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Journal of Virology, March 2008, p. 3078-3089, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.01812-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Lentiviral Vectors Encoding Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Receptor Genes Efficiently Convert Peripheral Blood CD8 T Lymphocytes into Cytotoxic T Lymphocytes with Potent In Vitro and In Vivo HIV-1-Specific Inhibitory Activity

Aviva Joseph,^1,2 Jian Hua Zheng,¹ Antonia Follenzi,³ Teresa DiLorenzo,^2,4 Kaori Sango,² Jaime Hyman,² Ken Chen,⁵ Alicja Piechocka-Trocha,^6,7 Christian Brander,^6,7 Erik Hooijberg,⁸ Dario A. Vignali,⁹ Bruce D. Walker,^6,7 and Harris Goldstein^1,2*

Departments of Pediatrics,¹ Microbiology & Immunology,² Pathology,³ Medicine,⁴ Developmental & Molecular Biology, Albert Einstein College of Medicine, Bronx, New York, 10461,⁵ Partners AIDS Research Center, Massachusetts General Hospital and Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115,⁶ Howard Hughes Medical Institute, Chevy Chase, Maryland 20185,⁷ Department of Pathology, VU University Medical Center, de Boelelaan 1117, NL-1081 HV Amsterdam, The Netherlands,⁸ Department of Immunology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105⁹

Received 17 August 2007/ Accepted 24 December 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The human immunodeficiency virus type 1 (HIV-1)-specific CD8 cytotoxic T-lymphocyte (CTL) response plays a critical role in controlling HIV-1 replication. Augmenting this response should enhance control of HIV-1 replication and stabilize or improve the clinical course of the disease. Although cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection in immunocompromised patients can be treated by adoptive transfer of ex vivo-expanded CMV- or EBV-specific CTLs, adoptive transfer of ex vivo-expanded, autologous HIV-1-specific CTLs had minimal effects on HIV-1 replication, likely a consequence of the inherently compromised qualitative function of HIV-1-specific CTLs derived from HIV-1-infected individuals. We hypothesized that this limitation could be circumvented by using as an alternative source of HIV-1-specific CTLs, autologous peripheral CD8⁺ T lymphocytes whose antigen specificity is redirected by transduction with lentiviral vectors encoding HIV-1-specific T-cell receptor (TCR) and β chains, an approach used successfully in cancer therapy. To efficiently convert peripheral CD8 lymphocytes into HIV-1-specific CTLs that potently suppress in vivo HIV-1 replication, we constructed lentiviral vectors encoding the HIV-1-specific TCR and TCR β chains cloned from a CTL clone specific for an HIV Gag epitope, SL9, as a single transcript linked with a self-cleaving peptide. We demonstrated that transduction with this lentiviral vector efficiently converted primary human CD8 lymphocytes into HIV-1-specific CTLs with potent in vitro and in vivo HIV-1-specific activity. Using lentiviral vectors encoding an HIV-1-specific TCR to transform peripheral CD8 lymphocytes into HIV-1-specific CTLs with defined specificities represents a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Several lines of evidence indicate that the human immunodeficiency virus type 1 (HIV-1)-specific CD8 cytotoxic T-lymphocyte (CTL) response plays a critical role in controlling HIV-1 replication (23, 33, 44). After infection, HIV-1 replication is initially controlled by the generation of CTLs specific for various HIV-1 epitopes (38, 51). Loss of specific CTL activity and/or viral escape from cytotoxic T-cell recognition is associated with an increase in HIV-1 viral load and accelerated progression to AIDS (24, 32, 52, 63). There is a strong correlation between the potency and specificity of the HIV-1-specific CTL response and the magnitude of the viral load and the clinical outcome in most HIV-1-infected individuals (47, 50), the delay or arrest in disease progression in long-term nonprogressors (19, 26, 53), and the protection of some HIV-1-exposed individuals from HIV-1 infection (18, 39, 54, 55). It is likely that increasing the quality and breadth of the HIV-1-specific CTL response would augment the control of HIV-1 replication in infected individuals and either stabilize or improve their clinical course. Immunodeficient patients can be provided with virus-specific immunity by adoptive transfer of ex vivo-expanded CTLs specific for cytomegalovirus (CMV) or Epstein-Barr virus (EBV) that can prevent or control established CMV and EBV infections, respectively (4). However, adoptive immunotherapy of ex vivo-expanded autologous HIV-1-specific CTLs into HIV-1-infected individuals had minimal effects on improving HIV-1 viral load and other surrogate markers of HIV replication (7, 43, 60). It is likely that the lack of impact of these transferred CTLs on disease course is a result of the compromised function and decreased replicative capacity of HIV-1-specific CTLs derived from HIV-1-infected individuals due to several mechanisms, including their decreased perforin production (2, 42), shortened telomere length (15), increased susceptibility to apoptotic death (41), and increased expression of PD-1 (68). An alternative technique for generating antigen-specific CTLs has been used to provide large quantities of tumor antigen-specific human T cells capable of delivering effective tumor-specific immunity to cancer patients: the genetic transfer with retroviral vectors of cancer antigen-specific T-cell receptor (TCR) - and β-chain genes into mature peripheral autologous CD8⁺ lymphocytes (31, 34, 46). This approach has recently been used successfully to augment tumor-specific immunity in melanoma patients by infusing them with CTLs genetically engineered to recognize MART-1, a tumor-associated antigen; the transferred CTLs displayed durable engraftment and were associated with the regression of some tumors (49).

