1. Volume 137, Issue 4, Pages 1280-1288.e6 (October 2009)
FOXP3 Expression in Hepatitis C Virus–Specific CD4+ T Cells During Acute Hepatitis C 
Malte H.J. Heeg, Axel Ulsenheimer, Norbert H. Grüner, Reinhart Zachoval, Maria–Christina Jung, J. Tilman Gerlach, Bijan Raziorrouh, Winfried Schraut, Sophia Horster, Teresa Kauke, Michael Spannagl, Helmut M. Diepolder 
Gastroenterology 
Volume 137, Issue 4, Pages e6 (October 2009)
DOI: /j.gastro
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2. Figure 1
Gating strategy for the phenotyping of FOXP3+CD4+ T cells. For the analysis of bulk CD4+ T cells, (A) first a gate was set for FOXP3+CD4+ T cells and (B) subsequently expression of CD127 and CD25 was analyzed. The sum of the right quadrants represents the percentage of FOXP3+CD4+ T cells expressing CD25, and the sum of the lower quadrants yields the percentage of FOXP3+CD4+ T cells that are negative for CD127. The lower right quadrant represents the percentage of FOXP3+CD4+ T cells that are both CD25+ and CD127−. The lower panels (C–E) show fluorescence-activated cell sorter stainings of PBMCs from a patient with chronic hepatitis C including the HCV-NS3 tetramers. (C) First a gate is set for Tet+CD4+ T cells. (D) The next gate is set on FOXP3-expressing Tet+CD4+ T cells, which are subsequently analyzed for the expression of (E) CD25 and CD127 in analogy to B. F–H show the staining of an HCV-specific T-cell line, which was generated by stimulation with recombinant HCV-NS3 antigen in the presence of interleukin-2 and transforming growth factor β. After 10 days of culture, expression of CD25 on FOXP3+CD4+ T cells increased to 77%, low expression of CD127 increased to 62%, and the CD25+CD127− phenotype was present on 28% of FOXP3+Tet+CD4+ T cells.
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3. Figure 2
Frequency and phenotype of putative Tregs in chronic hepatitis C. Patients with chronic hepatitis C (n = 9) had a significantly higher frequency of (A) FOXP3+ and (B) CD25+ expression on CD4+ T cells as compared with healthy controls. (C) There was a trend for lower expression of CD127 on CD4+ T cells (P = .15, all comparisons analyzed by Wilcoxon rank test), which is compatible with an overall higher frequency of Tregs in patients with chronic hepatitis C. Using triple staining for FOXP3, CD25, and CD127, patients with chronic hepatitis C had a significantly lower expression of CD25 on (D) FOXP3+CD4+ T cells and a significantly lower relative frequency of the (F) FOXP3+CD25+CD127− phenotype, whereas (E) CD127 expression was not affected. When the phenotype of bulk CD4+ T cells in patients with chronic hepatitis C was compared with HCV-specific CD4+ T cells, there were no significant differences for the expression of (A and B) FOXP3 or CD25 but a significantly higher frequency of the CD127− phenotype. When the phenotype of FOXP3+Tet+CD4+ T cells was further analyzed, there was a significantly lower expression of CD25, a higher expression of CD127, and a lower frequency of the CD25+CD127− phenotype on FOXP3+Tet+CD4+ T cells in comparison with bulk FOXP3+CD4+ T cells (paired Wilcoxon rank test). Significant P values are shown.
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4. Figure 3
Longitudinal course of regulatory T-cell markers in HCV-specific CD4+ T cells. (A and D) FOXP3, (B and E) CD25, and (C and F) CD127 expression were monitored during the first 24 weeks and up to week 192 in (A–C) resolving patients (AR1–5) and (D–F) chronically evolving patients (AC1–4). For FOXP3 and CD25, 3 distinct peaks (arrows) can be identified in patients AR1, AR2, and AC1. The decline of the CD127− phenotype correlates with spontaneous viral clearance in patients with acute resolving hepatitis C and with interferon-induced viral elimination in patients AC3 and AC4, whereas the CD127− phenotype is maintained in the majority of HCV-specific CD4+ T cells in patient AC1, who did not receive antiviral treatment and was still viremic at the end of follow-up.
