High levels and a broad specificity of anti-HIV-1 cytotoxic T lymphocytes (CTL) are important for long-term control of HIV-1 replication, disease progression, and viral load [1–11]. But as HIV-1 escapes the CD8+ T cells by mutations in the CTL epitopes [12,13], the immune system fails to control virus replication [14,15]. In contrast, long-term nonprogressors maintain strong HIV-1-specific CTL responses [1,16], often against conserved epitopes of HIV-1 proteins [17–19]. Alternatively, they start with a broad repertoire of T cells that recognize not only the original HIV-1 epitope, but also escape variants [9,20]. Most of the patients, however, have a narrow T-cell receptor (TCR) repertoire, and fail to control virus replication .
In-vitro expanded T lymphocytes can be used for adoptive immunotherapy of viral infections [22–26]. In clinical trials, expanded HIV-1-gag- and HIV-1-pol-specific CTL clones augmented antigen-specific lytic activity, and reduced the frequency of HIV-1-infected CD4+ T cells dramatically [27–30]. Despite these clinical successes, the employment of conventionally generated HIV-1-specific T cells for adoptive immunotherapy has not been further proceeded and optimized owing to the need for extended ex-vivo bulk culture of infected material and the difficulties in isolation and culturing of the antigen-specific CD8+ T cells .
An alternative strategy to generate specific T cells is the introduction of chosen TCR into bulk T lymphocytes by genetic means. CTL, retrovirally transduced with TCR specific for Epstein–Barr virus (EBV)-LMP2/human leukocyte antigen (HLA)-A2 , HIV-gag/HLA-A3 , and HIV-pol/HLA-B35 , recognized antigen specifically. However, the use of retroviral vectors and full-length TCR harbors certain hazards, like insertional mutagenesis, irreversible genetic manipulation, and generation of autoimmunity by mispairing of endogenous and introduced TCR chains [35–39]. This causes regulatory and other concerns.
Hence, we chose to transfer the TCR into T cells by RNA electroporation [40–45] to exclude any possibility of insertional mutagenesis. Here, we investigated for the first time the reprogramming of CD8+ T cells with a virus-specificity by TCR-RNA electroporation.
CD8+ T cells were isolated from whole blood of healthy volunteers (following informed consent and approved by the institutional review board) and electroporated as described previously . The transporter associated with antigen processing (TAP)-deficient T × B cell hybrid T2-A1  and the EBV-transformed B-cell lines DO5 were used as target cells.
Cloning of T-cell receptor genes
The TCR α and β chains of the HLA-A2/HIV-1-pol-specific CTL clone [HA (AJ238415)]  and the HLA-A2/HIV-1-gag-specific CTL clone were cloned into a pGEM4Z RNA production vector .
In-vitro transcription of T-cell receptor RNA
In-vitro transcriptions were performed as described before  using mMESSAGE mMACHINE T7 Ultra kit (Applied Biosystems/Ambion, Texas, USA), or MessageMAX T7 Capped Message Transcription Kit (Epicentre, Wisconsin, USA) according to the manufacturers' instructions.
Induction and determination of cytokine production by T-cell receptor-RNA-transfected T lymphocytes
T cells electroporated with TCR-encoding mRNA were cocultured with ultraviolet-irradiated (0.005 J/cm2) T2 cells, which were loaded with a control peptide, the ILKEPVHGV (IV9) peptide, and the SLYNTVATL (SL9) peptide (all at 1 or 10 μg/ml) for 1 h at 37 °C. T cells (50 000) were cocultured with 50 000 target cells in a volume of 100 μl of multilineage progenitor cell medium . Supernatants were harvested after 16–20 h and cytokine production was determined using a Human Th1/Th2 CBA Kit II (BD Biosciences, California, USA) according to the manufacturer's protocol.
Cytotoxicity was tested in standard 4–6 h 51Cr-release assays as described previously . When using EBV-transformed B cells (DO5) as targets (electroporated with 30 μg/100 μl HIV-1-gag RNA; electroporation settings 500V/3 ms), 51Cr labeling was performed overnight. After 4–6 h, the percentage cytolysis was calculated from the 51Cr release as follows: [(measured release − background release)]/[(maximum release − background release)] × 100%.
