After anti-IgM activation, the up-regulation of phosphorylation levels of the aforementioned molecules continued and prolonged over time. basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss ofCosmcconfers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate thatCosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance. == INTRODUCTION == B cells play pivotal roles in mediating the pathogenesis of autoimmune diseases (AIDs), which affect ~7 to 9% of the world population (13). A hallmark of autoimmunity is the breach of B cell tolerance, where B cells are unable to distinguish self-antigens from nonself-antigens and eventually lead to the emergence of pathogenic autoantibodies. Nonetheless, the molecular factors underneath the initial development and expansion of pathogenic autoreactive B cells remain incompletely understood. The B cell receptor (BCR) is the master regulator in controlling B cell development, differentiation, survival, and LY2794193 tolerance (1,4,5). It is essential for B cells to receive, process, and integrate both the antigenic signals delivered via BCR and environmental cues relayed via other receptors as they give rise to opposing outcomes. The B cells are either appropriately activated and generate high-affinity antibodies against foreign antigens, or they become tolerogenic upon interaction with self-antigens. Multiple physiological processes to keep autoreactive B cells in check have been found (69). Nonetheless, in the periphery, a notable percentage of B cells in healthy host exhibit autoreactivities (7,10), suggesting the existence of undefined mechanisms underlying the fine-tuned regulation of B cell activities. Glycosylation is an important posttranslational modification (PTM) that has WDFY2 been implicated in multiple AIDs (1117). Abnormally glycosylated immunoglobulins have been found in immunoglobulin A (IgA) nephropathy (IgAN), systemic lupus erythematosus, and other AIDs. Interactions between sialic acidbinding immunoglobulin type lectins (Siglecs) and sialic acids are involved in autoimmunity by regulating BCR and Toll-like receptor (TLR) signaling. In response to both developmental and environmental cues, dynamic glycosylation has been well documented on immune cells and molecules. In the context of AID pathogenesis, such dynamic glycosylation changes might play roles in mediating the initiation and progression of AID but are largely unexplored. A major PTM of surface proteins in B cells is the addition of O-glycans through formation of core 1 O-glycans (R-Gal1-3GalNAc1-Ser/Thr/Tyr). The generation of O-glycans, which are abundantly present on leukocytes (1822), requires expression of bothCosmc(C1GalT1C1), a LY2794193 molecular chaperone in LY2794193 the endoplasmic reticulum, and its only client, the LY2794193 Core 1 3galactosyl-tranferase (C1GalT1), also known as T-synthase. We recently demonstrated that deletion ofCosmcin murine B cells, which results in expression of the Tn antigen (GalNAc1-Ser/Thr/Tyr), greatly reduces their migration to lymph nodes (23). We also observed that the B cell-specificCosmc-knockout (BC-CosmcKO) mice displayed splenomegaly and hypergammaglobulinemia, indicating the potential development of AID.Cosmcis an especially interesting candidate gene, as it has been recently implicated in several autoimmune and inflammation-associated diseases, including inflammatory bowel diseases (24), IgAN (25), Tn syndrome (26), and Alzheimers disease (27). Here, we report thatCosmcdeficiency in B cells causes a breakdown of B cell tolerance. BC-CosmcKO mice spontaneously display multiple AID-like pathological phenotypes, in an age- and gender-associated manner.Cosmcdeficiency prolongs the retention of the BCR on cell surface and promotes stronger BCR signaling, which likely led to B cells hyperresponsiveness to stimuli. These results demonstrate that the presence ofCosmc, required for normal O-glycans on B cell glycoproteins, controls B cell tolerance by maintaining BCR signaling through regulating surface BCR internalization. == RESULTS == == AID-like pathological features in BC-CosmcKO mice == A range of obvious phenotypic abnormalities (Fig. 1) spontaneously developed in aged (11 to 20 months) BC-CosmcKO mice, although they appeared healthy at younger ages (8 weeks). Significant weight loss occurred in adult (3 to 7 months) female and both gender groups in aged BC-CosmcKO mice (Fig. 1A); however, the severity of the weight loss was greater in female mutant mice. Moreover, a substantial portion of mutant mice displayed LY2794193 ocular abnormalities in a syndromic fashion,.