Anne Astier

Alterations in CD46-mediated Tr1 regulatory T cells in patients with multiple sclerosis

Loss of Treg function appears to be a critical factor in the pathogenesis of human autoimmune diseases. Attention has focused on defects of CD4(+)CD25(high) Tregs, and techniques have been developed to determine their function. In contrast, the role of Tr1 regulatory T cells, which secrete the antiinflammatory cytokine IL-10, in autoimmune disease has not been well assessed. CD46 is a newly defined costimulatory molecule for T cell activation, and CD46-costimulated human T cells induce a Tr1 Treg phenotype with considerable amounts of IL-10 secretion. Here, we examined the role of Tr1 cells in patients with multiple sclerosis (MS) by stimulating CD4(+) T cells with anti-CD3 and -CD46 mAbs and measuring IL-10 secretion. There were striking defects in the induction of Tr1 cells with CD46 costimulation as measured by IL-10 but not IFN-gamma secretion in patients with MS compared with healthy subjects. This loss of Tr1 cell-associated IL-10 secretion was specific to CD46 and not CD28 costimulation and was associated with an altered regulation of the CD46-Cy2 isoform that differentially regulates T cell function in a CD46-transgenic murine model. These data demonstrate a second major Treg defect in human autoimmune disease associated with the CD46 pathway.

Measles Virus Induces Functional TRAIL Production by Human Dendritic Cells

ABSTRACT Measles virus infection induces a profound immunosuppression that can lead to serious secondary infections. Here we demonstrate that measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression in human monocyte-derived dendritic cells. Moreover, measles virus-infected dendritic cells are shown to be cytotoxic via the TRAIL pathway.

Linking innate and acquired immunity: divergent role of CD46 cytoplasmic domains in T cell–induced inflammation

CD46 is a widely expressed transmembrane protein that was initially identified as binding and inactivating C3b and C4b complement products. We used mice that were transgenic for one of two human CD46 isoforms that differ in their cytoplasmic domains (termed CD46-1 and CD46-2) to analyze the effect of CD46 stimulation on the immune response. We show here that CD46 can regulate inflammatory responses, either by inhibiting (CD46-1) or increasing (CD46-2) the contact hypersensitivity reaction. We found that engagement of CD46-1 or CD46-2 differentially affected CD8+ T cell cytotoxicity, CD4+ T cell proliferation, interleukin 2 (IL-2) and IL-10 production as well as tyrosine phosphorylation of Vav in T lymphocytes. These results indicate that CD46 plays a role in regulating the T cell–induced inflammatory reaction and in fine-tuning the cellular immune response by bridging innate and acquired immunity.

The Related Adhesion Focal Tyrosine Kinase Differentially Phosphorylates p130Cas and the Cas-like Protein, p105HEF1

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine-phosphorylated following β1 integrin or B cell antigen receptor stimulation in human B cells. Two substrates that are tyrosine-phosphorylated following integrin ligation in B cells are p130Cas and the Cas family member human enhancer of filamentation 1 (HEF1), both of which can associate with RAFTK. In this report we observed that RAFTK was involved in the phosphorylation of these two proteins. While a catalytically active RAFTK was required for both p130Cas and HEF1, phosphorylation of p130Cas, but not of HEF1, was dependent on an intact autophosphorylation site (Tyr402) on RAFTK. To determine if RAFTK phosphorylated p130Cas and HEF1 directly or through an intermediate, we assayed the ability of RAFTK and of a Tyr402 mutant to phosphorylate purified HEF1 and p130Cas domains. RAFTK was able to phosphorylate the substrate domains of both p130Cas and HEF1, but only the C-terminal domain of p130Cas. Furthermore, Tyr402, which mediates the binding of RAFTK to c-Src kinase, was required for the phosphorylation of the C-terminal domain of p130Cas. These data suggest that RAFTK itself is sufficient for HEF1 phosphorylation, whereas a cooperation between RAFTK and Src kinases is required for the complete phosphorylation of p130Cas.

Involvement of p130(Cas) and p105(HEF1), a novel Cas-like docking protein, in a cytoskeleton-dependent signaling pathway initiated by ligation of integrin or antigen receptor on human B cells.

The Crk-associated substrate p130Cas (Cas) and the recently described human enhancer of filamentation 1 (HEF1) are two proteins with similar structure (64% amino acid homology), which are thought to act as “docking” molecules in intracellular signaling cascades. Both proteins contain an N-terminal Src homology (SH), three domain and a cluster of SH2 binding motifs. Here we show that ligation of either β1 integrin or B cell antigen receptor (BCR) on human tonsillar B cells and B cell lines promoted tyrosine phosphorylation of HEF1. In contrast, Cas tyrosine phosphorylation was observed in certain B cell lines but not in tonsillar B cells, indicating a more general role for HEF1 in B cell signaling. Interestingly, pretreatment of tonsillar B cells with cytochalasin B dramatically reduced both integrin- and BCR-induced HEF1 phosphorylation, suggesting that some component of the BCR-mediated signaling pathway is closely linked with a cytoskeletal reorganization. Both HEF1 and Cas were found to complex with the related adhesion focal tyrosine kinase (RAFTK), and when tyrosine phosphorylated, with the adapter molecule CrkL. In addition, the two molecules were detected in p53/56Lyn immunoprecipitates, and Lyn kinase was found to specifically bind the C-terminal proline-rich sequence of Cas in an in vitro binding assay. These associations implicate HEF1 and Cas as important components in a cytoskeleton-linked signaling pathway initiated by ligation of β1 integrin or BCR on human B cells.

