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. 2003 Dec;110(4):513–518. doi: 10.1111/j.1365-2567.2003.01769.x

HCV-specific CD27− CD28− memory T cells are depleted in hepatitis C virus and Schistosoma mansoni co-infection

Mohamed Elrefaei *, Nabila El-sheikh †, Karim Kamal ‡, Huyen Cao *
PMCID: PMC1783079  PMID: 14632650

Abstract

Factors that influence the generation and maintenance of memory CD8+ T cells are not fully understood. The homeostasis of memory T cells is highly dynamic and tightly regulated by various stimuli, including cytokines and antigen–major histocompatibility complex ligands. We characterized the hepatitis C virus (HCV)-specific CD8+ T-cell responses in a cohort of HCV-infected individuals with or without Schistosoma mansoni co-infection from Egypt. We observed a significantly decreased CD27− CD28− (late differentiated) memory T-cell population in the HCV co-infected individuals compared to those with HCV infection alone. In contrast, there was no significant difference in the CD27+ CD28+ (early differentiated) memory T cells between the two groups. Analysis of human cytomegalovirus-specific CD8+ T-cell responses in the same individuals failed to reveal a similar pattern of altered memory T-cell differentiation. Thus, S. mansoni co-infection targets a specific subset of memory CD8+ T cells in HCV infection.

Introduction

The precise relationships between memory CD8+ T-cell subpopulations and the signals that modulate their generation and homeostasis remain undefined. The phenotypic distinction between different memory CD8+ T cells has been previously correlated with different in vitro functional characteristics.1–3 Stages of memory T-cell differentiation are associated with the expression of different co-receptor molecules.1,4–7 The CD27+ CD28+ phenotype is generally associated with an ‘early differentiated’ stage while CD27− CD28− T cells are thought to be ‘late differentiated’ memory cells. The generation of memory T cells in immune responses has been extensively studied over recent years, yet the requirements for production and persistence of the memory cell population are still unclear. There is now growing evidence that cytokines may play a major role in determining the fate of CD8+ memory T cells.8–10 The role of interleukin-15 (IL-15) has been particularly highlighted.8,11–14 The selective action of IL-15 on CD8+ memory T cells was also shown to correlate with high levels of IL-2/15 receptor β (IL-2/15Rβ; CD122) expression.11,13

Hepatitis C virus (HCV) infects an estimated 170 million people world-wide, with Egypt carrying the highest prevalence rate.15,16 Furthermore, concomitant HCV and Schistosoma mansoni infections are common in Egypt and have resulted in a negative impact on the course of liver disease this population. Co-infection leads to a more severe clinical course, higher incidence of cirrhosis and poor response to interferon-α (IFN-α) therapy.17–19 Individuals co-infected with HCV and S. mansoni have previously been shown to display a type 2-predominance cytokine profile.20 However, the effect of S. mansoni co-infection on the differentiation and maintenance of HCV-specific memory T cells has not yet been investigated. In this study, we characterized the phenotype of the HCV-specific IFN-γ responses in the presence or absence of S. mansoni co-infection in a cohort of individuals from Azhar University hospital in Cairo, Egypt. We demonstrated evidence for in vivo modulation of a specific subset of HCV-specific memory T cells by a parasitic co-infection.

Materials and methods

Study subjects and samples

Blood samples were taken from 24 patients with chronic HCV infection (>6 months) at Al Azhar University Hospital in Cairo, Egypt. All HCV seropositive volunteers were diagnosed based on the detection of antibodies against HCV (ELISA II, Abbott, Abbott Park, IL). HCV RNA was quantified by polymerase chain reaction as previously described.21 Viral titres were determined at the same time as blood samples were collected. Patients were classified into HCV infection alone (n = 11) or with those S. mansoni co-infection (n = 13). A summary of patient clinical data is shown in Table 1. Diagnosis of chronic schistosomiasis was based on the documented clinical history of repeated S. mansoni exposure and subsequent praziquantel treatment and all co-infected volunteers were found to have schistosomal antibodies by enzyme-linked immunosorbent assay (ELISA)22 or detection of S. mansoni ova in the stool. Blood samples were also taken from six age-matched, local control volunteers who had no evidence of HCV or S. mansoni infection. Other inclusion criteria for the study were: over 18 years of age and elevated alanine aminotransferase levels for at least 6 months. Exclusion criteria were: evidence of hepatocellular carcinoma; evidence of infection with hepatitis viruses A, B, or D, human immunodeficiency virus, or Epstein–Barr virus; pregnancy; and history of alcoholic liver disease or autoimmune hepatitis. None of the volunteers had received IFN-α therapy. Peripheral blood mononuclear cells (PBMC) were separated and cryopreserved onsite and subsequently shipped to the USA. The study protocol was approved by Al Azhar University Institutional Review Board, and each volunteer gave informed written consent for participation in the study.