In the present study, we examined whether we could increase the efficiency of this approach by using lentiviral vectors encoding the TCR for antigen and β chains as a single transcript linked with a self-cleaving peptide to enable the efficient conversion of primary human CD8 lymphocytes into HIV-1-specific CTLs that potently suppress in vivo HIV replication. CTLs that recognize the HIV-1 Gag epitope, SL9 (amino acids 77 to 85; SLYNTVATL), play an important role in controlling viral replication, as indicated by the inverse correlation between the amplitude of the SL9-specific CTL response and level of plasma viremia during the chronic phase of HIV-1 infection (47, 50). SL9-specific CTLs in particular may have potent anti-HIV activity because the SL9 epitope is efficiently processed and presented by infected cells (6, 25), is derived from a Gag region that must be conserved to maintain viral fitness (6, 9, 25), is independent of T-cell help (36), and is highly recognized by CTLs in gut-associated lymphoid tissues (58). Because HLA-A*0201 is the most prevalent HLA class I allele and is expressed by a large fraction of the population, we evaluated the effectiveness of genetic engineering to generate HIV-1-specific CTLs with potent in vivo anti-HIV activity by cloning out the TCR from 18030.D23.18, an HLA-A*0201-restricted SL9-specific CTL clone that exerts a potent inhibitory effect on HIV-1 replication (6, 25). To efficiently redirect primary CD8⁺ T lymphocytes to express an HIV-specific TCR, we combined the novel approach of expressing the TCR and TCR β chains as a single transcript linked with a self-cleaving peptide (30) with the robust capacity of third-generation lentiviral vectors to transfer genes into primary CD8⁺ T lymphocytes (12).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells and cell culture. The 18030.D23.18 (1803) CTL clone is a human HLA-A*0201-restricted CD8⁺ CTL that was cloned from an HIV-1-infected patient and is specific for the SL9 peptide (66). The 1803 CTL clone was expanded biweekly by adding irradiated (2,500 cGy) HLA-mismatched peripheral blood mononuclear cells (PBMCs; 6 x 10⁶ cells/well) to the CTL clones (1 x 10⁵ cells/well) cultured in a 12-well plate containing complete RPMI medium (RPMI 1640 supplemented with 10% [vol/vol] heat-inactivated fetal calf serum, penicillin [100 U/ml], streptomycin [10 µg/ml], HEPES buffer [10 mM], and glutamine [2 mM]) with added anti-CD3 (30 ng/ml; Orthoclone OKT3 [OrthBiotech Products, Raritan, NJ]) and interleukin-2 (IL-2; 50 U/ml). The 293T cell line used for lentiviral production was cultured in complete IMDM (Iscove's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum [10%, vol/vol], penicillin [100 U/ml], streptomycin [10 µg/ml], and glutamine [2 mM]) for less than 15 passages before being used in transfection. The Jurkat/MA cell line is a CD8⁺ T-cell line lacking expression of an endogenous TCR β chain and carrying an NFAT (nuclear factor of activated T cells)-regulated luciferase reporter gene that was maintained by culture in complete IMDM (56). Highly purified CD8⁺ lymphocytes (>90% purity) were isolated from donor leukopacks from HIV-naive, HLA-A2-positive donors using MACS CD8 microbeads (Miltenyi Biotec, Germany) according to the manufacturer's instructions. Expression of HLA A2 by lymphocytes was determined by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated anti-HLA-A2-specific antibody (BD Pharmingen, San Jose, CA).

Cloning of the TCR and TCR β chains from the 1803 CTL clone. After the AV2S1A1 and BV5S1A1T variable regions were identified as the specific TCR V and Vβ genes used by the 1803 CTL clone, respectively, the full-length TCR - and β-chain genes were cloned out by reverse transcription and PCR using primers specific for the TCR -chain AV2S1A1 and TCR β-chain BV5S1A1T sequences from 1803 CTL clone mRNA and inserted into TOPO TA vectors. Using the PCR strategy shown in Fig. 1A, the cDNAs for the TCR and β chains were combined into a single sequence linked by a picornavirus-like 2A "self-cleaving" peptide (TCR-2A-TCRβ) (3). Briefly, the first PCR amplification used a forward primer specific for the 5' leader sequence of the AV251A1 TCR chain with an added BamHI restriction site (primer 1) and a return primer which contains the sequence coding the P2A sequence followed by the terminal TCR -chain constant region (primer 3). The second PCR amplification used a forward primer containing the full P2A sequence followed by the BV531AT TCR β-chain leader sequence (primer 4) and a return primer specific for the terminal sequence of the TCR Vβ-chain constant region and an added XbaI restriction site (primer 2). The two PCR products were mixed, and the TCR-2A-TCRβ sequence was generated by PCR amplification with primer 1 and primer 2. The product was cloned into the BamHI/XbaI restriction site of a lentiviral transfer vector that was regulated either by the human phosphoglycerate kinase (hPGK) promoter (hPGK.ires.emcvwt.eGFP.Wpre) (20) or the spleen focus-forming virus (SFFV) promoter (SFFV.ires.emcvwt.eGFP.Wpre) (64) with an upstream internal ribosome entry site (IRES)-regulated green fluorescent protein (GFP) gene.

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FIG. 1. Jurkat/MA cells transduced with the 1803 TCR express functional high-avidity, SL9-specific TCR. (A) PCR cloning of the vectors expressing the TCR-2A-TCRβ construct was performed as described in Materials and Methods. (B) HLA-A*0201 SL9 tetramer binding was determined 5 days after Jurkat/MA cell transduction with the indicated lentiviral vector by staining cells with anti-human TCR (/β) and the HLA-A*0201 SL9 tetramer. The data shown are representative of three separate experiments. (C) Avidity of the TCR was calculated by generating Scatchard plots showing tetramer binding to the original 1803 CTL clone and SFFV-1803 lentiviral vector-transduced Jurkat/MA cells. GMF/concentration is plotted against GMF, with KD = 1/slope.

Transfection and lentiviral vector production. The vesicular stomatitis virus glycoprotein (VSV-g)-pseudotyped HIV-1-based third-generation lentivirus expressing the 1803 TCR and TCR β chains was generated by calcium phosphate-mediated cotransfection of 293T cells with four plasmids: a CMV promoter-driven packaging construct expressing the gag and pol genes, a Rous sarcoma virus promoter-driven construct expressing rev, a CMV promoter-driven construct expressing the VSV-g envelope, and a self-inactivating transfer construct driven either by the hPGK promoter or the SFFV promoter containing the HIV-1 cis-acting sequences and an expression cassette for the 1803 TCR-2A-TCRβ coding sequence inserted upstream of an IRES-regulated enhanced GFP (eGFP) as described previously (21). Control lentivirus was generated by transfecting 293T cells with the packaging, Rev-expressing, Env-expressing, and basal expression constructs. The culture medium was replaced 12 to 16 h after transfection, and 24 h and 48 h later, the supernatant was collected, filtered, and concentrated by ultracentrifugation. The viral pellet was resuspended in sterile phosphate-buffered saline (PBS) and frozen in aliquots at –80°C until use. The concentration of the lentiviral vector in the aliquots was determined by measuring the p24 concentration in the supernatant by enzyme-linked immunosorbent assay (ELISA) as described below.

Lentiviral transduction of CD8⁺ T lymphocytes. Purified CD8⁺ T lymphocytes were activated by incubating the cells with either phytohemagglutinin (PHA; 5 µg/ml) and IL-2 (50 U/ml) or in anti-CD3-coated wells with anti-CD28 (1 µg/ml) and IL-2 (25 U/ml) as described previously (48) for 24 h. The activated cells were harvested, washed, resuspended in 0.5 ml of complete IMDM with added IL-2 (50 U/ml) and Polybrene (4 µg/ml), and plated into 24-well plates (1 x 10⁵ cells/well). The indicated lentiviral vector (100 ng of p24) was added to each well, and the plates were centrifuged (2,500 rpm for 1 h) and incubated overnight at 37°C. The next day, complete IMDM with IL-2 (50 U/ml) was added to each well and incubated for an additional 2 to 5 days. Transduction efficiency and gene expression were determined by flow cytometry as described below.

Analysis of TCR expression and relative avidity by tetramer staining and flow cytometry. Expression of TCR and CD8 by transduced cells was determined by incubating the Jurkat/MA cells with phycoerythrin (PE)-labeled anti-human /β TCR (Biolegend, San Diego, CA) and/or Cy7-allophycocyanin (Cy7-APC; Biolegend) anti-human CD8 as described previously (59). Tetramer staining of the cells was performed using SL9 iTAg major histocompatibility complex (MHC) class I human tetramers (Beckman Coulter, Fullerton, CA) according to the manufacturer's instructions. For equilibrium binding, cells were stained with tetramer at concentrations that ranged from 0.001 to 10 nM. Briefly, Cy7-APC-labeled anti-human CD8 and PE-labeled tetramers were added to cells (1 x 10⁶ cells/0.2 ml) suspended in fluorescence-activated cell sorter buffer, incubated for 30 min at room temperature in the dark, washed, resuspended in 0.5 ml of phosphate-buffered saline (PBS) with 0.5% formaldehyde, and incubated for 1 h at 4°C in the dark. Fluorescent cells were enumerated using an LSRII (BD Biosciences, San Jose, CA), and the acquired data were analyzed using FlowJo software (Treestar, Ashland, OR). The avidity, K_D, was determined from Scatchard analysis of equilibrium binding data of geometric mean fluorescence (GMF)/concentration of the tetramer used in staining plotted against GMF using the formula K_D = 1/slope of the Scatchard plot (29).

Mice. The specific-pathogen-free male SCID mice (8 to 12 weeks old) used in this study were housed and maintained as described previously (62). All animal studies were approved by the Institutional Animal Care and Use Committee and were consistent with the guidelines for the care and use of laboratory animals.

ELISPOT assay to measure cellular production of IFN-. Antigen-induced gamma interferon (IFN-) secretion by CTL clones was measured by an enzyme-linked immunospot (ELISPOT) assay using a modification of a described technique (28). After wells of nitrocellulose-bottom 96-well plates (MultiScreen-HA; Millipore, Billerica, MA) were coated overnight with 50 µl monoclonal anti-IFN- antibody (10 µg/ml; R&D Systems, Minneapolis, MN), the plates were washed, 1% bovine serum albumin was added to each well to block nonspecific binding, and the plates were washed again. The HIV-1 peptides SL9 (SLYNTVATL) and IV9 (HIV reverse transcriptase amino acids 476 to 484; ILKEPVHGV) (66) were synthesized by the Albert Einstein College of Medicine Laboratory for Macromolecular Analysis with a PE Biosystems 433A peptide biosynthesizer using a standard solid-phase method. The target cells used were HLA-A*0201-positive T2 cells that are defective in their ability to present endogenous peptides in the context of MHC class I molecules, enabling efficient presentation of exogenously loaded HLA-A*0201-restricted peptides (10). T2 cells alone or with the indicated peptide (1 µM) were added in a total volume of 100 µl of complete RPMI medium to the wells and incubated at room temperature for 1 h. Following density centrifugation over a Hypaque/Ficoll cushion and overnight resting in complete medium (without IL-2), the indicated CTL clone or transduced CD8⁺ cells were added (1 x 10⁴ cells/well) in 100 µl of complete RPMI medium with added IL-2 (50 U/ml) and incubated for 48 h at 37°C with the T2 cells. After the plates were washed, captured IFN- was detected by incubation with 50 µl of biotin-conjugated goat anti-human IFN- (R&D systems) for 2 h at 37°C in the dark, and the plates were rewashed, incubated for 1 h at 37°C with streptavidin-alkaline phosphatase (Zymed, San Francisco, CA) diluted 1:10,000 in conjugate buffer (1% bovine serum albumin in PBS plus 0.05% Tween 20), washed, and incubated with 50 µl of FAST 5-bromo-4-chloro-3-indolylphosphate nitroblue tetrazolium (BCIP/NBT) substrate (Sigma) for 30 min at 37°C in the dark. After drying the plates overnight in the dark, spot-forming cells (SFC) corresponding to individual IFN--secreting cells were counted using an AID ELISPOT reader (Autoimmun Diagnostika GmbH, Strasbourg, Germany). The background number of SFC was determined by incubating the CD8⁺ cells with T2 cells alone or T2 cells loaded with irrelevant peptide (IV9 peptide), and wells containing CD8⁺ T cells stimulated with PHA (5 µg/ml) were included as a positive control. Each determination was performed in triplicate, and the results were expressed as SFC/number of plated CD8⁺ T cells.

