SUPPLEMENTAL MATERIAL AND METHOD

Ag-presenting CpG-activated pDCs prime Th17 cells that induce tumor regressionLeslie Guéry, Juan Dubrot, Carla Lippens, Dale Brighouse, Pauline Malinge, Magali Irla, Caroline Pot, Jean-Marc Waldburger, and Stéphanie Hugues Tumor supernatant extractionIn some experiments, tumors were dissociated using Miltenyi dissociator in culture medium with 1% BSA (1mL for 500mg of tumor). Chemokine production in tumor supernatant was assessed by luminex technology (R&D) on Bio-Plex 200 luminex device (Biorad).

Transwell migration assayMigration assays were performed using 24-well transwells with polycarbonate membranes (pore size 5uM). Tumor supernatants were placed in the lower chamber and 5.105 cells extracted from dLN were resuspended in 200μL of RPMI with 1% BSA and placed in the upper chamber. 2h later, migrated cells in the lower chamber were counted using flow cytometry (Accuri C6).

qPCR primers sequences Tnf forward, 5’-ACAGAAAGCATGATCCGCG-3’ and reverse, 5’-GCCCCCCATCTTTTGGG-3’; Il-6 forward, 5’-CTGCAAGAGACTTCCATCCAGTT-3’ and reverse, 5’-GAAGTAGGGAAGGCCGTGG-3’; Il-1β forward, 5’-CGGCACACCCACCCTG-3’ and reverse, 5’-AAACCGCTTTTCCATCTTCTTCT-3’; Ifn-β forward, 5’-ATGAGTGGTGGTTGCAGGC-3’ and reverse, 5’-TGACCTTTCAAATGCA GTAGATTCA-3’; Ifn-α4 forward, 5′-CCTGTGTGATGCAGGAACC-3′ and reverse, 5′-TCACCTCCCAGGCACAGA-3′; L32 forward, 5’-GAAACTGGCGGAAACCCA-3’ and reverse, 5´-GGATCTGGCCCTTGAACCTT-3’.

15.59%

15.08%

7.27%

CD4 T cellsB cells(CD11c-PDCA-1-)

pDCs(gated CD11cintPDCA-1+)

µMT:WT

µMT pIII+IV-/-:WT

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CD4

SS

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B22

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CIITA

cDCsMacrophagesmicroglial cells

B cellspDCsmTECs

mTECscTECsIFN-γ stimulated cells

pI pIII pIVExons 2-19

pIII+IV-/- mice

A

Supplemental Figure 1 Chimeric mice selectively lacking MHCII expression on pDCs. (A) In mice, CIITA gene expression is driven by three different cell specific promoters pI, pIII and pIV. pIII+IV-/- mice carry the deletion of promoters pIII and pIV. mTECs: medullary thymic epithelial cell, cTECs: cortical TEC. (B) Irradiated WT mice were reconstituted with BM cells from either µMT mice (µMT:WT) or µMT crossed with pIII+IV-/- mice (µMT pIII+IV-/-:WT). Frequency of CD4+ T cells (left panel) in WT, µMT:WT and µMT pIII+IV-/-:WT mice. Expression of MHCII on B cells (middle panel) and pDCs (right panel) in WT, µMT:WT and µMT pIII+IV-/-:WT mice. Results are representative of at least three experiments.

CFSE

CD

4

µMT:WT µMT pIII+IV-/-:WT

dLN

ndLN

83.9%±1.9

16.1%±1.9

91.3%±1.2

18.7%±1.2

18.3%±2.4

81.7%±2.3

20.5%±0.5

79.5%±0.5

Supplemental Figure 2Similar T cell proliferation in µMT:WT and µMT pIII+IV-/-:WT chimeric mice. µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized in the flank (s.c.) with CpG-B and OVAII peptide, and CFSE-labelled OT-II Rag2-/- cells were adoptively transferred 24h later. After 4 days, cells from dLN and ndLN were cultured with OVAII peptide for 18h. (A) Representative flow cytometry profiles showing OT-II Rag2-/- proliferation in dLN and ndLN. (B) Graphs show the means and SEM derived from 4 mice and are representative of 3 independent experiments.

