T cells play an important role in the adaptive immune response to acute hepatitis C virus (HCV) infection. In particular, broad and sustained CD4+ and CD8+ T-cell responses against HCV antigens after acute infection have been prospectively associated with subsequent immune clearance of HCV viremia.1,2 In individuals with chronic HCV infection, however, T-cell responses against HCV antigens are generally weak or even undetected despite ongoing viral replication.2,3 Furthermore, a large fraction of HCV-specific CD8+ T cells express cell surface markers of differentiation and exhaustion during chronic HCV infection, and these cells are susceptible to apoptosis.4 However, the broader impact of chronic HCV infection on overall T-cell differentiation and activation, including T cells that are not specific for HCV antigens, is not well understood. These relationships are important because nonspecific immune activation in individuals with chronic HCV infection has been hypothesized to contribute to the development of extrahepatic conditions, including diabetes and cardiovascular disease (CVD) that are found in excess among these individuals.5–9
Three recent studies examined T-cell phenotypes in HCV-viremic individuals and HCV-uninfected controls. All 3 studies reported lower percentages of naive CD4+ T cells, and 1 study also reported a lower percentage of naive CD8+ T cells in HCV-viremic individuals as compared with HCV-seronegative controls.10–12 The data from these studies were not consistent, although, regarding percentages of central memory (CM), effector memory (EM), and terminally differentiated effector (TE) T cells by HCV viremia status. EM and TE cells have shortened telomeres as compared with naive and CM cells and represent more differentiated T-cell phenotypes, with diminished capacity to replicate in response to antigenic stimulation.13
Studies by our group and others have additionally examined T-cell expression of activation markers (eg, CD38 and HLA-DR) in HCV-viremic individuals compared with uninfected controls. Most of these studies found no differences in the percentage of CD4+ and CD8+ T cells that were activated by HCV status,14–17 although 1 study found that CD4+ T-cell activation was higher in those with HCV viremia.18 Comparisons of the percentage of regulatory CD4+ T cells (Tregs) between individuals with HCV viremia and HCV-uninfected controls have also been conducted, but the data have conflicted.10,19–22
An important limitation of these previous studies is that the HCV-uninfected controls were often healthy individuals from the general population that differed in important ways from the subjects with HCV viremia. For example, cigarette smoking and injection drug use (IDU) are more common in HCV-seropositive individuals than in the general population and may have a strong influence on the immune response in general and on T-cell function in particular.23–28 Finally, although the association of HCV viremia with T-cell differentiation and activation in HIV-positive individuals has been studied,10,15–19,21,29 no previous investigation directly tested whether the relation of HCV viremia to T-cell differentiation and activation might differ according to the overall level of immunosuppression as measured by total CD4+ T cell count.
MATERIALS AND METHODS
The Women's Interagency HIV Study (WIHS) is a multicenter prospective study of HIV-infected and HIV-uninfected women. Women were recruited using similar methods at 6 US sites during 3 recruitment periods: 1994–1995 (N = 2623), 2001–2002 (N = 1143), and an ongoing recruitment period initiated in 2011. Detailed methods and characteristics of the study population have been described previously.30,31 At enrollment and then prospectively on a semiannual basis, interviews are conducted, a physical examination performed, and blood specimens collected. The protocol was approved by the Institutional Review Boards at each study site, and all participants provided written informed consent.
Structured interviews are conducted at each semiannual visit to obtain demographic, behavioral, and clinical information. This includes past and current IDU, alcohol use, cigarette smoking, and adherence to prescribed antiretroviral therapy regimens. The definition of highly active antiretroviral therapy (HAART) in WIH6S was guided by the DHHS/Kaiser Panel (DHHS/Kaiser 2008) guidelines and is defined as the reported use of 3 or more antiretroviral medications, 1 of which has to be a PI, an NNRTI, 1 of the NRTIs abacavir or tenofovir, an integrase inhibitor (eg, raltegravir), or an entry inhibitor (eg, maraviroc or enfuvirtide). A clinical examination is also performed at each study visit, which includes measurement of height and weight among other characteristics.
