10:15am - 10:30am
Role of Tracheal Inflammatory Dendritic Cells During Influenza Infection
1Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland; 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland
Influenza virus is responsible of high morbidity and mortality worldwide and a leading cause of death amongst young children, old people and the immuno-compromised. Despite the fact that the initial phase of the infection occurs in the upper respiratory tract, little is known about the immune reaction that follows infection and how it affects the outcome of the disease. Our research focused on elucidating the role of the inflammatory response in the mucosa of the trachea during early stages following influenza infection. Using confocal microscopy we observed a dense network of dendritic cells (DC) located under the mucosa of the trachea from mice, which increased significantly their number at day 3 post infection (p.i.) with influenza virus. The characterization of the tracheal subgroups of DC according to the expression of different surface markers indicated that at day 3 p.i. the majority of the infiltrated DC correspond to inflammatory dendritic cells (IDC) subtype. Our data suggest that IDCs are crucial during the initial immune response in trachea by the production of inflammatory chemokines. Furthermore we observed that the c-type lectin receptor SIGN-R1, that has the ability to bind influenza virus, was mainly expressed by IDC and its absence impaired the initial chemokine production leading to a significant lower presence and activation of NK cells. Finally, the absence of SIGN-R1, IDCs and NK cells corresponded with increased viral titers in the trachea at day 3p.i. leading to a major susceptibility after influenza infection. All this data suggested, that IDCs are key initiators of the immune reaction to influenza infection through the detection of viral particles and the secretion of immune chemokines that recruit and activate NK cells in the tracheal mucosa, which might help to the elimination of infected cells and the reduction of viral titers in the trachea.
10:30am - 10:45am
Susceptibility of Bona Fide Dendritic Cells to Porcine Reproductive and Respiratory Syndrome Virus
1Institute of Virology and Immunology, Vetsuisse Faculty, University of Bern, Switzerland; 2Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Switzerland; 3Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland
Bona fide dendritic cells (DCs) are important mediators of primary immune responses, and are composed of two subsets of conventional DCs (cDC1 and cDC2) as well as plasmacytoid DCs (pDCs). These cells have a very low frequency in the blood and survive poorly in culture, wherefore monocyte-derived dendritic cells (MoDCs) have often been employed to study dendritic cell functions. While bona fide DCs and MoDCs share a common DC-myeloid progenitor, along with several characteristics, their ontogenic lineages split early during differentiation, supporting that they are distinct lineages of cells. This is underpinned by distinctprofiles, when monocyte-derived cell’s (macrophage’s or MoDC’s) transcriptomes are compared to bona fide DC’s.
Following infection of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) delayed T-cell responses, as well as other signs of immunosuppression, are found. Published literature demonstrating high in vitro susceptibility of MoDCs to PRRSV, suggests that this may be caused by viral DC targeting. However, the susceptibility of bona fide DCs still remains to be elucidated. To clarify this issue, we first tested different cytokines (IL-3, GM-CSF & Flt3-ligand) to optimize the yield of DCs after culture. We identified cDC1 as CD135+CD14-CD172alowCADM1+, cDC2 as CD135+CD14-CD172a+CADM1+ and pDC as CD4+CD172a+. We found GM-CSF alone to create the most favourable culture condition.
We depleted the IL3R+ pDC-fraction, and infected the PBMCs with PRRSV or the known monocytotropic classical swine fever virus (CSFV) which also successfully proliferates in bona fide DCs. In contrast to CSFV, PRRSV was neither able to infect bona fide DCs, nor CD14+ monocytes. We also explored the susceptibility of DCs obtained from bone-marrow-derived precursor cells, but found the CD14- cells to be uninfected by PRRSV. These results would indicate that targeting of bona fide DCs by PRRSV is not a factor responsible for immunosuppression.