Measurement of the cytotoxic capacity of the genetically engineered SL9-specific CTLs. The cytotoxic capability of the TCR-engineered CTLs was measured by a standard 4-h ⁵¹Cr release cytotoxicity assay (35). Briefly, target HLA-A2 T2 cells were preincubated with 1 µM of the SL9 peptide or the control IV9 peptide and loaded with ⁵¹Cr (100 µCi/10⁶ cells) for 1 h in serum-free medium. Target cells were then washed twice, resuspended in complete medium, and plated at 10,000 cells/well in 96-well U-bottom plates together with effector cells and either mock-, empty vector-, or 1803-TCR-transduced CD8 cells, at an effector/target ratio of 50:1. After a 4-h culture period, 100 µl of supernatant was collected from each well and analyzed for ⁵¹Cr release. The percent specific lysis was calculated using the formula [(experimental release – spontaneous release)/(maximum release – spontaneous release)] x 100.

Evaluation of TCR function using a luciferase reporter gene assay. The Jurkat/MA cells have been stably transfected with an NFAT-luciferase reporter gene enabling them to be used to evaluate the functional activity of the expressed TCR as described previously (56). Briefly, T2 cells were preincubated with SL9 peptide or IV9 peptide at various concentrations ranging from 0.01 to 1 µM for 2 h and washed, and the peptide-loaded T2 cells were added to cultures of Jurkat/MA cells transduced with the indicated lentiviral vector. After 12 h, the cells were harvested and then lysed in 60 µl of lysis buffer and the luciferase activity in the cellular lysates was detected in duplicate cell lysate aliquots (25 µl) using the luciferase assay system (Promega, Madison, WI) and quantified using the 96-well MLX Multiplex luminometer (Dynex Technologies, Chantilly, VA).

Measurement of p24 antigen in culture supernant. HIV-1 p24 antigen production was measured using an ELISA. Maxisorp ELISA plates (NUNC, Rochester, NY) were coated for 1 h at 37°C with 100 µl of anti-p24 HIV-1 monoclonal antibody (1 µg/ml; ImmunoDiagnostics, Woburn, MA) in NaHCO₃ buffer (0.1 M, pH 8.5) and washed, and the wells were blocked for 2 h at 37°C with 200 µl of blocking buffer (NaCl [1.44 M], Tween 20 [0.5%], Trizma base [250 mM, pH 7.5], casein [0.1%]). After the wells were aspirated, samples diluted in blocking buffer were added in quadruplicate to wells and incubated for 1 h at 37°C, the plates were washed, and biotinylated rabbit anti-p24 antibody in blocking buffer was added to each well and incubated for 1 h at 37°C. The plates were washed again, streptavidin-horseradish peroxidase (BD Pharmingen) was added to each well and incubated for 1 h at 37°C, the plates were washed again, substrate was added (100 µl of Sigma FAST o-phenylenediamine) to each well, and the plates were incubated at room temperature for 30 min. The reaction was stopped by adding 100 µl of 4 N sulfuric acid to each well, and specific A₄₉₀ (above background of 650 nm) was quantified by using an ELISA microplate reader.

Measurement of the capacity of the genetically engineered HIV-specific CTLs to inhibit in vitro HIV infection. Human HLA-A2-positive PBMCs were infected by incubating about 100 x 10⁶ cells at 37°C in 1 ml of RPMI with a primary X4 isolate, HIV-1 BZ167 (1,600 50% tissue culture infective doses [TCID₅₀]) obtained from the NIH Research and Reference Reagent Program (8). Four hours later, complete medium was added (9 ml) and the cells were incubated overnight at 37°C. The next day, the cells were washed twice and plated at 5 x 10⁵ cells/well in a 24-well plate in complete medium (1 ml) with added IL-2 (50 U/ml). Equivalent numbers of primary human CD8⁺ lymphocytes transduced with the indicated lentiviral vector were added to the cultures, and an aliquot of medium was removed and replaced with fresh complete medium (with added IL-2) every 2 to 3 days for 14 days. The HIV concentration in the aliquots was measured using a p24 antigen ELISA.

