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

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.

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.