Mechanisms of regulation and role of dendritic cell subsets in antigen transfer and activation of B lymphocytes.

Supervisor: Fatah Ouaaz (see project 2)

Dendritic cells (DCs) are professional antigen-presenting cells, which sample antigens (Ags) in the periphery and migrate to the lymph node (LN) where they activate T cells and potentially B cells. Previousely, we have reported that human monocyte-derived DCs were able to store and to release native Ag internalized by macropinocytosis from the late endosomes in the extracellular medium by a process that was named “regurgitation”. Recently, we reported that murine DCs are important peripheral carriers of Ag to the LN B-cell zone and also potent B-cell activators both in vivo and in vitro. Importantly, we highlight that Ag released upon DC regurgitation is sufficient to efficiently induce early B-cell activation through the transcription factor NF-kB/cRel. However, the regulation of DC regurgitation as well as the respective role of the LN-resident DC subsets in Ag transfer and B-cell activation still need to be investigated.

On the basis of these findings, the PhD candidate will now explore: 1) the respective roles of the two distinct LN resident-DC subsets cDC1/CD8a⁺ and cDC2/CD11b⁺ in Ag transport, transfer and B-cell activation in vivo and in co-culture in vitro; 2) the mechanisms of regulation and control of DC regurgitation by investigating the role of NF-kB pathway, the intracellular traffic and the B-cell attracting chemokine CXCL-13; 3) Live imaging of Ag transfer from DC subsets to B cells in co-culture in vitro and in vivo.

The candidate will use murine specific anti-HEL B cells (from MD4 transgenic mice) and DCs (bone marrow-derived DCs; ex vivo purified spleen DC subsets) pulsed with Ag (HEL). The candidate will explore the Ag distribution, transfer by DCs and B cell activation in vivo and in co-culture in vitro by multi-color flow cytometry, immunohistochemistry and confocal/2-photon microscopy. The candidate will also measure by ELISA antibody production in mice sera following immunization with HEL-loaded DCs. NF-kB activation will be analyzed after cytosolic / nuclear extracts preparation followed by western blotting and confocal microscopy and its direct role will be approached by a specific chemical inhibition. All experimental approaches are available in the laboratory.

We expect to provide new mechanistic insights into Ag transfer and direct B-cell activation modalities by DCs and also new approaches for NF-kB manipulation and DC targeting for vaccination to elicit humoral immunity.