Cell-intrinsic roles of P110delta in primary and memory antibody responses

The production of antibodies is critical for immunity and is one of the key processes stimulated by vaccination. Vaccine failure, which becomes more common upon ageing, is largely due to a failure to elicit antibodies. Antibodies are secreted by specialised cells called plasma cells which are the descendents of the B-lymphocyte, a specialized (differentiated) form of white blood cell. Our study is aimed at understanding how the process of B lymphocyte differentiation is regulated in the mammal. Despite great effort, this cannot be mimicked by culturing cells in specialized media and incubators. Therefore we have to use animal models; in this case we use the mouse, partly because we have a great many research tools to study B lymphocyte differentiation in detail and also because we already know much about the system including how it is similar to other animals. This allows us to ask and examine sophisticated questions. We already know that when B lymphocytes are stimulated by antigen they may either quickly differentiate into antibody secreting plasma cells, which reside in a specific anatomical location. Alternatively they may become rapidly proliferating, but non-antibody secreting, germinal centre cells. Each of these two alternative cell fates is regulated by second type of cell called the T-lymphocyte. Our study is concerned with characterising the T-lymphocytes which promote each of these B cell fate decisions. To provoke an immune response, mice will be challenged with model antigens including an attenuated (not-virulent) form of salmonella. The responses to these challenges will be measured to count the numbers of each type of different cell and determine their precise anatomical location. The process will be perturbed by specifically targeting mutations of a cell activation pathway to the T cells; all other cells, including the B lymphocytes, will remain normal allowing us to conclude that any altered behaviour B lymphocytes display will be due to a defect in the T cells.

We will examine how PI3K activity in T cells regulates GC memory B cells and antibody secreting plasma cell output. To do this we will use the OX40Cre system to conditionally delete P110delta or PTEN in helper cells subsequent to development and following the initial antigen priming stage. In some experiments we will use antigen specific B and T lymphocyte populations which will permit us to follow the expansion of of very rare cells during immunisation. This study will investigate how the PI3K enzyme system in T cells regulates the ability of the B lymphocyte to differentiate and form antibody secreting cells in an extra-follicular (EF) response or to enter into and sustain the germinal center (GC) reaction. We will investigate whether the P110delta isoform of PI3K in T cells regulates the EF response by using mice in which conditional gene targeting is used to delete p110delta specifically in T cells. The mice will be immunised with model antigens and infectious micro-organisms, in this case Salmonella enterica serovar Typhimurium (S.typhimurium). We will use a similar system to test whether the chemokine receptor CXCR4 on T cells regulates the EF response. If this hypothesis is validated we will use additional mutants in the PI3K pathway to uncover the signalling elements downstream of CXCR4. However as we lack conditional mutants for these genes (P101 and P110gamma) we will use a mixed chimaera approach.

The research into the biology PI3K in general and P110delta and gamma in particular will be of interest to numerous biotechnology and pharma companies. A recent survey indicated over 20 patents had been filed on small molecule inhibitors of the PI3K pathway (see Patent watch Charlotte Harrison Nature Reviews Drug Discovery 8, 606-607 (August 2009). This includes patents form Calistoga, GSK, Roche, Genentech, Intellikine, Novartis and Wyeth. Companies in the U.K. that are also developing PI3K inhibitors include both giant (GSK) and small (Karus Therapeutics Ltd. in Southampton) ventures. Small molecule inhibitors of P110delta (Calistoga's CAL-101) have already been used in the first in-human trials. All of these companies will be interested in understanding the biological function of PI3K in the whole animal setting as will the regulatory authorities. Ultimately, the development of safe and efficacious medicines will benefit society as a whole. Our research on PI3K may provide new indications of where modulation of this pathway will be of use. Given the progress of this field these benefits could be realized within a decade. Our research into antibody production may provide insights into how to improve the nature of vaccines of adjuvants. We would argue from our preliminary data that one route to this goal would be to increase PI3K signalling in T cells. Our research using Salmonella as a model organism may ultimately provide insight relevant to reducing the morbidity and mortality associated with this disease. This is part of the global 'one-health' agenda where the study of zoonotic disease is part of the BBSRC strategy. The global impact of Salmonella infection, which is most acute in the third world, also justifies the choice of this model organism to study immunity. In this context the work from Birmingham and Malawi has shown that altered B cell (antibody) responses to Salmonella in AIDS sufferers is relevant to pathogenesis of salmonella infection. Post-doctoral training at the Babraham Institute involves the opportunity for the acquisition of generic skills in partnership with the University of Cambridge as well as public engagement.