Mouse Clec9a LOCUS NC_000072 13843 bp DNA linear CON 10-JUL-2007 DEFINITION Mus musculus 6, reference assembly (C57BL/6J). ACCESSION NC_000072 REGION: 129358881-129372723

Mouse Clec9a ORF sequence

Exon 1 ATGCATGCGGAAGAAATATATACCTCTCTTCAGTGGGACATTCCTACCTCAGAGGCCTCTCAGAAGTGCCAATCCCCTAGCAAA TGTTCAG Exon2 GAGCATGGTGTGTTGTGACGATGATTTCCTGTGTGGTCTGTATGGGCTTGTTAGCAACGTCCATTTTCTTGGGCATCAAGT Exon 3 TCTTCCAGGTATCCTCTCTTGTCTTGGAGCAGCAGGAAAGACTCATCCAACAGGACACAGCATTGGTGAACCTTACACAGTGG CAGAGGAAATACACACTGGAATACTGCCAAGCCTTACTGCAGAGATCTCTCCATTCAG Exon 4 GCACAGATGCTTCTACTGGACCAGTTCTTCTGACCTCTCCACAGATGGTTCCACAGACCCTGGACAGCAAGGAAACAG Exon 5 GTAGTGACTGCAGCCCTTGTCCACACAACTGGATTCAGAATGGAAAAAGTTGTTACTATGTCTTTGAACGCTGGGAAATG Exon 6 TGGAACATCAGTAAGAAGAGCTGTTTAAAAGAGGGCGCTAGTCTCTTTCAAATAGACAGCAAAGAAGAAATGGAGTTCATCAGC AGTATAGGGAAACTCAAAGGAGGAAATAAATATTGGGTGGGAGTGTTTCAAGATGGAATCAGTGGATCTTGGTTCTGGGAAGA TGGCTCTTCTCCTCTCTCTGACTT Exon 7 GTTGCCTGCAGAAAGACAGCGATCAGCCGGCCAGATCTGTGGATACCTCAAAGATTCTACTCTCATCTCAGATAAGTGCGATA GCTGGAAATATTTTATCTGTGAGAAGAAGGCATTTGGATCCTGCATCTGA

Mouse CLEC9a sequence. /Exon boundaries/ Cytoplasmic tail-transmembrane-neck-CTLD

MHAEEIYTSLQWDIPTSEASQKCQSPSKCS/GAWCVVTMISCVVCMGLLATSIFLGIK/FFQVSSLVLEQQERLIQQDTALVNLTQW QRKYTLEYCQALLQRSLHS/GTDASTGPVLLTSPQMVPQTLDSKET/GSDCSPCPHNWIQNGKSCYYVFERWEM/WNISKKSCLK EGASLFQIDSKEEMEFISSIGKLKGGNKYWVGVFQDGISGSWFWEDGSSPLSD/LLPAERQRSAGQICGYLKDSTLISDKCDSWKY FICEKKAFGSCI

Human CLEC9A gene LOCUS NC_000012 35290 bp DNA linear CON 30-AUG-2006 DEFINITION Homo sapiens , reference assembly, complete sequence. ACCESSION NC_000012 REGION: 10074543-10109832

Human CLEC9a ORF sequence Exon 4 ATGCACGAGGAAGAAATATACACCTCTCTTCAGTGGGATAGCCCAGCACCAGACACTTACCAGAAATGTCTGTCTTC CAACAAATGTTCAG Exon 5 GAGCATGCTGTCTTGTGATGGTGATTTCATGTGTTTTCTGCATGGGATTATTAACAGCATCCATTTTCTTGGGCGTCAAGT Exon 6 TGTTGCAGGTGTCCACCATTGCGATGCAGCAGCAAGAAAAACTCATCCAACAAGAGAGGGCACTGCTAAACTTTACAGAATGG AAGAGAAGCTGTGCCCTTCAGATGAAATATTGCCAAGCCTTCATGCAAAACTCATTAAGTTCAG Exon 7 CCCATAACAGCAGTCCTTGTCCAAACAATTGGATTCAGAACAGAGAAAGTTGTTACTATGTCTCTGAAATTTGGAGCATTTGGC ACACCAGTCAAGAGAATTGTTTAAAGGAAGGTTCCACGCTGCTACAAATAGAGAGCAAAGAAGAAATG Exon 8 GATTTTATCACTGGCAGCTTGAGGAAGATTAAAGGAAGCTATGATTACTGGGTGGGGTTGTCTCAGGATGGACACAGCGGACG CTGGCTTTGGCAAGATGGCTCCTCTCCTTCTCCTGGCCT Exon 9 GTTGCCAGCAGAGAGATCCCAGTCAGCTAACCAAGTCTGTGGATACGTGAAAAGCAATTCCCTTCTTTCGTCTAACTGCAGCA CGTGGAAGTATTTTATCTGTGAGAAGTATGCGTTGAGATCCTCTGTCTGA

