Critical Roles of a Dendritic Cell Subset Expressing a Receptor, XCR1 Chihiro Yamazaki, Masanaka Sugiyama, Tomokazu Ohta, Hiroaki Hemmi, Eri Hamada, Izumi Sasaki, Yuri Fukuda, This information is current as Takahiro Yano, Mikako Nobuoka, Takeshi Hirashima, of September 25, 2021. Akihiko Iizuka, Katsuaki Sato, Takashi Tanaka, Katsuaki Hoshino and Tsuneyasu Kaisho J Immunol 2013; 190:6071-6082; Prepublished online 13

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References This article cites 61 articles, 30 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/190/12/6071.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Critical Roles of a Dendritic Cell Subset Expressing a , XCR1

Chihiro Yamazaki,*,†,1 Masanaka Sugiyama,*,‡,x,{,1 Tomokazu Ohta,x,{ Hiroaki Hemmi,*,{ Eri Hamada,* Izumi Sasaki,*,{ Yuri Fukuda,*,{ Takahiro Yano,* Mikako Nobuoka,* Takeshi Hirashima,* Akihiko Iizuka,* Katsuaki Sato,‖ Takashi Tanaka,‡ Katsuaki Hoshino,*,{,# and Tsuneyasu Kaisho*,{

Dendritic cells (DCs) consist of various subsets that play crucial roles in linking innate and adaptive immunity. In the murine spleen, CD8a+ DCs exhibit a propensity to ingest dying/dead cells, produce proinflammatory cytokines, and cross-present Ags to generate CD8+ T cell responses. To track and ablate CD8a+ DCs in vivo, we generated XCR1-venus and XCR1-DTRvenus mice, in which genes

for a fluorescent protein, venus, and a fusion protein consisting of diphtheria toxin receptor and venus were knocked into the gene Downloaded from locus of a chemokine receptor, XCR1, which is highly expressed in CD8a+ DCs. In both mice, venus+ cells were detected in the majority of CD8a+ DCs, but they were not detected in any other cells, including splenic macrophages. Venus+CD8a+ DCs were superior to venus2CD8a+ DCs with regard to their cytokine-producing ability in response to TLR stimuli. In other tissues, venus+ cells were found primarily in lymph node (LN)-resident CD8a+, LN migratory and peripheral CD103+ DCs, which are closely related to splenic CD8a+ DCs, although some thymic CD8a2CD11b2 and LN CD1032CD11b2 DCs were also venus+. In response to dsRNAs, + +

diphtheria toxin–treated XCR1-DTR mice showed impaired CD8 T cell responses, with retained cytokine and augmented CD4 T cell http://www.jimmunol.org/ responses. Furthermore, Listeria monocytogenes infection and anti–L. monocytogenes CD8+ T cell responses were defective in diph- theria toxin–treated XCR1-DTRvenus mice. Thus, XCR1-expressing DCs were required for dsRNA- or bacteria-induced CD8+ T cell responses. XCR1-venus and XCR1-DTRvenus mice should be useful for elucidating the functions and behavior of XCR1-expressing DCs, including CD8a+ and CD103+ DCs, in lymphoid and peripheral tissues. The Journal of Immunology, 2013, 190: 6071–6082.

endritic cells (DCs) are specialized APCs that play crucial pathogenesis of immune disorders have been clarified by analyzing roles in linking innate and adaptive immunity (1). Critical mutant mice in which diphtheria toxin receptor (DTR) and diph- in vivo roles for DCs in various immune responses or the theria toxin (DT) A subunit are expressed under the control of the

D by guest on September 25, 2021 CD11c promoter (2–6). Those mice also enabled us to clarify ho-

*Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunol- meostatic roles for DCs in lymphocyte homing to lymph nodes ogy, Yokohama, Kanagawa 230-0045, Japan; †Department of Immunology, Graduate (LNs) or preventing autoimmunity (5–7). However, because DCs School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, are heterogeneous and can be divided into several subsets according Okayama, Okayama 700-8558, Japan; ‡Research Unit for Inflammatory Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230- to function and expression patterns of cell surface molecules, a DC x 0045, Japan; Laboratory of Immune Regulation, Department of Microbiology and subset–specific ablation system should be generated. Immunology, Graduate School of Medicine, Osaka University, Suita, Osaka 565- 0871, Japan; {Laboratory for Immune Regulation, World Premier International Re- Under steady-state conditions, murine splenic DCs consist of 2 search Center Initiative, Immunology Frontier Research Center, Osaka University, B220+CD11cdull plasmacytoid DCs (pDCs) and B220 CD11c+ Suita, Osaka 565-0871, Japan; ‖Laboratory for Dendritic Cell Immunobiology, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan; DCs. In the spleen, all DCs are resident DCs derived from blood 2 + + and #Department of Immunology, Faculty of Medicine, Kagawa University, Kita-gun, precursors, and B220 CD11c DCs can be divided into CD8a Kagawa 761-0793, Japan CD11b2 and CD8a2CD11b+ DCs. In lymphoid tissues, DCs con- 1 C.Y. and M.S. contributed equally to this work. sist of resident and migratory DCs, which can be defined as MHC Received for publication October 9, 2012. Accepted for publication April 4, 2013. class II (MHC-II)intCD11c+ and MHC-IIhighCD11c+ DCs, re- This work was supported by the Kishimoto Foundation, a Grant-in-Aid for Scientific spectively (8, reviewed in Ref. 9). As in the spleen, resident DCs Research (B, C), a Grant-in-Aid for Challenging Exploratory Research, a Grant-in-Aid for + 2 2 + Scientific Research on Priority Areas, a Grant-in-Aid for Scientific Research on Innova- consist mainly of CD8a CD11b and CD8a CD11b cells. Mi- tive Areas, the Uehara Memorial Foundation, and a Grant-in-Aid for Young Scientists. gratory DCs are derived from peripheral tissues, such as skin or C.Y., M.S., and I.S. were supported by a RIKEN Junior Research Associate grant. lamina propria, and can be divided into several subsets depending Address correspondence and reprint requests to Dr. Tsuneyasu Kaisho, Laboratory of on the expression patterns of CD103 and CD11b. Immune Regulation, World Premier International Research Center Initiative, Immunology + Frontier Research Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Among these DC subsets, resident CD8a DCs and migratory Japan. E-mail address: [email protected] CD103+ DCs are characterized by their ability to take up apoptotic Abbreviations used in this article: DC, dendritic cell; DT, diphtheria toxin; DTR, cells and to cross-present soluble and cell-associated Ags for diphtheria toxin receptor; EGFP, enhanced GFP; ES, embryonic stem; Flt3L, Flt3 + ligand; LC, Langerhans cell; L.m.-OVA, Listeria monocytogenes expressing OVA; generating CD8 cytotoxic T cells (10–12, reviewed in Ref. 13). LN, lymph node; MF, macrophage; MHC-II, MHC class II; mLN, mesenteric lymph This cross-presenting activity is important for establishing anti- node; Mo-DC, monocyte-derived dendritic cell; mOVA-Tg, C57BL/6-Tg(CAG-OVA) viral or antitumor immunity. Cross-presentation can be facilitated 916Jen/J; MZMMF, marginal zone metallophilic macrophage; pDC, plasmacytoid dendritic cell; poly(I:C), polyinosinic-polycytidylic acid; SDLN, skin-draining lymph by targeting Ags to several C-type lectins, such as DEC-205 node; Tip-DC, TNF-a/inducible NO synthase–producing dendritic cell; WT, wild-type; (CD205), Clec9a, and Langerin (CD207), expressed on these XCR1, XC chemokine receptor 1. DC subsets (14–16). Splenic CD8a+ DCs also show a propensity Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 to produce proinflammatory cytokines in response to a LPS sen- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202798 6072 IN VIVO FUNCTIONS OF XCR1-EXPRESSING CELLS sor, TLR4, and nucleic acid sensors, TLR3 and TLR9. It is im- Germline-transmitting chimeras were generated by injection of targeted ES portant to clarify the in vivo roles of this DC subset. clones to blastocysts from BALB/c mice and bred with C57BL/6J mice. Obtained Xcr1+/venus and Xcr1+/DTRvenus mice were backcrossed with Several mutant mice lacking this DC subset have been estab- C57BL/6J mice for an additional two to six or two to four generations, lished and analyzed. Although the mutant mice lacking a tran- respectively. C57BL/6J or Xcr1+/+ littermates were used as wild-type (WT) scription factor, IRF-8, show ablation of both CD8a+ DCs and mice. pDCs, spontaneous mutant mice (BXH2 mice) carrying a point Mice mutation on the Irf8 gene lack only CD8a+ DCs (17, 18). CD8a+ C57BL/6J mice were purchased from CLEA Japan. b2m-deficient DCs are also ablated in the mutant mice lacking a basic leucine tm1Unc zipper transcription factor, ATF-like 3 (BATF3) (19). Both tran- (B6.129P2-B2m /J) mice and transgenic mice ubiquitously expressing membrane-bound OVA (C57BL/6-Tg(CAG-OVA)916Jen/J [mOVA-Tg]) were scription factors are essential for the development of splenic purchased from The Jackson Laboratory and crossed to obtain mOVA- + + 2/2 CD8a DCs and their equivalent CD103 DCs in the periphery Tg:b2m mice, which carry OVA-expressing MHC class I–deficient (13, 20, 21). Batf3-deficient mice have been studied intensively; cells. All mice were bred and maintained in the Animal Facility of RIKEN their in vivo CD8+ T cell responses against virus, bacteria, parasites, Research Institute for Allergy and Immunology (Yokohama, Japan) and the Animal Resource Center for Microbial Diseases, Research Institute for and tumors are severely impaired (22–24). Ablation of splenic Infectious Diseases, Osaka University (Suita, Japan) under specific path- + CD8a DCs can also be achieved by injection of a proapoptotic ogen–free conditions and were used at 7–14 wk of age under institutional reagent, cytochrome C. Cytochrome C–injected mice show im- guidelines of RIKEN Institute and Osaka University. All animal experi- pairment of CD8+ T cell responses against soluble or cell-associated ments were approved by the Animal Research Committees of RIKEN Ags and subsequent immunity to tumor challenge (25). These find- Yokohama Research Institute and Osaka University. ings suggest that CD8a+ DCs are critical for cross-presentation. Reagents Downloaded from However, IRF-8 is also expressed in B cells and myeloid cells, DTwas purchased from Sigma-Aldrich. LPS-free OVAprotein and OVA257– such as monocyte/macrophages (MFs), and BXH2 mice manifest 264 peptide (SIINFEKL; OVA-I peptide) were purchased from Worthington an expansion of myeloid cells. BATF3 expression is not specific Biochemical and Toray Research Center, respectively. Polyinosinic-polycytidylic to CD8a+ DCs; it is also detected in a CD11b+ DC subset (19). In acid [poly(I:C)] was purchased from GE Healthcare Life Sciences. ODN1668 addition, cytochrome C may have some detrimental effects on (59-TCCATGACGTTCCTGATGCT-39, with phosphorothioate backbone) was synthesized by Hokkaido System Science and used as a TLR9 ligand, CpG DNA. other DC subsets or MFs, although it does not make them apo- LPS (O55:B5) was purchased from Sigma-Aldrich. http://www.jimmunol.org/ ptotic. Therefore, it is difficult to rule out the possibility that other cells besides CD8a+ and CD103+ DCs are functionally attenuated Depletion of DTRvenus-expressing cells in these mice. DT was injected i.p. to the mice at a dose of 25 ng/g body weight. PBS was A chemokine receptor, XC chemokine receptor 1 (XCR1), is used as control vehicle. The dose of DT was determined based on the selectively expressed in splenic CD8a+ and peripheral CD103+ DCs deletion efficiency after injection of graded doses of DT (data not shown). but is rare in other DC subsets or lymphocytes (26–31). Human Cell preparation XCR1 expression is restricted to CD141+ DCs, which correspond to murine splenic CD8a+ DCs (27–29, 32, 33). Thus, XCR1 is a suit- Spleen, thymus, skin-draining LNs (SDLNs; including inguinal, axillary,

