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Les nouvelles approches de l'analyse multi-paramétrique en cytométrie de masse : caractérisation des cellules réservoirs du VIH

Aurélien Corneau 1
1 PASS-CYPS - Cytométrie Pitié-Salpêtrière
PASS - Unité Mixte de Service Production et Analyse de données en Sciences de la vie et en Santé
Abstract : Mass Cytometry (MCM) has revolutionized the study of cell and phenotypic diversity, significantly increasing the number of markers that can be analyzed simultaneously (41 to date). By making it possible to precisely define the status of lymphocyte populations, particularly with regard to their differentiation, activation and entry into the cell cycle, CMM has uncovered small subsets previously unknown. In this study, MMC was used to try to better characterize HIV reservoirs. With the introduction of combination antiretroviral therapy (ART) in 1996, HIV infection has been transformed from a fatal fate to a manageable chronic disease with a normal lifespan due to a reduction in active viral replication (the amount of virus is below optimal detection limits). However, if treatment is interrupted, the patient's viral load increases again due to viable provirus reservoirs located in long-lived cell populations that cannot be eliminated by current therapies. These infected reservoir cells are a major obstacle to the eradication of HIV. The best characterized reservoir is that of CD4+ T lymphocytes and is mainly hosted in MCT, MST, MSCT, and Tfh. An initial study allowed us to evaluate the stages of the cell cycle in association with markers of differentiation, activation and depletion, leading to an in-depth evaluation of the quiescence status of CD4 T cells likely to harbour latent HIV reservoirs. This broad multiplex analysis demonstrates that certain subsets of LTCD4+CD25-HLA-DR - classically considered "at rest" - actually contain significant amounts of cells cycling or expressing inhibitory receptors, opening new avenues for redefining quiescent CD4 T cells in peripheral blood. A second study aimed to define in vivo HIV-producing CD4 LT populations. We developed a multiparametric analysis on cells from HIV+ patients on ART and in the therapeutic interruption phase (TIA). This study shows that CD3+CD4+CD32high cells express a high level of activation markers and receive important activation signals via cytokines, in contrast to CD32a cells. On the other hand, the analysis of HIV-producing LTCD4+ (expressing the p24 capsid protein), allowed us to detect a very low number of p24+ positive cells (less than 0.004% in ATI phase but none before). The phenotype of the producing cells was then highlighted. They are T lymphocytes that do not express CD8, enriched with a factor 4 in TSCM cells and a factor 2 in TFH. These populations are highly enriched in activated cells co-expressing 3 activation markers (increased by a factor of 20) and are cycling (Ki67+) and/or over-expressing immune control molecules (ICP) with an enrichment of a factor of 500. This allows us to detect producer cells with much higher frequencies in these TCD3+CD8- populations in cycle up to 0.08%, and in G2 phase (2.46%), but also in cells showing poly-expression of the 4 immune-checkpoints (2.27%). The advent of mass cytometry has exponentially increased the information we could obtain on a cell. Thanks to this tool, the identification of the cell cycle, in correlation with different phenotypic markers, makes it possible to explore previously inaccessible information, including the analysis of the latent and productive reservoirs of HIV. This work thus makes it possible to characterize as precisely as possible these HIV-producing cells, but also latent cells, and potential reservoirs of the virus.
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Aurélien Corneau. Les nouvelles approches de l'analyse multi-paramétrique en cytométrie de masse : caractérisation des cellules réservoirs du VIH. Immunité adaptative. PSL Research University, 2018. Français. ⟨tel-02534972⟩

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