Evaluation of circulating CD4 + CD25 + CD127 −/low regulatory T cells in newly diagnosed hepatitis C-infected patients

Abstract

Objectives

Hepatitis C virus (HCV) is one of the most global health problems with 2.5% prevalence worldwide. It seems that regulatory T (Treg) cells, which are able to modulate the host immune responses, play a substantial role in the immunopathogenesis of HCV infection. In this study, we evaluated the distribution of Treg cells in HCV-infected patients and its correlation with viral load and clinical manifestations.

Methods

Peripheral blood mononuclear cells (PBMCs) were collected from 14 newly diagnosed HCV-infected patients and 23 age- and sex-matched healthy subjects, and the frequency of CD4+CD25+CD127−/low Treg cells was determined by flow cytometry.

Results

Our results showed that the mean level of CD4+CD25+CD127−/low Treg cells in HCV-infected patients was significantly higher than that in healthy control subjects (8.2 ± 1.48% vs 5.4 ± 0.36%, p < .05). However, there was no statistical correlation between Treg cells frequency and viral load or clinical manifestations.

Conclusion

A higher proportion of Treg cells in HCV-infected patients might indicate their critical role in viral persistence and candidate them as a new target of immunotherapy to improve antiviral immunity.

Keywords

hepatitis C virus regulatory T cells viral persistence Hepatitis C virus-infection flow cytometry

Introduction

Hepatitis C virus (HCV) is an enveloped non-A non-B hepatitis agent, single-stranded, positive-sense RNA virus from the family Flaviviridae.¹ HCV is one of the most global health problems with a 2.5% prevalence (approximately 71 million infected individuals worldwide). In 60%–80% of those infected individuals, the virus persistence leads to chronic, long-term infection which can increase the risk of liver injuries, such as chronic hepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC).^2–5 Due to the pivotal role of the immune system in antiviral defense and its pathological role in viral infection, it is important to peruse the immunopathogenicity of HCV infection.⁶ Indeed, immunopathogenesis of HCV is complicated and cascades of immunological events determined the fate of HCV infection that might lead to viral clearance or viral persistence and long-lasting infection. Both innate and adaptive immune responses are necessary for the control of HCV infection, particularly HCV-specific CD4+ and CD8+ T cells which are able to clear the virus and limit the infection, whereas failure to respond can result in the development and persistence of a chronic HCV infection.^4,7 Several studies have reported impaired function of both innate and adaptive immune cells such as CD4+ T helper (Th) 1 cells, CD8+ cytotoxic T-lymphocyte (CTL) cells, innate lymphoid cells (ILCs), NK cells, regulatory T (Treg) cells in patients with a chronic HCV infection.^4,8,9 It has been revealed that HCV employs different immune escape mechanisms to evade the host immune system, including protein mutations, inducing the production of immunosuppressive cytokines (TGF-β and IL-10), altering the phenotype of effector T cells to exhausted T cells and increasing the frequency of Treg cells.^10,11 Zhai et al. have found that the core protein of hepatitis C virus (HCVc) is responsible for the expansion of CD4+CD25+ T cells, induction of IL-10, and secretion of TGF-β by CD4+CD25+ Treg cells. It has also been reported that it can inhibit the proliferation of CD4+ T cells and production of IFN-ϒ through the induction and activation of Treg cells.¹¹ Regulatory T-cells are a subset of T cells which mainly express the regulatory transcription factor Foxp3, glucocorticoid induced tumor necrosis factor receptor family-related protein (GITR), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), CD39, CD127 (IL7R-α), and high levels of CD25 (IL-2Rα). However, recently it has been characterized by the flowing markers CD4 (+) CD25 (+) CD127 (low/-). They have proved that these cells present the properties of T reg and have also FoxP3.^12,13 Treg cells mainly contribute to the induction of immune tolerance/homeostasis.^14,15 Moreover, Treg cells play a substantial role in immunopathogenesis of viral infection.¹⁶ It has been shown that the frequency of circulating and intra-hepatic Treg cells in the peripheral blood of patients with chronic hepatitis C was significantly higher than healthy controls.^17,18 Treg cells have been reported to inhibit the proliferation and function of HCV-specific CD4+ and CD8+ T cells via cell-to-cell contact and secretion of anti-inflammatory mediators such as tumor growth factor-beta (TGF-β) and interleukin (IL)-35 and modulate the immune responses to favor HCV infection.^4,17,19 Moreover, depletion of Treg cells from peripheral blood of HCV-infected patients increased the frequency and function of the HCV-specific CD8+ T cells that might be helpful in viral clearance.²⁰ Suppressive activity and frequency of Treg cells are mainly observed in long-term chronic subjects, while our knowledge about the suppressive effect and the proportional frequency of Treg cells in patients with acute HCV infection is limited.^21,22 As these cells are also dominant ones in the HCC and chronic hepatitis, targeting them could be a strategy of treatment.²³ Therefore, our study was designed to determine the frequency of Treg cells in newly diagnosed HCV-infected patients and its correlation with viral load and other clinical manifestations. Certainly, identification of Treg cells alteration following HCV infection might increase our knowledge about immunopathology of HCV.

