Supplementary material and methods

Tumor experiments B16-OVA were obtained from M. Swartz laboratory in 2012, and used at below passage 20. Cells were not authenticated, and were tested negative for mycoplasma. Dby-expressing MCA-101 (MCA-101-Dby), methylcholanthrene-induced fibrosarcoma expressing the MHCII-restricted male Dby (NAGFNSNRANSSRSS) (1), were obtained from O. Lantz laboratory in 2010, and used at below passage 20. Cells were not authenticated, and were tested negative for mycoplasma. Cells were cultured and implanted as described in the main material and methods. Dby HY peptide was purchased from Polypeptide. Mice were injected i.t. once tumors were established, at D10 post-tumor implantation, with CpG-B (30 µg), in the presence or not of HY peptide (10 µg), in PBS (40 µl).

CpG-B toxicity in vitro assay EG7-OVA cells were seeded at an initial density of 5 x 104 cells/ml in complete RPMI medium and cultured for three days, at 37°C and 5% CO2, in the presence of CpG-B at the indicated concentrations. Cells were counted every 24 hours.

Effect of i.t. CpG-B treatment on the growth of a distal tumor Tumor cells were implanted (as described in the main material and methods) in both flanks. Mice were injected i.t. in the left tumors (D12 post-tumor implantation) with CpG-B (30 µg), in the presence or not of

OVAII (10 µg) in PBS (40 µl). Tumor size was measured in the right flank (distal tumor), with a caliper [L (length) x l (width)] every one to two days over the indicated periods of time.

Reactive oxygen species (ROS) detection Flow cytometry cell sorted tumor-associated neutrophils (see main material and methods) were seeded in complete RPMI, treated with indicated CpG-B concentrations, and cultured for 24h at 37°C and 5% CO2. ROS production was assessed using the CellROX deep red flow cytometry Assay (Thermofisher), following manufacturer’s instruction. Anti-oxydant (N-acetyl cystein, NAC), and oxydant (tert-burtyl hydroperoxide, TBHP) were used as negative and positive controls, respectively.

Reference 1. Joncker NT, Marloie MA, Chernysheva A, Lonchay C, Cuff S, Klijanienko J, et al. Antigen- independent accumulation of activated effector/memory T lymphocytes into human and murine tumors. International journal of cancer. 2006;118(5):1205-14. Supplementary Figure 1. Effect of CpG-B on EG7 tumor cells in vitro. EG7 tumor cells were cultured for three days in the presence of CpG-B at 0.5 µM or 2 µM. The fold change corresponds to the ratio of cell counts the day of interest related to the day of cell seeding (D0). NT: non-treated. Supplementary Figure 2. Effect of intratumoral administration of CpG-B

±OVAII on B16-OVA tumor growth. B16-OVA cells were implanted s.c. in WT mice. Eight days later, mice received an i.t. injection of PBS, CpG-B alone or

CpG-B along with the OVAII peptide (CpG-B+OVAII). Tumor growth was measured every 1 to 2 days. Due to their tumor sizes, mice from the controls (PBS) were sacrificed earlier than the other groups, in accordance with the animal welfare guidelines. Results show the mean ± SEM derived from 6-8 mice and are representative of two independent experiments. Two-way ANOVA test with Bonferroni post Hoc test was performed. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns: non-significant. Supplementary Figure 3. Tumor evolution and effector T cell responses in LN five days after intratumoral injection of

