The TLR2 Binding Neisserial Porin PorB Enhances Antigen Presenting Cell Trafficking and Cross-presentation

TOLL-like receptor (TLR) ligands activate both innate and adaptive immune cells, while modulating the cellular immune response. The outer membrane protein (OMP) from Neisseria meninigitidis, PorB, is a naturally occurring TLR2 ligand and functions as an adjuvant. Here, we demonstrate that PorB increases the level of OVA in the endo-/lysosomal cellular compartment of BMDCs, increases antigen presenting cell (APC) trafficking to draining lymph nodes, and enhances antigen cross-presentation. PorB is capable of mounting an antigen specific T cell response by efficiently stimulating antigen cross-presentation in vivo and in vitro assessed by BMDC OT-I cocultivation assays. The enhanced antigen cross-presentation and the increased APC recruitment to secondary lymphoid tissues expand the scope of known adjuvant effects of PorB on the immune system. Our findings lead to a better understanding of how TLR-ligand based adjuvants can alter and modulate immune responses.

In vitro generated BMDCs derived from C57Bl/6 (a) or TLR2 -/mice (b) were left untreated (untr.) or were stimulated OVA protein or OVA+PorB for 1h, 2h or 4h and treated with Mitomycin C to prevent proliferation of BMDCs. Figure shows cytokine production in supernatant of BMDCs prior to coculture setup analyzed using TNFα and IL-6 ELISA. One out of 2 independent representative experiments is shown. Statistics were calculated using a Two-way 5 non-parametrical ANOVA with Tukey correction for multiple comparisons. ns P > 0.05, * P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001. were stimulated with OVA 257-264 CD8 T cell peptide ("SIINFEKL"), PorB alone or OVA 257-264 +PorB for 1h, 2h or 4h respectively and treated with Mitomycin C. After removal of superantant and extensive washing, BMDCs were cocultivated with OT-I splenocytes for 3 days in an effector to target ratio of 4:1. IFNγ production was determined as a readout for antigen recognition by OT-I splenocytes using ELISA. One out of 2 independent representative experiments is shown.

PorB Purification
PorB was purified from N. meningitidis strain H44/76 Δ-1/4 1 using protein extraction and column chromatography as previously described 2 . PorB was formed into protein micelles, termed proteosomes, for vaccine preparation or in vitro cell stimulations, as previously described 3,4 . Purified PorB was tested to stimulate TLR1/TLR2 expressing HEK cells as described previously 5 . Endotoxin content was determined using LAL assay (Pierce Thermo Scientific, USA) as described 6 . PorB preparations (10 μg per lane) were analyzed using non-denaturing and denaturing 12% polyacrylamide gel electrophoresis (PAGE). Non-denaturing PAGE samples were separated on a 12% PAGE with sample loading buffer containing 20% glycerol and bromophenol blue. Denatured SDS-PAGE was conducted on samples boiled for 5 minutes in βmercaptoethanol (5%) SDS containing loading buffer. Gels were stained in Coomassie Blue staining solution for 1h and afterwards de-stained using de-staining solution (10% methanol, 10% isopropanol, 80% ddH 2 O). Images were acquired on a Biorad Gel Doc XR imaging system (Biorad, USA).

Generation of single cell suspension of spleens and lymph nodes
Spleens and/or lymph nodes from either OVA transgenic mice (OT-I/OT-II) or vaccinated mice (C57Bl/6 or TLR2 -/-) were harvested and a single cell suspension was prepared as follows.
Briefly, spleens were pushed through a 70 μm cell strainer. Red blood cells in samples derived from spleens were lysed using ACK lysis buffer for 3 minutes, washed in PBS and afterwards ex vivo stimulated with peptides or cocultured with BMDCs, in the case of OT-I/OT-II splenocytes.
Lymph nodes were torn with tweezers and digested using 30 μl of 1 mg/ml Collagenase D (ThermoFisher, USA) in 1 ml RPMI-1640 for 30 minutes at 37°C with gentle agitation to allow easier separation of dendritic cells. Single cell suspension was generated by grinding the remaining tissue through a 70 μm nylon mesh. Cells were washed in PBS, counted and stained for analysis using flow cytometry. Briefly, cells were incubated for 15 minutes with CD16/CD32 F c block and afterwards stained for 30 minutes with CD11b -APC-Cy7 (Biolegend, USA), CD11c -APC (BD, USA). Antibodies were diluted 1:200 unless otherwise indicated.