Measurement of the capacity of the genetically engineered HIV-specific CTLs to inhibit in vivo HIV infection. The in vivo anti-HIV-1 activity of the CTLs was determined with an in vivo adoptive transfer system we developed. HLA-A2-positive PBMCs were activated with PHA (5 µg/ml) and IL-2 (50 U/ml) for 24 h and infected with HIV-1_JR-CSF (800 TCID₅₀). The next day, the HIV-1-infected PBMCs were washed and resuspended (5 x 10⁶ to 10 x 10⁶/ml) in 100 µl of sterile cold PBS and intrasplenically injected into SCID mice alone or mixed with the 1803 CTL clone or CD8 cells transduced with the indicated lentiviral vector (at a 1:1 ratio). This approach permits us to generate over 20 mice from the same donor and analyze the in vivo activities of several HIV-1-specific CTLs in a single experiment. The intrasplenically injected HIV-1-infected human PBMCs persist in the mouse for at least 2 weeks and are readily detectible by flow cytometry and coculture. After 1 week, the mice were sacrificed and the number of HIV-1-infected human PBMCs in the spleen was measured by limiting dilution coculture as described previously (62). Briefly, fivefold dilutions of splenocytes isolated from the injected mouse spleens that ranged from 1 x 10⁶ cells to 3.2 x 10² cells per well were cultured in quadruplicate at 37°C in 24-well culture plates with PHA-activated donor mononuclear cells (1.0 x 10⁶/well) in 2 ml of complete RPMI medium with added IL-2 (32 U/ml). The HIV-1 p24 antigen content of the culture supernatant was measured 1 to 2 weeks later with an ELISA as described above. The lowest number of added splenocytes that generated productive infection of at least half of the quadruplicate cultures with HIV-1 was determined. The data are reported as TCID/10⁶ splenocytes, which were calculated by determining the lowest number of HIV-1-infected splenocytes that generated productive infection by coculture/10⁶ splenocytes. We also used this in vivo adoptive transfer system to evaluate the in vivo capacity of the genetically engineered HIV-1-specific CTLs to protect activated human PBMCs from HIV-1 infection after injection into the spleens of SCID mice. The HLA-A2-positive PBMCs were activated with PHA (5 µg/ml) and IL-2 for 24 h and the next day washed, resuspended (5 x 10⁶ to 10 x 10⁶/ml) in 100 µl of sterile cold PBS mixed with 50 µl of HIV-1_JR-CSF (80 TCID₅₀) alone or with equivalent numbers of the indicated CTLs or transduced CD8 cells, and intrasplenically injected into SCID mice. After 7 days, the mice were sacrificed and the number of HIV-1-infected human PBMCs in the spleen was measured by limiting dilution coculture as described above.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Functional SL9-specific TCR can be generated by transduction of T cells with lentiviral vectors. As the first step to genetically engineer HIV-specific CTLs, we cloned out the TCR and TCR β chains from 18030.D23.18 (1803), a CTL clone that exhibits potent anti-HIV activity specific for the HLA-A*0201-restricted immunodominant HIV-1 Gag epitope, SL9 (SLYNTVATL) (66). Because the antigen-specific TCR consists of a heterodimeric and β chain, the most efficient cellular expression and function occur when equivalent levels of the TCR and TCR β chains are produced by a T lymphocyte (3). Therefore, we constructed a vector that combined the TCR -chain and TCR β-chain genes cloned from the SL9-specific 1803 CTL (Fig. 1A) into a single transcript linked with a picornavirus-like peptide (TCR-2A-TCRβ) that is spontaneously cleaved following translation, thereby yielding equivalent amounts of the TCR chain and TCR β chain proteins (3). The 1803 TCR-2A-TCRβ construct was cloned into a lentiviral vector regulated either by the hPGK promoter (20) (hPGK-1803) or the SFFV promoter (64) (SFFV-1803), with a reporter gene coding for eGFP regulated by a downstream IRES sequence. The expression and functional activity of the SL9-specific TCR encoded by the hPGK-1803 and SFFV-1803 lentiviral vectors were evaluated using the Jurkat/MA cell line, a TCR-negative, CD8⁺ T-cell line carrying a TCR-responsive luciferase reporter construct (56). After transduction with the hPGK-1803 or SFFV-1803 lentiviral vector, greater than 95% of the Jurkat/MA cells expressed GFP and greater than 85% expressed a TCR that bound to the HLA-A*0201 SL9 peptide tetramer (Fig. 1B). To determine the avidity of the SFFV-1803-encoded TCR, we performed binding studies by incubating SFFV-1803-transduced Jurkat/MA cells with various concentrations of the HLA*0201 SL9 peptide tetramer and used these data to generate Scatchard plots as described previously (29). The 1803 lentiviral vector-transduced Jurkat/MA cells exhibited high-avidity binding to A*0201 SL9 peptide tetramer with a K_D of (6.0 ± 0.97) x 10^–10 M that was comparable to that of the 1803 CTL clone, with a K_D of (5.2 ± 0.56) x 10^–10 M (Fig. 1C). These K_D values are consistent with those reported for other SL9-specific CTLs (25, 36, 57). We examined the functional activity of the expressed 1803 TCR by incubating SFFV-1803 lentiviral vector-transduced Jurkat/MA cells with T2 cells alone or T2 cells loaded with SL9 or a control HIV peptide, IV9, at concentrations that ranged from 0.01 µM to 10 µM and then measured the induction of the TCR-responsive luciferase construct that is stably transfected in the Jurkat/MA cells. Luciferase activity was potently induced after incubation of the 1803 lentiviral vector-transduced Jurkat/MA cells with T2 cells loaded with concentrations as low as 0.01 µM of the SL9 peptide, but not after incubation with T2 cells alone or T2 cells loaded with the control IV9 peptide (Fig. 2). This indicated that transduction with the 1803 lentiviral vector resulted in the expression of functional SL9-specific TCRs.

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FIG. 2. Jurkat/MA cells transduced with the SFFV-1803 lentiviral vector respond specifically to activation with SL9. Functional activity of the 1803 TCR expressed by the Jurkat/MA cells transduced with the indicated lentiviral vectors compared to Jurkat/MA cells transduced with a control lentiviral vector was determined by luciferase assay in duplicate, as described in Materials and Methods, after stimulation with the indicated concentrations of SL9 or a control peptide (IV9). The data shown are the means ± standard errors from an experiment performed in duplicate.

Transduction with 1803 lentiviral vectors converts primary CD8⁺ T lymphocytes into HIV-1-specific CTLs with potent in vitro anti-HIV-1 activity. The therapeutic use of genetically engineered CTLs would be greatly advanced by the capacity to efficiently convert primary peripheral CD8⁺ lymphocytes into HIV-1-specific CTLs. To determine whether transduction with 1803 lentiviral vectors could efficiently convert primary human CD8 T cells into SL9 peptide-specific CTLs, CD8⁺ lymphocytes were isolated from the peripheral blood of HIV-naïve individuals, activated and transduced with the SFFV-1803 lentiviral vector. The primary CD8⁺T lymphocytes were efficiently transduced by the 1803 lentiviral vector, as indicated by the expression of lentiviral vector-encoded GFP by over 30% of the SFFV-1803 lentiviral vector-transduced CD8⁺ lymphocytes, of which greater than 80% expressed a TCR that bound the HLA-A*0201 SL9 tetramer (Fig. 3). A fraction of the GFP-positive CD8 T cells did not express the 1803 TCR, which may be a consequence of the competitive association of the 1803 TCR and β chains with the endogenous TCR and β chains and expression of chimeric TCRs as described previously (27).