Table 1.

Characteristics of patients with chronic hepatitis C virus (HCV) infection

Patients infected with
Parameter HCV alone HCV and S. mansoni
No. 11 13
Age (years)mean ± SD 40·4 ± 8·6 41·6 ± 5
RNA titre* 39·3† 59·5†
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*

Values are mean HCV-RNA titre, × 104 copies per ml of plasma.

†

No significant difference in RNA titre between patients infected with HCV alone and those infected with HCV and Schistosoma. mansoni, P = 0·86.

Antigens

Peptides corresponding to the amino acid (aa) sequences of the HCV 4a core protein, aa 1–140,23 and non-structural protein 5B (NS5B), aa 2656–2725,24,25 were synthesized as free acids by Mitochor Mimotopes (Victoria, Australia). Peptides were 20 aa in length, overlapping adjacent peptides by 10 aa. A single pool of overlapping peptides corresponding to the aa sequence of the PP65 protein (BD Biosciences, Mountain View, CA) was used to detect the human cytomegalovirus (HCMV)-specific response.26

Flow cytometric analysis of memory T-cell subsets

PBMC were stained with saturating concentrations of different fluorescence-labelled monoclonal antibodies (mAb). For analysis of expression of surface markers, the following mAbs were used in different combinations: CD3 PerCP-Cy5.5, CD4 fluorescein isothiocyanate (FITC), CD8 phycoerythrin (PE), CD27 FITC, CD28 APC, CD45RA PE, CCR7 PE and CD122 PE (BD Pharmigen, San Diego, CA). For intracytoplasmic cytokine staining, cells were incubated for 2 hr with individual HCV peptides (10 μg/ml) at 37° in 5% CO2 in the presence of costimulatory anti-CD49d (1 μg/ml, Becton-Dickinson, San Jose, CA). Brefeldin A was subsequently added for 4 hr. Cells were fixed and permeabilized according to the Becton Dickinson protocol and subsequently stained with anti-IFN-γ PE (BD Pharmigen). All samples were analysed using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA) and flowjo software (TreeStar, San Carlos, CA). Percentages of peptide-specific IFN-γ-producing T cells were determined after subtraction of background activity. The percentage of IFN-γ-producing T cells without peptide stimulation was < 0·05%.

Statistical analysis

Statistical analysis and comparisons were performed with prism software version 2·0 (GraphPad, San Diego, CA). Mann–Whitney U-test was used for analysis. Statistical significance was defined as P < 0·05.

Results

Phenotypic analysis of memory T-cell subsets

To examine the effect of S. mansoni co-infection on HCV-specific T cells, a detailed phenotype analysis was performed of the overall memory T-cell subsets. The surface expression of CD3, CD27, CD28, CD45RA and CCR7 was evaluated. CD28 and CD27 are costimulatory molecules, which provide the signals needed for specific T-cell activation. CD3+ CD27+ CD28+ and CD3+ CD27− CD28− T-cell subsets expressing CD45RA and CCR7 were analysed (Fig. 1a, left). The majority of the CD27+ CD28+ T-cell subset was CD45RA− and CCR7+, while the CD27− CD28− T cells were mostly CD45RA+ and CCR7− (Fig. 1a, right). This is consistent with the phenotypic criteria defining CD27+ CD28+ CD45RA− CCR7+ as ‘early differentiated’ and CD27− CD28− CD45RA+ CCR7− as ‘late differentiated’ memory T cells.2,3,27,28 In addition, no significant difference was found in the distribution of the overall memory T-cell subsets, as defined by expression of costimulatory molecules CD27 and CD28 on CD3+ T cells between volunteers with chronic HCV alone and those with concomitant schistosomiasis (Fig. 1b).

Figure 1.

Figure 1

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Phenotypic analysis of memory T-cell subsets. Surface expression of CD3, CD27, CD28, CD45RA and CCR7 on lymphocytes was evaluated. (a) Samples were first gated on the CD3+ lymphocyte population. Further analysis of the CD27+ CD28+ and CD27− CD28− CD3+ T-cell subsets was performed to determine the pattern of CD45RA and CCR7 phenotypic expression. (b) No significant difference was found in the distribution of the overall memory T-cell subsets, as defined by expression of costimulatory molecules CD27 and CD28 on CD3+ T cells in volunteers with chronic HCV with and without concomitant schistosomiasis. Bars represent standard deviation (SD).