Material and methods

Study design

A case-control study was conducted on 37 individuals to evaluate the distribution of Tregs among HCV-infected patients and healthy controls. The study subjects included 14 patients with Hepatitis C virus (HCV-seropositive) and 23 age- and sex-matched healthy controls (HCV-seronegative) that were recruited from Motahari clinic, Shiraz University of Medical Sciences, Shiraz, Iran. The clinical characteristics of each patient shown in Table 1 were collected from electronic patient files in hospital. This study was approved by the ethics committee of Shiraz University of Medical Sciences (ethical code: 1396-01-01-14848), and an informed consent was obtained from all subjects in advance. Individuals who had a history of HAV, HBV, HEV, HDV and HIV infection, malignancy, cirrhosis, autoimmune diseases, acute hepatitis, alcoholic hepatitis and those who received anti-viral or immunosuppressive therapy for at least six months before sampling were excluded from the study.

Table 1.

The clinical characteristics of enrolled individuals.

Variables	HCV-infected patients (n = 14)	Healthy controls (n = 23)
Age (years±SD)	38.75 ± 0.548	36.17 ± 0.98
Gender (male/female)	12/2	22/1
AST (U/L)	44 ± 9.7	-
ALT (U/L)	72.6 ± 27.0	-
Albumin (g/dL)	4.4 ± 0.12	-
ALP (U/L)	225.6 ± 34.4	-
Total protein (g/dL)	7.4 ± 0.25	-
PLT (cells/µl)	218428.57 ± 35907.39	-
WBC (Cells/mm³)	6564.28 ± 486.81	-
Lymphocyte (%)	31.7 ± 2.21	-
HCV copy number (copy/ml)	279536.7 ± 189289.7	-

Data were expressed as mean ± SEM. AST; aspartate aminotransferase, ALT; alanine aminotransferase, ALP; alkaline phosphatase, PLT; platelet.

Peripheral blood mononuclear cells isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized blood by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, USA). Briefly, heparinized blood was mixed with an equal volume of phosphate buffer saline (PBS) and slowly layered over the Ficoll-Hypaque solution. After being centrifuged for 20 min at 400×g, 22°C, PBMCs were collected, washed, and suspended in RPMI-1640 medium ((Gibco, USA)) containing 10% fetal bovine serum (FBS). They were finally frozen in liquid nitrogen for further analyses.

Identification of treg cells using flow cytometry

PBMCs were re-suspended in FACS buffer (PBS containing 10% FBS) and stained with following monoclonal antibodies: Anti-human CD25 (Alexa Fluor 488-conjugated anti-CD25 clone: BC96, eBioscience, USA), anti-human CD4 (PE-conjugated anti-CD4 clone: OKT4, BD, USA), and anti-human CD127 (APC-conjugated anti-CD127 clone: A019D5, Bio legend, USA). Monoclonal antibodies cocktail was added to the cell suspension and incubated for 30 minutes in dark at 4°C. Isotype-matched control monoclonal antibodies were applied to delineate positive and negative cells in the appropriate channels. Flow cytometry analysis was done using BD FACS Calibur analyzer (BD Bioscience). Data were obtained as 10⁵ events per sample and analyzed by Flow Jo software version 7.6.2 (Tree Star, Inc., San Carlos, CA).

