Protective Non-neutralizing anti-N-terminal Domain mAb Maintains Fc-mediated Function against SARS-COV-2 Variants up to BA.2.86-JN.1 with Superfluous In Vivo Protection against JN.1 Due to Attenuated Virulence.

Substantial evidence supports that Fc-mediated effector functions of anti-spike Abs contribute to anti-SARS-Cov-2 protection. We have previously shown that two non-neutralizing but opsonic mAbs targeting the receptor-binding domain and N-terminal domain (NTD), Ab81 and Ab94, respectively, are protective against lethal Wuhan SARS-CoV-2 infection in K18-hACE2 mice. In this article, we investigated whether these protective non-neutralizing Abs maintain Fc-mediated function and Ag binding against mutated SARS-CoV-2 variants. Ab81 and Ab94 retained their nanomolar affinity and Fc-mediated function toward Omicron and its subvariants, such as BA.2, BA.4, BA.5, XBB, XBB1.5, and BQ1.1. However, when encountering the more heavily mutated BA.2.86, Ab81 lost its function, whereas the 10 new mutations in the NTD did not affect Ab94. In vivo experiments with Ab94 in K18-hACE2 mice inoculated with a stringent dose of 100,000 PFU of the JN.1 variant revealed unexpected results. Surprisingly, this variant exhibited low disease manifestation in this animal model with no weight loss or death in the control group. Still, assessment of mice using a clinical scoring system showed better protection for Ab94-treated mice, indicating that Fc-mediated functions are still beneficial. Our work shows that a protective anti-receptor-binding domain non-neutralizing mAb lost reactivity when BA.2.86 emerged, whereas the anti-NTD mAb was still functional. Finally, this work adds new insight into the evolution of the SARS-CoV-2 virus by reporting that JN.1 is substantially less virulent in vivo than previous strains.

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.

Neutrophil phenotypes in bronchial airways differentiate single from dual responding allergic asthmatics.

INTRODUCTION: Allergic asthmatics with both an early (EAR) and a late allergic reaction (LAR) following allergen exposure are termed 'dual responders' (DR), while 'single responders' (SR) only have an EAR. Mechanisms that differentiate DR from SR are largely unknown, particularly regarding the role and phenotypes of neutrophils. Therefore, we aimed to study neutrophils in DR and SR asthmatics. METHODS: Thirty-four allergic asthmatics underwent an inhaled allergen challenge, samples were collected before and up to 24 h post-challenge. Cell differentials were counted from bronchial lavage, alveolar lavage and blood; and tissue neutrophils were quantified in immune-stained bronchial biopsies. Lavage neutrophil nuclei lobe segmentation was used to classify active (1-4 lobes) from suppressive neutrophils (≥5 lobes). Levels of transmigration markers: soluble (s)CD62L and interleukin-1Ra, and activity markers: neutrophil elastase (NE), DNA-histone complex and dsDNA were measured in lavage fluid and plasma. RESULTS: Compared with SR at baseline, DR had more neutrophils in their bronchial airways at baseline, both in the lavage (p = .0031) and biopsies (p = .026) and elevated bronchial neutrophils correlated with less antitransmigratory IL-1Ra levels (r = -0.64). DR airways had less suppressive neutrophils and more 3-lobed (active) neutrophils (p = .029) that correlated with more bronchial lavage histone (p = .020) and more plasma NE (p = .0016). Post-challenge, DR released neutrophil extracellular trap factors in the blood earlier and had less pro-transmigratory sCD62L during the late phase (p = .0076) than in SR. CONCLUSION: DR have a more active airway neutrophil phenotype at baseline and a distinct neutrophil response to allergen challenge that may contribute to the development of an LAR. Therefore, neutrophil activity should be considered during targeted diagnosis and bio-therapeutic development for DR.

Prolonged inflammatory resolution in allergic asthma relates to dysfunctional interactions between neutrophils and airway epithelium.

