Potential conflict of interest: Nothing to report.
Hepatitis C virus (HCV) has been reported to elicit B and T cell immunity in infected patients. Despite the presence of antiviral immunity, many patients develop chronic infections leading to cirrhosis, hepatocellular carcinoma, and liver failure that can require transplantation. We have previously described the presence of HLA-A2–restricted, HCV NS3–reactive cytotoxic T lymphocytes (CTL) in the blood of HLA-A2− liver transplantation patients that received an HLA-A2+ liver allograft. These T cells are analogous to the “allospecific” T cells that have been described in hematopoietic stem cell transplantation patients. It has been speculated that allospecific T cells express high-affinity T cell receptors (TCRs). To determine if our HCV-reactive T cells expressed TCRs with relatively high affinity for antigen, we identified and cloned a TCR from an allospecific HLA-A2–restricted, HCV:NS3:1406-1415–reactive CD8+ T cell clone and expressed this HCV TCR in Jurkat cells. Tetramer binding to HCV TCR–transduced Jurkat cells required CD8 expression, whereas antigen recognition did not. In conclusion, based on the reactivity of the TCR-transduced Jurkat cells, we have identified a TCR that transfers anti-HCV reactivity to alternate effectors. These data suggest this high affinity HCV-specific TCR might have potential new immunotherapic implications. (HEPATOLOGY 2006;43:973–981.)
Chronic infection with hepatitis C virus (HCV) is a major cause of morbidity and mortality. It is estimated that over 3% of the worldwide population harbors chronic HCV infections, which can lead to cirrhosis and hepatocellular carcinoma in a significant proportion of patients.1, 2 HCV-related disease is the leading indication for liver transplantation worldwide.3, 4 Currently, the best medical therapy for HCV infection is pegylated interferon-α in combination with ribavirin.5 However, this treatment is effective in only half of chronically infected patients and has significant side effects.6 For those patients who fail antiviral therapy, there is no effective treatment, and these patients remain at risk for disease progression.
HCV is known to elicit both humoral and cellular immunity in humans.7 Despite the fact that HCV-reactive T cells have been isolated that recognize more than 50 known antigenic epitopes from HCV,8 the majority of patients exposed to HCV develop chronic infection.9 In some cases, patients develop immunity but are unable to clear the virus.10 HCV T cell epitopes have been shown to rapidly mutate, leading to antigen escape variants.11–13 These factors have made the development of preventive and therapeutic vaccines exceedingly difficult.14
We recently described the presence of HCV-reactive T cells in the peripheral blood of an HLA-A2− patient who received an HLA-A2+ liver allograft.15 These T cells are similar in nature to the “allospecific” T cells that have been described in hematopoietic stem cell transplantation patients.16 These allospecific T cells were likely normal T cells restricted by the donor HLA that cross-reacted with HLA-A2/HCV NS3:1406-1415 complexes, and their posttransplantation expansion was probably primed by the liver allograft.15
We and others have reported that retroviral vectors containing T cell receptor (TCR) genes can be used to redirect the reactivity of T cell lymphomas and normal T cells to recognize tumor cells (reviewed by Kaplan et al.17). Furthermore, if the TCR has sufficient affinity, it is possible to engineer CD8− effectors to recognize tumor cells.18–20 Therefore, we sought to determine if the TCRs from these HCV reactive T cells could transfer recognition of HCV+ cells to other effector cells and if CD8 expression was required for antigen recognition. The TCR genes from an HLA-A2–restricted, HCV NS3:1406-1415–reactive T cell clone were identified and cloned. This TCR bound HLA-A2/HCV NS3:1406-1415 tetramers when expressed in CD8+ but not CD8− cells, indicating that tetramer binding required CD8 expression. However, both CD8+ and CD8− HCV TCR–transduced Jurkat cells secreted interleukin 2 when stimulated with peptide-loaded targets or HCV+ cell lines, indicating that activation secretion did not require CD8 expression. Therefore, we concluded that the requirement of CD8 for tetramer staining was distinct from the requirement of CD8 for T cell activation. The ability to transfer anti-HCV reactivity to alternate effectors makes this HCV TCR gene transfer approach a potential new treatment for patients with chronic HCV infection or HCV-related malignancies.