CpG-B±OVAII and impact on distal tumor growth. EG7 cells were implanted s.c. in WT mice. At D8-D12 post-tumor cell implantation, mice received an i.t. injection of PBS, CpG-B alone or CpG-B along with the OVAII peptide, CpG-B+OVAII. A, The fold change corresponds to the ratio of the tumor size on the day of i.t. injection to that of five days later. B, Tumor weights were measured five days after i.t. injection. Results show the mean ± SEM derived from 12 mice and are representative of three independent experiments. C, D, Frequencies and absolute numbers of CD4+ T cells, IFN-γ+ and IL-17+ + + + + cells among CD4 T cells (C), IFN-γ and OVAI-specific (Pentamer ) cells among CD8 T cells (D) were measured in cells isolated from the TdLNs by flow cytometry five days after i.t. injection. Results show the mean ± the SEM derived from 4 (absolute numbers) or 8 (frequencies) and are representative of at least two independent experiments. One-way ANOVA tests with Bonferroni post Hoc tests were performed (A-D). E, EG7 cells were implanted s.c. in both flanks in WT mice. Twelve days later, mice were injected i.t. with PBS, CpG-B alone or CpG-B along with the OVAII peptide, CpG-B+OVAII, exclusively in the left flank. Tumor growth was measured every 1 to 2 days in the right flank. Results show the mean ± SEM derived from 8 mice and are representative of two independent experiments. Two-way ANOVA test with Bonferroni post Hoc test was performed. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns: non-significant. Supplementary Figure 4. Intratumoral CpG-B±Dby HY peptide controls MCA-101-Dby tumor growth and promote anti-tumor T cell immunity. MCA-101-Dby cells were implanted s.c. in WT mice. Ten days later, mice received an i.t. injection of PBS, CpG-B alone or CpG-B along with the Dby (HY) peptide (CpG-B+HY). A, tumor size was measured on the day of i.t. injection and five days later (left panel) and tumor weight was measured five days later (right panel). B and C, frequencies (left panel) and absolute numbers (middle panel) of Tregs (B), IFN-γ+ (C, upper panel) and IL-17+ (C, lower panel) among CD4+ T cells were measured in cells isolated from the tumor by flow cytometry five days after i.t. injection, right panel represent ratios [indicated cell number / mg tumor]. D, CD8+ T cells and IFN-γ+ among CD8+ T cells absolute numbers (left panel) were measured in cells isolated from the tumor by flow cytometry five days after i.t. injection. Ratios [indicated cell number / mg tumor] are shown (right panel). E, Ratios [indicated cell number / Treg number] are indicated. Results show the mean ± SEM derived from 4-7 mice and are representative of two independent experiments. One-way ANOVA test with Bonferroni post Hoc test were performed. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns: non- significant. Supplementary Figure 5. Plasmacytoid dendritic cell depletion. EG7 cells were implanted s.c. in BDCA2-DTR mice. One day prior i.t. injection, indicated mice were injected or not i.p. with diphtheria toxin (DT). Flow cytometry dot plots showing the frequency of pDCs (CD45hi CD11cint B220+ Siglec-H+ + PDCA1 ) in tumors on the day of CpG-B±OVAII delivery. Supplementary Figure 6. Effect of intratumoral administration of CpG-B -/- +OVAII on tumor growth in IFNAR mice. EG7 cells were implanted s.c. in IFNAR-/-. At D9 post-tumor cell implantation, mice received an i.t. injection (i.t.) of PBS or CpG-B along with the OVAII peptide (CpG-B+OVAII). Tumor growth was measured every 1 to 2 days. Results show the mean ± SEM derived from 6-7 mice and are representative of at least two independent experiments. Two-way ANOVA test with Bonferroni post Hoc test was performed. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns: non-significant. Supplementary Figure 7. Tumor-associated neutrophil phenotype and function after intratumoral injection of CpG-B±OVAII. EG7 cells were implanted s.c. in WT mice. A-B, at D11 post-tumor cell implantation, mice were injected i.t. with indicated reagents. The expression of CD62L, CD69, Fas-L and ICAM-1 by neutrophils (CD11b+ Ly6Ghi Ly6Cint) was measured in cells isolated from the tumor, TdLNs or non-draining LNs (ndLNs) by flow cytometry, 48h after i.t. injection. Flow cytometry histograms of neutrophil expression patterns in tumors, TdLNs and ndLNs after i.t. injection of PBS (A), and in tumors after i.t. injection of PBS, CpG-B or CpG-B along with the OVAII peptide, CpG-B+OVAII (B). C, Tumor- associated neutrophils sorted 9 days after tumor cell implantation were incubated for 24h with indicated CpG- B concentrations. ROS production was assessed by flow cytometry (CellROX deep red). Anti-oxydant (N- acetyl cystein, NAC), and oxydant (tert-butyl hydroperoxide, TBHP) were used as negative and positive controls, respectively. NT: Non-treated. Supplementary Figure 8. Neutrophil depletion. EG7 cells were implanted s.c. in WT mice. At D11 post- tumor cell implantation, mice were injected i.v. and i.t. with a depleting anti-Ly6G antibody or its isotype control . One day later, mice received an i.t. injection of PBS or CpG-B. A-B, Flow cytometry dot plots showing the frequency of neutrophils in blood (A) and in tumors (B) one day after i.t. injection (D2 after depleting anti- Ly6G antibody injection). Neutrophil depletion is evaluated using a gating strategy without the anti-Ly6G antibody (clone 1A8) for FACS as it is the same clone as the one injected in vivo, the epitope being hidden. Neutrophils are here defined as CD45+ CD11b+ Ly6Cint SSChi cells. Neutrophil depletion reaches 70-80% in the blood (A) and 60-70% in the tumor (B). C, Tumor weights were measured five days after i.t. injection. One-way ANOVA tests with Bonferroni post Hoc tests were performed. Results show the mean ± SEM derived from 5 mice, and are representative of at least two independent experiments. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns: non-significant. Supplementary Table 1: list of differenally expressed following intratumoral CpG-B administraon (classified into 3 categories)