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FIG. 3. Purified peripheral CD8+ lymphocytes transduced with the SFFV-1803 lentiviral vector express a TCR that binds HLA-A*0201 SL9 tetramer. After staining the cells with the indicated antibody and tetramer, the fluorescent cells were quantified on an LSR II flow cytometer and the results were analyzed using FlowJo software. The data shown are representative of three separate experiments.

The functional activity of the SFFV-1803 lentiviral vector-transduced primary human CD8⁺ lymphocytes was evaluated by measuring cellular IFN- production induced by SL9 peptide and was compared to the level of SL9-induced IFN- produced by the original 1803 CTL clone. The SFFV-1803 lentiviral vector-transduced primary human CD8⁺ lymphocytes produced IFN- in response to stimulation with the SL9 peptide but not in response to stimulation with the control peptide, IV9 (Fig. 4A). When corrected for the fraction of CD8 T lymphocytes expressing the 1803 TCR, the SL9-induced ELISPOT reactivity of the SFFV-1803-transduced CD8⁺ T lymphocytes was comparable to that of the original 1803 CTL clone. We further evaluated the functional activity of the SFFV-1803 lentiviral vector-transduced primary human CD8⁺ lymphocytes by determining their cytotoxic activity using a standard ⁵¹Cr killing assay. As shown in Fig. 4B, the SFFV-1803 lentiviral vector-transduced primary human CD8⁺ lymphocytes selectively killed T2 target cells loaded with SL9 peptide but not T2 cells either loaded with the control IV9 peptide or not loaded with peptide.

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FIG. 4. Peripheral CD8+ lymphocytes transduced with SFFV-1803 lentiviral vector display SL9-specific cytotoxic activity. (A) SL9-specific production of IFN- was determined by transducing purified peripheral CD8 lymphocytes with the indicated lentiviral vector and incubating with T2 cells (104 cells/well) without added peptide or loaded with SL9 or IV9 peptide in precoated (anti-human IFN- monoclonal antibody) and blocked ELISPOT plates. Forty-eight hours later, the wells were washed and IFN- was detected by ELISPOT. Positive controls (5 µg/ml PHA) and negative controls (T2 cells only) were included. The data presented are the mean ± standard error of two separate experiments done in duplicate. (B) SL9-specific lysis of target cells by peripheral CD8+ lymphocytes transduced with the 1803 TCR was determined by incubating purified peripheral CD8 lymphocytes transduced with the indicated lentiviral vector with 51Cr-loaded T2 cells (10,000 cells/well) incubated with the indicated peptide. Four hours later, supernatant was collected and the level of free 51Cr was measured. The data presented are the means ± standard error of an experiment done in duplicate.

The level of in vitro CTL activity assessed by ELISPOT assays that measure cognate peptide-induced IFN- secretion or by cytotoxicity assays that quantify the lysis of cell lines that are exogenously loaded with peptide or that express recombinant HIV-1 proteins may not correlate with their capacity to kill HIV-1-infected cells (65, 67). Several factors may increase the capacity of primary HIV-1-infected cells to resist lysis by HIV-1-specific CTLs compared to peptide-expressing target cells, including interference with peptide processing and loading into MHC class I molecules and inhibition of class I MHC molecule surface expression (1). Therefore, the physiologically relevant capacity of HIV-1-specific CTLs to inhibit HIV-1 infection may be better evaluated by determining their capacity to eliminate HIV-1-infected cells rather than by measuring their production of IFN- in response to exposure to lymphoid cell line target cells loaded with exogenous peptide or expressing recombinant HIV-1 proteins (2). Consequently, we evaluated the anti-HIV-1 activity and potency of the 1803 lentiviral vector-transduced primary CD8⁺ lymphocytes by quantifying their capacity to inhibit in vitro HIV-1 replication in PBMCs as described previously (1). HLA-A2-positive PBMCs (5 x 10⁵ cells/well) infected with HIV-1 ex vivo were incubated either with untransduced CD8⁺ lymphocytes or CD8⁺ lymphocytes transduced with a control lentiviral vector or the PGK-1803 lentiviral vector, and the p24 antigen concentration in the culture supernatant for the indicated day of culture was measured to evaluate the inhibitory effect of the untransduced or transduced CD8⁺ lymphocytes on productive HIV-1 infection. As shown in Fig. 5, CD8⁺ T lymphocytes transduced with the PGK-1803 lentiviral vector potently inhibited the replication of HIV-1 in the PBMCs, by over 90% on day 5 of culture and almost 99% by day 9 of culture. In contrast, the addition of CD8⁺ T lymphocytes transduced with a control lentiviral vector to the cultured HIV-1-infected PBMCs had minimal effects on HIV-1 replication. Although it appears that the PGK-1803 lentiviral vector-transduced CD8⁺ T lymphocytes were less effective than the 1803 CTL clone for inhibiting in vitro HIV-1 infection, this is likely due to expression of the 1803 TCR by only about 20 to 30% of the transduced peripheral CD8 lymphocytes. Taken together, these results indicated that transduction with the PGK-1803 lentiviral vector converts primary peripheral CD8⁺ T lymphocytes into HIV-1-specific CTLs that potently inhibit in vitro HIV-1 infection.

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FIG. 5. 1803 lentivirus-transduced peripheral CD8 lymphocytes potently inhibit in vitro HIV-1 infection. Primary human CD8+ lymphocytes transduced with the indicated lentiviral vector or the 18030.D23.18 CTL clone were added at the effector/target ratio of 1:1 to cultures of HLA-A2-positive PBMCs infected ex vivo with HIV-1-BZ167. During culture, the extent of HIV-1 infection was monitored by measuring the p24 antigen content in the culture supernatant on the indicated day of culture. Presented are the means ± standard errors of an experiment performed in duplicate.

