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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 2  |  Page : 383-388

PD-1 expression on peripheral CD8+ T cells closely correlated with hepatitis C virus viral load in chronic hepatitis C patients


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Hepatology, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, National Liver Institute, Menoufia University, Menoufia, Egypt

Date of Web Publication18-Oct-2016

Correspondence Address:
Hossam A Galbt
National Liver Institute, Menoufia University, Yassin Abdel-Ghaffar St, Shebin El-kom, Menoufia, 32511
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.192444

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  Abstract 

Objective:
Our objective was to evaluate the relationship between the expression of PD-1 as an inhibitory novel marker on CD8+ T cells and hepatitis C virus (HCV) viral replication and disease progression in chronic HCV-infected Egyptian patients.
Background:
Tight correlation between host circulating CD8+ T-cell-mediated immune response and control of viral replication is a classic characteristic of long-term HCV infection.
Patients and methods:
This study included 28 chronic hepatitis C patients without cirrhosis (17 male and 11 female), 25 chronic hepatitis C patients with cirrhosis (14 male and 11 female), and 15 healthy controls (10 male and five female). Laboratory investigations such as complete blood picture, liver function tests, evaluation of hepatitis viral markers (HBsAg and anti-HCV Ab), and HCV RNA PCR were performed on all participants. The level of PD-1 on CD8+ T cells was assessed using flow cytometry.
Results:
The data revealed a higher percentage of PD-1 on CD8+ T cells in HCV-infected patients compared with healthy controls. Our results also indicated a significant correlation between PD-1/CD8 level and HCV viral load in the studied groups.
Conclusion:
Our results suggested that PD-1 level on peripheral CD8+ was highly correlated with HCV viral load in chronic HCV-infected patients, which made PD-1 a novel indicator to evaluate the impairment and dysfunction of host CD8+ T-cell immunity as well as HCV viral persistence.

Keywords: CD8+ T cells, cirrhosis, hepatitis C virus, PD-1


How to cite this article:
Fathy AA, Khalefa KA, El-Sabawy MM, El Sharnoby AA, Galbt HA. PD-1 expression on peripheral CD8+ T cells closely correlated with hepatitis C virus viral load in chronic hepatitis C patients. Menoufia Med J 2016;29:383-8

How to cite this URL:
Fathy AA, Khalefa KA, El-Sabawy MM, El Sharnoby AA, Galbt HA. PD-1 expression on peripheral CD8+ T cells closely correlated with hepatitis C virus viral load in chronic hepatitis C patients. Menoufia Med J [serial online] 2016 [cited 2019 Nov 15];29:383-8. Available from: http://www.mmj.eg.net/text.asp?2016/29/2/383/192444


  Introduction Top


Hepatitis C virus (HCV) infection is responsible for many cases of chronic liver diseases worldwide. The consequences of chronic HCV infection represent compelling health problems, and is the most frequent cause of viral-related cirrhosis and liver cancer and the leading indication for liver transplantation worldwide [1].

HCV establishes persistent infection in ∼75–80% of patients. In these individuals, the function of HCV-specific CD8+ T cells is impaired by ligation of inhibitory receptors, the repertoire of which has expanded considerably in the past few years. It has been hypothesized that the coexpression of the negative regulatory receptors, T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) and programmed death 1 (PD-1), in HCV infection identifies patients at risk of developing viral persistence during and after acute HCV infection [2].

Some authors have postulated that high expression of the inhibitory PD-1 receptor seems to be a signature of functional CD8 cell exhaustion [3].

The PD-1 molecule is expressed on lymphocytes, especially on T and B cells, and is an inducible inhibitory regulator of T-cell activation [4].

Distinctive characteristics of PD-1 expression on T-cell regulators in HCV infection suggest that it is always associated with impaired adaptive immunity as well as with long-term viral persistence [5].

Recently, in 2011, Kasprowicz et al. [6] suggested the presence of high levels of PD-1 expression on HCV-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome.

The PD-1/PD-L pathway plays a major role in regulating T-cell exhaustion during chronic viral infections in animal models, as well as in humans, and blockade of this pathway can revive exhausted CD8+ T cells [7].

