Rallón, Norma I PhD*; Soriano, Vincent MD, PhD*; Restrepo, Clara MSc*; García-Samaniego, Javier MD, PhD†; Labarga, Pablo MD, PhD*; López, Mariola PhD*; Peris, Alejandra MSc*; González-Lahoz, Juan MD, PhD*; Benito, José M MD, PhD*
The course of hepatitis C virus (HCV)-related liver disease is accelerated in HIV/HCV-coinfected individuals, and thereby HCV has emerged as an important cause of morbidity and mortality in persons infected with HIV-1,1,2 especially because successful antiretroviral therapy has dramatically reduced the rate of opportunistic illnesses. The mechanisms involved in this enhanced HCV pathogenicity in HIV/HCV-coinfected subjects are unclear, although immunodeficiency clearly plays a role.3-6 Several previous studies have reported a diminished HCV-specific T-cell response in HIV/HCV-coinfected compared with HCV-monoinfected patients.3,7-11 However, these studies have been performed in HIV/HCV-coinfected patients with different levels of exposure to antiretroviral treatment and with variable degrees of HIV-associated immunodeficiency. Also, the majority have been limited to measure only interferon-γ (IFNγ) secretion with only few of them analyzing more than one cytokine simultaneously.9,10
Suppression of HIV replication with antiretroviral therapy has dramatically changed the course of HIV disease and in patients coinfected with HCV has reduced the liver-associated morbility and mortality.12,13 Given that the impairment of HCV-specific T-cell response in coinfected patients is associated with the degree of HIV-induced immunodeficiency,3 an improvement of this response after highly active antiretroviral therapy (HAART)-induced immune reconstitution may be involved in the benefit that antiretroviral therapy exerts on liver disease, although this immune restoration may also result in a transient worsening of liver inflammatory activity as a result of immune restoration disease in a small subset of patients.14 So far only a very recent study has explored, in a longitudinal design, to what extent HCV-specific response increases after control of HIV replication by HAART.15 That study, however, was limited by measuring only one cytokine and only against one HCV protein. Moreover, an HCV-monoinfected patient's control group was not included. In the present study, we have evaluated HCV-specific CD4+ and CD8+ T-cell responses against the full HCV proteome, measuring two different cytokines in HIV/HCV-coinfected patients on successful HAART with complete HIV suppression and we have compared it with a control group of HCV-monoinfected patients.
PATIENTS AND METHODS
A cross-sectional study was conducted in 50 patients with chronic hepatitis C, including 27 HCV-monoinfected and 23 HIV/HCV-coinfected patients on regular follow-up at Hospital Carlos III, a reference Infectious Diseases clinic located in Madrid, Spain. Twenty HCV- and HIV-seronegative healthy volunteers were included as control subjects to establish reference values for a positive HCV-specific T-cell response. All patients had detectable plasma HCV-RNA, and all were naive for interferon. HIV/HCV-coinfected patients were under antiretroviral treatment and had undetectable plasma HIV-RNA. To participate in the study, written informed consent for specific examinations was obtained from all individuals, and the study protocol was evaluated and approved by the Hospital Ethical Committee.
Viral Load and Hepatitis C Virus Genotyping
Plasma HCV-RNA was measured using a real-time polymerase chain reaction assay (COBAS TaqMan 48; Roche, Barcelona, Spain), which has a lower limit of detection of 10 IU/mL. HCV genotyping was performed on plasma using a commercial reverse transcription-polymerase chain reaction hybridization assay (Versant HCV Genotype 2.0 LiPA; Siemens, Barcelona, Spain). Plasma HIV-RNA was measured using Versant HIV-1 RNA Version 3.0 (Siemens), which has a lower limit of detection of 50 copies/mL.