HCV, hepatitis C virus; TCR, T cell receptor; MEL, melanoma; RCC, renal cell carcinoma; CMV, cytomegalovirus; IL, interleukin; cDNA, complementary DNA; AC, α constant; PCR, polymerase chain reaction; BV, β chain V; MHC, major histocompatiblity complex.
Materials and Methods
Jurkat, SupT1, and T2 cell lines were obtained from the American Type Culture Collection (Rockford, MD). Melanoma (MEL) cell lines were established from surgical specimens obtained from melanoma patients undergoing immunotherapy at the Surgery Branch of the National Cancer Institute.21, 22 Renal cell carcinoma (RCC) lines were obtained from patients undergoing radical nephrectomy at the Surgery Branch of the National Cancer Institute.23 All medium components were obtained from Mediatech (Herndon, VA) unless otherwise noted. MEL 624 (HLA-A2+), MEL 624-28 (HLA-A2−), RCC UOK131 (HLA-A2+), RCC 1764 (HLA-A2−), Jurkat, SupT1, and T2 cell lines were maintained in complete medium consisting of RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Carlsbad, CA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2.92 mg/mL glutamine. 293GP cells were maintained in DMEM supplemented as described above.
Tumor cell lines that have been engineered to express the HCV NS3:1406-1415 and CMV pp65:495-503 epitopes have been described elsewhere.15, 24 Briefly, retroviral vectors containing minigenes encoding the HCV NS3:1406-1415 or CMV pp65:495-503 epitopes were used to transduce HLA-A2+ and HLA-A2− MEL (MEL 624 and MEL 624-28) and RCC (RCC UOK131 and RCC 1764) lines. Cells were maintained in RPMI medium as described above supplemented with 500 μg/mL G418 (Research Products International, Mount Prospect, IL).
The isolation and characterization of HCV-reactive T cell clones have been previously described.15 Briefly, four HCV-reactive CD8+ T cell clones (designated HCV clones 1-4) were isolated from the peripheral blood mononuclear cells of an HLA-A2− recipient of an HLA-A2+ donor liver allograft. CMV-reactive T cell clones were isolated from the peripheral blood mononuclear cells of normal donors as follows: peripheral blood mononuclear cells from healthy donors were plated into 24-well flat-bottom tissue culture plates at a density of 3 × 106 cells per well in 2 mL AIM V medium (Invitrogen) supplemented with 10% heat-inactivated pooled human AB serum (Valley Biomedical, Winchester, VA), 100 U/mL penicillin, 100 μg/mL streptomycin, 2.92 mg/mL L-glutamine, 300 IU/mL recombinant human interleukin (IL)-2 (Chiron, Emeryville, CA), 10 μg/mL peptide CMV pp65:495-503, 5 μg/mL keyhole limpet hemocyanin (Sigma-Aldrich, St. Louis, MO), and 10 ng/mL recombinant human IL-7 (Biosource International, Camarillo, CA). The bulk culture was cloned in limiting dilution, and 13 CMV-reactive T cell clones (designated CMV1-13) were isolated. All T cell clones were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated pooled human AB serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 2.92 mg/mL glutamine, and 300 IU/mL recombinant human IL-2 in a 5% CO2 humidified incubator at 37°C. T cell clones were expanded using 30 ng/mL anti-CD3 monoclonal antibody (Ortho Biotech, Raritan, NJ) and 300 IU/mL IL-2 in the presence of irradiated pooled allogeneic peripheral blood mononuclear cells as feeders as previously described.22
HCV NS3:1406-1415 (KLVALGINAV), CMV pp65:495-503 (NLVPMVATV) and tyrosinase:368-376 (YMDGTMSQV) were obtained from Synthetic Biomolecules (San Diego, CA).
TCR α and β Chain Identification.