Ag presentaon-related genes

Gene_id Gene_name Gene_descripon 1 ENSMUSG00000026193 Fn1 fibronecn 1 2 ENSMUSG00000017760 Ctsa cathepsin A 3 ENSMUSG00000016256 Ctsz cathepsin Z 4 ENSMUSG00000016534 Lamp2 lysosomal-associated membrane 2 5 ENSMUSG00000036353 P2ry12 purinergic receptor P2Y, G-protein coupled 12 6 ENSMUSG00000005774 Rfx5 regulatory factor X, 5 7 ENSMUSG00000028111 Ctsk cathepsin K 8 ENSMUSG00000038642 Ctss cathepsin S 9 ENSMUSG00000068747 Sort1 sorlin 1 10 ENSMUSG00000029512 Ulk1 unc-51 like kinase 1 11 ENSMUSG00000030720 Cln3 ceroid lipofuscinosis, neuronal 3, juvenile 12 ENSMUSG00000007891 Ctsd cathepsin D 13 ENSMUSG00000025351 Cd63 CD63 angen 14 ENSMUSG00000031447 Lamp1 lysosomal-associated membrane protein 1 15 ENSMUSG00000021939 Ctsb cathepsin B 16 ENSMUSG00000025232 Hexa hexosaminidase A 17 ENSMUSG00000032359 Ctsh cathepsin H 18 ENSMUSG00000018774 Cd68 CD68 angen 19 ENSMUSG00000018012 Rac3 RAS-related C3 botulinum substrate 3 20 ENSMUSG00000021477 Ctsl cathepsin L 21 ENSMUSG00000021270 Hsp90aa1 heat shock protein 90, alpha, class A member 1 22 ENSMUSG00000033220 Rac2 RAS-related C3 botulinum substrate 2 23 ENSMUSG00000022504 Ciita class II transacvator 24 ENSMUSG00000022817 Itgb5 integrin beta 5 25 ENSMUSG00000024308 Tapbp TAP binding protein 26 ENSMUSG00000037649 H2-DMa histocompability 2, class II, locus Dma 27 ENSMUSG00000037548 H2-DMb2 histocompability 2, class II, locus Mb2 28 ENSMUSG00000079547 H2-DMb1 histocompability 2, class II, locus Mb1 29 ENSMUSG00000037321 Tap1 transporter 1, ATP-binding cassee, sub-family B (MDR/TAP) 30 ENSMUSG00000024339 Tap2 transporter 2, ATP-binding cassee, sub-family B (MDR/TAP) 31 ENSMUSG00000073421 H2-Ab1 histocompability 2, class II angen A, beta 1 32 ENSMUSG00000036594 H2-Aa histocompability 2, class II angen A, alpha 33 ENSMUSG00000007038 Neu1 neuraminidase 1 Immunogenicity-related genes (page 1/2)