Overlapping Hepatitis C Virus Peptides
Two genotype-matched peptide arrays of 460 overlapping peptides (Biodefense & Emerging Infections Research Resources Repository, Manassas, VA; www.beiresources.org) spanning all HCV proteins and corresponding to the HCV H77 strain (genotype 1a, beiresources catalog numbers: NR-3747 to NR-3756) and to the HCV K3a/650 strain (genotype 3a, beiresources catalog numbers: NR-4061 to NR-4070) were used to assess HCV-specific CD4+ and CD8+ T-cell immune responses. Each peptide was 12 to 19 aa long and overlapped 11 to 12 aa with the adjacent peptides. Overlapping peptides have been previously shown to be suitable for the measurement of both CD4+ and CD8+ T-cell responses.16 In the stimulation assays, peptides were grouped into eight pools according to HCV genotypes and proteins: core (28 peptides), E1 (29 peptides), E2/p7 (64 peptides), NS2 (32 peptides), NS3 (98 peptides), NS4A and NS4B (both together, 47 peptides), NS5A (71 peptides), and NS5B (91 peptides). The dimethylsulfoxide concentration in culture medium was less than 5% for all peptide pools. All individual peptides and peptide pools were aliquoted and stored at -80°C.
All immunologic studies were done using cryopreserved peripheral blood mononuclear cells (PBMCs). EDTA-anticoagulated blood was obtained by venipuncture; PBMCs were immediately isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma Chemical Co, St Louis, MO) and frozen in fetal calf serum plus 10% dimethylsulfoxide. Viability of thawed PBMCs was checked using tryptan blue and was always greater than 85%.
Intracellular Cytokine Production Assay
Two functions of CD4+ and CD8+ T-cells (production of IFNγ and tumor necrosis factor-α [TNFα]) were simultaneously measured in response to HCV genotype-matched overlapping peptides pools using multiparameter flow cytometry. A million of PBMCs were incubated for 6 hours at 37°C in 250 μL of complete medium (RPMI containing 10% fetal calf serum, l-glutamine, and streptomycin and gentamycin as antibiotics) with each pool of HCV genotype-matched peptides at a final concentration of 2 μg/mL for each individual peptide in the presence of 0.5 μg/mL each of anti-CD28 (CD28.2; BD Biosciences, San Diego, CA) and anti-CD49d (9F10; BD Biosciences) monoclonal antibodies (to provide exogenous costimulation). The secretion inhibitor Brefeldin A (GolgiPlug, 1 μL/mL; BD Biosciences) was added to the cultures during the second hour of incubation. Control conditions included stimulation with medium and the costimulators anti-CD28 and anti-CD49d as negative controls and stimulation with 50 ng/mL of PMA (Sigma) plus ionomycin (1 μM; Sigma-Aldrich, Madrid, Spain) and the costimulators anti-CD28 and anti-CD49d as positive controls.
After incubation, cells were harvested and stained for cell surface markers and intracellular cytokines using standard procedures with the following panel of monoclonal antibodies: anti-CD8α-ECD (Beckmann-Coulter, Miami, FL), anti-CD4-PECy5 (Cytognos, Salamanca, Spain), anti-IFNγ-FITC (BD Biosciences), and anti-TNFα-PECy7 (eBioscience, San Diego, CA). We did not use staining with CD3. However, in several samples, the percentage of HCV-specific CD8+ T-cells was analyzed by parallel staining with anti-CD8β (which stains only CD8+ T-cells) and anti-CD8α to be sure that these cells are truly CD8+ T-cells. No significant differences were observed in both assays. Acquisition was performed on a Cytomics FC 500 flow cytometer (Beckman Coulter). For each sample, a minimum of 50,000 CD4+ and 50,000 CD8+ events were acquired. Data analysis was performed using CXP software (Beckman Coulter). Gating was done on CD8+ bright and CD4+ T-cells. The percentage of CD8+ bright or CD4+ T-cells expressing any combination of cytokines was assessed using the negative control of each sample to establish quadrant boundaries.
