Person: Le Gall, Sylvie
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Le Gall
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Sylvie
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Le Gall, Sylvie
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Publication Variable Processing and Cross-presentation of HIV by Dendritic Cells and Macrophages Shapes CTL Immunodominance and Immune Escape(Public Library of Science, 2015) Dinter, Jens; Duong, Ellen; Lai, Nicole; Berberich, Matthew J.; Kourjian, Georgio; Bracho-Sanchez, Edith; Chu, Duong; Su, Hang; Zhang, Shao Chong; Le Gall, SylvieDendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8+ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.Publication A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells(BioMed Central, 2013) Vaithilingam, Archana; Lai, Nicole; Duong, Ellen; Boucau, Julie; Xu, Yang; Shimada, Mariko; Gandhi, Malini; Le Gall, SylvieBackground: Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. Results: In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. Conclusion: By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.Publication In vitro evaluation of digestive and endolysosomal enzymes to cleave CML-modified Ara h 1 peptides(John Wiley & Sons, Ltd, 2015) Mattison, Christopher P; Dinter, Jens; Berberich, Matthew J; Chung, Si-Yin; Reed, Shawndrika S; Le Gall, Sylvie; Grimm, Casey CAra h 1 is a major peanut allergen. Processing-induced modifications may modulate the allergenic potency of Ara h 1. Carboxymethyl lysine (CML) modifications are a commonly described nonenzymatic modification on food proteins. In the current study, we tested the ability of digestive and endolysosomal proteases to cleave CML-modified and unmodified Ara h 1 peptides. Mass spectrometric analyses of the digested peptides demonstrate that carboxymethylation of lysine residues renders these peptides refractory to trypsin digestion. We did not detect observable differences in the simulated gastric fluid or endolysosomal digestion between the parental and CML-modified peptides. One of the tested peptides contains a lysine residue previously shown to be CML modified laying in a previously mapped linear IgE epitope, but we did not observe a difference in IgE binding between the modified and parental peptides. Our findings suggest a molecular mechanism for the increased resistance of peanut allergens modified by thermal processing, such as Ara h 1, to digestion in intestinal fluid after heating and could help explain how food processing-induced modifications may lead to more potent food allergens by acting to protect intact IgE epitopes from digestion by proteases targeting lysine residues.Publication Mechanisms of HIV Protein Degradation into Epitopes: Implications for Vaccine Design(MDPI, 2014) Rucevic, Marijana; Boucau, Julie; Dinter, Jens; Kourjian, Georgio; Le Gall, SylvieThe degradation of HIV-derived proteins into epitopes displayed by MHC-I or MHC-II are the first events leading to the priming of HIV-specific immune responses and to the recognition of infected cells. Despite a wealth of information about peptidases involved in protein degradation, our knowledge of epitope presentation during HIV infection remains limited. Here we review current data on HIV protein degradation linking epitope production and immunodominance, viral evolution and impaired epitope presentation. We propose that an in-depth understanding of HIV antigen processing and presentation in relevant primary cells could be exploited to identify signatures leading to efficient or inefficient epitope presentation in HIV proteomes, and to improve the design of immunogens eliciting immune responses efficiently recognizing all infected cells.Publication Variable Fitness Impact of HIV-1 Escape Mutations to Cytotoxic T Lymphocyte (CTL) Response(Public Library of Science, 2009) Troyer, Ryan M.; McNevin, John; Krizan, Randall W.; Abraha, Awet; Tebit, Denis M.; Avila, Santiago; Lobritz, Michael A.; McElrath, M. Juliana; Mullins, James I.; Arts, Eric J.; Liu, Yi; Zhang, Shao Chong; Zhao, Hong; Le Gall, SylvieHuman lymphocyte antigen (HLA)-restricted CD8\(^+\) cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.