Allograft Tolerance CXCL16/CXCR6 in NKT

Cutting Edge: Critical Role of CXCL16/CXCR6 in NKT Cell Trafficking in Allograft Tolerance

This information is current as Xiaofeng Jiang, Takeshi Shimaoka, Satoshi Kojo, of October 2, 2021. Michishige Harada, Hiroshi Watarai, Hiroshi Wakao, Nobuhiro Ohkohchi, Shin Yonehara, Masaru Taniguchi and Ken-ichiro Seino J Immunol 2005; 175:2051-2055; ; doi: 10.4049/jimmunol.175.4.2051 http://www.jimmunol.org/content/175/4/2051 Downloaded from

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JOURNAL OF IMMUNOLOGY CUTTING EDGE

Cutting Edge: Critical Role of CXCL16/CXCR6 in NKT Cell Trafficking in Allograft Tolerance1 Xiaofeng Jiang,*† Takeshi Shimaoka,‡ Satoshi Kojo,* Michishige Harada,* Hiroshi Watarai,* Hiroshi Wakao,* Nobuhiro Ohkohchi,† Shin Yonehara,‡ Masaru Taniguchi,* and Ken-ichiro Seino2*

It is well-documented that certain chemokines or their re- ease development (6, 7) and anterior chamber-associated im- ceptors play important roles in the graft rejection. How- mune deviation (ACAID) (8). However, the in vivo behavior of ever, the roles of chemokines and their receptors in the NKT cells in such immune-regulatory responses has not been maintenance of transplantation tolerance remain un- fully understood.

clear. In this study, we demonstrate that blocking of the It has been known that CXCR6, one of the chemokine re- Downloaded from interaction between the chemokine receptor, CXCR6, ceptors, is expressed at a high level on NKT cells even under ؉ highly expressed on V␣14 NKT cells and its ligand, physiological conditions as compared with other lymphocytes (9–11). Its ligand, CXCL16, is identical to the scavenger recep- CXCL16, resulted in the failure to maintain graft toler- tor that binds phosphatidylserine and oxidized lipoprotein (SR- ance and thus in the induction of acceleration of graft re- PSOX), mainly expressed in macrophages and dendritic cells jection. In a mouse transplant tolerance model, the expres-

(9, 12–14). Interestingly, CXCL16 has unique structural prop- http://www.jimmunol.org/ sion of CXCL16 was up-regulated in the tolerated erties, such as membrane-anchored protein with a mucin stalk, allografts, and anti-CXCL16 mAb inhibited intragraft like fractalkine, and strict specificity to bind to CXCR6, al- accumulation of NKT cells. In vitro experiments further though other cytokines are generally released as a soluble form showed that blocking of CXCL16/CXCR6 interaction sig- and bind to more than one receptor (12). Similar to fractalkine, nificantly affected not only chemotaxis but also cell adhe- CXCL16 can also work as a soluble factor after cleaving by pro- sion of NKT cells. These results demonstrate the unique teolytic enzymes (9). role of CXCL16 and CXCR6 molecules in the mainte- Regarding their in vivo function, it has been reported that CXCL16-mediated CXCR6 activation is involved in patho-

nance of cardiac allograft tolerance mediated by NKT by guest on October 2, 2021 cells. The Journal of Immunology, 2005, 175: genic aortic smooth muscle cell proliferation or the develop- 2051–2055. ment of experimental autoimmune encephalomyelitis (15, 16). However, the impact of CXCL16 and CXCR6 in in vivo NKT cell function has not been elucidated. Thus, it is interesting to ϩ he V␣14 NKT cell is one of the immune regulatory investigate the role of CXCL16 and CXCR6 molecules in the cells expressing an invariant AgR ␣-chain encoded by a NKT cell-mediated transplantation tolerance, because various T V␣14-J␣281 gene segment (1). They recognize glyco- other chemokines and their receptors have been shown to play lipids such as ␣-galactosylceramide (␣-GalCer)3 presented by important roles in allogenic immune responses (17). CD1d (1). It has been reported that they play an important im- In this study, we examined the role of CXCL16/CXCR6 in- mune-regulatory role in the maintenance of transplant toler- teraction on NKT cell function in a murine cardiac transplant ance (2–4). In organ transplantation, when T cell costimula- model. The data indicate that, different from other chemokine/ tory signals through CD40L or CD28 are blocked, peripheral receptors, CXCL16/CXCR6 is prerequisite for the mainte- transplant tolerance can be induced in wild-type (WT) recipi- nance of transplant tolerance mediated by NKT cells. ents (5). However, the same treatment failed to maintain transplantation tolerance in NKT cell-deficient (NKT knockout Materials and Methods (KO)) recipients, indicating that, without NKT cells, periph- Mice eral tolerance induced by costimulation blockade could not be WT C57BL/6J (B6) (H-2b) and BALB/c (H-2d) mice were purchased from maintained (2). In addition to transplant tolerance, NKT cells Japan CLEA. NKT cell-deficient (B6.129S-J␣281tm, NKT KO) mice were have also been involved in the regulation of autoimmune dis- backcrossed to B6 mice by nine generations. All mice were bred and maintained

*Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunol- 1 This work was in part supported by a Grant-in Aid for Scientific Research from the Min- ogy, Kanagawa, Japan; †Department of Surgery, Institute of Clinical Medicine, University istry of Education, Culture, Sports, Science and Technology of Japan. of Tsukuba, Ibaraki, Japan; and ‡Graduate School of Biostudies and Institute for Virus 2 Address correspondence and reprints to Dr. Ken-ichiro Seino, Laboratory for Immune Research, Kyoto University, Kyoto, Japan Regulation, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Received for publication April 15, 2005. Accepted for publication June 1, 2005. Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan. E-mail address: seino@ rcai.riken.jp The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance 3 Abbreviations used in this paper: ␣-GalCer, ␣-galactosylceramide; WT, wild type; KO, with 18 U.S.C. Section 1734 solely to indicate this fact. knockout; MNC, mononuclear cell.

in our animal facilities under specific pathogen-free conditions and used at Results and Discussion 6–10 wk. The animals were used in accordance with the guidelines of the Effects of CXCL16 blockade on allograft tolerance RIKEN Research Center for Allergy and Immunology and the animal commit- tee of RIKEN approved the experiments. Treatment of B6 WT recipients with anti-CD40L mAb in- Reagents duced long-term BALB/c cardiac allograft survival, since 10 of 11 animals survived over 100 days (Fig. 1). In contrast, in B6 Fluorescence-conjugated or unconjugated mAbs were purchased from BD Bio- NKT KO recipients, only 3 of 11 allografts survived over 100 sciences. CXCL16-Fc fusion protein and control Fc were generated as described ϭ Ͻ previously (9). Rat IgG was purchased from Serotec. Anti-mouse CXCL16 days (n 11, p 0.01 vs anti-CD40L treated WT recipients, mAb (rat IgG1) was generated as described previously (18). Mouse CXCL16- Fig. 1). Interestingly, injection of anti-CXCL16 mAb to the specific polyclonal goat IgG and goat control IgG used for immunohistochem- WT recipients significantly shortened the allograft survival ical staining were purchased from R&D Systems. ␣-GalCer was kindly pro- ϭ vided by Kirin Brewery. time, similar levels to that observed in NKT KO recipients (n 5, p Ͻ 0.01 vs control Ab-treated recipients, Fig. 1). These re- Heart transplantation sults suggest that anti-CXCL16 mAb affected NKT cell traf- WT BALB/c or B6 mice were used as donors and WT or NKT KO B6 mice ficking in the recipients and impaired the graft acceptance. were used as recipients. Intra-abdominal heterotopic heart transplantation was performed according to the technique previously described (19). Rejection was Intragraft accumulation of NKT cells mediated by CXCL16/CXCR6 defined as a complete cessation of palpable beat and was confirmed by direct visualization after laparotomy. For tolerance induction, the recipients were i.v. To understand the cellular events in the graft rejection induced injected 0.25 mg/mouse anti-CD40L mAb on day 0 and i.p. injected on days 2 by anti-CXCL16 mAb, we attempted to quantify the accumu-

