Project Summary/Abstract. Despite the tremendous clinical success of immunotheraputics, little is known regarding IgG2-4 biology relative to IgG1. Our long-term goal is to understand how glycosylation of antibodies regulates, and is regulated, by immune responses. The overall objective of this application is to examine the regulation of extracellular sialylation of IgG, and understand how IgG sialylation exerts anti-inflammatory activity. Studies over the last decade have illuminated the importance of IgG1 glycosylation, little is known about the contribution of glycosylation to other IgG subclasses or the regulation of IgG sialylation. We recently demonstrated sialylation of IgG during inflammation occurs extracellularly in situ following administration of soluble glycosyltransferases, termed B4ST6Fc. Our central hypothesis is that extracellular sialylation by B4ST6Fc conveys type II FcγR binding and anti-inflammatory activity selectively to IgG1 and IgG3. Our hypothesis is informed by preliminary data shown here in the Approach subsection of the Research Strategy section. Extracellular sialylation in situ of pathogenic mouse IgG in the kidneys or paws during autoantibody-induced disease attenuates two distinct models of autoimmune disease. Further, we show sialylation of IgG1 and IgG3 results in anti-inflammatory activity in vivo, and DC-SIGN binding in vitro. The rationale that underlies the proposed research is understanding the contribution of Fc glycosylation to all IgG subclasses will enable new insights into IgG biology, and may lead to development of innovative antibody-based therapies. We will test our central hypothesis and, thereby, attain the objective of this application by pursuing the following specific aims using a combination of biophysical experiments, and in vitro and in vivo functional assays. 1) Define the regulation of extracellular IgG sialylation during autoimmune disease. Hypothesis: B4ST6Fc reduces SLE nephritis by sialylation of pathogenic IgG1 and IgG3 with the aid of nucleotide-sugar substrates released by deposited platelets. 2) Identify the molecular determinants of sialylated IgG anti-inflammatory activity. Hypothesis: a specific amino acid sequence that is unique to IgG1 and IgG3 and absent from IgG2 and IgG4, in combination with N297 sialylation, results in DC-SIGN binding ability and anti-inflammatory activity.