Although there is ample evidence that infection with HIV affects adversely the course of infection with hepatitis C virus (HCV) [1,2], the effect of HCV infection on the natural history of HIV infection is less clear. The available data are limited and contradictory [3–8]. In this study, we assessed the quantitative differences of T-lymphocyte subsets, T-cell activation state, and apoptosis comparing groups of HIV-infected and HIV/HCV coinfected patients with immune failure and those with immune reconstitution during suppressive antiretroviral therapy.
Patients and methods
The study was approved by the Institutional Review Board of the Perm Regional Center for Protection against AIDS and Infectious Diseases IRB00008964 (record N 8, 19 June 2012). All patients provided written informed consent.
Seventy-nine HIV-infected patients receiving HAART for more than 2 years were studied. All patients had a confirmed diagnosis of HIV infection, were judged by clinicians to be adherent to their HAART regimen, and had plasma HIV RNA levels below 50 copies/ml. HAART regimens included two nucleoside reverse transcriptase inhibitors (NRTIs), together with a ritonavir-boosted protease inhibitor or a non-nucleoside reverse transcriptase inhibitor (NNRTI). HCV coinfection was confirmed by a positive PCR assay in all HIV-infected patients (Quantitative RT-Gepatogen C kit; DNA-Technology, Moscow, Russia), and the absence of HCV was defined by negative tests for serum antibodies to HCV in the controls. The duration of HIV and HCV infections was determined according to the date of the first positive test. Patients who had been exposed to interferon/ribavirin treatment were excluded from the study. Immune success was defined as CD4+ T-cell counts exceeding 350 cells/μl, and immune failure was defined as CD4+ T-cell counts less than 350 cells/μl . We studied the following four groups:
- HIV+/HCV+, CD4+ T-cell count above 350/μl (n = 21);
- HIV+/HCV+, CD4+ T-cell count below 350/μl (n = 21);
- HIV+/HCV–, CD4+ T-cell count above 350/μl (n = 21);
- HIV+/HCV–, CD4+ T-cell count below 350/μl (n = 16).
An additional control group consisted of HIV–/HCV– age-matched healthy controls (n = 20).
HIV and hepatitis C virus levels in plasma
Plasma levels of HIV RNA were assessed using a Versant 440 amplifier (Siemens, Erlangen, Germany) and ‘Versant HIV 1 RNA 3.0 assay b’ kits. HCV RNA levels in plasma were measured using an iCycler IQ5 (BioRad, Moscow, Russia) and real-time PCR ‘Quantitative RT-Gepatogen C’ kits (DNA-Technology).
Blood samples for T-cell phenotyping
Approximately 30 ml of blood was taken from each participant in Vacutainer tubes containing EDTA (Becton Dickinson, San Jose, California, USA). CD4+ T-cell numbers were enumerated in real time. Peripheral blood mononuclear cells (PBMCs) were isolated using Diacoll-1077 (Dia-M, Moscow, Russia) density sedimentation. The percentage of apoptotic CD4+ and CD8+ cells (AnnexinV+7-AAD–) was measured on fresh PBMCs using flow cytometry (AnnexinV-FITC/7-AAD kit; Beckman Coulter, Marseilles, France). PBMCs were then cryopreserved in fetal calf serum and dimethyl sulfoxide (DMSO) stored at −196° C, then thawed for detailed immunophenotyping.
Fluorochrome-tagged monoclonal antibodies (anti-CD3-PerCP, anti-CD4-AF700, anti-CD8-PE-CF594, anti-CD27-APC-Cy7, anti-CD45RA-APC, anti-CCR7-PE-Cy7, anti-CD31-FITC, HLA-DR-FITC, CD38-PE) and isotype control antibodies were obtained from Becton Dickinson. LIVE/DEAD Fixable Yellow Dead Cell Stain Kit was obtained from Life Technologies (Grand Island, New York, USA).