We enrolled 658 WIHS women into a T-cell substudy conducted between October 2011 and March 2013. Specifically, women were enrolled sequentially into the substudy, as they presented for one of their routine semiannual follow-up visits, during which a fresh peripheral blood sample (10 mL) was drawn into a sodium heparinized vacutainer tube, which was then packed in a temperature-controlled shipping container and shipped overnight to Rush University for processing and phenotypic evaluation.
There is not a large body of literature examining the effects of cryopreservation on FACS results, but most studies suggest that the major lymphocyte subset percentages (eg, the total percentage of CD4+ T cells) are similar whether measured in fresh or frozen specimens.32,33 However, the data are conflicting as to whether more detailed phenotyping of T cells by FACS, including the percentage of naive T cells, Tregs, and other subsets, may be significantly altered by cryopreservation.32,34–36 Moreover, in many clinical/epidemiologic studies, including the WIHS, there is likely to be some variability in the adequacy cryopreservation, and cell viability is a concern. For these reasons, we measured T-cell phenotypes in fresh unfrozen blood samples in this study.
Flow Cytometry to Assess CD4+ and CD8+ T-Cell Phenotypes
Upon arrival at Rush University, peripheral blood samples were diluted in phosphate-buffered saline (PBS) without Ca++ and Mg++ and processed over Lymphocyte Separation Media (Mediatech Inc., Manassas, VA). The interface containing peripheral blood mononuclear cells (PBMCs) was collected, washed in PBS without Ca++ and Mg++, and stained for flow cytometric evaluation. PBMCs were stained with Fixable Aqua Dead Cell stain (Life Technologies, Grand Island, NY), washed, and stained with fluorochromeconjugated antibodies against the following cell surface markers: CD3, CD8, CD25, CD38, CD45RA, CCR7, HLA-DR (BD Biosciences, San Jose, CA), and CD4 (Life Technologies, Grand Island, NY). Cells were washed and stained intracellularly with fluorochrome-conjugated FoxP3 (eBioscience) antibody. After staining, the cells were washed, fixed, and acquired on a BD LSR II flow cytometer using FACS Diva v6.1.1 software. T-cell phenotypes were defined using Flow Jo v8.8.7 (Tree Star, Inc.) as the percentage of live (Aqua-) cells that expressed specific markers among total CD3+/CD4+ or CD3+/CD8+ T cells, including activated (CD38+ and HLA-DR+), naive (CD45RA (RA) + and CCR7 (R7) +), CM (RA−/R7+), EM (RA−/R7−), and TE (RA+/R7−) T-cell markers. T-regulatory cells (CD3+/CD4+/CD25+/FoxP3+) are expressed as a percentage of total CD4+ T cells.
Clinical Laboratory Measurements
HCV serostatus was determined at WIHS enrollment using a commercial second- or third-generation enzyme immunoassay, and HCV viremia was determined in HCV-seropositive women using either the COBAS Amplicor Monitor 2.0, which has a linear range of 600–5.0 × 105 IU/mL, as previously described,29 or the COBAS Taqman assay, which has a linear range of 10–2.0 × 108 IU/mL (both from Roche Diagnostics, Branchburg, NJ). Follow-up HCV antibody testing was conducted in 1998–1999 in most women who were HCV seronegative at their 1994–1995 enrollment visit. Follow-up HCV RNA testing has also been conducted on most women who were HCV RNA positive at enrollment using the Amplicor or Taqman assay, primarily in 1997–2000 and 2006–2007. Plasma HIV RNA levels were assessed by polymerase chain reaction assays in HIV-infected women using assays that had lower levels of detection of 80 copies per milliliter and total CD4+ and total CD8+ T-cell counts (cells per microliter) were determined in both HIV-infected and HIV-uninfected women by flow cytometry in laboratories participating in the DAIDS Quality Assurance Program at each study visit.37
In preliminary data analysis, we summarized selected demographic, behavioral, and clinical laboratory measurements by HIV serostatus and the presence of HCV viremia, at the time of specimen collection. Women who were enrolled in the T-cell substudy but lacked HCV serologic data (n = 6) or who were HCV seropositive but lacked HCV RNA data (n = 30) were excluded from the analysis. HCV viremia was defined based on the most recent HCV RNA data—for n = 63 women, the most recent HCV RNA test was in 2006–2007, whereas for n = 18 women, the most recent HCV RNA test was in 1997–2000. Usage of antiretroviral therapies and other medications, including medications used for the treatment of chronic HCV, are reported as a part of standard questionnaires at each semiannual WIHS visit. If a woman reported usage of an HCV medication at any time in the preceding 6 months, she was asked whether she had stopped taking this drug because the therapy was “successful.” Six HIV-positive women reported successful HCV antiviral therapy after their most recent HCV RNA test, and we considered the data set with and without inclusion of these women.
