Biology subjects

Lesage, S.

Publications and source records attributed to Lesage, S..

6 recordsLinked to original sources

bioRxiv2025

Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

Activation-induced marker (AIM) assays are a promising tool to track antigen-specific T cells, but methodological heterogeneity between research groups hinders their clinical utility. To evaluate AIM assay reproducibility, we conducted a multi-site study of SARS-CoV-2 and cytomegalovirus AIMs. We found inherent variability in AIM assays and optimized approaches to enhance reproducibility, including a standardized workflow to minimize technical variability and a generalizable Box-Cox transformation-based statistical method to optimize calculation of AIM stimulation responses. We further standardized AIM data analysis through development of automated flow cytometric gating software, which had superior reproducibility compared to manual analysis. We also characterized antigen-specific Tregs, finding that gating on a combination of CD134, CD137, FOXP3 and HELIOS optimally detects antigen-specific cells. The combined methodology results in a high degree of reproducibility within and between research groups and provides a comprehensive foundation from which standardized AIM assays can be implemented across diverse scientific and clinical settings. MOTIVATIONReliable detection of antigen-specific T cells is critical to understand immune responses to infection and vaccination, and has translational potential to monitor T cell responses across diverse clinical settings. Activation-induced marker (AIM) assays offer a variety of advantages over methods such as ELISPOT and tetramers, but are limited by methodological heterogeneity between research groups and a lack of standardized protocols. As such, the degree to which AIM assay results are reproducible is unknown. Key variables such as cell source, media, stimulation time, marker selection for CD4+ T cells, CD8+ T cells and regulatory T cells; as well as data analysis parameters such as flow cytometric gating strategies and mathematical correction for background AIM+ frequencies in unstimulated control samples, have not been rigorously studied. To address this, we comprehensively characterized variability in AIM assays, including within and between operators and across multiple research centres, and sought to optimize a standard AIM workflow to enhance reproducibility at both the experimental and analytical levels.

immunology↗

bioRxiv2021

Discovery of genes that modulate flavivirus replication in an interferon-dependent manner

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent way, opening new perspectives to target weakness points in the life cycle of these viruses.

microbiology↗

bioRxiv2020

Context-dependent effects of IL-2 rewire immunity into distinct cellular circuits

Interleukin 2 (IL-2) is a key homeostatic cytokine, with potential therapeutic applications in both immunogenic and tolerogenic immune modulation. Clinical application has been hampered by pleiotropic functionality and wide-spread receptor expression, with unexpected adverse events during trials. To characterize the IL-2 homeostatic network, we developed a novel mouse strain allowing IL-2 production to be diverted. Rewiring of IL-2 production to diverse leukocyte sources allowed the identification of contextual influences over IL-2 impact. Network analysis identified a priority access for Tregs, and a competitive fitness cost induced among both Tregs and conventional CD4 T cells for IL-2 production. CD8 T cells and NK cells, by contrast, exhibited a preference for autocrine IL-2 production. IL-2 sourced from dendritic cells amplified the Treg circuit, while IL-2 produced by B cells induced two context-dependent circuits: dramatic expansion of CD8+ Tregs and ILC2 cells. The former was associated with an unexpected concentration of rare CD8+ Tregs in B cell zones, while the latter drove a downstream, IL-5-mediated, eosinophilic circuit. The source-specific effects demonstrate the contextual influence of IL-2 function and potentially explain unexpected adverse effects observed during clinical trials of exogenous IL-2. Targeted IL-2 production therefore has the potential to amplify or quench particular circuits in the IL-2 network, based on clinical desirability. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2019

