Transduction with the Antioxidant Enzyme Catalase Protects Human T Cells against Oxidative Stress

Takashi Ando, Kousaku Mimura, C. Christian Johansson, Mikael G. Hanson, Dimitrios Mougiakakos, Charlotte Larsson, Telma Martins da Palma, Daiju Sakurai, Håkan Norell, Mingli Li, Michael I. Nishimura and Rolf Kiessling
J Immunol December 15, 2008, 181 (12) 8382-8390; DOI: https://doi.org/10.4049/jimmunol.181.12.8382

Abstract

Patients with diseases characterized by chronic inflammation, caused by infection or cancer, have T cells and NK cells with impaired function. The underlying molecular mechanisms are diverse, but one of the major mediators in this immune suppression is oxidative stress caused by activated monocytes, granulocytes, or myeloid-derived suppressor cells. Reactive oxygen species can seriously hamper the efficacy of active immunotherapy and adoptive transfer of T and NK cells into patients. In this study, we have evaluated whether enhanced expression of the antioxidant enzyme catalase in human T cells can protect them against reactive oxygen species. Human CD4+ and CD8+ T cells retrovirally transduced with the catalase gene had increased intracellular expression and activity of catalase. Catalase transduction made CD4+ T cells less sensitive to H2O2-induced loss-of-function, measured by their cytokine production and ability to expand in vitro following anti-CD3 stimulation. It also enhanced the resistance to oxidative stress-induced cell death after coculture with activated granulocytes, exposure to the oxidized lipid 4-hydroxynonenal, or H2O2. Expression of catalase by CMV-specific CD8+ T cells saved cells from cell death and improved their capacity to recognize CMV peptide-loaded target cells when exposed to H2O2. These findings indicate that catalase-transduced T cells potentially are more efficacious for the immunotherapy of patients with advanced cancer or chronic viral infections.

Patients and experimental animals with chronic bacterial and viral infections, autoimmune diseases including rheumatoid arthritis and lupus erythematosus, solid tumors, and hematologic malignancies have T cells and NK cells with impaired function and modified receptor repertoires (1, 2, 3). The magnitude of dysfunction is most severe in the local microenvironment of inflammatory lesions and tumors, but can extend to circulating T and NK cell populations (1, 4, 5). This immune dysfunction leads to diminished responses to recall Ags (6), decreased proliferative T cell responses (7), loss of cytokine production (7, 8, 9, 10, 11), and defective signal transduction in T cells and NK cells (2, 4, 7, 12, 13, 14, 15, 16, 17, 18, 19). There is also evidence for increased apoptosis among CD8+ T cells in PBL from cancer patients and tumor- bearing mice (7, 20). In cancer patients, these alterations correlate with disease severity and poor survival (4, 16, 21, 22, 23).

Several mechanisms may account for immune abnormalities. These include Fas-FasL interaction, resulting in T cell apoptosis involving caspase 3-mediated cleavage of CD3ζ (24) and selective loss of STAT5a/b expression (25). Tumor-derived gangliosides, inducing defective NF-κB activation (26), and absence of essential nutrients, such as l-arginine (27) and l-tryptophan (28), have also been shown to play major roles. Reactive oxygen species (ROS)4 produced by myelomonocytic cells have recently emerged as a potentially important immune suppressive mechanism in tumor-bearing hosts. Splenic macrophages from tumor-bearing mice (29), macrophages isolated from metastatic lesions of human melanomas (30), or activated granulocytes derived from peripheral blood of cancer patients (5) were found to induce loss of T cell and NK cell function. Concomitantly, defects in receptor-associated signaling molecules were induced. Oxidative stress can also induce defects in NF-κB activation in T cells (31, 32), which are reminiscent of those observed in T cells from cancer patients (19, 33). Oxidized lipids such as 4-hydroxynonenal that are present during oxidative stress have been reported to induce defects in NF-κB signaling as well as to induce apoptosis (34, 35). Furthermore, it has been shown that children with chronic hepatitis B or C have low levels of catalase, superoxide dismutase, and glutathione peroxidase activity in peripheral blood erythrocytes, indicating elevated levels of oxidative stress (36). It has also been demonstrated that glutathione levels is decreased in plasma, lung epithelial lining fluid, and T cells of HIV patients (37). Thus, treatments aimed at reversing immune suppression may involve targeting the altered redox status in patients with cancer or chronic viral infections, potentially normalizing the function of endogenous or adoptively transferred effector cells.

