Assessing Cross-Presentation of Necrotic Cell Antigens

H-2^bm1 MEFs were immortalized with SV40 T large antigen (bm1 T MEFs) and transduced to express a truncated and non-secreted OVA-GFP fusion protein. Before the assay, MEFs were UVC-irradiated and cultured overnight in complete medium to induce secondary necrosis and are referred to as UVC-treated cells in the figure legends. For cross-presentation assays in vitro, UVC-treated bm1 T OVA MEFs were cultured with purified CD8α⁺-like Flt3L BMDC and CFSE-labelled OVA-specific OT-I T cells. OT-I responses were quantified four days later by analysing IFN-γ staining and CFSE-dilution profiles by flow cytometry and by monitoring IFN-γ in supernatants. For in vivo experiments, UVC-treated bm1 T OVA MEFs were injected i.v. into clec9a^gfp/gfp or control CLEC9A⁺ littermates or, alternatively, C57BL/6 mice pre-treated with an i.p. injection of PBS, 400 μg isotype control (rat IgG1) or 1F6 anti-CLEC9A. CD8⁺ T cell responses were measured six days later by quantitating the number of H-2K^b-OVA tetramer positive cells and by IFN-γ production in response to OVA peptide restimulation.