Gene_id Gene_name Gene_descripon 1 ENSMUSG00000025779 Ly96 lymphocyte angen 96 2 ENSMUSG00000026073 Il1r2 interleukin 1 receptor, type II 3 ENSMUSG00000026177 Slc11a1 solute carrier family 11, member 1 4 ENSMUSG00000026321 Tnfrsf11a TNF receptor superfamily, member 11a, NFKB acvator 5 ENSMUSG00000042349 Ikbke inhibitor of kappaB kinase epsilon 6 ENSMUSG00000026478 Lamc1 laminin, gamma 1 7 ENSMUSG00000079164 Tlr5 toll-like receptor 5 8 ENSMUSG00000026773 P3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 9 ENSMUSG00000027399 Il1a interleukin 1 alpha 10 ENSMUSG00000017652 Cd40 CD40 angen 11 ENSMUSG00000027544 Nfatc2 nuclear factor of acvated T cells, cytoplasmic, calcineurin dependent 2 12 ENSMUSG00000040522 Tlr8 toll-like receptor 8 13 ENSMUSG00000044583 Tlr7 toll-like receptor 7 14 ENSMUSG00000036381 P2ry14 purinergic receptor P2Y, G-protein coupled, 14 15 ENSMUSG00000036362 P2ry13 purinergic receptor P2Y, G-protein coupled 13 16 ENSMUSG00000027776 Il12a interleukin 12a 17 ENSMUSG00000027995 Tlr2 toll-like receptor 2 18 ENSMUSG00000027947 Il6ra interleukin 6 receptor, alpha 19 ENSMUSG00000056054 S100a8 S100 calcium binding protein A8 (calgranulin A) 20 ENSMUSG00000056071 S100a9 S100 calcium binding protein A9 (calgranulin B) 21 ENSMUSG00000027962 Vcam1 vascular cell adhesion molecule 1 22 ENSMUSG00000028163 Nb1 nuclear factor of kappa polypepde enhancer in B cells 1 23 ENSMUSG00000073889 Il11ra1 interleukin 11 receptor, alpha chain 1 24 ENSMUSG00000028364 Tnc tenascin C 25 ENSMUSG00000039005 Tlr4 toll-like receptor 4 26 ENSMUSG00000048806 Ifnb1 interferon beta 1, fibroblast 27 ENSMUSG00000028859 Csf3r colony smulang factor 3 receptor (granulocyte) 28 ENSMUSG00000028599 Tnfrsf1b tumor necrosis factor receptor superfamily, member 1b 29 ENSMUSG00000039936 Pik3cd phosphadylinositol 3-kinase catalyc delta polypepde 30 ENSMUSG00000028965 Tnfrsf9 tumor necrosis factor receptor superfamily, member 9 31 ENSMUSG00000002944 Cd36 CD36 angen 32 ENSMUSG00000025746 Il6 interleukin 6 33 ENSMUSG00000051498 Tlr6 toll-like receptor 6 34 ENSMUSG00000029373 Pf4 platelet factor 4 35 ENSMUSG00000029426 Scarb2 scavenger receptor class B, member 2 36 ENSMUSG00000042121 Ssh1 slingshot homolog 1 (Drosophila) 37 ENSMUSG00000004849 Ap1s1 adaptor protein complex AP-1, sigma 1 38 ENSMUSG00000032766 Gng11 guanine nucleode binding protein (G protein), gamma 11 39 ENSMUSG00000049093 Il23r interleukin 23 receptor 40 ENSMUSG00000000628 Hk2 hexokinase 2

Immunogenicity-related genes (page 2/2)

