Localization and persistence of vaccine carrier BLS in dendritic cells is dependent on TLR4

ROSSI ANDRÉS; FARIAS ANA; FERNANDEZ JAVIER; ALZOGARAY VANINA; GOLDBAUM FERNANDO; BERGUER PAULA

Tours

Simposio; 13th International Symposium on Dendritic Cells; 2014

Brucella spp. Lumazine Synthase (BLS) is a highly immunogenic and stable decameric protein, successfully used as a carrier for several proteins. BLS immunization activates murine dendritic cells (DC) and recruits DC, B cells, CD8+ and CD4+ cells at the draining lymph nodes (LN) via TLR4. We have shown that BLS induces the cross-presentation of associated peptides and a TLR4-dependent specific cytotoxicity in mice. Here we show that after immunization in the hind footpad with BLS-Fluorophore, BLS is found at 4 h in 30±4% of DC and in 10±2% of B lymphocytes at popliteal LN, and in 14±3% of the DC at the inguinal LN. At 120 h BLS is associated within DC at the inguinal LN. In TLR4-deficient mice similar results are observed until 4 h; at longer times BLS cannot be detected. These results show that BLS resides for longer periods in DC and suggest that DC carry BLS to the inguinal LN. To measure the duration of antigen exposure to CD8+ T cells, mice were immunized with BLS-OVA(257-264) and at different time points after vaccination adoptive transfer of OT-I cells was performed. Proliferation of OT-I cells is observed even at 17 days after immunization, indicating that antigen presentation still takes place and suggesting a prolonged antigen availability. Subcellular localization of BLS was studied by confocal microscopy. BMDC were incubated with BLS-Fluorophore. Only minor colocalization is observed with classical antigen presentation markers. BLS colocalizes with TLR4 at the cell membrane at 5 min and with cytoplasmic TLR4 at 6 h. BLS is present at least until 6 h in BALB/c BMDC; in TLR4-deficient BMDC it can be found only for 1 h. These results suggest that BLS binds to TLR4 at the plasma membrane and subsequently enters to the cytoplasm. BLS would escape the endocytic pathway of antigen processing and its persistence is dependent on TLR4 signaling.