Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection

Abstract

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8(+) T cells. The role of CD4(+)CD25(+) T regulatory (T(reg)) cells in priming and expanding virus-specific CD8(+) T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8(+) T-cell proliferation and gamma interferon (IFN-gamma) frequency were analyzed with/without depletion of T(reg) cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4(+)CD25(+) T(reg) cells inhibited anti-CD3/CD28 CD8(+) T-cell proliferation and perforin expression. Depletion of CD4(+)CD25(+) T(reg) cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-gamma-expressing CD8(+) T cells. Although stimulated CD8(+) T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4(+)CD25(+) regulatory T cells on CD8(+) T-cell proliferation. In conclusion, marked CD4(+)CD25(+) regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8(+) T-cell responses and viral persistence.

FIG. 1.

Function and phenotype of CD4⁺CD25⁺ cells from patients with persistent HCV infection. (A) CD4⁺CD25⁺ T_reg cells, CD4⁺CD25⁻ T cells, or a combination of these at a ratio of 1:1 were stimulated with anti-CD3 and cell proliferation was measured using [³H]thymidine incorporation. (B and C) Intracellular CTLA-4 expression of (B) CD4⁺CD25⁺ cells and (C) CD4⁺CD25⁻ cells. (D) Percentage of CD8⁺ and CD4⁺CD25⁺ cells within the intrahepatic lymphocyte population. (E) Histological staining for Foxp3 within the liver of an HCV-infected individual.

FIG. 2.

Inhibition of CD8⁺ T-cell proliferation and perforin expression by CD4⁺CD25⁺ T_reg cells. (A) CD8⁺ T cells or (B) CD8⁺ T cells in the presence of CD4⁺CD25⁺ T_reg cells were stimulated with anti-CD3/CD28 and stained with anti-Ki-67, a nuclear marker of proliferation. Histograms are gated on CD8⁺ T cells. (C) The proportion of Ki-67-positive CD8⁺ T-cell was reduced in the presence of CD4⁺CD25⁺ T_reg cells (coculture; n = 6) and was not restored upon addition of anti-IL-10 or anti-TGF-β antibodies. (D) Intracellular perforin expression of anti-CD3/CD28 activated CD8⁺ T cells with (grey filled) or without CD4⁺CD25⁺ T_reg cells (black line) at a 1:1 ratio. (E) The proportion of intracellular perforin-positive CD8⁺ T cells was reduced in the presence of CD4⁺CD25⁺ T_reg cells (coculture; n = 6). Horizontal bars represent median values, the box represents the interquartile range, and the error bars represent the range.

FIG. 3.

CD4⁺CD25⁺ T_reg cells suppress the expansion of virus-specific CD8⁺ T cells during HCV infection. Proliferation in response to peptide stimulation was measured by tetramer frequency. Examples of individual patients are shown: (A) PBMC alone (left-hand panels) and CD4⁺CD25⁺ T_reg-depleted PBMC (right-hand panels) were restimulated with HCV-specific (top panels), CMV-specific (middle panels), or EBV-specific (bottom panels) peptides, and the extent of proliferation was measured by tetramer frequency. (B) Overall results are shown for HCV (top panel), CMV (middle panel), and EBV (bottom panel). The left-hand box represents the responses in unfractionated PBMC, the right-hand box represents the responses following depletion of CD4⁺CD25⁺ T_reg cells. Horizontal bars represent median values, the box represents the interquartile range, and the error bars represent the range.

FIG. 4.

Repletion of CD4⁺CD25⁺ T_reg cells to CD4⁺CD25⁺ T_reg cell-depleted PBMC inhibits CD8⁺ T-cell peptide-induced proliferation. Proliferation in response to peptide stimulation was measured by tetramer frequency. Examples are shown with HCV peptide-specific (left-hand panels) or CMV peptide-specific (right-hand panels) expansions. CD4⁺CD25⁺ T_reg-depleted PBMC (top panels), repletion of CD4⁺CD25⁺ T_reg-depleted PBMC with CD4⁺CD25⁺ T_reg cells at 1:10 (middle panels), or 1:2 (CD4⁺CD25⁺ T_reg cell: PBMC) (bottom panels). (B) Group data (n = 12) of HCV-specific tetramer frequency following peptide restimulation with or without the addition of CD4⁺CD25⁺ T_reg cells. Repletion of CD4⁺CD25⁺ T_reg cells was also performed with the addition, on day 0, of IL-2 at concentrations of 200 U/ml and 400 U/ml.

FIG. 5.

Virus-specific responses are suppressed by CD4⁺CD25⁺ T_reg cells in HCV patients compared to healthy individuals. (A) The ratio of the specific CD8⁺ T-cell increase following CD4⁺CD25⁺ T_reg cell depletion over PBMC alone of all patients studied. The EBV- and CMV-specific responses in healthy individuals are in the left column, the EBV- and CMV-specific responses in HCV-infected patients are in the middle column, and the HCV-specific responses in HCV-infected patients are in the right column. The horizontal line in each column represents the median. (B) Depletion of CD4⁺CD25⁺ T_reg cells enhances IFN-γ secretion by virus-specific CD8⁺ T-cell in HCV-infected patients. PBMC alone (PBMC) and following depletion of CD4⁺CD25⁺ T_reg cells (Depleted) were used in an IFN-γ ELIspot assay using a pool of three HCV peptides or (C) the EBV peptide.

FIG. 6.

Expression of the TGF-β receptor type II and IL-10 receptors on virus-specific CD8⁺ T cells. (A) TGF-β receptor type II and (B) IL-10 receptor expression on HCV-specific CD8⁺ T cells ex vivo. (C) TGF-β receptor type II and (D) IL-10 receptor expression on CMV-specific CD8⁺ T cells ex vivo. (E) TGF-β receptor type II and (F) IL-10 receptor expression on CMV-specific CD8⁺ T cells after peptide restimulation.