Figure 1 shows a representative example of flow cytometry data with the gating strategy used. On the basis of expression of the two cytokines examined, three unique CD4+ and CD8+ T-cells subsets were defined (cells not producing any of the two cytokines were not considered). For each of these subsets, a threshold for a positive response was defined as the 95th percentile of 160 pooled values obtained from eight different stimulations (with the eight HCV pools) in 20 HCV- and HIV-seronegative healthy donors. This threshold was applied to each sample after background subtraction (the response of the negative control in that particular sample). The global CD4+ and CD8+ HCV-specific T-cell response was calculated as the sum of each unique cell subset response against the different HCV proteins. To asses the functional profile of HCV-specific response, the contribution of each unique cell subset to the global HCV-specific CD4+ and CD8+ T-cell response was calculated. Moreover, the contribution of single cytokine-producing cells (IFNγ+ TNFα-or IFNγ- TNFα+ cells) and double cytokine-producing cells (IFNγ+TNFα+ cells) as well as the contribution of all cells producing one particular cytokine (IFNγ or TNFα) was further analyzed.
The main characteristics of the study population and the different parameters evaluated are expressed as median (interquartile range). Comparisons between groups were done using Mann-Whitney U test and between different parameters in the same group of patients using Wilcoxon test. Associations between qualitative variables were explored using the chi-square test or Fisher exact test as appropiate. All statistical analyses were performed using the SPSS software Version 13 (SPSS Inc, Chicago, IL). All P values were two-tailed and were considered as significant only when <0.05.
Table 1 summarizes the main characteristics of HCV-monoinfected and HIV/HCV-coinfected patients. Both groups were comparable in terms of median plasma HCV-RNA (6.5 [6.2-7.1] versus 6.6 [6.4-7.3] log IU/mL, respectively; P = 0.80). The median CD4+ count in HIV/HCV-coinfected patients was relatively high (486 [378-740] cells/μL), most likely reflecting that all of them were under antiretroviral treatment and had undetectable plasma HIV-RNA. Median time since HIV diagnosis and median time on HAART in coinfected patients were 16 (9-19) years and 78 (15-99) months, respectively. HCV genotype 1 was the most prevalent (93%) in HCV-monoinfected patients, whereas in HIV/HCV-coinfected patients, HCV genotype 1 and genotype 3 represented 74% and 26% of cases, respectively.
Prevalence, Intensity, and Breadth of Hepatitis C Virus-Specific CD4+ and CD8+ T-Cell Responses
More than half of patients presented CD4+ (60%) or CD8+ (57%) -positive responses to at least one HCV protein, with core being the most frequently targeted by both CD4+ (35% of patients) and CD8+ T-cells (25% of patients). For each HCV protein, the prevalence of patients presenting a CD4+ T-cell response was similar to prevalence of CD8+ T-cell response. When comparing HCV-monoinfected and HIV/HCV-coinfected patients, there were no significant differences in the prevalence of CD4+ and CD8+ response to any HCV protein. In both groups of patients, the most frequently targeted protein was core (Fig. 2).
The intensity of T-cell responses was defined as the percentage of CD4+ and CD8+ T-cells that responded to the different HCV proteins. In patients with a detectable response, levels of response to each protein were uniformly low and of similar magnitude in both groups of patients (Fig. 3). The sum of individual CD4+ and CD8+ T-cell responses against each HCV protein did not differ comparing HCV-monoinfected and HIV/HCV-coinfected patients (total CD4+ response: 0.72% [0.17-1.50] versus 0.10% [0.03-3.74], P = 0.84; total CD8+ response: 0.25% [0.08-0.70] versus 0.41% [0.21-0.62]; P = 0.94). Core was the protein that most contributed to total CD4+ response in both groups of patients (11% [0-35] and 10% [0-100], respectively), whereas for CD8+ response, there was no clear difference in the contribution of the different HCV proteins.
The breadth of immune response, defined as the number of different HCV proteins with a positive CD4+ or CD8+ T-cell response, was not significantly different between HCV-monoinfected and HIV/HCV-coinfected patients. It was 1 (0-3) versus 1 (0-2), respectively (P = 0.51) for CD4+ T-cells and 1 (0-3) versus 0 (0-3), respectively (P = 0.15) for CD8+ T-cells.
