Abstract
We investigated the ability of CD8+ T cells to inhibit hepatitis C virus (HCV) replication in peripheral blood mononuclear cells (PBMCs). PBMCs isolated from 11 of 20 HCV-infected subjects had no detectable HCV RNA. Removal of CD8+ T cells from these PBMCs resulted in detection of HCV RNA, and depletion of CD8+ T cells from PBMCs that had detectable HCV RNA amplified HCV replication. Reconstitution of CD8− PBMCs with autologous CD8+ T cells led to inhibition of HCV replication. Interferon-γ produced by CD8+ T cells was partially responsible for CD8+ T cell–mediated noncytotoxic anti-HCV activity in PBMCs. This noncytotoxic anti-HCV activity was confirmed in HCV replicon cells. Supernatants from CD8+ T cell cultures inhibited HCV RNA expression in the replicon cells. These findings may have important implications for the immunopathogenesis of HCV in both immune and hepatic cells and are relevant to the development of host innate immunity–based anti-HCV interventions
CD8+ T cells play a major role in the innate immune response to viral infections. The majority of viral-specific cytotoxic T lymphocytes (CTLs) are CD8+ T cells that specifically recognize and lyse virus-infected cells [1–3 ]. CD8+ T cells also secrete antiviral cytokines that inhibit virus replication. CD8+ T cells release antiviral factors to inhibit HIV replication in CD4+ T cells [4–7 ]. Loss of CD8+ T cells or their functional impairment results in increased viremia and a rapid progression to AIDS in simian immunodeficiency virus infection [8]. CD8+ T cells inhibit hepatitis B virus (HBV) gene expression and replication in the liver of transgenic mice by producing antiviral cytokines that interrupt the HBV life cycle [9]. CD8+ T cells have direct antiviral activity against cytomegalovirus [10]. Functional loss of Epstein-Barr virus (EBV)–specific CD8+ T cells is associated with an increase in EBV load [11]. CD8+ T cells also play a critical role in the control of HCV infection. HCV-specific CTLs are present in the peripheral blood and liver of chronically infected subjects [12]. That most patients fail to resolve HCV infection suggests that the CD8+ T cell response is compromised. Subjects with chronic HCV infection have a relative paucity of circulating CD8+ T effector cells, which may contribute to HCV persistence in the liver [13]. Although CD8+ T cell–mediated killing of virus-infected target cells is the predominant mechanism of viral inhibition, noncytotoxic antiviral effects mediated by cytokines produced by CD8+ T cells are also critical for the clearance of HCV [14]. Among the cytokines produced by CD8+ T cells, interferon (IFN)–γ plays a significant role in the control of viral infections [15, 16]. IFN-γ inhibits the replication of subgenomic and genomic HCV RNA [17]. CD8+ T cells have an impaired capacity to produce IFN-γ in several persistent viral infections, including HCV infection [18, 19]
HCV is found not only in the liver, where viral replication is expected [20], but also in the serum and peripheral blood mononuclear cells (PBMCs) [21–24 ] of many patients with persistent HCV infection. Several lines of evidence indicate that PBMCs can serve as an extrahepatic site for HCV replication and may represent a reservoir for HCV. HCV negative-strand RNA (a viral replicative form) has been detected in PBMCs [24, 25], including monocytes/macrophages [26] and T and B cells [27]. Because PBCMs are a more readily available alternative to hepatic cells, PBMCs from chronically HCV-infected patients have been used as the primary target cells for in vitro HCV studies, although HCV replication in these cells occurs at a very low level [22]. In the present study, we investigated the role that CD8+ T cells play in the control of HCV replication in PBMCs from chronically HCV-infected patients
Subjects, Materials, and Methods
