Spleen-Resident CD4+ and CD4− CD8α− Dendritic Cell Subsets Differ in Their Ability to Prime Invariant Natural Killer T Lymphocytes

One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α+ and CD8α− cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4− and CD4+ CD8α− cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4− and CD4+ cDC are equivalent in their capacity to prime and direct CD4+ and CD8+ T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4− and CD4+ cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4+ counterparts, CD4− cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4+ and CD4− cDC subsets that may be important in immune responses

Dendritic Cells and Hepatocytes Use Distinct Pathways to Process Protective Antigen from Plasmodium in vivo

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2Kb epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase

Exploiting the Role of Endogenous Lymphoid-Resident Dendritic Cells in the Priming of NKT Cells and CD8+ T Cells to Dendritic Cell-Based Vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d−/− BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose

TLR1/2 Activation during Heterologous Prime-Boost Vaccination (DNA-MVA) Enhances CD8+ T Cell Responses Providing Protection against Leishmania (Viannia)

Leishmania (Viannia) are the predominant agents of leishmaniasis in Latin America. Given the fact that leishmaniasis is a zoonosis, eradication is unlikely; a vaccine could provide effective prevention of disease. However, these parasites present a challenge and we do not fully understand what elements of the host immune defense prevent disease. We examined the ability of vaccination to protect against L. (Viannia) infection using the highly immunogenic heterologous prime-boost (DNA-modified vaccinia virus) modality and a single Leishmania antigen (TRYP). Although this mode of vaccination can induce protection against other leishmaniases (cutaneous, visceral), no protection was observed against L. (V.) panamensis. However, we found that if the vaccination was modified and the innate immune response was activated through Toll-like receptor1/2(TLR1/2) during the DNA priming, vaccinated mice were protected. Protection was dependent on CD8 T cells. Vaccinated mice had higher CD8 T cell responses and decreased levels of cytokines known to promote infection. Given the long-term persistence of CD8 T cell memory, these findings are encouraging for vaccine development. Further, these results suggest that modulation of TLR1/2 signaling could improve the efficacy of DNA-based vaccines, especially where CD8 T cell activation is critical, thereby contributing to effective and affordable anti parasitic vaccines

Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells.

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8(+) T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and alpha-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants