This grant will continue to support our ongoing discovery antd functional characterization of molecular mechanisms of lymphocyte homing to the Gl tract. We propose the following specific Aims: Aim 1. GPR15 in intestinal immunobiology: role in intestinal immune responses and mechanisms and effects of species specific differences in expression. We showed expression of GPR15 by effector T cells (Teff) in colitis, and demonstrated that this novel colon homing receptor can play a pathogenic role. We also discovered major differences in GPR15 expression in humans vs mice that have the potential to underlie differences in inflammation, vaccination responses, and intestinal immunity. We propose that aberrant GPR15 expression in mice is due to absence of GATA3 consensus binding sites that regulate GPR15 in other species. We will use CyTOF and flow cytometry to compare the expression and regulation of GPR15 on gut homing lymphocyte subsets in mouse vs man. We will use standard reporter assays, site directed mutagenesis and Chip-seq data to test the hypothesis that the species differences in Th vs Treg subset expression are controlled by loss of GATA3 binding to the mouse Gpr15 enhancer. Most importantly, we propose to analyze the effects of "humanized" Gpr15 expression patterns on small intestinal Teff vs Treg subsets, and on intestinal immune responses in vivo. Mice in which GPR15 is driven under native human sequence control (BAG transgenics; 5 founder lines have been generated already), or in which GATA3 TFBS are introduced or repaired in the mouse enhancer sequence by targeted mutagenesis or knockin strategies, will be studies under homeostatic and inflamed/immunized conditions. The effects of altered, 'human-like' GPR15 expression patterns on intestinal lymphocyte subset frequencies and immune responses will be assessed. Significance: The studies will directly test the hypothesized role of GATA3 TFBS mutations in human vs mouse differences in GPR15 expression and in intestinal immunobiology. If, as hypothesized, mice with a "humanized Ggr15 enhancer" display colon immunobiology that much more closely reflects that of humans, the studies will have fundamental implications for our understanding of colitis and for drug discovery for IBD. Aim 2: To define the role and significance of CD22 as a novel intestinal lymphocyte homing receptor Our experiments show that CD22 functions as a B cell homing receptor for HEV in Peyer's patches. Short term homing assays will be used to confirm this, and to assess critically a hypothesized role of CD22 in homing of follicular T helper cell subsets to GALT as well. We will also assess the role of CD22 in lymphocyte recruitment to the lamina propria and colon patches. Finally, we observe significant residual Sfga/6-dependent homing of CD22-deficient lymphocytes to PP: we hypothesize that this may be mediated by the closely related B cell lectin, SiglecG, which binds similar carbohydrates. We propose to use readily avail...