In this study, we suggest adoptive transfer of lymphocytes, rendered resistant to ROS by transduction with antioxidative enzymes, as an improved therapeutic modality for cancer and chronic viral infections. Using a retroviral vector, we increased the intracellular levels of the antioxidant enzyme catalase in human CD4+ and CD8+ T cells and thereby enhanced their resistance to ROS. Catalase transduction made CD4+ T cells less sensitive to H2O2-induced loss-of-function as measured by their cytokine production and ability to expand in vitro following anti-CD3 stimulation. Transduced T cells also displayed increased resilience to oxidative stress-induced cell death after coculture with activated granulocytes and exposure to the oxidized lipid 4-hydroxynonenal or H2O2. Expression of catalase by CMV-specific CD8+ T cells precluded cells from cell death and improved their capacity to recognize CMV peptide-loaded target cells when exposed to H2O2. These findings represent the first “proof-of-principle” that gene therapy approaches can be used to modify human T cells to be more resistant to ROS-mediated immune suppression.

Materials and Methods

Reagents

AIM-V, RPMI 1640, and DMEM cell medium, FBS (FCS), Lipofectamine, PLUS Reagent, NBT, trypan blue stain (0.4%), and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Invitrogen. X-Vivo 15 cell medium was purchased from Lonza. The following reagents were purchased from Sigma-Aldrich: Tween 20, hydrogen peroxide (H2O2), penicillin-streptomycin, human serum albumin, geneticin (G418), DMSO, paraformaldehyde, Triton X-100, EDTA, BSA, PGE2, p-nitrophenylpalmitate, human catalase, HRP, 3-methoxy-4-hydroxyphenylacetic acid (HVA), (E)-4-hydroxynonenal (HNE), PMA, and gelatin. Ficoll-Paque was purchased from GE Health Care and human AB serum was purchased from Valley Biomedical Products & Services. MACS LS columns, human Pan T Cell Isolation kit II, and human CD4 microbeads were purchased from Miltenyi Biotec. Anti-CD3 Ab (OKT-3) was purchased from eBioscience. IL-2 (Proleukin) was purchased from Chiron/Novartis. GM-CSF, IL-4, IL-6, IL-1β, and TNF-α were purchased from PeproTech and CMV peptide (NLVPMVATV, CMV pp65 (495–503)) was purchased from AnaSpec. 7-Aminoactinomycin-D (7-AAD) and annexin V as well as anti-CD3-allophycocyanin and anti-CD4-FITC mAbs were purchased from BD Biosciences. A human IFN-γ ELISA kit, human IFN-γ ELISPOT kit, and streptavidin-ALP-PQ were purchased from Mabtech.

Cells

Peripheral blood was collected from healthy volunteers with informed consent as per a protocol approved by the institutional ethics committee. PBMC were isolated from buffy coats using Ficoll-Paque gradient centrifugation. Monocytes from a CMV-positive HLA-A2 healthy donor were enriched by plastic adherence for 90 min at 37°C. Adherent cells were cultured in X-Vivo 15 medium supplemented with 1000 U/ml GM-CFS and 1000 U/ml IL-4. On day 5, immature dendritic cells (DC) were treated with TNF-α (10 ng/ml), PGE2 (1 μg/ml), IL-1β (10 ng/ml), and IL-6 (1000 U/ml) for 2 days. Mature DC were pulsed with CMV peptide (20 μg/ml) for 4 h at 37°C and subsequently coincubated with autologous PBMC at 1:10 ratio in X-Vivo 15 medium supplemented with 20 IU/ml IL-2 for 48 h. CD8+ T cells were selected from the cocultures using two consecutive cycles of negative selection with immunomagnetic beads enriching T cells and depleting CD4+ cells, respectively. This procedure generated CMV-specific CD8+ T cell lines as determined by ELISPOT. The target cells T2 and C1RA2 were cultured in complete RPMI 1640 medium containing 100 μg/ml l-glutamine, 10% heat-inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin. For autologous coculture experiments, granulocytes were isolated from the pellet obtained after Ficoll-Paque gradient centrifugation of buffy coats. Erythrocytes present in this fraction were depleted in a buffer containing 10 mM KHCO3, 155 mM NH4Cl, and 8.9 μM EDTA.

