FUNCTIONAL CHARACTERIZATION OF MEMORY B CELLS – BcellMemory

1-Scientific background and rationale Immune responses to infectious agents have different out-comes that can lead either to protection from re-infection or may be inefficient in disease control. The factors controlling these different out-comes are in part yet unknown. Protection against infection requires the establishment of efficient secondary immune responses. Immunological memory requires the generation of antigen-specific 'memory' B and T lymphocytes. Memory B cells express receptors with increased affinity for antigen, selected during primary immune responses in specialized areas of the lymphoid organs. Memory B cell generation and selection requires specific T cell help and involves distinct molecular mechanisms: immunoglobulin isotype switch and somatic hypermutation. However, the program of B cell differentiation imprinted during these responses and the biological properties that ensure a more efficient secondary antibody response and the long-term persistence of memory have not yet been completely established. 2-Description of the project methodology To investigate the biological properties that ensure more efficient secondary antibody responses and persistence of memory, we propose to compare homogeneous populations of naïve and memory B cells of known antigen specificity, belonging to the same clone and to correlate these properties to their gene expression patterns. So far these studies have not been possible due to our inability to generate relatively high numbers of memory B cells with known antigen specificity. In current BCR Tg mice the transgene insertion occurs randomly and do not permit Ig class switch and the generation of memory B cells. To circumvent this problem we will use SWHEL mice where anti-HEL B cells are capable of class switch recombination and somatic hypermutation (SHM). These mice will be in a Rag-deficient background where only a pure population of HEL-specific B cells will be present. Since T cell help is required for the induction of B cell memory, we will use monoclonal HEL-specific helper CD4 T cells from TCT Tg mice specific for HEL in the context of MHC I-Ab. These mice will also be in a Rag-deficient background. Using these mouse models we will generate isotype switched memory B cells in sufficient numbers to allow their functional characterization. The behavior of these memory B cells will be compared to equivalent populations of naïve B cells of the same clone. We believe the unique innovative character of our experimental approaches will allow us to achieve cutting edge information on several fundamental questions, such as: homeostasis and survival requirements of memory B cells, kinetics of memory B cell responses to antigen, role of antigen-specific T cell help in the survival of memory B cells, tissue localization, memory B cell differentiation and mechanisms of the preferential selection of high-affinity memory B cells and gene expression patterns of naïve and memory B cells. 3- Expected results In this application we propose to compare the biological properties of monoclonal populations of naïve and memory B cells with known antigen specificity. We this approach we expect to increase our current understanding about the mechanisms that lead to the generation and selection of memory B cells and to the long term persistence of secondary antibody responses. Results from the proposed studies may offer a number of potential practical benefits. These include the development of a) approaches to the generation of memory B cells, which may be used in therapy of immune-compromised individuals, for example following BM transplantation and b) new vaccination strategies.