Effect of S. mansoni co-infection on HCV-specific memory CD8+ T-cell subsets

PBMC were stimulated with individual HCV peptides and evaluated for HCV-specific responses by IFN-γ intracellular flow cytometry assay.26 CD8-specific responses against HCV core or NS5B peptides were detected in four of 11 (median 0·34%; range 0·19–0·57%) and six of 13 (median 0·37%; range 0·2–0·62%) individuals with HCV alone or concomitant schistosomiasis, respectively (data not shown). There was no significant difference in the total HCV-specific response rate (P = 0·99) nor in the HCV RNA titre (P = 0·86, Table 1) between the two cohorts. Similarly, the HCV RNA titre did not differ among individuals demonstrating an HCV-specific response in these cohorts (data not shown).

Next, the effect of concomitant schistosomiasis on the differentiation of HCV-specific memory CD8+ T-cell subsets was evaluated by measuring the number of HCV-specific IFN-γ-positive T cells that express either CD27+ CD28+ or CD27− CD28− phenotypes. We found no significant difference in the CD27+ CD28+ (P = 0·06; Fig. 2a, c) or the CD27+ CD28− (P = 0·11; data not shown) memory subsets of HCV-specific T cells between the two HCV cohorts. In contrast, individuals with S. mansoni co-infection demonstrated a significant decrease in the number of HCV-specific T cells that are CD27− CD28− compared to those who did not have schistosomiasis (Fig. 2b, c).

Figure 2.

Figure 2

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Magnitude of IFN-γ responses aganist HCV in early and late differentiated memory T-cell subsets. Representative plots of the percentage of HCV-specific T cells that produce IFN-γ in HCV infection alone (a) or co-infected with S. mansoni (b). The numbers represent the percentage of IFN-γ-positive T cells that express either CD27+ CD28+ (early differentiated) or CD27− CD28− (late differentiated) phenotypes. (c) Percentage of IFN-γ-positive CD3+ T cells that are CD27+ CD28+ or CD27− CD28− in the S. mansoni (−) and (+) groups. Bars represent median values. Differences between S. mansoni (−) versus (+) in the CD27− CD28− T-cell subset were statistically significant (P = 0·015) but were not statistically significant in the CD27+ CD28+ T-cell subset (P > 0·05). *Percentage values represent the fraction of HCV-specific T cells expressing either CD27+ CD28+ or CD27− CD28− phenotypes over the total number of HCV-specific CD3+ T cells (equivalent to 100%) as measured by IFN-γ production assay.

Effect of S. mansoni co-infection on HCMV-specific memory CD8+ T-cell subsets

To determine whether S. mansoni co-infection selectively impairs the ability of the HCV-specific CD27− CD28− memory T-cell subset to produce IFN-γ, a similar analysis was performed on the HCMV responses in the same volunteer cohort. CD8-specific responses against HCMV-PP65 were detected in 17 of the 24 volunteers and there was no significant difference in the total HCMV-specific response rate between volunteers with HCV infection alone and those with S. mansoni co-infection (P = 0·54; Fig. 3a). Similarly, no significant effect on the CD27+ CD28+ and CD27− CD28− memory subsets of HCMV-specific T cells was observed in HCV-positive individuals with or without concomitant schistosomiasis (Fig. 3b). These results suggest that co-infection with S. mansoni specifically modulates the development and generation of HCV-specific memory CD8+ T cells.

Figure 3.

Figure 3

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Magnitude of IFN-γ responses to HCMV PP65 in early and late differentiated memory T-cell subsets. (a) Lymphocytes were stimulated with HCMV PP65 and the percentage of IFN-γ-producing CD8+ T cells was calculated. Bars represent median values. Differences between S. mansoni (−) vs. (+) were not statistically significant (P > 0·05). (b) Phenotypes of HCMV PP65-specific memory T cells as measured by IFN-γ production assay. Percentages represent the fraction of HCMV PP65-specific CD3+ T cells that express either CD27+ CD28+ (early differentiated) or CD27− CD28− (late differentiated) phenotypes over the total number of HCMV-specific CD3+ T cells. Bars represent median values. Differences between S. mansoni (−) versus (+) group in either the CD27+ CD28+ or CD27− CD28− T-cell subsets were not statistically significant (P > 0·05).

Effect of S. mansoni co-infection on CD122 expression

IL-15 has been implicated in the generation and maintenance of memory CD8+ T cells,8,9,13 and the selective action of IL-15 on CD8+ memory T cells correlated with high expression of CD122.11,13 An elevated serum level of IL-15 has been reported in chronic HCV infection.29 To determine if concomitant schistosomiasis is associated with decreased IL-15 production, serum IL-15 levels were measured and no significant difference was found between HCV-positive individuals with or without concomitant schistosomiasis (data not shown).

To determine the effect of concomitant schistosomiasis on CD122 expression, the ex vivo CD122 surface expression on lymphocytes was assessed. Expression of CD122 was significantly increased in chronically HCV-infected patients without schistosomiasis compared to the control group (Fig. 4). In contrast, expression of CD122 in individuals with concomitant schistosomiasis did not differ from controls and was generally lower than in individuals with HCV alone, although the difference did not reach statistical significance (P = 0·07). Analysis of CD122 surface expression on HCV-specific CD8+ T cells could not be performed because of the low frequency of these cells.

