Unique ErkMAPkinase checkpoint signatures drive differential responses during the immature-mature B cell transition?

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

B-lymphocytes are cells that produce proteins known as antibodies that play important roles in the body s fight against infection. In order to fight any infection, vast numbers of randomly different B cells are made. However, many of these B cells can potentially make antibodies capable of attacking molecules within the body (self molecules), resulting in autoimmune diseases like Rheumatoid Arthritis. The immune system has therefore evolved checkpoints to prevent production of autoimmune B cells by killing immature cells that attach themselves to self molecules in a process known as tolerance. By contrast, when mature tolerant B cells encounter infections, many of these B cells develop into antibody-secreting cells to fight the disease. In addition, some become memory B cells that can produce faster, better antibody responses if the host becomes re-infected with this same disease. ?Memory? is the basis underlying the development of vaccines against diseases such as tetanus and polio. The aim of this application, therefore, is to work out the key molecular events regulating destruction of autoreactive B cells and retention of useful B cell specificities as identification of these has obvious potential for the development of new vaccine, cancer and autoimmunity therapies.

Technical Summary

B-lymphocytes are the principal cellular mediators of the antibody response to infection. Moreover, the advent of B cell-targeting reagents such as rituximab, which have been demonstrated to have therapeutic potential in several autoimmune inflammatory diseases, has revealed that B cells also play pivotal roles in antigen presentation and regulation of the immune response. The molecular mechanisms that constitute developmental checkpoints preventing the development of pathogenic B cells are not fully understood. However, we have shown that the precise signature of ErkMAPkinase signalling, in terms of the kinetics, signal strength, localisation and maturation stage-specific expression and activation of downstream effectors, can direct either cell death or survival and proliferation of B cells in vitro. We have therefore hypothesised that differential ErkMAPKinase signalling signatures act as developmental checkpoints during B cell maturation in vivo and the core aims of the proposal are therefore to define the (i) precise ErkMAPkinase signatures differentially directing positive and negative selection by antigen during the immature to mature B cell transition in vivo and (ii) impact of dysregulation of such selection checkpoints on immune phenotype and potential development of autoimmune, lymphoproliferative or immunodeficient B cell disorders.

B cell signalling has traditionally been investigated using biochemical, broken cell analysis of purified cells, cell lines or by characterization of transgenic/knockout mice. These approaches, however, can mask identification of multiple functional roles of individual signals dependent on their strength, duration and subcellular localization and hence are unlikely to reflect the responses of individual antigen-specific B cell progenitors within their physiological microenvironment in vivo. We have therefore exploited our complementary expertise in tracking and imaging of antigen-specific signalling responses, B cell development models and instant transgenesis , involving adoptive transfer of B cell progenitors genetically modified by retroviral gene transfer, to develop techniques to quantify and image the subcellular localization of signalling events in individual antigen-specific B cells within their physiological microenvironment in vivo, using laser-scanning cytometry (LSC). This approach addresses the strategic priority of 3Rs (replacement, reduction, refinement) obviating the need for the labour- and animal-intensive, time consuming and expensive process of generating Tg, knockout and knockin mice on appropriate genetic backgrounds. Importantly, by providing a mix of wild type and modified cells within a single animal, with the target gene expressed over a range of signal strengths, this approach allows us to determine the differential antigen-dependent selection of the B cell repertoire by signal strength.
 
Title In situ analysis of instant transgenesis systems 
Description We have further developed our novel techniques of combining instant transgenesis using trackable retroviral and lentiviral vectors for dissecting the molecular mechanisms underlying B cell development in vitro and in vivo that can be analysed in situ at the single cell level within their physiological anatomical niche by laser scanning cytometry 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact these systems have only been optimised for B cell development since the RA started in July but are expected to have impact, in terms of publications, in 2010. This approach has greatly reduced the numbers of animals required to study signalling in the immune responses for 2 major reasons. 1. Traditionally, genetic modification has required transgenic or knockout procedures which require extensive crossbreeding onto the required genetic backgrounds whilst this instant transgenesis, allows analysis of wild type and modified cells within a single animal without any breeding. 2. Signalling assays have traditionally required large numbers of animals to prepare sufficient cells for biochemical analysis. Development of in situ laser scanning cytometry assays have the negated the need for large numbers of cells as this analysis allows investigation at the single cell level in individual animals. In addition, development of laser scanning cytometry assays allows such analysis within the physiological lymphoid tissue environment, rather than in a test-tube. 
 
Description B cell development in health and disease 
Organisation University of Glasgow
Department Institute of Cancer Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution we applied the understanding of B cell development and function gained in this MRC award in recent studies (funded by a Wellcome Trust Programme grant and Wellcome Trust Ph D studentships) investigating dysregulation of the homeostatic balance of regulatory/effector B cells in allergic and autoimmune inflammatory disease and how this can be modulated therapeutically by the parasitic worm-derived product, ES-62. In addition, we have recently begun to extend this to working on the molecular mechanisms underlying dysregulation of B cell development in cancer
Collaborator Contribution in vitro and in vivo models of B cell development and instant transgenesis systems, in vivo models of allergic and autoimmune inflammatory disease and leukaemia
Impact Publications and current grant submissions
 
Description B cell development in health and disease 
Organisation University of Strathclyde
Country United Kingdom 
Sector Academic/University 
PI Contribution we applied the understanding of B cell development and function gained in this MRC award in recent studies (funded by a Wellcome Trust Programme grant and Wellcome Trust Ph D studentships) investigating dysregulation of the homeostatic balance of regulatory/effector B cells in allergic and autoimmune inflammatory disease and how this can be modulated therapeutically by the parasitic worm-derived product, ES-62. In addition, we have recently begun to extend this to working on the molecular mechanisms underlying dysregulation of B cell development in cancer
Collaborator Contribution in vitro and in vivo models of B cell development and instant transgenesis systems, in vivo models of allergic and autoimmune inflammatory disease and leukaemia
Impact Publications and current grant submissions
 
Description Regulation of B cell signalling during development 
Organisation University of Glasgow
Department Institute of Infection, Immunity and Inflammation
Country United Kingdom 
Sector Academic/University 
PI Contribution Assisting with experiments, troubleshooting between groups in common areas of research
Collaborator Contribution Co-supervision of post-graduate and undergraduate students. Sharing of equipment and reagents
Impact Enabled award of an MRC grant: G0800167
 
Description Open days/interviews 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 1. Describing work to potential postgraduate students at open days and/or "research sessions" 2. interviewed by undergraduate students on science communication course in order that they could prepare a "newspaper"-type article

recruited a Ph D student
Year(s) Of Engagement Activity 2006,2007
 
Description Symposium speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact World Autoimmunity Congress Granada 2012

Talk delivered to academic peers and members of relevant patient groups (eg SLE)
Year(s) Of Engagement Activity 2012
 
Description open days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact The co-PI AMM regularly gives presentations, interviews with patient groups on B cells in health and disease

patient groups raise funds for parallel studies of AMM in B-CLL
Year(s) Of Engagement Activity 2009,2010,2011
 
Description open days/fund raising for research 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Participants in your research and patient groups
Results and Impact Co-PI routinely meets with relevant patient groups/fundraisers for B-CLL to explain research

n/a
Year(s) Of Engagement Activity 2012