Successful HCV treatment with pegylated interferon/ribavirin or blocking PD-1/PDL-1 engagement ex vivo led to reduced PD-1 expression and improved interleukin-12 production as well as STAT-1 activation in M/Mφ from HCV-infected individuals. These results suggest that the PD-1 inhibitory pathway may negatively regulate interleukin-12 expression by limiting STAT-1 phosphorylation in M/Mφ during chronic HCV infection [8].

The cross-talk between CD8 T cells and PD-1-induced inhibition in chronic HCV infection warrants further exploration for HCV infection-associated immune pathogenesis. Further manipulation of these suggestions may represent a rational target for novel immunotherapeutic approaches.


  Patients and Methods Top


Study population

This study was conducted on 53 individuals, including 28 chronic HCV noncirrhotic and 25 chronic HCV cirrhotic patients. They were presented at the Hepatology Department, National Liver Institute, Menoufia University, between December 2011 and April 2012.

Diagnosis was based on clinical examination, laboratory tests, and ultrasound. Fifteen age-matched and sex-matched individuals, who constituted a control group, were also included in the study.

All patients and control groups were subjected to the following

  1. Full history taking.
  2. Complete clinical examination.
  3. Abdominal ultrasonography.
  4. Laboratory investigations including:


  1. Complete blood picture.
  2. Liver function tests for aspartate transaminase, alanine transaminase (ALT), serum bilirubin, serum albumin (Alb), serum g-glutamyl transpeptidase, and serum total proteins.
  3. Evaluation of viral markers (HBsAg, HCV-Ab).
  4. Quantitative HCV RNA PCR.
  5. Evaluation of the level of PD-1 on CD8+ T cells by flow cytometry.


Written consent was obtained from each individual and the protocol was approved by the ethical committee of Menoufia Faculty of Medicine.

Sample collection

Venous blood samples of 15 ml were taken by sterile venipuncture after minimal venous stasis using sterile disposable syringes. The blood samples were used as follows.

A volume of 5 ml of venous blood was taken in a vacutainer plain test tube and left for a sufficient amount of time to clot. Serum was then separated after centrifugation at 3000 rpm/min for 10 min, and then liver function tests and evaluation of hepatitis viral markers were performed.

Another 5 ml of venous blood was taken in a vacutainer plain test tube and left for a sufficient amount of time to clot. Serum was then separated after centrifugation at 3000 rpm/min for 10 min, and HCV RNA PCR was performed.

A volume of 5 ml of venous blood was taken in a vacutainer plastic tube containing EDTA for complete blood count, PD-1, and CD8 analysis.

PD-1 expression assay

Peripheral blood mononuclear cells were separated using Ficoll hypaque solution.

Thereafter, the following steps were performed:

  1. The concentration of cell suspension was adjusted to 2 × 107/ml with washing buffer.
  2. A volume of 50 Ul of the cell suspension was added to each one of two FACS tubes.
  3. A volume of 5 Ul of both FITC-labelled PD-1 and PE-labelled CD8 was added to the first tube and a second tube was used as negative control.
  4. Mixing and incubation for 30 min at 4°C was performed.
  5. Washing with PBS was performed and the supernatant was discarded.
  6. The cells were resuspended in 0.5 ml PBS.
  7. Analysis was performed on a FACS brand flow cytometer. The number of events to be analyzed was adjusted to 50 000.
  8. The sample was mixed properly before acquisition.


Statistical analysis

Data were statistically analyzed using SPSS (Statistical Package for Social Science) version 13 for Windows for all analyses (V.13; SPSS Inc., Chicago, Illinois, USA). P values less than 0.05 were considered statistically significant.


  Results Top


[Table 1] shows that all of the studied groups were homogenous regarding age and sex, as there was no statistically significant difference between them.
Table 1: Comparison between the studied groups as regards age and sex

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[Table 2] shows the results of the liver function tests among all studied groups using the Mann–Whitney U-test and the t-test. The aspartate transaminase level showed highly significant difference in the noncirrhotic and cirrhotic groups when compared with the control group, as well as between the noncirrhotic group and the cirrhotic group. The ALT level showed highly significant difference in the noncirrhotic and cirrhotic groups when compared with the control group. The total bilirubin level showed highly significant difference in the noncirrhotic and cirrhotic groups when compared with the control group, as well as between the noncirrhotic group and the cirrhotic group. With regard to the total protein and albumin levels, the lowest levels of total protein and albumin (mean values 5.47 and 2.25, respectively) were detected in HCV-cirrhotic patients.
Table 2: Comparison between the three studied groups as regards liver functions

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[Table 3] shows that there was no statistically significant difference between HCV-noncirrhotic and HCV-cirrhotic groups as regards HCV viral load.
Table 3: Comparison between the three studied groups as regards viral load

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[Table 4] shows comparison between the three studied groups as regards PD-1, CD8, and PD-1/CD8.
Table 4: Comparison between the three studied groups as regards PD-1 and CD8

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The Mann–Whitney U-test was used to compare each of the diseased groups with the control as regards the level of both PD-1 and PD-1/CD8: a highly significant increase (P<0.001) in PD-1 and PD-1/CD8 was found in HCV-noncirrhotic patients when compared with controls.

PD-1 and PD-1/CD8 showed a highly significant increase (P<0.001) in HCV-cirrhotic patients when compared with controls, but when both HCV-noncirrhotic and HCV-cirrhotic groups were compared only PD-1/CD8 showed a significant increase (P < 0.05).

There was no significant difference between the three groups as regards CD8 level.

[Table 5] shows a correlation between PD-1/CD8 level and other parameters in the studied groups.
Table 5: Pearson's correlation between PD-1/CD8 level and other parameters in the studied groups

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There was significant correlation (P<0.05) between PD-1/CD8 level and HCV viral load in the studied groups.

No significant correlation was noted between PD-1/CD8 levels and serum albumin, bilirubin, and aminotransferases in any of the groups, suggesting that PD-1/CD8 reflects neither the hepatic synthetic functions nor the inflammatory activity.


  Discussion Top


The present study was conducted on 53 HCV patients recruited from the Hepatology Department and outpatient clinic at National Liver Institute in Menoufia University, during the period from December 2011 to April 2012.

HCV persists with impaired antigen-specific CD8 T-cell function and progressive immune-mediated liver disease. Because PD-1 expression has been linked with virus-specific effector T-cell dysfunction in chronic viral infections, we examined the extent to which PD-1 signaling might contribute to immune regulation in HCV-infected patients, particularly within the liver. As expected, PD-1 expression in circulating HCV-specific CD8 T cells was increased in HCV-infected patients in association with their effector dysfunction, and PD-1/PD-L blockade could enhance HCV-specific CD8 T-cell function in some cases [9].

In our study, there was a significant increase in the level of PD-1/CD8 in noncirrhotic and cirrhotic patients compared with the control group.

These results agreed with those of Shen et al. [0], who measured PD-1 expression on CD8+ T cells and results were shown as mean fluorescence intensity and percentage of positive cells. Significantly higher PD-1 (P<0.0001) expression was observed in HCV-infected patients [0].

High levels of PD-1 expression on HCV-specific CD8 T cells represent a profound functional exhaustion refractory to PD-1/PD-L blockade that is particularly prominent in HCV-infected liver or during acute hepatitis C, perhaps reflecting active antigenic exposure. These findings provide new insights into HCV-specific CD8 T-cell dysfunction with therapeutic relevance [9].

According to Lucy et al. [1], PD-1 expression was significantly higher on CD8 T cells in patients with chronic HCV compared with those with spontaneous viral eradication, patients with non-HCV liver disease, and normal controls [1].

Lucy et al. [2] also reported that comparison of total CD4 and CD8 T-cell subsets revealed that PD-1 expression was two-fold higher on CD8 than on CD4 T cells. Moreover, HCV-infected patients had significantly higher PD-1 expression on both T-cell subsets compared with normal individuals [2].

