The long term objective of this application is to understand the immunological mechanisms responsible for initiating allergic inflammation, with an eye to identifying novel therapeutic targets upstream of the response itself. This proposal aims to develop novel techniques and a new avenue of investigation which will allow the investigator to identify and characterize a rare population of leukocytes that plays a central role in the events that occur immediately following re-exposure to allergen and are necessary for the later development of the full allergic inflammatory response. In recent years, research has clearly demonstrated that contact sensitivity and delayed type hypersensitivity allergic responses are dependent on a novel immune circuit that involves a number of rare leukocyte subsets. Of these, a subpopulation of non-classical B cells plays a particularly central role. These B cells produce antibodies that, upon re-exposure to allergen, initiate a cascade of pro-inflammatory events required for the development of the later and more severe T cell-mediated hypersensitivity response. Importantly, interference with this pathway or these B cells prevents the development of the development of severe inflammation. While the B cells responsible for initiation of contact sensitivity have been shown to be similar to B-1 cells, they have not yet been fully characterized. Further understanding of these types of allergic responses is dependent on the identification and characterization of these cells. The activity of these cells is dependent on the enzyme activation-induced deaminase (AID), which is involved in a number of steps in the assembly of the B cell receptor gene. The dependence on AID distinguishes the initiating B cell from the general B-1 cell population. Therefore, the specific aims of this proposal are to: 1) Identify AID expressing subpopulations of B-1 cells and 2) determine the mechanism by which AID contributes to the initiation response. Multicolor flow assisted cytometery analysis using multiple cell surface markers and a new strain of mice that expresses a fluorescent marker in cells that have expressed AID will be used to identify these cells. In vivo adoptive transfer experiments will be used to characterize their biological activity. Finally, molecular genetic analysis of B cell receptor genes from individual cells will be developed and performed to identify the role of AID in the generation of the relevant initiating antibodies.Allergic diseases such as contact sensitivity and delayed type hypersensitivity can be very debilitating and are a rapidly increasing and costly burden on public health systems in the United States. This proposal will develop new technology to facilitate the characterization of the immune events upstream of the severe, delayed inflammation associated with these disorders. This will potentially lead to the identification of novel therapeutic targets to effectively prevent responses before they develop fully.
