Role of memory B cell migration through the lymph node subcapsular sinus

Lead Research Organisation: University of Birmingham
Department Name: Institute of Immunology & Immunotherapy

Abstract

Vaccination programmes are a core element of public health strategy worldwide and are key to preventing a wide range of life threatening infections. How vaccines induce immune memory to bacteria and viruses are still poorly understood. We know that vaccination and infection induces B cells to mutate and adapt their genes coding for antibody specific to structures seen on the vaccine. These mutations then will lead to the production of highly specific antibody that can neutralize bacteria or viruses. During this process are also long-lived memory B cells generated, which preserve these mutations for a long time.
We recently observed that memory B cells not only spread through the body and preserve information on vaccines encountered, they also seem to harvest vaccine particles they encounter and transport them back to the places where B cells mature.
The purpose of this project is to understand why memory B cells do this antigen harvest, and which factors regulate it. To understand the regulation, we will produce B cells that cannot produce a range of receptors that direct memory B cell migration. To understand the function of antigen transport, we will disrupt the process and test whether efficient B cell maturation is dependent on continued antigen harvest and transport by memory B cells.
Aged people have problems efficiently producing good antibody responses to vaccines. Some of the defects seen during their B cell maturation may be due to a defect in antigen harvest by memory B cells, and we plan to test whether this process is disrupted in the aged immune system.
Results from this project will help to better understand how vaccines induce protective immune responses and memory. We may better understand how vaccines should be designed or delivered. Better understanding why elderly do not react efficiently to vaccination may lead to ideas on how to correct this process.

Technical Summary

The relevance of migration of germinal centre (GC)-derived memory B cells (mBC) entering the lymph node (LN) subcapsular sinus (SCS), interaction with macrophages and reentry into the GC is tested. Five migratory receptors (CCR7, CXCR3, CCR6, EBI2, S1PR1) are induced during at this stage. We have shown that LN reentry is directed by CCR7.

Objectives:
1) Identify roles of migratory receptors: Cg1Cre mTmG mice, where GC-derived mBC are GFP positive will be used. Cg1Cre mTmG CCR6ko mice are already available and used for CRISP-mediated deletion to produce CCR6 CXCR3 double ko mice. CXCR3 single ko mice will be generated by backcrossing onto wt (Cxcr3 is X-chromosomal). Cg1Cre-EBI2-floxed mice will be generated from available ES cells. S1PR1 will be blocked chemically, which allows study of blockage of combinations of migratory receptors.
Models will be used to study effects on mBC entry into the SCS, entry into other lymphoid and non-lymphoid tissues, and effects on antigen rechallenge.
2) Why is mBC return into the GC is happening? Four hypotheses will be tested:
I. mBC reentry replaces antigen consumed in the GC and sustains the GC while new antigen is produced at the site of entry.
II. unstable membrane bound antigens need to be continuously replaced to support continued GC B cell selection against native undenatured versions of the antigen
III. continued supply of antigen safeguards against escape mutants and supports selection towards antigenic variations
IV. interaction with SCS mBC represents an additional selection step during mBC generation
This will be done using mice generated earlier, by deleting SCS macrophages, or by using Cg1Cre mTmG Ackr4ko mice (available, and deficient in CCR7-dependent mBC reentry).
3) Aged lymph nodes show defects in immune complex deposition in the GC and CCR7 dependent migration. We will test whether reduced vaccine-induced antibody responses are related to reduced SCS mBC - macrophage interaction and mBC reentry.

Planned Impact

There is a broad range of potential beneficiaries of the research that can be summarised as follows:

The scientific community.
The project will produce fundamental new understanding on how vaccine or pathogen antigens are delivered to the place where they induce affinity maturation and the generation of antibody neutralizing pathogens and preventing infection. Understanding these fundamental processes may benefit vaccinology and our understanding how antigens should be delivered and presented in order to produce efficient vaccines that induce antibody that targets the relevant structures on pathogen antigens. Understanding how memory is generated is relevant many different immunological disciplines as it is relevant for T cell immunology, memory, inflammation, autoimmunity, and ageing.