Functional Profile of Hepatitis C Virus-Specific CD4+ and CD8+ T-Cell Responses
The contribution of each T-cell subset, the contribution by number of functions, and the contribution by cytokine to the global HCV-specific CD4+ and CD8+ T-cell response are summarized in Figure 4. There were no significant differences in the functional profile of global HCV-specific CD4+ response between HCV-monoinfected and HIV/HCV-coinfected patients. In both groups, this response was mainly mediated by monofunctional subsets, although there was also a moderate contribution of cells producing both IFNγ and TNFα (median contribution of IFNγ+TNFα+ cells was 20% [0-54] and 9% [0-100] in monoinfected and coinfected patients, respectively; P = 0.31). In both groups, the contribution of TNFα+ cells was slightly higher than IFNγ+ cells.
Regarding global CD8+ response, the functional profile was also similar in HCV-monoinfected and HIV/HCV-coinfected patients. However, this functional profile showed some differences with respect to the functional profile of CD4+ response. In both groups, global CD8+ response was mediated almost exclusively by IFNγ+TNFα- cells with much lower contribution of IFNγ+TNFα+ cells and no contribution at all of IFNγ-TNFα+ cells. Thus, most of the response was mediated by monofunctional cells with much lower contribution of bifunctional cells. Also, most of the response was mediated by IFNγ+ cells with only a slight contribution of TNFα+ cells.
Characteristics of Hepatitis C Virus-Specific T-Cell Responses According to Hepatitis C Virus Genotype
To compare differences in HCV immune responses according to HCV genotype, the analysis was restricted to the HIV/HCV-coinfected group, in which both HCV genotype 1 (HCV-1, n = 17) and HCV genotype 3 (HCV-3, n = 6) were represented. There were no significant differences between HCV-1 and HCV-3 patients in terms of age and CD4+ count, whereas HCV-1 patients presented a significantly higher level of HCV plasma viremia than HCV-3 patients (6.7 [6.4-7.2] versus5.8 [5.1-6.3] log IU/mL; P = 0.02). More than 50% of individuals in both groups had detectable CD4+ and/or CD8+ responses against at least one HCV protein. Moreover, the prevalence of response to each HCV protein was similar in both groups, except for CD8+ T-cells responding against E1 that were more frequent in HCV-3 than HCV-1 patients (75% versus 6%; P = 0.01). Levels of total CD4+ and CD8+ responses were higher in HCV-3 compared with HCV-1 patients (CD4+ response: 1.76% [0.02-5.10] versus 0.03% [0-0.30], P = 0.1; CD8+ response: 0.45% [0.09-0.62] versus 0% [0-0.17], P = 0.01). Finally, the cytokine profile of HCV-specific CD4+ and CD8+ T-cell response was dominated by single cytokine producing cells, regardless HCV genotype.
To investigate the HCV-specific T-cell response in HIV/HCV-coinfected patients with good control of HIV replication, we examined the cytokine profiles at single cell level in peripheral blood CD4+ and CD8+ lymphocytes. A group of HCV-monoinfected patients was taken as controls. We found that HCV-specific CD4+ and CD8+ T-cell responses are not only low and of limited specificity, but also characterized by a predominantly monofunctional cytokine secretion profile. More than 80% of the contribution to the global HCV-specific T-cell response was mediated by single cytokine-producing cells with no differences between both groups of patients. These findings suggest that the lack of polyfunctionality of HCV-specific T-cells could be on the basis of HCV persistence, as it has also been suggested for HIV disease.17
Using the comprehensive screening strategy with HCV-derived overlapping peptides to analyze the complete HCV genome, HCV-specific CD4+ T-cell responses were detected in a similar proportion as CD8+ T-cell responses. This methodological strategy allowed the recognition of a higher rate of T-cell responses in comparison with other studies that used a limited number of optimal epitopes.18-20 Moreover, our results are in agreement with those obtained in other studies, which have also used overlapping HCV peptides.3,8-10,21 We should acknowledge, however, as a limitation of our study (as most others so far) that we may have underestimated anti-HCV responses because consensus peptide sequences may not always represent the autologous HCV peptides circulating in infected individuals.
