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1 Supporting Information Fukaya et al /pnas SI Materials and Methods Generation of Cd205 dtr/dtr Mice. The targeting vector for Cd205 dtr/dtr mice was constructed in the pbluescript vector by using a 2.0-kb genomic fragment (left arm) upstream of the Cd205 exon 35, and a 4.3-kb genomic fragment (right arm) downstream of exon 35 cloned from a modified BAC clone RP23-10L17 (Children s Hospital Oakland Research Institute) containing the complete Cd205 gene. The left arm was generated by PCR with the use of the following oligonucleotides: left arm forward (5 -CGC CTC GAG ATC TTC TGA TAC ATA TGC AGC TAG AGT CAA- 3 ) and left arm reverse (5 -CGC GTC GAC CAG TAT ACA TAT CTT TGA AAT AGT ATA AGA AAC AT-3 ). The 2.0-kb PCR fragment was digested with XhoI and SalI, and ligated into each site of pbluescript. The right arm was generated by PCR with the use of the following oligonucleotides: right arm forward (5 -CGC GTC GAC GTA TTA ATT AAT TGT TAC ATT TCT GTT TTT CAT TG-3 ) and right arm reverse (5 -CGC ATC GAT CAT CAA TTG TTC TAT AAG ACT GTG TTT TCC-3 ), digested with SalI and ClaI, and ligated into each site of the targeting vector. Each of the 5 - and 3 -primers were also tagged (indicated in italics) with XhoI and SalI sites for the left arm or SalI and ClaI sites for the right arm, respectively. A SalI restriction site was engineered 560-bp downstream of the stop codon in the 3 UTR of Cd205 upstream of the polyadenylation signal. The pires2-dtregfp-loxp-cre/neo r -loxp auto-deleter cassette was cloned into the SalI site inserted into the targeting vector. Finally, the targeting construct was abutted to a PMC1-DTa negativeselection cassette and linearized. The linearized targeting construct was introduced by electroporation into C57BL/6-derived Bruce4 ES cells and neomycin-resistant clones were first screened for homologous recombination by PCR using a pair of the following oligonucleotides corresponding to a sequence outside of the 5 left arm and to the enhanced GFP (EGFP) site: Primer 3: 5 -TCT GTA ATT TGT ACA GGA CTT TGT ATT TTT-3, and Primer 4: 5 -ATA TAG ACG TTG TGG CTG TTG TAG TTG TA-3. EcoRI-digested genomic DNA of positive clones was then screened by Southern blotting with a 3 external singlecopy probe corresponding to a 0.6-kb fragment, which was amplified by PCR using 5 -AGC ATA TTC AAA CCA CCA CAG TTA T-3 and 5 -AAA AGT GTC TCC TCA CAG TAA ATG G-3. When tested on XbaI-digested DNA, it hybridized either to a 11.6-kb WT fragment or to a 14.2-kb recombinant fragment. ES clones bearing the correctly targeted locus were injected into BALB/c blastocysts, and chimeric male offspring, in which the auto-deleter cassette was self-excised during the male germ-line transmission, were mated to female C57BL/6 mice to obtain heterozygotes, which were then crossed to obtain homozygotes. Transmission of the targeted allele was confirmed by PCR with the following oligonucleotides corresponding to a sequence downstream of exon 35: Primer 1: 5 -ATT TAG CTA ATG TGA ATT TTC TCT GTA AAA-3, andprimer2:5 -AAA CAA AAA CAA AAA CAA CAA AAC AAA AC-3. CRE-mediated deletion of the floxed Neo r cassette can be visualized by the presence of a 10.5-kb fragment using XbaI-digested DNA hybridized with the 3 external single-copy probe. Although the diphtheria toxin (DT) receptor (DTR) sequence present in the IRES2-DTREGFPloxP cassette was fused in-frame to an EGFP sequence, EGFP expression was not observed in the resulting Cd205 dtr/dtr mice. The mutant mice were cross-mated for nine more generations with C57BL/6 mice, and Cd205 +/+ littermates were used as WT mice. Cell Isolation. To prepare single-cell suspensions from thymus (Thy), spleen (Spl), mesenteric