Conservation of a Chemokine System, XCR1 and Its Ligand, XCL1, Between Human and Mice

Biochemical and Biophysical Research Communications 397 (2010) 756–761

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Biochemical and Biophysical Research Communications

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Conservation of a chemokine system, XCR1 and its ligand, XCL1, between human and mice

Chihiro Yamazaki a,c,1, Rie Miyamoto b,1, Katsuaki Hoshino a, Yuri Fukuda a, Izumi Sasaki a,c, Masuyoshi Saito a, Hironori Ishiguchi a, Takahiro Yano a, Takahiro Sugiyama a, Hiroaki Hemmi a, Takashi Tanaka d, Eri Hamada a,e, Takeshi Hirashima a, Ryuichi Amakawa b, Shirou Fukuhara b, Shosaku Nomura b, Tomoki Ito b,*, Tsuneyasu Kaisho a,c,e,** a Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunology, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan b First Department of Internal Medicine, Kansai Medical University, Fumizono 10-15, Moriguchi, Osaka 570-8506, Japan c Department of Allergy and Immunology, Graduate School of Medicine, Osaka University, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan d Laboratory for Inﬂammatory Regulation, RIKEN Research Center for Allergy and Immunology, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan e Department of Supramolecular Biology, International Graduate School of Bionanoscience, Yokohama City University, Suehiro-cho 1-7-29, Tsurumi-ku, Kanagawa 230-0045, Japan article info abstract

Article history: Understanding dendritic cell (DC) subset functions should lead to the development of novel types of vac- Received 2 June 2010 cine. Here we characterized expression of XC chemokine receptor 1 (XCR1) and its ligand, XCL1. Murine Available online 10 June 2010 XCR1 was the only chemokine receptor selectively expressed in CD8a+ conventional DCs. XCL1 was constitutively expressed in NK cells, which contribute to serum XCL1 levels. NK and CD8+ T cells increased Keywords: XCL1 production upon activation. These expression patterns were conserved in human blood cells, Chemokine including the BDCA3+ DC subset. Thus, in human and mice, certain DC subsets should be chemotactic Dendritic cells towards NK or activated CD8+ T cells through XCR1. XCR1 Ó 2010 Elsevier Inc. All rights reserved. XCL1

1. Introduction tionally regulated should contribute to the development of novel immunoregulatory reagents. Dendritic cells (DCs)2 sense immune adjuvants through patho- We found that Xcr1 was the only chemokine receptor gene that gen sensors and are critical for linking innate and adaptive immunity is highly expressed in CD8a+ cDC (Table 1). We examined how the [1,2]. DCs consist of various subsets including plasmacytoid DC chemokine system, XCR1 and its ligand, is conserved in human and (pDC) or conventional DC (cDC) and exhibit subset-specific functions mice. [3]. One cDC subset, CD8a+ cDC, is characterized by high ability to + ingest necrotic cells and to cross-present antigens to CD8 T cells 2. Materials and methods [4]. Notably, CD8a+ cDC is the only DC subset that detects double- stranded RNA through Toll-like receptor (TLR) 3 [5,6]. TLR3 signaling 2.1. Mice can induce inflammatory cytokines and augment cross-presenting + + activity in CD8a cDC [7]. In human, BDCA3 DC corresponds to mur- C57BL/6J mice (CLEA Japan) were maintained under the specific + ine CD8a cDC according to expression of certain genes including a pathogen free conditions in the RIKEN animal facility and utilized C-type lectin, CLEC9A [8–10]. Understanding how this subset is func- under institutional guidelines.

2.2. Cell sorting * Corresponding author. Fax: +81 6 6994 8344. ** Corresponding author at: Laboratory for Host Defense, RIKEN Research Center Antibodies were described in Supplementary Table 1. All the for Allergy and Immunology, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, Kanag- awa 230-0045, Japan. Fax: +81 45 503 7064. sorting was performed with FACSAria or FACSVantage (BD E-mail addresses: [email protected] (T. Ito), [email protected] (T. Kaisho). Biosciences). 1 Chihiro Yamazaki and Rie Miyamoto contributed equally to this work. 2 Abbreviations used: DCs, dendritic cells; pDC, plasmacytoid DC; cDC, conventional 2.3. Gene chip analysis DC; mDC, myeloid DC; PBMC, peripheral blood mononuclear cells; XCR1, XC chemokine receptor 1; IFN, interferon; TLR, Toll-like receptor; MEF, mouse embryonic ﬁbroblast; poly(I:C), polyinosinic:polycytidylic acids; ODN, oligonucleotides; PMA, Splenic single cell suspensions were prepared by density phorbol 12-myristate 13-acetate. gradient centrifugation using Histopaque-1083 (Sigma). Flt3L- or