and 4 posttransplant. Anti-CXCL16 mAb or control Ab (0.2 mg/mouse) was lation of NKT cells and the expression of CXCL16 in the trans- Downloaded from i.p. injected daily for 14 days posttransplant and every 5 days until 40 days planted cardiac allografts. The grafts were harvested from thereafter or until rejection was complete. For analyses of real-time PCR, im- munohistochemistry, and in situ hybridization, the grafts or spleens were har- groups treated with or without anti-CXCL16 mAb and were vested as follows: in the syngenic group and the group with treatment of anti- estimated for amounts of the invariant V␣14-J␣281 and CD40L mAb and control Ab (rat IgG), they were harvested 60 days after CXCL16 mRNAs by a quantitative real-time RT-PCR. The ex- transplant; and in the group with treatment of anti-CD40L plus anti-CXCL16 pression of CXCL16 in the cardiac allografts was higher both in mAbs, the grafts and spleens were obtained at the time point of rejection or 60 days after transplant. the groups treated with and without anti-CXCL16 mAb than http://www.jimmunol.org/ in the syngenic grafts (p Ͻ 0.05, Fig. 2). In contrast, the expres- Real-time RT-PCR analysis sion of V␣14-J␣281 mRNA was significantly higher in the ac- At the indicated time points, cardiac allografts were harvested and total RNA cepted cardiac allografts than that in syngenic grafts (p Ͻ 0.05, was extracted using Isogen (Nippon Gene). After cDNA synthesis, the quantity Fig. 2). These results indicate that allogeneic heart transplanta- ␣ ␣ of mRNA for rearranged V 14-J 281 and CXCL16 was examined with the tion plus tolerance induction with CD40L-blockade induces an ABI PRISM 7000 Sequence Detector System (Applied Biosystems), which was normalized using 18S expressions. Real-time PCR primers and probes for expression of CXCL16 in the allogeneic grafts, and subse- V␣14-J␣281 were prepared as described previously (20) and those for CXCL16 quently recruits NKT cells in the tolerated grafts. Consistently, were purchased from Applied Biosystems. The 18S-normalized value of syn- the administration of anti-CXCL16 mAb led to an 8-fold de- genic transplant group was designated as the calibrator and expressed as 1. Final ␣ ␣ by guest on October 2, 2021 relative quantity of mRNA was expressed relative to the calibrator. crease in V 14-J 281 gene expression in the grafts (Fig. 2). Thus, the blocking with anti-CXCL16 mAb impeded the ac- Immunohistochemical analysis and in situ hybridization cumulation of NKT cells, resulting in the failure of mainte- Anesthetized mice were perfusion-fixed with a tissue fixative (GenoStaff) and 6 nance of tolerance. This up-regulation of CXCL16 and accu- ␮m of the neighboring frozen sections were prepared after being embedded in mulation of NKT cells observed in the accepted grafts was not paraffin. For immunohistochemical analysis, the slides were stained overnight seen in the spleens (p Ͼ 0.05 vs syngenic, Fig. 2), indicating that at 4°C with 1 ␮g/ml goat anti-mouse CXCL16 or control Ab in blocking re- agent. The slides were then incubated for 2 h with HRP-labeled anti-goat IgG the recruitment of NKT cells in the grafted tissue is important. at room temperature and incubated with Fluorophore Tyramide (PerkinElmer Life Science). Subsequently, the slides were incubated with anti-fluorescein- HRP (PerkinElmer) for 30 min at room temperature and immersed in diami- nobenzidine-H2O2. For in situ hybridization, a 0.35-kb fragment of mouse rearranged V␣14-J␣281 gene, amplified with the above-mentioned PCR primers, was subcloned into the pBluescript2 KSϩ vector (Stratagene) and was used for the generation of RNA probes. Digoxigenin-labeled RNA probes were prepared with DIG RNA Labeling Mix (Roche). Hybridization was performed as described (21) with some modifications. Coloring reactions were performed with NBT/5-bromo-4-chloro-3-indolyl phosphate, an alkaline phosphatase color substrate, and tissue sections were counterstained with Kernechtrot stain solution (Muto Pure Chemicals).

