Enhanced functions of peripheral γδ T cells in chronic hepatitis B infection during interferon α treatment in vivo and in vitro

Abstract

Background: γδ T cells play an important role in infectious, autoimmune, or neoplastic diseases. Here, a study was conducted to investigate the dynamic changes in phenotype and function of peripheral γδ T cells in patients with chronic hepatitis B (CHB) during pegylated-interferon (pegIFN)-α treatment, and to explore their roles in IFN-α therapy.

Methods: Total 15 CHB patients with pegIFN-α therapy and 6 healthy controls (HC) were enrolled in this study. Flow cytometry was used for the study of frequency of peripheral γδ T cells, subtypes, effector or memory γδ T cells, and also the IFN-γ+, TNF-α+, CD107a+ or Granzyme B+ γδ T cells in 10 patients at week 0, 4, 8, 12, 24, 36 and 48 of treatment. Another 5 CHB patients and 6 HC were recruited for the γδ T cell isolation, and gene expression in γδ T cells was evaluated before or after IFN-α treatment in vitro.

Results: Although γδT cells decreased in CHB patients during pegIFN-α therapy, their capacities to produce TNF-α and to express CD107a were enhanced. More effector γδT cells (CD27-CD45RA+) were found in the response group than in non-response group. Furthermore, IFN-α boosted the expression of Mx2 and cytokine genes in γδT cells from CHB patients in vitro.

Conclusion: IFN-α could enhance the cytokine production or cytotoxicity potential of γδT cells in vivo and in vitro. The enhanced function of γδT cells might contribute to the effect of IFN-α treatment.

Fig 1. The changes in the proportion of total γδ T cells or the subtype of Vδ1 T or Vδ2 T cells from 10 CHB patients during pegIFN-α treatment.

The FACS figures were the representatives of gated lymphocytes (A), CD3 T cells (B), γδ T cells (C), or Vδ1 T or Vδ2 T cells (D). The time curves showed the proportion of γδ T cells (E), Vδ1 T (F) or Vδ2 T (G) cells during the treatment for 48 weeks. The statistical analyses were performed between the response (5 patients) and non-response group (5 patients), and also among different time points in each group. When comparing a certain timepoint to others, there showed ★ (p<0.05) or ★★ (p<0.01) in response group and ▲ (p<0.05) or ▲▲ (p<0.01) in non-response group.

Fig 2. The dynamic change in naïve or effector γδ T cells from 10 CHB patients during pegIFN-α therapy.

FACS gating strategy was shown in A, B and C. The time curves showed the proportion of CD45RA+CD27+ γδ T cells (naïve) (D), CD45RA+CD27- γδ T cells (effector) (E), CD45RA-CD27- γδ T cells (effector memory) (F), or CD45RA-CD27+ γδ T cells (central memory) (G) during the treatment for 48 weeks. The statistical analyses were performed between the response (5 patients) and non-response group (5 patients), and also among different time points in each group. When comparing a certain timepoint to others, there showed ★ (p<0.05) or ★★ (p<0.01) in response group and ▲ (p<0.05) or ▲▲ (p<0.01) in non-response group. When comparing response to non-response group, there showed ※ (p<0.05) or ※※ (p<0.01) at a certain timepoint.

Fig 3. The changes in function of γδ T cells from 10 CHB patients during pegIFN-α therapy.

FACS figures showed IFNγ+, TNFα+, CD107a+ or Granzyme B+ γδ T cells (A-D). The curves showed dynamic changes in proportion of IFNγ+ γδ T cells (E), TNFα+ γδ T cells (F), CD107a+ γδ T cells (G), or Granzyme B+ γδ T cells (H) during the pegIFN-α therapy for 48 weeks. The statistical analyses were performed between the response (5 patients) and non-response group (5 patients), and also among different time points in each group. When comparing a certain timepoint to others, there showed ★ (p<0.05) or ★★ (p<0.01) in response group and ▲ (p<0.05) or ▲▲ (p<0.01) in non-response group. When comparing response to non-response group, there showed ※ (p<0.05) or ※※ (p<0.01) at a certain timepoint.

Fig 4. Gene expression of γδ T cells with (B, D) or without (A, C) IFN-α stimulation in vitro.

Peripheral γδ T cells were isolated from 6 healthy controls (HC) or 5 patients with chronic HBV infection (CHB). The expression of Isg15, Ifit1, Mx1 and Mx2 was shown in A and B, and expression of Ifnγ, Tnfα, Il10, Il12a, or Cxcl10 was shown in C and D. All genes expression from peripheral γδ T cells was normalized to that of GAPDH and results of 5 CHB patients are presented with fold (Mean±SD) relative to the average of 6 HCs. The difference in the gene expression was significant when it was higher than 2-fold or lower than 0.5-fold.