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. 2016 May 19;6:26296.
doi: 10.1038/srep26296.

Functional Dichotomy of Vδ2 γδ T Cells in Chronic Hepatitis C Virus Infections: Role in Cytotoxicity but Not for IFN-γ Production

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Free PMC article

Functional Dichotomy of Vδ2 γδ T Cells in Chronic Hepatitis C Virus Infections: Role in Cytotoxicity but Not for IFN-γ Production

Wenwei Yin et al. Sci Rep. .
Free PMC article

Abstract

Vδ2 γδ (Vδ2) T cells, a major human γδ T cell subset, exhibit broad anti-tumor and anti-infective activity; however, their precise role in chronic hepatitis C virus (HCV) infections remains unclear. In this study, we analyzed the phenotype and function of Vδ2 T cells in 43 HCV-infected patients compared to 39 healthy controls (HCs). Vδ2 T cells from HCV-infected patients were activated and differentiated into effector cells. Vδ2 T cells in patients expressed significantly higher levels of natural killer (NK) cell markers CD56 and CD16 than in HCs, acquiring cytotoxic NK-like phenotype. The Vδ2 T cell phenotype was associated with increased cytolytic effector molecules expression in HCV-infected patients with elevated serum ALT levels. Surprisingly, Vδ2 T cells in patients had a markedly impaired capacity to produce IFN-γ. Further in vitro and in vivo analysis showed that interferon-α, which was induced during HCV infection, caused Vδ2 T cell function bias toward cytotoxicity. These results suggest a functional dichotomy for Vδ2 T cells in chronic HCV infections: a role in cytotoxicity but not for IFN-γ production, which may contribute to both the liver inflammation and HCV persistence.