Peripheral blood mononuclear cells were thawed and stained, and viable cells were enumerated using a Becton Dickinson LSR II Flow Cytometer. Naive cells were identified as CD27+CD45RA+CCR7+. Naive CD4+CD31+ cells were considered to be recent thymic emigrants (RTEs). CD27+CD45RA–CCR7+ lymphocytes were defined as central memory cells, CD27–CD45RA–CCR7– cells as effector memory cells; and CD27–CD45RA+CCR7– as terminally differentiated effectors. CD38+HLA-DR+ T cells were considered to be activated. At least 100 000 events in the lymphocyte gate were collected for each sample. Relative values were received from the cytometer data. Absolute lymphocyte subpopulations were calculated based on CD4+ T-cell numbers detected in the fresh blood.
Data were reported as medians and interquartile ranges, and groups were compared by means of the Mann–Whitney method. The impact of HIV infection, immune failure on HAART and HCV coinfection was evaluated by variance analysis with the calculation of effect confidence using the Fisher's test. Correlation analysis was performed using Spearman correlations. All statistical analyses were performed using ‘STATISTICA 6.0’ software (StatSoft Software, Tulsa, Oklahoma, USA).
Patient clinical characteristics
As per our patient selection strategy, the ages of the HIV-infected patient groups and healthy controls were comparable (Table 1), averaging between 31 and 35 years. The average age in the uninfected group was 31 (26–35) years. Men were over-represented among HIV/HCV coinfected patients (P < 0.001) reflective of the demographics of HCV infection in Russia . In contrast, the HIV-monoinfected patients in this study were predominantly women. In the healthy control group 40% were men. The known duration of infection was longer in dually infected patients than in HIV-monoinfected immune success patients (11 vs. 8 years, respectively). As per our selection criteria, CD4+ T-cell numbers were higher in immune successes than in immune failures. In addition, plasma HIV level before HAART was higher (320285 vs. 95195 copies/ml; P < 0.001) and nadir CD4+ T-cell level was lower (97 vs. 140 cells/μl; P < 0.001) in nonresponders than among responders. In HIV/HCV coinfected patients, median HCV RNA levels exceeded 1 000 000 copies/ml and liver enzymes were elevated compared to levels in HIV-monoinfected patients. Platelet counts in coinfected immune failures were lower than in coinfected immune successes and lower than in monoinfected immune failures. The aspartate aminotransferase (AST)-to-platelet ratio index (APRI) for predicting fibrosis and cirrhosis  was highest in coinfected immune failures, and higher in both coinfected groups than in the corresponding monoinfected groups.
In all HIV+ patient populations, there were significant decreases in the absolute numbers of circulating CD4+ T cells when compared to numbers in healthy individuals (P < 0.001 when HIV+ groups are compared to the control group; P < 0.05 when HCV+ immune successes are compared to HCV– immune successes; Table 1). In immune failures whether coinfected or not, profound CD4+ T-cell cytopenia was associated with significant decreases in the absolute numbers of naive, central memory, and effector memory CD4+ T cells (Fig. 1).
HIV-infected patients had decreased proportions of naive CD4+ T cells (35.11% in the HIV+ group vs. 48.36% in the controls, P < 0.01) and increased percentages of effector memory CD4+ T cells (16.37% in the HIV+ group vs. 11.91% in the controls; P < 0.01). Immune failures had decreased absolute numbers of all CD4+ T-cell maturation subsets when compared to numbers in healthy controls, but in immune successes, only numbers of the less mature CD4+ T-cell populations (naive and central memory CD4+ T cells) were lower than among healthy controls (P < 0.001). The HCV coinfected patients did not differ from the singly infected patients in relative or absolute numbers of any of these maturation subsets.
Activation and apoptosis
Expression of CD38 and HLA-DR on circulating T cells has been linked to increased risk of disease progression in untreated HIV infection . We therefore analyzed the expression of these activation antigens on CD4+ T cells in our patient groups and also examined the binding of Annexin V as a marker of cells that were prone to programmed cell death (Fig. 2). Our data indicated, and as shown earlier , that immune failure was associated with increased CD4+ T-cell activation and here we also show that Annexin V binding is also increased in immune failure. In these patients, HCV infection was associated with a small but no significant (HIV+HCV+ vs. HIV+HCV–; P > 0.05) increase in CD4+ T-cell activation and had no impact on Annexin V binding.
