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

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.

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.

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.
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