Supplemental Material and Methods
Mice. C57BL/6, BALB/c, DO11.10 or CD90.1+, OVA T-cell receptor transgenic OT-II mice 1
were obtained from the Bundesamt für Risikobewertung (Berlin, Germany) or Charles River
(Wilmington, MA). TF-OVA mice expressing membrane-bound OVA under control of the TF
promoter in hepatocytes 2 and OT-I mice 3 were bred at the Charité animal facility (Berlin,
Germany). Vitamin A-deficient mice were generated as previously described 4. Briefly,
pregnant BALB/c mice and subsequently their offspring received a defined diet lacking
vitamin A. Vitamin A-deficiency was confirmed by dramatically decreased percentages of
47 integrin+ T cells within intestine and GALT compared to mice that were fed with
vitamin A-containing diet. All animals received human care according to the national criteria
published by the National Institutes of Health (Bethesda, MD).
Cell isolation. LSEC were isolated as previously described 5,6. Briefly, livers were perfused
in situ with digestion medium containing Collagenase IV (Sigma Aldrich, Steinheim,
Germany). To enrich NPC, removed livers were digested and single cell suspensions were
subjected to a density-gradient centrifugation with 26% Nycodenz (AXIS-SHIELD, Oslo,
Norway). LSEC were purified by magnetic cell sorting using anti-CD146 antibody 5
(Deutsches Rheumaforschungszentrum; DRFZ, Berlin, Germany) to a purity of at least 95%.
Prior to in vitro co-culture, LSEC were allowed to adhere over night. Subsequently, LSEC
were vigorously washed increasing the purity to more than 99%. For mRNA analysis, ex vivoisolated LSEC were enriched by FACSAria cell sorter (BD Biosciences, Heidelberg,
Germany) to a purity of 99%.
To isolate MHCII+LSEC- liver APC, NPC were depleted from CD146+ LSEC and sorted for
MHCII+ cells using anti-MHCII antibody (DRFZ) resulting in 99% MHCII+LSEC- liver APC.
Professional APC were isolated from spleen and lymph nodes and depleted from CD90+ cells
using anti-CD90.2 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). For isolation
of DC, pLN or mLN were digested with Collagenase D (Roche, Grenzach-Wyhlen, Germany)
and the cell suspension was enriched for CD11c+ DC using anti-CD11c MicroBeads (Miltenyi
Naive CD4+CD62L+ T cells were prepared using anti-CD4 antibody (BD Biosciences) and
anti-CD62L MicroBeads (Miltenyi Biotec), resulting in a purity of 99%. Naive CD8+CD44T cells were purified using anti-CD8 and anti-CD44 antibody (Miltenyi Biotec).
In vitro polarization of naive CD4+ T cells.
To generate TLSEC or TSAPC, 5x105 naive CD4+ T cells from OT-II mice were co-cultured with
1x106 LSEC or SAPC in the presence of 5 µg/ml OVA peptide (ISQAVHAAHAEINEAGR;
Humboldt-Universität, Berlin, Germany) for 6 days. To demonstrate comparable proliferation,
naive CD4+ T cells were labelled with 5 µM carboxyfluorescein diacetate succinimidyl ester
(CFSE; MoBiTec, Göttingen, Germany) prior co-culture with LSEC or SAPC (Supplemental
Fig. 1). DC isolated from mLN (mLN DC; 1x105), pulsed for 2 h with 1.8 µg/ml OVA, were
co-cultured with 2x105 naive CD4+ T cells from OT-II mice for 4 days to generate TmLN DC.
To obtain in vitro-polarized Th1 cells, 6.5x105 OVA-specific, naive CD4+ T cells were cocultured with 1.3x106 SAPC in the presence of 5 µg/ml OVA peptide, 5 µg/ml interleukin-4depleting antibody (11B11; DRFZ), 10 ng/ml interleukin-12 and 20 ng/ml interferon  (both
R&D systems, Wiesbaden, Germany) resulting in 70-80% interferon γ+ cells.
In vitro and in vivo induction of 47 integrin.
