| Dendritic cells (DCs) internalize antigens, process proteins, and present peptides complexed with major histocompatibility (MHC) molecules to naive T cells. As such, they are crucial for the initiation of adaptive immune responses. The presentation of exogenous antigens on MHC class I for the activation of CD8 T cells is known as cross presentation, and DCs appear to cross present antigen most efficiently of all antigen presenting cells. Understanding this process is critical, particularly for therapy where robust CD8 responses are a desired outcome. To address this, we used three different receptors to target clinically relevant antibody-antigen conjugates selectively to early endosomes or to late endosomes and lysosomes. In both primary human BDCA1 and monocyte-derived DCs, CD40 and mannose receptor targeted antibody-conjugated antigens to early endosomes, although CD40 endocytosis was less efficient. DEC205 targeted antigen primarily to late compartments, and internalized more antigen than CD40. Interestingly, the receptor that was the least efficient at antigen internalization, CD40, was the most efficient at facilitating cross presentation. This appeared to be due to its poor uptake or poor endosomal-lysosomal degradation, which would likely decrease the amount of antigen available for egress to the cytosol, rather than any activating effects of the anti-CD40. Thus, while we find that both early and late endosomes support cross presentation in human DCs, poor uptake and an early endocytic localization results in greater cross presentation due to reduced proteolytic capacity.;BDCA3 DCs have been shown recently to be superior for inducing CD8 responses via cross presentation as compared to other DC subsets, and may be the functional equivalent of the mouse CD8 DCs. We optimized a large scale method for isolating this cell type, and tested their cross presentation capacity as compared to BDCA1 DCs using our antibody-antigen conjugate to DEC205. We confirmed that BDCA3 DCs were able to present antigen from late endosomally targeted DEC205 conjugates more efficiently than BDCA1 DCs. This was not due to an enhanced capacity for surface or endogenous MHC class I presentation by BDCA3 DCs, nor was it due to the levels of internalization, accumulation, or localization of anti-DEC205 antibodies in this cell type. Instead, the differences appear to be at the level of the late endosome, where BDCA3 DCs may possibly exhibit differences in antigen egress or degradation. Interestingly, targeting CD40, which is lowly internalized to early endosomes, resulted in superior cross presentation in multiple DC types, including BDCA3 DCs, as compared to DEC205. Taken together, these data indicate that internalization and intracellular trafficking are key criteria that should be considered when selecting candidate receptors for targeting cross presentation by human DCs.;Other collaborative efforts were undertaken to understand the potential role of MHC class I localization in plasmacytoid DCs (PDCs) in the rapid presentation of viral peptides, as well as the role of endocytosis in the sensing of complexed and uncomplexed self-DNA by PDCs. In the first study, we found that MHC class I was found in vesicles that were positive for early and recycling markers such as transferrin. This, combined with data generated by Connolly and co-workers, suggested that PDCs are able to rapidly present antigen from viral sources by loading MHC class I found in the recycling pathway. In the second study, we assisted with the imaging of uncomplexed and complexed DNA in PDCs, and found that the complexing of DNA by anti-microbial peptides resulted in the retention of this aggregate in early endosomes, providing a mechanism for the production of pathogenic Type I interferon by PDCs in autoimmunity. |