Cell adhesion assay A total of 1 ϫ 106 liver mononuclear cells (MNCs; the percentage of CD1d- tetramerϩ NKT cells was 15.5–21.6%) were transferred to 6-well plates where 1 ϫ 106 CHO-CXCL16 cells or control CHO cells were preseeded. After in- cubation at 37°C in 5% CO2 for the indicated times, nonadherent cells were removed by washing, and adherent cells were collected and stained with anti- TCR␤ mAb and CD1d tetramer. The number of NKT cells attached to the preseeded cells was estimated using FACSCalibur. In the blocking experiment, FIGURE 1. Administration of anti-CXCL16 mAb impaired the prolonged ␮ anti-CXCL16 mAb was added (20 g/ml). cardiac allograft survival induced by anti-CD40L mAb. Anti-CD40L mAb measuring 0.25 mg/mouse was i.v. injected on day 0 and i.p. injected on days 2 Statistical analysis and 4 posttransplant. Anti-CXCL16 mAb or control Ab (rat IgG) measuring Comparisons were performed using Mann-Whitney U test. A value of p Ͻ 0.05 0.2 mg/mouse was i.p. injected daily for 14 days posttransplant, and every 5 was considered significant. days until 40 days thereafter or until rejection was completed. The Journal of Immunology 2053

migration in the grafts and resulted in the failure of tolerance induction.

Adhesion of NKT cells to membrane-bound CXCL16 Unlike other chemokines, CXCL16 is expressed on the cell membrane and works as an adhesion molecule to CXCR6-expressing cells (18). Thus, we examined adhesion of NKT cells to membrane-bound CXCL16 in vitro. CHO cells transfected with CXCL16 cDNA (CHO-CXCL16) were prepared as described previously (13) and the cell surface expression was confirmed by FACS (data not shown). Liver MNCs were cocul- tured with control CHO or CHO-CXCL16 cells, and were examined for their attachment by counting cell numbers of ϩ CD1d-tetramer NKT cells in the adherent cells. The results indicated that NKT cells selectively attached to the CHO- CXCL16 cells, which was inhibited to the control level by the addition of anti-CXCL16 mAb (Fig. 4). Because it has been reported that NKT cells can migrate to soluble CXCL16 (10), Downloaded from the results indicate that the CXCL16/CXCR6 can induce not only migration but also adhesion of NKT cells. However, several chemokines other than CXCL16, such as SDF-1 or FIGURE 2. Quantitative analysis for NKT cells and CXCL16 expression. CXCL9, can also induce chemotaxis in NKT cells (10). Thus, The cardiac allografts and spleens were harvested from the recipients with the those non-CXCL16 chemokines and their receptor pairs might indicated treatments at time points as described in Materials and Methods. The

also be involved in the accumulation of NKT cells in a http://www.jimmunol.org/ ␣ ␣ quantity of mRNA for rearranged V 14-J 281 and CXCL16 were examined CXCL16/CXCR6-independent manner. In fact, a significant by real-time PCR and normalized with 18S expressions of each samples. The ␣ ␣ 18S-normalized value of gene expression for syngenic combination was desig- increase in the V 14-J 281 mRNA in the cardiac allografts was nated as the calibrator, and the final relative quantity of mRNA was expressed observed with injection of anti-CXCL16 mAb (3.4–4.8 times relative to the calibrator. The number of mice tested in each group is five. higher than syngenic, p Ͻ 0.05, Fig. 2). However, it seems that the role of CXCL16/CXCR6 is dominant in regulating NKT cell trafficking in vivo, as the accumulation of NKT cells in the To confirm the results of real-time RT-PCR, we further per- accepted grafts was mostly inhibited by anti-CXCL16 mAb in- formed immunohistological and in situ hybridization analyses. jection. This inhibition seems to be consistent with a recent re- In keeping with the results of real-time RT-PCR analysis, graft- port regarding CXCR6-deficient (cxcr6gfp/gfp) mice, which indi- by guest on October 2, 2021 infiltrating cells in the cardiac allografts, regardless of the graft cated that CXCR6 controls an accumulation of NKT cells in acceptance or rejection, showed a significant staining of vivo: CXCR6-deficient mice showed a selective and severe re- CXCL16, while those from syngenic grafts showed no staining duction of hepatic NKT cells (22). Re-evaluation of the prin- (Fig. 3). In situ hybridization of the grafts with a specific probe cipal experiments in this study using such gene-manipulated for the V␣14-J␣281 gene detected significant signals in the ac- mice that lack CXCL16/CXCR6 interaction can verify these cepted grafts (Fig. 3). However, no V␣14-J␣281 signals were results. Such a study is now under way at our laboratory. detected in grafts from recipients treated with anti-CXCL16 To investigate whether blocking of the CXCL16 and mAb, confirming that CXCL16 blockade inhibited NKT cell CXCR6 interaction would also affect the cytokine production

