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Cross-presentation of tumor or viral antigens by pDC

ABG-92280 Sujet de Thèse
28/05/2020 < 25 K€ brut annuel
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Institut Cochin
Paris - Ile-de-France - France
Cross-presentation of tumor or viral antigens by pDC
  • Biologie
  • Santé, médecine humaine, vétérinaire
Dendritic cells, human, mouse, cross-presentation, uptake, cell biology, immunology

Description du sujet

Dendritic cells (DC) are exquisitely sensitive sentinels for viral infection or tumoral transformation, with unique innate antiviral and cytotoxic capacities, in addition to their antigen presentation capacities. We previously found that human plasmacytoid DC (pDC) perform cross presentation (Hoeffel Immunity 2007). We also showed that monocyte-derived DC cross-present antigens from live cells (Marañon PNAS 2004), as well as bone-marrow derived DC, with a protective effect in vivo in mice (Matheoud Blood 2010).  We found that pDC also cross-present antigens from live cells (Isnard, Hatton, EMBO APP10, Paris 2019). We would like to put this in use to kill HIV reservoirs or residual tumor cells.

1. Cross presentation of antigens from live cells by pDC. This well-advanced and promising publication, which raised lots of interest in international congress presentations, will need to be completed at the beginning of the project.

2. We will test in which conditions human pDC can cross-present tumoral Ag to CD8 T cells in vitro. Until now we show that HIV-1 antigens from live cells can be cross-presented by pDC. We need to dissociate antigen from viral stimulation to determine whether this viral stimulation is required for cross-presentation or whether it can be replaced by other stimuli or by other viruses, like oncolytic viruses. We will use influenza or measles virus or Toll-like Receptor ligands and cytokines (Isnard, in preparation; Hatton, in preparation) to induce cross-presentation of HIV-1 Gag or of Melan-A antigens to specific CD8 T cell clones. We will use DC isolated from human blood, donor cells carrying HIV or melanoma Ags, and CD8+ T cell clones directed agains HIV-1 Gag or against Mela-A, which are available in the laboratory (Isnard, Methods in Enzymol 2020). We will characterize the mechanism of antigen capture. We will inactivate potential candidate molecules for antigen entry from our preliminary experiments using shRNAs. We will determine which molecules allow Ag transfer from live cells to DC by flow cytometry, live microscopy (Incucyte), flow imaging (Image Stream with 3 lasers), and then confocal microscopy (both in a L2) and how they can be stimulated to crosspresent residual tumor cells or latent HIV reservoirs (Month 18).

3. We will test whether endogenous DC really crosspresent tumoral Ag specific CD8 T cells in vivo. We will use mice with endogenously fluorescent and hDTR-encoding DC from the different lineages developed by Marc Dalod (CIML). We will analyse spleen slices by real time microscopy as in (Matheoud Blood 2010). We will seek contacts between live tumor cells and CD8+ T cells and Calcium flux responses in these T cells. We will test the hypothesis that the molecules designated by the shRNA screening above mediate this Ag transfer and that each DC population can be stimulated to crosspresent residual tumor cells or HIV reservoirs (Month 30).

Methods: cell immunology, flow cytometry (Aria cell sorter, Aurora analyzer), multianalyte cytokine measurement (Quanterix), cytokine and transduction signal transcriptomic analysis (RNA seq), real time confocal microscopy, Incucyte, tumoral challenge in mice

In summary, we will try and identify agents that can be used for immunotherapy to stimulate the antigen presenting cells that are able in vivo to eliminate latently infected HIV reservoirs or residual tumor cells.

Funding: Ligue de Recherche contre le Cancer, Labex IBEID

Safety rules: L2, Institut Cochin's Safety Service rules

Prise de fonction :

01/10/2020

Nature du financement

Contrat doctoral

Précisions sur le financement

Urgent: deadline for BioSPC studentship applications 29 Mai noon !!! Otherwise, ANRS, Ligue...

Présentation établissement et labo d'accueil

Institut Cochin

Institut Cochin (680 persons) develops interactions between fundamental and clinical research on the Cochin-Port-Royal campus. It is a lively and interactive research campus in the center of Paris. It offers multiple scientific seminars and an active young researcher association (Jecco). Technical platforms offer state of the art facilities, including flow, imaging  and soon spectral cytometry analysis and sorting, immunobiology, confocal and live fluorescence microscopy, pathology, sequencing… Facilities located in L2 or L3 facilities allow study of infected human samples.

 

The "Dendritic cell biology" group is part of the « DC, B cells and cytokines in viral infection and cancer» team, in the Infection, Immunology and Inflammation Department at Institut Cochin. The group has evidenced that dendritic cells were able to cross-present antigens from live cells in vitro and in vivo (Proc Natl Acad Sci USA 2004; Blood 2010), and that plasmacytoid dendritic cells were able to perform antigen cross-presentation (Immunity 2007, JEM 2010, Meth Enzymol 2020, article in revision), and currently from live cells (EMBO workshop 2019, ICI 2019).  The group also works on the roles of dendritic cells and interferons in the hyperactivation of the immune system found during viral infections.

The Director has trained more than 10 PhD students, many of them are now sucessful research team leaders, Professors, or other good carriers.

The team is an associate member of the Integrative Biology of Emerging Infectious Disease Labex.

Intitulé du doctorat

Doctorat d'Immunologie

Pays d'obtention du doctorat

France

Etablissement délivrant le doctorat

Université de Paris

Ecole doctorale

BioSPC (ED 562)

Profil du candidat

The candidate should have a strong academic record,  including in Immunology, a former practical experience in a laboratory, if possible in cellular Immunology, and a strong motivation for research in general and for this fascinating and demanding topic.

Date limite de candidature

28/07/2020
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