Introduction
Hepatitis C virus (HCV) is a single-stranded RNA virus that affects 130–150 million people worldwide as a chronic infection [
1]. In HCV-infected patients, viral replication has been demonstrated not only in hepatocytes but also in cells of the immune system, such as B cells, monocytes, and CD4
+ and CD8
+ T lymphocytes [
2‐
6]. In this laboratory, it was uncovered that primary T lymphocyte cultures, generated by ex vivo treatment of peripheral blood mononuclear cells (PBMC) from healthy individuals with a T cell-inducing mitogen phytoheamagglutin (PHA), are susceptible to wild-type (patient-derived) HCV and capable of supporting its replication at a level comparable to that of in vivo infected lymphoid cells [
6,
7]. Furthermore, these cells were able to produce infectious virions that de novo infected lymphocytes [
7]. It was also uncovered that patient-derived HCV is significantly more infectious to primary T cells than laboratory-derived clonal strains of HCV [
8]. As well, HCV infection of T cells requires surface expression of CD5, and transfection of HCV non-susceptible T cell lines with CD5 renders these cells susceptible to infection [
9].
HCV infection causes chronic hepatitis C (CHC) in up to 85 % of those afflicted, while acute HCV infection is thought to spontaneously resolve in 15-25 % of cases [
1]. Resolution of hepatitis C appears to be a result of a robust HCV-specific T cell-mediated response. In HCV-infected chimpanzees, representing the closest animal model of human HCV infection, recovery from hepatitis C and a drop in plasma HCV loads to levels undetectable by clinical assays requires the activity of both CD4
+ T helper cells and CD8
+ cytotoxic T cells [
10,
11]. In patients with CHC, T cells display markers of exhaustion and are defective in their ability to produce interferon-γ (IFN-γ) and interleukin-2 (IL-2) [
12]. In vitro, the functional consequences of T cell infection in Molt-4 and Jurkat T cell lines and primary T cells have been investigated only using laboratory-adapted clonal strains of HCV [
4,
13,
14]. It was found, among others, that infection with a HCV SB strain of Molt-4 cells suppressed IFN-γ signalling through the STAT-1 pathway [
4]. This virus strain was also able to infect primary CD4
+ T cells and this infection was associated with a decrease in their proliferation [
14].
Stemming from the previous observation that infection of naïve lymphoid cells with patient-derived HCV can lead to changes in the CD4
+ to CD8
+ T cell ratio [
7], we asked in the current study whether the virus can differentially alter CD4+ or CD8+ T cell proliferation and/or their apoptosis resulting in a shift in the T cell phenotypic characteristics. We found that the exposure of T lymphocytes to a naturally occurring HCV, although not necessarily molecularly evident active virus replication in these cells, can be sufficient to alter the CD4
+ to CD8
+ T cell ratio. This phenotypic change was due to the selective inhibition of CD4+ but not CD8+ T lymphocyte proliferation and was not related to differential apoptotic death of either T cell subset.
Discussion
In this study, we examined the effect of authentic HCV on the proliferative capacity, apoptosis and phenotype of T lymphocytes in primary cultures. Exposure of lymphoid cells to 2 of 3 HCV inocula caused a decrease in the frequency of CD4+ T cells compared to virus-untreated cultures with a relative increase in CD8+ T cells. This relative CD8
+ T cell enrichment was a result of a significant reduction in CD4
+ T cell proliferation compared to the cultures exposed to normal healthy plasma. On this note, the effect appeared to correlate with HCV cell uptake, since the shift in the CD4
+ to CD8
+ ratio was highest in the cultures in which virus exposure lead to quantifiable levels of HCV RNA positive strands and, in the case of CHC-1, negative strands in test cells (Table
1). This was consistent with the observation that in cultures exposed to CHC-1 inoculum, which induced the highest levels of HCV RNA positive strand and detectable HCV RNA negative strand, the inhibition in CD4
+ T cell proliferation appeared earlier, i.e.
, 4 d.p.i. In the cells exposed to CHC-2 inoculum, T cell proliferative and phenotypic changes were seen in the absence of detectable HCV replicative intermediate and after 10-fold lower multiplicity of infection compared to cells exposed to CHC-1. In the case of CHC-3, while the plasma viral load was higher than CHC-2 and comparable to CHC-1, there did not appear to be much virus uptake by cells, as indicated by undetectable HCV RNA by our high sensitivity assays. This may suggest that the amount of virus taken by cells exposed to CHC-3 was minuscule. HCV genotype may also play a role in the anti-proliferative effects observed, as CHC-1 carried an HCV genotype 1a/1b mix, CHC-2 genotype 3a, and CHC-3 genotype 2b. It is of note that there was no impact of TLP treatment on the change in the CD4+ and CD8+ T cell frequency, which suggests that exposure to HCV and uptake of the virus by cells rather than replication itself may be driving these phenotypic changes (Fig.
5).
Previously, infection of T cell lines and primary T cells with laboratory-derived HCV clones led to the impairment of T cell proliferation and IFN-γ production [
4,
14]. In the current study, we show an anti-proliferative effect using patient-derived HCV as a virus source. The biological impact of HCV lymphotropism on immune cell function is a topic that is still not well understood. However, it was shown that HCV infection can led to disruption of immunological activity of different types of immune cells, including T cells, B cells, dendritic cells, monocytes and macrophages [
4,
14,
18,
19].
