Antigen-Based Immune Therapeutics for Type 1 Diabetes: Magic Bullets or Ordinary Blanks?

The ideal drug of modern medicine is the one that achieves its therapeutic target with minimal adverse effects. Immune therapy of Type 1 diabetes (T1D) is no exception, and knowledge of the antigens targeted by pathogenic T cells offers a unique opportunity towards this goal. Different antigen formulations are being considered, such as proteins or peptides, either in their native form or modified ad hoc, DNA plasmids, and cell-based agents. Translation from mouse to human should take into account important differences, particularly in the time scale of autoimmune progression, and intervention. Critical parameters such as administration route, dosing and interval remain largely empirical and need to be further dissected. T1D staging through immune surrogate markers before and after treatment will be key in understanding therapeutic actions and to finally turn ordinary blanks into magic bullets

P17-16. Anti-Langerin-HIV Gag p24 fusion protein targeting Langerhans cells as a new anti-HIV vaccine strategy

International audiencen.

T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP206-214. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results

Nouveaux éléments dans l'apprêtement des antigènes présentés par les molécules de classe 1 du Complexe Majeur d'Histocompatibilité

La présentation de peptides antigéniques par les molécules du CMH-I est la base de la surveillance de l'organisme, de la détection et de l'élimination de cellules infectées ou transformées, par les cellules T CD8+. Les composants de la machinerie complexe d'apprêtement des Ags et de leur présentation par les molécules du CMH-I, ont été identifiés par plusieurs groupes de recherche. Cependant, même si la séquence des événements de l'apprêtement des Ags pour la présentation par les molécules du CMH-I est actuellement bien définie, il subsiste des parties non résolues. Le sujet de ma thèse vise à élucider certains aspects peu compris de la voie d'apprêtement et de présentation des Ags par des molécules du CMH-I. La description complète des étapes d'apprêtement des Ags dans le cytoplasme ou dans d'autres compartiments cellulaires, du transport des peptides vers les molécules du CMH-I par le système complexe de chargement, de l'expression des complexe CMH I/peptide à la surface cellulaire et de l'induction d'une réponse par les cellules T CD8+, permettrait d'améliorer nos connaissances fondamentales de cette voie et de développer de nouvelles stratégies pour la lutte contre les maladies infectieuses, tumorales et auto-immunes. L'introduction de cette thèse traite trois aspects fondamentaux de la voie d'apprêtement et de présentation des Ags par les molécules du CMH-I: le rôle du protéasome, qui constitue l'un des éléments majeurs impliqués dans la génération des épitopes de classe I (sa structure, ses fonctions et son rôle dans l'apprêtement); la présentation croisée qui représente la voie de présentation des Ags exogènes par les molécules du CMH-I; l'enzyme de dégradation de l'insuline ou IDE (son rôle dans la dégradation de l'insuline). La partie expérimentale est illustrée par l'article publié ainsi que la discussion concernant l'article soumis et des résultats non publiés. Il y est exposé comment la calreticuline favorise la formation des molécules du CMH-I à basse température, capables de fixer un peptide ; les résultats que nous avons obtenus sur le rôle dans la dégradation cytoplasmique de l'insuline / proinsuline par l'enzyme de la dégradation de l'insuline ; et finalement, une stratégie de double queue TAP/Tag que nous avons développée dans le but de comprendre l'interaction entre les différents éléments de la voie de présentation.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

COMPLEXE IMMUNOMODULATEUR ET SES APPLICATIONS POUR LA THÉRAPIE

L’invention se situe dans le domaine des immunothérapies modulatrices. L’invention concerne une complexe moléculaire consistant en au moins un ligand d’un sucre sulfaté de la famille des glycosaminoglycanes et au moins un ligand d’une autre molécule de surface de cellules présentatrices d’antigène liés entre eux, pour utilisation en tant que médicament immunomodulateur, en particulier en 10 immunothérapie du cancer et des maladies infectieuses

Antigen-based immune therapeutics for type 1 diabetes: magic bullets or ordinary blanks? Clin Dev Immunol

The ideal drug of modern medicine is the one that achieves its therapeutic target with minimal adverse effects. Immune therapy of Type 1 diabetes (T1D) is no exception, and knowledge of the antigens targeted by pathogenic T cells offers a unique opportunity towards this goal. Different antigen formulations are being considered, such as proteins or peptides, either in their native form or modified ad hoc, DNA plasmids, and cell-based agents. Translation from mouse to human should take into account important differences, particularly in the time scale of autoimmune progression, and intervention. Critical parameters such as administration route, dosing and interval remain largely empirical and need to be further dissected. T1D staging through immune surrogate markers before and after treatment will be key in understanding therapeutic actions and to finally turn ordinary blanks into magic bullets

Electroporation as a vaccine delivery system and a natural adjuvant to intradermal administration of plasmid DNA in macaques