and brachial LNs), and mesenteric LNs (mLNs) were digested with 400 by guest on September 25, 2021 able marker for defining this DC subset in both humans and mice. To Mandl units/ml Collagenase D (Roche) at 37˚C for 20 min. EDTA (5 mM) generate the mutant mice in which this DC subset can be specifically was added for the last 5 min. To prepare DCs in the skin, epidermal sheets tracked and deleted, we knocked a gene encoding a YFP derivative, were separated from ear skin by digestion with 2 mg/ml Dispase II (Roche) venus (34), or a fusion protein consisting of DTR and venus for 2.5 h at 37˚C. Epidermal sheets and dermal tissues were cut into small (DTRvenus) into the Xcr1 locus. In these knock-in mice, designated pieces and digested with 400 U/ml Collagenase D for 30 min at 37˚C. EDTA (5 mM) was added for the last 5 min. as XCR1-venus or XCR1-DTRvenus mice, venus fluorescence sig- 2 For isolation of lamina propria DCs, fat and Peyer’s patches were re- nal was detected mainly in resident CD8a+CD11b and migratory moved from the small intestine, which were opened longitudinally and CD103+CD11b2 DCs in lymphoid tissues and CD103+CD11b2 stirred in RPMI 1640 containing 2% FCS, 2 mM EDTA for 20 min at 37˚C DCs in peripheral tissues. Upon DT injection to XCR1-DTRvenus to remove epithelial cells and intraepithelial lymphocytes. After additional stirring in RPMI 1640 supplemented with 2% FCS, intestinal tissues were mice, DTRvenus-expressing cells were transiently and efficiently minced and stirred in 170 U/ml Collagenase (Wako Pure Chemical In- ablated. Using XCR1-DTRvenus mice, we investigated the in vivo dustries) and 10 mg/ml DNase I (Sigma-Aldrich) for 20 min at 37˚C, and roles of XCR1-expressing DCs in CD8+ T cell responses. floating cells were collected. Collagenase digestion was repeated three times. Low-density cells were enriched by centrifugation with 40 and 75% Percoll (GE Healthcare) density gradient. Materials and Methods To obtain monocyte-derived DCs, bone marrow (BM) monocytes (MHC- 2 2 2 Generation of XCR1-venus and XCR1-DTRvenus mice II Ly6G CD11c Ly6C+CD11b+) were sorted and cultured with RPMI 1640 containing 5% FCS, 100 mM 2-ME, penicillin, streptomycin, 20 ng/ml Targeting vectors were designed to replace the entire murine Xcr1 coding recombinant mouse GM-CSF (R&D Systems), and 20 ng/ml recombinant sequences with a gene encoding venus or DTRvenus (Figs. 1A, 4A). The mouse IL-4 (BD Biosciences) (35, 36). To obtain BM-derived DCs, BM cells genes carry a polyadenylation signal derived from bGHpA. A neomycin were cultured with 10 ng/ml recombinant mouse GM-CSF or 100 ng/ml resistance gene (neo) driven by the MC1 promoter and flanked by bacte- recombinant human Flt3 ligand (Flt3L; PeproTech), as described previ- riophage P1 loxP and yeast FRT sequence was used as a selection marker. ously (37, 38). An HSV thymidine kinase gene (HSV-TK) was inserted for negative se- lection. To construct the DTRvenus cassette, genes for DTR and venus FACS analysis were amplified from HB-EGF cDNA (kindly provided by Dr. Eisuke Mekata, Osaka University) and venus/pCS2 vector (kindly provided by Dr. Single-cell suspensions were incubated with anti-CD16/32 Ab (BD Bio- Atsushi Miyawaki, RIKEN Brain Science Institute, Wako, Japan), re- sciences or eBioscience) to block nonspecific binding of Abs. The cells were spectively, and the 39 end of human DTR open reading frame was linked in stained with fluorochrome-conjugated Abs and biotinylated Abs against frame with a gene encoding venus. A C57BL/6-derived embryonic stem mice CD3ε (145-2C11), CD4 (RM4-5), CD8a (53-6.7), CD11b (M1/70), (ES) cell line, Bruce4 (kindly provided by Drs. Colin L. Stewart, Institute CD11c (N418 or HL3), CD24 (M1/69), B220 (RA3-6B2), CD62L (MEL- of Medical Biology, Singapore, and Masaki Hikida, Kyoto University, 14), CD49b (DX5), CD103 (M290), CD206 (C068C2), CD207 (eBioL31), Kyoto, Japan), was transfected with the linearized targeting vector by Ly6C (HK1.4), Ly6G (1A8), I-A/I-E (M5/114.15.2), PDCA-1/BST-2 electroporation and selected with G418 (Nacalai Tesque) and ganciclovir (JF05-1C2.4.1 or eBio927), IFN-g (XMG1.2), and IL-12p40 (C15.6). (Mitsubishi Tanabe Pharma). Doubly resistant clones were screened for Biotinylated Abs were visualized by fluorochrome-conjugated streptavidin. homologous recombination by PCR and verified by Southern blot analysis. Abs and fluorochrome-conjugated streptavidin were purchased from BD The Journal of Immunology 6073

Biosciences, eBioscience, BioLegend, and Miltenyi Biotec. CD206 and min. Low-density cells were enriched by centrifugation with 40 and 80% CD207 were stained intracellularly using a Cytofix/Cytoperm (BD Percoll density gradient. Bioscience). In Figs. 2A, 3, 4D, and 7B, dead cells were excluded by staining with a LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). Statistical analysis To monitor cytokine production after in vitro stimulation of DCs, + The data were analyzed using the two-tailed unpaired Student t test, with or CD11c splenic DCs were enriched by MACS sorting with anti-CD11c without a Welch correction, depending on whether the data had possible m beads (Miltenyi Biotec). Then the cells were stimulated with 50 g/ml unequal or equal variances, respectively. For differences in CFU, data were m poly(I:C) or 1 M CpG DNA (ODN1668). After 30 min, GolgiStop (BD analyzed by the Mann–Whitney U test. The p values ,0.05 were con- Bioscience) was added, and the cells were cultured for another 6 h. The sidered statistically significant. All statistical analyses were performed cells were stained with mAbs to CD11c, CD8a, and PDCA-1, fixed with using GraphPad Prism software (GraphPad). fixation/permeabilization solution (Cytofix/Cytoperm Kit), and stained with anti-IL12p40 mAb, according to the manufacturer’s instructions. PDCA-12CD11c+ cells were analyzed for IL-12p40 production. Results To evaluate OVA-specific CD8+ T cell responses, splenocytes were first Expression pattern of venus in XCR1-venus mice analyzed by H-2Kb/OVA-I peptide tetramer (MBL) staining. For intra- cellular IFN-g staining, splenocytes were restimulated with 1 mg/ml OVA-I We first replaced the coding region of Xcr1 with a gene encoding peptide for 6 h in the presence of GolgiStop or brefeldin A (10 mg/ml; venus (Fig. 1A). The replacement was confirmed by Southern blot Sigma-Aldrich), as described previously (38). Subsequently, the cells were analysis (Fig. 1B). Xcr1+/venus mice were born at an expected stainedwithmAbstoCD8a, CD62L, and CD49b, fixed with fixation/ Mendelian ratio and did not show any obvious developmental ab- permeabilization solution (Cytofix/Cytoperm Kit), and stained with anti- +/venus IFN-g mAb. CD49b2DTRvenus2CD8+ cells were analyzed for IFN-g normalities. Throughout this study, Xcr1 mice were used as production. For CD4+ T cell responses, splenocytes were restimulated with XCR1-venus mice.

100 mg/ml OVA protein for 12 h. Brefeldin A (10 mg/ml) was added to the Next, we investigated venus expression in the spleen of XCR1-venus Downloaded from culture for the last 6 h. Subsequently, the cells were stained with mAbs to mice. Less than 1% of splenocytes showed venus expression (Fig. CD4, CD62L, and CD49b, fixed with fixation/permeabilization solution, + 2 2 + 1C). More than 85% of venus cells expressed CD8a and CD11c, and stained with anti–IFN-g mAb. CD49b DTRvenus CD4 cells were + analyzed for IFN-g production. The cells were analyzed by FACSCalibur, suggesting that venus was selectively expressed in CD8a DCs FACSCanto II, FACSAria II, or FACSVerse (BD Biosciences). Data were (Fig. 1C). Meanwhile, venus expression was not observed in analyzed with FlowJo software (TreeStar). CD11c2 cells, including T, B, and NK cells, granulocytes, and + Histology monocytes (Fig. 1D). The expression pattern of venus in CD8a http://www.jimmunol.org/ DCs (Fig. 1C) suggested that CD8a+ DCs consisted of two subsets: Spleen and inguinal LNs were embedded in OCT compound (Sakura XCR1+ and XCR12 DCs. We then asked whether venus expression Finetek) or FSC22 frozen section compound (Leica Microsystems), and 5- + mm cryosections were prepared. Spleen sections were stained with rabbit correlates with the cytokine-producing abilities of CD8a DCs. anti-GFP polyclonal Ab (MBL), allophycocyanin-conjugated anti-F4/80 Splenic DCs were stimulated with a TLR3 agonist [poly(I:C)] or (BM8; eBioscience), and biotinylated anti-CD169 (MOMA-1; Abcam) a TLR9 agonist (CpG DNA), and IL-12p40 production was moni- and then developed with Alexa Fluor 480–conjugated anti-rabbit IgG tored by FACS (Fig. 1E). When stimulated with poly(I:C), IL- (H+L) (Invitrogen) and streptavidin–Pacific Blue. LN sections were stained 12p40 production was strongly induced in CD8a+ DCs but not in with rabbit anti-GFP polyclonal Ab (MBL), Pacific Blue–conjugated anti- 2 + B220, and biotinylated anti-CD169 and then developed with Alexa Fluor CD8a DCs. Among CD8a DCs, IL-12p40 production was ob- 480–00conjugated anti-rabbit IgG (H+L) and streptavidin-APC. Stained served primarily in venus-expressing cells. When stimulated with by guest on September 25, 2021 sections were analyzed by FV10i (Olympus). a TLR9 agonist, both CD8a+ and CD8a2 DCs produced IL-12p40. + + Measurement of serum cytokine level Among CD8a DCs, venus cells mainly responded to CpG DNA. Thus, venus is dominantly expressed in mature CD8a+ DCs with XCR1-DTRvenus mice were injected i.p. with DT at days 22 and 21of poly(I:C) injection. At day 0, PBS or 150 mg poly(I:C) was injected i.v. a high capacity to produce cytokines. into the mice, and serum samples were collected at the indicated times. We further analyzed venus expression in various lymphoid tis- Serum cytokine levels were analyzed by Bio-Plex Suspension Array sues, such as spleen, thymus, SDLNs, and mLNs (Fig. 2A). In these System (Bio-Rad) or ELISA. ELISA kits for IFN-a and IFN-b were from organs, similar ratios of DC subsets were observed between WTand PBL InterferonSource. XCR1-venus mice (Fig. 2A), and venus was expressed only in + + Immunization MHC-II CD11c cells (data not shown). In the spleen, venus was expressed in 70–90% of CD8a+CD11b2 DCs. Venus was not 2 2 Mice were injected with PBS or DT on days 1 and 0 of immunization. At expressed in CD8a CD11b+ DCs, although a small fraction of day 0, each hind footpad was immunized with soluble OVA (50 mg) with 2 2 10 mg poly(I:C). OVA-specific CD4+ and CD8+ T cell responses were CD8a CD11b DCs was positive for venus. In the thymus, venus + 2 monitored 7 d later. For cross-presentation of cell-associated Ags, OVA- expression was observed in 70% of CD8a CD11b DCs and 50% of 2/2 2 2 expressing MHC class I–deficient (mOVA-Tg:b2m ) thymocytes and CD8a CD11b DCs. Although only resident DCs are present in the splenocytes were subjected to gamma radiation (15 Gy) to induce apo- spleen, SDLNs carry both resident MHC-IIintCD11c+ and migratory ptosis. Irradiated dying cells (1.3–2 3 107 cells) were immediately MHC-IIhighCD11c+ DCs. Migratory DCs were characterized by the injected i.v. into the mice with 50 mg poly(I:C) at day 0. OVA-specific + CD8+ T cell responses were monitored 7 d later. expression profile of CD103 and CD11b. In the SDLNs, venus cells were found mainly in CD8a+CD11b2 and CD103+CD11b2 cells in L. monocytogenes infection the resident and migratory DCs, respectively. In the mLNs, migra- + + 2 L. monocytogenes expressing OVA (L.m.-OVA) was originally established tory CD103 DCs contained CD11b DCs, as well as CD11b DCs. by Dudani et al. (39) and was purchased from DMX. DT or PBS was As in SDLNs, venus+ cells were found primarily in resident CD8a+ injected i.p. at days 21 and 0 of infection. L.m.-OVA was grown in brain– CD11b2 DCs and migratory CD103+CD11b2 DCs, whereas 30% of 2 2 heart infusion broth to an OD600 of 0.1–0.2. An OD600 of 0.1 is equivalent CD8a CD11b cells were positive for venus. We further assessed to 2 3 108 bacteria/ml. To measure bacterial loads, mice were infected i.v. with the indicated numbers of L.m.-OVA and sacrificed 3 d postinfection to venus expression in the peripheral tissues, such as the skin and small collect spleens and livers, which were lysed in PBS containing 0.05% intestine lamina propria (Fig. 2B, 2C). In the skin, epidermal Triton X-100. Bacterial titers were determined by plating serial dilutions Langerhans cells (LCs) did not express venus but dermal CD103+ on brain–heart infusion agar plates containing 1 mg/ml erythromycin 2 + CD11b DCs did. In the small intestinal lamina propria, venus was (Sigma-Aldrich). To assess L.m.-OVA–specific CD8 T cell responses, 2 expressed only in CD103+CD11b DCs, but not in CD103+CD11b+ mice were analyzed 7 d after i.v. infection. To isolate liver leukocytes, the 2 + liver was perfused with PBS via the portal vein and digested with 400 U/ml or CD103 CD11b DCs, as in the case of migratory DCs in the Collagenase D for 35 min at 37˚C. EDTA (5 mM) was added for the last 5 mLNs (Fig. 2A). Thus, venus is abundantly expressed in resident 6074 IN VIVO FUNCTIONS OF XCR1-EXPRESSING CELLS

FIGURE 1. Expression of venus in XCR1-venus mice. (A) Schematic dia- grams of the mouse Xcr1 WT allele, a targeting vector, and venus knocked-in allele. Filled and open boxes denote cod- ing and noncoding exons of Xcr1,re- spectively. (B) Southern blot analysis of Xcr1 WT (+/+), Xcr1+/venus (+/v), and Xcr1venus/venus (v/v) mice. Genomic DNAs were isolated from mice tails, digested with EcoRI and EcoRV, electrophoresed, and hybridized with a radiolabeled probe indicated in (A). Southern blot gave a 14.3- and a 7.5-kbp band for WT and knock-in allele, respectively. (C) Spleno- cytes from WT or XCR1-venus mice were stained with Abs against the indicated cell surface markers. Whole or CD192 splen- ocytes are shown. C57BL/6J mice were used as WT mice. (D) Venus expression Downloaded from was analyzed in B cells, T cells, NK cells, granulocytes, and monocytes in the spleen of XCR1-venus mice. Each pop- ulation was defined as indicated. The number in each panel indicates the per- centage of venus+ cells. (E) Splenic DCs

from XCR1-venus mice were left unstim- http://www.jimmunol.org/ ulated (medium) or were stimulated with 50 mg/ml of poly(I:C) or 1 mMofCpG DNA for 6 h. IL-12p40 production from PDCA-12CD11c+ or PDCA-12CD8a+ CD11c+ cells was analyzed by intracel- lular staining with anti–IL-12p40 Ab and FACS. In (C)and(E), the numbers rep- resent the percentage of the cells within the indicated gates or each quadrant; data are representative of at least two inde- by guest on September 25, 2021 pendent experiments. EI, EcoRI; EV, EcoRV.