10:45am - 11:00am
Polarization of Macrophages in Epidural Inflammation Induced by Canine Intervertebral Disc Herniation
1Department of Veterinary Clinical Medicine, Vetsuisse Faculty, University of Bern, Switzerland; 2Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, Switzerland; 3Department of Infectious Diseases and Pathobiology, Institute of Virology and Immunology, Vetsuisse Faculty, University of Bern, Switzerland
Introduction: Canine interverterbral disc (IVD), although being physiologically acellular, displays a cell population consisting almost exclusively of macrophages (Mφ) when herniated. Mφ encompass a heterogenous cell population which can be roughly divided into classically (M1) or alternatively activated (M2)Mφ. The polarization into M1 Mφ is mainly driven by IFN-γ and leads to strong antimicrobial activity characterized by a pro-inflammatory signature. In contrast, M2 Mφ require IL-4/IL-13 and exibit anti-inflammatory function and regulate wound healing. The purpose of this study was to characterize the phenotype of the Mφ population present in naturally occuring herniated IVD.
Materials and Methods: IVD material of dogs with IVD disease were collected during decompressive surgery. A negative control group consisting of IVD material of dogs without disc degeneration that were euthanised for reasons unrelated with this study was collected. A positive control group consisting of canine liver and lymph node samples euthanised for reasons unrelated to this study was also included.
All samples were immediately embedded in OCT, shock frozen and stored at -80ºC. 8μm cryostat sections were prepared, air dried and immunostained without prefixation or permeabilization. CD14 was used as Mφ marker , MHCII for M1Mφ and CD206 for M2Mφ.
Results: 15 samples of dogs with IVD herniation, 10 negative and 5 positive control samples were obtained.
No CD14, CD206 or MHCII positive cell was found in the negative control group. The positive control group displayed several MHCII and CD206 positive cells, all of them being simultaneously positive to CD14.
All herniated samples displayed a mixed population of M1Mφ and M2Mφ, and some sparse Mφ displaying markers for both M1 and M2Mφ simultaneously.
Clinical Relevance: The mixed Mφ phenotype encountered shows the plasticity and dynamism of Mφ and evidences the chronic component of IVD disease despite its acute clinical presentation.
11:00am - 11:15am
Tumor-Associated CD90+ Mesenchymal Precursor Cells Are Part of an Immunosuppressive Tumor Microenvironment in Early-Stage Non-Small Cell Lung Cancer
1Division of General Thoracic Surgery, Inselspital, Bern University Hospital, Switzerland; 2Department for BioMedical Research, University of Bern, Switzerland; 3Interfaculty Bioinformatics Unit, University of Bern, Switzerland; 4Institute of Pathology, University of Bern, Switzerland; 5Boerhinger Ingelheim, Vienna, Austria
Rationale: Blockade of immune-checkpoint PD-1/PD-L1 pathway using antibodies has shown durable antitumor responses in patients with advanced non-small cell lung cancer (NSCLC). Despite this initial success, immune checkpoint blockade remains ineffective in the majority of NSCLC patients with documented PD-L1 expression.
Objective: To analyze the functional orientation of tumor infiltrating lymphocytes (TILs) using a multiparametric flow cytometric approach. In parallel, we interrogated the tumor mesenchymal compartment within the tumor microenvironment in early-stage NSCLC specimens and matched nonadjacent normal lung tissue.
Measurement and Main Results: We found that CD4+ and CD8+ TILs with increased PD-1 expression show a lack of degranulation while also downregulating CD127 expression in both lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) patients. The majority of CD4 and CD8 PD-1hi TILs were found in the central memory and effector memory compartment with increased TIM3 expression, with diminished or absent CD127 expression. We found coexpression of PD-L1 and CD47 in tumor-associated CD90+ mesenchymal precursor cells, despite elevated PD-L1 and CD47 also being coexpressed in the tumor epithelial fraction. Using machine learning we were able to identify a combination of features that were predictive of a smoking signature, tumor recurrence and survival. Last, TNFα/IFN-γ immune primed tumor-associated CD90+ mesenchymal precursor cells display an immunosuppressive secretory phenotype and target T cells for immunosuppression, irrespective of PD-L1 expression.
Conclusions: Our observations suggest that an immune reactive tumor microenvironment may fine tune and enhance the immunosuppressive signaling in constituent mesenchymal precursor cells in early-stage NSCLC. Therefore, a multi-targeted approach may be necessary to overcome the immunological barrier in advanced NSCLC.