Analysis of DNM3 and VAMP4 as genetic modifiers of LRRK2 Parkinsons disease

ObjectiveTo assess genetic modifiers of Parkinsons disease (PD) age at onset (AAO) penetrance in individuals carrying common and rare LRRK2 risk alleles\n\nMethodsWe analysed reported genetic modifier DNM3 rs2421947 in 724 LRRK2 p.G2019S heterozygotes using linear regression of AAO. We meta-analysed our data with previously published data (n=754). VAMP4 is in close proximity to DNM3 and is associated with PD. We analysed the effect of the rs11578699 VAMP4 variant on pG2019S penetrance in 786 LRRK2 p.G2019S heterozygotes. We also evaluated the impact of VAMP4 variants using AAO regression in 4882 patients with PD carrying a common LRRK2 risk variant (rs10878226).\n\nResultsThere was no evidence for linkage disequilibrium between DNM3 rs2421947 and VAMP4 rs11578699. Our linear regression AAO of 724 p.G2019S carriers showed no relationship between DNM3 rs2421947 and AAO (beta = -1.19, p = 0.55, n =708). Meta-analysis with previously published data did not indicate a significant effect on AAO (beta = -2.21, p = 0.083, n = 1304), but there was significant heterogeneity in the analyses of new and previously published data. VAMP4 rs11578699 was nominally associated with AAO in patients dichotomized by the common LRRK2 risk variant rs10878226 (beta=1.68, se=0.81 p=0.037).\n\nInterpretationAnalysis of DNM3 in previously unpublished data does not show an interaction between DNM3 and LRRK2 G2019S for AAO, however the inter-study heterogeneity may indicate ethnic-specific effects of DNM3 rs2421947. Analysis of sporadic PD patients stratified by the PD risk variant rs10878226 indicates a possible interaction between LRRK2 and VAMP4.

genetics↗

bioRxiv2018

Parkinson disease age of onset GWAS: defining heritability, genetic loci and a-synuclein mechanisms

Increasing evidence supports an extensive and complex genetic contribution to Parkinsons disease (PD). Previous genome-wide association studies (GWAS) have shed light on the genetic basis of risk for this disease. However, the genetic determinants of PD age of onset are largely unknown. Here we performed an age of onset GWAS based on 28,568 PD cases. We estimated that the heritability of PD age of onset due to common genetic variation was ~0.11, lower than the overall heritability of risk for PD (~0.27) likely in part because of the subjective nature of this measure. We found two genome-wide significant association signals, one at SNCA and the other a protein-coding variant in TMEM175, both of which are known PD risk loci and a Bonferroni corrected significant effect at other known PD risk loci, INPP5F/BAG3, FAM47E/SCARB2, and MCCC1. In addition, we identified that GBA coding variant carriers had an earlier age of onset compared to non-carriers. Notably, SNCA, TMEM175, SCARB2, BAG3 and GBA have all been shown to either directly influence alpha-synuclein aggregation or are implicated in alpha-synuclein aggregation pathways. Remarkably, other well-established PD risk loci such as GCH1, MAPT and RAB7L1/NUCKS1 (PARK16) did not show a significant effect on age of onset of PD. While for some loci, this may be a measure of power, this is clearly not the case for the MAPT locus; thus genetic variability at this locus influences whether but not when an individual develops disease. We believe this is an important mechanistic and therapeutic distinction. Furthermore, these data support a model in which alpha-synuclein and lysosomal mechanisms impact not only PD risk but also age of disease onset and highlights that therapies that target alpha-synuclein aggregation are more likely to be disease-modifying than therapies targeting other pathways.

genetics↗

bioRxiv2018

Parkinson’s disease genetics: identifying novel risk loci, providing causal insights and improving estimates of heritable risk

We performed the largest genome-wide association study of PD to date, involving the analysis of 7.8M SNPs in 37.7K cases, 18.6K UK Biobank proxy-cases, and 1.4M controls. We identified 90 independent genome-wide significant signals across 78 loci, including 38 independent risk signals in 37 novel loci. These variants explained 26-36% of the heritable risk of PD. Tests of causality within a Mendelian randomization framework identified putatively causal genes for 70 risk signals. Tissue expression enrichment analysis suggested that signatures of PD loci were heavily brain-enriched, consistent with specific neuronal cell types being implicated from single cell expression data. We found significant genetic correlations with brain volumes, smoking status, and educational attainment. In sum, these data provide the most comprehensive understanding of the genetic architecture of PD to date by revealing many additional PD risk loci, providing a biological context for these risk factors, and demonstrating that a considerable genetic component of this disease remains unidentified.

genetics↗