Shp1 selectively inhibited 7 endocytosis, enhancing surface 47 display and lymphocyte homing to GALT. cells expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface 47 and in homing to GALT. Consistent with the specialized role of 47 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses. Introduction The integrin 47 functions as a B PF-4878691 and T cell adhesion receptor for the mucosal vascular addressin (MAdCAM-1) expressed by postcapillary high endothelial venules (HEVs) in GALT, and by, lamina propria venule sites of effector cell recruitment, and by stromal cells in GALT1C3. GALT are the major sites of B cell activation and humoral immune induction for intestinal immunity. Activated B cells undergo isotype class switching in Peyers patches (PPs) and differentiate into migratory immunoglobulin A PF-4878691 (IgA)-secreting plasmablasts that use 47 to home to mucosal PF-4878691 surfaces4 where local production of secretory IgA provides immune protection. Presumably in support of this role, B cells home preferentially to and predominate in murine PPs, contrasting with peripheral lymph nodes (PLNs) where T cells are the majority5. This homing preference correlates with higher surface expression of 47 on B cells than on T cells6, but the mechanisms responsible for this differential 47 surface expression and its essential role in intestinal immunity have not been defined. The tyrosine phosphatase Shp1 (Src homology region 2 domain-containing phosphatase-1, encoded by the gene as described in the mice (mutant mice (mutant mice (mice (B and T cells. a, Flow cytometry of WT or (gene expression and protein synthesis. Intracellular Shp1 spots co-localized with cell surface 7 in wild-type B cells more often than in with 2-6 sialic acid(2-6 Sia)-decorated glycoproteins such as CD2217 or CD4518, affecting its distribution and motility on the B cell surface19. To assess a potential role for the CD22 lectin-carbohydrate interactions in cell autonomous 47 regulation, we assessed integrin cell surface levels by B cells expressing a mutated lectin PF-4878691 domain (CD22R130E) that prevents 2-6 Sia binding16 (Fig. 2a). CD22R130E B cells expressed CD22 at wild-type levels16, and displayed a significant reduction in 47 levels (Fig. 2b), although the effect was less severe than that of CD22 deficiency or ITIM mutations. Intermediate reduction in 47 was also observed in B cells from sialidase at a concentration that retained cell viability while reducing Sambucus Nigra Lectin (SNA) (2-6 Sia binding lectin) staining by ~60% (Extended Data Fig. 3). Sialidase-treated wild-type B cells showed significantly reduced association between CD22 and 7 compared to untreated wild-type cells (Fig. 3a). Together, the data show cell surface sialic acid-dependent CD22 association with integrin 7 but not 1. Open in a separate window Figure 3 Direct physical association of CD22 Rabbit Polyclonal to p38 MAPK and 7. a, Association between CD22 and integrin 7 was assessed by proximity ligation assay (PLA) using purified B cells from wild-type either untreated (WT) or sialidase-treated (WT+ Sialidase), or video microscopy experiment. Representative movie pictures are shown with WT B cells (green) and for three days to titer the quantity of secreted CTB-specific IgA, total IgA, and CTB-specific IgA normalized to total IgA (e) and CTB-specific IgG, total IgG, and CTB-specific IgG normalized to total IgG (f) produced by ELISA and expressed as a percentage of the WT group mean. Shown are pooled data (mean SEM) from n=2 experiments with 5-6 mice per group total. Groups were compared using Two-way ANOVA with Sidaks multiple comparison test (a-d), and two-tailed Students t-test (e,f). *P 0.05, **P 0.01, ***P 0.001 and ****P0.0001. ns: not significant. We also analyzed CTB-specific IgA and IgG production from cultures of small intestine (SI) segments. Intestinal CTB-specific IgA (Fig. 7e) and IgG (Fig. 7f) were decreased in culture of SI segments. We found a two-fold decrease in the RV-specific IgA and IgG titers in CD22-deficient animals as compared to wild-type controls (Fig. 8g and ?and8h),8h), which correlates with higher viral shedding in the feces at day 12 p.i. as mentioned above (Fig. 8c). Together, these data highlight a significant role of B cell CD22 in the mucosal pathogen response. Discussion We report a previously unappreciated contribution for the Shp1/CD22 complex in the cell autonomous regulation of the gut integrin receptor 47. We show that Shp1 inhibits 47 endocytosis and maintains cell surface 47 expression. We show that B.