Statistical analysis

Results were shown as mean ± SEM, and mean values were compared between the studied groups by either unpaired t test or ANOVA, followed by Tukey and Bonferroni correction.²⁴ GraphPad Prism (La Jolla, CA, USA) version 8.0.2 was used for data analysis. The Kolmogorov-Smirnov (K-S) test was used to evaluate the normal distribution of data.²⁵ Also, Spearman’s correlation analysis was use to evaluate the relationships between quantitative parameters. p-values equal to or less than 0.05 were considered significant.

Result

Treg cells distribution in the peripheral blood of HCV-infected patients

To investigate the frequency of Treg cells in HCV-infected patients and healthy controls, we examined Tregs distribution in peripheral blood of the participants. Treg cells were identified as CD4+CD25+CD127−/low population; the gating strategy for detection of these cells is shown in Figure 1. Our results demonstrated that the statistical differences between the groups regarding the frequency of Treg cells. CD4+CD25+CD127−/low regulatory cells accounted for 8.2 ± 1.48% of the total CD4+ cells pool in the peripheral blood of newly diagnosed HCV-infected patients, which was significantly higher compared to healthy controls (5.4 ± 0.36%, p = .031). Moreover, CD4+ T cells and CD4+CD25+ T cells populations were more frequent in HCV-infected patients compared to healthy controls (38.54 ± 1.67 vs 30.9 ± 3.14, p = .025 and 12.42 ± 1.80 vs 8.76 ± 0.64, p = .031, respectively) (Figure 2).

Figure 1.

Gating strategy for detection of Treg cells in PBMCs of HCV-infected patients and healthy controls. The cells were isolated from the peripheral blood of HCV-infected patients and healthy controls, stained with conjugated monoclonal antibodies against the cell surface markers CD4 (PE), CD25 (Alexa Fluor 488), CD127 (APC), and analyzed by flow cytometry. Representative dot plots were gated on forward versus side scatter (FSC/SSC) to determine the lymphocyte population. Then, the CD4+ cells were gated versus SSC and selected to show the percentage of CD4+CD25+ CD127−/low subsets which are considered as Treg cells markers.

Figure 2.

Altered frequencies of CD4+, CD4+CD25+ and CD4+CD25+ CD127−/low cells in the peripheral blood of HCV-infected patients. PBMCs were isolated from the whole blood of HCV-infected patients and healthy controls. CD4+, CD4+CD25+ and CD4+CD25+ CD127−/low populations were more significantly frequent in HCV-infected patients compared to healthy controls. Data are shown as mean ± SEM, and p < .05 was considered statistically significant.

Association of treg cells frequency with clinical manifestations of HCV-infected patients

The results showed that there was no significant correlation between percentage of Treg cells and clinical manifestations in HCV-infected patients (p > .05).

