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Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

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jws-hep.21498.fig1.pdf15K Suppl Fig. 1:Cytokine-responses to ConA at different timepoints of rechallenge after prestimulation. To assess the effects of ConA-pretreatment on ConA-induced cytokine expression, mice were rechallenged at indicated timepoints after pre-treatment. Plasma cytokine concentrations and hepatic cytokine mRNA levels were analyzed in plasma and liver samples taken 8hrs after ConA rechallenge by ELISA and quantitative RT-PCR, respectively. To enable direct comparisons of cytokine responses at the different timepoints in spite of normal experimental day-to-day variations, the relative plasma cytokine levels from ConA-treated mice were normalized for their concentrations in saline controls of the same timepoint, with the latter concentration being defined as ?1?. With respect to cytokine mRNA levels in liver tissue normalization to mRNA in control samples is already immanent in the method of quantitative RT-PCR. (All data presented as mean±SEM)
jws-hep.21498.fig2.pdf134K Suppl Fig. 2:Development of ConA tolerance is not associated with depletion of NKT cells, T cells or Kupffer cells upon ConA-pretreatment. Frequency and total numbers of T cells and NKT cells in the liver of saline or ConA-pretreated mice were measured by FACS analysis, detecting CD3 and NK1.1 expression on the surface of intrahepatic lymphocytes as defined by their light scatter characteristics. Representative dot blots of one representative sample from the saline- or ConA-pretreated group, revealing the significant increase in intrahepatic NKT cell frequency upon ConA-pretreatment (Panel A), is shown together with the relative intrahepatic T- and NKT-cell numbers (relating to the total number of T-cells in saline-control mice) (Panel B; mean ± SEM; n ≥ 3; *, p ≤ 0.05 vs. saline-pretreated control. C) After a short transient decrease of the numbers of detectable NKT cells directly after ConA-pretreatment, their total numbers (bars) as well as their frequencies among lymphocytes (lines/symbols) increased both in liver and liver spleen and even exceeded their base levels, as detected by FACS analysis. D) Presence of intrahepatic Kupffer cells eight days after ConA- or saline-pretreatment was verified by immunofluorescent staining of liver sections from tolerized or non-tolerized mice with anti-mouse-macrophage antibody BM8 and FITC-labelled secondary anti-rat-IgG antibody.
jws-hep.21498.fig3.pdf9K Suppl Fig. 3:The characteristic cytokine profile of ConA tolerance is maintained in CD1d knockout mice. NKT-cell deficient CD1d-/- mice were injected intravenously with ConA or saline eight days prior to ConA-restimulation. Eight hours after ConA-re-challenge, plasma cytokine levels were measured by ELISA, revealing significantly reduced IFNg, IL-4 and IL-6 levels and increased IL-10 concentration in ConA tolerized CD1d-/- mice. The figure depicts one of three independent experiments with corresponding results (mean ± SEM; n = 4; *, p ≤ 0.05 vs. saline controls)
jws-hep.21498.fig4.pdf38K Suppl Fig. 4:ConA pretreatment induces a decline in the frequency of CD45RBhigh population among CD4+ T cells. Eight days after ConA-pretreatment liver mononuclear cells were isolated and CD45RB expression on CD4+ T cells was determined by flow-cytometry. Therefore, cells were gated on viable lymphocytes by their light-scatter characteristics, on CD4-positive cells, and additionally on NK1.1-negative cells to exclude CD4-positive hepatic NKT cells from the analysis. The specified percentages represent the frequency of CD45RBhigh cells - as indicated by gray shading in the 3D-plots - within the CD4+ T-cell population.
jws-hep.214985.pdf30KA discussion of tolerization-induced IL-2 suppression

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