The TCR α chain from the four HCV-reactive T cell clones was identified as previously described.25, 26 Briefly, total RNA was isolated from 1 to 5 million cells using TRIzol (Invitrogen), and TCR complementary DNAs (cDNAs) were amplified using the 5′ RACE (Rapid Amplification of cDNA Ends) system (Invitrogen) using an α constant (AC) region reverse primer. Polymerase chain reaction (PCR) products were cloned and sequenced, and two productively rearranged α chains (AV38s2 and AV41s1) were identified (Fig. 1). Both full-length α chains were amplified from cDNA using AV forward (AV38s2 forward 5′-AAAGTCGACCTGTGAGCATGGCATGCCCTGGCTTCCTG-3′; AV41s1 forward 5′-AAAGTCGACTAATAATGGTGAAGATCCGGCAATTT-3′) and AC reverse (5′-AAAGTCGACCCTCAGCTGGACCACAGCCGCAGCGTCATGAGCAGA-3′) primers containing Sal I restriction sites for subsequent subcloning. PCR products were ligated into the pCR 2.1 TA cloning vector (Invitrogen), and transformed into Escherichia coli TOP 10 competent cells (Invitrogen). Bacterial clones were screened for presence of the α chain cDNA via PCR and were sequenced to ensure that no errors had occurred during PCR amplification.
The TCR β chain from the four HCV-reactive T cell clones was identified via reverse-transcriptase PCR using a panel of TCR β chain V region (BV) degenerate subfamily specific primers as previously described.23 Briefly, total RNA was isolated from 1 to 5 million cells using TRIzol. First-strand cDNA was synthesized from 1 μg of total RNA using Superscript II reverse-transcriptase and oligo (dT)12-18 (Invitrogen). Ten nanograms of cDNA was PCR-amplified in a 50-μL reaction consisting of 1× PCR buffer, 1.5 mmol/L MgCl2, 200 μmol/L dNTP, 400 nmol/L TCR BV subfamily-specific forward primer, 400 nmol/L TCR β chain C region–specific reverse primer, and 1 U Taq DNA polymerase (all PCR reagents obtained from Invitrogen). A BV11 band obtained from all four HCV-reactive T cell clones was cloned, sequenced, and identified as BV11s1 based on known genomic DNA sequences. The full-length β chain was amplified from cDNA using forward and reverse primers containing Xho I restriction sites (forward 5′-AAACTCGAGCCCCAACTGTGCCATGACTATCAGGCT-3′; reverse 5′-AAACTCGAGCTAGCCTCTGGAATCCTTTCTCTTGACCATTGCCAT-3′), ligated into the pCR 2.1 TA cloning vector, and transformed into Escherichia coli TOP 10 competent cells. Bacterial clones were screened for presence of the β chain gene, and recombinant clones were sequenced to ensure that no errors had occurred during PCR amplification.
Full-length CD8 α and β chains were amplified via reverse-transcriptase PCR from human T cell cDNA. The cloning primers used to amplify the CD8 α (forward 5′-AAACTCGAGCGCGTCATGGCCTTACCAGTGACCG-3′; reverse 5′-AAACTCGAGTTAGACGTATCTCGCCGAAAG-3′) and β (forward 5′-AAAGTCGACGCCACGATGCGGCCGCGGCTGTGGCT-3′; reverse 5′-GTCGACAATAAACACTTCAACAAAGCACTC-3′) chains contained Xho I or Sal I restriction sites, respectively, for subsequent subcloning. PCR products were ligated into the PCR 2.1 TA cloning vector and transformed into Escherichia coli TOP 10 competent cells. Bacterial clones were screened for presence of the full-length CD8 α or α chain genes, and recombinant clones were sequenced to ensure that no errors had occurred during PCR amplification.
Retroviral Vector Construction.
The SAMEN CMV/SRα retroviral vector has been previously described18 and was used as the backbone for all retroviral constructs. The HCV TCR α and β chain genes and the CD8 TCR α and β chain genes were inserted into the Xho I and Sal I restriction sites, respectively, of the retrovirus using a rapid ligation strategy as previously described18 to create three retroviral constructs (Fig. 2). One retrovirus contained the AV38s2 α chain and the BV11s1 β chain (designated HCV TCR). A second retrovirus contained the AV41s1 α chain and the BV11s1 β chain (designated Alt TCR). A third retrovirus contained the CD8 α and β chains.