Table 1 2216 (InvivoGen) at 2 Â 104 cells/100 ll for 24 h. Cells were Expression of chemokine receptors in splenic DC subsets. stained with FITC-labeled CD86 (2331, BD Biosciences) and ana- Probe ID Accession pDC CD8a+ CD8aÀ Common lyzed by FACSCalibur (BD Biosciences). number DC DC name Blood cells were sorted into CD4+ T, CD8+ T, B, and NK cells as 1419609_at AV231648 0.6 4.7 1.5 Ccr1 described in Supplementary data, and cultured for 24 h with med- 1419610_at AV231648 0.2 1.5 0.3 Ccr1 ium alone, 2 lg/ml precoated CD3 antibody (OKT3) + 1 lg/ml 1421187_at BB148128 40.6 27.7 27.4 Ccr2 CD28 antibody (28.2), 25 ng/ml phorbol 12-myristate 13-acetate 1421186_at BB148128 24.1 15.0 16.2 Ccr2 (PMA) + 1 lg/ml Ionomycin (IONO), 100 U/ml IFN-a, 10 ng/ml IL- 1421188_at BB148128 18.9 11.9 8.4 Ccr2 1460067_at BB324415 3.3 2.1 2.0 Ccr2 12, 10 or 100 ng/ml IL-2, 50 ng/ml IL-4 + 10 lg/ml anti-human 5 1422957_at NM_009914 0.6 0.1 0.4 Ccr3 IgM Ab (KPL), or 1 lg/ml LPS (InvivoGen) at 5 Â 10 /500 ll in 48- 1421655_a_at NM_009916 0.2 0.5 0.2 Ccr4 well plates. 1424727_at D83648 67.2 10.3 5.5 Ccr5 1422259_a_at X94151 31.5 4.6 1.6 Ccr5 1422260_x_at X94151 37.2 2.7 1.8 Ccr5 2.7. DC transwell migration assay 1450357_a_at NM_009835 3.4 2.3 13.9 Ccr6 1423466_at BB204380 1.8 97.7 71.6 Ccr7 One million of murine Flt3L-induced BM DCs and 105 of human 1422291_at NM_007720 0.0 0.0 0.1 Ccr8 bulk DCs were placed into the upper chamber of a 24- and 96-well 1421920_a_at NM_009913 146.1 15.5 3.0 Ccr9 1421919_a_at NM_009913 143.5 14.4 2.4 Ccr9 plate of Transwell (5.0 lm pore size) (Corning), respectively. The 1427419_x_at AJ131357 73.6 7.8 0.4 Ccr9 lower chamber was filled with RPMI1640 with 10% FCS containing 1440432_at BB314873 2.1 1.5 1.3 Ccr9 murine XCL1, human XCL1, CCL3 or CXCL12 (all chemokines from 1442758_at BE691372 7.7 1.2 0.7 Ccr9 R&D Systems) and the cells were cultured for 2 h (mice) or 3 h (hu- 1450019_at BC012653 0.1 4.3 4.0 Cx3cr1 1450020_at BC012653 0.0 3.9 6.6 Cx3cr1 man). Cells in the lower chamber were analyzed with FACSCalibur. 1449925_at NM_009910 56.2 19.3 3.8 Cxcr3 1448710_at D87747 88.0 96.5 96.5 Cxcr4 2.8. Gene expression analysis 1425832_a_at AF301018 0.9 0.5 1.2 Cxcr6 1422812_at NM_030712 0.5 0.4 0.6 Cxcr6 1422294_at NM_011798 0.4 57.7 2.6 Xcr1 Quantitative RT-PCR (qRT-PCR) was performed as described in Supplementary data. PDCA-1+B220+CD11c+, CD11c+B220ÀCD8+, and CD11c+B220ÀCD8À cells in the spleen were sorted as pDC, CD8a+ cDC, and CD8aÀ cDC, respectively. The data have been deposited in RCAI RefDIC (Sample ID: pDC, RSM01658; CD8a+ cDC, RSM01586; 2.9. Measurement of cytokine production CD8aÀ cDC, RSM01659). The production of murine XCL1, murine IFN-c, human