Genetically engineered HIV-1-specific CTLs generated from primary CD8⁺ T lymphocytes potently inhibit in vivo HIV-infection. In vitro assays of CTL function may not correlate with the in vivo capacity of CTLs to eliminate HIV-1-infected cells and predict their therapeutic effect when given to HIV-1-infected individuals. Therefore, we examined the in vivo capacity of SFFV-1803-transduced primary CD8⁺ T cells to eliminate HIV-1 infected PBMCs using a novel in vivo mouse model we developed that uses as target cells HIV-infected human PBMCs injected into the spleens of SCID mice that persist in the mouse spleens for at least 2 weeks after injection. This permits us to evaluate the in vivo efficacy of anti-HIV-1 therapies by measuring the capacity of defined therapeutic interventions to reduce the number of HIV-1-infected human PBMCs in the spleens as quantified by limiting dilution coculture (14). Human PBMCs from HLA-A2-positive donors were ex vivo infected with HIV-1 and then injected intrasplenically into SCID mice either alone, coinjected with CD8⁺ T lymphocytes transduced with the indicated lentiviral vector, or coinjected with the original 1803 CTL clone. One week later, as measured by limiting dilution coculture, the spleens of mice injected with primary CD8⁺ T lymphocytes transduced with the control lentiviral vector contained sufficient HIV-1-infected human PBMCs to generate productive HIV-1 infection after coculture of only 320 splenocytes, which was equivalent to the numbers of HIV-1-infected human PBMCs detected in the spleens of mice injected with HIV-1-infected PBMCs alone (Fig. 6A). In contrast, in the spleens of SCID mice also coinjected with SFFV-1803 lentivirus-transduced primary CD8⁺ T lymphocytes, we did not detect any HIV-1-infected PBMCs even after coculture of up to 10⁶ splenocytes. This potent in vivo suppression of HIV-1 infection by SFFV-1803 lentivirus-transduced primary CD8⁺ T lymphocytes was similar to the suppressive effect observed in mice intrasplenically injected with the original 1803 CTL clone (Fig. 6A).

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FIG. 6. Potent in vivo anti-HIV activity displayed by 1803 lentivirus-transduced peripheral CD8 lymphocytes. (A) HLA-A2-positive activated PBMCs were infected with HIV-1JR-CSF overnight in complete RPMI. The next day, cells were harvested and injected (5 x 106 to 10 x 106 cells/mouse) with or without equivalent numbers of the 1803 CTLs or the CD8 cells transduced with the indicated lentiviral vector into the spleens of SCID mice. (B) HIV-1JR-CSF (80 TCID50) and 5 x 106 to 10 x 106 HLA-A*0201-positive activated PBMCs were coinjected with or without equivalent numbers of CD8 cells transduced with the indicated lentiviral vector into the spleens of SCID mice. One week later, serial fivefold dilutions, starting at 1 x 106 cells were added to 1 x 106 activated PBMCs in a total of 2 ml of complete medium plus IL-2. Seven days later, supernatants were collected and the p24 ELISA was done. The results presented are the means ± standard errors of the number of infectious cells (TCID)/106 splenocytes from three experiments done in triplicate (with different HLA-A2 donors each time).

We used a modification of this in vivo mouse system to determine whether the SFFV-1803-transduced primary CD8⁺ T cells could inhibit the initiation of in vivo HIV-1 infection. SCID mice were intrasplenically injected with PBMCs from HLA-A2-positive donors together with the indicated lentiviral vector-transduced CD8⁺ T lymphocytes and with an aliquot of infectious HIV-1. Seven days later, limiting dilution coculture demonstrated that the spleens of mice also injected with SFFV-1803-transduced primary CD8⁺ T cells contained 500-fold-lower numbers of HIV-1-infected PBMCs than did the spleens of mice intrasplenically injected either with added CD8 lymphocytes transduced with a control lentiviral vector or with no added CD8 lymphocytes (Fig. 6B). Thus, lentiviral vectors encoding the 1803 TCR can efficiently convert primary peripheral CD8⁺ lymphocytes into CTLs with potent in vivo anti-HIV-1 activity.

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The HIV-1-specific CTL response mounted by HIV-1-infected patients following infection is ultimately unsuccessful in controlling HIV-1-infection. We postulated that the immunity of HIV-1-infected individuals could be reconstituted by genetic engineering to redirect the antigen specificity of mature peripheral autologous CD8⁺ lymphocytes by transduction with vectors encoding HIV-1-specific TCR -chain and TCR β-chain genes as described for the successful augmentation of tumor-specific immunity (31, 34, 46). Genetically engineered lymphocytes transferred into melanoma patients have recently been shown to display sustained engraftment that conferred tumor antigen-specific CTL immunity and tumor regression (46). The success of this approach in some cancer patients supports the extension of this approach to HIV-1-infected individuals to augment their T-cell immunity to pathogens such as HIV-1 and thereby stabilize or improve the clinical course of the disease. Investigators have previously reported the use of genetic engineering to redirect antigen specificity of CD8 lymphocytes to HIV-1, but these studies used a less efficient approach of sequentially transducing peripheral CD8 lymphocytes with one retroviral vector expressing the TCR chain and another retroviral vector expressing the TCR β chain specific for either an HIV Gag peptide (13) or an HIV-1 Pol peptide (61). The low efficiency of this technique to generate primary CD8⁺ lymphocytes expressing both the TCR chain and the TCR β chain required either serial drug selection (13) or sorting by flow cytometry using HLA-Pol peptide tetramers (61), followed by T-cell cloning and in vitro expansion to generate sufficient numbers of genetically engineered HIV-specific T lymphocytes to demonstrate their in vitro capacity to lyse HIV-1-expressing target cells. Although one of these studies demonstrated that the genetically engineered HIV-1-specific CD8⁺ T lymphocytes inhibited in vitro HIV-1 replication, the target cells used were an HIV-1-infected CD4-expressing EBV-transformed B-cell line that may display HIV-1 replication kinetics different from those of the primary PBMCs that are the targets of HIV-1 infection in HIV-1-infected individuals (61).

