Mechanisms of leukocyte and tumour cell trafficking in the lymphatic system

Lead Research Organisation: MRC Human Immunology Unit

Abstract

The lymphatic network is a second circulatory system as extensive as the blood system that acts as a conduit for fluid drainage as well as a pipeline for the delivery of foreign antigens to the lymph nodes - the sites where protective immune responses are generated. The lymphatic system is also exploited by cancers which frequently use it to spread from the primary tumour to distant organs. Understanding how cells enter and exit the lymphatics and learning how to block these processes would benefit treatments for inflammation _ where the immune response needs to be damped down, as well as treatments for bowel, breast and other common cancers where lymphatic spread prevents control of the disease. The work being carried out by this group involves studying the mechanisms for cell entry to lymphatic vessels at the molecular level using materials isolated from patients as well as animal models for inflammation and cancer.

Technical Summary

This project addresses the fundamental question of how leukocytes - particularly antigen presenting dendritic cells, memory T cells and neutrophils enter the lymphatics and migrate to draining lymph nodes in the course of the immune response to foreign antigen. It also concerns the important related issue of how tumour cells disseminate to lymph nodes and distant organs via the lymphatics during the process of metastasis. The methodology involves the isolation and culture of lymphatic endothelial cells from patients and their analysis in a range of cell migration, cell adhesion, chemotaxis and gene chip microarray (transcriptional profiling) studies. In addition, the research involves immunohistochemical and microscopic studies of lymphatic vessel organization in human and murine cancer specimens, lymphatic trafficking studies in mouse models of inflammation and cancer, and gene knockout analyses. The work involves collaboration with surgeons, clinicians and pathologists at the Oxford Radcliffe site, as well as in-house expertise from clinician D.Phil students and DoH Clinical Research Development Awardees (RDAs). The work is likely to reveal targets for the blockade of immune activation in the treatment of inflammation and the induction of immunosuppression as well as providing information for optimizing vaccine delivery. Furthermore, the work is likely to identify new prognostic markers for prediction of lymphogenic tumour metastasis in head and neck squamous cell carcinoma and colorectal carcinoma and may also identify targets for blockade of lymph node metastasis in these as well as other human cancers.

Publications

10 25 50
 
Description Academy of Finland
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
Impact Advised on appointment of prestigious Professorship
 
Description Lund University
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
Impact Advised on appointment of prestigious Professorship
 
Description Membership of MRC Infection and Immunity Board
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description NIH Study Section
Geographic Reach North America 
Policy Influence Type Participation in advisory committee
Impact Reviewed applications for research funding submitted to NIH
 
Description Polish Science Foundation
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
Impact Reviewed biomedical science grant proposals submitted to Polish Government Research Board
 
Description Swiss National Science Foundation
Geographic Reach Asia 
Policy Influence Type Participation in advisory committee
 
Description MRC Research Grant Neutrophil trafficking in lymphatics
Amount £500,000 (GBP)
Funding ID G1100134 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2011 
End 10/2014
 
Description MRC Research Grant Streptococcal dissemination
Amount £592,795 (GBP)
Funding ID MR/L008610 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 04/2014 
End 03/2017
 
Description NHS RCD Fellowships
Amount £372,000 (GBP)
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2006 
End 10/2009
 
Title Crystal structure of CD44 receptor ligand complex 
Description First crystal showing molecular details of interaction between a hyaluronan receptor and its ligand hyaluronan. Determined structure in the case of the primary receptor CD44 that is involved in inflammatory leukocyte trafficking and tumour metastasis 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Major boost to fundamental biology. Template for design of small molecule inhibitors of CD44 function with possible therapeutic potential 
 
Title Function blocking antibodies 
Description Defined properties of mAbs generated against lymphatic vessel receptor as being function blocking for leukocyte trafficking in lymphatics 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Use of mAbs identifies lymphatic receptor as important for leukocyte movement from tissues to lymph nodes in inflammation 
 
Title Primary lymphatic endothelial cells 
Description Primary lymphatic endothelial cells isolated by immunomagnetic bead selection from human skin, mouse skin and human colorectal tissue 
Type Of Material Cell line 
Provided To Others? No  
Impact Ability to analyze molecular details of lymphatic vessel physiology and to establish various assays for cell adhesion / transmigration studies with cells of authentic phenotype 
 
Title Tumour-induced lymphatic vessel markers 
Description Identified molecular markers induced in lymphatic vessels of murine metastatic tumours by comparison with normal tissue lymphatics. Confirmed expression also in human cancers. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Used lymphatics markers to predict metastasis in colorectal/head and neck cancers. Identified receptor molecules that are liely to be mechanistically involved in lymphatic metastasis of tumours. 
 