Simona et al. [3] reported that, in patients with chronic evolution of infection, CD8 cells maintained high levels of PD-1 and remained functionally impaired, with no switch from an effector to a memory phenotype. Restoration of antiviral CD8 function concurrent with PD-1 decline in self-limited HCV infection and persistence of CD8 impairment concomitant with persistent expression of high levels of PD-1 by HCV-specific CD8 cells support a role for the PD-1/PDL-1 pathway in modulating CD8 function under conditions of sustained high levels of HCV antigen stimulation. This is confirmed by the observation that blockade of PD-1/PDL-1 interaction enhances proliferation of HCV-specific CD8 cells [3].

Nobuhiro et al. [9] showed that, in peripheral blood, CTLA-4 expression levels were uniformly low in CD8 T cells during chronic or resolved HCV infection; this contrasts with PD-1 expression, which is elevated in circulating HCV-specific CD8 T cells from chronic compared with resolved patients [9].

The study by Victoria et al. also found significant differences in PD-1 expression on bulk CD8 T cells during the early phase of infection, whereas we observed similar bulk CD8 and CD4 T-cell expression levels regardless of the stage of infection [4].

In this study, there was no significant correlation between CD8+ T cells and HCV viral load.

Shen et al. [0], in agreement with our study, showed that no correlation was found between total CD8+ T cells or naïve/TCM subsets and HCV viral load [0].

The obtained results showed that there was significant correlation between PD-1/CD8 cells and HCV viral load.

These results agreed with those of Alleluiah et al. [5], who showed that levels of virus (HIV or HCV) and PD-1 expression on virus-specific T cells are positively correlated; the levels of PD-1 on CD8 T cells ranged widely at all HCV RNA levels during both early and late infection. Levels of virus (HIV or HCV) and PD-1 expression on CD8 T cells are positively correlated [5].

Shen et al. [0] also reported that PD-1 expression on CD8 T cells was positively correlated with HCV viral load in HCV infection [0].

Unfortunately, we did not find any correlation between mean fluorescence intensity of PD-1, CD38, HLA-DR, or CD127 and ALT levels in HCV-infected patients [0].

In persistent HCV infection, specific cytotoxic response is weak and unable to clear the virus [6].

To assess the role of PD-1 and CD127 molecules on HCV-specific CTL reactivity, Urbani et al. [3] conducted a deeper analysis into the effect of these molecules on peripheral T-cell expansion ability. Interestingly, in chronic infection [6] the PD-1/CD127 phenotype on circulating pentamer+ cells correlated with the level of viremia. HCV-specific CTL submitted to high viral load displayed an anergic PD-1+/CD127- phenotype, probably due to persistent ineffective CTL triggering as it has been shown in other viral infections. The higher the viral load in chronic patients, the more intense the PD-1+/CD127- phenotype on HCV-specific CTL. A negative correlation was seen between CD127 expression and HCV viremia [6].

The level of persistent HCV antigenemia was able to regulate the expression of these two molecules on HCV-specific CD8+ cells, with this modulation being more intense in the HCV replication site where the antigenemia is higher. Clearly this could be an HCV evolutionary mechanism to escape from immune control [6].

In peripheral blood, CTLA-4 expression levels were uniformly low in CD8 T cells during chronic or resolved HCV infection; this contrasts with PD-1 expression, which is elevated in circulating HCV-specific CD8 T cells from chronic compared with resolved patients [9].

Interestingly, although combined blockade enhanced both HCV-specific T-cell interferon-c and tumor necrosis factor-a (TNF-a) production, the increase was particularly evident for TNF-a, suggesting that PD-1/CTLA-4 blockade may promote a cytokine profile that differs from a preferential (albeit weak) interferon-c rather than TNF-a production by dysfunctional HCV-specific CD8 T cells in chronic hepatitis C. The combined PD-1/CTLA-4 blockade also enhanced intrahepatic HCV-specific CD4 T-cell function, an important consideration given the relevance of CD4 T cells in immune regulation [9].


