Download 1 Supplementary Information Materials and Methods Animals Inbred

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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
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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
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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.
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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.
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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).
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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.
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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
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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.
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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
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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.