Mouse CLEC9a protein sequence. /Exon boundaries/ Cytoplasmic tail-transmembrane-neck-CTLD

MHEEEIYTSLQWDSPAPDTYQKCLSSNKCS/GACCLVMVISCVFCMGLLTASIFLGVK/LLQVSTIAMQQQEKLIQQERALLNFTEW KRSCALQMKYCQAFMQNSLSS/AHNSSPCPNNWIQNRESCYYVSEIWSI/WHTSQENCLKEGSTLLQIESKEEMDFITGSLRKIKGS YDYWVGLSQDGHSGRWLWQDGSSPSPG/LLPAERSQSANQVCGYVKSNSLLSSNCSTWKYFICEKYALRSSV

Fig. S2. Sequence alignment

Mus m. 1 MHAEEIYTSLQWDIPTSEASQKCQSPSKCSGAWCVVTMISCVVCMGLLAT 50 Homo s. 1 MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTA 50 Rattus n. 1 MHEEEIYTSLQWDIPTSEASQKCPSLSKCPGTWCIVTVISCVVCVGLLAA 50 Macaca m. 1 MHEEEIYTSLQWDSPAPNTYQKCLSSNKCSGAWCLVMAISCIFCMGLLTA 50 Pan t. 1 MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTA 50 Canis f. 1 MQEEETYTSLRWDSPTPSFYQKHLSSTKYSGAWCLVTVITCILCVGSIAT 50 *. ** ****.** *. ** * * *. *.* *.*. *.* ...

Mus m. 51 SIFLGIKFFQVSSLVLEQQERLIQQDTALVNLTQWQRKYTLEY--CQALL 98 Homo s. 51 SIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFM 100 Rattus n. 51 SIFLGIKFSQVSSLVMEQRERLIRQDTALLNLTEWQRNHTLQLKSCQASL 100 Macaca m. 51 SIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSHVLQMKFCQTFM 100 Pan t. 51 SIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFM 100 Canis f. 51 SVFLGLKLFQVSTIAMKQREKLILQDRALLNFTQWERNHNLQMKYCQTLM 100 *.***.* ***.. . *.*.** *. **.* *.* * *. **. .

Mus m. 99 QRSLHSGTDASTGPVLLTSPQMVPQTLDSKETGSDCSPCPHNWIQNGKSC 148 Homo s. 101 QNSLSS------AHNSSPCPNNWIQNRESC 124 Rattus n. 101 QRSLRS------GSNCNPCPPNWIQNGKSC 124 Macaca m. 101 QSSFSS------AHNCSPCPNNWIQNRESC 124 Pan t. 101 QNSLSS------AHNSSPCPNSWIQNRESC 124 Canis f. 101 QNSFSS------AHNCSPCPDNWIQNGESC 124 * * * . *** **** **

Mus m. 149 YYVFERWEMWNISKKSCLKEGASLFQIDSKEEMEFISS-IGKLKGGNKYW 197 Homo s. 125 YYVSEIWSIWHTSQENCLKEGSTLLQIESKEEMDFITGSLRKIKGSYDYW 174 Rattus n. 125 YYAFDRWETWNNSKKSCLKEGDSLLQIDSKEEMEFINLSIWKLKGGYEYW 174 Macaca m. 125 YYVSEHWKIWHTSQENCLKEGSTLLQIESEEEMDFITGSLRKIRGSYDYW 174 Pan t. 125 YYVSEIWSIWHTSQENCLKEGSTLLQIESKEEMDFITGSLRKIKGSYDYW 174 Canis f. 125 YHVFENWKIWHTSKEDCLKEGSNLLQIDSKEEMDFITGSLKKVKSGFDYW 174 * . * *. *. ***** * **.* ***.** . *.. **

Mus m. 198 VGVFQDGISGSWFWEDGSSPLSDLLPAERQRSAGQICGYLKDSTLISDKC 247 Homo s. 175 VGLSQDGHSGRWLWQDGSSPSPGLLPAERSQSANQVCGYVKSNSLLSSNC 224 Rattus n. 175 VGVFQDGPSGSWFWEDGSSPLSDLLPTDRQLSASQICGYLKDHTLISDNC 224 Macaca m. 175 VGLSQDGHSGRWLWQDGSSPSPGLLPVEISQSTNQVCGYIKNSSLLSSNC 224 Pan t. 175 VGLSQDGHSGRWLWQDGSSPSPGLLPVERSQSANQVCGYMKSNSLLSSNC 224 Canis f. 175 VGLSQDGLSKPWLWQDGSSPSPDLSPVQTLQSTNQLCGYLKDKFLSSANC 224 **. *** * * *.***** * * *. *.***.* * * *

