doi: 1.138/nature775 4 O.D. (595-655) 3 1 -ζ no antibody isotype ctrl Plated Soluble 1F6 397 7H11 Supplementary Figure 1 Soluble and plated anti- Abs induce -! signalling. B3Z cells stably expressing! were cultured overnight in the presence of anti- (1F6, 397 or 7H11) or isotype-matched control antibodies in soluble form (soluble) or previously adsorbed onto plastic (plated). Lac Z activity was measured at the end of the culture. 1
doi: 1.138/nature775 a long arm (6.6 Kb) short arm ( Kb) 1 EGFP Neo Tk Targeting Vector b clec9a +/+ 1 3 4 5 6 7 knock out (1.35 Kb) clec9a gfp/gfp 1 EGFP Neo clec9a gfp/gfp.19 1.78 short arm ( Kb) 3 Fw1Rv1.14Kb FwRv.35Kb c clec9a +/+ 43.6 6.1 Genomic Locus Targeted Locus clec9a gfp/gfp.7.35 CD11c d 1 FL4-H: CD11C APC 1 Splenocytes (x1 6 ) 1 8 4.4 1 1 GFP % of total splenocytes 7 6 5 4 3 1 LB LT % of total splenocytes 1 1 FL-H: 1 PE 13 85.1 1 CD8 3 1 DC NK NKT e 1 Thymocytes (x1 6 ) 47.8 175 15 15 1 75 5 5.61 1 GFP 1 48 49.3 1 + - f % of CD3 + NK1.1 - Splenocytes IL-1p4 (ng/ml) 7 6 5 4 3 1 145 85 5 5 15 1 5 CD4 CD8 - Cdlan + - Zym % of CD11c + I-A b+ Splenocytes CpG 6 4 LPS CD4 CD8 DN IL-6 (ng/ml) 7 5 3 1 1 - Cdlan + - % of total Thymocytes 8 45 1 1 TNF (pg/ml) DP DN CD4 SP 5 4 3 1 - Cdlan Supplementary Figure Generation and characterization of clec9agfp/gfp mice - (see next page for full legend). Zym CpG LPS Zym CpG CD8 SP LPS
doi: 1.138/nature775 Supplementary Figure Generation and characterization of clec9a gfp/gfp mice. a, Targeting strategy used for the generation of clec9a gfp/gfp mice. Correct targeting leads to the insertion of a farnesylated form of EGFP in frame with the two first aminoacids of. Half of the exon 1 and the entire exon are eliminated and transcription following GFP is terminated by a polyadenylation signal. b, GFP expression in the spleen of clec9a gfp/gfp mice is restricted to CD8" + DC and correlates with lack of expression of. Splenocytes from clec9a gfp/gfp and clec9a +/+ mice were stained for, CD8" and CD11c. The relative expression of CD11c and GFP by total splenocytes is depicted in the left two panels. The dot plot on the right shows CD8" vs expression by CD11c + GFP + splenocytes from clec9a gfp/gfp mice. c, Lack of expression of by CD8" + splenic DC from clec9a gfp/gfp mice. Splenocytes from clec9a gfp/gfp and clec9a +/+ mice were stained for, CD8" and CD11c. The relative expression of and GFP by CD11c + CD8" + cells of each genotype is shown. d, Spleen composition is unaltered by the absence of. Total splenocytes and frequency of T and B lymphocytes, NK and NKT cells, CD4 + and CD8 + T cells and CD4 +, CD8" + and CD4 - CD8" - DCs were compared between clec9a +/+ ( + ) and clec9a gfp/gfp ( - ) mice. e, Thymocyte differentiation is not affected by deficiency. Absolute number of thymocytes and frequency of CD4 + CD8 + double positive (DP), CD4 + CD8 - single positive (CD4 SP), CD4 - CD8 + single positive (CD8 SP) and CD4 - CD8 - double negative (DN) populations is depicted. f, Functional response of DC to innate stimulis is not affected by deficiency. Purified splenic DCs from clec9a +/+ ( + ) and clec9a gfp/gfp ( - ) mice were stimulated overnight with curdlan (1 µg/ml; Cdlan), zymosan (1 µg/ml; Zym), CpG-containing DNA oligonucleotide (.5 µg/ml; CpG) or LPS (1 µg/ml). Supernatants were analyzed for the presence of IL-1 /3 p4, TNF and IL-6 by ELISA. Data in (b, e-f) are the arithmetic mean ± SEM of three mice per group from one representative experiment out of three. c and d: one representative mouse per group is shown. 3
doi: 1.138/nature775 a clec9a +/+ 85. 4.13 clec9a gfp/gfp 7. 11 1 1 b 1..41 1 31.6 5. 1 GFP Sancho et al. 175 ( - ) mice were stimulated overnight 6 with curdlan (1 µg/ml; Cdlan), 5 zymosan (1 µg/ml; 15 5 4 Zym), 15CpG-containing DNA oligonucleotide 4 (.5 µg/ml; CpG) or LPS (1 µg/ml). Supernatants 1 3 3 were 75 analyzed for the presence of IL-1 /3 p4, TNF and IL-6 by ELISA. Data in (b, e-f) are the 5 arithmetic mean ± SEM of three mice per group from one representative experiment 5 1 1 out of three. c IL-1p4 (ng/ml) IL-6 (ng/ml) and d: one representative mouse per group is shown. - Cdl Zym CpG LPS - Cdl Zym CpG LPS - Cdl Zym CpG LPS TNF (ng/ml) Supplementary Figure 3 Phenotypic and functional characterization of Flt3L BMDC generated from clec9a gfp/gfp mice. Flt3L BMDC were differentiated from bone marrow progenitors isolated from clec9a gfp/gfp mice or control wild type littermates. a, GFP and expression by Flt3L BMDC after gating on CD8" + -like DC (CD4 hi CD11b lo B neg ). One representative mouse per group is shown. b, Functional response of Flt3L BMDC to innate stimuli is not affected by deficiency. Flt3L BMDC of the indicated genotypes were cultured overnight with curdlan (1 µg/ml; Cdlan), zymosan (1 µg/ml; Zym), CpG-containing DNA oligonucleotide (.5 µg/ml; CpG) or LPS (1 µg/ml). Culture supernatants were analysed for the presence of IL-1 /3 p4, TNF and IL-6 by ELISA. One representative experiment is shown. Data represent the arithmetic mean ± SEM of three independent Flt3L BMDC cultures from different mice. Supplementary Figure 4 Uptake of dead cell-derived material by CD8"-like DC is not affected by loss of. Flt3L BMDC, generated from wild type ( + ) or clec9a gfp/gfp ( - ) littermates, were allowed to interact with PKH6-labelled UVC-treated cells for the indicated periods of time (min) at 4ºC or 37ºC. The association of PKH6-labelled cell material with the CD11c + CD4 high CD8"-like subpopulation was then analyzed by flow cytometry. Representative pseudo-color contour plots are depicted. Supplementary Figure 5 recruits and signals via Syk kinase 4
doi: 1.138/nature775 3 1 Sancho et al. ( - ) mice were stimulated overnight with curdlan (1 µg/ml; Cdlan), zymosan (1 µg/ml;.6. Zym), CpG-containing DNA 1 oligonucleotide (.5 1 µg/ml; CpG) or LPS 1(1 µg/ml). Supernatants 4ºC were analyzed for the presence of IL-1 /3 p4, 1 TNF and IL-6 by ELISA. 1 Data in (b, e-f) are the 1 1 1 1 arithmetic mean ± SEM of three mice per group from one representative experiment out of 4 three. c + and d: one representative mouse per group is shown. 37ºC. 8.7 48.8 1 1 1 1 Supplementary Figure 3 Phenotypic and functional characterization 1 of Flt3L BMDC 1 1 1 1 generated from clec9a gfp/gfp mice. Flt3L BMDC were differentiated from bone marrow progenitors 4 isolated from clec9a gfp/gfp mice or control wild type littermates. a, GFP and expression by 4ºC 1 Flt3L BMDC after gating on CD8" + -like DC (CD4 hi 1 CD11b lo B neg 1). One representative mouse.49 3.9 PKH6 per group is shown. b, Functional response of Flt3L BMDC to innate stimuli is not affected by 1 1 - deficiency. Flt3L BMDC of the indicated genotypes were cultured overnight with curdlan 37ºC.1 1 (1 µg/ml; Cdlan), zymosan (1 µg/ml; Zym), CpG-containing DNA oligonucleotide (.5 µg/ml; 1 1 1 CpG) or LPS (1 µg/ml). Culture supernatants were analysed for the presence of IL-1 /3 p4, TNF 35.7 46.6 1 and IL-6 by ELISA. One representative experiment is shown. Data represent the arithmetic mean ± 1 SEM of three independent Flt3L BMDC cultures from different mice. CD4 1 Supplementary Figure 4 Uptake of dead cell-derived material by CD8"-like DC is not affected by loss of. Flt3L BMDC, generated from wild type ( + ) or clec9a gfp/gfp Supplementary Figure 4 ( - ) littermates, were allowed to interact with PKH6-labelled UVC-treated cells for the indicated periods of time (min) at 4ºC or 37ºC. The association of PKH6-labelled cell material with the CD11c + CD4 high CD8"-like subpopulation was then analyzed by flow cytometry. Representative pseudo-color contour plots are depicted. Supplementary Figure 5 recruits and signals via Syk kinase 5