BACKGROUND: Allergic asthma is a heterogeneous disorder involving chronic airway inflammation, reversible airflow limitation, and tissue remodeling, causing chronic airflow limitation. Most of the asthma research has been focused on elucidating the proinflammatory pathways underlying disease pathogenesis. Paradoxically, the necessity of appropriate termination and resolution of inflammation has not been recognized until recently. The latter has led to the concept of chronic inflammation developing as a result of lack of specific "stop" signals for the inflammatory process. OBJECTIVE: To investigate the interaction between neutrophils and airway epithelium during inflammatory resolution in patients with allergic asthma. METHODS: An in vitro scratch assay with cultured epithelial cells, based on live-imaging microscopy, was used to evaluate regeneration and the influence of neutrophils on resolution. Epithelial cells and autologous neutrophils were derived from healthy donors and patients with allergic asthma. Supernatants and cells were collected for enzyme-linked immunosorbent assay and transcriptional analyses at the end of the experiment. RESULTS: Healthy epithelial cells regenerated faster than epithelial cells from patients with allergic asthma. Autologous neutrophils improved the regeneration of healthy epithelial cells but not asthmatic epithelial cells. Interleukin (IL)-8 and ß-catenin were down-regulated in healthy epithelial cells after resolution, but not in allergic asthmatic epithelial cells. CONCLUSION: The prolonged duration of inflammation in the respiratory tract in patients with allergic asthma could be due to the impaired healing pattern of epithelial cells and their compromised interactions with the neutrophils.

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.

Substance P represents a novel first-line defense mechanism in the nose.

BACKGROUND: Neuropeptides, such as substance P (SP), have long been seen as mediators of widespread continuous airway inflammation, a process known as neurogenic inflammation. However, this has been difficult to demonstrate clinically, suggesting an alternative role for these signaling molecules. OBJECTIVES: We sought to examine the role of SP in nasal infection by assessing the release of SP in response to viral stimulation and characterizing the effects of SP on innate immunity, with the latter reflected in changes in local Toll-like receptor (TLR) expression. METHODS: The distribution of SP and TLRs in the nasal mucosa and local airway neurons was assessed with immunohistochemistry. The TLR7 agonists R-837 and R-848 were used to mimic a viral insult in the upper airways represented by primary human nasal epithelial cells (HNECs) and murine nasal epithelial cells (MNECs) and isolated murine trigeminal ganglial neurons. SP release from HNECs, MNECs, and trigeminal ganglial neurons was quantified with EIA. The effects of SP on TLR expression on HNECs were determined by using flow cytometry and confocal microscopy. RESULTS: SP was released from the sensory neurons, MNECs, and HNECs within 15 minutes of local TLR7 stimulation. Subsequently, stimulation with SP induced upregulation of TLR expression in HNECs within 30 minutes through induction of TLR movement within HNECs. Upregulation of TLR expression was not evident when cells were treated with the neurokinin 1 receptor antagonist aprepitant before SP stimulation. CONCLUSIONS: This highlights a novel role for sensory neuropeptides as acute and local mediators of pathogen-driven inflammation, rapidly priming innate immune defenses in the airway.

NET-producing CD16high CD62Ldim neutrophils migrate to tumor sites and predict improved survival in patients with HNSCC.

The concept of functional neutrophil subsets is new and their clinical significance in malignancies is unknown. Our study investigated the role of CD16dim CD62Lhigh , CD16high CD62Lhigh and CD16high CD62Ldim neutrophil subsets in head and neck squamous cell carcinoma (HNSCC) patients. These neutrophil subsets may play different roles in immune-related activity in cancer, based on their profile, activation state and migration ability within a tumor site, which may be important in predicting cancer prognoses. Tumor biopsies and blood were obtained from newly diagnosed untreated HNSCC patients and healthy controls. Neutrophil subsets and their phenotype were characterized using flow cytometry. Isolated granulocytes were assessed for anti-tumor immune functions. Compared to controls HNSCC patients exhibited increased CD16high CD62Ldim neutrophils in blood; this subset displayed a distinct phenotypes with high expression of CD11b and CD18. This subset was prone to migrate into the tumor facilitated by tumor-derived IL-8. Furthermore, IL-8 was also found to activate neutrophils and thereby promoting subset transition. Various assays demonstrated that activated CD16high CD62Ldim neutrophils inhibited migration, proliferation and induced apoptosis of FaDu cancer cells. Neutrophil elastase detected in activated CD16high CD62Ldim neutrophils and tumor biopsies suggested that CD16high CD62Ldim neutrophils impart anti-tumoral activity via neutrophil extracellular traps. Furthermore, increased fraction of CD16high CD62Ldim neutrophils was shown to correlate with an increased survival rate. Our study demonstrates the clinical relevance of the CD16high CD62Ldim neutrophil subset, providing evidence for its increased migration capacity, its anti-tumor activity including increased NET formation and finally its correlation with increased survival in HNSCC patients.

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.

Intralymphatic immunotherapy of pollen-induced rhinoconjunctivitis: a double-blind placebo-controlled trial.