IFN-a, and human XCL1 in the culture supernatants was determined by ELISA (PBL Biomedical Laboratories for human IFN-a, R&D Systems GM-CSF-induced bone marrow (BM) DC or mouse embryonic fibro- for the other cytokines). The production of human IL-12p70 was blasts (MEF) from day 11.5–14.5 embryos were generated as de- determined by Cytometric Beads Array (BD Biosciences). scribed previously [11–13]. For gene chip analysis, see Supplementary data. 3. Results and discussion 2.4. Murine NK and T cell culture 3.1. A CD8a+ cDC specific chemokine receptor, XCR1 Murine splenic CD8+ T cells and NK cells were prepared as described in Supplementary data (Fig. 3D). The cells were cultured Several chemokine receptors are differentially expressed in DC for 24 h with medium alone, 1 lg/ml precoated anti-CD3e mAb subsets and play critical roles in DC subset functions. For example, (clone: 145-2C11) with 1 lg/ml precoated anti-CD28 mAb CCR9 is selectively expressed in pDC and crucial for homing to the (37.51), 2 ng/ml IL-12 (R&D Systems) and/or 20 ng/ml IL-18 small intestine [14]. We first performed gene chip analysis on sple- (MBL), 300 ng/ml IL-15 (Peprotech), 100 ng/ml IL-2 (GT), 1000 U/ nic DC subsets. Xcr1 was the only chemokine receptor gene ex- ml IFN-aA (PBL Biomedical Laboratories) at 1 Â 105/200 ll in 96- pressed abundantly in CD8a+ cDC (Table 1) [15]. BM DC can be well plates. generated by GM-CSF or Flt3L. GM-CSF-induced BM DCs do not NK1.1+, NK1.1ÀCD11cÀCD8a+, and NK1.1ÀCD11cÀCD4+ cells contain any equivalents to CD8a+ cDC. Meanwhile, Flt3L-induced were sorted as NK, CD8a+ T, and CD4+ T cells, respectively BM DCs include pDC, CD24high cDC and CD11bhigh cDC, correspond- (Fig. 3E). Then cells were cultured with 100 ng/ml IL-2 or with pre- ing to splenic pDC, CD8a+ cDC, and CD8aÀ cDC, respectively [16]. coated 10 lg/ml anti-CD3e mAb and 1 lg/ml anti-CD28 mAb at As in the case of splenic DC subsets, Xcr1 was the only chemokine 2 Â 105/150 ll in 96-well plates. receptor gene substantially expressed in CD24high cDC (Supple- mentary Table 3). We then examined Xcr1 gene expression in var- 2.5. NK cell depletion in vivo ious splenocytes by qRT-PCR (Fig. 1A). Xcr1 was specifically expressed in CD8a+ cDC, consistent with the microarray analysis. Mice were injected intraperitoneally with rabbit anti-asialo To verify whether XCR1 functions in DCs, Flt3L-induced BM DCs GM1 polyclonal Ab (Wako Pure Chemical) or normal rabbit serum were subjected to transwell migration assay (Fig. 1B). CD24high (Wako Pure Chemical). After 24 h, mice were bled and serum XCL1 cDCs, but not pDC or CD11bhigh cDCs, migrated to XCL1 in a levels were measured by ELISA. dose-dependent manner.

