1 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 47 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. 2 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 Biotec). 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. 3 In vitro and in vivo induction of 47 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 47 integrin by flow cytometry. Antibodies and FACS analysis. Cells were stained with anti-CCR9 (242503; R&D Systems), anti-47 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 4 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: RALDH1: 5′-AGACAGGCTTTCCAGATTGGCTCT-3′ / 5′-GCGACACAACATTGGCCTTGATGA-3′, 736 bp; RALDH2: 5′-ACCGTGTTCTCCAACGTCACTGAT-3′ / 5′-TGGAAGGACTCAAAGCCACTGTCA-3′, 859 bp; RALDH3: 5′-TGGCACGAATCCAAGAGTGGAAGA-3′ / 5′-TTGAAGAACACTCCCTGGTGAGCA-3′, 832 bp; RALDH4: 5′-TGCTTCCCACGGTGATAACAGACA-3′ / 5′-TGAGTCATCTCCCAGGCCTTTGTT-3′, 629 bp; GAPDH: 5′-CATCCTGCACCACCAACTGC-3′ / 5′-ACGCCACAGCTTTCCAGAGG-3′, 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: RALDH1: 5´-AGCAGCAGACGATCTCTCTCCATTA-3´, 107 bp; RALDH2: 5´-TCCAAAGTCTGAGTTATTGGCTCTTTC-3´, 129 bp; RALDH3: 5´-AGCCTTGTCCACATCGGGCTTATCT-3´, 105 bp; RALDH4: 5´-TCCTCTTCACTATCAAACGGAACAACA-3´, 103 bp. 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 5 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. 6 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. 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