Multivariate linear regression models were used to examine associations between HCV viremia and the percentage of CD4+ and CD8+ T cells that were naive, CM, EM, or TE cells, the fraction that were Tregs, as well as the fraction defined as activated. Distributions that were not normally distributed, including percentage of CD4+ activated, CD8+ CM, and CD8+ activated T cells were log10 transformed before analysis. Analyses were conducted separately in HIV-positive and HIV-negative women. Specifically, in the HIV-negative women, we adjusted for age (continuous term), race/ethnicity (non-Hispanic black, Hispanic, non-Hispanic white/other), and other factors known to be associated with inflammation and immune dysregulation: cigarette smoking (current, former/never) and body mass index (BMI—thin/normal, overweight, obese). Only 3 HIV-negative women reported recent IDU, and we assessed the results both including and excluding these individuals.
In HIV-positive women, all multivariate models included adjustment for age, race/ethnicity, cigarette smoking, and BMI but additionally included current and/or nadir CD4+ T-cell counts (<350, 350–500, and >500), HIV RNA viral load (undetectable, lower limit of detection: 4000, 4001–20,000, 20,001–100,000, and >100,000 copies/mm3), HAART use (yes/no), and percent self-reported adherence with the prescribed HAART regimen (no HAART, <75% adherence, 75%–95%, >95%). Finally, because we hypothesized that associations between HCV viremia and T-cell phenotypes might differ by immune status in HIV-positive women, in a second set of models, we stratified by 2 different binary thresholds to define high vs. low total CD4+ count; that is, using a total CD4+ count of ≤500 or ≤350 CD4+ T cells per microliter.
Selected Characteristics of the Study Population
The data set included 158 HIV-negative and 464 HIV-positive women, of whom 18 and 63, respectively, were HCV viremic. Table 1 shows selected subject characteristics at the time of T-cell phenotyping. In both HIV-negative and HIV-positive groups, the HCV viremic women were older than HCV nonviremic women, and were more likely to have been recruited in 1994–1995 (the first WIHS recruitment period). Cigarette smoking, history of IDU and, among the HIV-positive women, recent IDU, and alcohol use, also differed by HCV viremia (all P < 0.05). Of the factors studied only in HIV positives, none differed significantly between those with and without HCV viremia, including current and nadir CD4+ T-cell count, HIV RNA viral load, and recent HAART use (Table 1). The median percentage of live lymphocytes observed in blood samples of study women was 98.4 and the interquartile range was 97.1–99.0.
Table 2 and Table S1 (see Supplemental Digital Content,http://links.lww.com/QAI/A557) show the relationship between HCV viremia in HIV-negative women and the percentage of naive, CM, EM, TE, and activated CD4+ and CD8+ T cells and Tregs. In both unadjusted analyses (Table 2 and Fig. 1) and in analyses adjusted for age, race/ethnicity, smoking, and BMI (Table 2), HCV viremic women had lower percentages of naive CD4+ cells (25% lower in adjusted analyses vs. the mean percentage (28.8%) in HCV nonviremics; P = 0.03) and higher percentages of EM CD4+ cells (33% higher in adjusted analyses vs. the mean percentage (38.7%) in HCV nonviremics; P = 0.0002) than women without HCV viremia. In addition, HCV viremia was associated with a significantly lower percentage of CM CD8+ T cells in multivariate models (37% lower vs. the mean percentage (0.46%) in HCV nonviremics; P = 0.02). Excluding the 3 women who reported recent IDU had no impact on the findings (data not shown).