Retroviral supernatants were prepared using a transient transfection protocol as described.18 Briefly, 100-cm2 tissue culture dishes were coated with 0.02% type B bovine skin gelatin (Sigma-Aldrich) in Hank's basic salt solution for 15 minutes at room temperature. 293GP cells were plated at sufficient density to provide 60% to 70% confluence after 24 hours. Cells were transiently cotransfected with 3 μg retroviral vector and 3 μg plasmid containing the vesicular stomatitis virus envelope gene using Lipofectamine and Plus reagents (Invitrogen). Transfection medium was replaced with complete medium, and retroviral supernatants were collected after 24 and 48 hours.
Jurkat and SupT1 cells were transduced by spinoculation as described.27 Briefly, cells were resuspended at 1 × 106 cells/mL in retroviral supernatant supplemented with 8 μg/mL polybrene (Sigma-Aldrich). Cells were added to 24-well flat-bottom tissue culture plates (1 mL/well), and the plates were centrifuged at 1,000g for 90 minutes at 32°C. Cells were resuspended following spinoculation, incubated for 4 hours at 37°C, and then 1 mL fresh complete medium was added to each well. This spinoculation procedure was repeated the next day using fresh retroviral supernatant. After 24 hours, transduced cells were selected by adding G418 to each culture (2 mg/ml for Jurkat cells and 2.5 mg/mL for SupT1 cells).
Cytokine Release Assays.
Antigen reactivity by the HCV-reactive T cell clones and TCR-transduced Jurkat cells was measured in cytokine release assays as described.28 Briefly, 1 × 105 responder and stimulator cells were cocultured in a 1:1 ratio in 96-well U-bottom tissue culture plates in 200 μL complete medium. For the Jurkat experiments, 10 ng/mL PMA (Sigma-Aldrich) was added to each well. As a positive control for Jurkat stimulation, maximal cytokine release was obtained by the addition of 1 μg/mL ionomycin (Sigma-Aldrich). Cocultures were incubated at 37°C for 20 hours, and then supernatants were harvested. The amount of cytokine released was measured via ELISA using monoclonal antibodies to interferon-γ (Pierce, Rockford, IL) or IL-2 (R&D Systems, Minneapolis, MN).
T2 cells were loaded with peptide by incubating 1 × 106 cells/mL in complete medium containing varying concentrations of peptide at 37°C for 2 hours. Peptide-loaded T2 cells were washed with fresh complete medium before coculture with responders.
The cell surface expression of the TCR and other T cell markers was measured via immunofluorescence staining and quantified via flow cytometry as described.24 The following antibodies were used: anti–CD3-PE, anti–CD8-FITC, anti–TCR αβ-PE (BD Biosciences, San Diego, CA), and anti–TCR Vβ11-FITC (Beckman Coulter, Brea, CA). The following PE-labeled HLA-A*0201 tetramers were used: HCV NS3:1406-1415 (KLVALGINAV) and CMV pp65:495-503 (NLVPMVATV) (Beckman Coulter). Flow cytometry was performed using a FACScan flow cytometer (BD Biosciences), and data were analyzed with the CellQuest program (BD Biosciences).
Identification of HCV TCR.
We have previously described four HLA-A2–restricted, HCV NS3:1406-1415–reactive T cell clones that could secrete interferon-α when stimulated with peptide-loaded T2 cells and HCV+ cell lines.15 TCR analysis indicated that all four of the HCV-reactive T cell clones expressed a single Vβ chain (BV11s1) and two Vα chains (AV38s2 and AV41s1). DNA sequence analysis revealed that all four T cell clones used the same Jα (AJ30 and AJ49) and Dβ/Jβ (BD2s1/BJ2s7) segments and had identical sequences across the CDR3 region, indicating they were sister clones (Fig. 1). Given the source of these HCV-reactive T cells,15 their frequency would be expected to be quite low. Thus, the finding that they were sister clones was not surprising.
Cell Surface Expression of HCV TCR.
