Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcγRγ and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.

Toll-like receptor (TLR) 3 is an endosomal TLR that mediates immune responses against viral infections upon activation by its ligand double-stranded RNA, a replication intermediate of most viruses. TLR3 is expressed widely in the body and activates both the innate and adaptive immune systems. However, little is known about how TLR3 intracellular trafficking and maturation are regulated. Here we show that newly synthesized endogenous TLR3 is transported through the ER and Golgi apparatus to endosomes, where it is rapidly cleaved. TLR3 protein expression is up-regulated by its own ligand, leading to the accumulation of its cleaved form. In agreement with its proposed role as a transporter, UNC93B1 expression is required for TLR3 cleavage and signaling. Furthermore, TLR3 signaling and cleavage are sensitive to cathepsin inhibition. Cleavage occurs between aa 252 and 346, and results in a functional receptor that signals upon activation. A truncated form of TLR3 lacking the N-terminal 345 aa also signals from acidic compartments in response to ligand activation. Screening of the human cathepsin family by RNA interference identified cathepsins B and H as key mediators of TLR3 processing. Taken together, our data indicate that TLR3 proteolytic processing is essential for its function, and suggest a mechanism of tight control of TLR3 signaling and thus immunity.Toll-like receptor trafficking | proteolysis | proteases | poly(I:C)

The uptake of immune complexes by FcRs on APCs augments humoral and cellular responses to exogenous Ag. In this study, CD11c+ dendritic cells are shown to be responsible in vivo for immune complex-triggered priming of T cells. We examine the consequence of Ab-mediated uptake of self Ag by dendritic cells in the rat insulin promoter-membrane OVA model and identify a role for the inhibitory FcγRIIB in the maintenance of peripheral CD8 T cell tolerance. Effector differentiation of diabetogenic OT-I CD8+ T cells is enhanced in rat insulin promoter-membrane OVA mice lacking FcγRIIB, resulting in a high incidence of diabetes. FcγRIIB-mediated inhibition of CD8 T cell priming results from suppression of both DC activation and cross-presentation through activating FcγRs. Further FcγRIIB on DCs inhibited the induction of OVA-specific Th1 effectors, limiting Th1-type differentiation and memory T cell accumulation. In these MHC II-restricted responses, the presence of FcγRIIB only modestly affected initial CD4 T cell proliferative responses, suggesting that FcγRIIB limited effector cell differentiation primarily by inhibiting DC activation. Thus, FcγRIIB can contribute to peripheral tolerance maintenance by inhibiting DC activation alone or by also limiting processing of exogenously acquired Ag.

Plasmacytoid dendritic cells (pDCs) are key regulators of the innate immune response, yet their direct role as APCs in the adaptive immune response is unclear. We found that unlike conventional DCs, immune complex (IC) exposed murine pDCs neither up-regulated costimulatory molecules nor activated Ag-specific CD4+ and CD8+ T cells. The inability of murine pDCs to promote T cell activation was due to inefficient proteolytic processing of internalized ICs. This defect in the IC processing capacity of pDCs results from a lack of activating FcγR expression (FcγRI, III, IV) and the dominant expression of the inhibitory receptor FcγRIIB. Consistent with this idea, transgenic expression of the activating human FcγRIIA gene, not present in the mouse genome, recapitulated the human situation and rescued IC antigenic presentation capacity by murine pDCs. The selective expression of FcγRIIB by murine pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. The unexpected difference between the mouse and human in the expression of activating/inhibitory FcγRs has implications for the role of pDCs in Ab-modulated autoimmunity and anti-viral immunity.

The type I interferon (IFNα) response is crucial for viral clearance during primary viral infections. Plasmacytoid dendritic cells are important early responders during systemic viral infections and, in some cases, the sole producers of IFNα. However, their role in IFNα production during memory responses is unclear. We found that IFNα production is absent during a murine viral memory response despite colocalization of virus and pDCs to the splenic marginal zone. The absence of interferon was dependent on circulating antibody, and reversed by the transgenic expression of the activating human FcγRIIA receptor on pDCs. Furthermore, FcγRIIB was required for Sendai Virus immune complex (SeV IC) uptake by splenic pDCs in vitro and internalization via FcγRIIb prevented cargo from accessing TLR signaling endosomes. Thus, pDCs bind viral immune complexes via FcγRIIB, and prevent IFNα production in vivo during viral memory responses. This antibody-dependent, IFNα regulation maybe an important mechanism by which the potentially deleterious effects of IFNα are prevented during a secondary infection.

Plasmacytoid DC's (pDCs) in their role as Type I IFN producers, are key regulators of the innate immune response, yet their role in the adaptive immune response remains unclear. The ability of murine pDCs to present antigen in immune complexes (ICs) to T cells was examined. Unlike conventional DCs, ovalbumin‐IC exposed murine pDCs failed both to upregulate costimulatory molecules and to activate OVA specific CD4+ and CD8+ T cells. The failure of murine pDCs to promote T cell activation through either the exogenous or cross‐presentation pathways occurred despite IC binding and internalization. ICs internalized by pDCs however were inefficiently delivered to an intracellular degradative compartment. This defect in the IC processing capability is likely the consequence of a lack of activating Fc Receptor expression (FcRI, III, IV) and the exclusive expression of the inhibitory receptor FcRIIB. This selective expression of FcRIIB by pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. Thus, pDCs are unable to effectively process ICs for antigen presentation, and may not be able to directly activate naive T cells in vivo.

Type I diabetes (T1D) results from autoimmune attack of the pancreatic β cell islet by inflammatory cells, autoreactive T cells being the primary effectors. However, B cells are necessary for disease in nonobese diabetic (NOD) mice, the best studied model of human T1D. B cells may participate pathogenically as antigen presenting cells and/or as producers of autoantibodies (autoAbs). We have shown in the RIP‐mOVA peripheral tolerance model (RO) that auto‐Abs induce a loss of T cell tolerance in a manner dependent on activating Fc receptors on cross‐priming dendritic cells (DCs). Therefore, we have targeted the spleen tyrosine kinase (Syk), which is required for both BCR‐ and FcR‐mediated signaling, in order to broadly inhibit B cell‐mediated as well as autoAb‐enhanced antigen presentation. Using the oral Syk inhibitor R406 (Rigel Pharmaceuticals) we show that Syk blockade abrogates both B cell‐ and DC‐mediated Ag presentation in vitro, as well as effector T cell differentiation and diabetes in RO mice (100% healthy‐treated vs. 20% healthy‐untreated mice, p=0.02). Finally, Syk inhibition decreases autoAb production, prevents diabetes (60% healthy‐treated vs 14% healthy‐untreated mice at 32 weeks, p<0.01) and prolongs survival in NOD mice (p<10−5). Thus pharmacologic blockade of Syk is a promising therapeutic strategy to inhibit the humoral component of autoimmunity and prevent T1D.This work was supported by the training grant of the Integrated Graduate Program in Cellular, Molecular, Structural and Genetic Studies at Columbia University, NY and by the Juvenile Diabetes Research Foundation, NY.