lymph nodes (MLNs), and pooled subcutaneous LNs, tissue samples were digested with collagenase type III (Worthington Biochemical) at 37 C for 20 min, and ground between glass slides. Bone marrow (BM) cells were flushed from the femurs and tibias. Splenocytes and BM cells were treated with RBC lysis buffer (Sigma-Aldrich) before their suspension. Single-cell suspensions were obtained by forcing through a 100-μm cell strainer (BD Bioscience). CD4 + T cells and CD8 + T cells were purified from Spls with mouse CD4 T lymphocyte Enrichment Set-DM and mouse CD8 T lymphocyte Enrichment Set-DM (both from BD Bioscience). CD4 + T cells from Foxp3 EGFP CD OT-II mice were sorted into Foxp3 EGFP T cells and Foxp3 EGFP+ T cells by FACSVantage (BD Biosciences). CD11c + dendritic cells (DCs) were purified using AutoMACS with mouse CD11c (N418) microbeads. For the isolation of lamina propria (LP) lymphocytes, intestines were opened longitudinally, washed to remove fecal content, and cut into small pieces. Small intestinal segments were treated in PBS containing 10% (vol/vol) FCS, 20 mm Hepes, 100 U/mL penicillin, 100 μg/ml streptomycin, 1 mm sodium pyruvate, 20 mm EDTA, and 10 μg/ml polymyxin B (Calbiochem) with continuous stirring at 37 C for 20 min in a water bath for the removal of epithelial cells and fat tissue, and then washed extensively with PBS. Small intestinal segments were digested with 400 U/mL collagenase type III, 500 μg/ml DNase I (Roche Diagnostics) and 250 mu/ml dispase (Invitrogen) in RPMI 1640/2% (vol/vol) FCS with continuous stirring at 37 C for 40 min in a water bath. EDTA was added (10 mm final concentration) and the digested tissue was incubated for an additional 5 min at 37 C. The cell suspension was prepared by forcing it through a 100-μm cell strainer, washed with PBS, resuspended in 10 ml of 30% (vol/vol) Percoll (GE Healthcare), and overlaid on 2 ml of 70% (vol/vol) Percoll in a 15-mL tube. Percoll gradient separation was performed by centrifugation at 780 g for 20 min at 25 C. The LP lymphocytes were collected at the interface of the Percoll gradient and washed with RPMI 1640/10% (vol/vol) FCS, and used immediately for experiments. BM Chimeric Mice and DT Treatment. Recipient WT mice received 10 Gy of total body irradiation, split into two doses separated by 4 h to minimize gastrointestinal toxicity, and intravenously with BM cells ( /mouse) from WT mice or Cd205 dtr/dtr mice. The BM chimeric mice were allowed to rest for 8 wk before they were used in experiments. For the systemic in vivo ablation of CD205 + conventional DCs (cdcs), Cd205 dtr/dtr WT chimeras were injected intraperitoneally with DT (0.1 1 μg/mouse; Sigma-Aldrich). Flow Cytometry. Cells were stained with fluorescein-conjugated mabs to mouse CD4 (RM4-5), CD8α (53-6.7), CD11c (HL3), CD44 (IM7), CD45.1 (A20), mouse Vα2 TCR (B20.1), IFN-γ (XMG1.2), Mouse Vβ TCR Screening Panel, isotype-matched control mab (BD Bioscience), mpdca-1/bst2 (JF05-1C2.4.1) (Miltenyi Biotec), CD205 (NLDC-145) (Serotec), CD8α (KT15), H-2K b ovalbumen (OVA) tetramer, and H-2K b HSV-1 gb tetramer (MBL). For the intracellular expression of cytokines, cells were incubated for 4 h with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml; Sigma-Aldrich) plus ionomycin (500 ng/ml; Sigma-Aldrich), OVA peptide (SIINFEKL; 10 μm), or HSV-1 gb peptide (SSIEFARL; 10 μm) plus GolgiPlug (BD Biosciences) during the final 2 h. Subsequently, the cells were resuspended in Fixation-Permeabilization solution (BD Cytofix/ 1of9

