Macroscopic nodules of gut associated lymphoid tissue (GALT) are distributed over the length of the gastrointestinal tract. However, their frequency at some sites is greater than can be accounted for by random distribution. Similarly, anodular areas also occur at specific sites. Such a non-random distribution of GALT suggests the presence of a microenvironmental/stromal element in both the small and large intestine, which may play a role in the formation and localization of GALT. In addition, grafts of fetal intestine from areas of high GALT frequency develop normal lymphoid architecture with associated stroma when transplanted ectopically, even in the absence of luminal antigen. Although their role is not yet clear, stromal cells associated with GALT may play an important role in the formation, localization and function of this component of the immune system. It is the objective of this proposal to better characterize this stromal population. To do this, we will use light and electron microscopy along with immunohistochemical techniques, while taking advantage of the steroid sensitivity of the lymphocytes with proximal colonic lymphoid tissue to enrich for stromal elements. We will examine the ontogeny of these stromal elements, to determine whether they are intrinsic to sites of high GALT frequency or whether they are an immigrant population. We propose that location, development and cellular composition of these nodules are microenvironmentally determined. To test this, we will transplant small pieces of fetal gut from sites which we predict will develop lymphoid nodules, versus adjacent anodular regions, under the kidney capsule of syngeneic recipients and evaluate the yield of nodules from the former vs the latter. We will attempt to purify GALT stromal elements utilizing techniques of low temperature organ culture and deoxyguanosine treatment to deplete lymphocytes. We have had considerable success with these techniques in the purification of thymic epithelium, and predict similar results with GALT stroma. Finally, we will examine the origin of the lymphoid component of GALT using grafts of fetal gut or purified GALT stroma. Grafts will be placed into chimeric mice which have distinguishable allelic isotypic markers and evaluated using flow cytometry and immunohistochemistry. These studies will provide a more complete understanding of GALT microenvironments and their role in the ontogeny and localization of GALT. Clearly, these studies may be important in the understanding of a variety of clinical situations including digestive and autoimmune disorders, intestinal transplantation, as well as tumors arising in GALT.
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