Figures

Figure 1
Figure 1. Vδ2 T cells in HCV-infected patients show an activated terminally differentiated effector phenotype.
(A) Representative flow cytometry panels depict the expression of the activation markers CD38 and CD69 on Vδ2 T Cells from HCs and HCV-infected patients. (B) Frequency of Vδ2 T cells expressing CD38 and CD69 in HCs and HCV-infected patients. n = 21 and 27 for HC and HCV, respectively. (C) Distribution of Vδ2 T cell subsets in representative individuals from HCs and HCV-infected patients. Differentiation was analyzed by monitoring CD27 and CD45RA expression. (D) Vδ2 T cell differentiation profile from HCs and HCV-infected patients. n = 33 and 36 for HC and HCV, respectively. Horizontal lines indicate the median. (E,F) Correlation analysis of the percentages of Vδ2 TEffector cells and the serum ALT levels (E) or HCV RNA loads (F) in HCV-infected patients. n = 36. *p < 0.05, **p < 0.01.
Figure 2
Figure 2. Comparison of the expression of NK cell-associated antigens on Vδ2 T cells in HCs and HCV-infected patients.
(A) Representative dot plots show the expression of CD56, CD16, FasL,NKG2A and NKG2D on Vδ2 T cells from HCs and HCV-infected patients. (B) Frequency of Vδ2 T cells displaying the indicated surface markers in HCs and HCV-infected patients. CD56, n = 25 and 35, respectively; CD16, n = 27 and 36, respectively; FasL, n = 19 and 21, respectively; NKG2A n = 18 and 25, respectively; NKG2D, n = 21 and 32, respectively. Horizontal lines indicate the median. (C,D) Correlation analysis of the percentages of CD56 + Vδ2 T cells and the serum ALT levels (C) or HCV RNA loads (D) in HCV-infected patients. n = 35. *p < 0.05, **p < 0.01.
Figure 3
Figure 3. Enhanced cytolytic enzymes production by Vδ2 T Cells associates with liver damage in HCV-infected patients.
(A) Representative dot plots depict the expression of GrA, GrB, Granulysin and perforin on Vδ2 T cells from HCV-infected patients with elevated ALT levels and from those with normal ALT levels and HCs. (B) Frequency of Vδ2 T cells expressing GrA, GrB, Granulysin (gated on CD3 + T cells) and perforin (gated on lymphocytes) in each group. GrA, n = 15 and 12 and 22, respectively; GrB, n = 16 and 12 and 22, respectively; Granulysin, n = 13and 13 and 28, respectively; perforin, n = 12 and 14 and 26, respectively. Horizontal lines indicate the median. (C,D) Correlation analysis of the percentages of GrB + Vδ2 T cells and the serum ALT levels (C) or HCV RNA loads (D) in HCV-infected patients. n = 34. *p < 0.05, **p < 0.01.
Figure 4
Figure 4. Vδ2 T cells display enhanced degranulation but decreased IFN-γ production in patients with chronic HCV.
(A) Representative dot plots depict CD107a expression on Vδ2 T cells from HCs and HCV-infected patients. (B) Frequency and mean fluorescence intensity (MFI) of Vδ2 T cells expressing CD107a in HCs and HCV-infected patients. n = 15 and 18 for HC and HCV, respectively. (C) Representative dot plots depict IFN-γ expression on Vδ2 T cells from HCs and HCV-infected patients. (D) Frequency and mean fluorescence intensity (MFI) of Vδ2 T cells expressing IFN-γ in HCs and HCV-infected patients. n = 17 and 20 for HC and HCV, respectively. (E) A quantitative analysis of IFN-γ produced by Vδ2 T cells from HCs and HCV-infected patients was performed by ELISA assay. Results (pg/ml of IFN-γ) were normalized for 1000 Vδ2 T cells. n = 14 for each group. Horizontal lines indicate the median. *p < 0.05.
Figure 5
Figure 5. Expression of activation and cytotoxic markers by hepatic Vδ2 T cells is higher than those in peripheral blood in HCV-infected patients.
(A,B,D) The proportions of Vδ2 T cells expressing CD38 (A), CD56 (B), GrB (D) were compared in HCV-infected patients with paired liver and blood samples. n = 8 for each group. (C,E) The proportions of Vδ2 T cells producing CD107a (C) and IFN-γ (E) upon zoledronate stimulation are also shown. n = 8 for each group. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6. Vδ2 T cells of HCs are activated and upregulate cytolytic activity but not IFN-γ production after in vitro exposure to IFN-α.
(A–C,E) PBMCs from HCs were preincubated with or without IFN-α for 24 h. (A,B) Expression of activation markers CD38 and CD69 (A), and cytolytic enzymes GrB and perforin (B) on Vδ2 T cells was assessed by flow cytometry. (C,E) Expression of CD107a (C) and IFN-γ (E) on Vδ2 T cells upon zoledronate stimulation was analyzed by flow cytometry. n = 6 for each group. (D) Percentages of K562 and HepG2 lysis byVδ2T cell lines preincubated with or without IFN-α for 24 h. n = 5 for each group. *p < 0.05, **p < 0.01.
Figure 7
Figure 7. IFN-α could directly modulate Vδ2 T-cell phenotype and function.
(A) PBMCs from HCs were stained with an unlabeled primary antibody against IFNAR2 or an isotype-matched control antibody (mouse IgG2a), and then by a phycoerythrin (PE)-conjugated anti- mouse IgG2a secondary antibody. IFNAR2 expression on Vδ2 T cells was analyzed. A representative staining profile is shown. (B) PBMCs of healthy blood donors were separated for Vδ2 T cells and non- Vδ2 T cells using magnetic beads. Purified cells were stained with anti- Vδ2 T mAb and anti-CD3 mAb and analyzed by flow cytometry. The representative dot plots from six experiments are shown. (C–F) Purified Vδ2 T cells were preincubated with or without IFN-α for 24 h. (C,D) Expression of activation markers CD38 (C), and cytolytic enzymes GrB (D) on Vδ2 T cells was assessed by flow cytometry. (E,F) Expression of CD107a (E) and IFN-γ (F) on Vδ2 T cells upon zoledronate stimulation was analyzed by flow cytometry. n = 6 for each group. (G) Purified Vδ2 T cells were cultured in medium containing PBS or 100 U ⁄ml IFN-α, a blocking antibody against type I IFN receptor (anti-IFNAR2, 5 μg ⁄ml) or an isotype control antibody was added to the medium. After 24 hr of culture, the expression of CD38 on Vδ2 T cells was assessed. n = 5. *p < 0.05, **p < 0.01.
Figure 8
Figure 8. In vivo IFN-α administration further enhances Vδ2 T cell activation and cytolytic activity but decreased Vδ2 T cell IFN-γ production in HCV-infected patients.
(A–C) The frequency of Vδ2 T cells (A), and expression of activation markers CD38 and CD69 (B), and cytolytic enzyme perforin (C), on Vδ 2 T cells was detected by flow cytometry before and after one month HCV therapy. n = 7 for each group. (D,E) Expression of CD107a (D) and IFN-γ (E) on Vδ2 T cells before and after one month HCV therapy following zoledronate stimulations was also analyzed by flow cytometry. n = 7 for each group. (F) Supernatant levels of IFN-γ released in culture by Vδ2 T cells before and after one month HCV therapy after zoledronate stimulation were quantified by ELISA. Results (pg/ml of IFN-γ) were normalized for 1000 Vδ2 T cells. n = 7 for each group. *p < 0.05.

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