Mouse Clec9a ORF sequence

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Mouse Clec9a gene LOCUS NC_72 13843 bp DNA linear CON 1-JUL-27 DEFINITION Mus musculus chromosome 6, reference assembly (C57BL/6J). ACCESSION NC_72 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 protein sequence. /Exon boundaries/ Cytoplasmic tail-transmembrane-neck-ctld

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

Human CLEC9A gene LOCUS NC_12 3529 bp DNA linear CON 3-AUG-26 DEFINITION Homo sapiens chromosome 12, reference assembly, complete sequence. ACCESSION NC_12 REGION: 174543-119832 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 5 Homo s. 1 MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTA 5 Rattus n. 1 MHEEEIYTSLQWDIPTSEASQKCPSLSKCPGTWCIVTVISCVVCVGLLAA 5 Macaca m. 1 MHEEEIYTSLQWDSPAPNTYQKCLSSNKCSGAWCLVMAISCIFCMGLLTA 5 Pan t. 1 MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTA 5 Canis f. 1 MQEEETYTSLRWDSPTPSFYQKHLSSTKYSGAWCLVTVITCILCVGSIAT 5 *. ** ****.** *. ** * * *. *.* *.*. *.*... Mus m. 51 SIFLGIKFFQVSSLVLEQQERLIQQDTALVNLTQWQRKYTLEY--CQALL 98 Homo s. 51 SIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFM 1 Rattus n. 51 SIFLGIKFSQVSSLVMEQRERLIRQDTALLNLTEWQRNHTLQLKSCQASL 1 Macaca m. 51 SIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSHVLQMKFCQTFM 1 Pan t. 51 SIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFM 1 Canis f. 51 SVFLGLKLFQVSTIAMKQREKLILQDRALLNFTQWERNHNLQMKYCQTLM 1 *.***.* ***... *.*.** *. **.* *.* * *. **.. Mus m. 99 QRSLHSGTDASTGPVLLTSPQMVPQTLDSKETGSDCSPCPHNWIQNGKSC 148 Homo s. 11 QNSLSS--------------------------AHNSSPCPNNWIQNRESC 124 Rattus n. 11 QRSLRS--------------------------GSNCNPCPPNWIQNGKSC 124 Macaca m. 11 QSSFSS--------------------------AHNCSPCPNNWIQNRESC 124 Pan t. 11 QNSLSS--------------------------AHNSSPCPNSWIQNRESC 124 Canis f. 11 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 mdngr-1 L S VS CD4 + DC CD8α + DC pdc DN DC 1 8 7 6 5 9 β-actin

Fig. S4A. Phylogenetic tree of Clec9a CTLD.5 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 492252D21Rik LOC67744 Clec2f Clec2i Clec2d Clec2g Clec2e Clec2h

Fig S4B. Percentage identity and similarity of Clec9a CTLD. Protein % id % sim Clec9a 1 1 Klrk1 34.7 4.3 Clec7a 33.6 38.3 Clec12b 3.7 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 3.4 Klrg2 24 32.8 Klrg1 24 28 Clec1a 23.6 31.5 Klrd1 23.4 32.3 Olr1 23.4 31.3 LOC67744 22.4 31.2 Clec2g 21.6 28.8 Clec2d 21.6 29.6 Clec2f 21.6 29.6 Cd69 21.6 32 492252D21Rik 21.6 28.8 Clec12a 2.8 28 Clec5a 2.6 26.2 Clec2i 2 27.2

Fig. S5 MFI 1 4 22.1 15.3 18.2 1236 1 4 1 4 17.9 35 18.9 1324 1 4 1 3 1 3 1 3 1 3 rat IgG 1 2 1 1 1F6 1 2 1 1 397 1 2 1 1 7H11 1 2 1 1 1 1 1 1 EGFP

Fig. S6. CD11c - CD4 + cdc CD8α + cdc DN cdc pdc 1 1 1 1 1 % of Max 8 6 4 2 8 6 4 2 8 6 4 2 8 6 4 2 8 6 4 2 CD11c 1 4 1 3 1 2 1 1 rat IgG1 / DNGR-1 cdc.4.4 skin DC % of Max 1 8 6 4 2 skin-dc 1 8 6 4 2 CD8α - cdc 1 8 6 4 2 CD8α + cdc 1 CD4 rat IgG1 / DNGR-1

Fig S7 1 4 5.51.23 1 4 5.11 1.35 1 4 3.2 4.27 1 3 1 3 1 3 CD11c 1 2 1 1 1 2 1 1 1 2 1 1 1 93.9.38 IgG1-A488 1 92.7.85 αdngr-1-a488 1 56.3 36.4 αdec25-a488

A B H2Kb SIINFEKL % Specific lysis 1 8 1 3 1 2 1 1 6 4 2 CD8 p <.1 2 nm 2nM Peptide (nm) S1 peptide + αcd4 1 3 1 2 1 1 IgG1 S1 + αcd4 peptide S1 + αcd4 IgG1 S1 + αcd4 DNGR-1 S1 + αcd4 1 99.9 1 99.9 αdngr-1 S1 + αcd4 1 4 1 4.1 1.91 4 2.4 1 3 1 2 1 1 1 97.6 % Tetramer + CD8 + 3 2 1 S1 peptide + αcd4 p <.5 IgG1 S1 αdngr-1 S1 + αcd4 + αcd4 Fig. S8

p =.3 Tumors per mouse 2 15 1 5 IgG1-Endo + Adj αdngr-1-endo + 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 proteins 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 14-16, 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-25 mabs. (A) Mice were injected i.v. with 1 µg of Alexa-488 conjugated anti-dngr-1 (7H11), anti-dec-25 (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-

25 (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 (1 fold excess compared to the amount present in 2µg of the antibody conjugates) were injected s.c. with anti-cd4 (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-cd4. (B) H-2K b -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.

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