Recent thymic emigrants
With the profound decrease in naive CD4+ T-cell counts in all HIV-infected populations studied here, we examined the frequencies of naive CD4+ T lymphocytes that also expressed CD31+ (Fig. 3), as these cells are thought to be enriched for recent thymic emigrants . While proportions of CD31+ cells among naive CD4+ T lymphocytes were comparable in singly and dually infected patients (pooling immunological responders and nonresponders), singly infected immune failures had proportionally more naive CD4+CD31+ T cells than singly infected immune successes or dually infected immune failures (P < 0.05). When absolute numbers were compared, however, all patient populations had fewer naive RTE in circulation than did the healthy controls, and immune failures had fewer RTEs than immune successes, whether singly infected (P < 0.01) or dually infected (P < 0.001). Analysis performed on pooled immune success and immune failure groups demonstrated that coinfected patients had fewer CD4+ RTEs in circulation than singly infected patients, suggesting the possibility that thymic production is less effective in HCV/HIV infection than in HIV infection alone (P < 0.05). Not surprisingly, when analyzing the entire population, there was a significant negative correlation between the absolute number of naive CD4+CD31+ T lymphocytes and age (R = −0.323, P < 0.01). Sex also had a significant impact on the number of RTEs as women had 1.6 times more CD4+ RTE/μl of blood than men did (mean values 128 vs. 80; P < 0.02). We evaluated the impact of the initial (pretreatment) level of CD4+ T cells, response to HAART, the known duration of HIV infection, and duration of HAART on CD4+ RTE counts in the combined group of HIV-infected patients. Naive CD4+CD31+ T-cell numbers were directly related to pretreatment CD4+ T-cell counts (R = 0.332, P < 0.01). The impact of the response on HAART was significant: immune failures had 2.3 times lower CD4+ RTE number than immune successes (mean values 46 vs. 108; P < 0.001). At the same time, no influence of known duration of infection or duration of HAART exposure was registered.
CD4+ RTE counts in HIV+/HCV+ women were more than two times higher than those among coinfected men, and in this combined population of immune failures and immune successes when age was not linked to RTE numbers, the mean ages of the men and women in this group were similar (33 and 35 years; P > 0.05, respectively).
To explore further the relationship between liver disease and RTE numbers, we performed several correlation analyses combining the two HCV-infected groups: HIV+/HCV+ immune success and HIV+/HCV+ immune failure (Fig. 3). We found significant negative correlations between levels of AST, alanine aminotransferase (ALT) and APRI, and the absolute number of naive CD4+CD31+ T cells. These liver function abnormalities were not related to patients’ age, to sex or to known duration of HIV or HCV infection, or duration of HAART exposure. They were, however, each directly related to the levels of HCV in blood: R AST-HCV = 0.527 (P < 0.001), R ALT-HCV = 0.483 (P < 0.01), R APRI-HCV = 0.361 (P < 0.05).
In this study, we enumerated CD4+ T-cell maturation subsets in four convenience cohorts of ‘singly’ HIV-infected or ‘dually HIV/HCV coinfected’ patients who experienced either successful (CD4+ >350/μl) or unsuccessful (CD4+ <350/μl) CD4+ T-cell restoration after at least 2 years of virologically suppressive combination antiretroviral therapy. As reported earlier , immune failure patients had diminished numbers of all CD4+ T-cell maturation subsets. Here we show that this was the case whether HCV coinfected or not. The cause for the failure of CD4+ T-cell restoration in immune failure patients is not known. Low-level viral production in tissues may somehow account for this failure, but if so, this is not likely directly related to sustained productive infection of CD4+ T cells as the frequency of infection in this compartment is low. In numerous studies, systemic immune activation has been linked to failure of CD4+ T-cell restoration . Although HAART reduces the activation level of CD4+ and CD8+ T cells, the percentage of lymphocytes expressing CD38 and HLA-DR in treated patients remains higher than in healthy controls . Failure of cellular restoration on HAART may be related to an accelerated CD4+ T-cell turnover  or failure to respond to homeostatic signals . Coinfection with other pathogens (such as cytomegalovirus)  or HCV  has also been linked to increased immune activation in this setting and those findings prompted the design of this study.