International audienceIn vivo electroporation (EP) is used to enhance the uptake of nucleic acids and its association with DNA vaccination greatly stimulates immune responses to vaccine antigens delivered through the skin. However, the effect of EP on cutaneous cell behavior, the dynamics of immune cell recruitment and local inflammatory factors, have not been fully described. Here, we show that intradermal DNA vaccination combined with EP extends antigen expression to the epidermis and the subcutaneous skin muscle in non-human primates. In vivo fibered confocal microscopy and dynamic ex vivo imaging revealed that EP promotes the mobility of Langerhans cells (LC) and their interactions with transfected cells prior to their migration from the epidermis. At the peak of vaccine expression, we detected antigen in damaged keratinocyte areas in the epidermis and we characterized recruited immune cells in the skin, the hypodermis and the subcutaneous muscle. EP alone was sufficient to induce the production of pro-inflammatory cytokines in the skin and significantly increased local concentrations of Transforming Growth Factor (TGF)-alpha and IL-12. Our results show the kinetics of inflammatory processes in response to EP of the skin, and reveal its potential as a vaccine adjuvant. Among the various vaccination approaches against infectious diseases such as human immunodeficiency virus (HIV), deoxyribonucleic acid (DNA) vaccines have several advantages: they are easily produced, provide opportunities for molecular engineering, lack anti-vector immunity, and have the potential to promote both cellular and humoral immune responses 1. However, despite their high immunogenicity in murine models, DNA vaccines have shown poor efficacy in large animal models and humans 2. New strategies to improve DNA vaccines include the optimization of transcriptional control elements and codons 3-5 , the use of adjuvants, such as Toll-like receptor (TLR) ligands 6 , cytokine expressing plasmids 7-9 or apoptosis-based adjuvants 10-12 and the choice of an appropriate delivery system such as local electroporation (EP) 13-15. In particular, EP has been largely used to enhance plasmid DNA uptake and increase the number of antigen-producing cells 16, 17. In addition, EP modifies blood vessel permeability and facilitates leukocyte extrava-sation in the exposed area 18. However, the effects of EP on cutaneous antigen presenting cells (APCs) and on the dynamics of cell recruitment at the vaccine site have not been fully described. In a previous study, we demonstrated that intradermal (id) administration of the auxo-GTU ®-multiHIV plasmid (GTU for Gene Transport Unit) combined with noninvasive EP induces a strong and persistent poly-functional T-cell response in macaques 19. Here, we investigated the early events that occur in the skin and the 1 CEA-Université Paris Sud 11-INSERM U1184, DRF/Jacob/Immunology of Viral infections and Autoimmune Diseases (IMVA), IDMIT infrastructure, 92265, Fontenay-aux-Roses

Deletion of the fission yeast homologue of human insulinase reveals a TORC1-dependent pathway mediating resistance to proteotoxic stress.

Insulin Degrading Enzyme (IDE) is a protease conserved through evolution with a role in diabetes and Alzheimer's disease. The reason underlying its ubiquitous expression including cells lacking identified IDE substrates remains unknown. Here we show that the fission yeast IDE homologue (Iph1) modulates cellular sensitivity to endoplasmic reticulum (ER) stress in a manner dependent on TORC1 (Target of Rapamycin Complex 1). Reduced sensitivity to tunicamycin was associated with a smaller number of cells undergoing apoptosis. Wild type levels of tunicamycin sensitivity were restored in iph1 null cells when the TORC1 complex was inhibited by rapamycin or by heat inactivation of the Tor2 kinase. Although Iph1 cleaved hallmark IDE substrates including insulin efficiently, its role in the ER stress response was independent of its catalytic activity since expression of inactive Iph1 restored normal sensitivity. Importantly, wild type as well as inactive human IDE complemented gene-invalidated yeast cells when expressed at the genomic locus under the control of iph1(+) promoter. These results suggest that IDE has a previously unknown function unrelated to substrate cleavage, which links sensitivity to ER stress to a pro-survival role of the TORC1 pathway

Efficiency of IDE down-regulation by siRNA.

<p><b>A</b>, percentage of IDE mRNA inhibition, measured by qRT-PCR, in HeLa HHD cells transfected with different concentrations (4×100 or 4×400 nM) of specific siRNA (left panel), and at different time points after transfection by 4×100 nM siRNA (right panel). <b>B</b>, fluorescence microscopy analysis of HeLa HHD cells transfected with 400 nM of siIDE and siNTG 72 h after transfection. IDE expression was detected by staining with anti-IDE mAb 9B12. <b>C</b>, IDE expression by HeLa cells (left panel) and HEK293 cells (right panel) transfected with 4×100 nM of siIDE or siNTG and probed 48 h later by immunoblot. β_2-m served as a loading control. siNTG, small interfering RNA, non-targeted; siIDE, small interfering RNA, IDE-specific; β_2-m, beta 2-microglobulin. One out of 5 (<b>A, C</b>) and 2 (<b>B</b>) experiments is shown.</p

HeLa Tet-on cells expressing H-2K^b were nucleofected with 4×100 nM siRNA.

<p>Twenty-four hours later, the cells were electroporated with inducible plasmids encoding amyloid beta preceded by a signal peptide (+SP) or not (−SP), or encoding proinsulin, all tagged with the S8L peptide at the C-terminus. Protein expression was induced immediately by addition of 1 µg/ml doxycylin. Forty-eight hours later, K^b/S8L complexes on the cell surface were detected using mAb 25D1.16 (Ab1) followed by FITC-labeled goat anti-mouse Abs (Ab2) and Alexa488-labeled goat anti-FITC Ab (Ab3). Control samples were HeLa-K^b cells pulsed for 2 h with 10⁻⁸M S8L and stained with Abs 1, 2 and 3 or with Abs 2 and 3 only, as well as peptide-pulsed HeLa cells expressing H-2K^d stained with Abs 1, 2 and 3. One of two experiments is shown.</p