Increased hepatitis C virus (HCV)-specific CD4+CD25+ regulatory T lymphocytes and reduced HCV-specific CD4+ T cell response in HCV-infected patients with normal versus abnormal alanine aminotransferase levels

Abstract

CD4+CD25+ T regulatory cells may play a role in the different clinical presentations of chronic hepatitis C virus (HCV) infection by suppressing CD4+ T cell responses. Peripheral CD4+CD25+ T cells from chronic HCV carriers with normal and abnormal alanine aminotransferase (ALT) were analysed for specificity and effect on HCV-specific CD4+ T cell reactivity by flow cytometry for intracellular cytokine production and proliferation assay. HCV-specific CD4+CD25(+high) T cells consistently produced transforming growth factor (TGF)-beta but only limited amounts of interleukin (IL)-10 and no IL-2 and interferon (IFN)-gamma. The HCV-specific TGF-beta response by CD4+CD25(+high) T cells was significantly greater in patients with normal ALT compared to patients with elevated ALT. In addition, a significant inverse correlation was found between the HCV-specific TGF-beta response by CD4+CD25(+high) T cells and liver inflammation. In peripheral blood mononuclear cells (PBMC), both HCV antigen-induced IFN-gamma production and proliferation of CD4+ T cells were greater in patients with elevated ALT compared with patients with normal ALT. Depletion of CD4+CD25+ cells from PBMC resulted in an increase of both IFN-gamma production and proliferation of HCV-specific CD4+ T cells that was significantly greater in patients with normal ALT levels compared with patients with elevated ALT. In addition, CD4+CD25+ T cells from patients with normal ALT levels proved to be significantly more potent to suppress CD4+ T cell reactivity with respect to those from patients with elevated ALT. In conclusion, these data support the hypothesis that CD4+CD25+ cells may play a role in controlling chronic inflammatory response and hepatic damage in chronic HCV carriers.

Fig. 1

Hepatitis C virus (HCV)-specific transforming growth factor (TGF)-β secretion by CD4⁺ CD25⁺ T cells in patients with normal and elevated ALT. Pure CD4⁺CD25⁺ T cells (1 × 10⁵) were stimulated with pooled HCV antigens (core, NS3, NS4, NS5: 2 µg/ml each). The cells were stained with Cychrome-anti-CD4, fluorescein isothiocyanate (FITC)-anti-CD25 monoclonal antibody and phycoerythrin (PE)-anti-TGF-β monoclonal antibodies (mAbs). The cells were gated on CD4⁺ and analysed on FL2 (FITC) versus FL1 (PE) two-dimensional plots to discriminate positive cells. The percentage of positive cells was calculated with respect to total CD4⁺CD25⁺ T cells. The number of positive events calculated in the same samples in the absence of antigen stimulation was constantly below 0·01%. No significant production of interleukin (IL)-2, interferon (IFN)-γ, IL-10 and TGF-β was detected in healthy controls.

Fig. 2

Correlation analysis between hepatitis C virus (HCV)-specific transforming growth factor (TGF)-β response by CD4⁺CD25⁺ T cells and histological inflammatory grade. A significant negative correlation was observed (r = –0·84, P < 0·001) by the non-parametric Spearman's rank test.

Fig. 3

Hepatitis C virus (HCV)-specific transforming growth factor (TGF)-β and interleukin (IL)-10 production by CD25^+high T cells from a single patient. (a) CD4⁺CD25⁺ T cells, stimulated with medium alone, gated on CD25^high population. (b) American Type Culture Collection CRL-2159 cells (positive staining for TGF-β). (c) CD4⁺CD25⁺ T cells, stimulated with phorbol myristate acetate (PMA) plus ionomycin (IO) (positive staining for IL-10). (d) CD4⁺CD25⁺ T cells, stimulated with pooled HCV antigens (core, NS3, NS4, NS5: 2 µg/ml each) and stained with phycoerythrin (PE)-anti-IL-10 monoclonal antibodies (mAbs), gated on CD25^high population. (e) CD4⁺CD25⁺ T cells, stimulated with pooled HCV antigens (core, NS3, NS4, NS5: 2 µg/ml each) and stained with PE-anti-TGF-β mAbs, gated on CD25^high population.

Fig. 4

Hepatitis C virus (HCV)-specific interferon (IFN)-γ secretion in patients with elevated and normal alanine aminotransferase (ALT). HCV-specific IFN-γ responses by CD4⁺ T cells were assessed by flow cytometry in whole peripheral blood mononuclear cells (PBMC) that were mixed with HCV antigens (core, NS3, NS4, NS5: 2 µg/ml each) and anti-CD28 and anti-CD49d monoclonal antibodies or positive controls [phytohaemagglutinin (PHA)] or negative controls (medium). The percentage of IFN-γ-CD4⁺ T cells in PHA-stimulated controls was 12·2% and 14·5% in patients with elevated and normal ALT, respectively, P > 0·05. The number of positive events in the absence of antigen stimulation was constantly below 0·01%.

Fig. 5

CD4⁺CD25⁺ T cell suppression of hepatitis C virus (HCV)-specific interferon (IFN)-γ secretion in patients with elevated and normal alanine aminotransferase (ALT). HCV-specific IFN-γ responses by CD4⁺ T cells were assessed by flow cytometry in peripheral blood mononuclear cells (PBMC) depleted of CD4⁺CD25⁺ T cells that were mixed with HCV antigens (core, NS3, NS4, NS5: 2 µg/ml each) and positive controls (PHA) or negative controls (medium) and anti-CD28 and anti-CD49d monoclonal antibodies. The percentage of IFN-γ-CD4⁺ T cells in PHA-stimulated controls was 24·3% and 26·4% in patients with elevated and normal ALT, respectively, P > 0·05. The number of positive events in the absence of antigen stimulation was constantly below 0·01%.

Fig. 6

Inhibition of CD4⁺ T cell proliferation by CD4⁺CD25⁺ T cell in patients with normal and elevated alanine aminotransferase (ALT). CD25⁺ depleted CD4⁺ T cells (4 × 10⁴) were stimulated with pooled recombinant hepatitis C virus (HCV) antigens (core, NS3, NS4, NS5: 2 µg/ml each) and anti-CD28 and anti-CD49d monoclonal antibodies. HCV-specific cell proliferation was measured by [³H]-thymidine incorporation. The counts per minute (cpm) (× 10³) in phytohaemagglutinin (PHA)-stimulated controls were 323·9 × 10³ and 354·1 × 10³ in patients with elevated and normal ALT, respectively, P > 0·05. In the absence of stimuli and in normal subjects stimulated with HCV antigens the cpm was constantly below 10³.

Fig. 7

Dose–response inhibition of hepatitis C virus (HCV)-specific interferon (IFN)-γ production by CD4⁺CD25⁺ T cell in patients with normal and elevated alanine aminotransferase (ALT). CD4⁺ T cells were used as effector cells to examine HCV-specific IFN-γ activity as measured by flow cytometry in response to pooled HCV antigens (core, NS3, NS4, NS5: 2 µg/ml each) and anti-CD28 and anti-CD49d monoclonal antibodies in co-culture with CD4⁺CD25⁺ T cells at various ratios. Duplicate wells without stimuli were also included to determine the background level of cytokine production. The number of positive events in the absence of antigen stimulation was constantly below 0·01%.