FIGURE 3. Identification of CXCL16 and NKT cells in the cardiac grafts. A neighboring section of cardiac allografts treated with anti-CD40L mAb Ϯ anti-CXCL16 mAb or syngenic grafts were prepared at time points as described in Ma- terials and Methods. Expression of CXCL16 and NKT cells were identified by immunohistochemical method and in situ hybridization, respectively. The bars indicate the scale of the images. The arrows represent NKT cells. 2054 CUTTING EDGE: CXCL16/CXCR6 AND NKT CELLS IN TRANSPLANT TOLERANCE

ACAID system, blocking CXCL16/CXCR6 with anti- CXCL16 mAb completely impeded the suppression of subse- quent delayed-type hypersensitivity induction (K. Sonoda, Ky- ushu University, personal communication). Although, it is not clear where NKT cells accumulate in ACAID, this information combined with the present data strongly suggest that CXCL16/ CXCR6 is generally involved in the immune-regulatory responses controlled by NKT cells. Given the fact that there seems to be an inflammation to some extent in the tolerated grafts, it is highly conceivable that the accumulated NKT cells are chronically stimulated. Because recent findings have indicated that chronic or repeated stimu- FIGURE 4. Adhesion of NKT cells to membrane-bound CXCL16. A total lation of NKT cells renders them immune regulatory (Ref. 26 of 1 ϫ 106 hepatic MNCs were added into 6-well plates, where 1 ϫ 106 and our unpublished data), NKT cells in the tolerated allografts CXCL16-transfected CHO or control CHO cells were preseeded. After incu- may regulate the transplant tolerance in this manner. Consis- bation at 37°C for the indicated times, nonadherent cells were removed by tently, we have recently found that NKT cells in the tolerated washing and adherent cells were collected and counted with FACSCalibur. The recipients changed their cytokine profile to produce higher IL- percent adherent NKT cells of each well was calculated. For blocking, anti- CXCL16 mAb was added to some wells. Representative data from three inde- 10, one of immune-regulatory cytokines, than those from re- Downloaded from pendent experiments. cipients undergoing rejection (K. Seino et al., manuscript in preparation). Further studies elucidating more detailed cytokine profile of NKT cells and their interaction with other im- by NKT cells, we injected 200–1000 ␮g of anti-CXCL16 mAb mune cells are now under way to clarify these issues. or control Ab into mice receiving 2 ␮gof␣-GalCer and subsequently measured IFN-␥ and IL-4 levels in the serum. How- Disclosures http://www.jimmunol.org/ ever, no significant changes in the level and time course of cy- The authors have no financial conflict of interest. tokine production with the CXCL16 blockade were observed (our unpublished data). Therefore, it appears that interaction of References 1. Taniguchi, M., M. Harada, S. Kojo, T. Nakayama, and H. Wakao. 2003. The regu- CXCL16 and CXCR6 molecules mainly influence the traffick- latory role of V␣14 NKT cells in innate and acquired immune response. Annu. Rev. ing of NKT cells but not their cytokine production. Immunol. 21: 483–513. Regarding the role of accumulated NKT cells in the tolerated 2. Seino, K. I., K. Fukao, K. Muramoto, K. Yanagisawa, Y. Takada, S. Kakuta, Y. Iwakura, L. Van Kaer, K. Takeda, T. Nakayama, et al. 2001. Requirement for nat- allografts, their precise mechanisms on regulatory function have ural killer T (NKT) cells in the induction of allograft tolerance. Proc. Natl. Acad. Sci. not been clarified so far. However, it is noted that there exist USA 98: 2577–2581.

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