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Supplemental Figure 3Selective MHCII abrogation on pDCs in μMT pIII+IV-/-:WT mice does not affect MHCII expression and upregulation in other cell subtypes. µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized in the flank (s.c.) with CpG-B and cells were analyzed in the dLN 15h later (A) Representative flow cytometry profiles of MHCII expression after gating on CD11cint SiglecH+ cells (pDCs), CD11c+ CD8+ cDCs (CD8+ DCs) and F4/80+ CD11b+ CD11c- cells (macrophages). (B) Graphs show the MHCII MFI and SEM derived from 4 mice and are representative of 3 independent experiments.

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CT Imiquimod CpG-B

CT Imiquimod CpG-B

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CT CpG-A

B

Supplemental Figure 4MHCII deficient pDCs exhibit normal TLR responses in vitro. MHCII deficient pDCs produce normal levels of inflammatory cytokines after in vitro TLR stimulation. (A,B) BM-pDCs from WT, H2-Aα-/- and pIII+IV-/- mice were activated or not with TLR ligands. (A) Tnf-α, Il-6, Il-1β and Ifn-α4 mRNA expression were quantified by qPCR after 3h of treatment with Imiquimod or CpG-B. (B) Ifn-β and Ifn-α4 mRNA expression after 3h of treatment with CpG-A. (A-B) Results are pooled from 4 to 8 mice.

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-α4

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Supplemental Figure 5Vaccination using OVA protein + CpG-B significantly inhibits tumor growth after Ag presentation by pDCs. (A) µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized s.c. with OVA protein and CpG-B. 7 days later, EG7 cells were implanted s.c. in an ipsilateral manner. Tumor growth was measured every 2-3 days. Results show the mean and SEM derived from 6 mice and are representative of 2 independent experiments. (B) EG7-tumor bearing WT mice were immunized s.c. in a contralateral manner with either OVAII peptide or OVA protein and CpG-B. Tumor growth was measured every 1-2 days. Results show the mean and SEM derived from 6 mice and are representative of 2 independent experiments.

*VAX

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OVAII + CpG-B

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Supplemental Figure 6Characterisation of in vitro generated Th0 and Th17 OT-II T cells. Naive OT-II T cells were differentiated in vitro into Th0 or Th17 cells using α-CD3 + α-CD28 (Th0) in addition with IL-6 and TGF-β (Th17). (A) Representative flow cytometry profiles of IL-17 and IFN-γ expression after gating on CD4+ TCR+ T cells. (B) Percentages of CD4+ T cells expressing IL-17 or IFN-γ. Graphs show the means and SEM derived from 3 mice and are representative of 3 independent experiments.

IL-17 IFN-γ

IFN-γ

IL-1

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Supplemental Figure 7Impaired cell migration toward tumor supernatant in mice lacking MHCII on pDCs. µMT:WT and µMT pIII+IV-/-:WT chimeras were immunized s.c. with OVAII peptide and CpG-B. 7 days later, Eg7 cells were implanted s.c. in an ipsilateral manner, and tumor supernatant was collected 8 days later. (A) WT LN cells were implanted in the upper chamber of a transwell and migrated toward tumor supernatant form µMT:WT or µMT pIII+IV-/-:WT mice. Graphs shows the number of LN cells collected in the lower chamber after 2h of migration. (B) Chemokines were measured by multiplex in the tumor supernatant. Graphs show the concentration of CCL-2, CCL-5, CXCL-1 and CXCL-2. (A-B) Graphs show the means and SEM derived from 3 mice and are representative of 3 independent experiments.

NS

Supplemental Figure 8CD8+ T cell depletion in EG7 bearing mice.EG7 cells were implanted s.c. in WT mice. CD8+ T cells were depleted using anti-CD8 mAbs at day 8, 11 and 14. CD8+ T cell depletion was analyzed in the blood 3 days after each mAb injection and an example (day 11) is provided. (A) Representative flow cytometry profiles of CD3 and CD8 expression with (anti-CD8) or without (CT) treatment with anti-CD8 mAbs. (B) Graphs show the mean and SEM derived from 3 mice and are representative of 2 independent experiments.

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