Subjects and isolation of immune cellsPeripheral blood samples were obtained from 20 HCV-infected adults who tested positive for both HCV antibody and plasma HCV RNA (table 1). The Institutional Review Board of the Children’s Hospital of Philadelphia approved this study, and informed consent was obtained from all subjects. PBMCs were isolated from blood by use of lymphocyte separation medium (Amersham Pharmacia Biotech), as described elsewhere [28]. PBMCs were then subjected to CD8+ or CD4+ T cell purification using magnetic-activated cell sorting CD8 or CD4 microbeads (Miltenyi Biotec) in accordance with the manufacturer’s instructions. The purified CD8+ T cells were activated by anti-CD3 antibody (1 μg/mL) for 72 h. After filtration through 0.22-μm-pore filters, aliquots of cell-free supernatants collected from the activated CD8+ T cell cultures were stored at −70°C as CD8+ T cell supernatant–conditioned medium (CD8+ CM). PBMCs or CD8− PBMCs were cultured in RPMI 1640 containing 10% fetal bovine serum and activated with 1% (vol/vol) phytohemagglutinin (Gibco) for 72 h in T-75 flasks. The activated cells were then cultured in medium containing interleukin (IL)–2 (10 U/mL) for 9 days. If the cells needed to be treated with CD8+ CM and/or antibodies against IFN-γ or IFN-γ receptors, CD8+ CM or antibodies were replaced every 3 days during the course of cultivation. Monocytes were isolated from PBMCs as described elsewhere [28]
Hepatitis C virus (HCV) RNA copies in plasma, peripheral blood mononuclear cells (PBMCs), and CD8− PBMCs from HCV-infected subjects
Hepatitis C virus (HCV) RNA copies in plasma, peripheral blood mononuclear cells (PBMCs), and CD8− PBMCs from HCV-infected subjects
ReagentsIL-2 was purchased from Roche Molecular Biochemical. Recombinant human IFN-γ and antibodies against CD3, IFN-γ, and IFN-γ receptors (IFN-γR1 and IFN-γR2) were obtained from R&D Systems. The monoclonal antibody against HCV NS5 was a gift from B. Sun (Thomas Jefferson University, Philadelphia, PA). Horseradish peroxidase–conjugated goat anti–mouse IgG antibody was purchased from Jackson ImmuneResearch Labs. ELISA kits for HCV antibody were purchased from Kehua Biotechnology. ELISA kits for IFN-γ were obtained from Endogen. ELISAs were performed in accordance with the manufacturer’s instructions
Cell linesJurkat (human T cell line), IM-9 (human B cell line), and U937 (promonocytic cell line) cells were obtained from the American Type Culture Collection. Huh.8 cells were obtained from C. Rice (Rockefeller University, New York, NY, and Apath, St. Louis, MO). Huh.8 cells contain a G418-selectable HCV RNA replicon with a wild-type HCV nonstructural NS5A protein sequence [29]. Using a real-time reverse-transcription polymerase chain reaction (RT-PCR) assay that we have recently developed [30], we were able to detect 2500–5000 copies of HCV mRNA/Huh.8 cell. FCA-1 cells were obtained from C. Seeger (Fox Chase Cancer Center, Philadelphia, PA). FCA-1 cells contain a subgenomic replicon from a known infectious HCV clone [31] that has several consensus mutations in NS3 as well as in NS5A (NS3 E177G; NS5A D1229E and I1299V) [31]. Huh.8 and FCA-1 cells were maintained as described elsewhere [32]. CD8+ CM had no cytotoxic effect on Huh.8 or FCA-1 cells, as assessed by trypan blue staining (data not shown). Cell viability was also measured by cell proliferation assay. In all cases, the limulus amebocyte lysate assay demonstrated that medium and reagents were endotoxin free
HCV RNA quantificationTotal RNA (1 μg) was extracted from PBMCs, CD8− PBMCs, Huh.8 cells, and FCA-1 cells using Tri-Reagent (Molecular Research Center), as described elsewhere [32, 33]. An HCV real-time RT-PCR assay that we have developed was used for the quantification of HCV RNA [30]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA was run in parallel for each sample, to normalize the level of HCV RNA, and CD8+ CM had no effect on GAPDH expression (data not shown). For detection of the 5′-noncoding region of HCV negative-strand RNA, we also adapted a strand-specific RT-PCR assay that was described elsewhere [25, 26, 34]
