Genetic analysis of the role of the PI3K signalling in humoral immunity

Antibodies are produced by B lymphocytes when they differentiate into plasma cells following exposure to antigen. Antibodies are proteins that are key mediators of immunity required to protect us from pathogenic micro-organisms such as bacteria, fungi and viruses. The production of potent antibodies is central to immunity and the principle of vaccination and it has been known for many decades that during the course of an infection the quality of antibodies produced increases. Revealing the mechanism by which this takes place may provide information which will aid in vaccine design. The study of B lymphocytes in health and disease is directly relevant to our attempts to improve vaccine design; to develop new treatments for autoimmune disease; and to develop therapies to combat B cell malignancies. This project seeks to uncover the basic mechanisms that control the development and function of B cells when they are activated. The research will use genetic models in which the function of individual genes that we propose to be vital for antibody production is specifically switched off. This can be achieved in a specific cell type or at a particular stage of the immune response. By deleting candidate genes in specific cells or at specific times and testing the consequences for B cell differentiation and the ability to produce and sustain antibody production we will obtain good evidence for the function of the genes.

The intracellular signalling pathways that regulate the generation, maintenance and activation of memory B cells and plasma cells are poorly understood. We have obtained data suggesting the PI3K pathway plays a role in the maintenance of the germinal centre reaction from which memory and plasma cells arise. To test the hypothesis that the PI3K pathway regulates survival or activation of memory B cells and plasma cells we propose to study mice in which Cre mediated recombination of conditional (floxed) alleles of PTEN or p110delta is regulated in activated germinal centre B cells. This will employ the recently described Cgamma1Cre system in which Cre is expressed from constant region of the gamma1 gene. To test if p110delta is required for plasma cell survival we propose to study p110delta flox/flox mice containing the Rosa26 ERT2-Cre allele in which Cre is activated upon addition of 4-OH-tamoxifen (4HT). The persistence of antibody and antibody secreting cells will be tested following 4HT administration. Collaboration between T and B cells is vital for a proper germinal centre reaction and memory formation. We predict that the ability of 110delta-?deficient CD4 T cells to provide help to B cells will be defective. To date, no experiments have been performed to asses the contribution of defective T cell help to the impaired humoral immune responses of p110delta-deficient mice. We will therefore immunise p110delta flox/flox CD4Cre mice and measure antibody production and the formation of memory cells. In each mouse model we will immunise with the model antigen 4-hydroxy-3-nitrophenyl-acetyl conjugated to chicken gamma globulin (NP-CGG) in alum. This non-replicating antigen has been extensively studied in the C57BL/6 mouse and it is possible to identify antigen specific memory and plasma cells by flow cytometry; measure antigen specific antibody titres of high and low affinity by ELISA; and enumerate antibody secreting plasma cells by EISPOT.