Here, while immune failure patients had higher CD4+ T-cell activation than did immune successes, we find only modest and nonsignificant increases in CD4+ T-cell activation in HCV-coinfected patients. We also find here that the proportion of CD4+ T cells that bind Annexin V is increased in HIV infection and this is especially demonstrable in immune failure patients as has been shown by others . But in this study, Annexin V binding does not seem to be related to the presence of HCV coinfection. Conceivably, an increased tendency to programmed cell death is related to the increased T-cell activation and cycling seen in immune failure and is reflective of the accelerated CD4+ T-cell turnover that is seen in chronic HIV infection .
In addition to a potential role of HAART in controlling the level of T-cell activation through control of HIV, in the setting of HIV/HCV coinfection, HAART-induced immune restoration may indirectly lead to enhanced immune surveillance over HCV. In fact, it is known that clearance of chronic HCV infection can occur after exposing HIV/HCV coinfected patients to HAART [22,23]. One possible explanation may be a beneficial immune status being a result of HAART . We find no differences in HCV level comparing HAART responders to immune failure patients, perhaps indicating the situation is more complex during chronic HCV infection.
The role of thymic function in adult T-cell homeostasis is uncertain. Although thymic involution is demonstrable in young adulthood, remnants of thymic tissue may remain active and may release newly minted T cells into the systemic circulation. To this end, estimates of thymic function based upon imaging, detection of T-cell receptor excision circles and phenotypic markers of recent thymic emigration have reported variable degrees of thymic dysfunction in untreated and treated HIV infection [25,26]. Hereby enumerating CD31+ naive cells that are thought to be enriched for recent thymic emigrants, we find that the proportions of these cells are relatively preserved, whereas their absolute numbers are diminished substantially in the two immune failure groups, and in this analysis, absolute numbers of naive CD4+ RTEs were lower in coinfected patients than in singly infected patients and were lower in all HIV-infected populations than among healthy controls (Fig. 3). We suspect that this reflects a true biologic phenomenon in HIV and HCV coinfection as there is a consistent relationship between abnormalities in liver function (AST, ALT and APRI) and decreased numbers of naive CD4+ RTE in circulation (Fig. 3e). Yonkers et al.  found a similar relationship between circulating naive CD4+ T-cell numbers and serum ALT levels in chronic hepatitis C infection. Whereas it is difficult to identify a role of naive RTE in protection against hepatocellular dysfunction, these findings suggest that HCV infection may impair thymic T-cell production. Earlier study has suggested as much as the frequency of T cells containing T-cell receptor excision circles , and the frequency of CD31+ naive CD4+ T cells has been found to be diminished in HCV infection [27,29]. If this is the case, homeostatic expansion of RTE is likely unimpaired in this setting as naive CD4+ T-cell numbers did not appear to be affected by HCV infection in this study nor in earlier studies by Al-Harthi et al.  and by Roe et al. . In our study, RTE numbers were related inversely to indices of hepatocellular function, but not to levels of HCV RNA in plasma, suggesting that if HCV infection affects thymic function, it may do so indirectly via hepatic impairment rather than as a direct consequence of HCV levels or replication. In fact, lower RTE levels have been observed in uninfected individuals with cirrhosis than in controls . Conceivably, hepatocellular dysfunction blocks residual thymic function in adults or somehow promotes the loss or maturation of these cells. Additional study is warranted to explore the relationship between hepatocellular function and RTE.
Funding: National Institute of Allergy and Infectious Diseases under National Science Foundation Cooperative Agreement No. OISE-9531011; CRDF Global under Agreement No. RUB1–31089-PE-12; Russian Foundation for Basic Research under Agreement No. 12–04–91441-НИЗ_а; Presidium of the Ural Branch of the Russian Academy of Sciences under Agreement No.12-С-4-1033.
Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of CRDF Global, NIAID or the National Science Foundation
Conflicts of interest
The authors have no conflict of interests.
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