Table 3 and Table S2 (see Supplemental Digital Content,http://links.lww.com/QAI/A557) show similar data in HIV-positive women. HCV viremia was significantly associated with higher percentages of activated CD4+ T cells and Tregs in both unadjusted analyses (Table 3 and Fig. 1) and in models adjusted for age, race/ethnicity, cigarette smoking, BMI, current and nadir CD4+ T-cell counts, HIV viral load, HAART use in the past 6 months, and adherence with the prescribed HAART regimen. For example, in adjusted analyses, activated CD4+ T cells were 26% higher [vs. the mean percentage (0.5%) in HCV nonviremics; P = 0.01], whereas Tregs were 17% higher [vs. the mean percentage (5.2%) in HCV nonviremics; P = 0.02] in women with vs. without HCV viremia. The percentage of activated CD8+ T cells was also marginally associated with HCV viremia in adjusted analysis (P = 0.06). These results were unaffected by additional adjustment for alcohol use, study site, and recruitment wave, or by exclusion of women who reported successful HCV antiviral therapy (data not shown).
Results Stratified by CD4+ T-Cell Count in HIV-Positive Women
To determine whether the associations of HCV viremia with T-cell differentiation and activation might vary by the level of immunosuppression in HIV-positive women, we conducted multivariate analyses stratified by current total CD4+ T-cell count. Among HIV-positive women with diminished immune status (whether defined as ≤500 or ≤350 CD4+ T cells/μL), HCV viremia was associated with an increased percentage of cells that were Tregs and the fraction of CD4+ cells that were activated (Table 4). The percentage of EM CD4+ cells was also significantly higher in women with vs. without HCV viremia with diminished immune status, regardless of whether immune status was defined as ≤500 CD4+ cells per microliter (14% higher EM CD4+ cells vs. the mean percentage (45.7%) in HCV nonviremics; P = 0.03) or as ≤350 CD4+ cells per microliter (18% higher EM CD4+ cells vs. the mean percentage (49.2%) in HCV nonviremics; P = 0.01). Conversely, no significant associations between HCV viremia and CD4+ T-cell phenotypes were seen in HIV-infected women with good immune status (defined as either >500 or >350 CD4+ cells/μL). CD8+ T-cell phenotype percentages were not associated with HCV viremia in any analyses in HIV-positive women. Exclusion of women who reported successful HCV antiviral therapy did not affect the findings, with the exception of associations of HCV viremia with percentage of CD4+ EM cells among women with total CD4+ ≤500 cells per microliter (P = 0.03 to P = 0.09) and with percentage of activated CD4+ cells among women with total CD4+ ≤350 cells per microliter (P = 0.05 to P = 0.15).
The relationship of HCV viremia with T-cell differentiation and activation phenotypes varied greatly by HIV status in this study. In HIV-negative women, we found that HCV viremia was significantly associated with lower percentage of naive CD4+ and higher percentage EM CD4+ T cells, as well as a lower percentage of CM CD8+ T cells. In contrast, among HIV positives, we observed only 1 of these 3 relationships, namely, a higher EM CD4+ percentage in those with HCV viremia. Furthermore, this relationship was limited to the subset of HIV positives with diminished immune status (eg, total CD4+ count ≤350 T cells/μL). In the women with diminished immune status, we additionally observed associations of HCV viremia with higher percentages of activated CD4+ T cells and Tregs, whereas among HIV-positive women with high CD4+ count, we found no significant associations between HCV viremia and T-cell phenotype percentages. Thus, the findings not only differed by HIV-serostatus but also by CD4+ count among HIV-positive women.
The low percentage of naive CD4+ T cells in HIV-negative women with versus without HCV viremia is consistent with data reported in all 3 previous studies that examined this relationship.10–12 However, this article is the first to involve a comparison group from the same low-income population with high rates of smoking and alcohol use as the HCV viremic individuals, instead of healthy controls from the general population. These factors can affect T-cell differentiation and activation and, therefore, the current data clarify the relationship between HCV viremia and low-naive CD4+ T-cell percentages.
Furthermore, the low percentage of naive CD4+ cells in HCV viremic HIV-negative women, in combination with the low percentage of CM CD8+ T cells (another early differentiation phenotype) but high percentage of EM CD4+ (a late differentiation phenotype), suggests that T-cell differentiation may be accelerated in the setting of chronic HCV infection. Although not all associations of HCV viremia with naive, CM and EM CD4+ and CD8+ T-cell phenotypes were statistically significant, the pattern of these associations was consistent with this interpretation; that is, accelerated T-cell differentiation in HCV-viremic individuals (Table 2). The lack of statistical significance may relate to the small number of women with HCV viremia among the HIV negatives.