OVA-specific, naive CD4+ T cells or in vitro-polarized Th1 cells (both 5x105) were cocultured with LSEC, MHCII+LSEC- liver APC or SAPC (all 1x106) in the presence of
5 µg/ml OVA peptide. Naive CD4+ T cells were harvested after 6 days, Th1 cells after three
days. Naive CD8+ T cells (2.5x105) from OT-I mice were co-cultured with 1x106 LSEC in the
presence of 20 ng/ml OVA peptide (SIINFEKL, Humboldt-Universität) for 6 days. For RAR
blockage, the RAR pan-antagonist LE 540 (1 µM; Wako, Richmond, VA) was added to the
cultures 4. To study the effect of exogenously added vitamin A in cultures with LSEC from
vitamin A-deficient mice, TLSEC were generated in the presence of 50 nM all-trans retinol
(Sigma Aldrich).
CFSE-labelled, OVA-specific, naive CD4+ T cells (1x107) or Th1 cells (5x106) were
adoptively transferred into TF-OVA or C57BL/6 WT mice. Naive CD4+ T cells were reisolated after three days, Th1 cells after two days. Donor cells bearing the congenic marker
CD90.1 were distinguished from CD90.2+ recipient cells by staining for CD4 and CD90.1.
CD90.1+ donor cells were analyzed for proliferation and expression of 47 integrin by flow
Antibodies and FACS analysis. Cells were stained with anti-CCR9 (242503; R&D Systems),
anti-47 integrin (DATK32), anti-CD4 (RM4-5), anti-CD25 (PC61), anti-CD44 (IM7) antiCD90.1 (OX-7; all BD Biosciences), anti-CD45 (30-F11), anti-CD62L (MEL-14), anti-CD8
(53-6.7; all BioLegend, Fell, Germany), anti-CXCR3 (CXCR3-173), anti-CCR7 (4B12), antiCD11c (N418; all eBiosciences, Frankfurt, Germany), anti-CD146 (ME-9F1), anti-CD31
(3E2) or anti-MHCII antibody (M5/114; all DRFZ), mouse P-selectin/human IgG Fc chimera
protein (DRFZ) and anti-human IgG Fc (Dianova, Hamburg, Germany).
Data were acquired using a FACS Canto II (BD Biosciences) flow cytometer, and analyzed
by FlowJo software (Tree Star, Ashland OR, USA). Cells were incubated with propidium
iodide (PI; 1 µg/ml; Sigma Aldrich) prior to acquisition to discriminate between PI- live and
PI+ dead cells. Between 10,000 and 20,000 PI- live cells were acquired in a combined gate set
in forward and side light scatter.
Reverse transcription PCR analysis. Total RNA was extracted and reversely transcribed into
cDNA. PCR reactions were set up with 25 ng cDNA for RALDH1, RALDH4 and GAPDH or
100 ng cDNA for RALDH2 and RALDH3. Sequences of the forward and reverse primers
(TIB Molbiol, Berlin, Germany) were as follows:
629 bp;
143 bp. Products were separated by agarose gel electrophoresis and visualized by ethidium
bromide staining.
Quantitative RT-PRC analysis. PCR reactions were set up with 250 ng cDNA for all
RALDH isoforms and GAPDH. Sequences of the forward primers were the same used for
RT-PCR analysis. Sequences of the reverse primers (TIB Molbiol) were as follows:
Primers of GAPDH were the same used for RT-PCR analysis. Quantitative PCR was
performed using a SYBR® Green PCR MasterMix (Applied Biosystems, Darmstadt,
Germany). Using the 2-∆∆CT method 7, the data for liver RALDH expression were presented as
the fold change in gene expression normalized to the housekeeping gene GAPDH and relative
to the normalized RALDH expression in mLN. Expression of the RALDH isoforms by LSEC
was determined in relation to GAPDH expression (ΔCT). For relative quantification,
correlation coefficients of experimental titration curves for individual and pooled cDNA
samples were R2≥0.997 in the exponential range between 250 ng and 7.8 ng total cDNA.
Maximum slope deviations for control and test target were 5%.