BACKGROUND: Allergen-specific immunotherapy represents the only disease-modifying treatment for allergic diseases. We and others have previously demonstrated that intralymphatic immunotherapy (ILIT), a less time-consuming alternative to conventional subcutaneous immunotherapy (SCIT), is safe and effective. However, this has recently been disputed. The aim of this study was therefore to expand our previous trial, further assessing the safety and efficacy of ILIT. METHODS: Thirty-six patients with pollen-induced rhinoconjunctivitis were randomised to receive three intralymphatic inguinal injections of active allergen (1000 SQ-U birch- or grass-pollen) or placebo. Clinical effects, safety and circulating immunological markers were assessed before, 4 weeks after treatment and at the end of the consecutive pollen season. RESULTS: No moderate or severe reactions were recorded following ILIT. Patients receiving active ILIT experienced a significant improvement in self-recorded seasonal allergic symptoms, as compared to placebo (p = 0.05). In a subgroup of these patients ("improved"), a reduction in nasal symptoms following nasal allergen provocation was also demonstrated. No changes in total IgE or IgG4 were found. However, the affinity of allergen specific IgG4 following active treatment was significantly increased, as compared to non-improved patients (p = 0.04). This could be correlated with clinical improvement, on an individual level. CONCLUSIONS: This double-blinded placebo-controlled study confirms that ILIT is a safe and effective treatment for pollen-induced rhinoconjunctivitis, markedly reducing seasonal allergic symptoms. TRIAL REGISTRATION: EudraCT: 2009-016815-39.

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.

Allergic sensitization does not influence advancement or survival in oral cancer.

A history of allergies has been said to be associated with a lower risk of head and neck cancer compared to the general population. However, it is not known whether having an allergic sensitization influences the prognosis and advancement of cancer disease. Thus, the aim of the study was to investigate the relationship between allergic sensitization and oral cancer advancement and patient survival. Allergen-specific IgE antibodies were investigated by ImmunoCAP™ Rapid in consecutive 80 patients with oral cancer. ImmunoCAP Rapid system tests a mix of representative inhalant allergens such as birch, timothy grass, mugwort, house dust mite, cat, dog, cockroach, olive (pollen), wall pellitory and mold. Eighty patients met the inclusion criteria for the study. Fifteen patients (19%) had positive ImmunoCAP test. There was no statistically significant difference in primary tumour size (T-stage) between groups (60% in allergy vs 68% in non-allergy had T1-T2 stage and 40% vs 32% T3-T4, respectively, p = 0.570). 27% of patients with allergy had nodal metastases compared with 37% of patients without allergy (p = 0.557). Both groups had comparable short-term survival. In conclusion, allergic sensitization does not seem to influence either the advancement or the short-term survival of patients with oral squamous cell carcinoma.

A Novel Method Using Fine Needle Aspiration from Tumor-Draining Lymph Nodes Could Enable the Discovery of New Prognostic Markers in Patients with Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell cancer (cSCC) is the second most common form of skin cancer, characterized by abnormal, accelerated growth of squamous cells. When caught early, most cSCCs are curable. About 5 percent of the cSCC cases have advanced to such an extent, generally metastatic, that they are far more dangerous, with very poor prognosis and challenging to treat. All efforts to find biomarkers, in blood or in the tumor itself, for early identification of patients with a risk for metastasis have so far failed. The present study describes a novel method that enables the identification of lymphocyte markers in tumor-draining lymph nodes. Six patients with advanced cSCC were analyzed using a combination of a sentinel lymph node biopsy (SLNB) protocol, fine needle aspiration (FNA), and flow cytometry. Immunological results from the sentinel nodes were combined with corresponding data from peripheral blood and unfixed tumor tissues. The result demonstrates a striking difference between the subsets of T-cells from the three compartments. Our interpretation of this first pilot study is that the ability to follow specific immunological markers on lymphocytes in tumor-draining lymph nodes will enable the identification of novel prognostic biomarkers not detectable in material from blood and tumor tissues.

A prolonged innate systemic immune response in COVID-19.

Despite the introduction of vaccines, COVID-19 still affects millions of people worldwide. A better understanding of pathophysiology and the discovery of novel therapies are needed. One of the cells of interest in COVID-19 is the neutrophil. This cell type is being recruited to a site of inflammation as one of the first immune cells. In this project, we investigated a variety of neutrophils phenotypes during COVID-19 by measuring the expression of markers for migration, maturity, activation, gelatinase granules and secondary granules using flow cytometry. We show that neutrophils during COVID-19 exhibit altered phenotypes compared to healthy individuals. The activation level including NETs production and maturity of neutrophils seem to last longer during COVID-19 than expected for innate immunity. Neutrophils as one of the drivers of severe cases of COVID-19 are considered as potential treatment targets. However, for a successful implementation of treatment, there is a need for a better understanding of neutrophil functions and phenotypes in COVID-19. Our study answers some of those questions.