CocultureFor coculture experiments, 0.4-μm-pore transwell tissue culture plates (Costar) were used. Huh.8 and FCA-1 cells were incubated in the lower compartment, and CD8+ T cells were incubated in the upper compartment. Cells in the lower compartment were collected for RNA extraction and were subjected to HCV real-time RT-PCR 48 h after coculture
ImmunoassaysThe immunofluorescent and immunoblotting assays were performed as described elsewhere [33]
Statistical analysisWhere appropriate, data were expressed as the mean±SD. For comparison of the means of different groups (treated group vs. untreated controls), statistical analysis was performed using Student’s t test. Calculations were performed with Stata 8 statistical software (StataCorp). P<.05 was considered to be statistically significant
Results
Detection of HCV RNA in PBMCsWe first examined whether HCV RNA is detectable in PBMCs isolated from chronically HCV-infected subjects. PBMCs isolated from 11 of 20 HCV-infected subjects had no detectable HCV RNA. However, after we removed CD8+ T cells from these PBMCs, HCV RNA became readily detectable (table 1). In the other 9 PBMC samples that had detectable HCV RNA, the removal of CD8+ T cells led to a significant increase in HCV RNA expression (table 1). HCV RNA was detected in the plasma of all 20 subjects with HCV infection (table 1). There was no correlation between the levels of HCV RNA in plasma and those in PBMCs or CD8− PBMCs isolated from the same subjects (table 1)
Evidence of HCV replication in CD8− PBMCsTo confirm that HCV has the ability to replicate in PBMCs, we examined whether there was detectable HCV negative-strand RNA, a viral replicative intermediate, in PBMCs. Of the 9 PBMC samples that had detectable HCV positive-strand RNA (table 1), we selected 4 (from subjects 6, 7, 8, and 11) that had sufficient cells available for the experiments shown in figures 1, 2, and 7. None of the 4 PBMC samples had detectable HCV negative-strand RNA or HCV core RNA. Three of the 4 PBMC samples, however, tested positive for HCV negative-strand RNA after we removed CD8+ T cells (figure 1). In addition, HCV replication was evidenced by the presence of HCV core RNA in all 4 CD8− PBMC samples (figure 1)
Detection of hepatitis C virus (HCV) negative-strand RNA and core RNA in CD8− peripheral blood mononuclear cells (PBMCs). PBMCs and CD8− PBMCs isolated from 4 subjects with HCV infection were activated and cultured as described in Subjects, Materials, and Methods. Cells were collected for RNA extraction at day 9 after activation. HCV RNA levels were analyzed by strand-specific reverse-transcription polymerase chain reaction (RT-PCR) assay, for HCV negative-strand RNA expression, or by regular RT-PCR, for HCV core RNA expression
Detection of hepatitis C virus (HCV) negative-strand RNA and core RNA in CD8− peripheral blood mononuclear cells (PBMCs). PBMCs and CD8− PBMCs isolated from 4 subjects with HCV infection were activated and cultured as described in Subjects, Materials, and Methods. Cells were collected for RNA extraction at day 9 after activation. HCV RNA levels were analyzed by strand-specific reverse-transcription polymerase chain reaction (RT-PCR) assay, for HCV negative-strand RNA expression, or by regular RT-PCR, for HCV core RNA expression
Effect of CD8+ T cells on hepatitis C virus (HCV) replication in cultured peripheral blood mononuclear cells (PBMCs). PBMCs, CD8− PBMCs, and CD8+ T cells isolated from 4 subjects with HCV infection were activated and cultured as described in Subjects, Materials, and Methods. At day 3 after activation, CD8− PBMC cultures were reconstituted with autologous CD8+ T cells. HCV RNA levels were quantified by real-time reverse-transcription polymerase chain reaction at indicated time points during the course of cultivation
Effect of CD8+ T cells on hepatitis C virus (HCV) replication in cultured peripheral blood mononuclear cells (PBMCs). PBMCs, CD8− PBMCs, and CD8+ T cells isolated from 4 subjects with HCV infection were activated and cultured as described in Subjects, Materials, and Methods. At day 3 after activation, CD8− PBMC cultures were reconstituted with autologous CD8+ T cells. HCV RNA levels were quantified by real-time reverse-transcription polymerase chain reaction at indicated time points during the course of cultivation
