Route Specific Effects of Lipoprotein Agonists of TLR2 on CD4 T Cell Priming
Johndrow, Christopher Taylor
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Vaccines are currently the most successful prophylactic intervention against many infectious diseases. Subunit vaccines are a promising strategy for the development of novel effective vaccines, however, protein subunits are poorly immunogenic alone. Successful subunit vaccines require formulation with adjuvant compounds in order to stimulate cells of the innate immune system and generate protective adaptive immune responses. The development of vaccine adjuvants is critical for subunit vaccine development, but requires improved high throughput screening methods that reliably predict in vivo immune responses. Pattern recognition receptor (PRR) agonists are a growing class of potential vaccine adjuvants that are able to shape both the scale and character of the immune response to subunit vaccines through the direction of CD4 T cell polarization. We have applied a high-throughput in vitro assay to assess the CD4 T cell polarization potential of a panel of PRR agonists. Using this system, we identified lipopeptide TLR2 agonists as exceptional Th1 polarizing adjuvants. In vivo, we demonstrated that i.v. administration of TLR2 agonists replicates the findings from in vitro screening by promoting strong Th1 polarization. In contrast, these TLR2 agonists were found to inhibit priming of Th1 responses when administered intradermally in mice. We found that this route-specific suppression was mediated by infiltrating CCR2+ cells in the skin draining lymph nodes and was independent of skin-derived dendritic cells. We further demonstrated that CD4 T cell priming after immunization against Mycobacterium tuberculosis with BCG, a lipoprotein-rich bacteria recognized by TLR2, was dependent on immunization route, and Th1-like responses generated by intradermal administration of BCG were significantly improved when administered intravenously. We performed further studies to characterize the CD4 T cell priming environment in the skin draining lymph node, particularly dendritic cell and other innate myeloid cell trafficking into secondary lymphoid organs and expression of cytokines and co-inhibitory signals after treatment with TLR2 and TLR9 agonists. We assessed the expression of pro-inflammatory and anti-inflammatory cytokines by lymph node resident cells, expression of co-inhibitory molecules by dendritic cells, antigen trafficking and myeloid cell infiltration, and the effects of IL-10 and TGFβ neutralization on Th1 suppression. We also explored the effects of mycobacterial lipoproteins, either enriched directly from BCG or purified from recombinant expression on TLR2 activation and describe aspects of in vivo responses which suggest mycobacterial lipoproteins function similarly in the skin as synthetic TLR2 ligands and suppress optimal Th1 priming. Complete understanding of route-dependent TLR2 responses are critical for informed design of novel subunit vaccines and improvement of BCG and other vaccines based on live-attenuated organisms that express TLR2 ligands. It is the goal of this work to advance understanding of downstream effects of TLR2 signaling in various contexts and to contribute to future vaccine design strategies.