Additional file 1: of Excellent local control and tolerance profile after stereotactic body radiotherapy of advanced hepatocellular carcinoma

Supplementary table showing the fractionation regimes used. (DOCX 13 kb

Functional profile of HCV-specific CD8+ T cells.

<p>(A) Cytokine production and CD107a mobilization of CTL lines, derived from chronically HCV infected subjects specific for HLA-A*02-restricted epitopes (n = 4, black bars) and the immunodominant HLA-B*27-restricted epitope NS5B₂₈₄₁ (n = 4, white bars). Prior to intracellular multi-cytokine staining CTL lines were stimulated for 5 hours with specific peptides. The background value from the negative control (without peptide) was subtracted from all measured response frequencies (cytokine+ CD8+/total CD8+). Data are presented as mean ± SD. (B) Pie charts showing the mean multifunctionality of HLA-A*02-restricted (n = 4) and HLA-B*27-restricted (n = 4) HCV-specific CD8+ T-cell lines (one to five functions: CD107a, IFN-γ, IL-2, MIP-1β and TNF-α). Responding cells are grouped by the number of functions indicated by the numbers in the pie charts and matched to the color code in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003042#ppat-1003042-g002" target="_blank">figure 2C</a>. (C) Detailed functional profile of HLA-A*02-restricted (n = 4, black bars) and HLA- B*27-restricted (n = 4, white bars) HCV-specific CD8+ T-cell lines. Bars represent frequency of CD8+ T cells displaying the indicated combination of functions within the total population of responding CD8+ T cells. P-values were calculated using the Mann-Whitney U-test. Data are presented as mean ± SD.</p

Functional avidity of HCV-specific CD8+ T cells.

<p>(A) Representative flow cytometry plots showing intracellular IFN-γ staining of a CTL line exposed to target cells pulsed with a range of peptide concentrations. PBMCs from a subject chronically infected with HCV were stimulated for two weeks with the HLA-A*02-restricted NS3₁₀₇₃ epitope. Prior to intracellular IFN-γ staining, the CTL line was cocultured for 5 hours with HLA-A*02+ EBV-immortalized B-cell lines (B-LCLs) pulsed with serially diluted concentrations of the cognate peptide. The background value from the negative control (B-LCLs without peptide loading) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+). (B) Representative data from intracellular IFN-γ staining of a CTL line specific for the HLA-A*02-restricted NS3₁₀₇₃ epitope (filled circles) and a CTL line specific for the HLA-B*27-restricted NS5B₂₈₄₁ epitope (open circles) across a range of peptide concentrations. The background value from the negative control (B-LCLs without peptide loading) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+), which were then normalized to the maximum response by defining the smallest value as 0% and the largest value as 100%. (C) The functional avidity of CTL lines, derived from chronically HCV infected subjects, specific for HLA-A*02-restricted epitopes (n = 7, filled circles) and the immunodominant HLA-B*27-restricted epitope NS5B₂₈₄₁ (n = 6, open circles), as well as of CTL lines derived from subjects with resolved infection specific for HLA-A*02-restricted epitopes (n = 3, filled squares) and the immunodominant HLA-B*27-restricted epitope NS5B₂₈₄₁ (n = 4, open squares). Functional avidity was determined as the concentration of peptide required to achieve half-maximal IFN-γ induction (EC₅₀). P-values were calculated using the Mann-Whitney U-test. Horizontal bars represent mean values.</p

Proteasomal digestion of HCV-specific epitope-containing regions.

<p>(A) Peptides of 24 (A2-NS3₁₀₇₃) and 25 (A2-NS5B₂₅₉₄ and B27-NS5B₂₈₄₁) amino acids in length spanning the regions containing the respective epitopes were used for incubation with constitutive proteasomes and immunoproteasomes. Epitope sequences are underlined. (B) Production of epitope-containing and non-epitope-containing cleavage products of the 24-mer (A2-NS3₁₀₇₃) and two 25-mer (A2-NS5B₂₅₉₄ and B27-NS5B₂₈₄₁) peptides incubated with constitutive proteasomes and immunoproteasomes for 1 to 6 hours using standard conditions. The sum of all fragment intensities was set at 100%. Data are representative of triplicate experiments. (C) The relative production of all cleavage products of the B27-NS5B₂₈₄₁ peptide by constitutive proteasomes and immunoproteasomes for 1 to 6 hours using standard conditions. All fragments were between 4 and 9 amino acids long. Data are representative of triplicate experiments. (D) The relative production of all cleavage products of the B27-NS5B₂₈₄₁ peptide by constitutive proteasomes and immunoproteasomes for 1 to 6 hours using a 4× dilution of both of the proteasomal forms. The abundance of B27-NS5B₂₈₄₁-containing fragments ending in a C-terminal ²⁸⁵⁷D represents a maximum as this corresponds to the end of the 25-mer peptide substrate. All fragments containing the NS5B₂₈₄₁ epitope are highlighted using black symbols. The NS5B₂₈₄₁ epitope sequence is underlined. Data are representative of triplicate experiments.</p

Processing and presentation of HCV-specific CD8+ T-cell epitopes.

<p>(A) APCs were infected with vaccinia virus constructs carrying the relevant part of the HCV polyprotein (vHCV 827). After a defined time period (0, 2, 4, 8, 12, 16, 20 and 24 hours) these APCs were added to HCV-specific CTL lines and antigen-specific IFN-γ production was assessed by flow cytometry. (B) Representative flow cytometry plots of a CTL line stimulated with infected APCs. Prior to intracellular IFN-γ staining, the epitope-specific CTL line was stimulated for 5 hours with APCs previously infected with vHCV 827 and pre-incubated for 2, 4, 8 and 12 hours respectively. The background value from the negative control (T7 RNA polymerase (vTF7)) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+). (C) Representative time course showing intracellular IFN-γ staining of an HLA-A*02-restricted CTL line (filled circles) and an HLA-B*27-restricted (open circles) CTL line stimulated with infected APCs pre-incubated for 0 to 24 hours. The background value from the negative control (vTF7) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+), which were then normalized to the maximum response by defining the smallest value as 0% and the largest value as 100%. The complete kinetic (0 to 24 hours) was performed for all subjects, and the normalized responses were used to generate the data shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003042#ppat-1003042-g008" target="_blank">Figure 8D and E</a>. (D) Responses of CTL lines against APCs infected with vHCV 827 and pre-incubated for 2, 4 and 8 hours. Normalized IFN-γ responses of CTL lines specific for HLA-A*02-restricted epitopes (n = 9, filled circles) and for the immunodominant HLA-B*27-restricted NS5B₂₈₄₁ epitope (n = 7, open circles) are shown. Responses were normalized to the maximum response, as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003042#ppat-1003042-g008" target="_blank">Figure 8C</a>. Horizontal bars represent mean values. (E) Calculated peptide concentration present at the surface of infected APCs after 2, 4 and 8 hours of incubation with vHCV 827. The concentration of presented peptide restricted by HLA-A*02 (n = 9, filled circles; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003042#ppat-1003042-t001" target="_blank">Table 1</a>) and HLA-B*27 (NS5B₂₈₄₁; n = 7, open circles) was calculated by correcting for the individual functional avidity of the respective peptide-specific CTL line. For details of the calculations, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003042#ppat.1003042.s003" target="_blank">Figure S3</a>. P-values were calculated using the Mann-Whitney U-test. Horizontal bars represent mean values. (F) Representative time course showing intracellular IFN-γ staining of an HLA-B*27-restricted CTL line stimulated with infected APCs incubated for 0 to 8 hours and pre-incubated with (triangles) or without (squares) the proteasome inhibitor epoxomicin. The background value from the negative control (vTF7) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+). Data are representative of experiments performed with two different HLA-B*27-restricted CTL lines and one HLA-A*02-restricted CTL line.</p

Epitope precursor degradation rates of natural HLA-A*02- and HLA-B*27-restricted epitopes as well as of an artificial chimeric epitope.

<p>The percentage of undigested 25-mer precursor peptide left following 1, 2, 4, and 6 hours of constitutive and immunoproteasomal digestion, respectively. The amino acid sequences of the examined 25-mer peptides are shown with imbedded optimal epitopes underlined and the amount of purified proteasomal solution is indicated. B27-in-A2 signifies the B27-NS5B₂₈₄₁ epitope surrounded by the A2-NS5B₂₅₉₄ flanking regions, while A2-in-A2 and B27-in-B27 show these epitopes in their natural sequence contexts. The grey line indicates 50% degradation of the precursor peptide. Data are representative of triplicate experiments.</p

Antiviral efficacy of HCV-specific CD8+ T cells.

<p>Antiviral efficacy (white bars) and functional avidity (filled circles) of CTL lines, derived from subjects with a chronic HCV infection specific for (A) HLA-A*02-restricted epitopes (n = 3) and (B) the immunodominant HLA-B*27-restricted epitope NS5B₂₈₄₁ (n = 3) as well as of CTL lines derived from subjects with resolved infection specific for (C) HLA-A*02-restricted epitopes (n = 3) and (D) the immunodominant HLA-B*27-restricted epitope NS5B₂₈₄₁ (n = 3). HuH7_A2HCV and HuH7_B27HCV cell lines were pulsed with a range of cognate peptide concentrations and cocultured for 24 hours with CTL lines at an E∶T ratio of 1∶1. Inhibition of viral replication was measured by luciferase activity. As a reference, intracellular IFN-γ staining at corresponding peptide concentrations is indicated. Measured RLUs and IFN-γ responses were normalized to maximum viral replication or maximum IFN-γ response, respectively, by defining the smallest value as 0% and the largest value as 100%. Data are presented as mean ± SD.</p

HLA-A^*02 and -B^*27 restricted epitopes.

*<p>IC50 was determined by using the immune epitope database (IEDB, <a href="http://www.immuneepitope.org/" target="_blank">http://www.immuneepitope.org/</a>).</p

Precursor frequency of HCV-specific CD8+ T cells.

<p>(A) Representative flow cytometry plots of PBMCs from a healthy donor displaying staining with the NS5B₂₈₄₁/HLA-B*27 tetramer before and after magnetic-bead enrichment. Percentage of tetramer+ CD8+ T cells is indicated. (B) Precursor frequencies of CD8+ T cells specific for NS3₁₄₀₆ (HLA-A*02 tetramer, filled circles) and NS5B₂₈₄₁ (HLA-B*27 tetramer, open circles) detected in healthy donors after tetramer staining, magnetic-bead enrichment and multiparametric flow cytometric analysis. The enriched tetramer+ CD8+ T-cell populations were stained for CD45RA, CD27, CCR7 and CD11a. The number of phenotypically naïve (CD45RA^hi, CD27^hi, CCR7^hi, CD11a^low) tetramer+ CD8+ T cells relative to the number of total CD8+ T cells is indicated. Horizontal bars represent mean values.</p

Cross-recognition patterns of all B27-NS5B₂₈₄₁-containing fragments derived from immunoproteasomal digestion.

<p>(A) Representative flow cytometry plots showing intracellular IFN-γ staining with CD8+ T cells from one subject across a range of peptide concentrations. PBMCs from HLA-B*27+ subjects chronically infected with HCV were enriched for CD8+ T cells and expanded non-specifically for 14 days. The background value from the negative control (without peptide loading) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+). Tested peptide sequences are indicated. (B) Representative cross-recognition patterns of all naturally processed B27-NS5B₂₈₄₁-containing fragments by enriched and non-specifically expanded CD8+ T cells (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003042#ppat-1003042-g007" target="_blank">Figure 7A</a>). Responses were measured by intracellular IFN-γ staining against serial dilutions of all 7 B27-NS5B₂₈₄₁-containing peptide forms. The background value from the negative control (without peptide loading) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+). Symbol code for peptide sequences is indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003042#ppat-1003042-g007" target="_blank">Figure 7C</a>. (C) Representative cross-recognition patterns of the NS5B₂₈₄₁- peptide by PBMCs specifically expanded by all naturally processed B27-NS5B₂₈₄₁-containing fragments. PBMCs from subjects chronically infected with HCV were stimulated for two weeks with all 7 B27-NS5B₂₈₄₁-containing peptide forms separately (see indicated symbol code). Responses were measured by intracellular IFN-γ staining against serial dilutions of the NS5B₂₈₄₁- peptide. The background value from the negative control (without peptide loading) was subtracted from all measured response frequencies (IFN-γ+ CD8+/total CD8+).</p