a, CLEC9A-dependent enrichment for phospho-Syk at the contact area between DC and dead cells. Quantification of conjugates showing phospho-Syk enrichment at the contact area between CD8α⁺-like DC and dead cell is shown as mean± SEM of three independent experiments. b-c, CLEC9A signalling is required for efficient cross-presentation of dead cell-associated material to CD8⁺ T cells. Bone marrow cells from CLEC9A⁻ mice were retrovirally transduced with a vector encoding only for GFP (GFP) or encoding additionally for the wild-type (CLEC9A WT) or a mutant form (CLEC9A Y7F) of CLEC9A. After culture in the presence of Flt3L, GFP⁺ CD8α⁺-like Flt3L BMDC were purified by cell sorting and co-cultured with UVC-treated H-2^bm1 MEFs expressing (bm1T OVA), or not (bm1T), OVA in the presence of CFSE-labelled OVA-specific OT-I cells. OT-I proliferation and IFN-γ production were measured. OVA-coated beads were used as a control source of antigen. b, one representative experiment out of three. c, Data are mean ± SEM of three independent experiments. *, p<0.05 and **, p<0.01 two way ANOVA.

a-c, clec9a^gfp/gfp (CLEC9A⁻) or control clec9a^+/+ or clec9a^gfp/+ (CLEC9A⁺) littermates were immunised i.v. with 7.5×10⁵ UVC-treated bm1T OVA MEFs. Six days later, the frequency of OVA-specific CD8⁺ T cells was determined in the spleen (a-b). a, each dot corresponds to an individual mouse; the data are pooled from seven independent experiments. b, the average response in each litter for clec9a^gfp/gfp or control CLEC9A⁺ littermates. Lines link sex-matched (female) littermates. c, IFN-γ production in response to OVA peptide restimulation ex vivo. Data indicate frequency of IFN-γ⁺ cells within the CD8⁺ T cell population. Individual mice and average for one representative experiment (out of three) is shown. d-e, C57BL/6 mice untreated or treated with anti-CLEC9A antibody or an isotype-matched control (IgG1) were immunised i.v. with 7.5×10⁵ UVC-treated bm1T OVA MEFs. The frequency of OVA-specific CD8⁺ T cells (d) or of IFN-γ producing CD8⁺ T cells in response to OVA peptide restimulation (e) was measured six days later. Data show individual mice and average response from three different litters. (a-e). p values were determined using Student's t test (paired for b)

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).

a, BWZ cells stably expressing mouse or human CLEC9A-ζ or Dectin-1-ζ were plated at different concentrations and allowed to grow for 48h. The frequency of TO-PRO-3⁺ cells was determined and equivalent numbers of live cells were transferred to fresh medium. After overnight culture, LacZ activity was measured using a colorimetric assay and expressed relative to the frequency of TO-PRO-3⁺ cells. b, Untreated or UVC-treated MEFs were cultured with BWZ cells expressing CLEC9A-ζ or Dectin-1-ζ. Where indicated, Fab fragments of control or anti-mCLEC9A or anti-hCLEC9A antibodies were added. Reporter activity in BWZ cells was measured as in (a). Ctrl., BWZ cells alone. c, LK cells were exposed to increasing doses of UVC before culturing with BWZ cells expressing, or not, mCLEC9A-ζ. LacZ activity in BWZ cells was measured as in (a) and is expressed as a function of the percentage of TO-PRO-3⁺ LK cells at each dose of radiation. d, PE-labelled tetramers of CLEC9A or Dectin-1 CTLD were used to stain untreated or UVC-treated MEFs or zymosan particles. e, BWZ cells expressing mouse CLEC9A were cultured overnight in medium alone (Ctrl) or together with LK cells that had been untreated (−) or treated 16h before with UVC (UV) or mitoxantrone (Mtx), or cultured overnight without serum (serum deprivation; SD) or subjected to freeze / thawing (FT). LacZ activity (left y axis) is depicted, together with the frequency of TO-PRO-3⁺ and CLEC9A CTLD tetramer⁺ LK cells at the beginning of the co-culture (right y axis). f, CLEC9A ligand is a preformed signal. LK cells were fixed with formaldehyde and permeabilized, or not, with Triton-X-100 before labelling with TO-PRO-3 and PE-tetramers of Dectin-1 or CLEC9A CTLD. g, Fixed and permeabilized MEFs were labelled with Dectin-1 or CLEC9A CTLD monomers and counterstained with TO-PRO-3 before confocal microscopy. h, Fixed and permeabilised MEFs were left untreated or were treated with acid (pH 3.5), peptido N-glycosidase F (PNGase F), O-Glycosidase (O-Glyc), proteinase K (Ptase K), trypsin, pronase-E or benzonase, or were heated for 5 min at 80°C. Cells were then labelled with Dectin-1 or CLEC9A CTLD tetramers and analysed by flow cytometry. Data are ratio between the mean fluorescence intensity (MFI) of the CLEC9A CTLD staining and the Dectin-1 CTLD staining for each of the treatments. a-h, data are from one representative experiment of at least three. All error bars are shown in a-c, e, h and represent mean ± SD of triplicate measurements.