Etude de l’efficacité et des mécanismes de la présentation croisée d’antigènes cellulaires tumoraux intacts par les cellules dendritiques

Abstract : Dendritic cells (DC) are specialized in the capture, processing and antigen presentation. They have developed a special antigen presentation mechanism, known as cross-presentation, allowing them to internalize exogenous antigens, to digest and associate them to MHC class I molecules for presentation to CD8+ T lymphocytes. The cross-presentation is essential to the presentation of antigens that are not directly synthesized by the DC (self antigens, tumor antigens, microorganisms that don’t infect DC) and therefore to establish anti-infectious or anti-tumoral CD8+ T cell responses. His study is therefore essential for vaccination and immunotherapy involving a presentation by the DC. Our team showed that, like apoptotic cells, living cells are an efficient antigen source for cross-presentation by DC in vitro and in vivo. We have shown that immunization of mice with DCs that have captured material from living cells could protect effectively against a B16 melanoma challenge in a prophylactic model. During my PhD, I have shown that immunization was also very effective in a therapeutic model. Surprisingly, the protection and the CD8+ T cell response obtained using living cells as antigen source, are better than those obtained with apoptotic cells. DCs cultured with live or apoptotic antigen donor cells, expressed equivalent levels of costimulatory molecules. In contrast, DCs cultured with apoptotic cells secrete more IL- 10, giving them a tolerogenic phenotype. Furthermore, we have also shown that tumor antigens were better preserved within living cells than apoptotic cells, and the amount of MHC-I/peptide complexes at the surface of DC after culture with living cells was greater than after culture with apoptotic cells. In a second part of my thesis, I tried to characterize the receptors and mechanisms involved in the transfer of antigen from living cells to DCs. I have shown that this transfer is not dependent on exosomes transfer, nor on "cross-dressing". However, it is initiated after a close contact with the DC that seems to depend at least in part in scavenger receptors (SR) and calreticulin. The microscopy images obtained suggest the passage of large molecules in a structure, which may be similar to annular junctions (Annular Gap Junctions). Indeed, we observe the passage of connexin 43 (Cx3) and cellular material in a native conformation (GFP 70 kDa protein) from the living cell that partially colocalize with the early endosome marker EEA-1 in DCs. However, the use of an shRNA specific for Cx43 indicates that the cross-presentation does not require its expression. Our results suggest the existence of a mechanism of intercellular communication allowing the passage of large antigen, which could then be processed by DCs.
Keywords : Immunology
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Camille Baey. Etude de l’efficacité et des mécanismes de la présentation croisée d’antigènes cellulaires tumoraux intacts par les cellules dendritiques. Médecine humaine et pathologie. Université René Descartes - Paris V, 2013. Français. ⟨NNT : 2013PA05T054⟩. ⟨tel-00930283⟩

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