Mus m. 248 DSWKYFICEKKAFGSCI 264 Homo s. 225 STWKYFICEKYALRSSV 241 Rattus n. 225 SNWKYFICEKKAFGSCI 241 Macaca m. 225 STWKYFICEKYALRSSV 241 Pan t. 225 STWKYFICEKYALRSSV 241 Canis f. 225 SIWKYFICEKYALRSSN 241 ******** * *. Fig. S3

+ DC + DC α

CD4 CD8 pDC DN DC 1000 L 900 S 800 mDNGR-1 700 600 VS 500

β-Actin Fig. S4A. Phylogenetic tree of Clec9a CTLD

0.05 Clec9a Clec7a Clec12a Clec12b Clec1a Clec1b Klrc2 Klrc1 Klrc3 Klrk1 Klrd1 Klre1 Gm156 Klri2 Klri1 Olr1 Klrg1 Klrg2 Clec2l Clec5a Klrb1f Klrb6 Klrb1d Klrb1a Klrb1c Klrb1b Cd69 4922502D21Rik LOC677440 Clec2f Clec2i Clec2d Clec2g Clec2e Clec2h Fig S4B. Percentage identity and similarity of Clec9a CTLD.

Protein % id % sim Clec9a 100 100 Klrk1 34.7 40.3 Clec7a 33.6 38.3 Clec12b 30.7 37.8 Klre1 29 33.1 Gm156 28.8 32 Klrc3 27.4 33.9 Klrc2 27.4 33.1 Klrc1 26.6 33.9 Klri1 25.6 29.6 Clec1b 25.6 31.2 Clec2e 25.6 31.2 Klrb1d 25.6 33.6 Klrb1f 25.6 32.8 Klri2 24.8 28 Clec2h 24.8 31.2 Clec2l 24.8 34.4 Klrb1b 24 32 Klrb1c 24 32 Klrb1a 24 31.2 Klrb6 24 30.4 Klrg2 24 32.8 Klrg1 24 28 Clec1a 23.6 31.5 Klrd1 23.4 32.3 Olr1 23.4 31.3 LOC677440 22.4 31.2 Clec2g 21.6 28.8 Clec2d 21.6 29.6 Clec2f 21.6 29.6 Cd69 21.6 32 4922502D21Rik 21.6 28.8 Clec12a 20.8 28 Clec5a 20.6 26.2 Clec2i 20 27.2

Fig. S5

MFI 22.1 15.3 18.2 1236 17.9 305 18.9 1324 4 4 10 104 104 10

3 3 10 103 103 10

2 2 10 102 102 10 397 1F6 7H11

rat IgG 1 1 10 101 101 10

0 0 10 100 100 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10 EGFP Fig. S6.

CD11c- CD4+ cDC CD8α+ cDC DN cDC pDC

100 100 100 100 100

80 80 80 80 80

60 60 60 60 60

40 40 40 40 40

% of Max 20 20 20 20 20

0 0 0 0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 rat IgG1 / DNGR-1 skin-DC CD8α- cDC CD8α+ cDC 104 cDC 100 100 100 0.4 103 80 80 80

60 60 60 102 0.4 40 40 40 CD11c skin 101 DC % of Max 20 20 20

0 10 0 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 10 10 10 10 10 100 101 102 103 104 CD40 rat IgG1 / DNGR-1 Fig S7

CD11c 10 10 10 10 10 0 1 2 3 4 10 5.51 0 93.9 IgG1-A488 10 1 10 2 10 3 0.38 0.23 10 4 10 10 10 10 10 0 1 2 3 4 10 5.11 0 92.7 α DNGR-1-A488 10 1 10 2 10 3 0.85 1.35 10 4 10 10 10 10 10 0 1 2 3 4 10 3.02 0 56.3 α DEC205-A488 10 1 10 2 10

3

36.4 4.27 10 4 A

peptide S1 + αCD40 IgG1 S1 + αCD40 p < 0.001 DNGR-1 S1 + αCD40 100 80 60 40 20

% Specific lysis 0 20 nM 200nM Peptide (nM) B S1 peptide IgG1 S1 αDNGR-1 S1 p < 0.05 + αCD40 + αCD40 + αCD40 3 4 4 + 10 10 104 0 0.1 0 0.091 0 2.4

3 10 103 3 CD8 10 + 2

2 2 10 10 102 1 1 1 10 10 101 H2Kb SIINFEKL 0 0 99.9 0 0 99.9 0 97.6 % Tetramer 10 10 100 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 100 101 102 103 104 0 S1 peptide IgG1 S1 αDNGR-1 S1 CD8 + αCD40 + αCD40 + αCD40

Fig. S8 p = 0.0003

200

150

100

50

Tumors per mouse Tumors 0 IgG1-Endo αDNGR-1-Endo + Adj + Adj Fig. S9 Supplementary figure legends

Figure S1. Mouse Clec9a and human CLEC9A sequences. Gene localization is indicated using Genbank nomenclature. The open reading frame (ORF) and predicted protein sequence is depicted. The exon boundaries are mapped onto the protein sequence and the protein domains are highlighted: cytoplasmic tail (red), transmembrane region

(green), stalk region (black) and CTLD (blue).