Effect of CD8+ T cell–produced interferon (IFN)–γ on hepatitis C virus (HCV) replication. A IFN-γ production by purified CD8+ T cells. CD8+ T cell supernatants were prepared as described in Subjects, Materials, and Methods. IFN-γ produced by CD8+ T cells from 4 HCV-infected subjects was determined by ELISA. Results shown are the mean ± SD of triplicate samples. B Effect of antibodies (Abs) against IFN-γ or IFN-γ receptors (IFN-γR) on HCV replication in peripheral blood mononuclear cells (PBMCs). PBMCs from 4 HCV-infected subjects were incubated with or without Abs to IFN-γ (10 μg/mL) or IFN-γR (10 μg/mL IFN-γR1 and IFN-γR2). HCV RNA levels in PBMCs were determined at day 9 after Ab treatment. Results shown are the mean±SD of triplicate cultures. C Effect of Abs to IFN-γ or IFN-γR on mediated anti-HCV activity in HCV replicon cells. Huh.8 cells were cultured in CD8+ T cell supernatant–conditioned medium (CD8+ CM) preincubated with or without Ab against IFN-γ (10 μg/mL) for 1 h at 37°C. For the experiments using antibodies against IFN-γR, Huh.8 cells were incubated with or without Abs to IFN-γR (10 μg/mL IFN-γR1 and IFN-γR2) for 1 h before the addition of CD8+ CM. Mouse IgG2A and goat IgG were used as control Abs for Abs against IFN-γ and IFN-γR, respectively. HCV and glyceraldehyde-3-phosphate dehydrogenase RNA levels in Huh.8 cells were determined at 48 h after exposure to CD8+ CM. The data are expressed as HCV RNA levels in treated Huh.8 cells relative (% of inhibition) to that in untreated Huh.8 cells (defined as 0). The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments. +, present; −, absent
Effect of CD8+ T cell–produced interferon (IFN)–γ on hepatitis C virus (HCV) replication. A IFN-γ production by purified CD8+ T cells. CD8+ T cell supernatants were prepared as described in Subjects, Materials, and Methods. IFN-γ produced by CD8+ T cells from 4 HCV-infected subjects was determined by ELISA. Results shown are the mean ± SD of triplicate samples. B Effect of antibodies (Abs) against IFN-γ or IFN-γ receptors (IFN-γR) on HCV replication in peripheral blood mononuclear cells (PBMCs). PBMCs from 4 HCV-infected subjects were incubated with or without Abs to IFN-γ (10 μg/mL) or IFN-γR (10 μg/mL IFN-γR1 and IFN-γR2). HCV RNA levels in PBMCs were determined at day 9 after Ab treatment. Results shown are the mean±SD of triplicate cultures. C Effect of Abs to IFN-γ or IFN-γR on mediated anti-HCV activity in HCV replicon cells. Huh.8 cells were cultured in CD8+ T cell supernatant–conditioned medium (CD8+ CM) preincubated with or without Ab against IFN-γ (10 μg/mL) for 1 h at 37°C. For the experiments using antibodies against IFN-γR, Huh.8 cells were incubated with or without Abs to IFN-γR (10 μg/mL IFN-γR1 and IFN-γR2) for 1 h before the addition of CD8+ CM. Mouse IgG2A and goat IgG were used as control Abs for Abs against IFN-γ and IFN-γR, respectively. HCV and glyceraldehyde-3-phosphate dehydrogenase RNA levels in Huh.8 cells were determined at 48 h after exposure to CD8+ CM. The data are expressed as HCV RNA levels in treated Huh.8 cells relative (% of inhibition) to that in untreated Huh.8 cells (defined as 0). The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments. +, present; −, absent
Inhibition of HCV replication in cultured PBMCs by CD8+ T cells To determine whether CD8+ T cells have the ability to inhibit HCV replication in PBMCs, after activation for 3 days, PBMCs and CD8− PBMCs isolated from the same subjects were cultured in vitro for 9 days. The cells were collected at different time points during cultivation and were subjected to HCV real-time RT-PCR. The level of HCV RNA in cultured PBMCs isolated from all 4 subjects (subjects 6, 7, 8, and 11) was very low (figure 2). A significant level of HCV RNA, however, was observed in CD8− PBMCs isolated from all 4 subjects (figure 2). The subsequent reconstitution of CD8− PBMCs with autologous CD8+ T cells led to the inhibition of HCV replication (figure 2)