2 Cytoperm kit; BD Biosciences), and intracellular cytokine staining was done according to the manufacturer s directions. Fluorescence staining was analyzed with a FACSCalibur flow cytometer and CELLQuest Software (both from BD Biosciences). Antigen Presentation Assay. CD4 + T cells ( ) were cultured with irradiated (15 Gy) CD11c + DCs ( ) in the presence or absence of OVA protein (100 μg/ml; Sigma-Aldrich) for 3 d in 96-well flat-bottomed plates (BD Bioscience). [ 3 H]thymidine (GE Healthcare) incorporation was measured on day 3 for the last 18 h. In another experiment, the cells and the culture supernatants were collected to detect the production of cytokines. Immunization. For the analysis of antigen-specific CD4 + T-cell responses (1), chimeric mice were immunized subcutaneously with 100 μg of OVA protein emulsified in complete Freund s adjuvant (CFA) (Difco), and Spl was obtained 14 d after the immunization. For the generation of antigen-specific cytotoxic T lymphocytes (CTLs), chimeric mice received an intraperitoneal injection of 500 μg of OVA protein in combination with 50 μg of poly (I:C) (Amersham Biosciences) plus 10 μg of anti-cd40 mab (clone 1C10; ebioscience), and Spl was obtained from the mice 6 d later. In another experiment, serum samples were collected 3 h after injection. Detection of Cytokines. Culture supernatants and sera were assayed for IFN-β (PBL), IFN-γ, IL-6, and IL-12p40 (Invitrogen) by ELISA kits according to the manufacturer s instructions. Bacteria, Virus, and Infection. For bacterial infections, chimeric mice were intraperitoneally infected with Listeria monocytogenes expressing OVA (LM-OVA; CFU/mouse or CFU/mouse; DMX) (2). Survival was then monitored for 14 d or serum samples were collected 24 h after infection. For the determination of bacterial burden, splenic growth of LM-OVA was quantified by plating serial dilutions of homogenates on Oxford- Listeria-Selective-Agar (Merck) 2 d or 6 d after infection, and colonies were counted after an overnight incubation at 37 C. In some experiments, Spl was obtained from the chimeric mice 6 d after infection. WT HSV-1 (3) was propagated and titered by a plaque assay with Vero cells. For viral infections, chimeric mice were intravenously infected with HSV-1 (10 7 plaque-forming units per mouse), and serum samples were collected 6 h after infection. In some experiments, Spl was obtained from the chimeric mice 5 d after infection. Viral titers within splenic tissue samples were determined by homogenizing the tissue followed by freeze-thawing, and clarifying the supernatant, then subjecting the supernatant to a plaque assay on Vero cells. Adoptive Transfer. CD OT-I CD8 + T cells or CD OT- II CD4 + T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes; 2.5 μm) at 37 C for 10 min, and washed twice with cold PBS. Subsequently, CFSE-labeled CD OT-II CD4 + T cells or CD OT-I CD8 + T cells ( /mouse) were intravenously injected into chimeric mice 24 h before the intraperitoneal injection with OVA protein (50 μg/mouse) alone for systemic steady-state immunization. In some experiments, recipient mice were i.p. infected with LM-OVA ( CFU/mouse). After 3 d, the gated CD OT-I CD8 + T cells and CD OT-II CD4 + T cells in Spl were analyzed for CFSE dilution to detect the dividing cells by flow cytometry. For generation of antigen-specific CD4 + Foxp3 + itregs, CD OT-II CD4 + Foxp3 EGFP- T cells ( /mouse) were intravenously injected into mice 24 h before the intraperitoneal injection with OVA protein (50 μg/mouse), and the expression of Foxp3 EGFP among gated CD OT-II CD4 + Tcells in Spl was analyzed by flow cytometry 8 d after immunization. 1. Takagi H, et al. (2011) Plasmacytoid dendritic cells are crucial for the initiation of inflammation and T cell immunity in vivo. Immunity 35: Foulds KE, et al. (2002) Cutting edge: CD4 and CD8 T cells are intrinsically different in their proliferative responses. J Immunol 168: Arii J, et al. (2010) Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1. Nature 467: of9