Figure S2. Sequence alignment of CLEC9 in different species. CLEC9A sequences from Mus musculus, Homo sapiens, Rattus norvegicus, Macacca mulata, Pan troglodytes and Canis familiaris were aligned using Clustal W. Conserved features are highlighted: cytoplasmic tyrosine (red); transmembrane domain (grey); Asn81, putative site for N-glycosylation (green); putative Cys involved in stabilization of dimer (blue);

Ser 104-106, putative site for glycosaminoglycan binding (green); conserved CTLD residues, including six Cys involved in intramolecular disulphide bonds (yellow). Identity is represented by an asterisk (*) and similarity with a dot (.).

Figure S3. Distribution of mouse DNGR-1 transcripts. mRNA from subsets of spleen

DC were subjected to RT-PCR using mouse DNGR-1 specific primers (upper lanes) or β- actin primers (lower lanes). Arrows indicate the long (L), short (S) and very short (VS) isoforms detected for mouse DNGR-1.

Figure S4. Phylogenetic analysis of mouse CLEC9A. (A) Phylogenetic tree of mouse

CLEC9A. NK receptor-like lectins were identified by searching the mouse proteome with the domain alignment sourced from NCBI's Conserved Domain Database (CDD).The identified proteins were aligned to Clec9a and the tree was generated in Clustal W using the CDD domain as a guide. The distance data represents the minimum number of substitutions required to convert one sequence into another. (B) Percentage identity and similarity was calculated from the alignment using MacBoxshade.

Figure S5. Generation of rat anti-mouse DNGR-1 mAbs. mAb were generated as indicated in the Materials and Methods and were used for staining a mixture of DNGR-1- expressing B3Z cells (GFP positive) and parental cells (GFP negative). MFI of the GFP positive (DNGR-1+) and GFP negative (parental) is indicated for the rat IgG irrelevant control or the anti-DNGR-1 1F6, 397 and 7H11 mAbs.

Figure S6. Expression of DNGR-1 in peripheral lymph nodes cells. Analysis of

DNGR-1 expression (blue) compared to the isotype control (red) in the indicated subsets of peripheral lymph nodes cells. Upper and lower panels represent different experiments comparing DNGR-1 expression in blood-derived DC subsets and skin-derived DC subsets (defined as shown on the dot plot), respectively.

Figure S7. Comparison of in vivo labeling with anti-DNGR-1 and anti-DEC-205 mAbs. (A) Mice were injected i.v. with 100 µg of Alexa-488 conjugated anti-DNGR-1

(7H11), anti-DEC-205 (NLDC-145) or isotype-matched control (rat IgG1) and lymph nodes were analyzed one day later. Dot plots show CD11c versus Alexa488 in anti-DEC- 205 (right panel), anti-DNGR-1 (middle panel) or rat IgG1 (left panel) injected mice.

Numbers represent % events in the indicated quadrant.

Figure S8. Comparison of anti-OVA CTL priming by free OVA peptide versus peptide targeted via anti-DNGR-1. 2µg S1 conjugated anti-DNGR-1 or rat IgG1 isotype-matched control mAb or 2µg of free S1 peptide (100 fold excess compared to the amount present in 2µg of the antibody conjugates) were injected s.c. with anti-CD40 (25

µg). Target cells were injected five days later and mice analyzed on day 6 as in Figure

6A. (A) In vivo CTL activity as measured by target cell elimination. Graph shows mean

± SEM of % specific lysis in one experiment of two (n=3 mice/group). All groups are shown but the only one in which killing was detectable was that receiving anti-DNGR-1-

S1 + anti-CD40. (B) H-2Kb-SIINFEKL tetramer staining of splenocytes. Left panel: representative dot plots of tetramer staining vs. CD8 in gated CD8+ CD3+ T cells. Right panel: frequency of tetramer+ CD8+ T cells in one experiment of two (n=3 mice/group). p values were calculated using Student’s t test.

Figure S9. Immunoprophylaxis of B16 melanoma via targeting of tumor antigens to

DNGR-1. Experiments were carried out as in Figure 8 except that the vaccine was given one day prior to infusion of B16 cells. Data show the number of lung tumors per mouse.

Data are pooled from two independent experiments (n=7 mice/group) and each point represents one mouse. p values were calculated using the Mann Whitney U test.