CD8a+CD11b2 DCs and migratory CD103+CD11b2 DCs in lym- We further confirmed venus expression in in vitro Mo-DCs or phoid organs and peripheral CD103+CD11b2 DCs. In the thymus BM-derived DCs (Fig. 3C, 3D). In vitro Mo-DCs were generated or LNs, venus was also expressed in a subset of CD8a2CD11b2 by culturing purified BM monocytes with GM-CSF and IL-4. or CD1032CD11b2 DCs. Venus expression was not observed in in vitro Mo-DCs (Fig. Distinct populations of DCs are generated transiently and ac- 3C). In vitro BM-derived DCs were generated in the presence of cumulate in the inflamed tissues and lymphoid organs in response GM-CSF or Flt3L. GM-CSF–induced BM DCs failed to show to microbial infection or inflammatory stimuli. We analyzed venus expression (Fig. 3D). Flt3L-induced BM DCs include whether venus was expressed in TNF-a/inducible NO synthase– B220+CD11c+ and B2202CD11c+ cells. The former population producing DCs (Tip-DCs) detected in the spleen after systemic corresponds to pDCs. B2202CD11c+ cells can be further divided infection of L. monocytogenes and in monocyte-derived DCs (Mo- into CD24highCD11blow DCs (CD24high DCs) and CD24low DCs) observed in LNs after LPS injection or Gram-negative bac- CD11bhigh DCs (CD11bhigh DCs), and CD24high DCs are thought terial infection (35, 40). To analyze Tip-DCs, mice were infected to be the equivalents of splenic CD8a+ DCs. Venus expression with L.m.-OVA. Two days postinfection, splenocytes were ana- was detected in CD24high DCs but not in CD11bhigh DCs or pDCs lyzed for venus expression. L.m.-OVA infection induced the gen- (Fig. 3D). eration and accumulation of Tip-DCs (Ly6C+CD11b+MHC-II+ CD11c+) in the spleen (Fig. 3A). Ly6C2 DCs contained venus- Depletion of a DC subset that expresses XCR1 expressing cells, .90% of which were CD8a+CD11b2 DCs (data To achieve conditional ablation of XCR1+ DCs in vivo, we next not shown). However, the majority of Tip-DCs was negative for replaced the coding region of Xcr1 with the DTRvenus cassette venus. To analyze Mo-DCs, mice were injected i.v. with LPS. After (Fig. 4A). The replacement was confirmed by Southern blot anal- 24 h, Mo-DCs, which can be defined as CD206+CD11c+ cells, ysis (Fig. 4B). Xcr1+/DTRvenus mice were born at an expected were increased in the LNs. Although CD2062CD11c+ cells con- Mendelian ratio and did not show any obvious developmental ab- tained venus+ cells, which should correspond to resident CD8a+ normalities. Throughout this study, Xcr1+/DTRvenus mice were used DCs and migratory CD103+ DCs, Mo-DCs were negative for venus as XCR1-DTRvenus mice. (Fig. 3B). Thus, the majority of inflammatory DCs, such as Tip- In XCR1-DTRvenus mice, subsets of CD11c+ DCs and other DCs and Mo-DCs, did not express XCR1. cellularities were similar to those in WT mice, suggesting that the The Journal of Immunology 6075 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Expression of venus in lymphoid and peripheral DCs from XCR1-venus mice. Cells from the indicated lymphoid (A) and peripheral (B, C) tissues in WT or XCR1-venus mice were stained with Abs against the indicated markers. (B) Epidermal LCs and dermal DCs from ear skin were defined as CD207+CD11b+MHC-II+CD11c+ and MHC-II+CD11c+ cells, respectively. (C) MHC-II+CD11c+ cells are shown as lamina propria DCs in the small in- testine. The number in each graph indicates the percentage of venus+ cells. The numbers in the dot plots indicate the percentages of the cells within the indicated gates or each quadrant. C57BL/6J mice were used as WT mice. Data are representative of at least two independent experiments. expression of DTRvenus itself was not toxic to cells or mice. DTRvenus-expressing cells in the spleen (Fig. 4C). DTRvenus- Although the fluorescence intensity of venus in XCR1-DTRvenus expressing CD8a+ DCs were rapidly depleted at days 1 and 2 after mice was lower than that in XCR1-venus mice, the venus-expressing DT injection. Then, DTRvenus-expressing cells began to increase cell population was similar between these two mutant mice (data not around day 4 and had nearly recovered at day 8. Both resident and shown). migratory DTRvenus-expressing DCs were depleted with similar We next assessed the ablation of DTRvenus-expressing cells kinetics in SDLNs and mLNs, as shown by the frequency and after DT injection. XCR1-DTRvenus mice were injected i.p. with absolute cell numbers (Fig. 4D). Other cells, such as B, T, and NK DT (25 ng/g body weight) and monitored for the presence of cells, granulocytes, monocytes, and DTRvenus2 DC subsets, in- 6076 IN VIVO FUNCTIONS OF XCR1-EXPRESSING CELLS Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Expression of venus in DCs induced by bacterial infection or LPS injection. (A) WT or XCR1-venus mice were infected with 5 3 103 L.m.-OVA. Two days later, splenocytes were stained with Abs against the indicated molecules. Tip-DCs, defined as Ly6C+CD11b+MHC-II+CD11c+ cells, and Ly6C2 MHC-II+CD11c+ DCs were monitored for venus expression. (B) WT or XCR1-venus mice were injected i.v. with PBS or 10 mg of LPS. Twenty-four hours later, cells from SDLNs were stained with Abs against the indicated molecules. Mo-DCs, defined as CD206+CD11c+ cells, and CD2062CD11c+ cells were monitored for venus expression. (C) MHC-II+CD11c+ in vitro Mo-DCs were analyzed for venus expression. (D) BM-derived DCs were analyzed for venus expression. In Flt3L-induced BM-derived DCs, pDCs, CD24high DCs, and CD11bhigh DCs were identified as B220+CD11clow,B2202CD24highCD11blow CD11c+, and B2202CD24highCD11blowCD11c+ cells, respectively. Numbers in the dot plots and the graphs represent the percentage of cells within the in- dicated gates and venus+ cells, respectively. Xcr1+/+ littermates (A, C, D) or C57BL/6J (B) mice were used as WT mice. Data are representative of two in- dependent experiments. cluding pDCs and CD11b+ DCs, were unaffected (Fig. 4C, Table I, presenting Ags to T cells. Therefore, we further analyzed venus data not shown). expression in subsets of CD169+ cells in LNs (Fig. 4F). CD169+ In the murine spleen, MF subsets include CD169+ marginal CD11c2 and CD169+CD11cdull cells did not express DTRvenus, but zone metallophilic MFs (MZMMFs) and F4/80+ red pulp MFs. about a half of CD169+CD11chigh cells expressed DTRvenus. The CD169+ MZMMFs are known to contribute to cross-presentation DTRvenus+ cells included MHC-IIhigh and MHC-IIint cells (data not of blood-borne Ags by splenic DCs (41). In the LNs, CD169+ cells shown), which should correspond to migratory and resident cells, are found in the sinus and have the ability to cross-present Ags respectively. All of the DTRvenus+ cells were ablated in DT-treated from dead cells (42). CD169+ cells in the spleen and LNs were XCR1-DTRvenus mice (Fig. 4F). ablated after DT injection to CD11c-DTR mice (43). We then We next examined dsRNA-induced immune responses in XCR1- asked whether MFs in the spleen and sinus CD169+ cells in the DTRvenus mice. In mice, there are two systems to detect dsRNAs, LNs of XCR1-DTRvenus mice were affected by DT injection. including poly(I:C) and viral RNAs. One is endosomal recognition DTRvenus was detected in the T cell area and red pulp of the by TLR3, and the other is cytoplasmic recognition by RIG-I–like spleen and T cell area, sinus, and medulla of the inguinal LNs receptors, including RIG-I and MDA5 (44–47). TLR3 and RIG-I– (Fig. 4E). After DT injection, these DTRvenus+ cells were de- like receptors activate the distinct signaling pathways through the pleted. In contrast, the majority of CD169+ MZMMFs and F4/80+ adaptors TRIF and IPS-1, respectively (48). dsRNA-mediated in- red pulp MFs in the spleen and CD169+ cells in the inguinal LNs duction of proinflammatory cytokines, such as IL-12p40, TNF-a, was present in XCR1-DTRvenus mice after DT injection. According and IL-6, and type I IFNs is dependent on TLR3–TRIF and RLR– to Asano et al. (42), the CD169+CD11c+ cell fraction in LNs shows IPS-1 pathways, respectively (46, 49) (data not shown). TLR3 is a high capacity to ingest dead cells and is responsible for cross- expressed only in CD8a+ DCs among DC subsets (50), and venus+ The Journal of Immunology 6077 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Depletion of XCR1+ cells in XCR1-DTRvenus mice. (A) Schematic diagrams of the mouse Xcr1 WT allele, a targeting vector, and venus knocked-in allele. Filled and open boxes denote coding and noncoding exons of Xcr1, respectively. (B) Southern blot analysis of XCR1 WT (+/+) and Xcr1+/DTRvenus mice. Genomic DNAs were isolated from mice tails, digested with HincII and PstI, electrophoresed, and hybridized with a radiolabeled probe indicated in (A). Southern blot gave a 4.3- and a 3.1-kbp band for WT and knock-in allele, respectively. (C) XCR1-DTRvenus mice were injected i.p. with DT and analyzed on the indicated days. Whole splenocytes were analyzed for the expression of B220 and CD11c, and the percentages of pDCs (B220+ CD11cdull) and conventional DCs (B2202CD11c+) are shown (upper panels). Expression of CD8a and DTRvenus in B2202CD11c+ DCs was analyzed, and the percentages of cells within the indicated quadrants are shown (lower panels). (D) Numbers and percentages of DTRvenus+ cells in the MHC-II+ CD11c+ population from the spleen or in migratory and resident DCs are shown. Cells were prepared from the indicated lymphoid tissues at the indicated days after DT injection. The line connects the mean of each day (n = 6 for days 0, 1, 2; n = 5 for day 4; n = 3 for day 8). (E) Spleen or inguinal LN cryosections from PBS- or DT-treated XCR1-DTRvenus mice were stained with Abs against GFP (DTRvenus, green) and the indicated molecules. Scale bar, 100 mm. (F) LN cells from PBS- or DT-treated XCR1-DTRvenus mice were stained with Abs against the indicated surface molecules. Whole LN cells were gated on CD169+CD11c2 (R1), CD169+CD11clow (R2), and CD169+CD11chigh (R3) and analyzed for DTRvenus expression. Numbers in the dot plots and the graphs indicate the percentages of cells in each gate and DTRvenus+ cells, respectively. Data in (C)–(F) are representative of at least two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. H, HincII; P, PstI. 6078 IN VIVO FUNCTIONS OF XCR1-EXPRESSING CELLS

