Adhesion Molecule Targeted Therapy for Non-Infectious Uveitis

Non-infectious uveitis (NIU) is an inflammatory eye disease initiated via CD4+ T-cell activation and transmigration, resulting in focal retinal tissue damage and visual acuity disturbance. Cell adhesion molecules (CAMs) are activated during the inflammatory process to facilitate the leukocyte recruitment cascade. Our review focused on CAM-targeted therapies in experimental autoimmune uveitis (EAU) and NIU. We concluded that CAM-based therapies have demonstrated benefits for controlling EAU severity with decreases in immune cell migration, especially via ICAM-1/LFA-1 and VCAM-1/VLA-4 (integrin) pathways. P-selectin and E-selectin are more involved specifically in uveitis related to vasculitis. These therapies have potential clinical applications for the development of a more personalized and specific treatment. Localized therapies are the future direction to avoid serious systemic side effects.

The Critical Importance of Spatial and Temporal Scales in Designing and Interpreting Immune Cell Migration Assays

Intravital microscopy and other direct-imaging techniques have allowed for a characterisation of leukocyte migration that has revolutionised the field of immunology, resulting in an unprecedented understanding of the mechanisms of immune response and adaptive immunity. However, there is an assumption within the field that modern imaging techniques permit imaging parameters where the resulting cell track accurately captures a cell’s motion. This notion is almost entirely untested, and the relationship between what could be observed at a given scale and the underlying cell behaviour is undefined. Insufficient spatial and temporal resolutions within migration assays can result in misrepresentation of important physiologic processes or cause subtle changes in critical cell behaviour to be missed. In this review, we contextualise how scale can affect the perceived migratory behaviour of cells, summarise the limited approaches to mitigate this effect, and establish the need for a widely implemented framework to account for scale and correct observations of cell motion. We then extend the concept of scale to new approaches that seek to bridge the current “black box” between single-cell behaviour and systemic response.

935 Attraction of immune cells by head and neck cancer cell lines and primary tumor-conditioned supernatants

BackgroundHead and neck squamous cell carcinomas (HNSCC) are classified in human papillomavirus (HPV)-positive and HPV-negative tumors. In general, HPV-negative HNSCC are genetically characterized by many chromosomal gains and losses.1 Previously, we and others identified a HPV-negative subgroup with few or absent copy number alterations (CNA-silent), and a more favorable prognosis.2 3 Tumors with low copy number changes have generally been associated with high immune infiltration scores,4 but for CNA-silent versus CNA-high HPV-negative HNSCC such data are lacking.In this study we aim to unravel by functional assays immunological differences between HPV-negative and HPV-positive HNSCC, as well as between CNA-silent and CNA-high HPV-negative HNSCC. We analyzed the immune cell subsets attracted by HNSCC cell lines and by tumor-conditioned supernatants.MethodsEight HNSCC cell lines (3 HPV-positive, 3 HPV-negative CNA-high, 2 HPV-negative CNA-silent) and 24-hour supernatants of thirteen HNSCC biopsies were used to characterize their ability to attract immune cells in a transwell migration system. A chemokine mixture was used as a positive control, while medium alone was used to determine spontaneous migration. Peripheral blood mononuclear cells (PBMCs) of various healthy donors were plated in the upper compartment and after six hours the transwell migration was quantified by flow cytometry.ResultsMost HNSCC cell lines induced migration of monocytes, B cells and CD4+ T-cells up to maximal 12%, whereas CD8+ T-cells and conventional dendritic cells (cDCs) were not attracted, irrespective of the donor. Notably, one HPV-negative CNA-silent cell line induced significantly more migration compared to the negative control and other cell lines. Tumor-conditioned supernatants promoted immune cell migration with no apparent differences between tumor sites or HPV-status. Remarkably, up to 31% of monocytes migrated to these supernatants, 9x more than the chemokine control. Also cDC migration was induced, whereas lymphocytes were not attracted.ConclusionsHNSCC cell lines induced monocyte, B-lymphocyte and CD4+ T-lymphocyte migration, whereas tumor-conditioned supernatants attracted monocytes and cDCs only. No difference in immune cell attraction between HPV-positive and -negative HNSCC was observed. Interestingly, one HPV-negative CNA-silent cell line induced robust immune cell migration. Currently we perform a comprehensive chemokine analysis to explain the observed migration. The noted lack of CD8+ T-cell attraction may explain why current treatments with PD-1 inhibitors are effective in only a minority of HNSCC patients. Our data could provide a means to identify patients who might most likely respond to immune checkpoint blockade and to find clues to improve CD8+ T-cell attraction.ReferencesLeemans CR, Snijders PJF, Brakenhoff RH. The molecular landscape of head and neck cancer. Nat Rev Cancer 2018;18:269–82.Smeets SJ, Brakenhoff RH, Ylstra B, van Wieringen WN, van de Wiel MA, Leemans CR, et al. Genetic classification of oral and oropharyngeal carcinomas identifies subgroups with a different prognosis. Cell Oncol 2009;31:291–300.Cancer Genome Atlas N. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 2015;517:576–82.Davoli T, Uno H, Wooten EC, Elledge SJ. Tumor aneuploidy correlates with markers of immune evasion and with reduced response to immunotherapy. Science 2017;355.Ethics ApprovalWritten informed consent was obtained from all patients from whom fresh tumor biopsies were used for research, as part of the HNcol protocol at the Department of Otolaryngology|Head and Neck Surgery of Amsterdam UMC (VUmc) as approved by the Institutional Review Board (2008.071|A2016.035). Buffy coats, with written consent from the donors, were purchased from the Dutch blood bank (Sanquin) and used to isolate PBMC.