Most previous studies on HCV-specific T-cell responses in HIV/HCV-coinfected patients have been limited to measure only IFNγ secretion.3,7,8 Two recent studies have analyzed the functional profile of HCV-specific T-cells in HIV/HCV-coinfected and HCV-monoinfected patients by measuring the simultaneous production of IFNγ and interleukin-2.9,10 These studies showed an impairment of HCV-specific T-cell responses in HIV/HCV-coinfected compared with HCV-monoinfected patients. However, they included a heterogeneous population of HIV/HCV-coinfected patients with respect to antiretroviral treatment and degree of immune deficiency. Our study demonstrates that in HIV/HCV-coinfected patients with maximal HIV suppression under HAART, several characteristics of the anti-HCV T-cell response are similar to those found in HCV-monoinfected patients, suggesting that successful HAART might improve HCV-specific T-cell responses in HIV/HCV-coinfected patients. We may speculate about the driving factor for this improvement in HCV-specific responses. In our study, all coinfected patients had undetectable HIV viremia and therefore any improvement of T-cell responses could result from suppressed HIV replication. Alternatively, this improvement could be more directly related to the immune restoration induced by therapy.3 Interestingly, we did not find an association between CD4 counts and HCV-specific T-cell responses, arguing against this hypothesis. However, all these conclusions must be cautious given that our coinfected population did not include individuals with very low CD4 counts in whom HCV-specific responses could be worse despite complete viral suppression.
It is also important to highlight that the approach used in our study differs substantially from others that have examined the same question9,10 because we defined polyfunctionality as the ability to simultaneously produce multiple cytokines by a single cell, which is in line with most studies in different virus models,17,22-25 whereas they defined polyfunctionality as heterogeneity of the response (presence of different T-cell subsets: single cytokine and dual cytokine-producing cells). Another difference is that we measured TNFα and IFNγ instead of interleukin-2 and IFNγ. The inclusion of TNFα in the simultaneous assessment of cytokine production together with IFNγ is important because TNFα is a crucial antiviral cytokine that amplifies Type 1 antiviral immune responses by inducing the synthesis of interleukin-12 and interleukin-18, which ultimately upregulate IFNγ production. TNFα can alternatively kill virally infected cells by binding its cognate receptor on the cell surface.22 Accordingly, TNFα has recently been associated with HCV clearance or with a transient drop in serum HCV-RNA in patients with acute hepatitis C.26
Regarding the clinical significance of our results, the beneficial effect of HAART on HCV-specific responses is in parallel with the beneficial effect of HIV suppression on the natural course of HCV-induced liver disease.12,13 It is interesting that our cohort of HIV/HCV-coinfected patients presented levels of HCV plasma viremia similar to those found in HCV-monoinfected patients, suggesting a potential relationship between improvement in HCV-specific T-cell response and decrease in HCV replication. Because we did not include a group of HAART-naïve coinfected patients, we cannot positively demonstrate such an association, although our results are in agreement with the study of Rohrbach et al showing a concomitant reduction in HCV-RNA levels in parallel with an increase in HCV core-specific T-cell responses.15
Finally, because HCV-specific cellular immune responses might differ for distinct HCV genotypes and most previous studies have used only HCV genotype 1 peptides,3,8-10,21,27,28 an underestimation of T-cell responses could have occurred. To avoid this bias, we used genotype-matched peptides in the stimulation assays. Using this approach, some subtle differences in the anti-HCV response between HCV-1- and HCV-3-coinfected patients were noted. Some characteristics of HCV-specific T-cell response such as prevalence against E1 protein as well as intensity of global CD4+ and CD8+ T-cell response were increased in patients infected with HCV-3, highlighting the importance of using genotype-matched peptides when comparing patients with distinct HCV genotypes. Given the relatively small sample size of our study population infected with HCV genotype 3, our results should be interpreted cautiously, and the trend recognized should be confirmed testing a larger number of patients. Because patients with HCV-3 respond better to anti-HCV therapy than patients with HCV-1, our results suggest a potential association between T-cell responses and response to treatment as has been previously suggested by other authors.29
We thank Sara Lozano for her excellent technical assistance and all patients and volunteers who participated in the study.
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