The antiinflammatory activity of intravenous immunoglobulin (IVIG) is dependent on the presence of sialic acid in the core IgG fragment crystallizable domain (Fc) glycan, resulting in increased conformational flexibility of the C H 2 domain with corresponding modulation of Fc receptor (FcR) binding specificity from type I to type II receptors. Sialylated IgG Fc (sFc) increases the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory receptor FcγRIIB. We have found that the structural alterations induced by sialylation can be mimicked by specific amino acid modifications to the C H 2 domain. An IgG Fc variant with a point mutation at position 241 (F→A) exhibits antiinflammatory activity even in the absence of sialylation. F241A and sFc protect mice from arthritis in the K/BxN-induced model and, in the T cell-mediated experimental autoimmune encephalomyelitis (EAE) mouse model, suppress disease by specifically activating regulatory T cells (T reg cells). Protection by these antiinflammatory Fcs in both antibody-and T cell-mediated autoimmune diseases required type II FcRs and the induction of IL-33. These results further clarify the mechanism of action of IVIG in both antibody-and T cellmediated inflammatory diseases and demonstrate that Fc variants that mimic the structural alterations induced by sialylation, such as F241A, can be promising therapeutic candidates for the treatment of various autoimmune disorders.IgG Fc sialylation | conformational change | antiinflammatory | T reg cells

The immediate presentation of peptide epitopes on MHC class I (MHC I) after antigen expression has led to the concept that MHC I ligands are mostly derived from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are rapidly degraded by the proteasome. Whether and to what extent mature proteins contribute to the antigenic pool, however, has remained elusive. Here, we developed a conditional antigen expression system that allows studying antigen presentation from mature proteins by inducing their rapid proteasomal degradation in the absence of further antigen synthesis. Target cells in which expression of two Epstein-Barr virus (EBV) antigens was induced were rapidly recognized by antigen-specific CD8 + T cells in a time-and dosage-dependent manner, demonstrating that antigen presentation was linked to antigen synthesis. By contrast, T cells failed to recognize target cells containing large amounts of mature protein even after induction of their rapid proteasomal degradation. Thus, the presentation of these antigens proved to be strictly dependent on protein synthesis whereas mature proteins failed to furnish the antigenic pool. These results have implications for the design of immunotherapeutic strategies that aim at targeting proteins with increased half-lives and are hence overexpressed in tumors. Immunol. 2012. 42: 3167-3173 antigenic peptides originate from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are defective in some manner and rapidly degraded [3,4]. Evidence in support of the DRiP hypothesis has been provided by functional studies that linked peptide generation with protein translation [5][6][7][8], and by the finding that a significant proportion of all newly synthesized proteins is rapidly degraded [9]. Following these seminal observations, a number of ensuing studies performed with multiple antigens and in different cell types with different allomorphs pointed to DRiPs as the major, if not the sole source of antigenic peptides [4,10,11]. However, the contribution of DRiPs versus mature proteins to the antigenic peptide pool is typically assessed by abrogating protein synthesis with translation-inhibiting drugs such as cycloheximide (CHX), that might not only affect precursor pool size but also other features of the antigen presentation pathway, for example, protein degradation as well as synthesis, loading, and transport of MHC molecules [5,12]. Also, the use of chimeric proteins as the source of antigen and viruses for antigen expression may have led to an overestimation of the contribution of DRiPs to the immunopeptidome; the fusion of genes may decrease the overall fidelity of protein synthesis or protein folding [13] and differences in viral translation mechanisms can greatly increase the fraction of DRiPs [14]. Likewise, studies performed with antigens with very long half-lives and hence, very slow turnover, might have led to an underestimation of the contribution of mature proteins to the antigenic pool [7,10,15]. Moreove...

Peptides presented on major histocompatibility complex (MHC) class I molecules are generated via cytosolic proteolysis. However, the nature of the endogenous peptide precursors and the intracellular processing steps preceding protein degradation remain poorly defined. Here, we assessed whether ubiquitination is an essential signal for proteasomal cleavage of antigen substrates in human cells. Conversion into antigenic peptides occurred in the absence of any detectable N-terminal ubiquitination of the model antigens, and did not require the presence of any of the four types, nor a minimum number of ubiquitinatable amino acids within the antigen substrate. However, the knockdown of ubiquitin, expression of a lysine 48 (K48) ubiquitin mutant, or inhibition of proteasomeassociated deubiquitinases significantly impaired antigen presentation. The results presented here are consistent with a model in which the binding of the antigen substrate by an adaptor protein leads to its K48-polyubiquitination and the subsequent delivery of the antigen cargo for degradation by the 26S proteasome. Altogether, these findings show an important but indirect role of K48-polyubiquitination in preproteasomal antigen sampling.