doi: 1.138/nature775 a Cytoplasmic Tail - Dectin-1 7Y 7F 7Yp 15Y 15Yp rsyk Syk b t (min) plated Ab P-Syk - anti- rat IgG 1F6 397 7H11 c Syk O.D. (595-655).5. 1.5 1..5..1.1 1 Plated antibody (μg/ml) Syk wt Syk Y7F wt Supplementary Figure 5 recruits and signals via Syk kinase a, Binding of Syk to phosphorylated peptides corresponding to the cytoplasmic domains of or of Dectin-1. Biotinylated peptides bearing the critical tyrosine residue in unphosphorylated or phosphorylated form or mutated to a phenylalanine were incubated with recombinant Syk kinase before precipitation with streptavidin. Precipitates were subjected to SDS- PAGE followed by Western blotting with anti-syk antibodies. rsyk and (-) mark lanes loaded with recombinant Syk or left bank as a positive and negative control, respectively. b, Syk activation in response to triggering in LK cells stably transfected with m. Cells were cultured for or min on plates coated with the indicated antibodies before lysis and SDS-PAGE and Western blotting with anti-phospho-syk or anti-syk antibodies. c, signals in a Tyr7- and Syk-dependent fashion. B3Z cells stably expressing the wild type or Y7F mutated form of m and co-expressing or not Syk kinase were cultured in plates coated with antim. NFAT activity was measured using a colorimetric assay for the Lac Z reporter. 6
doi: 1.138/nature775 4 O.D. (595-655) 3 1 WT Syk/ WT Syk/ Y7F no antibody isotype ctrl Plated Soluble 1F6 397 7H11 Supplementary Figure 6 Signalling through requires Syk and a tyrosine at position 7. B3Z cells stably expressing ( WT), Syk and (Syk/ WT) or Syk and Y7F (Syk/ Y7F) were cultured overnight in the presence of anti- (1F6, 397 or 7H11) or isotype-matched control antibodies in soluble form (soluble) or previously adsorbed onto the plate (plated). Lac Z activity was measured at the end of the culture. 7
doi: 1.138/nature775 a O.D (595-655) TO-PRO-3 rsctld 1. 1 O.D. (595-655).5 75 5 5 % CTLD % TO-PRO-3 + cells. Ctrl - UV Mtx SD FT MEFs treatments b 1.5 1. O.D. (595-655).75.5.5. Syk/ wt Syk/ Y7F CTRL LK LK UV 1F6 Fab LK UV + rat IgG1 LK UV + rat IgGa 1F6 397 7H11 Supplementary Figure 7 Necrotic cells expose ligands for that provoke signalling through Syk kinase. a, Different necrosis-inducing treatments generate dead cells that trigger /Syk signalling. BWZ cells stably co-expressing Syk and wild type were cultured overnight in medium alone (Ctrl) or together with bm1 T MEFs that had been untreated (-) or treated 16h before with UVC (UV) or mitoxantrone (Mtx), MEFs cultured overnight without serum (serum deprivation; SD) or MEFs subjected to freeze / thawing (FT) just before the assay. LacZ activity (left y axis) is depicted, together with the frequency of TO-PRO-3 + and CTLD + MEFs at the beginning of the co-culture (right y axis). b, Necrotic cell-induced signalling via /Syk can be blocked by soluble anti- mabs. B3Z cells stably co-expressing 8
doi: 1.138/nature775 Syk and wild type (Syk/ wt) or a mutated version (Syk/ Y7F) were cultured overnight in the absence (CTRL) or presence of LK cells either untreated (LK) or UVCirradiated 4h before (4 mj/cm, LK UV). Where indicated, monovalent Fab fragments of antim (1F6) or bivalent intact antibodies, including isotype-matched controls (rat IgG1, rat IgGa) and anti-m (1F6, 397, 7H11), were added to the culture. Lac Z activity was measured at the end of the culture. 9
doi: 1.138/nature775 a % of PKH6 + CD8α-like DC 5 4 3 1 WT n.s. Syk KO b 5 Syk KO DC 5 WT DC 1 WT Syk KO *** Cell number 15 1 5 15 1 5 IFN-γ (ng/ml) 5 * ** 1 1 CFSE no DC. 1 5 1 OVA (mg/ml) Supplementary Figure 8. Syk is necessary for efficient cross-presentation of dead cellassociated antigens. a, Syk-deficient CD8" + -like DC are not impaired in their ability to capture dead cell material. Purified wild type or Syk-deficient CD8" + -like Flt3L BMDC were incubated at 37ºC for 1 min with PKH6-labelled and UVC- treated H-bm1 splenocytes at a 1 to 5 ratio. Uptake of dead cell material was determined using an ImageStream multispectral imaging flow cytometer. Results are mean ± SEM of two independent experiments. b, Impaired crosspresentation of dead cell-associated antigen by Syk-deficient DC. Purified wild type or Syk-deficient CD8!