1. Volume 138, Issue 1, Pages 336-346 (January 2010)
Virally Infected Mouse Liver Endothelial Cells Trigger CD8+ T-Cell Immunity 
Michaela Kern, Alexey Popov, Kai Scholz, Beatrix Schumak, Dominik Djandji, Andreas Limmer, Daniela Eggle, Torsten Sacher, Rainer Zawatzky, Rafaela Holtappels, Matthias J. Reddehase, Gunther Hartmann, Svenja Debey–Pascher, Linda Diehl, Ulrich Kalinke, Ulrich Koszinowski, Joachim Schultze, Percy A. Knolle 
Gastroenterology 
Volume 138, Issue 1, Pages (January 2010)
DOI: /j.gastro
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2. Figure 1
LSECs functionally express PRRs. (A) LSECs were mock treated or stimulated with TLR2-L (Pam3Cys, 1 μg/mL), TLR3-L (polyriboinosinic, 10 μg/mL), TLR4-L (LPS, 100 ng/mL), TLR7-L (sense strand of small interfering RNA 9.2, 6 μg/mL), and TLR9-L (ODN1668, 2.5 nmol/mL). After 24 hours, IL-6/IFN-β were determined in supernatants by enzyme-linked immunosorbent assay. n.d., not detectable. (B and C) LSECs were incubated with IFN-β (30 U, 3 hours) before lipofection with (B) 3pRNA (600 ng) or (C) poly-dAdT (10 μg/mL). IL-6 was determined as in A. Values in A–C are expressed as mean ± SD; one representative experiment out of 3 is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

3. Figure 2
PRR stimulation does not influence the tolerogenic phenotype of LSECs. (A) OVA-pulsed LSECs were stimulated with TLR ligands as in Figure 1A before CFSE-labeled naive OT-1 T cells were added. After 3 days, flow cytometric determination of T-cell proliferation and phenotype was performed. (B) Antigen-presenting LSECs were stimulated with TLR ligands, pretreated with IFN-β (30 U), and subjected to lipofection with 3pRNA (600 ng) or poly-dAdT (2.5 μg) or combinatorial stimulation for 24 hours before naive TCR-transgenic CD8+ T cells were added. After 5 days, equal numbers of viable CD8+ T cells were restimulated (αCD3ε) and IFN-μ production was determined by enzyme-linked immunosorbent assay. Splenic CD11c+ DCs were used as positive and untreated LSECs as negative control. APC/effectors, 1:2; mean ± SD of one out of 3 representative experiments.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
MCMV infection of LSECs. CD146+ LSECs were infected with MCMV-GFP (MOI = 1) for 24 hours and analyzed for (A) GFP expression or (B) release of IL-6/IFN-β. Splenic DCs were used for comparison. (C) LSECs infected with MCMV-OVA (MOI = 1) or with UV-irradiated MCMV-OVA (5000 J/cm2) were cocultured with B3Z cells. IL-2 production was determined 24 hours later. Values in B and C are expressed as mean ± SD. (D) LSECs infected with MCMVΔvRAP26 (MOI = 1) were cocultured with M45-Db peptide-specific T cells.26 IFN-γ production was determined by intracellular cytokine staining. The numbers indicate the percentage of positive cells; APC/effectors = 1:2. In A–D, one out of at least 2 independent experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Transition from tolerogenic to immunogenic LSECs by MCMV infection. (A) CFSE-labeled naive OT-1 cells were cocultured with uninfected/OVA-pulsed, MCMV-OVA–infected LSECs, or OVA-pulsed splenic CD11c+ DCs. After 3 days, analysis was performed as in Figure 2A for T-cell proliferation (upper panel) and activation markers (lower panel). Numbers indicate proliferation indices. (B) LSECs pulsed with antigen or virus were cocultured for 5 days with naive OT-1 cells. Equal numbers of viable T cells were restimulated and analyzed for IFN-γ, IL-2, and tumor necrosis factor α expression. (C) Cytotoxicity of OT-1 cells was activated as in B. (D) H-2Kb+ LSECs infected with MCMV-GFP or incubated with UV-irradiated MCMV-GFP were cocultured for 5 days with naive DesTCR CD8+ T cells. Analysis of T-cell function was performed as in Figure 2B. (E) LSECs were infected with LCMV for 48 hours and cocultured with DesTCR T cells for 4 days. Analysis was performed as in B; APC/effectors 1:2. In A–D, one of 3 representative experiments is shown. Values in B–E are expressed as mean ± SD.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions

6. Figure 5
LSEC-triggered CD8+ T-cell immunity is mediated by distinct pathways. (A) LSECs from wild-type or CD80/86−/− mice (mock treated or MCMV infected) were incubated with naive CD8+ T cells for 4 days and restimulated as in Figure 2B (left) or used for cytotoxicity assays as in Figure 4C (right); splenic DCs served as controls (right). (B) IL-12 release after MCMV infection of DCs and LSECs. (C) T-cell stimulation by LSECs in the presence of IFN-β (40 U/mL). MCMV-infected LSECs were positive control, and analysis of T-cell function was performed as in Figure 2B. (D) MCMV-OVA–infected LSECs (MOI = 1) were cocultured with OT-1 cells with antagonistic α-IL-7 or isotype control (5 μg/mL). T-cell analysis was performed as previously described. Values in A–D are expressed as mean ± SD. (E) Venn diagram illustrating the intersection of DC-associated pathogen core response genes (left) and MCMV-regulated LSEC-associated genes (right). Numbers indicate significantly regulated genes.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
MCMV infection of LSECs leads to CD8+ effector cells in vivo. (A) C57BL/6 mice were injected with phosphate-buffered saline, 2.5 × 104 plaque-forming units MCMV-GFP or MCMV-OVA 1 day before transfer of 5 × 106 Thy1.1+OT-1 cells. Four days later, flow cytometric determination of OT-1 cell expansion (left) and IFN-μ expression by intracellular cytokine staining after restimulation (right). (B and C) As in A, OT-1 cell transfer into MCMV-OVA–infected (B6.CH-2bm1 → C57Bl/6) chimeras and analysis of T-cell function in (B) spleen and (C) liver. Mean ± SEM (n = 6) from 2 independent experiments (n = 3). (D) A total of 106 OT-1 cells were transferred into C57BL/6 mice or chimeras 1 day before infection with MCMV-OVA or 1 × 107 plaque-forming units Adeno-OVA. Cytotoxic T lymphocyte activity was determined 5 days later in vivo. (E) OT-1 cell transfer into wild-type or CD80/86−− mice before infection and determination of cytotoxicity as previously described. Values in D and E are expressed as mean ± SEM (n = 3) from one out of 2 representative experiments.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Early T-cell activation occurs predominantly in the liver in vivo. Thy1.1+C57BL/6 mice were infected with MCMV-OVA 1 day after adoptive transfer of 5 × 106 Thy1.2+ OT-1 cells. Six hours postinfection, OT-1 cells were analyzed for CD69 expression (black lines; isotype controls, shaded areas). Histograms of 2 representative mice from 2 independent experiments (n = 3). LN, pooled lymph nodes.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions