T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Abstract

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.

FIG. 1.

Depletion of CD4⁺ cells enhances HCV-specific CD8⁺ T-cell responses. Tetramer staining of a T-cell line after 7 days in culture. PBMC (white bars) and PBMC depleted of CD4⁺ cells (black bars) were stimulated with HCV peptides and cultured for 7 days. In all cases, in vitro stimulated T-cell lines showed an enhanced expansion after depletion of CD4⁺ cells.

FIG. 2.

CD4⁺CD25⁺ regulatory T cells suppress HCV-specific CD8⁺ T-cell response. (A) PBMC depleted of CD4⁺ cells were cultured alone (grey bars), in the presence of CD4⁺CD25⁻ (black bars) or CD4⁺CD25⁺ (white bars) for 7 days. Cells were stained with HLA-A2 tetramers. (B) PBMC depleted of CD4⁺ cells were cultured alone, in the presence of CD4⁺CD25⁻ or CD4⁺CD25⁺ for 7 days. Representative density plots of cells stained with tetramer and anti-CD8 are shown.

FIG. 3.

Treg cells inhibit proliferation of HCV specific tetramer-positive CD8⁺ T cells. (A) CD4-depleted PBMC were stimulated with HCV peptide 3 prior to culture with decreasing numbers of CD4⁺CD25⁺ regulatory T cells (CD4-depleted PBMC: Treg cells). After 7 days in culture, cells were tested for virus-specific responses by tetramer staining. (B) CD4-depleted PBMC were labeled with CFSE- and stimulated with HCV peptide 3 prior to culture with high (ratio 3:1) or low (ratio 100:1) numbers of CD4⁺CD25⁺ regulatory T cells. Representative histograms are shown. The cells are gated on CD8⁺ HCV tetramer positive cells.

FIG. 4.

Treg cell-mediated suppression is cell-cell contact dependent but independent of IL-10 and TGF-β. (A) PBMC were depleted of CD4⁺ cells and cultured alone (left), in the presence of CD4⁺CD25⁺ regulatory T cells but separated through a microporous membrane (no cell-cell contact) (middle), and unseparated together in one well (cell-cell contact) (right). (B) Addition of neutralizing antibodies against IL-10 and TGF-β had no effect on the suppressive capacity of CD4⁺CD25⁺ regulatory T cells. Data are the means of duplicate wells.

FIG. 5.

Inhibition of HCV and influenza virus-specific CD8⁺ T-cell proliferation by Treg cells in chronically HCV-infected patients and healthy blood donors. (A) T-cell lines from HCV-infected patients were tested for Treg-mediated suppression (ratio 3:1) of HCV-specific (patients C1 [two epitopes], C2, C3, and C4) and influenza virus-specific (patients C1, C2, C3, C4, and C5) CD8⁺ T-cell proliferation after 7 days of antigen-specific stimulation by tetramer staining. To calculate the percentage of CD4⁺CD25⁺ cell-mediated inhibition after 7 days in culture the following formula was used: 100 − (percentage of tetramer-positive CD8⁺ T cells in the presence of CD4⁺CD25⁺ cells divided by the percentage of tetramer-positive CD8⁺ T cells in the presence of CD4⁺CD25⁻ cells) × 100. Patients with resolved HCV infection and healthy blood donors were tested in the same way for Treg-mediated suppression of HCV-specific (patients R1, R2 [two epitopes], R3, R7, and R8 [two epitopes]) and influenza virus-specific (patients R2, R3, R4, R5, and R6 and healthy blood donors) CD8⁺ T-cell proliferation. Importantly, a significant difference was observed between the inhibition of HCV-specific and influenza virus-specific CD8⁺ T cells in chronically HCV-infected patients compared to patients with resolved HCV infection and healthy blood donors (only influenza virus). (B) Frequency of CD4⁺CD25⁺ T cells in the peripheral blood of chronically HCV-infected patients, patients with resolved HCV infection, and healthy blood donors.