Release of noncytotoxic anti-HCV factors by CD8+ T cellsTo directly link the noncytotoxic action of CD8+ T cells with the inhibition of HCV RNA expression, we used recently developed HCV replicon cells. The HCV replicon system is the first effective cell model for the investigation of the dynamics of virus replication [29, 35, 36]. We used this cell model to investigate whether CD8+ T cells release anti-HCV soluble factors. HCV RNA expression was markedly inhibited (up to 90%) in Huh.8 and FCA-1 cells cocultured with CD8+ T cells (figure 3). The degree of inhibition was positively correlated with the number of CD8+ T cells added to the HCV replicon cell cultures (figure 3). The role that CD8+ T cell–released soluble factors play in controlling HCV RNA expression in HCV replicon cells was further demonstrated by the observation that CD8+ CM has anti-HCV activity. The anti-HCV effect of the CD8+ T cell–released factors was concentration dependent (figure 4). In addition, this anti-HCV activity of CD8+ T cell–released factors was specific, because the medium conditioned with supernatants from other immune cells, such as CD4+ T cells and macrophages, had little effect (<10% inhibition) on HCV RNA expression in HCV replicon cells (figure 5). Because HCV NS5 protein plays a critical role in HCV infection and replication, we examined NS5 protein expression in HCV replicon cells cultured with or without CD8+ CM. HCV NS5 protein expression was inhibited in cells treated with CD8+ T cell supernatants, as determined by immunofluorescent staining (figure 6A) and immunoblotting assay (figure 6B)
Effect of cocultivation of hepatitis C virus (HCV) replicon cells with CD8+ T cells on HCV replication. CD8+ T cells and Huh.8 (A) and FCA-1 (B) HCV replicon cells were separated by a transwell insert in a 24-well plate on which HCV replicon cells were cultured in the lower compartment and the indicated no. of CD8+ T cells was added in the upper compartment. After 48 h of coculture, total cellular RNA extracted from hepatic cell cultures was subjected to real-time reverse-transcription polymerase chain reaction for HCV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA quantification. The data are expressed as no. of HCV RNA copies per reaction and were normalized on the basis of the ratio of HCV RNA:GAPDH RNA amplified in the same plate. The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments
Effect of cocultivation of hepatitis C virus (HCV) replicon cells with CD8+ T cells on HCV replication. CD8+ T cells and Huh.8 (A) and FCA-1 (B) HCV replicon cells were separated by a transwell insert in a 24-well plate on which HCV replicon cells were cultured in the lower compartment and the indicated no. of CD8+ T cells was added in the upper compartment. After 48 h of coculture, total cellular RNA extracted from hepatic cell cultures was subjected to real-time reverse-transcription polymerase chain reaction for HCV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA quantification. The data are expressed as no. of HCV RNA copies per reaction and were normalized on the basis of the ratio of HCV RNA:GAPDH RNA amplified in the same plate. The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments
Effect of CD8+ T cell supernatant–conditioned medium (CD8+ CM) on hepatitis C virus (HCV) replication. Huh.8 (A) and FCA-1 (B) cells were cultured in the presence or absence of CD8+ CM at indicated concentrations for 48 h. Total cellular RNA extracted from replicon cells was subjected to real-time reverse-transcription polymerase chain reaction for HCV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA quantification. The data are expressed as no. of HCV RNA copies per reaction and were normalized on the basis of the ratio of HCV RNA:GAPDH RNA amplified in the same plate. The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments
Effect of CD8+ T cell supernatant–conditioned medium (CD8+ CM) on hepatitis C virus (HCV) replication. Huh.8 (A) and FCA-1 (B) cells were cultured in the presence or absence of CD8+ CM at indicated concentrations for 48 h. Total cellular RNA extracted from replicon cells was subjected to real-time reverse-transcription polymerase chain reaction for HCV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA quantification. The data are expressed as no. of HCV RNA copies per reaction and were normalized on the basis of the ratio of HCV RNA:GAPDH RNA amplified in the same plate. The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments
Comparison of anti–hepatitis C virus (HCV) activity of human immune cells. Huh.8 (A) and FCA-1 (B) cells were cultured in the presence or absence of medium conditioned with supernatants (Cell CM; 25% vol/vol) from different immune cell cultures as indicated for 48 h. Total cellular RNA extracted from HCV replicon cells was subjected to real-time reverse-transcription polymerase chain reaction for HCV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA quantification. The data are expressed as no. of HCV RNA copies per reaction and were normalized on the basis of the ratio of HCV RNA:GAPDH RNA amplified in the same plate. The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments. M/Φ, macrophages
Comparison of anti–hepatitis C virus (HCV) activity of human immune cells. Huh.8 (A) and FCA-1 (B) cells were cultured in the presence or absence of medium conditioned with supernatants (Cell CM; 25% vol/vol) from different immune cell cultures as indicated for 48 h. Total cellular RNA extracted from HCV replicon cells was subjected to real-time reverse-transcription polymerase chain reaction for HCV and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA quantification. The data are expressed as no. of HCV RNA copies per reaction and were normalized on the basis of the ratio of HCV RNA:GAPDH RNA amplified in the same plate. The results shown are the mean±SD of triplicate cultures and are representative of 3 separate experiments. M/Φ, macrophages
Effect of CD8+ T cell supernatant–conditioned medium (CD8+ CM) on hepatitis C virus (HCV) NS5 protein expression. A Immunofluorescence staining of NS5 protein. Huh.8 and FCA-1 cells were cultured on glass coverslips in the presence (c and d) or absence (a and b) of CD8+ CM (25% vol/vol). The cells were then fixed with 75% ice-cold acetone and were stained with a monoclonal antibody against NS5 72 h after treatment. B Immunoblotting analysis of NS5 protein. Huh.8 and FCA-1 cells were plated on a 24-well plate and were cultured with or without CD8+ CM. The cells were then subjected to protein extraction at 72 h after treatment. Total protein (10 μg) from Huh.8 and FCA-1 cells was transferred to a nitrocellulose membrane for immunoblotting analysis using anti-HCV NS5 monoclonal antibody. A representative result is shown
Effect of CD8+ T cell supernatant–conditioned medium (CD8+ CM) on hepatitis C virus (HCV) NS5 protein expression. A Immunofluorescence staining of NS5 protein. Huh.8 and FCA-1 cells were cultured on glass coverslips in the presence (c and d) or absence (a and b) of CD8+ CM (25% vol/vol). The cells were then fixed with 75% ice-cold acetone and were stained with a monoclonal antibody against NS5 72 h after treatment. B Immunoblotting analysis of NS5 protein. Huh.8 and FCA-1 cells were plated on a 24-well plate and were cultured with or without CD8+ CM. The cells were then subjected to protein extraction at 72 h after treatment. Total protein (10 μg) from Huh.8 and FCA-1 cells was transferred to a nitrocellulose membrane for immunoblotting analysis using anti-HCV NS5 monoclonal antibody. A representative result is shown
