lymph node involvement, but not with the size of the primary tumour.

Toll-like receptor ligands LPS and poly (I:C) exacerbate airway hyperresponsiveness in a model of airway allergy in mice, independently of inflammation.

It is well-established that bacterial and viral infections have an exacerbating effect on allergic asthma, particularly aggravating respiratory symptoms, such as airway hyperresponsiveness (AHR). The mechanism by which these infections alter AHR is unclear, but some studies suggest that Toll-like receptors (TLRs) play a role. In this study, we investigated the impact of TLR3 and TLR4 ligands on AHR and airway inflammation in a model of pre-established allergic inflammation. Female BALB/c mice were sensitised and challenged intranasally (i.n.) with either PBS or ovalbumin (OVA) and subsequently i.n. challenged with poly (I:C) (TLR3) or LPS (TLR4) for four consecutive days. The response to methacholine was measured in vivo; cellular and inflammatory mediators were measured in blood, lung tissue and broncheoalveolar lavage fluid (BALF). OVA challenge resulted in an increase in AHR to methacholine, as well as increased airway eosinophilia and TH2 cytokine production. Subsequent challenge with TLR agonists resulted in a significant increase in AHR, but decreased TLR-specific cellular inflammation and production of immune mediators. Particularly evident was a decline in LPS-induced neutrophilia and neutrophil-associated cytokines following LPS and poly (I:C) treatment. The present data indicates that TLRs may play a pivotal role in AHR in response to microbial infection in allergic lung inflammation. These data also demonstrate that aggravated AHR occurs in the absence of an exacerbation in airway inflammation and that allergic inflammation impedes a subsequent inflammatory response to TLRs. These results may parallel clinical signs of microbial asthma exacerbation, including an extended duration of illness and increased respiratory symptoms.

Toll-like receptor (TLR) 7 decreases and TLR9 increases the airway responses in mice with established allergic inflammation.

Toll-like receptor (TLR) 7 and TLR9 recognise microbial products of viral descent. Since viruses are a common trigger of asthma exacerbations these TLRs have emerged as interesting therapeutic targets. Even though their effects on allergic inflammation have been evaluated in several models their effects on established allergic airway inflammation remains to be described. Therefore, mice with an on-going ovalbumin (OVA)-induced allergic airway inflammation were given R848 or CpG (TLR7 and TLR9 agonists, respectively) intranasally during four consecutive days. At day five, the R848 treatment had reduced OVA-induced airway hyperresponsiveness (measured as the increased resistance to methacholine), counteracted the accompanying influx of eosinophils and macrophages, and decreased the OVA-enhanced release of interleukin (IL)-5 and leukotriene (LT) B4 in bronchoalveolar lavage fluid. CpG, which by itself caused airway hyperresponsiveness, did not influence the OVA-induced airway hyperresponsiveness, and release of IL-5 and LTB4, but decreased the OVA-induced influx of cells in bronchoalveolar lavage fluid, and increased the amount of pro-inflammatory mediators like IL-12, CXCL1 and CXCL9. To conclude, TLR7 dampens the allergic airway reactivity and local inflammation, whereas TLR9 that causes airway hyperresponsiveness and increased cellular response per se, do generally not interfere with the effects induced by allergic inflammation.

Intranasal administration of poly(I:C) and LPS in BALB/c mice induces airway hyperresponsiveness and inflammation via different pathways.

BACKGROUND: Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role. MATERIALS AND METHODS: To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days. RESULTS: When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation. CONCLUSION: In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another.