The presence of two TCR α chains in these T cell clones necessitated constructing two retroviral vectors to determine which TCR mediated HCV NS3 antigen recognition (Fig. 2). The TCR β chain from HCV clone 3 was first inserted in the upstream cloning site of SAMEN CMV/SRα under the transcriptional control of the MMLV LTR. Then, each of the HCV clone 3 TCR α chains were inserted into the downstream cloning site of SAMEN CMV/SRα under the transcriptional control of the SRα promoter. The resulting retroviral vectors (AV38s2/BV11s1 and AV41s1/BV11s1) were used to transduce SupT1 cells. SupT1 cells are a CD4+/CD8+ human T cell lymphoma cell line that does not naturally express CD3 (Fig. 3A,C) or TCR αβ (Fig. 3B,D) that we use to validate the expression of a cloned TCR. SupT1 cells transduced with either TCR restored CD3 (Fig. 3A,C) and TCR αβ (Fig. 3B,D) expression, indicating both forms of the HCV clone 3 TCR are capable of forming stable TCR/CD3 complexes on the surface of T cells.
Because the parent HCV T cell clones stain with HLA-A*0201/HCV NS3:1406-1415 tetramers,15 SupT1 cells transduced with the AV38s2/BV11s1 and AV41s1/BV11s1 forms of the HCV TCR were stained with HLA-A2/HCV NS3:1406-1415 tetramers to determine which TCR α chain mediated antigen recognition. HCV clone 3 T cells exhibited uniform and high levels of staining with the HLA-A*0201/HCV NS3:1406-1415 tetramers (Fig. 4A; MFI 526). In contrast, a bulk population of SupT1 cells expressing the AV38s2/BV11s1 TCR had heterogeneous levels of tetramer staining (MFI 167) (Fig. 4B). This heterogeneous tetramer staining was the result of variability in the expression of the introduced TCR from cell to cell. Tetramer staining was specific, because AV38s2/BV11s1 TCR transduced SupT1 cells did not stain with an irrelevant tetramer (HLA-A*0201/CMV pp65:495-503) (Fig. 4D), and SupT1 cells expressing a different TCR (TIL 1383I TCR) did not stain with the HLA-A*0201/HCV NS3:1406-1415 tetramer (Fig. 4E). SupT1 cells transduced to express the HCV TCR AV41s1/BV11s1 did not stain with the HLA-A*0201/HCV NS3:1406-1415 tetramers (Fig. 4C). Based on tetramer binding, we concluded that the AV38s2/BV11s1 TCR mediated HCV antigen recognition and the AV41s1 α chain represented a second α chain rearrangement with unknown antigen reactivity.
Peptide Recognition by HCV TCR–Transduced Jurkat Cells.
The binding of peptide/major histocompatiblity complex (MHC) tetramers to TCR-transduced cells was insufficient to convincingly demonstrate that the AV38s2/BV11s1 TCR mediated HCV NS3:1406-1415 recognition. Given that SupT1 cells do not secrete cytokines when stimulated by antigen, nor do they mediate lysis of antigen-expressing targets, we transduced Jurkat cells to verify the function of the AV38s2/BV11s1 HCV TCR. Jurkat cells are a CD8− human T cell lymphoma line that expresses its native TCR, meaning that any introduced TCR would have to compete with the endogenous TCR. Furthermore, Jurkat cells expressing a foreign TCR secrete IL-2 upon antigen stimulation in an antigen-specific fashion.19, 28 Therefore, Jurkat cells represent a model T cell that can be used to evaluate the function of any cloned TCR.
Jurkat cells transduced with the HCV TCR secreted significant quantities of IL-2 when stimulated with T2 cells loaded with HCV NS3:1406-1415 peptide (Fig. 5). These cells were considered to be HCV-reactive because HCV TCR–transduced Jurkat cells did not recognize T2 cells alone or T2 cells loaded with irrelevant peptide (CMV pp65:495-503 or tyrosinase:368-376). As a control, Jurkat cells transduced with a TCR that mediates HLA-A2–restricted recognition of tyrosinase (TIL 1383I TCR)18 only secreted IL-2 when stimulated with T2 cells loaded with tyrosinase:368-376 and not HCV NS3:1406-1415 or CMV pp65:495-503. Therefore, HCV NS3:1406-1415 peptide recognition was mediated by the AV38s2/BV11s1 TCR cloned from HCV clone 3 cells.