Increased IL-23 secretion and altered chemokine production by dendritic cells upon CD46 activation in patients with multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). In MS, myeloid dendritic cells (mDCs) secrete elevated amounts of IL-23, a potent proinflammatory cytokine, compared to healthy donors. Here, we examined the role of CD46, a complement binding factor, in mDCs by analyzing cytokine and chemokine production in healthy donors and patients with MS. There were striking differences between these groups with increased IL-23p19, CCL3 and CCL5 production, but decreased CCL2 levels in patients. This demonstrates major differences of DC activation upon CD46 activation, with a potential role in the pathogenesis of MS.

Regulation of integrin-mediated p130(Cas) tyrosine phosphorylation in human B cells. A role for p59(Fyn) and SHP2.

Engagement of β1 integrins in terminally differentiated human B cell lines, such as ARH-77, leads to prominent tyrosine phosphorylation of the p130 Crk-associated substrate (Cas). Cas regulates the assembly of several SH2 and SH3 domain-containing proteins into signaling complexes, which are potentially involved in the propagation of downstream signals. We demonstrate here that immunoprecipitated Cas from β1 integrin-stimulated ARH-77 cells was associated with tyrosine kinase and phosphatase activities and that integrin ligation led to the recruitment of at least p59Fyn tyrosine kinase and SHP2 tyrosine phosphatase in Cas immune complexes. Cotransfection studies in COS-7 cells further indicated that Fyn/Cas physical interaction and Fyn-mediated Cas phosphorylation required amino acids 638–889 in the C-terminal region of Cas. This sequence contains both c-Src SH2 and SH3 domain-binding motifs. In vitro binding studies using glutathioneS-transferase fusion proteins derived from the SH2 or SH3 domains of Fyn suggested that both Fyn domains can participate in Fyn/Cas interaction. These data implicate Fyn and SHP2 as potential modulators of Cas signaling complexes in B cells.

Soluble Fc gamma receptors.

Soluble FCγ receptors have been identified in biological fluids of mice and humans. They are produced either by alternative splicing of the exon encoding the transmembrane region of the receptor (FcγRII) or by proteolytic cleavage at the cell membrane (FCγRII and FC7RIII). They inhibit B cell proliferation and immunoglobulin production. Their concentrations in plasma seem to be modified during the development of certain diseases, as for instance in multiple myeloma, where plasma concentrations oi soluble FCγKIll are correlated with the stage of the disease.

The Dynamic Processing of CD46 Intracellular Domains Provides a Molecular Rheostat for T Cell Activation

Background Adequate termination of an immune response is as important as the induction of an appropriate response. CD46, a regulator of complement activity, promotes T cell activation and differentiation towards a regulatory Tr1 phenotype. This Tr1 differentiation pathway is defective in patients with MS, asthma and rheumatoid arthritis, underlying its importance in controlling T cell function and the need to understand its regulatory mechanisms. CD46 has two cytoplasmic tails, Cyt1 and Cyt2, derived from alternative splicing, which are co-expressed in all nucleated human cells. The regulation of their expression and precise functions in regulating human T cell activation has not been fully elucidated. Methodology/Principal Findings Here, we first report the novel role of CD46 in terminating T cell activation. Second, we demonstrate that its functions as an activator and inhibitor of T cell responses are mediated through the temporal processing of its cytoplasmic tails. Cyt1 processing is required to turn T cell activation on, while processing of Cyt2 switches T cell activation off, as demonstrated by proliferation, CD25 expression and cytokine secretion. Both tails require processing by Presenilin/γSecretase (P/γS) to exert these functions. This was confirmed by expressing wild-type Cyt1 and Cyt2 tails and uncleavable mutant tails in primary T cells. The role of CD46 tails was also demonstrated with T cells expressing CD19 ectodomain-CD46 C-Terminal Fragment (CTF) fusions, which allowed specific triggering of each tail individually. Conclusions/Significance We conclude that CD46 acts as a molecular rheostat to control human T cell activation through the regulation of processing of its cytoplasmic tails.

T-cell regulation by CD46 and its relevance in multiple sclerosis.

CD46 is a complement regulatory molecule expressed on every cell type, except for erythrocytes. While initially described as a regulator of complement activity, it later became a ‘magnet for pathogens’, binding to several viruses and bacteria. More recently, an alternative role for such complement molecules has emerged: they do regulate T‐cell immunity, affecting T‐cell proliferation and differentiation. In particular, CD46 stimulation induces Tr1 cells, regulatory T cells characterized by massive production of interleukin‐10 (IL‐10), a potent anti‐inflammatory cytokine. Hence, CD46 is likely to control inflammation. Indeed, data from CD46 transgenic mice highlight a role for CD46 in inflammation, with antagonist roles depending on the cytoplasmic tail being expressed. Furthermore, recent data have shown that CD46 is defective in multiple sclerosis, IL‐10 production being severely impaired in these patients. This lack of IL‐10 production probably participates in the inflammation observed in patients with multiple sclerosis. This review will summarize the data on CD46 and T cells, and how CD46 is likely involved in multiple sclerosis.