T cells, transfected with an HIV-1-specific T-cell receptor, produce cytokines in an antigen-specific manner
To test whether ex-vivo generated CD8+ T cells could be functionally reprogrammed with the specificity for HIV-1-pol and HIV-1-gag, these T cells were transfected with RNA encoding either the HIV-1-pol-specific TCR, the HIV-1-gag-specific TCR, or a control TCR, and were stimulated with T2 cells loaded either with the HIV-1-pol-peptide ILKEPVHGV, the HIV-1-gag-peptide SLYNTVATL, or with a control peptide. T cells, transfected with HIV-1-pol-TCR RNA or HIV-1-gag-TCR RNA, specifically produced interferon-γ (IFNγ) (Fig. 1a, b), tumor necrosis factor-α (TNFα), and interleukin 2 (IL-2) (data not shown) after stimulation with T2 cells loaded with the corresponding peptides, whereas the use of a control peptide or a control TCR resulted in substantially lower production of these cytokines (Fig. 1a and b, and data not shown). Furthermore, TCR-transfected T cells produced different cytokines (i.e. TNFα and IFNγ) simultaneously, specifically proliferated after stimulation, and 70% of the T cells upregulated CD25 surface expression after antigen-specific stimulation (data not shown). Taken together, these data indicate that the HIV-1 specificity of the original CTL clones, from which the HIV-1-pol and HIV-1-gag-specific TCR were generated, was indeed functionally transferred to ex-vivo generated bulk T cells by RNA electroporation.
T-cell receptor-RNA-electroporated T cells obtain an HIV-1-specific lytic capacity
The main aim of the generation of reprogrammed CD8+ T cells is to facilitate lysis of HIV-1-infected target cells. Therefore, we tested whether TCR-RNA-transfected CD8+ T cells were able to lyse targets that presented the HIV-1-pol-derived or the HIV-1-gag-derived peptide. The TCR-reprogrammed T cells were used in a chromium-release assay with peptide-loaded T2 target cells. HIV-1-pol-peptide-loaded and HIV-1-gag-peptide-loaded T2 cells were specifically lysed at all targets to effector ratios by HIV-1-pol-TCR-transfected (Fig. 2a) and HIV-1-gag-TCR-transfected (Fig. 2b) T cells, respectively, whereas presentation of a control peptide or use of a control receptor resulted in no lysis (Fig. 2a, b). Furthermore, HIV-1-pol-TCR-positive T cells lysed peptide-loaded target cells over 3 days, but no specific lysis was detected 1 week after electroporation (data not shown). These data demonstrate that the HIV-1-TCR-transfected T cells also obtained a new antigen-specific cytolytic capacity.
T-cell receptor-reprogrammed CD8+ T cells respond to target cells presenting endogenously processed antigen with cytokine production and lysis
As TCR transfer can result in T cells with lower avidity compared with the parental T-cell clone [40,48], the avidities of the HIV-1-pol- and HIV-1-gag-TCR-RNA-transfected CD8+ T cells and the parental CTL were directly compared in peptide-dilution assays. The peptide concentration corresponding to the arithmetic mean between background lysis and maximum lysis (i.e. ED50) was taken as a measure for avidity. The ED50 of the HIV-1-pol- and HIV-1-gag-TCR-RNA-transfected T cells was about 20 and about 3 ng/ml, respectively, whereas the ED50 of the corresponding parental CTL was about 2 and about 0.3 ng/ml, respectively (data not shown). As this lower functional avidity of the TCR-reprogrammed T cells might not be sufficient to recognize endogenously processed HIV-1 epitopes, we electroporated EBV-transformed B cells with HIV-1-gag-encoding RNA and used these as target cells. HIV-1-gag-TCR-RNA-transfected T cells specifically produced IFNγ (Fig. 1c), TNFα, and IL-2 (data not shown) after stimulation with the HIV-1-gag-RNA-electroporated EBV-transformed B cells. As a positive control, the target cells were loaded with the HIV-1-gag peptide (Fig. 1c). Background levels of cytokines were produced by HIV-1-gag-TCR-transfected T cells stimulated with control-peptide-loaded target cells, and by control-TCR-transfected T cells (Fig. 1c). Furthermore, we determined the cytolytic capacity of HIV-1-gag-TCR-transfected T cells against these B cells presenting the endogenously processed HIV-1-gag epitope. These TCR-reprogrammed T cells specifically recognized and lysed the HIV-1-gag-RNA-transfected target cells (Fig. 2c). Control-TCR-transfected T cells did not lyse the HIV-1-gag-RNA-transfected target cells, and control-peptide-loaded targets were not recognized (Fig. 2c). From these data, we conclude that the HIV-1-specific TCR-reprogrammed T cells did even recognize target cells that present the naturally processed epitope, resulting in cytokine production and cytolysis.