If correct, this conclusion has important implications. Specifically, the reduction in naive and CM T cells would be expected to result in a reduced capacity to mount T-cell responses against novel and previously encountered antigens, including vaccine antigens. This could explain in part why HCV positives have lower response rates to hepatitis B virus vaccination.38 However, this hypothesis assumes that it is in fact individuals with naive T-cell lymphopenia that respond less well to hepatitis B virus vaccination, which would need to be verified.
Nonspecific (generalized) accelerated T-cell differentiation in HCV infection might also play a role in extrahepatic conditions that occur at higher rates in HCV positives, such as CVD. For example, a high percentage of EM CD4+ T cells was associated with higher levels of carotid atherosclerosis and with carotid artery disease in a recent study.39 Although these biologic relationships are not completely characterized, it is clear that understanding the origins of generalized immune differentiation and inflammation in CVD patients is a clinically relevant topic.40
We had hypothesized that associations of HCV viremia in HIV positives with high total CD4+ T cell count but not those with low total CD4+ count would be similar to associations in HIV negatives, but this was not the case. Instead, the associations of T-cell phenotypes with HCV viremia was limited to HIV positives with low CD4+, although 31 and 41 women with HCV viremia (of a total of 63 among HIV positives) were among those with >500 and >350 CD4+ T cells per microliter, respectively. Specifically, among HIV positives with diminished immune status, we observed a significant association of HCV viremia with high percentage of activated CD4+ T cells. A similar association was seen in a study of Scandinavian patients with HIV on stable HAART,18 although it was not observed by us in our previous studies in the WIHS cohort in the pre-HAART era.15,29 We also observed an association of HCV viremia with high Treg percentage in HIV positives with diminished immune status, a finding that is inconsistent with data from 2 previous studies.10,17 However, this study was much larger than these previous studies, and as mentioned above, ours was the first to study differences in associations by total CD4+ count.
We were also surprised by the absence of associations with CD8+ T-cell phenotypes in HIV positives because a significant association of HCV viremia with CD8+ T-cell activation was observed in our previous WIHS studies in the pre-HAART era15,29 and in the Scandinavian HAART users.18 Although a marginal association (P = 0.06) of HCV viremia with CD8+ activation was observed in this study of HIV positives, it is possible that HAART use attenuates the relationship between HCV viremia and CD8+ T-cell activation. The conflicting findings between the current investigation and the Scandinavian study could relate to differences in the definition of T-cell activation (CD38+ in the previous study18 vs. CD38+ and HLA-DR+ in the current investigation) or from population (eg, sex or racial) differences.
Several limitations to this study must be considered in the interpretation of these data. First, HCV viremia status was not ascertained at the same WIHS visit at which we measured T-cell phenotypes. Although all women who were categorized as HCV viremic had at least 2 positive HCV RNA tests during follow-up, it is possible that a small fraction of these women cleared HCV spontaneously after their most recent HCV RNA test, conducted either in 1997–2000 or in 2006–2007. Furthermore, although some WIHS women who were HCV seronegative at enrollment were tested for HCV antibody again a few years later, HCV antibody testing is not conducted at each WIHS visit. IDU declined dramatically among WIHS women after enrollment, but even if some women acquired new HCV infections after enrollment, or if a few women classified as having chronic HCV were actually HCV RNA negative at the T-cell phenotype testing visit, the results would still be biased toward rather than away from the null (ie, increasing the likelihood of false-negative but not false-positive findings). Second, liver function biomarkers had not been measured on all women at the time of analysis, and we were therefore unable to examine T-cell phenotypes in relation to liver disease. Finally, the current data are cross-sectional, and prospective investigations are therefore needed to verify that HCV infection is indeed antecedent to changes in T-cell phenotype percentages.
In conclusion, the data suggest that HCV viremia is associated with changes in the general distribution of T-cell phenotypes in circulation. The changes involved, however, differ by HIV status and by total CD4+ T-cell count in HIV positives. If correct, further efforts to define the T-cell changes that are not specific for HCV may help inform efforts to reduce extrahepatic disease among HCV-viremic individuals.
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hepatitis C virus; HIV; T cell; phenotype; activation; differentiation
Supplemental Digital Content
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