+like Flt3L BMDC were cultured with CFSE-labelled OVA-specific OT-I cells and UVC- treated H- bm1 splenocytes loaded with the indicated amounts ofova. After four days, OT-I proliferation was analyzed by flow cytometry (left panel) and IFN-" levels in the supernatant were determined by ELISA (right panel). Results are mean ± SEM of four independent biological replicates (cells from different mice) and are representative of four independent experiments. p values were determined using one way ANOVA for comparation between different treatments. *, p<.5, **, p<.1, ***, p<.1, post-hoc Bonferroni test. b. Left panel. One representative experiment is shown out of four performed. 1
doi: 1.138/nature775 H-K b :SIINFEKL Tetramers UVC bm1 T OVA UVC bm1 T - IgG1 α.13 1.79 1.64.37 1 1 1 1 1 1 1 3 4 1 1 CD8 UVC bm1 T OVA UVC bm1 T - IgG1 α.1 1.35 1.3. IFN-γ 1 1 1 FL4-H: IFNg APC 1 Sancho et al. 5 four performed. 1 1 1 3 4 1 1 CD8 Supplementary Figure 9 Supplementary Figure 9. Blockade of reduces crosspriming to dead cell-associated antigen in vivo. C57BL/6 mice either untreated or injected with isotype-matched control (rat IgG1) or anti- antibody were immunised i.v. with 7.5x1 5 UVC-treated H- bm1 transformed MEFs expressing OVA (UVC bm1t OVA) or not (UVC bm1t). Top: six days later, the frequency of OVA-specific splenic CD8 + T cells was determined by staining with H-K b / OVA tetramers. Bottom: IFN-# production by splenic CD8 + T cells was measured by staining after ex vivo restimulation for 5 h with OVA peptide. Data from one representative mouse per group is shown. Supplementary Figure 1. is associated with necrotic cell material that does not enter lysosomal compartments. a, but not DEC-5 is routed to non-lysosomal compartments after internalisation. #g of Alexa488-coupled anti- or anti-dec5 antibody were injected i.v. into C57BL/6 mice. 1h later, spleen DC were isolated, stained with Lysotracker and plated on fibronectin-coated coverslips. Confocal sections were collected and the intracellular distribution of and DEC5 was analysed. b, colocalises with necrotic cell material away from lysosomes. CD8" + splenic DC from C57BL/6 mice were purified by cell sorting and co-incubated for h with Alexa633SE-labelled necrotic bm1t cells at a 1: ratio. The intracellular distribution of necrotic cells material associated, or not, with was then 11
doi: 1.138/nature775 a DEC-5 Lysotracker Lysotracker % DC with receptor pattern 1 8 6 4 Non Lysosomal Both DEC5 Lysosomal only b Necrotic material associated with Necrotic material not associated with necrotic material Lysotracker Necrotic cell DC % necrotic granules/dc Lysosomal Non Lysosomal % necrotic granules/dc Lysosomal Non Lysosomal Supplementary Figure 1. is associated with necrotic cell material that does not enter lysosomal compartments. a, but not DEC-5 is routed to non-lysosomal compartments after internalisation. #g of Alexa488-coupled anti- or anti-dec5 antibody were injected i.v. into C57BL/6 mice. 1h later, spleen DC were isolated, stained with Lysotracker and plated on fibronectin-coated coverslips. Confocal sections were collected and the intracellular distribution of and DEC5 was analysed. b, colocalises with necrotic cell material away from lysosomes. CD8" + splenic DC from C57BL/6 mice were purified by cell sorting and co-incubated for h with Alexa633SE-labelled necrotic bm1t cells at a 1: ratio. The intracellular distribution of necrotic cells material associated, or not, with was then analysed by confocal microscopy. Arrows show co-localization of + vesicles with necrotic material, whereas arrowheads point at Lysotracker + vesicles containing necrotic material. a and b. Quantification of the results shows the mean ± SEM of three experiments (n > 1 DCs/experiment). All the differences between white and black bars are statistically significant (p <.1, Student s t test). 1