Discussion

The present study was designed to determine the frequency of Treg cells in newly diagnosed HCV-infected patients. In this study, we defined Treg cells as CD4+CD25+CD127−/low cells. CD4+CD25+ CD127−/low has been proposed as Treg cells specific marker, and several studies demonstrated that CD4+CD25+CD127−/low T cells expressed the highest level of FoxP3 and had the strongest correlation with CD4+CD25+FoxP3+ T cells.^13,26 On the other hand, there was a close correlation between FoxP3 expression and the lack of CD127.²⁷ Therefore, to determine the percentage of Treg cells in the peripheral blood, we analyzed the expression of CD4, CD25, and CD127 in PBMCs using flow cytometry. Our study revealed that CD4+CD25+ and CD4+CD25+ CD127−/low populations were more frequent in newly diagnosed HCV-infected patients compared to healthy subjects. These observations were in the same line with those of a previous studies that showed more proportions of CD4+ CD25+ Treg cells in the peripheral blood of patients with chronic and also acute hepatitis C (AHC).^28,29 It has also been observed that AHC patients who had undergone chronic infection had higher levels of CD4+ CD25+ Treg cells compared to those who cleared the infection.²⁹ Indeed, it has been proposed that Treg cells probably mediate viral persistence through suppression of specific T cells responses. Several studies have demonstratedthe dysregulated function of CD4+ and CD8+ T cells in chronic HCV-infected patients.^30,31 Sugimoto et al. have revealed that isolated CD4+CD25+ T cells from patients with chronic infection were immunosuppressive and could inhibit the secretion of IFN-γ from HCV-specific CD8+ T cells ex vivo³² Another in vitro study revealed that this suppressive effect of CD4+CD25+ T cells from patients with chronic HCV infection was significantly higher than those with cleared HCV infection or healthy donors.³³ It has also been shown that levels of transforming growth factor beta (TGF-β1) and IL-10 in the supernatants of PBMCs co-cultured with CD4+CD25+ T cells from chronically HCV-infected patients were significantly higher than the whole PBMCs culture and PBMCs depleted of CD4+CD25+ cells culture.²⁰ Moreover, liver biopsies reported that CD4+FoxP3+ Treg cells were more frequent in the HCV-infected livers, particularly near the portal tract areas, while they were almost absent in the biopsies from healthy livers. These cells also exhibited a fully differentiated and highly activated phenotype.³⁴ All these findings suggested that Treg cells might have a key role in HCV-specific CD4+ and CD8+ T cells dysfunction and the suppression of antiviral immune responses which can result in the development and persistence of a chronic infection. However, Heeg and colleagues have revealed that although HCV-specific FOXP3+CD4+ T cells were present in both acute and chronic HCV groups, these cells were not associated with persistence of a chronic HCV infection.³⁵ Similarly, Pearson et al. have reported that there were not any significant differences regarding the frequency and phenotype of CD4+CD25high regulatory T cells between AHC patients who progressed to chronic infection and those who resolved the infection.²¹ These controversial results may be explained by discrepancies in characterization. Some studies have focused on CD4+CD25+ T cells as Tregs, while others have added other specific markers such as FoxP3 or CD127.

Several studies have shown a positive correlation between the viral load and Treg cells proportion in HCV-infected patients.^18,20,36 However, we found no correlation between the percentage of Treg cells and HCV RNA copy number. In to the same line with our study, Pearson et al. have reported no significant association between HCV RNA titer and Treg cells percentage.²¹ These differences between studies might be related to different reagents and methods used or the sample size. In this study, we also examined the correlations between Treg cells frequency and clinical parameters, such as alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) levels and platelet counts; the results revealed that clinical manifestations did not correlate with Treg frequency. Our study was in accordance with those of previous studies, which reported no correlation between the percentage of Treg cell; serum ALT and AST levels; and platelet counts.^18,36,37

The main limitation of our study was small sample size. The other one was that we were unable to isolate and assess the function of CD4+CD25+CD127−/low T cells during co-culture with PBMCs. Therefore, further experiments, which focus more on the phenotype and function of Treg cells during acute HCV infection, with increased sample size are needed to increase our knowledge about the role of Treg cells in the resolution or persistence of HCV infection.

Study limitations

Different Treg subsets in hepatitis C-infected patients are not evaluated in this study which can be applied in future studies.

Conclusion

In conclusion, we demonstrated elevated proportions of CD4+ CD25+ CD127−/low Treg cells in the initiation of HCV infection. This result suggests that Treg cells may play a substantial role in HCV viral persistence and the establishment of long-lasting infection. However, precise mechanisms of Treg cells in HCV-infected patients remain completely clarified and large-scale studies are needed to define the exact mechanisms of Treg cells in HCV infection.

Footnotes

Acknowledgments

The authors would like to thank Shiraz University of Medical Sciences, Shiraz, Iran and also Center for Development of Clinical Research of Nemazee Hospital and Dr Nasrin Shokrpour for editorial assistance.

Author contributions

K.K and S.Kh and Z.F devised the study plan, M.A and M.A.H collected the data, M.E.V and S.KH were responsible for data analysis, Z.F F.R.K interpreted the data, MR.A and K.SH drafted the manuscript, and K.K and M.AH supervised the whole process and gave constructive advice. All authors have made contributions to the current work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Shiraz University of Medical Sciences under grant number 14848.

ORCID iD

Kurosh Kalantar

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