Cell scientist to watch – Tim Lämmermann

ABSTRACT Tim Lämmermann studied molecular medicine at the Friedrich-Alexander-University, Erlangen-Nuremberg, Germany and the Lund University, Sweden. He then joined the lab of Michael Sixt at the Max Planck Institute of Biochemistry in Martinsried, where he earned his PhD in 2009 for studying the role of integrins and cytoskeletal forces in immune cell migration. Tim then moved to the National Institute of Allergy and Infectious Diseases in Bethesda, USA for his postdoc with Ron Germain. There, he worked on the mechanisms of neutrophil swarming during infection, and received the Robert-Koch Postdoctoral Award in 2014. Since 2015, Tim has been a Group Leader at the Max Planck Institute of Immunobiology and Epigenetics in Freiburg, where his lab investigates the single-cell and population dynamics of immune cells. He was awarded an ERC Starting Grant in 2016.

Krüppel-like Factor 2 (KLF2) in Immune Cell Migration

Krüppel-like factor 2 (KLF2), a transcription factor of the krüppel-like family, is a key regulator of activation, differentiation, and migration processes in various cell types. In this review, we focus on the functional relevance of KLF2 in immune cell migration and homing. We summarize the key functions of KLF2 in the regulation of chemokine receptors and adhesion molecules and discuss the relevance of the KLF2-mediated control of immune cell migration in the context of immune responses, infections, and diseases.

Activation of NLRP3 Inflammasome Complexes by Beta-Tricalcium Phosphate Particles and Stimulation of Immune Cell Migration in vivo

Beta-tricalcium phosphate (β-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of β-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of β-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of β-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that β-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1β production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, β-TCP increased also IL-18 production, and NLRP3 inflammasome activation by β-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of β-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected β-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of β-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.

Extracellular Matrix Enzymes and Immune Cell Biology

Remodelling of the extracellular matrix (ECM) by ECM metalloproteinases is increasingly being associated with regulation of immune cell function. ECM metalloproteinases, including Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinases (ADAMs) and ADAMs with Thombospondin-1 motifs (ADAMTS) play a vital role in pathogen defence and have been shown to influence migration of immune cells. This review provides a current summary of the role of ECM enzymes in immune cell migration and function and discusses opportunities and limitations for development of diagnostic and therapeutic strategies targeting metalloproteinase expression and activity in the context of infectious disease.

Hyaluronan and Its Receptors: Key Mediators of Immune Cell Entry and Trafficking in the Lymphatic System

Entry to the afferent lymphatics marks the first committed step for immune cell migration from tissues to draining lymph nodes both for the generation of immune responses and for timely resolution of tissue inflammation. This critical process occurs primarily at specialised discontinuous junctions in initial lymphatic capillaries, directed by chemokines released from lymphatic endothelium and orchestrated by adhesion between lymphatic receptors and their immune cell ligands. Prominent amongst the latter is the large glycosaminoglycan hyaluronan (HA) that can form a bulky glycocalyx on the surface of certain tissue-migrating leucocytes and whose engagement with its key lymphatic receptor LYVE-1 mediates docking and entry of dendritic cells to afferent lymphatics. Here we outline the latest insights into the molecular mechanisms by which the HA glycocalyx together with LYVE-1 and the related leucocyte receptor CD44 co-operate in immune cell entry, and how the process is facilitated by the unusual character of LYVE-1 • HA-binding interactions. In addition, we describe how pro-inflammatory breakdown products of HA may also contribute to lymphatic entry by transducing signals through LYVE-1 for lymphangiogenesis and increased junctional permeability. Lastly, we outline some future perspectives and highlight the LYVE-1 • HA axis as a potential target for immunotherapy.