Western blot analysis. Cells were lysed and separated by SDS-polyacrylamide gel
electrophoresis according to Laemmli 8. Proteins were transferred to a polyvinylidene fluoride
membrane (Millipore, Billerica, MA) by tank blotting (Biometra, Göttingen Germany).
Membranes were incubated with anti-RALDH1 (H-85; Santa Cruz Biotechnology, Santa
Cruz, CA) or anti-β-actin (AC-15; Sigma Aldrich). The secondary antibody (Dako, Glostrup,
Denmark) was conjugated to horseradish peroxidase and binding was visualized by
chemiluminescence (GE Healthcare, Munich, Germany) using the ECLTM system (Roche).
Analysis of RALDH activity. RALDH activity was assessed using the ALDEFLOUR ®
staining kit (StemCell Technologies, Vancouver, Canada). Cells (1x106) were incubated in
assay buffer containing ALDEFLOUR substrate with or without the RALDH inhibitor
DEAB. ALDEFLOUR+ cells were detected by flow cytometry.
Transmigration assay. Assays were performed as previously described 6. TLSEC or TSAPC
(5x105) were added to the upper chamber of the transwell with or without 300 nM CCL25
(R&D Systems, Wiesbaden, Germany) in the lower chamber. Cells from the lower chamber
or from the input were mixed with Fluoresbrite beads (Polysciences, Eppelheim, Germany)
and analyzed by flow cytometry. Absolute cell numbers were determined by gating on CD4 +
T cells in relation to a defined numbers of beads.
In vivo homing assay. Radioactively labelled cells (1-3x106; 20 µCi/ml 51Cr; GE Healthcare,
Braunschweig, Germany) were intravenously transferred into C57BL/6 mice. After 24 h,
radioactivity of the PP, mLN, small and large intestines as well as the remaining body was
counted using a Wizard gamma counter (Wallac, Turku, Finland). The percentage of organspecific radioactivity in relation to the total recovered radioactivity reflects the percentage of
cells that have migrated into the respective organ 9, 10.
Data analysis. Data were analyzed using the GraphPad Prism software (GraphPad software,
San Diego, CA). Statistical comparison was carried out using the nonparametric two-tailed
Mann-Whitney test or the One-sample t test.
Barnden MJ, Allison J, Heath WR, Carbone FR. Defective TCR expression in
transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the
control of heterologous regulatory elements. Immunol Cell Biol 1998;76:34-40.
Derkow K, Loddenkemper C, Mintern J, Kruse N, Klugewitz K, Berg T, Wiedenmann
B, et al. Differential priming of CD8 and CD4 T-cells in animal models of
autoimmune hepatitis and cholangitis. Hepatology 2007;46:1155-1165.
Hogquist KA, Jameson SC, Heath WR, Howard JL, Bevan MJ, Carbone FR. T cell
receptor antagonist peptides induce positive selection. Cell 1994;76:17-27.
Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, Song SY. Retinoic acid
imprints gut-homing specificity on T cells. Immunity 2004;21:527-538.
Schrage A, Loddenkemper C, Erben U, Lauer U, Hausdorf G, Jungblut PR, Johnson J,
et al. Murine CD146 is widely expressed on endothelial cells and is recognized by the
monoclonal antibody ME-9F1. Histochem Cell Biol 2008;129:441-451.
Schrage A, Wechsung K, Neumann K, Schumann M, Schulzke JD, Engelhardt B,
Zeitz M, et al. Enhanced T cell transmigration across the murine liver sinusoidal
endothelium is mediated by transcytosis and surface presentation of chemokines.
Hepatology 2008;48:1262-1272.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402-408.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 1970;227:680-685.
Hamann A, Klugewitz K, Austrup F, Jablonski-Westrich D. Activation induces rapid
and profound alterations in the trafficking of T cells. Eur J Immunol 2000;30:32073218.
Kruse N, Neumann K, Schrage A, Derkow K, Schott E, Erben U, Kuhl A, et al.
Priming of CD4+ T cells by liver sinusoidal endothelial cells induces CD25low
forkhead box protein 3- regulatory T cells suppressing autoimmune hepatitis.
Hepatology 2009;50:1904-1913.