Relative Avidity of HCV TCR–Transduced Jurkat Cells.
It has been reported that one critical feature of viral-reactive29 and tumor-reactive30, 31 T cells that influences recognition of processed antigen is relative avidity. Relative avidity is defined as the relative sensitivity of the T cell to antigen stimulation as measured by the minimum amount of peptide required to elicit significant cytokine release (at least 100 pg/mL and twice background) and/or the amount of peptide required to elicit half maximum cytokine production. To determine the contribution of the HCV TCR to relative avidity, the parental HCV-reactive T cell clones and the TCR-transduced Jurkat cells were stimulated with T2 cells loaded with decreasing amounts of HCV NS3:1406-1415 peptide, and the amount of cytokine released was measured via ELISA (Fig. 6). The HCV T cell clones secreted significant quantities of interferon-γ (>100 pg/mL and at least twice background) when stimulated with T2 cells loaded with less than 1 ng/mL concentrations of peptide and required that T2 cells were loaded with between 1 and 10 ng/mL peptide to elicit half the maximum production of interferon-γ (Fig. 6A). In contrast, the HCV TCR–transduced Jurkat cells required T2 cells to be loaded with 10 ng/mL or greater concentrations of peptide to elicit significant (>100 pg/mL and at least twice background) IL-2 release (Fig. 6B). The half-maximum response was between 20 and 30 ng/mL regardless of the number of HCV TCR–transduced Jurkat cells used in the assays. Both the relative avidity and half-maximum response for the HCV TCR–transduced Jurkat cells were at least 10-fold lower than that of the parent T cell clones. Given that these comparisons were made between T cell clones secreting interferon-γ and T lymphoma cells secreting IL-2, we expected TCR-transduced Jurkat cells to exhibit lower relative avidity compared with T cell clone cells. TCR-transduced Jurkat cells probably express lower levels of HCV TCR relative to the parent T cell clone because of competition between the exogenous and endogenous TCR chains in Jurkat cells, which does not exist in normal T cells. As a result, the differences in relative avidity were not surprising and were consistent with results we have obtained with other TCRs.19, 28
Recognition of Processed Antigen by HCV TCR–Transduced Jurkat Cells.
The critical feature for any T cell or TCR-modified cell is its ability to recognize processed antigen on the surface of cells. However, human cells infected with HCV were not available for our experiments. We have previously shown that human MEL and RCC cells could be engineered to express the HCV NS3:1406-1415 peptide epitope and that the parent HCV-reactive T cell clones recognized these cell lines.15 Therefore, we used this panel of HCV+ targets to assess the ability of our HCV TCR–transduced Jurkat cells to recognize endogenously encoded antigen presented through the MHC class I pathway. As shown in Fig. 7, HCV TCR transduced Jurkat cells secreted significant amounts of IL-2 (greater than 100 pg/ml and at least twice background) when cocultured with HLA-A2+ HCV NS3:1405-1415+ but not HLA-A2− HCV NS3:1405-1415+ or HLA-A2+ CMV pp65:495-503+ tumor cells. As a control, TIL 1383I TCR transduced Jurkat cells, secreted IL-2 only when stimulated with HLA-A2+ MEL cells and not with HLA-A2− MEL cells or RCC cells. These results indicate that the HCV TCR can transfer the ability to recognize processed antigen to other effector cells. Furthermore, the absence of CD8 expression on Jurkat cells did not preclude the HCV TCR Jurkat cells from recognizing HCV+ cells. Therefore, we conclude that CD8 expression was not required for recognition of processed antigen.
Role of CD8 in Tetramer and Antigen Recognition.