2.6. Human cell preparation and culture 3.2. Human XCR1 is highly expressed in BDCA3+ DC

Human DC subsets were prepared as described in Supplemen- Human peripheral blood includes BDCA4+ pDCs and myeloid tary data. Cells were cultured for 24 h with medium alone, DCs (mDCs), including CD11c+BDCA3+ DCs (BDCA3+ DCs) and 10 lg/ml polyinosinic:polycytidylic acids [poly(I:C)] (InvivoGen), CD11c+BDCA3À DCs (BDCA3À DCs) (Fig. 1C). qRT-PCR analysis 1 lg/ml R848 (InvivoGen), or 5 lM CpG oligonucleotides (ODNs) revealed that XCR1 was selectively expressed in BDCA3+ DCs 758 C. Yamazaki et al. / Biochemical and Biophysical Research Communications 397 (2010) 756–761

Fig. 1. Murine and human XCR1 expression and function in DC subsets. (A) Indicated cells in the murine spleen were sorted. For CD8+ and CD4+ T cells, CD11cÀCD3+CD4ÀCD8+ and CD11cÀCD3+CD4+CD8À cells were sorted, respectively. These cells were subjected to qRT-PCR. Data are shown as mean ± SD. (B) Murine Flt3L-induced BM DCs were subjected to transwell migration assay. The migrated cells in lower chambers were analyzed by FACS. (C) Human blood DC subsets were sorted as indicated and stained by May-Giemsa solution. Original magniﬁcation, 100Â. (D) Indicated human cells were subjected to qRT-PCR. Data are shown as mean ± SD. (E) Transwell migration assay of sorted human blood bulk DCs was performed as described in (B). (F) The percentages of migrated DC subsets per input cells were calculated (numbers of migrated cells/ numbers of input cells Â 100). Data are shown as mean ± SEM of three independent experiments.

(Fig. 1D). Purity of BDCA3+ DCs was veriﬁed by selective expression 3.3. Characterization of human BDCA3+ DC of CLEC9A. To examine whether XCR1 expression in BDCA3+ DCs is functional, we next performed a transwell migration assay. From In mice, CD8a+ cDCs express TLR3 and TLR9, but not TLR7 [5]. total blood DCs (lineageÀ CD4+ population containing 4% BDCA3+ We tested whether the nucleic acid sensing TLRs, including TLR3, DCs, 55% BDCA3À DCs, and 44% pDCs) (Fig. 1E; ‘‘input bulk DCs”), TLR7, TLR8, and TLR9, are expressed and functional in BDCA3+ BDCA3+ DCs substantially migrated upon addition of increasing DCs [17]. BDCA3+ DCs expressed TLR3 and TLR8, but neither TLR7 amounts of recombinant XCL1 (Fig. 1E and F). XCL1 failed to induce nor TLR9 (Fig. 2A). Although TLR8 was equivalently expressed in migration of the other two DC subsets, although CCL3 and CXCL12 both two mDC subsets, TLR3 was more abundantly expressed in enhanced the migration of BDCA3À DCs and pDCs, respectively. BDCA3+ DCs than BDCA3À DCs. Consistent with the gene expres- These ﬁndings indicate a selective migration of BDCA3+ DCs sion, BDCA3+ DCs upregulated CD86 expression in response to a through their functional XCR1 expression. TLR3 agonist, poly(I:C), and a TLR7/8 agonist, R848, but not to a C. Yamazaki et al. / Biochemical and Biophysical Research Communications 397 (2010) 756–761 759

Fig. 2. Responses of BDCA3+ DC to nucleic acid TLR ligands. (A) TLR3, 7, 8, and 9 expression in isolated human blood DC subsets and CD4+ T cells were analyzed by qRT-PCR. (B) BDCA3+ DCs were cultured with the medium alone, poly(I:C), R848, and CpG2216. After 24 h, CD86 expression was analyzed by ﬂow cytometry. The results of a representative data in three independent experiments are shown. (C) After 24 h culture with different stimuli, IFN-a and IL-12 p70 levels in the culture supernatants of each DC subsets were measured. Data are shown as mean ± SEM of three independent experiments. n.d., not detected.

Fig. 3. Murine XCL1 expression. (A, B) Splenic cells were sorted and subjected to qRT-PCR. Data are shown as mean ± SD. (C) Mice were injected with anti-asialo GM1 Ab (n = 3) or normal rabbit serum (n = 5). Serum XCL1 levels were measured by ELISA. Means are shown by bars. Student’s t test was used to determine statistical signiﬁcance between groups. (D) CD8+ T or NK cells were unstimulated or stimulated for 24 h with anti-CD3e and anti-CD28 mAbs, IL-12 and/or IL-18, IL-15, IL-2 or IFN-a. XCL1 and IFN-c levels in the culture supernatants were measured by ELISA. (E) NK or CD4+ cells were stimulated with IL-2. CD4+ or CD8+ T cells were stimulated with immobilized anti-CD3e and anti-CD28 mAbs. At 2, 6, 12, or 24 h after stimulation, cells were harvested and subjected to qRT-PCR. Data are shown as mean ± SD. Error bars are smaller than the plot symbols. 760 C. Yamazaki et al. / Biochemical and Biophysical Research Communications 397 (2010) 756–761

