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1 Supplementary Information Materials and Methods Animals Inbred DA (RT1AaBa) and Lewis (RT1AlBl) rats were supplied by SLC Co. (Shizuoka, Japan). Both were bred under specific pathogen-free conditions. Animal handling and care was approved by the Dokkyo Medical University Animal Experiments Committee, and was in accordance with the Dokkyo University’s Regulations for Animal Experiments and with Japanese Governmental Law (No. 105). Donor liver non-parenchymal cells, hepatic lymph cells, and liver grafts were obtained from male DA rats. Male Lewis rats were the liver transplant recipients. Liver Transplantation (LTx) Orthotopic LTx was performed using a cuff method (1) under isoflurane anesthesia without revascularization of the hepatic artery. At various time points during the 7 days after LTx, host rats received an intravenous injection of 5-bromo-2'-deoxyuridine (BrdU, 2 mg/100 g body weight; Sigma Chemical Co., St. Louis, MO) and were sacrificed 1 hour later. Graft liver and host secondary lymphoid organs, including the spleen, skin LNs (including cervical, axillary, and brachial LNs), parathymic LNs, and Peyer's patches were excised and fresh-frozen for immunostaining (2). Some were 2 also used for FACS or microarray analysis. Collection of DCs from Hepatic Lymph The celiac LNs, draining the liver and upper gastrointestinal tract, were surgically removed, resulting in a direct influx of peripheral hepatic lymph containing DCs into the thoracic duct after regeneration of lymphatic vessels (3). DA rats received routine thoracic duct cannulation (4) ~6–8 weeks after the celiac lymphadenectomy. Thoracic duct lymph was collected overnight at 4ºC and processed for FACS analysis. With this method the isolated DCs are derived mainly from the liver (3). The isolated DCs were used for FACS analysis. Peritoneal Fluid Cell Collection and Preparation of Tissue Spreads On days 1–3 after LTx, recipient rats were sacrificed and injected intraperitoneally with 30 ml chilled PBS solution containing 10 IU/ml heparin. After mechanically massaging the flank several times, peritoneal fluid was collected and processed for FACS. Spreads of recipient omentum and diaphragm were then prepared and immunostained. Antibodies and Reagents All antibodies used for immunohistology and FACS are listed in 3 Supplementary Table 1. Some monoclonal antibodies were purified from culture supernatants. Those used for FACS analysis were coupled to fluorochromes or biotin in-house. A polyclonal antibody to forkhead box P3 (FoxP3)(5) was kindly donated by Dr. S. Ueha, Tokyo University. FACS Analysis For donor DC cell yields and phenotype analysis, the donor livers and recipient skin LNs, and parathymic LNs were digested with collagenase D (Roche Diagnostics, Indianapolis, IN), and isolated cells were analyzed for FACS as described previously (2). For each LN-DC isolation, LNs from 2–3 rats were pooled. These cells were then purified as a low-density fraction using 12% OptiPrep solution (AXIS-SHIELD, Oslo, Norway). For hepatic lymph and peritoneal wash analysis, donor-cell yields were low, especially in the Irr(+) group. Accordingly, whole cell suspensions were used. The isolated donor MHCI+ cells were stained using anti-donor MHCII-Alexa Fluor 647-conjugated and anti-CD103-PerCP/Cy5.5-conjugated, FITC- and R-phycoerythrin-conjugated mAbs. Cells were washed twice then analyzed on a FACS Calibur (BD Biosciences) using Cell Quest software (BD Biosciences). Donor MHCIIhighCD103high cells were gated as the DC fraction and analyzed for expression of CD172a, CD11b, CD25, or CD86. 4 Immunohistological Analysis To investigate the recipient’s immune response, cryosections were triple-immunostained (2) for CD8, FoxP3, recipient MHCI (I169.1+)(6), or donor MHCII (alkaline phosphatase-blue), type IV collagen (peroxidase-brown), and BrdU (alkaline phosphatase-red). The T-cell areas of the spleen (periarterial lymphocyte sheath, PALS) or those of other secondary lymphoid organs were examined to determine the number of BrdU+ cells or BrdU+CD8β+ T-cells as reported previously (2). For the regulatory T-cell response, cells with a red BrdU+ nucleus superimposed with a blue FoxP3+ nucleus were registered as FoxP3+ proliferating cells. The phenotype of donor MHCII+ cluster-forming cells, was analyzed as reported previously (2) in serial fresh 2-m cryosections of the parathymic LNs and graft liver. One section was stained for donor MHCII (blue), type IV collagen (brown), and BrdU (red). A neighboring section was stained for CD103, CD11c, CD25, or CD86 (blue), type IV collagen (brown), and BrdU (red). The corresponding areas in two neighboring sections were photographed, and the percentage of either CD103+ or CD11c+ cluster-forming cells was calculated relative to total donor MHCII+ cluster-forming cells. 5 Mixed Leukocyte Reaction Three DC subsets were sorted from the low-density fractions of non-parenchymal cells of DA rat liver by a FACS Aria (BD Biosciences) to obtain a purity of >90%. Since there were very few sorted cells in each experiment, only the CD172a+CD11b+ subset with/without irradiation and the non-irradiated CD172a-CD11b+ subset were used as stimulators. Irradiated hepatic lymph DC fractions, which made up 10% of the DCs, were also used after mitomycin C treatment. As a positive stimulator control, mitomycin C-treated splenic DCs from DA rats were used at varying concentrations (3x102–1x104 cells/well). Responder T-cells were isolated from Lewis skin LNs by collagenase digestion and nylon-wool passage to obtain a purity of >90% (3). DC fractions (3x102–3x103 cells/well) were cultured with responder T-cells (1x105 cells/well) in flat bottom, 96-well plates with a final volume of 200 L. The proliferation of responder T cells (relative light unit/sec, rlu/s) was measured after 4 days using a cell proliferation ELISA kits, BrdU (Roche Applied Science, Mannheim, Germany) following the manufacturer’s instructions. Because of low yields of the stimulators, each culture was performed either in triplicate or duplicate and representative data from 3 separate experiments (each with duplicate or triplicate cultures) were expressed as the mean ± SD (n=3). 6 Microarray Analysis and Quantitative RT-PCR The microarray analysis was performed by Filgen (Nagoya, Japan). For some genes that were either upregulated or downregulated after graft irradiation, we further examined the expression levels by quantitative RT-PCR. Briefly, total RNA was isolated from approximately 50 mg of graft liver on day 2 or 3 after LTx using TRIzol reagent (Life Technologies Japan, Tokyo, Japan), and further purified using the RNeasy Mini kit (QIAGEN, Tokyo, Japan). The RNA quality was assessed using an Agilent 2100 Bioanalyser (Agilent Technologies, Santa Clara, CA). An equivalent amount of total RNA from three individual animals was pooled in each group. The biotin-conjugated aRNA target was synthesized and 10 g was hybridized to the CodeLinkTM Rat Whole Genome Bioarray (Applied Microarrays, Tempe, AZ) for 18 h at 37oC. Arrays were washed and stained with streptavidin-Cy5 conjugate to detect hybridization. Arrays were scanned with a GenePix 4000B Array Scanner (Molecular Devices, Sunnyvale, CA), and images were quantified and normalized using CodeLinkTM Expression Analysis Software v5.0 (Applied Microarray). Microarray data was analyzed using the Microarray Data Analysis Tool ver3.2 (Filgen). A gene was defined as upregulated when the Irr(+)/Irr(-) intensity ratio was >3.0 with P<0.05, and down-regulated when the ratio was <0.33 with P<0.05. 7 The quantitative RT-PCR method has been described previously (7). All primer sequences used in this paper are available upon request. Statistical Analysis Each parameter was measured in a blinded fashion and expressed as the mean ± SD (n=3). Image analysis was performed on a personal computer using the public domain NIH Image program (Image J1.36b) to estimate surface areas. For FACS analysis, each assay was repeated 3 times. Statistical analysis was performed using the Student’s t-test. Discussion Similar Mouse DC Subsets and Other Possible Subsets. Mouse DC subsets can also be grouped based on expression of CD103, CD172a, and CD11b. However, CD103 expression is more restricted and related to CD172a-CD8+ DCs, and in most nonlymphoid tissues, CD172a+CD11b+ DCs are CD103- except in the lamina propria DCs (8). Cyclophosphamide treatment increases the potency of mouse DCs by selectively depleting CD8+ LN-resident DCs but not lymph DCs or plasmacytoid DCs (9). Drug-resistant DCs that migrate in lymph are immunogenic, whereas drug-sensitive CD8+ DCs show 8 regulatory effects. The radioresistant DC subset might also be resistant to other cytotoxic agents, as is the case for the stably immunogenic drug-resistant mouse DC subset. These cytotoxic agent-resistant DC subsets may be exploited clinically for use in an immunogenic DC vaccine. In an experimental pathological state in which rats received intravenous latex or carbon particles, DCs that took in these particulates soon appeared in the hepatic lymph. These DCs were MHCI+MHCII+CD11b+CD25-ICAM1+, CD103+(70%), CD4+(50%), and Thy1+(50%) (10). We speculate that these cells are DC precursors or immature DCs recruited from the bone marrow in response to danger signals (11). Similar DC precursors have been reported in mouse liver after injection of Propionibacterium acnes (12). Their relevance to the liver DC subsets in the present study is currently under investigation. 9 References 1. Miyanari N, Yamaguchi Y, Matsuno K, Tominaga A, Goto M, Ichiguchi O, et al. Persistent infiltration of CD45RC- CD4+ T cells, Th2-like effector cells, in prolonging hepatic allografts in rats pretreated with a donor-specific blood transfusion. Hepatology 1997;25:1008-1013. 2. Ueta H, Shi C, Miyanari N, Xu XD, Zhou S, Yamashita M, et al. Systemic transmigration of allosensitizing donor dendritic cells to host secondary lymphoid organs after rat liver transplantation. Hepatology 2008;47:1352-1362. 3. Matsuno K, Kudo S, Ezaki T, Miyakawa K. Isolation of dendritic cells in the rat liver lymph. Transplantation 1995;60:765-768. 4. Ford WL: The preparation and labelling of lymphocytes (Chapter 23). In: Weir DM, ed. Handbook of experimental immunology, 3rd edition: Blackwell Scientific Publications, 1978; 23.21-23.22. 5. Ueha S, Yoneyama H, Hontsu S, Kurachi M, Kitabatake M, Abe J, et al. CCR7 mediates the migration of Foxp3+ regulatory T cells to the paracortical areas of peripheral lymph nodes through high endothelial venules. J Leukoc Biol 2007;82:1230-1238. 10 6. Smilek DE, Boyd HC, Wilson DB, Zmijewski CM, Fitch FW, McKearn TJ. Monoclonal rat anti-major histocompatibility complex antibodies display specificity for rat, mouse, and human target cells. J Exp Med 1980;151:1139-1150. 7. Tanaka T, Yamamoto J, Iwasaki S, Asaba H, Hamura H, Ikeda Y, et al. Activation of peroxisome proliferator-activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome. Proc Natl Acad Sci U S A 2003;100:15924-15929. 8. Ginhoux F, Liu K, Helft J, Bogunovic M, Greter M, Hashimoto D, et al. The origin and development of nonlymphoid tissue CD103+ DCs. J Exp Med 2009;206:3115-3130. 9. Nakahara T, Uchi H, Lesokhin AM, Avogadri F, Rizzuto GA, Hirschhorn-Cymerman D, et al. Cyclophosphamide enhances immunity by modulating the balance of dendritic cell subsets in lymphoid organs. Blood 2010;115:4384-4392. 10. Matsuno K, Ezaki T, Kudo S, Uehara Y. A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph. J Exp Med 1996;183:1865-1878. 11. Uwatoku R, Suematsu M, Ezaki T, Saiki T, Tsuiji M, Irimura T, et al. Kupffer 11 cell-mediated recruitment of rat dendritic cells to the liver: roles of N-acetylgalactosamine-specific sugar receptors. Gastroenterology 2001;121:1460-1472. 12. Yoneyama H, Matsuno K, Zhang Y, Murai M, Itakura M, Ishikawa S, et al. Regulation by chemokines of circulating dendritic cell precursors, and the formation of portal tract-associated lymphoid tissue, in a granulomatous liver disease. J Exp Med 2001;193:35-49. 12 Supplementary Figure Legends Fig. S1. Donor MHCII+ cells migrated via blood to the recipient’s lymphoid organs, such as the spleen (A) and the Peyer's patches (B), in the Irr(-) group but not the Irr(+) group (C, D) on day 2 after liver transplantation. In contrast, the parathymic lymph nodes (LNs) in both groups contained donor MHCII+ cells (E, F). Triple immunostaining for donor MHCII (blue), type IV collagen (brown), and BrdU (red). Donor DC-like cells formed clusters with recipient proliferating cells (insets in panels A, C, E, and F). Fig. S2. CD8+ T-cell responses in the splenic PALS and the T-cell area of the parathymic lymph nodes (LNs) on day 3 after liver transplantation. Triple immunostaining for CD8 (blue), type IV collagen (brown), and BrdU (red). While there were many BrdU+CD8+ T-cells in tissues from the Irr(-) group (arrows, A, C), there were fewer in the spleens of the Irr(+) group (B). There were comparable numbers of BrdU+CD8+ T-cells present in the parathymic LNs of the Irr(+) group (arrows, D). Fig. S3. FoxP3+ regulatory T-cell responses in the Irr(+) and Irr(-) groups after liver transplantation (LTx). The number of BrdU+FoxP3+ cells/mm2 in the splenic PALS (A) and 13 the portal area (B) are shown, as are the number of total FoxP3+cells cells/mm2 in the portal area (C). Mean ± SD (n=3). Fig. S4. Quantitative RT-PCR analysis of relevant genes. Of the genes examined, the macrophage inhibitory factor (MIF) gene showed a significant increase in expression after irradiation. Mean ± SD (n=3). *P<0.05 versus the Irr(-) group.