3 Fig. S1. Generation and identification of Cd205 dtr/dtr mice. (A) Strategy used to produce the Cd205-DTREGFP knock-in mice: (1) Partial restriction map of the WT Cd205 gene. Exons are depicted as black filled boxes. The restriction site indicated is X: XbaI. (2) Targeting vector used for the introduction of the mutations in the Cd205 gene. A SalI site engineered in the 3 UTR of the Cd205 was used to clone the IRES-DTRGFP-Cre/Neo r cassette. Dta, diphtheria toxin a expression cassette; I, internal ribosome entry site; D, DTR; E, EGFP. The Cre/Neo r auto-deleter cassette is shown bracketed by Lox P sites (filled triangles); it directs its own excision as it passes through the male germline. (3) Structure of the targeted allele following homologous recombination in recombinant ES clones. (4) Structure of the Cd205 dtr allele following expression of the Cre recombinase and excision of the Neo r cassette in mutant mice. The 3 external single-copy probe (hatched box) and the PCR primers at the 5 end (black arrows) used to verify proper homologous recombination events are shown. (B and C) DNA-PCR (B) and Southern blot (C) analysis of WT and recombinant ES clones. (D and E) Genotyping of tail DNA from WT mice and from heterozygous or homozygous mice for the Cd205 dtr allele by DNA-PCR (D) and Southern blot (E) analysis. In the Southern blot analysis, genomic DNA was digested by XbaI and hybridized to the 3 external single-copy probe. The results are representative of three independent experiments. 3of9

4 Fig. S2. Conditional ablation of CD205 + cdcs in skin-draining LNs in Cd205 dtr/dtr mice. WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) were injected with DT (0.5 μg/mouse), and subcutaneous LNs were obtained 2 d after the injection. The frequency (A) and the absolute number (B) of CD205 + cdcs was analyzed by flow cytometry. Data are represented by a dot plot, and numbers represent the proportion of CD8α + CD205 + cdcs and CD8α CD205 + cdcs among CD11c + cells in each quadrant (A). *P < 0.01 compared with WT WT chimeras. The results are representative of three independent experiments. Fig. S3. Ablation of CD205 + cdcs affects the constituency of CD11c + DCs in vivo. WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) were injected with DT (0.5 μg/mouse), and Spl were obtained 2 d (A) or at the indicated days (B) after the injection. The absolute number of CD11c + CD8α + cdcs, CD11c + CD8α cdcs, and CD11c + BST2 + pdcs were analyzed by flow cytometry. *P < 0.01 compared with WT WT chimeras. Data are the mean ± SD, and the results are representative of three independent experiments. 4of9

5 Fig. S4. Cellularity of Spl in the absence of CD205 + cdcs. WT WT chimeras (n =6)andCd205 dtr/dtr WT chimeras (n = 6) were injected with DT (0.5 μg/mouse), and Spl was obtained 2 d after the injection. The frequency of CD11c + BST2 cdcs (A), CD11c + BST2 + pdcs (B), CD4 + T cells (C), CD8 + T cells (D), B220 + B cells (E), CD3 + CD49b + NKT cells (F), CD3 CD49b + NK cells (G), or CD11b + CD11c macrophages (H) was analyzed by flow cytometry. Data are the percentage of positive cells among leukocytes. Data are the mean ± SD, *P < 0.01 compared with WT WT chimeras. The results are representative of three independent experiments. 5of9