CD8+ SupT1 cells were capable of binding HCV tetramers, whereas CD8− Jurkat cells could not. These results suggest that binding of tetramers to HCV TCR–transduced cells requires CD8 expression despite the fact that CD8 expression was not required for tumor cell recognition. To confirm this observation, HCV TCR–transduced Jurkat cells were transduced to express human CD8 and stained with anti-CD8 monoclonal antibody or HLA-A2/ HCV NS3:1406-1415 tetramers (Fig. 8). Untransduced and HCV TCR Jurkat cells do not express CD8 (MFI 4.4 and 5.9, respectively) (Figs. 8D-E) and do not bind tetramers (0.3% and 0.6%, respectively) (Figs. 8A-B). In contrast, HCV TCR Jurkat cells transduced with the CD8 retrovirus express high levels of CD8 (MFI 108) (Fig. 8F), and some of the cells could bind tetramers (7.0%) (Fig. 8C). We speculate that the failure of the remainder of the CD8+ HCV TCR Jurkat cells to bind tetramers was due to the level of HCV TCR expression in those cells. When cocultured with T2 cells loaded with 1 μg/mL HCV NS3:1406-1415 peptide, the CD8+ HCV TCR Jurkat cells secreted 53,284 pg/mL IL-2 and CD8− HCV TCR Jurkat cells secreted 30,822 pg/mL IL-2. In contrast, when cocultured with T2 cells loaded with a control tyrosinase peptide, these cells secreted 88 and 48 pg/mL of IL-2, respectively. These results confirm that tetramer binding to the HCV TCR requires CD8 expression. Although CD8 expression is not necessary for IL-2 production, the expression of CD8 augments the sensitivity of HCV TCR Jurkat cells to antigen stimulation by HCV+ target cells.
It has been reported that almost one third of HLA-A2+, HCV-exposed individuals have T cells that stain with HLA-A2 tetramers loaded with HCV peptides.32 Recently, we have shown that HLA-A2/HCV tetramers can detect HCV-reactive T cells in the blood of HLA-A2− recipients of an HLA-A2+ liver allografts.15 We speculated that these T cells, being of host origin, would express high-affinity TCRs. To test this hypothesis, we cloned and characterized a TCR isolated from one of these HLA-A2–restricted cytotoxic T lymphocyte clones reactive with HCV NS3:1406-1415. A retroviral vector encoding this TCR can transfer anti-HCV reactivity to CD8− Jurkat cells, which can recognize the HCV NS3:1406-1415 peptide loaded exogenously onto T2 cells or loaded through the endogenous MHC class I pathway on tumor cells. Although we recognize that protective immunity is likely mediated by a multispecific HCV-specific T cell response,13 this TCR represents a potential new immunotherapy option in patients with chronic HCV infection who have failed conventional therapy.
There are several features of this TCR that distinguish it from the many other TCRs that have been cloned and expressed. Most notable is its ability to transfer recognition of processed HCV antigen to CD8− cells. CD8 helps stabilize the TCR/peptide/MHC complex by binding to the α3 domain of the MHC molecule.33, 34 We have speculated that any TCR that can bind peptide/MHC complexes without CD8 would have higher relative affinity than a TCR that requires CD8 for binding.19, 27, 28 CD8 expression is clearly not required for antigen stimulation, because CD8− Jurkat cells secrete IL-2 when stimulated with peptide-loaded targets or endogenously processed and presented antigen. Based on functional assays, we would conclude that the affinity of this HCV TCR is higher than other TCRs that require CD8 for target cell recognition.
In conclusion, we have identified and characterized a TCR isolated from an HLA-A2–restricted, HCV NS3–reactive CD8+ T cell clone. Using a retroviral vector, we expressed this TCR in Jurkat cells. The resulting HCV TCR–transduced Jurkat cells have lower relative avidity than the parental T cell clone, yet they secrete substantial amounts of IL-2 when stimulated with peptide-loaded T2 cells. More importantly, their ability to recognize tumor cells expressing the HCV NS3:1406-1415 epitope does not require CD8 expression. Based on this antigen recognition data, we conclude that this TCR is a useful reagent for engineering human CD4+ and CD8+ T cells for treating patients with HCV infection and HCV-related diseases.
The authors would like to thank Barbara L. F. Kaplan and Alexander Langerman for their helpful advice during the completion of these experiments and for valuable comments during the preparation of this manuscript.