IFN-γ and CD8+ T cell actionTo identify the soluble anti-HCV factors released by CD8+ T cells, we examined several soluble factors that are related to the antiviral ability of CD8+ T cells. Antibodies against tumor necrosis factor (TNF)–α, IL-1, IL-6, and β-chemokines (macrophage inflammatory protein [MIP]–1α, MIP-1β, and RANTES) failed to neutralize CD8+ CM–mediated anti-HCV ability (data not shown). Because IFN-γ, a primary antiviral cytokine produced by CD8+ T cells, has the ability to inhibit the replication of subgenomic and genomic HCV RNA [17, 37], we determined whether IFN-γ is responsible for CD8+ T cell–mediated anti-HCV activity. We first examined whether CD8+ T cells isolated from HCV-infected subjects produce IFN-γ. We showed that these CD8+ T cells produce IFN-γ at the nanogram level (figure 7A). To directly link CD8+ T cell–produced IFN-γ with the anti-HCV activity of CD8+ T cells, we used neutralizing antibodies against IFN-γ or IFN-γR1 and IFN-γR2 to treat PBMCs isolated from HCV-infected subjects. HCV replication was dramatically enhanced, by 4–30-fold, in PBMCs treated with antibodies (figure 7B). In addition, antibody against IFN-γ partially neutralized CD8+ CM–mediated anti-HCV activity (85% inhibition of HCV RNA expression was reduced to 30% inhibition of HCV RNA expression) in HCV replicon cells (figure 7C). The contribution of IFN-γ to the anti-HCV activity of CD8+ T cells was confirmed in experiments using antibodies against IFN-γR1 and IFN-γR2 that block IFN-γ binding to its receptors on HCV replicon cells. Preincubation of replicon cells with these antibodies reduced the anti-HCV activity of CD8+ T cell supernatants by 60% (figure 7C)
Discussion
The results of the present study demonstrate that CD8+ T cells release noncytotoxic soluble factors that inhibit HCV replication in PBMCs. Our data support the notion that HCV infects not only hepatic cells but also PBMCs, an extrahepatic target for HCV infection [38, 39]. Both HCV positive-strand and negative-strand RNA were present in PBMCs [40]. The presence of the replicative form (negative-strand RNA) of HCV in lymph nodes and PBMCs [26] suggests that immune cells are a possible HCV reservoir. We showed that PBMCs isolated from 11 of 20 HCV-infected subjects had no detectable HCV RNA (table 1) and that removal of CD8+ T cells from these PBMC samples led to the detection of HCV RNA (table 1). In the PBMC samples that had detectable HCV RNA, the depletion of CD8+ T cells amplified HCV RNA expression (table 1). In addition, significant HCV replication was observed in CD8− PBMCs during in vitro cultivation, which was evidenced by the detection of negative-strand RNA (figure 1) and the appearance of an HCV replication curve (figure 2). These data provide direct evidence that PBMCs are a replication site for HCV. Most importantly, we have demonstrated that CD8+ T cells play a key role in the control of HCV replication in PBMCs, a possible HCV reservoir
Cellular immune responses, particularly those mediated by CD8+ T cells, are important in the immunopathogenesis and control of HCV infection [41–43 ]. In acute HCV infection, a strong CD8+ T cell response is associated with spontaneous viral clearance [43, 44], whereas, in chronic infection, the role of CD8+ T cells is not defined [45]. Although direct killing of virus-infected cells by antigen-specific CTLs is the primary mechanism of virus inhibition, noncytotoxic soluble factors released by CD8+ T cells also play an important role. In HIV infection, the soluble factors secreted by CD8+ T cells from HIV-positive and HIV-negative subjects are potent inhibitors of HIV replication [4, 46–48 ]. These factors, although they have not yet been identified, are designated by the term “CD8+ T lymphocyte antiviral factor” (CAF) [4, 5, 49]. CAF-associated antiviral activities are correlated with delayed disease progression in HIV-infected subjects [50–52 ]. Several lines of evidence from both in vitro and ex vivo studies have indicated that CAF is an innate, rather than an acquired, immune response [53–56 ]. We examined the possibility that CD8+ T cells produce noncytotoxic anti-HCV soluble factors and observed that CD8+ T cells isolated from HCV-infected subjects release soluble factors that