Analysis of alternative triggers for PI-9 expression.

<p>A) The up-regulation of PI-9 was also found to be triggered by other inflammatory cytokines. HepG2 cells were treated for 16 hours with either IFN-γ at 100 IU/ml, or IL-1β at 50 ng/ml. GAPDH expression was used as a positive control. B) Stimulation of the HepG2 cell line with either IFN-γ (open squares) or IL-1β (open triangles) also inhibited CTL killing compared to the un-stimulated HepG2 cells (closed circle). C) HepG2 cells were left un-infected (Nil), or infected with either a baculovirus expressing a sub-genomic replicon (NS-replicon), or with a control baculovirus expressing LacZ (+Control). PI-9 expression was analysed by RT-PCR. PI-9 was strongly up-regulated only in the cells expressing the sub-genomic replicon. GAPDH expression was used as a positive control.</p

IFN- treatment did not protect a B-cell line.

<p>Treatment of a BCL with 1000 IU/ml IFN-α (open inverted triangles) prior to the cytotoxicity assay, did not reduce CTLs ability to kill the treated BCL compared to the untreated BCL (closed circles).</p

Comparison of differentiation profiles of Gag specific effector CD8+ T cells between progression groups.

<p>All Gag specific responses are shown in panel A. Only immuodominant responses (the response with the highest magnitude for each individual) are shown in panel B. The line in each column represents the median value. The differentiation phenotype is referenced beneath each column.</p

Expression of PI-9 in liver tissue from patients with chronic hepatitis C.

<p>Liver specimens obtained from diagnostic biopsy were stained for PI-9 expression as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000791#s4" target="_blank">methods</a>. A) Representative negative isotype control. B–D) PI-9 was detected in the majority of hepatocytes. Stronger PI-9 expression, as expected, was seen within the mononuclear infiltrate (highlighted by red arrows), while all the hepatocytes stained positively, albeit at a lower level (the strongest are highlighted by blue arrows).</p

IFN-α treated hepatocytes remain susceptible to FASL-induced apoptosis.

<p>Aii) Induction of apoptosis by FASL was assessed across a concentration range. HepG2 cells, either stimulated with IFN-α (1000 IU/ml) (closed inverted triangles) or left un-stimulated (open diamonds), were incubated with cross-linked rFASL at concentrations ranging from 0.1 pg/ml to 2 µg/ml. Apoptosis was assessed by Annexin V binding and propidium iodine (P.I) incorporation. Ai) Raw FACS data is shown. Bii) HepG2 cells, either un-stimulated (open and closed circles), stimulated with 100 IU/ml (open and closed squares) IFN-α or 1000 IU/ml (open and closed inverted triangles) IFN-α, were incubated with 1 µg/ml (left, open symbols) or 2 µg/ml (right, closed symbols) cross-linked rFASL over a time course of up to 6 hours. Apoptosis was assessed by the cytosolic presence of the activated form of caspase 3. Bi) Raw FACS data.</p

IFN-α reduces hepatocyte sensitivity to CTL cytotoxicity.

<p>Hepatocytes have been described as expressing low to no MHC Class I. As expected, IFN-α treatment increased the levels of MHC Class I on HepG2 (A) and HHL (B); filled curve represents an isotype control, the solid line represents the MCH Class I expression. C) HepG2 cells were stimulated for 16 hours with a serial dilution of IFN-α at 0 IU/ml (closed circles), 10 IU/ml (open diamonds), 100 IU/ml (open squares), and 1000 IU/ml (open inverted triangles), prior to cytotoxicity assay with the CTL line 2. Treatment with IFN-α reduced the HepG2 cells sensitivity to CTL cytotoxicity in a dose dependent manner. D) This phenomenon was also found with the novel human hepatocyte cell lines (HHL). HHL-17 cells were either left untreated (closed circles) or stimulated for 16 hours with 1000 IU/ml (open inverted triangles) prior to co-incubation with the CTL line 1.</p

Patient cohort characteristics.

a<p>Log viral load.</p>b<p>H = Hispanic; AA = African American; M = Male; F = Female; Race was not available for 1 subject in the NS group and 2 subjects in the SS group.</p><p>The numbers in parentheses represent 95% confidence intervals.</p

Clinical data of HCV patients at time of biopsy.

<p>Clinical data of HCV patients at time of biopsy.</p

Distribution of HLA class I alleles in study Cohort.

<p>The frequency of expression of HLA class I A alleles (A) and B alleles (B) among the two progression groups in the cohort was compared to the expected frequency in a Hispanic and African American cohort (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021135#s4" target="_blank">methods</a>). Associations between HLA Class I alleles and clinical characteristics are shown in panels C–F. Associations between log viral load (LCL) of all subjects and class I HLA-A alleles (C) and B alleles (D), are ordered from lowest LVL to highest. Associations between CD4% values and HLA Alleles (E) and B alleles (F) are ordered from highest CD4% to lowest. The solid line in each column is the median value for that HLA allele. The dotted line is the median value of the total cohort.</p

Association between Gag specific responses and disease progression.

<p>The magnitude of the total observed Gag specific responses (the sum of all the epitope- specific responses) is compared between the two groups in panel A. Correlations between LVL and the magnitude of the total observed Gag response are displayed for both progression groups in panels B and C.</p