Transferrin receptor (TfR) has been identified as a candidate IgA1 receptor expressed on human mesangial cells (HMC). TfR binds IgA1 but not IgA2, co-localizes with mesangial IgA1 deposits, and is overexpressed in patients with IgA nephropathy (IgAN). Here, structural requirements of IgA1 for its interaction with mesangial TfR were analyzed. Polymeric but not monomeric IgA1 interacted with TfR on cultured HMC and mediates internalization. IgA1 binding was significantly inhibited (>50%) by soluble forms of both TfR1 and TfR2, confirming that TfR serves as mesangial IgA1 receptor. Hypogalactosylated serum IgA1 from patients with IgAN bound TfR more efficiently than IgA1 from healthy individuals. Serum IgA immune complexes from patients with IgAN containing aberrantly glycosylated IgA1 bound more avidly to TfR than those from normal individuals. This binding was significantly inhibited by soluble TfR, highlighting the role of TfR in mesangial IgA1 deposition. For addressing the potential role of glycosylation sites in IgA1-TfR interaction, a variety of recombinant dimeric IgA1 molecules were used in binding studies on TfR with Daudi cells that express only TfR as IgA receptor. Deletion of either N- or O-linked glycosylation sites abrogated IgA1 binding to TfR, suggesting that sugars are essential for IgA1 binding. However, sialidase and beta-galactosidase treatment of IgA1 significantly enhanced IgA1/TfR interaction. These results indicate that aberrant glycosylation of IgA1 as well as immune complex formation constitute essential factors favoring mesangial TfR-IgA1 interaction as initial steps in IgAN pathogenesis.

IgA nephropathy (IgAN), the most common primary glomerulonephritis in the world, is characterized by IgA immune complex-mediated mesangial cell proliferation. The transferrin receptor (TfR) was identified previously as an IgA1 receptor, and it was found that, in biopsies of patients with IgAN, TfR is overexpressed and co-localizes with IgA1 mesangial deposits. Here, it is shown that purified polymeric IgA1 (pIgA1) is a major inducer of TfR expression (three- to four-fold increase) in quiescent human mesangial cells (HMC). IgA-induced but not cytokine-induced HMC proliferation is dependent on TfR engagement as it is inhibited by both TfR1 and TfR2 ectodomains as well as by the anti-TfR mAb A24. It is dependent on the continued presence of IgA1 rather than on soluble factors released during IgA1-mediated activation. In addition, pIgA1-induced IL-6 and TGF-beta production from HMC was specifically inhibited by mAb A24, confirming that pIgA1 triggers a TfR-dependent HMC activation. Finally, upregulation of TfR expression induced by sera from patients with IgAN but not from healthy individuals was dependent on IgA. It is proposed that deposited pIgA1 or IgA1 immune complexes could initiate a process of auto-amplification involving hyperexpression of TfR, allowing increased IgA1 mesangial deposition. Altogether, these data unveil a functional cooperation between pIgA1 and TfR for IgA1 deposition and HMC proliferation and activation, features that are commonly implicated in the chronicity of mesangial injuries observed in IgAN and that could explain the recurrence of IgA1 deposits in the mesangium after renal transplantation.

DC cross-present exogenous antigens on MHC class I molecules, a process required for the onset of anti-tumor immune responses. In order to study the cross-presentation of tumor antigens by human DC, we compared the pathways of cross-presentation of long peptides requiring internalization and intracellular processing with the direct presentation of short peptides, which does not require intracellular processing. We found that, after brief incubations with DC, short peptides were presented to CD8 1 T cells with higher efficiencies than long peptides. After longer times of chase in the absence of peptide, however, the efficiency of presentation of the two types of peptides was reversed. After 2-3 days, DC pulsed with long peptides still activated T cells efficiently, while DC pulsed with short peptides failed to do so. Long-lasting presentation of the long peptides was, at least in part, due to a stored persistent pool of antigen, which was still available for loading on MHC class I molecules after several days of chase. These results show that the use of long synthetic peptides allows the efficient, long-lasting, presentation of tumor antigens, suggesting that long peptides represent an interesting approach for active anti-tumor vaccination.Key words: Cross-presentation . CTL . DC . Long peptides . Melanoma-associated antigen Supporting Information available online IntroductionAdaptative immune responses, especially through CD8 1 T cells, can induce the rejection of solid tumors [1][2][3]. Endogenously expressed tumor-associated antigens (TAA) expressed in tumor cells are recognized by CTL through cognate interactions of the TCR with class I MHC molecules associated with 8-10 amino acid long antigenic peptides. These tumor cell/T-cell interactions, however, are not sufficient to activate naïve T lymphocytes and to induce a memory response. Indeed, professional APC are required for the induction of effector and memory tumor-specific immune responses [4,5]. As most often APC do not endogenously express TAA, uptake and presentation of antigen via an exogenous pathway is required. This so-called ''cross-presentation'' pathway is mainly performed by DC [6,7]. Following the initial observations of cross-priming of T cells by bone-marrowderived cells [8,9], numerous studies investigated the role of cross-priming in the induction of anti-tumor immune responses [10][11][12][13][14] and the strategies to manipulate cross-priming to induce and amplify tumor-specific immune responses [15][16][17][18][19]. Because 380they display a unique capacity to efficiently cross-present exogenous antigens and to prime naïve CD8 1 T cells, DC are good candidates for cancer immunotherapy. The DC-based cancer vaccines that have been tested successfully in mouse models include DC loaded with different forms of tumor antigens, including short peptides, tumor extracts, tumor-derived membrane vesicles and tumor-derived RNA [20][21][22][23]. Nevertheless, these cell-based vaccines present some disadvantages, mainly related to their cost and the...

Plasmacytoid predendritic cells (pDCs) play a key role in antiviral immunity through their capacity to produce large amounts of type I interferons in response to Toll-like receptor triggering, and to differentiate into dendritic cells (DCs). However, their antigen processing and presentation pathways remain poorly characterized. In this study, we analyzed major histocompatibility complex class II (MHC II) synthesis and transport in primary human pDCs. We show that stimulation of pDCs with influenza virus leads to a sustained neosynthesis of MHC II molecules, which rapidly accumulate in antigen loading compartments organized around the microtubule organization center. MHC II endocytosis as well as antigen internalization remain active during the entire process of pDC differentiation into DCs, suggesting a capacity to constantly renew surface peptide-MHC II complexes. IntroductionHuman peripheral blood contains 2 main populations of dendritic cells (DCs): the "conventional" DCs (cDCs), and the plasmacytoid predendritic cells (pDCs). 1 These 2 subsets express different sets of Toll-like receptors (TLR) and differentially respond to TLR ligands, indicating that they display nonredundant functions. 2 pDCs play a central role in the immune system by linking the innate with the adaptive immune responses. 3 First, pDCs rapidly produce large amounts of type I interferons (IFNs) after microbial stimulation. 4,5 pDC activation occurs through the recognition of viral nucleic acids (single-stranded RNA) by TLR7, and viral and bacterial DNA by TLR9. 6 Second, depending on the stimuli, pDCs can differentiate into cells with a dendritic phenotype. 3,7 This differentiation is accompanied by a strong up-regulation of the surface expression of major histocompatibility complex class I (MHC I) and class II (MHC II), as well as T-cell costimulatory molecules, suggesting that they become potent antigen-presenting cells. 3,7,8 However, the capacity of pDCs to act as antigenpresenting cells remains controversial. Depending on the stimuli, 3,9,10 the source of pDCs, 11,12 the way the antigen is internalized, 13,14 and the dose of antigen, 12 pDCs are able or not to activate naive CD4 T cells. It has also been proposed that pDCs are endowed with tolerogenic capacities by inducing regulatory T cells producing interleukin-10 (IL-10) both in vitro [15][16][17][18][19][20] and in vivo. 21,22 It is well established that antigen presentation by MHC II heavily depends on their intracellular traffic. The biosynthetic route followed by nascent MHC II has been well characterized in various antigen-presenting cells. 23 MHC II are synthesized in the endoplasmic reticulum (ER) where they associate with the chaperone invariant chain (Ii) to form ␣␤Ii complexes. Such complexes exit from the ER and traffic through the Golgi apparatus to most probably reach the plasma membrane where they are rapidly internalized into endosomes. There, Ii is degraded, allowing MHC II to acquire their peptide load. MHC II peptide-loaded complexes are then exported to the c...