  Conclusion Top


It can be concluded that PD-1 level on peripheral CD8+ was highly correlated with HCV viral load in chronic HCV-infected patients, which made PD-1 a novel indicator for evaluating the impairment and dysfunction of host CD8+ T-cell immunity as well as HCV viral persistence.

Conflicts of interest

There are no conflicts of interest.[16]

 
  References Top

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2.
McMahan RH, Golden-Mason L, Nishimura MI, et al. Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity. J Clin Invest 2010; 120:4546-4557.  Back to cited text no. 2
    
3.
Urbani S, Amadei B, Tola D, Massari M, Schivazappa S, Missale G, Ferrari C. PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion. J Virol 2006; 80:11398–11403.  Back to cited text no. 3
    
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Shen T, Zheng J, Liang H, et al. Characteristic and PD-1 expression of peripheral CD4+ CD127loCD25hiFoxP3+ Treg cells in chronic HCV infected-patients, Virol J 2011; 8:279.  Back to cited text no. 5
    
6.
Kasprowicz V, Schulze Zur Wiesch J, Kuntzen T, Nolan BE, Longworth S, Berical A, et al. High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome. J Virol 2008; 82:3154-3160.  Back to cited text no. 6
    
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Blackburn SD, Crawford A, Shin H, Polley A, Freeman GJ, Wherry EJ. Tissue-specific differences in PD-1 and PD-L1 expression during chronic viral infection: implications for CD8 T-cell exhaustion. J Virol 2010; 84:2078–2089.  Back to cited text no. 7
    
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Ma CJ, Ni L, Zhang Y, Zhang CL, Wu XY, Atia AN, et al. PD-1 negatively regulates interleukin-12 expression by limiting STAT-1 phosphorylation in monocytes/macrophages during chronic hepatitis C virus infection. Immunology 2011; 132:421–431.  Back to cited text no. 8
    
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Nobuhiro N, Hyosun C, Abraham S, et al. Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA4 blockade. PLoS Pathog 2008; 5:e1000313.  Back to cited text no. 9
    
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Shen T, Zheng J, Xu C, Liu J, Zhang W, Lu F, Zhuang H. PD-1 expression on peripheral CD8+ TEM/TEMRA subsets closely correlated with HCV viral load in chronic hepatitis C patients. Virol J 2010; 7:310.  Back to cited text no. 10
    
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Golden-Mason L, Palmer B, Klarquist J, Mengshol JA, Castelblanco N, Rosen HR. Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction. J Virol 2007; 81:9249–9258.  Back to cited text no. 11
    
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Golden-Mason L, Klarquist J, Wahed AS, Rosen HR. Cutting edge: programmed death-1 expression is increased on immunocytes in chronic hepatitis C virus and predicts failure of response to antiviral therapy: race-dependent differences. J Immunol 2008; 180:3637–3641.  Back to cited text no. 12
    
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Urbani S, Amadei B, Tola D, Massari M, Schivazappa S, Missale G, Ferrari C. PD-1 expression in acute hepatitis C virus (HCV) infection is associated with HCV-specific CD8 exhaustion. J Virol 2006; 80:11398–11403.  Back to cited text no. 13
    
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Victoria K, Julian S, Thomas K, et al. High level of PD-1 expression on hepatitis c virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection irrespective of clinical outcome. J Virol 2007; 82:3154.  Back to cited text no. 14
    
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Rutebemberwa A, Ray SC, Astemborski J, Levine J, Liu L, Dowd KA, et al. High-programmed death-1 levels on hepatitis C virus-specific T cells during acute infection are associated with viral persistence and require preservation of cognate antigen during chronic infection. J Immunol 2008; 181:8215–8225.  Back to cited text no. 15
    
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Larrubia JR, Benito-Martinez S, Miquel J, Calvino M, Sanz-de-Villalobos E, González-Praetorius A, et al. Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1(+)/CD127(–) HCV-specific CD8(+) cells targeting the virus in chronic hepatitis C virus infection. Cell Immunol 2011; 269:104–114.  Back to cited text no. 16
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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