Figure 4.

Figure 4

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Ex vivo expression of CD122 on lymphocytes. Lymphocytes from HCV-infected volunteers or seronegative controls were analysed for surface expression of CD122 (black line). Gating was based on the isotype control (shaded histogram). A representative histogram of CD122 expression is shown for each subgroup. Statistically significant differences as determined by Mann–Whitney U-test: S. mansoni (−) versus controls, P = 0·016. Differences between S. mansoni (−) versus (+) and S. mansoni (+) versus controls were not statistically significant (P > 0·05).

Because CD122 also shares domains with the IL-2 receptor complex, the IL-2 levels were assessed in the plasma and culture supernatant following stimulation of PBMC with phytohaemagglutinin. No significant difference was found in IL-2 levels in the plasma or in the culture supernatant between the two HCV cohorts (data not shown). These results suggest that decreased CD122 expression by patients with concomitant schistosomiasis is not caused by decreased IL-2 production.

Discussion

Stages of memory T-cell differentiation are associated with the expression of different co-receptor molecules.1,4–7 CD28 and CD27 are costimulatory molecules that provide signals needed for specific T-cell activation. Down-regulation of CD27 is believed to be irreversible.2 CCR7 is a homing molecule that can be re-induced upon in vitro stimulation.30 In the current study, early and late differentiated T cells were defined as CD27+ CD28+ CD45RA− CCR7+ and CD27− CD28− CD45RA+ CCR7−, respectively, similar to previous reports.2,3,27,28 Examination of the total pattern of memory T-cell subsets in patients infected with HCV alone compared to individuals with concomitant schistosomiasis demonstrated that S. mansoni co-infection had no effect on the general distribution of memory T-cell subsets.

Previous studies have demonstrated that patients co-infected with HCV and S. mansoni have a more severe clinical course, higher incidence of cirrhosis and poorer response to IFN-α therapy.17–19 In addition, individuals with concomitant schistosomiasis demonstrate increased serum levels of type 2 cytokines (IL-10 and IL-4), decreased type 1 cytokines (IFN-γ), and decreased HCV-specific CD4+ T-cell proliferative responses compared to individuals with HCV alone.20 In the current study, the possibility was investigated whether the adverse prognosis of a patient co-infected with S. mansoni is associated with alterations in the HCV-specific memory CD8+ T cells. Our data suggest that concomitant schistosomiasis leads to a significant decrease in the number of late differentiated HCV-specific CD8+ T cells. In contrast, we observed no significant difference in the early differentiated HCV-specific CD8+ T cells between the two HCV cohorts. Similarly, the net HCV-specific CD8+ T-cell responses did not differ between the volunteers with HCV infection alone and those with S. mansoni co-infection. We performed similar analysis on HCMV responses in the same groups and found that S. mansoni co-infection had no effect on either the total HCMV-specific response rate or the early and late differentiated memory subsets of HCMV-specific CD8+ T cells. These results suggest that S. mansoni specifically targets and alters the development and generation of the HCV-specific memory CD8+ T-cell population.

IL-15 has been implicated in the generation and maintenance of memory CD8+ T cells,8,9,13 and the selective action of IL-15 on CD8+ memory T cells correlated with high expression of CD122.11,13 We did not detect significant differences in serum IL-15 levels in our cohorts. However, ex vivo CD122 surface expression on lymphocytes in the chronic HCV infection alone group was significantly increased compared to the control group. In contrast, expression of CD122 in individuals with concomitant schistosomiasis did not differ from controls. This interesting finding leads us to postulate that the altered CD122 expression in S. mansoni co-infection is associated with the modulation of the late stages of HCV-specific CD8+ T-cell differentiation.

The generation and regulation of antigen-specific T cells remain a central question of immunology. The evolution of phenotypic and functional criteria in delineating memory/effector T cells has revolutionized the understanding of immunological memory. A specific effector function has been ascribed to phenotypically distinct HCV-specific T cells.31 However, factors that govern T-cell homeostasis in vivo have remained undefined. We report here the modulation of a specific subset of memory T cells in HCV infection by a parasitic infection, possibly contributing to HCV pathogenesis. Interestingly, the early differentiated T-cell population that is HCV-specific is not altered, which suggests that distinct factors and functions are implicated at selective stages of T-cell maturation. The fact that HCMV-specific memory T-cell subpopulations are not perturbed by S. mansoni co-infection may reflect on differences in viral-specific memory maturation and ongoing viral replication,3,32,33 as well as specific viral pathogenesis.

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