Table I. Analysis of XCR12 cell population in the spleen of XCR1-DTRvenus mice injected with PBS or DT

% of Whole Splenocytes Cell No.

Cells PBS DT PBS DT T cells 32.5 6 2.79 35.6 6 2.18 3.22 6 0.39 3 107 3.26 6 0.36 3 107 B cells 45.0 6 4.31 49.1 6 1.89 4.44 6 0.49 3 107 4.49 6 0.39 3 107 NK cells 2.93 6 0.31 2.43 6 0.22 2.95 6 0.54 3 106 2.22 6 0.27 3 106 Granulocytes 1.44 6 0.15 1.87 6 0.31 1.54 6 0.50 3 106 1.65 6 0.12 3 106 Monocytes 1.20 6 0.42 0.90 6 0.06 0.95 6 0.49 3 106 1.10 6 0.24 3 106 CD8a2CD11b+ DCs 0.53 6 0.10 0.60 6 0.09 5.59 6 0.83 3 105 5.57 6 0.29 3 105 pDCs 0.57 6 0.03 0.90 6 0.22 6.81 6 0.26 3 105 9.36 6 2.24 3 105 XCR1-DTRvenus mice were injected with PBS or DT. One day after injection, splenocytes were stained with CD3ε, CD11b, CD19, CD115, DX5, and Ly6G. T cells (CD3ε+CD192), B cells (CD3ε2CD19+), NK cells (CD3ε2DX5+), granulocytes (Ly6G+CD11b+), monocytes (Ly6G2CD115+ CD11b+), CD8a2CD11b+ DCs (CD8a2CD11b+MHC-II+CD11c+), and pDCs (PDCA-1+CD11clow) were analyzed by FACS. Data represent the mean 6 SEM (n = 3 or 4). No values were significantly different between PBS- and DT-injected mice. cells were potent IL-12p40–producing cells in response to poly(I: presence or absence of poly(I:C). Seven days postimmunization, C) stimulation (Fig. 1D). Therefore, we measured serum cytokine CD8+ T cell responses in the spleen were monitored by H-2Kb/

levels in XCR1-DTRvenus mice after poly(I:C) injection. The OVA-I peptide tetramer staining and IFN-g production. In PBS- Downloaded from serum cytokine levels were similar in DT-treated XCR1-DTRvenus treated XCR1-DTRvenus mice, H-2Kb/OVA-I tetramer+ and IFN- mice and PBS-treated XCR1-DTRvenus mice (Fig. 5). Thus, g–producing CD62L2CD8+ T cells were increased when immunized XCR1-expressing cells are dispensable for in vivo cytokine in- with poly(I:C). However, the responses were severely diminished in duction by dsRNA. DT-treated XCR1-DTRvenus mice (Fig. 6E–G). Thus, XCR1- expressing cells were crucial for dsRNA-induced CD8+ Tcellre- XCR1-expressing cells were required for cross-presentation of sponses against dead/dying cell–associated Ags. soluble or cell-associated Ags http://www.jimmunol.org/ We next checked dsRNA-induced cross-presentation activity in XCR1- XCR1-expressing cells were required for infection of DTRvenus mice. DT-treated WT mice and PBS- or DT-treated XCR1- L. monocytogenes in the spleen and development of DTRvenus mice were immunized with soluble OVA protein in the L. monocytogenes–specific T cell responses presence or absence of poly(I:C). Seven days postimmunization, CD8+ Analyses of various mutant mice revealed that splenic CD8a+ DCs T cell responses in the spleen were monitored by H-2Kb/OVA-I are crucial for L. monocytogenes to enter into and replicate in the peptide tetramer staining and IFN-g production (Fig. 6A–C). In spleen (23, 51). We then examined the roles of XCR1-expressing both DT-treated WT and PBS-treated XCR1-DTRvenus mice, H-2Kb/ cells in L. monocytogenes infection in XCR1-DTRvenus mice. + +

OVA-I peptide tetramer and IFN-g–producing CD8 T cells were DT- or PBS-treated XCR1-DTRvenus mice were infected i.v. with by guest on September 25, 2021 2 clearly observed in a CD62L activated fraction after immunization the indicated numbers of L.m.-OVA, and bacterial titers were mea- with OVA and poly(I:C) but not with OVA only. However, these sured at day 3 postinfection. Bacterial burden in the spleen was responses were severely impaired in DT-treated XCR1-DTRvenus severely diminished in DT-treated mice compared with PBS-treated mice. On the contrary, after immunization with OVA and poly(I:C), mice (Fig. 7A). Meanwhile, bacterial burden in the liver was com- IFN-g production from CD4+ T cells after restimulation with OVA parable between PBS- and DT-treated XCR1-DTRvenus mice. We protein in vitro was enhanced in DT-treated XCR1-DTRvenus mice then evaluated DTRvenus expression and depletion of DTRvenus+ compared with PBS-treated XCR1-DTRvenus mice (Fig. 6D). Thus, cells in the liver after DT injection to XCR1-DTRvenus mice (Fig. XCR1-expressing cells were essential for dsRNA-induced CD8+,but 7B). DTRvenus was primarily expressed in CD103+MHC-II+CD11c+ not CD4+, T cell responses against soluble Ags. cells, and DTRvenus+ cells were efficiently ablated after DT injec- We further analyzed dsRNA-induced cross-presentation of cell- tion, excluding the possibility that comparable bacterial loads in the associated Ags. PBS- or DT-treated XCR1-DTRvenus mice were liver of DT-injected XCR1-DTRvenus mice was due to incomplete 2/2 + immunized with dying cells from mOVA-Tg:b2m mice in the deletion of hepatic DTRvenus cells, which could be target cells of L. monocytogenes infection. We further evaluated OVA-specific T cell responses (Fig. 7C–F). Expansion of Ag-specific activated T cells was clearly impaired in DT-treated mice compared with PBS-treated mice (Fig. 7C, 7E). IFN-g production from OVA-I peptide–stimulated CD8+ T cells was also severely impaired in DT-treated mice (Fig. 7D, 7F). Thus, XCR1-expressing cells were essential for the establishment of L. monocytogenes infection in the spleen and for the develop- ment of L. monocytogenes–specific CD8+ T cell responses.

Discussion We generated mutant mice by knocking the venus or DTRvenus gene into the Xcr1 locus. In these mice, venus was selectively FIGURE 5. dsRNA-induced cytokine production in XCR1-DTRvenus + mice. XCR1-DTRvenus mice were treated with PBS or DT on days 22 expressed in splenic CD8a DCs and related subsets in lymphoid 2 organs, such as thymus and LNs, and in the peripheral tissues, and 1 and then poly(I:C) (150 mg/mouse) was injected i.p. Sera samples 2 were collected at the indicated times, and the concentration of IFN-a,IFN-b, although it was also expressed in a significant fraction of CD8a 2 2 2 IL-12p40, IL-12p70, TNF-a, and IL-6 was measured. Data are mean 6 SEM CD11b or CD103 CD11b DCs in the thymus or LNs. The 2 2 (n =6). percentages of venus-expressing cells among CD8a CD11b The Journal of Immunology 6079 Downloaded from http://www.jimmunol.org/

FIGURE 6. dsRNA-induced immune responses against soluble (A–D) or cell-associated (E–G) Ags in XCR1-DTRvenus mice. WT or XCR1-DTRvenus mice were injected with PBS or DT on days 21 and 0, immunized s.c. in the footpads with soluble OVA in the presence or absence of 10 mg of poly(I:C), and then analyzed for CD8+ (A–C) or CD4+ (D) T cell responses 7 d later. (A) Numbers indicate the percentages of H-2Kb/OVA-I tetramer+CD62L2 cells in CD49b2venus2CD8+ splenocytes from DT-treated WT, PBS-treated XCR1-DTRvenus, or DT-treated XCR1-DTRvenus mice immunized with soluble OVA plus poly(I:C). (B) Whole splenocytes were further restimulated with (+) or without (-) OVA-I peptide and subjected to intracellular cytokine staining. (C) After in vitro restimulation with OVA-I peptide, IFN-g production was analyzed by intracellular staining. Percentages of IFN-g–producing CD62L2 cells in CD49b-DTRvenus-CD8+ splenocytes gated as in (B) are shown. (D) Whole splenocytes were restimulated with OVA protein and subjected to by guest on September 25, 2021 intracellular cytokine staining. Percentages of IFN-g–producing CD62L2 cells in CD49b2DTRvenus2CD4+ splenocytes are shown. (E–G) XCR1- DTRvenus mice were injected with PBS or DT on days 21 and 0 and immunized i.v. with cell-associated OVA (Cell-OVA) derived from dying mOVA- 2/2 + Tg:b2m cells in the presence or absence of 50 mg of poly(I:C). Seven days later, CD8 T cell responses were analyzed. (E) Numbers indicate the percentages of H-2Kb/OVA-I tetramer+CD62L2 cells in CD49b2venus2CD8+ splenocytes from PBS- or DT-treated XCR1-DTRvenus mice immunized with cell-OVA plus poly(I:C). (F) Whole splenocytes were further restimulated with (+) or without (-) OVA-I peptide and subjected to intracellular cytokine staining. (G) After in vitro restimulation with OVA-I peptide, IFN-g production was analyzed by intracellular staining. Percentages of IFN-g–producing CD62L2 cells in CD49b2venus2CD8+ splenocytes, gated as in (F), are shown. In (C), (D), and (G), each symbol represents an individual mouse, and the horizontal lines represent the means. Data in (A), (B), (E), and (F) are representative of at least three mice. In (C), (D), and (G), data were combined from three independent experiments. *p , 0.05, **p , 0.01. Imm., Immunization.

