Generation of human intestinal IgA plasma cells: roles of innate and adaptive immunity.

The majority of cells belonging to the immune system are located in the wall of the intestine. Despite this, infections of the intestine kill millions of people each year throughout the world and if we are to fight such diseases we need to know how to direct the intestinal immune system to identify and halt the bugs that cause disease. At the moment this is problematic. We believe that the reason for this is that we have failed to answer a fundamental question, specifically, 'How is the normal human intestinal antibody response driven?' The cells that have the capability to make antibodies are called B cells, which are made fresh in the bone marrow all the time. Each antibody has its own individual shape and there is a massive variety of potential individual shapes. Each B cell has antibody with only a single shape on its surface. B cells put antibody on their cell surface but do not develop into cells that release antibodies unless the B cell is activated. B cells can be activated by a number of routes. One requires that the exact shape of the antibody has to fit, like a lock and key, with the shape of a bug. With the help of T cells, which are organisers of the immune response, the B cells become activated. The B cells divide very fast. Their antibodies can adapt to generate a better fit with the shape that activated them. This T cell dependent activation of B cells that allows the shape of the antibody to adapt, is called a germinal centre response and it generates antibodies with best fit and also memory B cells that are at the ready in case the same shape turns up again; hence this type of response is most effective for vaccination against infections. Another route of antibody production is not so dependent on the shape of the antibody itself, but rather involves the recognition of characteristic shapes on the surfaces of bugs by molecules on the B cell surface. This is possible because the surfaces of bacteria for example are very different to the surfaces of mammalian cells. The receptors for bacterial components on the B cell surface are known, but as yet, the status of human intestinal B cells is not known. The first aim of this project is to determine this. These responses are enhanced by T cells to some degree, but do not involve generation of germinal centres. They do not make best fit antibodies or memory cells and therefore this is not a useful pathway to activate for effective vaccination. A recent experiment using mice noted that if specific antibody is removed from the surface of B cells, as expected, germinal centres do not form in lymph nodes or spleen. However, surprisingly, germinal centres did form in the gut. This implies that the factors that drive germinal centres in the gut are different to those in the lymph nodes and spleen, and might not generate specific antibodies. Therefore we believe that attempts to effectively vaccinate the gut have failed because the rules that govern germinal centre formation in the gut are different and do not necessarily generate specific responses or memory under normal conditions. When B cells are activated, the cells have to convert the stimulus at the cell surface into appropriate action, which may differ according to the nature of the stimulus as described above. This is achieved via signalling molecules that form a chain of messages from the cell surface to the nucleus. The nucleus contains all the information to change behaviour, such as start dividing or start antibody production. The components and state of these signals differ according to the nature of the stimulus at the cell surface. The second aim of this project is to analyse these signals to determine what is driving the antibody response in the normal human intestine. We will then be in a much better position create a vaccine that would effectively protect against intestinal infection.

We have shown that germinal centres (GC) of normal human gut associated lymphoid tissue (GALT) generate intestinal plasma cells. The classical route to GC formation involves cognate interaction between B and T cells. B cells specific for protein process it and present peptide to specific T cells. T cells then provide help for GC formation, resulting in production of specific antibodies and memory cells. Recent studies using a mouse model suggested that GC's in GALT are not necessarily driven by cognate B/T interaction, but may be part of an innate response that is also dependent on T cells and the flora. We hypothesise that the IgA response in healthy human gut is not a response to specific antigen, but a highly diversified innate response. This has profound implications for mucosal immunisation, which requires specificity and memory. FIRST AIM: To analyse expression of receptors of the innate immune response (TLR's and RP105 and associated molecules MD-1 and MD-2) by mucosal B cells. Although their relative expression on peripheral B cells is known, the status of mucosal B cells is not. We present preliminary data suggesting that B cells in GALT may be different to those in blood. We propose to isolate B cell subsets from blood (controls) and human appendix and terminal ileum, to analyse expression of innate receptors by quantitative PCR and to compare gene expression using gene chips. We propose to use available antibodies to locate receptors using immunohistochemisty and confocal microscopy. SECOND AIM: To analyse down stream intracellular events associated with signalling through different surface receptors, in freshly isolated cells. This will enable us to deduce the stimlus that initated the response in vivo. The status of signalling molecules in response to receptor engagement will also be tested in vitro using isolated cells stimulated either through innate receptors or the BCR.