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5. Figure 4
The peaks of FOXP3 expression in patients AR1, AR2, and AC1 are in relation to T-cell proliferation in response to NS3–NS4 antigen and viremia (upper panels) as well as to the other regulatory T-cell markers CD25 and CD127 (lower panels). Each peak of FOXP3 expression correlated with an albeit smaller peak of CD25 and an increase of the CD127− phenotype. On all 3 occasions, T-cell responses were lost and on 2 occasions, in patients AR1 and AC1, transient recrudescence of viremia was observed. The right part of the figure shows individual dot blots for FOXP3 expression in bulk CD4+ T cells (upper rows) and HCV-specific CD4+ T cells (lower rows) on the time point before (left), at (middle), and after (right) the peak of FOXP3 expression. No obvious change in the frequency of bulk FOXP3+CD4+ T cells was observed.
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6. Figure 5
Changes in NS3–NS4—specific T-cell proliferation and FOXP3 expression on HCV-specific CD4+ T cells at time points of viral recrudescence. Four patients (AR1, AR3, AC1, and AC4) had fluctuating levels of HCV RNA with 1 or 2 episodes of transient viral suppression followed by relapse. While all 6 episodes correlated with a decline in HCV-specific T-cell proliferation (P = .03), an increase in FOXP3 expression was observed on only 2 occasions, both of which were rapidly followed again by viral control that led to resolution in AR1 and to another phase of transient viral control in AC1, respectively.
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7. Supplementary Figure 1
Selection of epitopes for synthesis of HLA-DR15 tetramers. To be able to detect FOXP3 expression in hepatitis C virus specific CD4+ T cells, we designed a new set of HLA-DR15 tetramers which were based on previously characterized, immunodominant HLA-DR15 restricted CD4+ T cell epitopes, NS3-aa and NS3-aa Among 13 HLA-DR15 positive patients with acute hepatitis C, epitope yielded the strongest mean proliferative response with the NS3-4 region (A) and was most frequently recognized (8/13) (B) whereas the other epitope ranked third, also both for strength (A) and frequency (7/13) (B) of proliferative response.
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8. Supplementary Figure 2
Staining of specific CD4+ T cell clones with HLA-DR15 tetramers. The HLA-DR15 tetramers Tet1411 and Tet1582 were initially validated by staining epitope specific CD4+ T cell clones. Strong and specific staining was demonstrated with the specific tetramer (upper right and lower right panel) whereas the control staining was negative (upper left and lower left panel).
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9. Supplementary Figure 3
Validation of HLA-DR15 tetramers. Epitope specific CD4+ T cell clones were titrated into non-antigen-specific PBMC (A and B) and the number of added clone cells was correlated to the number of cells detected by specific HLA class II tetramer (C), showing a strong correlation between added and detected cells. To show that the enrichment step did not influence the detection rate of HCV-specific FOXP3+ CD4+ T cells we analyzed all samples with tetramer frequencies >0.05% before and after the enrichment step. R2 values were >0.95 for both overall frequency of tetramer-positive CD4+ T cells and FOXP3 expression (D and E). In ten DR15-positive healthy controls and five non-DR15 patients with acute hepatitis C tetramer frequencies were always <0.001% (data not shown).
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10. Supplementary Figure 4
Longitudinal course of FOXP3 expression in virus specific CD4+ T cells in patients with self-limited acute hepatitis C. The top panels show the course of HCV-RNA (grey shade) and proliferation (open circles). The bottom panels show the frequency of tetramer-positive CD4+ T cells (open squares) and the percentage of FOXP3 expressing tetramer-positive cells (closed triangles). The middle panels in patients AR2-5 display the frequency of IFN-g (closed triangles) and IL-10 (open circles) producing CD4+ T cells.
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11. Supplementary Figure 5
Longitudinal course of FOXP3 expression in virus specific CD4+ T cells in patients with acute hepatitis C and viral persistence or early antiviral treatment. The top panels show the course of HCV-RNA (grey shade) and proliferation (open circles). The bottom panels show the frequency of tetramer-positive CD4+ T cells (open squares) and the percentage of FOXP3 expressing tetramer-positive cells (closed triangles). The middle panels in patients AC2-4 and AE1 display the frequency of IFN-gamma (closed triangles) and IL-10 (open circles) producing CD4+ T cells.
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12. Supplementary Figure 6
FOXP3 expression in bulk CD4+ T cells in acute hepatitis C. The time course of FOXP3 expression in bulk CD4+ T cells from (A) patients with acute self-limited hepatitis C and (B) patients with evolving chronic hepatitis C.
Gastroenterology  , e6DOI: ( /j.gastro )
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