TLR9 agonist, CpG ODN (Fig. 2B). In addition, BDCA3+ DCs re- failed to increase IFN-c production. Meanwhile, IL-2 could not ele- sponded to poly(I:C) and R848 by producing IFN-a and IL-12 vate XCL1 production from CD8+ T cells. (Fig. 2C). Of note, BDCA3+ DCs produced larger amounts of IFN-a Next, kinetics of Xcl1 induction was tested. Xcl1 expression in than BDCA3À DCs in response to poly(I:C). Thus, TLR3 is the only NK cells was augmented at 6 h after the addition of IL-2 (Fig. 3E). nucleic acid sensing TLR that are expressed in both murine Activation by anti-CD3/28 Abs signiﬁcantly upregulated Xcl1 gene CD8a+ cDC and human BDCA3+ DC. expression from CD8+, but not CD4+ T cells (Fig. 3E). This induction was more rapid and prominent than that of IL-2-stimulated NK 3.4. Murine XCL1 expression cells.

We next tested expression of an XCR1 ligand, XCL1, in wildtype 3.5. Human XCL1 expression splenocytes. CD11c+B220+ cells dominantly expressed Xcl1 + + (Fig. 3A). CD11c B220 cells include not only pDC but also NK cells. + + We then prepared purified human CD4 T, CD8 T, NK, and B To distinguish this, further sorting analysis was performed. Xcl1 cells and measured XCL1 and XCL2 expression (Fig. 4A). XCL1 and gene expression was detected in Gr-1À, DX5+, NK1.1+, and PDCA- À XCL2 were highly expressed in unstimulated NK cells. We further 1 cells (Fig. 3B), indicating that NK cells, but not pDC, are respon- examined XCL1 production from those cells with various stimuli sible for Xcl1 expression. To clarify in vivo roles of NK cells, NK cells (Fig. 4B). NK cells showed enhanced production of XCL1 in re- were depleted in mice by the treatment with anti-asialo GM1 Ab sponse to IL-2, but not to IFN-a or IL-12. CD8+, but not CD4+, T cells (Fig. 3C). In the spleen, NK cell percentages from anti-asialo increased XCL1 production upon activation with anti-CD3/28 Abs. GM1-treated mice (n = 3, 0.17 ± 0.02%) were decreased compared PMA and IONO increased XCL1 production much more promi- with those from control mice (n = 5, 1.52 ± 0.64%). Serum XCL1 lev- nently from CD8+ T cells than from CD4+ T cells, indicating that els were also significantly decreased after the treatment, indicating CD8+ T cells intrinsically possess high ability to produce XCL1. that NK cells contribute to in vivo XCL1 production. We further Meanwhile, B cells failed to produce XCL1 even after activation examined the effects of various cytokines on XCL1 production from + with anti-IgM + IL-4 or LPS. Thus, expression pattern of XCL1 as NK and CD8 T cells (Fig. 3D). Only IL-2 could increase XCL1 pro- well as XCR1 is conserved in human and mice. duction from NK cells in a dose-dependent manner, although it Murine XCR1 was selectively expressed on a splenic CD8a+ cDC among various immune cells, consistent with the previous report [18]. Among Flt3L-induced BM DC subsets, only CD24high cDC, which is analogous to CD8a+ cDC [16,19], expressed functional XCR1. An XCR1 ligand, XCL1 [20–23], was constitutively highly expressed in NK cells and in vivo XCL1 production depended on NK cells. XCL1 production from NK cells was increased by IL-2, but not by other stimuli. Furthermore, XCL1 production was promi- nently induced in activated CD8+, but not CD4+, T cells. These expression patterns in mice were conserved in human, implying the importance of this chemokine system in innate and adaptive cytotoxic responses. Our data, together with the recent findings [24–27], would provide useful insights for the immune-based ther- apeutic strategy using XCR1 or its ligand.

Acknowledgments

We thank N. Iwami and E. Haga for technical assistance and S. Haraguchi for secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and Japan Society for the Promotion of Sci- ence (JSPS), the Takeda Science Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and Japan Intractable Diseases Research Foundation. C.Y. and I.S. are RIKEN Junior Research Associates.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.06.029.

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