In cultures exposed to CHC-3 inoculum, the level of HCV RNA positive strand detected during the 10-d.p.i. culture period was unquantifiable by a real time RT-PCR assay (below 50 vge/μg total RNA). However, HCV RNA signals were detected, as confirmed by NAH analysis of amplicons produced by real-time RT-PCR (data not shown). In this situation, detection of virus RNA negative strand would not be feasible, and, therefore, was not attempted (Table
1). As indicated, CHC-3 inoculum did not induce changes in T cell proliferation or phenotype, similarly as NHP-1-3, suggesting that regardless of the viral load in the inocula, if the virus cannot be taken up by cells, then the cells’ proliferation is not affected. We have previously shown that not all patient-derived HCV inocula are infectious to ex vivo pre-stimulated lymphoid cells [
7,
20]. The viral and host factors determining infectivity of T cells by wild-type HCV have yet to be recognized and are under investigation in this laboratory [
8,
9,
21].
In experiments leading to this study, alternating stimulation with PHA and IL-2 was applied to upregulate lymphoid cell susceptibility to HCV infection and to augment virus replication [
7]. In the current work, repeated stimulation with PHA after the initial 48-h treatment was not employed to prevent excessive cell activation that may mask virus pro- or anti-proliferative or apoptotic effects. The removal of this periodical stimulation resulted in a decreased HCV replication and, therefore, a reduced detection of both virus RNA positive and negative strands. This finding is consistent with previous observations in regard to HCV replication in lymphoid cell cultures [
22] and in in vitro infections with other viruses [
23‐
25].
It is well recognized that HCV-specific CD4
+ T cell response in individuals chronically infected with HCV is poor or absent [
26]. In HCV-infected patients who transiently control viremia and have fluctuating plasma viral load, including periods of apparent HCV RNA negativity, this transient viral control is accompanied by increased virus-specific CD4
+ T cell response [
26]. In patients with CHC, T cells have been found to be impaired in the production of IFN-γ and IL-2 [
12]. As well, studies investigating CD4
+ T cell function during a symptomatic persistent HCV infection have shown a significant loss of IL-2 secreting cells compared to individuals who spontaneously resolved viremia, as well as a weak IFN-γ production by HCV-specific CD4
+ T cells upon stimulation [
27]. In the latter study, HCV-specific IFN-γ production by CD4
+ T cells was rescued after in vitro culture with exogenous IL-2, but the effect of IL-2 on the CD4+ T cell proliferation was not measured. These findings may suggest that impaired proliferation of CD4
+ T cells observed in our study could be due to weakened IL-2 production. However, only CD4+ T cells but not CD8+ T cells were affected. Due to limitations in cell numbers we were not able to evaluate whether there was indeed impaired IL-2 and/or IFN-γ cytokine production by in vitro infected T cells. This issue requires further investigation. Preliminary data obtained in this regard in the present study indicate that there were no significant differences between T cells exposed or not to HCV in terms of IFN-γ, IFN-5α and IL-2 mRNA expression. However, TNF-α transcription appeared to be transiently but severely down-regulated shortly (i.e., 1 d.p.i.) after exposure to CHC-1 or CHC-2. This finding warrants further examination. Nonetheless, it needs to be taken under consideration that the level of cytokine gene expression may not accurately reflect the cytokine protein production.
The results of this study should be interpreted with certain limitations taking under consideration that PBMC exposed to authentic HCV were isolated from a single healthy donor. The observations made might be limited to this particular donor and experiments with cells from other healthy individuals are needed to confirm the findings. However, we have previously reported that a comparable shift in the CD4+ to CD8+ T cell ratio also occurred after exposure to native HCV of T cells from another healthy donor [
7].
Overall, our findings imply that exposure to HCV can modify the T cell phenotype due to a relative decrease in the proliferation capacity of CD4
+ T cells. Furthermore, exposure to naturally occurring HCV in vitro did not appear to differentially augment apoptotic death of lymphocyte subsets, although apoptotic effects of a HCV clone on T cells have been described previously [
18]. In this context, it needs to be determined whether primary CD4+ and CD8+ T cells derived from patients with CHC display similar characteristics. The results from the current study raise a possibility that HCV may exert a direct effect on the overall T cell phenotypic properties and, in consequence, on the function of T cells as a whole in HCV-infected patients.
Acknowledgements
The authors thank Dr. C.S. Coffin from the Liver Unit, Division of Gastroenterology, Faculty of Medicine, University of Calgary, Calgary, Alberta and Dr. S.B. Reddy and Ms. D. King, a hepatology nurse practitioner, from the Gastroenterology Clinic, Eastern Health, St. John’s, Newfoundland and Labrador for providing clinical samples. They also thank Mr. Adam K.M. Jenkins for help with evaluations of T cells.
This work was supported by the research operating grant MOP-126056 to TIM from the Canadian Institutes of Health Research (CIHR). SAM was a recipient of graduate fellowship awards from the National CIHR Research Training Program in Hepatitis C (NCRTP-HepC) and the Canadian Liver Foundation. TNQP was supported by postdoctoral fellowship awards from the NCRTP-HepC and the Canadian Association for the Study of the Liver/Hoffmann-La Roche/Astellas Pharma Canada. TIM is supported by the Canada Research Chair Program and funds from the CIHR and the Canada Foundation for Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SAM participated in the design and conducted majority of experiments, analyzed the data and wrote a draft of the manuscript; AYC contributed to infection experiments and performed treatments with telaprevir; CPC, contributed to acquisition and analysis of flow cytometric data; TNQP contributed to analysis of the data and assisted with writing; TIM conceived the study, supervised its execution and helped to draft the final manuscript. All authors read and approved the final manuscript.