6 Fig. S5. Cellularity of MLNs in the absence of CD205 + cdcs. WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) were injected with DT (0.5 μg/mouse), and MLNs were obtained 2 d after the injection. The frequency of CD11c + BST2 cdcs (A), CD11c + BST2 + pdcs (B), CD4 + T cells (C), CD8 + T cells (D), B220 + B cells (E), CD3 + CD49b + NKT cells (F), CD3 CD49b + NK cells (G), or CD11b + CD11c macrophages (H) was analyzed by flow cytometry. Data are the percentage of positive cells among leukocytes. Data are the mean ± SD. The results are representative of three independent experiments. Fig. S6. Constituency of thymocytes in the absence of CD205 + cdcs. WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) were injected with DT (0.5 μg/mouse), and Thy was obtained 2 d after the injection. The frequency of CD4 CD8 thymocytes (A), CD4 + CD8 + thymocytes (B), CD4 + CD8 thymocytes (C) or CD4 CD8 + thymocytes (D) was analyzed by flow cytometry. Data are the percentage of positive cells among thymocytes. Data are the mean ± SD. The results are representative of thee independent experiments. 6of9

7 Fig. S7. Vβ repertoire of thymic and peripheral T cells in the absence of CD205 + cdcs. WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) were injected with DT (0.5 μg/mouse), and Thy and Spl were obtained 12 d after the injection. Vβ distribution of CD4 + T cells (A and C) and CD8 + T cells (B and D) in Thy (A and B) and Spl (C and D) was analyzed by flow cytometry. Data are the percentage of positive cells among CD4 + T cells and CD8 + T cells. Data are the mean ± SD, and the results are representative of six independent experiments. Fig. S8. Cytokine production in WT WT chimeras and Cd205 dtr/dtr WT chimeras. (A and B) Spl CD4 + T cells obtained from WT WT chimeras (n =6)and Cd205 dtr/dtr WT chimeras (n = 6) that had been treated with DT (0.5 μg/mouse) were cultured with WT CD11c + DCs in the presence of OVA protein for the measurements of production of IFN-γ by ELISA (A). (B) Intracellular expression of IFN-γ in the cultured CD4 + T cells was analyzed by flow cytometry. Data are represented by a dot plot, and numbers represent the proportion of IFN-γ + cells among gated CD4 + T cells in each quadrant. (C) Serum levels of IFN-β, IFN-γ, IL-6, and IL-12p40 in WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) that had been treated with DT (0.5 μg/mouse) were measured by ELISA; n.d., not determined. 7of9

8 Fig. S9. Ablation of CD205 + cdcs affects the T-cell homeostasis in vivo. WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) were injected with DT (0.5 μg/mouse), and Thy, Spl, and LP were obtained 12 d after the injection. The proportion (A) and the absolute number (B) of CD4 + Foxp3 + Tregs or intracellular IFN-γ and IL-17 producing cells among gated CD4 + T cells in Thy, Spl, and LP were analyzed by flow cytometry. Data are represented by a dot plot, and numbers represent the proportion of Foxp3 + cells, IFN-γ + cells, or IL-17 + cells among gated CD4 + T cells in each quadrant (A). *P < 0.01 compared with WT WT chimeras. Data are the mean ± SD, and the results are representative of six independent experiments. 8of9

9 Fig. S10. Ablation of CD205 + cdcs impairs the generation of antiviral CTLs in vivo. WT WT chimeras (n = 6) and Cd205 dtr/dtr WT chimeras (n = 6) that had been treated with DT (0.5 μg/mouse) were uninfected or infected with HSV-1. (A and B) Serum levels of IFN-β (A) and IL-12p40 (B) were measured at 6 h after infection by ELISA. (C and D) At 5 d after infection, splenocytes were analyzed for the generation of MHC I-HSV-1 gb-tetramer + CD44 high CD8 + T cells (C) and for intracellular IFN-producing CD8 + T cells (D) byflow cytometry. Data are represented by a dot plot, and numbers represent the proportion of MHC I-HSV-1 gbtetramer + CD44 high cells (C) and IFN + cells (D) among gated CD8 + T cells in each quadrant. (E) Viral burden in the Spl was determined as PFU 5 d after infection. n.d., not detected. *P < 0.01 compared with WT WT chimeras. Data are the mean ± SD, and the results are representative of three independent experiments. 9of9