powerfully inhibit HCV replication. Our results demonstrate that (1) removal of CD8+ T cells from PBMCs led to HCV RNA detection (table 1 and figure 1) and the amplification of viral replication (figure 2); (2) inhibition of HCV RNA expression was restored after autologous CD8+ T cells were replaced in CD8− PBMC cultures (figure 2); and (3) coculture with CD8+ T cells (figure 3) and CD8+ CM (figure 4) inhibited HCV RNA expression in HCV replicon cells. In addition, the anti-HCV effect of the CD8+ T cell–released soluble factors is highly specific, because medium conditioned with supernatants from other immune cells (CD4+ T cells, macrophages, and Jurkat, IM-9, and U937 cells) had little effect on HCV RNA expression in replicon cells. These data also rule out the possibility that the inhibitory effect of CD8+ CM was due to a general metabolic effect as a result of decreased nutrients for the replicon cells
To identify the noncytotoxic anti-HCV factors released by CD8+ T cells, we examined several soluble factors that have antiviral potential in the HCV replicon system. Among CD8+ T cell–produced cytokines (IL-1, IL-6, TNF-α, and β-chemokines), IFN-γ is the only one partially responsible for CD8+ T cell–mediated noncytotoxic anti-HCV activity (data not shown) (figure 7). The anti-HCV ability of recombinant IFN-γ has been documented in HCV replicon systems [17]. In the experiments with PBMCs from HCV-infected subjects, we demonstrated that PBMCs treated with antibodies against IFN-γ or IFN-γ receptors had increased levels of HCV RNA (figure 7B). In addition, antibodies against IFN-γ or IFN-γ receptors partially neutralized the anti-HCV activity of CD8+ T cells in the replicon system (figure 7C). These data suggest that IFN-γ released by CD8+ T cells plays an important role in CD8+ T cell activity. Liu et al. [37] reported that the CD8+ antiviral effect in HCV replicon cells was markedly reduced by blocking IFN-γ but was unaffected by blocking TNF-α. These data highlight the critical role that CD8+ T cell–produced IFN-γ plays in the control of HCV replication. An important question is why CD8+ T cells are unable to produce sufficient amounts of IFN-γ to eradicate HCV in vivo. It has been suggested that CD8+ T cells are “stunned” and therefore unable to produce IFN-γ during the peak of viremia [57, 58]. HCV-specific CD8+ T cells from persistently HCV-infected subjects do not significantly expand and do not produce IFN-γ in response to HCV peptides [59]. The majority of patients who acquire HCV progress to chronic infection, suggesting that HCV-induced cellular immunity is insufficient to eliminate the virus. Furthermore, because IFN-γ is only partially responsible for the noncytotoxic anti-HCV activity of CD8+ T cells, other unknown soluble factors are also probably responsible for this activity. It is possible that multiple soluble factors are involved in CD8+ T cell–mediated anti-HCV activity
Our findings are relevant to the understanding of CD8+ T cell–mediated host defense mechanisms against HCV infection. CD8+ T cells capable of secreting soluble anti-HCV factors such as IFN-γ may provide protection against both HCV infection and HCV disease progression. CD8+ T cell–produced noncytotoxic anti-HCV factors have the ability to eradicate viruses from infected cells without lysing them, which is an attractive strategy to protect the liver from excessive immune-mediated damage. The identification of these unknown soluble factors and an understanding of the interaction between HCV and these factors may lead to the development of novel antiviral agents as well as a better understanding of the determinants of HCV disease progression. Thus, the delineation of the precise mechanism of the noncytotoxic anti-HCV activity of CD8+ T cells in chronic HCV infection should be a main focus of future studies, to facilitate the development of host innate immunity–based anti-HCV interventions
Acknowledgment
We acknowledge Stephen Jasionowski, for his excellent editorial work on this article