DCs were highest in the thymus. This might be due to the lower The XCR1+ DCs were efficiently ablated in XCR1-DTRvenus expression of CD8a in the thymic DCs and the difficulty in dis- mice. Conditional ablation of CD8a+ DCs was also achieved in criminating CD8a+CD11b2 DCs from CD8a2CD11b2 DCs. several DTR knock-in mice (52, 53). Clec9A, also known as DC Venus expression was also detected in a CD24high DC subset, NK lectin group receptor-1, is expressed dominantly in CD8a+ which can be generated in vitro with Flt3L from BM cells and is DCs (15). Clec9A-DTR mice were generated by transfecting ES equivalent to splenic CD8a+ DCs. Meanwhile, the other DC cells with recombineered bacterial artificial clones subsets among Flt3L-induced BM DCs, in vitro and in vivo Mo- carrying insertion of human DTR cDNA with its polyA site into DCs, as well as Tip-DCs, failed to show venus expression. Fur- the Clec9a locus (52). Upon injection of DT in Clec9A-DTR thermore, venus was not expressed in CD11c2 cells, including T, mice, splenic CD8a+ DCs can be efficiently depleted, but pDCs B, and NK cells, granulocytes, and monocytes. These expression are also partially depleted. This is consistent with the low ex- patterns are consistent with the previous findings based on the pression of Clec9A in pDCs. CD205, also called DEC-205, is a C- expression pattern of LacZ knocked into the Xcr1 locus or type lectin–like molecule that is expressed abundantly in CD8a+ analysis with anti-XCR1 Ab (26, 27, 30, 31). Twenty to thirty DCs (9, 54). CD205-DTR enhanced GFP (EGFP) mice were percent of splenic CD8a+ DCs did not express venus. We found generated by inserting an internal ribosome entry site followed by that venus+CD8a+ DCs produced much greater amounts of cy- cDNA encoding human DTR fused to EGFP into the Cd205 locus tokines than did venus2CD8a+ DCs in response to TLR agonists, (53). Although DT injection in CD205-DTREGFP mice depleted which indicates that XCR1+ DCs are more mature than XCR12 splenic CD8a+ DCs efficiently, it also depleted CD205-expressing DCs and that functional CD8a+ DCs can be tracked in our mutant cells in CD8a2 DCs. Furthermore, the irradiated WT mice recon- mice. It is also notable that the XCR1 expression level was un- stituted with BM of CD205-DTREGFP mice had to be analyzed, changed after injection of LPS or poly(I:C) (Fig. 3B, data not because CD205-DTREGFP mice die within 10 d after DT injection, shown). probably as a result of DTR expression on certain radioresistant 6080 IN VIVO FUNCTIONS OF XCR1-EXPRESSING CELLS Downloaded from http://www.jimmunol.org/

FIGURE 7. Immune responses against L.m.-OVA infection in XCR1-DTRvenus mice. XCR1-DTRvenus mice received PBS or DT at days 21 and 0 of A L.m.-OVA infection. ( ) Mice were infected i.v. with the indicated numbers of L.m.-OVA. Three days postinfection, bacterial load in the spleen or liver was by guest on September 25, 2021 evaluated. (B) Depletion of liver DTRvenus-expressing cells. XCR1-DTRvenus mice were injected with PBS or DT at days 21 and 0. One day after the last injection, liver leukocytes were prepared and stained with the indicated Abs. MHC-II+CD11c+ cells were gated, and their expression profiles of CD103 and DTRvenus are shown (left panels). Numbers represent the percentages of the cells in each quadrant. Percentages of DTRvenus+ cells in MHC-II+CD11c+ cells are also shown (right panels). PBS- or DT-treated XCR1-DTRvenus mice were infected i.v. with 1 3 103 (C, D) or the indicated numbers (E, F)ofL.m.- OVA and analyzed for CD8+ T cell responses 7 d later. (C and E) H-2Kb/OVA-I tetramer+ cells in CD49b2DTRvenus2CD8+ splenocytes were monitored by FACS. (E) The percentages of H-2Kb/OVA-I tetramer+CD62L2 cells in CD49b2DTRvenus2CD8+ splenocytes, gated as in (C), are shown. (D) Whole splenocytes were further restimulated with (+) or without (-) OVA-I peptide and subjected to intracellular cytokine staining. (F) After in vitro restimulation with OVA-I peptide, IFN-g production was analyzed by intracellular staining. The percentages of IFN-g–producing CD62L2 cells in CD49b2DTRvenus2 CD8+ splenocytes, gated as in (D), are shown. Data in (C)and(D) are representative of six PBS-treated and three DT-treated mice, respectively. Data were combined from three (A, E, F)ortwo(B) independent experiments. In (A), (B), (E), and (F), each symbol represents an individual mouse, and the horizontal lines represent the means. *p , 0.05, **p , 0.01, ***p , 0.001. cells (53). Meanwhile, the XCR1-DTRvenus mice were viable after of type I IFNs by dsRNA was retained in DT-treated XCR1- injection of up to 100 ng/g body weight of DT (data not shown). DTRvenus mice. This is consistent with the finding that splenic Langerin is expressed in splenic CD8a+ DCs and subsets of CD8a+ DCs show faint expression of cytosolic dsRNA sensors that resident/migratory LNs and dermal DCs, as well as LCs in the can lead to type I IFN production (50). Furthermore, induction of epidermis (8, 55–58). Langerin+ LN and dermal DCs contain both proinflammatory cytokines, including IL-12p40, which depends on CD103+ and CD1032 DCs (59). A cDNA encoding a fusion pro- a TLR3-TRIF axis, was also preserved in DT-treated XCR1- tein consisting of DTR and EGFP was knocked into the Langerin DTRvenus mice, although DTRvenus+CD8a+ DCs are mainly locus to generate Langerin-DTREGFP mice (8, 55). Langerin+ responsible for dsRNA-induced IL-12p40 production among DC DCs, including both XCR1+ DCs (dermal and LN CD103+ DCs) subsets. It can be assumed that TLR3-expressing DTRvenus2 cells and XCR12 DCs (dermal and LN CD1032 DCs and LCs), can be compensate for dsRNA-induced production of proinflammatory depleted upon DT injection in Langerin-DTREGFP mice (8, 55, cytokines in vivo. Thus, XCR1-expressing cells were dispensable 56). Thus, each mouse shows a distinct pattern of cell ablation. In for dsRNA-induced cytokine induction in vivo. terms of cell tracking, fluorescence protein expression can be Type I IFN is required for optimal induction of cross-priming monitored in XCR1-DTRvenus and Langerin-DTREGFP mice but (24, 37, 60). In DT-treated XCR1-DTRvenus mice, dsRNA- not in Clec9A-DTR or CD205-DTREGFP mice. Thus, XCR1- induced CD8+ T cell responses were impaired under the con- DTRvenus mice are useful mutant mice for analyzing splenic ditions in which the cytokines are normally induced. This suggests CD8a+ DCs and their relatives. that dsRNA-stimulated XCR12 DCs fail to induce CD8+ T cell Using XCR1-DTRvenus mice, we clarified the roles of XCR1- responses, even in the presence of cytokines, including type I expressing cells in dsRNA-induced immune responses. Induction IFNs. Notably, dsRNA-induced CD4+ T cell responses were en- The Journal of Immunology 6081 hanced in DT-treated XCR1-DTRvenus mice. CD4+ T cell responses cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. should be provoked by other APCs that can be activated by cytosolic Immunity 17: 211–220. 2 3. Zammit, D. J., L. S. Cauley, Q. M. Pham, and L. Lefranc¸ois. 2005. Dendritic sensors or type I IFNs. CD8a DCs should be the candidate APCs, cells maximize the memory CD8 T cell response to infection. 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