Description Anne Dell collboration 
Organisation Imperial College London
Department Division of Molecular Biosciences
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Preparation of materials for glycan analysis
Collaborator Contribution Expertise in analysis of regulatory carbohydrate components of key glycoproteins in our research
Impact Expertise in glycan analysis for core projects and future publications
Start Year 2007
 
Description Awen Gallimore collaboration 
Organisation Cardiff University
Department Infection and Immunity
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Advice on experimental design and evaluation of experimental data from mouse tumour model
Collaborator Contribution Carried out studies into mouse fibrosarcoma tumour development and role of different vasculatures in T cell recruitment to tumour
Impact Manuscript in International Journal of Cancer PMID 24142504
Start Year 2014
 
Description David Greaves collaboration 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Intellectual input and experimental design Animal experiments involving cell trafficking
Collaborator Contribution Advice and expertise in measurement of endothelial cell junctional permeability Contribution of transgenic reporter mice
Impact Manuscript in Blood; describing utility of CD68 transgenic reporter mice for macrophage trafficking studies Manuscript in preparation for J Biol Chem
Start Year 2012
 
Description Dietmar Vestweber collaboration 
Organisation Max Planck Society
Department Max Planck Institute for Molecular Biomedicine
Country Germany, Federal Republic of 
Sector Charity/Non Profit 
PI Contribution Application of antibody reagents to characterization of tumour lymphatics. Intellectual input into experiments
Collaborator Contribution Allowed us to obtain transgenic mice for future mechanistic studies
Impact Generated publication PMID 18794116 Allowed us to obtain transgenic mice for future mechanistic studies
Start Year 2006
 
Description Fiona Powrie collaboration IBD 
Organisation University of Oxford
Department Nuffield Department of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Intellectual input, experimental design. Provision of function blocking mAbs and gene knockout mice
Collaborator Contribution Expertise and advice in Mouse models of inflammatory bowel disease. Intellectual input.
Impact In progress
Start Year 2012
 
Description Fluorescence and EM imaging of lung 
Organisation Medical University of Vienna
Department Clinical Institute of Pathology
Country Austria, Republic of 
Sector Academic/University 
PI Contribution Provision of reagents and intellectual input
Collaborator Contribution Performed immunoelectron microscopy and immunofluorescence imaging of thin sections of mouse alveolae
Impact Obtained important data for inclusion in manuscript under preparation for publication in scientific journal
Start Year 2012
 
Description High Resolution confocal Lung Imaging 
Organisation University of California, San Francisco
Department Department of Anatomy
Country United States of America 
Sector Academic/University 
PI Contribution Provided reagents and intellectual input
Collaborator Contribution Carried out perfusion fixation and inflation of lungs and prepared sections for confocal imaging and z sectioning to identify critical cell population in alveolae
Impact Obtained critical data for inclusion in a manuscript under preparation for submission to scientific journal
Start Year 2012
 
Description Jesus Angulo NMR collaboration 
Organisation University of Seville
Department Department of Bioorganic Chemistry
Country Spain, Kingdom of 
Sector Academic/University 
PI Contribution Provision of recombinant soluble CD44 protein for STD NMR experiments
Collaborator Contribution Performed STD NMR analyses
Impact In progress
Start Year 2012
 
Description Lung lymphatics mAbs 
Organisation Medical University of Vienna
Department Clinical Institute of Pathology
Country Austria, Republic of 
Sector Academic/University 
PI Contribution Generation of mAbs for imaging vasculature in lung sections
Collaborator Contribution EM and confocal imaging of lung vasculature
Impact Manuscript in preparation
Start Year 2012
 
