The role of the low-density lipoprotein receptor family in the pathology of the antiphospholipid syndrome

The results in this thesis show that the interaction between ß2GPI and LDL-R family members is based on the recognition of a cationic patch within domain V of ß2GPI by LDL-R homologous. Molecular studies have identified a specific region within domain V of ß2GPI (Lys282, Lys284, Lys286 and Lys287) that is recognized by the human platelet receptor apoER2'. Furthermore, the studies demonstrate that the apoER2' recognition site does not overlap the phospholipid-binding site (the flexible loop located at Ser311-Lys317) within domain V of ß2GPI. These observations may learn us how ß2GPI binds to cellular surfaces containing anionic phospholipids. First, low-affinity binding of ß2GPI to anionic phospholipids via the flexible loop located at Ser311-Lys317 occurs, inducing a conformational change within ß2GPI. These structural changes result in binding of anti-ß2GPI antibodies to an exposed epitope in domain I of ß2GPI followed by protein dimerization. Now, high-affinity binding of dimerized ß2GPI to LDL-R family members takes place, probably via the residue Lys282-Lys287 within domain V of ß2GPI. However, several questions still not to be answered. So far, the studies described in this thesis show the relevance of ß2GPI dimerization in cellular activation in in vitro and in biochemical experiments. Animal models, in which thrombosis can be studies, should be used to investigate the physiological role of ß2GPI dimerization. In addition, future studies must be performed to investigate the role of the LDL-R family in ß2GPI/anti- ß2GPI induced thrombosis. This can be accomplished using LDL-R knockout mice

Discovery of a selective islet peptidome presented by the highest-risk HLA-DQ8trans molecule

Transplantation and autoimmunit

Human islets and dendritic cells generate post-translationally modified islet auto-antigens

Initiation of type 1 diabetes (T1D) requires a break in peripheral tolerance. New insights into neo-epitope formation indicate that post-translational modification of islet auto-antigens, for example via deamidation, may be an important component of disease initiation or exacerbation. Indeed, deamidation of islet auto-antigens increases their binding affinity to the T1D highest-risk HLA haplotypes HLA-DR3/DQ2 and -DR4/DQ8, increasing the chance that T-cells reactive to deamidated auto-antigens can be activated upon T-cell receptor ligation. Here we investigated human pancreatic islets and inflammatory and tolerogenic human dendritic cells (DC and tolDC) as potential sources of deamidated islet auto-antigens and examined whether deamidation is altered in an inflammatory environment. Islets, DC and tolDC contained tissue transglutaminase, the key enzyme responsible for peptide deamidation, and enzyme activity increased following an inflammatory insult. Islets treated with inflammatory cytokines were found to contain deamidated insulin C-peptide. DC, heterozygous for the T1D highest-risk DQ2/8, pulsed with native islet auto-antigens could present naturally processed deamidated neo-epitopes. HLA-DQ2 or -DQ8 homozygous DC did not present deamidated islet peptides. This study identifies both human islets and DC as sources of deamidated islet auto-antigens and implicates inflammatory activation of tissue transglutaminase as a potential mechanism for islet and DC deamidation. This article is protected by copyright. All rights reserved

Phosphoinositide phosphatase SHIP-1 regulates apoptosis induced by edelfosine, Fas ligation and DNA damage in mouse lymphoma cells

International audienceS49 mouse lymphoma cells undergo apoptosis in response to the alkyl-lysophospholipid (ALP) edelfosine, Fas/CD95 ligand (FasL) and DNA damage. Cells made resistant to ALP (S49AR) are defective in sphingomyelin synthesis and ALP uptake, and also have acquired resistance to FasL and DNA damage. However, these cells can be resensitized following prolonged culturing in the absence of ALP. The resistant cells show sustained ERK/Akt activity, consistent with enhanced survival signalling. In search of a common mediator of the observed cross-resistance, we found that S49AR cells lacked the PtdIns(3,4,5)P3 phosphatase SHIP-1, a known regulator of the Akt survival pathway. Resensitization of the S49AR cells restored SHIP-1 expression as well as phosphoinositide and sphingomyelin levels. Knockdown of SHIP-1 mimicked the S49AR phenotype in terms of apoptosis cross-resistance, sphingomyelin deficiency and altered phosphoinositide levels. Collectively, our results suggest that SHIP-1 collaborates with sphingomyelin synthase to regulate lymphoma cell death irrespective of the nature of the apoptotic stimulus

Posttranslational modification of HLA-DQ binding islet autoantigens in type 1 diabetes

Transplantation and autoimmunit

Epitope Stealing as a Mechanism of Dominant Protection by HLA-DQ6 in Type 1 Diabetes

Proteomic

Dendritic cells guide islet autoimmunity through a restricted and uniquely processed peptidome presented by high-risk HLA-DR

Abstract Identifying T cell epitopes of islet autoantigens is important for understanding type 1 diabetes (T1D) immunopathogenesis and to design immune monitoring and intervention strategies in relationship to disease progression. Naturally processed T cell epitopes have been discovered by elution from HLA-DR4 of pulsed B lymphocytes. The designated professional APC directing immune responses is the dendritic cell (DC). To identify naturally processed epitopes, monocyte-derived DC were pulsed with preproinsulin (PPI), glutamic acid decarboxylase (65-kDa isoform; GAD65), and insulinoma-associated Ag-2 (IA-2), and peptides were eluted of HLA-DR3 and -DR4, which are associated with highest risk for T1D development. Proteome analysis confirmed uptake and processing of islet Ags by DC. PPI peptides generated by DC differed from those processed by B lymphocytes; PPI signal-sequence peptides were eluted from HLA-DR4 and -DR3/4 that proved completely identical to a primary target epitope of diabetogenic HLA-A2–restricted CD8 T cells. HLA-DR4 binding was confirmed. GAD65 peptides, eluted from HLA-DR3 and -DR4, encompassed two core regions overlapping the two most immunodominant and frequently studied CD4 T cell targets. GAD65 peptides bound to HLA-DR3. Strikingly, the IA-2 ligandome of HLA-DR was exclusively generated from the extracellular part of IA-2, whereas most previous immune studies have focused on intracellular IA-2 epitopes. The newly identified IA-2 peptides bound to HLA-DR3 and -DR4. Differential T cell responses were detected against the newly identified IA-2 epitopes in blood from T1D patients. The core regions to which DC may draw attention from autoreactive T cells are largely distinct and more restricted than are those of B cells. GAD65 peptides presented by DC focus on highly immunogenic T cell targets, whereas HLA-DR–binding peptides derived from IA-2 are distinct from the target regions of IA-2 autoantibodies.</jats:p