The A-Z of APCs

Cancers develop through a complex interplay of factors such as the accumulation of oncogenic mutations, epigenetic and posttranslational modifications, and interactions with the extracellular matrix and the immune system. Advancing cancers recruit host cells and remodel the vasculature and extracellular matrix, thereby shaping the tumor microenvironment. Immune cells are an important component of the tumor microenvironment. Several therapies, such as immune checkpoint inhibitors, have been developed to intervene in the immune dysregulating actions exerted by the tumor. Therapeutic cancer vaccines incorporating cancer antigens can be applied to enhance priming of cancer antigen-specific T cells. Several cancer vaccines are under investigation and these consist of different combinations of cancer antigens, immunostimulatory adjuvants and vaccination platforms. Most cancer vaccines rely on nonspecific uptake by DCs in order to elicit an immune response, but our previous studies indicated that targeting antigen to CD169/Siglec-1 on macrophages significantly enhanced T cell priming. In this thesis, we have investigated whether targeting of cancer vaccines to different receptors on DCs and macrophages (DEC-205, CLEC9A, DC-SIGN, and CD169) can enhance their ability to elicit an anti-tumor immune response. First, we outlined the function of CD169+ macrophages in health and disease and discussed their potential as targets for vaccination strategies in different settings (chapter 2). Next, we examined liposomes that bind to CD169 or DC-SIGN via the incorporation of natural ligands or nanobodies and compared the immune response after targeting these antigen-containing liposomes to CD169 or DC-SIGN-expressing APCs in vivo. Here, we discovered that CD169-specific nanobody and natural ligand liposomes elicit an increased T cell response compared to untargeted control liposomes, while DC-SIGN-specific nanobody or natural ligand liposomes did not induce an increased immune response (chapter 3). In chapter 4, we investigated three liposomes incorporating different CD169-specific nanobody clones and one CLEC9A-specific nanobody liposome. We observed high uptake of the CD169- and CLEC9A-specific nanobody liposomes by receptor-overexpressing cell lines and identified several CD169+ or CLEC9A+ cell subsets in human blood which specifically took up the nanobody liposomes. Interestingly, only one CD169-specific nanobody liposome elicited increased T cell activation. Besides liposomes as vaccination platform, we also investigated antibodies and successfully conjugated antigens to antibodies specific for DEC-205 or CD169 through proximity-based Sortase A-mediated ligation. Both model and neoantigens could successfully be attached to antibodies and these antibody-antigen vaccines were specifically taken up by DEC-205- or CD169-expressing cell subsets in vitro and in vivo, which led to increased T cell priming in vivo (chapter 5). To increase our understanding of vaccine uptake and processing by CD169+ APCs, we explored the intracellular routing of CD169-binding nanobodies, antibodies, nanobody liposomes and natural ligand liposomes. We observed that antibodies and nanobodies are retained on the cell surface of a CD169-expressing monocytic cell line with some recycling to and from the early endosomes. In comparison, CD169 nanobody or natural ligand liposomes are internalized rapidly and accumulate in the lysosomes, but not the virus-containing compartments (chapter 6). In the bone marrow, DCs develop from hematopoietic stem cells into different subsets with unique functions. While transcriptional regulation of DC development is well established, whether epigenetic mechanisms are involved is still unknown. In chapter 7 we investigated DC development in the absence of epigenetic regulator DOT1L and discovered changes in the development of DC2s and pDCs after Dot1l knock out in vitro or in vivo. In addition, Dot1l knock out resulted in an increased inflammatory status. Finally, we discussed the findings of this thesis in relation to other developments in the cancer vaccine field and outlined challenges and key steps to take to improve vaccine availability for patients in the future (chapter 8).