Description Luxheng Xue collaboration 
Organisation University of Oxford
Department Nuffield Department of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Advice and experimental design. Discussion of data and manuscript editing
Collaborator Contribution Performance of experiments to investigate neutrophil : T cell crosstalk
Impact Manuscript in J allergy and Clinical Immunol PMID 25441644
Start Year 2013
 
Description Melody Swartz collaboration 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Department Institute of Bioengineering
Country Switzerland, Swiss Confederation 
Sector Academic/University 
PI Contribution Travel to partners laboratory for training
Collaborator Contribution Training in the immunoisolation of lymphatic endothelial cells from mice using podoplanin mAbs. Training in intravital imaging of lymphatics.
Impact Transfer of specialist expertise
Start Year 2013
 
Description Neil Barclay and Clive Metcalf CD44 labile sulphydryl collaboration 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Intellectual input. Experimental design. Analysis of CD44 molecules before and after selective reduction to identify labile sulphydryls and the effects of their reduction on ligand (HA) binding by surface plasmon resonance measurement and cellular assays
Collaborator Contribution Intellectual input. Experimental design. Mass spectrometric analysis of reduced CD44 protein adducts and reagent sharing.
Impact Manuscript that is about to be submitted to J Biol Chem
Start Year 2011
 
Description Nick Athanasou collaboration 
Organisation Oxford University Hospitals NHS Foundation Trust
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Generated and provided antibodies for immunohistochemistry Provided advice and expertise in microscopy and intellectual contribution towards publications
Collaborator Contribution Research into clinicopathology of lymphatics in connective tissue
Impact Several co-authored publications PMID 19727710; 18813946;18484257;17904616
 
Description Peter Mortimer collaboration 
Organisation St George's Hospital
Department Department of Dermatology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Hospitals 
PI Contribution Generated antibodies and provided advice and intellectual input
Collaborator Contribution Broadened knowledge of clinical issues in lymphatic disease
Impact Ongoing characterization of pathology of lymphoedemas
Start Year 2006
 
Description Simon Milling collaboration 
Organisation University of Glasgow
Department Institute of Infection, Immunity and Inflammation
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Design of experiments to assess lymphatic trafficking from intestine by sampling afferent lymph in mice. Contribution of function blocking mAbs and knockout mice
Collaborator Contribution Provision of expertise in surgical techniques and sampling of pseudoafferent lymph in mice. Intellectual input. Mouse models of gut inflammation.
Impact In progress
Start Year 2012
 
Description Sriskandan collaboration 
Organisation Imperial College London
Department Faculty of Medicine
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Design of experiments and advise on performing experiments to assess interactions between hyaluronan encapsulated Group A strep and lymphatic endothelial cells leading to possible transport to lymph nodes
Collaborator Contribution Provision of wild-type and mutant strains of Strep pyogenes and participation of post-doctoral scientist
Impact Publication in PLoS Pathogens 2015 PMID 26352587 Multidsciplinary Immunology Bacteriology Infectious Diseases
Start Year 2012
 
Description Super resolution microscopy 
Organisation Medical Research Council (MRC)
Department MRC Human Immunology Unit
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Public 
PI Contribution Preparation of primary cells and transfectants, and appropriate mAbs and conjugates for STED microscopic evaluation of receptor surface distribution
Collaborator Contribution Performing STED microscopy and analysis of data
Impact Multidisciplinary collaboration Manuscript submitted to Scientific Reports 2016
Start Year 2014
 
Description Surface Plasmon resonance (Biacore) analysis of receptor ligand binding affinity 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom of Great Britain & Northern Ireland (UK) 
Sector Academic/University 
PI Contribution Generated recombinant soluble hyaluronan receptors and mutants thereof and performed Biacore analyses using hyaluronan coated chip to determine binding affinities
Collaborator Contribution Mathematical evaluation of binding data from kinetic analyses and derivation of accurate Kd values
Impact Manuscript that is about to be submitted to J Biol Chem
Start Year 2014
 
Title Licensing of monoclonal antibodies 
Description Hybridomas producing monoclonal antibodies to human LYVE-1 were licensed to Merck Millipore on a non-exclusive basis.The mAbs will be included in their catalogue for non-medical use. 
IP Reference  
Protection Protection not required
Year Protection Granted 2016
Licensed Yes
Impact N.A.