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Stimulator of Interferon Genes (STING) is a cytosolic sensor of cyclic dinucleotides (CDNs). The activation of dendritic cells (DC) via the STING pathway, and their subsequent production of type I interferon (IFN) is considered central to eradicating tumours in mouse models. However, this contribution of STING in preclinical murine studies has not translated into positive outcomes of STING agonists in phase I & II clinical trials. We therefore questioned whether a difference in human DC responses could be critical to the lack of STING agonist efficacy in human settings. This study sought to directly compare mouse and human plasmacytoid DCs and conventional DC subset responses upon STING activation. We found all mouse and human DC subsets were potently activated by STING stimulation. As expected, Type I IFNs were produced by both mouse and human plasmacytoid DCs. However, mouse and human plasmacytoid and conventional DCs all produced type III IFNs (i.e., IFN-λs) in response to STING activation. Of particular interest, all human DCs produced large amounts of IFN-λ1, not expressed in the mouse genome. Furthermore, we also found differential cell death responses upon STING activation, observing rapid ablation of mouse, but not human, plasmacytoid DCs. STING-induced cell death in murine plasmacytoid DCs occurred in a cell-intrinsic manner and involved intrinsic apoptosis. These data highlight discordance between STING IFN and cell death responses in mouse and human DCs and caution against extrapolating STING-mediated events in mouse models to equivalent human outcomes., Competing Interests: HH is an employee of Bavarian Nordic GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pang, Daraj, Balka, De Nardo, Macri, Hochrein, Masterman, Tan, Shoppee, Magill, Jahan, Bafit, Zhan, Kile, Lawlor, Radford, Wright and O’Keeffe.)

Background: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8 + T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141 + DCs, the human cDC1 equivalent. CD141 + DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8 + T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141 + DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8 + T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs., Methods: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8 + T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141 + DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8 + T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, tumor necrosis factor and CD107a and by lysis of target tumor cells., Results: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141 + DCs for activation of NY-ESO-1-specific CD8 + T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8 + T cells with polyclonal effector function and potent tumor killing capacity in vitro., Conclusions: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141 + DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)