Society
The importance of this work extends significantly beyond supporting academic research. By understanding how antibody responses develop we are helping to understand how to improve vaccine development. This is vital. In our highly mobile society the risks from infectious diseases are increasing due to increased travel and altered global migration and extend also into the food-chain. This is because many of our infections are actively acquired from food (e.g. Salmonella) or livestock rearing acts as an incubator and enables pathogen diversity (e.g. influenza virus). In parallel, we have a decreasing efficacy of anti-microbial treatments due to resistance and only a modest number of anti-virals available. Vaccination is a cost-effective approach that can protect against infection at the extremes of age in those groups that are most susceptible. Furthermore, vaccination is an acceptable intervention to society at large, and as the media response to the recent measles outbreak demonstrates, is one that is diminishing in controversy. Indeed, the measles outbreak demonstrates the importance of vaccination programmes to protecting society and the consequences when there is insufficient vaccine coverage. Theoretical background how vaccines work, what signals regulate the emergence of high affinity B cells and antibody producing cells, and how immunological memory cells are regulated, is still in its infancy.
Industry

Publications

10 25 50
 
Description Gene expression in memory B cells 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provision of mRNA from in vivo differentiated memory B cells
Collaborator Contribution Gene expression analysis from different in vivo differentiated memory B cells by RNAseq and bioinformatic analysis
Impact New data on gene expression in new memory B cell subsets.
Start Year 2015
 
Description In silico modelling of germinal centres 
Organisation Helmholtz Association of German Research Centres
Department Helmholtz Centre for Infection Research (HZI)
Country Germany 
Sector Public 
PI Contribution Data provision for in silico models Work on the in silico model
Collaborator Contribution In silico modelling Predictions for optimal experimental in vivo setups
Impact The collaboration has led to several publications. It has produced a new in silico model to analyse the regulation of affinity maturation in germinal centres. This has lead to several further collaborations and publications for our collaborators.
Start Year 2006
 
Description Industrial partnership 
Organisation MedImmune
Department MedImmune Cambridge
Country United Kingdom 
Sector Private 
PI Contribution We are analysing GM mouse strains with altered B cell receptor signalling in germinal centres for their suitability as tools to produce new therapeutic monoclonal antibody drugs. The prediction is that the GM mice will easier produce specific high affinity antibodies to difficult target antigens. Further, we are analysing effects of these genetic modification on the regulation of high affinity antibody responses.
Collaborator Contribution The industrial partners design and produce the initial vectors for genetic manipulation of mice. They transfect these into embryonic stem cells and produce GM mice. After this step the mice are transferred to us. At a later stage they will test the mice for practical usability as vehicles to produce high affinity B cells to difficult targets for hybridoma fusions. They will use the mice and test whether they ca produce monoclonal antibodies using some of there identified human therapeutic targets.
Impact Three GM mouse strains have been produced and are currently analysed in our lab, publications in preparation. New iCASE PhD studentship to work on 4th strain.
Start Year 2014
 
Description Intravital imaging of memory B cell migration 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of GMO mice,
Collaborator Contribution Immunisation of GMO mice and intravital imaging of migrating memory B cells. Training visits for UoB staff on intravital microscopy in preparation.
Impact Early results show new patterns of memory B cell migration and roles of chemokine receptors directing this.
Start Year 2016
 
Description Intravital imaging of plasma cell differentiation 
Organisation Leibniz Association
Department German Rheumatism Research Centre
Country Germany 
Sector Charity/Non Profit 
PI Contribution A postdoctoral research fellow spent twice 2 months at DRFZ to learn intravital imaging techniques and study plasma cell differentiation from germinal centres
Collaborator Contribution Hosting, training, and help with experiments. Provision of experimental animals
Impact Training of postdoctoral research fellow Preliminary data on plasma cell differentiation Submission of funding proposal to Wellcome Trust for an intravital microscope
Start Year 2011
 
Description Memory B cell migration and activation of secondary responses to antigenic variation 
Organisation University of Basel
Department Department of Biomedicine
Country Switzerland 
Sector Academic/University 
PI Contribution Advise and provision of virus vectors that can be used to study variable virus antigens
Collaborator Contribution The partner plans to provide virus samples that can be used at UoB for immunisation experiments
Impact none yet
Start Year 2018
 
Description Provision of GM mice 
Organisation RIKEN
Department RIKEN Center for Integrative Medical Sciences (IMS)
Country Japan 
Sector Hospitals 
PI Contribution Provision of mice that can be used to track germinal centre B cells
Collaborator Contribution Provision of mice that have an inducible expression of Cre recombinase under the control of the S1PR2 promotor
Impact We currently use these mice to generate new unique mouse models to study germinal centre B cell differentiation.
Start Year 2018