Gene_id Gene_name Gene_descripon 41 ENSMUSG00000067599 Klra7 killer cell lecn-like receptor, subfamily A, member 7 42 ENSMUSG00000058709 Egln2 egl-9 family hypoxia-inducible factor 2 43 ENSMUSG00000030786 Itgam 44 ENSMUSG00000025498 Irf7 interferon regulatory factor 7 45 ENSMUSG00000019970 Sgk1 serum/glucocorcoid regulated kinase 1 46 ENSMUSG00000044199 S1pr4 sphingosine-1-phosphate receptor 4 47 ENSMUSG00000020184 Mdm2 transformed mouse 3T3 cell double minute 2 48 ENSMUSG00000025383 Il23a interleukin 23, alpha subunit p19 49 ENSMUSG00000004567 Mcoln1 mucolipin 1 50 ENSMUSG00000000791 Il12rb1 interleukin 12 receptor, beta 51 ENSMUSG00000031903 Pla2g15 phospholipase A2, group XV 52 ENSMUSG00000000303 Cdh1 cadherin 1 53 ENSMUSG00000041515 Irf8 interferon regulatory factor 8 54 ENSMUSG00000021948 Prkcd protein kinase C, delta 55 ENSMUSG00000037405 Icam1 intercellular adhesion molecule 1 56 ENSMUSG00000032175 Tyk2 tyrosine kinase 2 57 ENSMUSG00000039115 Itga9 integrin alpha 9 58 ENSMUSG00000004296 Il12b interleukin 12b 59 ENSMUSG00000089669 Tnfsf13 tumor necrosis factor (ligand) superfamily, member 13 60 ENSMUSG00000020826 Nos2 nitric oxide synthase 2, inducible 61 ENSMUSG00000035042 Ccl5 chemokine (C-C mof) ligand 5 62 ENSMUSG00000000982 Ccl3 chemokine (C-C mof) ligand 3 63 ENSMUSG00000038067 Csf3 colony smulang factor 3 (granulocyte) 64 ENSMUSG00000021367 Edn1 endothelin 1 65 ENSMUSG00000067586 S1pr3 sphingosine-1-phosphate receptor 3 66 ENSMUSG00000021457 Syk spleen tyrosine 67 ENSMUSG00000047415 Gpr68 G protein-coupled receptor 68 68 ENSMUSG00000021190 Lgmn legumain 69 ENSMUSG00000001729 Akt1 thymoma viral proto-oncogene 1 70 ENSMUSG00000003882 Il7r interleukin 7 receptor 71 ENSMUSG00000053137 Mapk11 mitogen-acvate d protein kinase 11 72 ENSMUSG00000022901 Cd86 CD86 angen 73 ENSMUSG00000023830 Igf2r insulin-like growth factor 2 receptor 74 ENSMUSG00000090877 Hspa1b heat shock protein 1B 75 ENSMUSG00000091971 Hspa1a heat shock protein 1A 76 ENSMUSG00000024401 Tnf tumor necrosis factor 77 ENSMUSG00000023947 Nbie nuclear factor of kappa light polypepde gene enhancer in B cells inhibitor, epsilon 78 ENSMUSG00000005824 Tnfsf14 tumor necrosis factor (ligand) superfamily, member 14 79 ENSMUSG00000051439 Cd14 CD14 angen 80 ENSMUSG00000024621 Csf1r colony smulang factor 1 receptor 81 ENSMUSG00000024798 Htr7 5-hydroxytryptamine (serotonin) receptor 7 DC migraon-related genes

Gene_id Gene_name Gene_descripon 1 ENSMUSG00000026581 Sell selecn, lymphocyte 2 ENSMUSG00000027002 Nckap1 NCK-associated protein 1 3 ENSMUSG00000027087 Itgav 4 ENSMUSG00000002108 Nr1h3 nuclear receptor subfamily 1, group H, member 3 5 ENSMUSG00000017009 Sdc4 syndecan 4 6 ENSMUSG00000015340 Cybb cytochrome b-245, beta polypepde 7 ENSMUSG00000001131 Timp1 ssue inhibitor of metalloproteinase 1 8 ENSMUSG00000031391 L1cam cell adhesion molecule 9 ENSMUSG00000033721 Vav3 vav 3 oncogene 10 ENSMUSG00000001847 Rac1 RAS-related C3 botulinum substrate 1 11 ENSMUSG00000030672 Mylpf myosin light chain, phosphorylatable, fast skeletal muscle 12 ENSMUSG00000025355 Mmp19 matrix metallopepdase 19 13 ENSMUSG00000031740 Mmp2 matrix metallopepdase 2 14 ENSMUSG00000000957 Mmp14 matrix metallopepdase 14 (membrane-inserted) 15 ENSMUSG00000050578 Mmp13 matrix metallopepdase 13 16 ENSMUSG00000049723 Mmp12 matrix metallopepdase 12 17 ENSMUSG00000010051 Hyal1 hyaluronoglucosaminidase 1 18 ENSMUSG00000020469 Myl7 myosin, light polypepde 7 19 ENSMUSG00000017466 Timp2 ssue inhibitor of metalloproteinase 2 20 ENSMUSG00000020592 Sdc1 syndecan 1 21 ENSMUSG00000020580 Rock2 Rho-associated coiled-coil containing protein kinase 2 22 ENSMUSG00000002289 Angptl4 angiopoien-like 4