Figure 2. CLEC9A is required for crosspresentation of dead cell-associated antigens in vitro.

a-b, Flt3L BMDC from clec9a^gfp/gfp (CLEC9A⁻) or control (CLEC9A⁺) mice were incubated at 4°C or 37°C for 0, 30 or 120 min with PKH26-labelled and UVC-treated H-2^bm1 splenocytes at different ratios. (a) Acquisition of PKH26 label by the CD8α⁺-like DC subset was quantified by flow cytometry and normalised by subtracting the signal obtained at 4°C from the one obtained at 37°C. Data are mean ± SD of two biological replicates. (b) Uptake of dead cell material by CD8α⁺-like DC was determined using multispectral imaging flow cytometry to discriminate between “true” (uptake) and “false” (binding) positive events. Results are mean ± SD of two independent biological replicates from one experiment and are representative of two independent experiments. c, clec9a^gfp/gfp (KO) or control clec9a^+/+ or clec9a^egfp/+ (WT) mice were injected with PKH26-labelled UVC-treated H-2^bm1 splenocytes. 2h later, the frequency (left) and mean fluorescence (right) of CD8α⁻ and CD8α⁺ splenic DC that acquired dead cell material was determined by flow cytometry (left panel). Results are the mean ± SD of three mice. Differences between WT and KO in (a-c) are not statistically significant. d-e, Purified CD8α⁺-like Flt3L BMDC from clec9a^egfp/egfp (CLEC9A⁻) or CLEC9A⁺ littermates were cultured with CFSE-labelled OVA-specific OT-I cells and UVC-treated H-2^bm1 MEFs expressing (bm1 T OVA), or not (bm1 T), OVA. OT-I proliferation (d and e, top panel) and IFN-γ in the supernatant were quantified (e, bottom panel). Soluble OVA and OVA-coated beads were used as a control source of antigen. Results are mean ± SEM of three independent biological replicates from one experiment and are representative of three independent experiments. *, p<0.05, **, p<0.01 and ***, p<0.001 (Student's t test).