Until now, the method of choice for the transfer of HIV-1-specific TCR into patient-derived T cells was retroviral transduction [33,34]. The drawback of retroviral transduction in clinical settings is the introduction of stable genetic alterations with all associated risks. Therefore, we used the method of RNA electroporation, already described for tumor-specific TCR [40–45], which completely excludes the possibility of chromosomal integration. As a proof of principle, we used two TCR specific for the highly conserved HIV-1-pol sequence ILKEPVHGV and HIV-1-gag sequence SLYNTVATL [17,19].
The functional reprogramming of CD8+ T cells against HIV-1 peptides was proven by specific secretion of proinflammatory cytokines (IL-2, IFNγ, and TNFα) and specific cytotoxic activity (Figs 1 and 2). The time-span of 3 days, during which this lytic capacity was maintained, would suffice to enter lymph nodes, where HIV-1-infected targets would be present .
The lytic threshold of the TCR-RNA-transfected T cells required an approximately 10-fold higher peptide concentration than the parental CTL. A similar decrease in avidity has been observed previously with cancer-antigen-specific TCR, transferred with retroviral systems,  or by RNA electroporation . A possible explanation may be that CTL clones are highly differentiated effector T cells. It is known that these cells only need small amounts of presented peptide to lyse their target cells [49,50]. In contrast, we electroporated unstimulated bulk T cells comprising all different phenotypes, and these cells may have a higher stimulation threshold. Nevertheless, we found that the HIV-1-gag-TCR-RNA-transfected T cells recognized endogenously processed antigen, and subsequently produced cytokines and lysed the target cells (Fig. 2). A possible approach to increase the percentage of T cells with an effector phenotype would be to stimulate the T cells prior to electroporation, for example, with cytokines and/or CD3 and CD28 binding beads. This approach will also expand the T cells, providing larger numbers for electroporation [51,52]. This may be necessary to generate sufficient quantities of CD8+ T cells from immunologically impaired HIV-1-infected patients.
A potential threat in immunotherapy, and especially in adoptive T-cell and TCR transfer, is the generation of autoimmunity. The introduction of an allogeneic TCR can create autoimmune specificities in different ways: mispairing of exogenous and endogenous TCR chains resulting in an unpredictable, possibly autoreactive specificity [35–38,48,53], the introduced TCR is cross-reactive with host antigens or major histocompatibility complex haplotypes, or the introduced TCR activates otherwise silent autoreactive T cells. All these possibilities of autoimmunity can occur by retroviral transduction as well as by RNA transfection. However, while the former could generate lasting autoimmunity (exemplified by a clinical trial using T cells retrovirally transduced with a chimeric receptor, which lysed cells of the bile ducts unexpectedly expressing the antigen ), the latter strategy results in loss of the exogenous TCR chain after few days. The RNA approach allows a rapid and safe identification of clinically relevant TCR in small clinical trials, and selected TCR could then be further pursued.
In the present study, it was shown for the first time that it is possible to generate functional virus-specific CTL by electroporation with RNA, which encoded the α and β chains of a virus-specific TCR. The generated HIV-1-specific T cells will probably lead to a better understanding and an increased knowledge about the role of CD8+ T cells in HIV-1 infection and AIDS, and this technology represents an innovative, safe, and easy method to produce virus-specific T cells.
We thank the ELAN-Fonds of the Friedrich-Alexander-University Erlangen-Nuremberg (DE-06.03.29.1), the DFG– German Research Foundation (Collaborative Research Centre SFB643, Project C1), the DFG-Graduiertenkolleg 1071 (Viruses of the Immune System, Project B1), DFG-grant HA 2331/2-1, the German Competence Network for HIV/AIDS (HIVNET), and the IZKF Erlangen (T. Harrer, project A27) for financial support. We thank Argos Therapeutics for providing us with HIV-gag RNA. We thank Stefanie Baumann, Ina Müller, Tanja Schunder, and Verena Wellner for excellent technical assistance, Katrin Birkholz and Stefanie Hoyer for fruitful discussions, and Gabi Theiner and Steve Voland for providing reagents. We thank Martina Schmid, Michael Erdmann, Stina Rosenheinrich, Doris Schuster, Sandra Schiemann, and Hans Simon for acquiring blood samples. Principle contributions made by the authors: Christian Hofmann (study design, conception, and performance), Thomas Harrer (study design and conception), Verena Kubesch (study performance), Katja Maurer (study performance), Karin J. Metzner (study performance) Kathrin Eismann (study performance), Silke Bergmann (study performance), Matthias Schmitt-Haendle (study performance), Gerold Schuler (study design and conception), Jan Dörrie (study design, conception, and performance), Niels Schaft (study design, conception, and performance).
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