This grant effort has recently discovered that the Peyer's patches, which are the major mucosal inductive sites in the gastrointestinal (GI) immune system, are not strictly required for mucosal IgA antibody (Ab) responses after oral immunization. Mice treated in utero with lymphotoxin-beta receptor (LTbR)-Ig do not develop Peyer's patches ; however, these mice exhibit mesenteric lymph nodes which drain the small intestine. In this regard, oral immunization of LTbR-Ig treated mice with protein and cholera toxin (CT) as mucosal adjuvant resulted in significant IgA Ab responses in the GI tract. Preliminary studies suggested that the mesenteric lymph nodes served as a compensatory site for these IgA Ab responses. In addition, we discovered that Peyer's patches are a strict requirement for oral tolerance to proteins such as ovalbumin (OVA), but not to haptens such as trinitrophenyl sulfonic acid (TNBS). Clear proof that Peyer's patches are required for oral tolerance to proteins was provided by an experiment where TNBS conjugated to OVA (TNP-OVA) failed to induce oral tolerance to either TNBS or to OVA in LTbR-Ig treated mice. These experiments reveal fundamental differences in a requirement for Peyer's patches in mucosal immunity versus oral tolerance and provide us with a platform to simultaneously study these responses in the GI tract. In this renewal application, the first specific aim will characterize the mesenteric lymph nodes of LTbR-Ig treated, Peyer's patch null mice as alternate IgA inductive sites. We will characterize B cells and plasma cells with emphasis on u > a switches, and for TGF-b 1 expression. The second specific aim will focus on CD4+ T helper (Th) cells and especially Th1-type cells for mucosal IgA Ab responses in the GI tract. Major emphasis will be placed on recombinant Salmonella expressing OVA for initiation of mucosal immunity in Peyer's patch-null mice. The third specific aim will assess the role of antigen-presenting cells (APCs) in Peyer's patches for IgA immunity versus oral tolerance induction. Emphasis will be placed on dendritic cells (DCs), in a natural environment or after activation with Flt3 ligand. The fourth specific aim will employ OVA peptide-specific tetramers to track CD4+ T cells from Peyer's patches to the periphery under conditions for mucosal IgA or oral tolerance responses. The last specific aim will bridge mucosal IgA Ab responses with oral tolerance induction. In this regard, we will study the mechanism of the switch from oral tolerance to IgA immunity which occurs when CT is used as an oral adjuvant. The seminal findings which have emanated from this grant effort since 1982 will again remain focused on the cellular and molecular events required for induction of mucosal immunity or oral tolerance in the GI tract.
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