In the present study, we describe the development of a novel system that efficiently converts primary CD8 T lymphocytes into HIV-1-specific CTLs with defined specificities that could be used as a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients. Specifically, we demonstrated that transduction with a single lentiviral vector encoding the TCR and β chains derived from an SL9-specific CTL as a single transcript linked with a self-cleaving peptide efficiently converted peripheral CD8⁺ lymphocytes from an HIV-1-naïve individual into SL9-specific CTLs that exhibited specific and high-avidity binding to the SL9 peptide-HLA-A*0201 complex and potently suppressed in vitro HIV-1 infection of primary human PBMCs. In addition, we demonstrated that the genetically engineered SL9-specific CTLs suppressed in vivo HIV-1 infection of human PBMCs that had been transferred into the spleens of SCID mice, indicating that they could inhibit HIV-1 infection in a lymphoid environment, the major location of HIV-1 replication in HIV-1-infected individuals. To our knowledge, this is the first demonstration of the in vivo efficacy of this approach for HIV-1 infection.

Use of this approach may circumvent several factors that limit the effectiveness of the naturally induced HIV-1-specific CTL response or of treatment by adoptive transfer of ex vivo-expanded autologous HIV-1-specific CTLs (7, 43, 60). First, HIV-1-specific CTL function may be compromised by the prolonged activation and extensive clonal expansion associated with chronic HIV-1 infection that adversely affect the lifespan of HIV-1-specific CTLs by shortening their telomere length (15), increasing their susceptibility to apoptotic death (41), and increasing their expression of PD-1 (68). This limitation could be overcome by generating new HIV-1-specific CTLs from naïve CD8⁺ lymphocytes using lentiviral vectors encoding HIV-1-specific TCRs that also encode hTERT, or small interfering RNA constructs targeting Fas or PD-1 to block inhibitory or death-inducing signals. Second, the CTL response generated by HIV-1 infection is narrowly focused on only a small number of HIV-1 epitopes (16) due to the phenomenon of immunodominance, a consequence of the relative abundance of peptides presented by HLA class I molecules on antigen-presenting cells and the specific antigenic repertoire of the TCRs expressed by naïve T cells generated during thymopoiesis and the capacity of these naïve T cells to undergo clonal expansion (17, 69, 70). This narrow HIV-1-specific CTL response enables the rapidly mutating HIV-1 to evade the HIV-1-specific CTL response by generating escape variants that are mutated in the region recognized by the CTLs (5). Infusing patients with autologous CD8 lymphocytes transduced with a mixture of lentiviral vectors that each encode a TCR specific for a different HIV-1-specific epitope should circumvent this limitation by rapidly populating the immune system with CTLs that recognize a broad repertoire of HIV-1 peptides, including predicted escape mutant epitopes. Conceptually, this is similar to the advantage of highly active antiretroviral activity over monotherapy in reducing the emergence of drug-resistant isolates. Third, although CTL responses to immunodominant HIV-1 epitopes predominate during infection, less frequently observed responses to highly conserved subdominant epitopes such as TV9, a p24 epitope described in HIV-1-resistant African sex workers, may exert potent in vivo control of viral replication (55). Modalities that induce these CTLs in HIV-1-infected individuals may increase the control of viral replication (22). Genetic engineering would permit the generation of CTLs recognizing these subdominant epitopes by bypassing the phenomenon of immunodominance as well as the inability to respond to an epitope in the absence of naïve T cells carrying TCRs specific for these protective subdominant epitopes. Fourth, CTL function in HIV-1-infected individuals may be compromised by their expression of low-avidity TCR. Murine studies have demonstrated that CTLs expressing TCRs with high avidity for HIV-1 epitopes were 100- to 1,000-fold more effective in clearing vaccinia virus/HIV-1 infection than CTLs expressing low-avidity TCRs (1). Consequently, control of HIV-1 replication should be enhanced by increasing the avidity of HIV-specific CTLs in infected individuals by transducing autologous CD8 T lymphocytes with a lentiviral vector encoding a high-avidity TCR either isolated from a potent HIV-1-specific CTL clone or molecularly engineered from a natural HIV-1-specific TCR to display increased avidity for the HIV-1 peptide-class I complex(45).

The clinical application of utilizing lentiviral vectors to genetically engineer peripheral CD8⁺ T lymphocytes into HIV-1-specific CTLs may be restricted by the factors that have limited the successful application of gene therapy. However, as stated above, the approach of using CTLs genetically engineered with retroviral vectors has already been successfully applied to cancer patients where CTLs are not as effective as they are for controlling viral infections (46). In addition, the safety of lentiviral vectors is indicated by their successful use in a phase I clinical trial, which treated HIV-1-infected individuals with infused autologous CD4⁺ T lymphocytes carrying an anti-HIV-1 gene that was transferred and expressed using a lentiviral vector (40). Because the genetically engineered CTLs are CD8⁺ lymphocytes, they should not be infectible with HIV-1 and subject to subsequent HIV-1-mediated depletion or to mobilization of the lentiviral vector by HIV-1-mediated packaging of the vector genomic transcript and transfer to another cell (37). Although not reported for cancer patients treated with CTLs genetically engineered to express MART-1-specific TCRs (46), it is possible that alternative pairing may occur between the 1803-encoded TCR and β chains and the endogenous TCR and β chains expressed by the transduced primary CD8⁺ T lymphocytes to generate TCRs that are autoreactive. If this is a concern, we would engineer the TCR and β chains encoded by the lentiviral vector to express leucine zipper motifs to favor their heterodimeric pairing and prevent alternative pairing with the endogenous TCR and β chains (11). Thus, using lentiviral vectors encoding HIV-specific TCR to transform peripheral CD8 lymphocytes into HIV-1-specific CTLs with defined specificities represents a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients.

 
This work was supported by the National Institutes of Health (National Institute of Allergy and Infectious Diseases AI67136 and the Einstein/MMC Center for AIDS Research AI51519).

 
* Corresponding author. Mailing address: Albert Einstein College of Medicine, Forchheimer Building, Room 408, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-2156. Fax: (718) 430-8972. E-mail: hgoldste{at}aecom.yu.edu

Published ahead of print on 9 January 2008.

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Journal of Virology, March 2008, p. 3078-3089, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.01812-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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