Retroviral construct

A plasmid (SAMEN-CMV-hCAT) containing the human wild-type catalase gene (hCAT, original plasmid containing the hCAT cDNA sequence was provided by Dr. P. Lemarchand (Medical Faculty, Paris, France)) and components for retroviral production were constructed in the laboratory. The SAMEN-CMV vector is composed of a Moloney murine leukemia virus backbone. The 5′ long terminal repeat (LTR) was modified by replacing the R and U5 segments with a CMV immediate early promoter enhancer (CMV/5′ LTR) to promote high-level transient expression in 293GP packaging cells. A ψ+ packaging signal to allow proper insertion of viral DNA into virus capsules, an internal ribosomal entry site for expression of a geneticin (G418) resistance gene (neo), 3′ LTR for proper insertion of the DNA into T cell genome, and ampicillin resistance gene (AmpR) for propagation of the plasmid in bacteria were all present (see Fig. 1⇓ for details).

FIGURE 1.
FIGURE 1.

Structure of the retroviral vector plasmid and hypothesized insertion of retroviral vector’s DNA into the genomic DNA of T cells. Retroviral vector encoding for the cDNA of wild-type hCAT was used to transduce primary T cells to express the wild-type catalase protein. A, A schematic illustration of the retroviral vector plasmid is depicted. The hCAT retroviruses were created by inserting the hCAT wild-type gene into the SAMEN CMV retroviral vector. The vector is composed of a Moloney murine leukemia virus backbone. The 5′ LTR has been modified by replacing the R and U5 segments with a CMV immediate early promoter enhancer (CMV/5′ LTR) to promote high-level transient expression in 293GP producer cells. Other key elements include the ψ+ packaging signal for proper insertion of viral DNA into the virus capsule, an internal ribosomal entry site (IRES) for expression of a geneticin resistance gene (neo), 3′ LTR for proper insertion of the DNA in to T cell genome, and ampicillin resistance gene (AmpR) for propagation of the plasmid in bacteria. Retroviral vector lacking the hCAT gene was used as control. B, A schematic illustration of the hypothesized insertion of the retroviral vector’s DNA into the genomic DNA of T cells is depicted.

Generation of retroviral supernatants

293GP cells were seeded onto gelatin-coated, 100- mm cell culture dishes in DMEM supplemented with 10% FCS plus 1% penicillin-streptomycin (DMEM-PS) at sufficient density to provide ∼70% confluence after 24 h (∼5 × 106 cells). Culture medium was then removed and replaced with 5 ml of antibiotic-free DMEM with 1% FCS. Reagents for transfection were mixed in two tubes: tube 1: 750 μl of additive-free DMEM, 3 μg of vesicular stomatitis virus envelope plasmid DNA, 3 μg of SAMEN-CMV-hCAT or SAMEN-CMV (control), and 20 μl of PLUS reagent were mixed and tube 2: 750 μl of additive-free DMEM and 30 μl of Lipofectamine were mixed. Both tubes were incubated for 15 min at 20°C, mixed, and incubated for an additional 15 min at 20°C. The mixture was added dropwise to the cells and incubated for 3–5 h at 37°C. Ten milliliters of DMEM-PS was then added to the dish and the cells were cultured overnight. After 24 h, medium was discarded and replaced with 10 ml of DMEM-PS. Retroviruses were harvested twice from these cultures by collecting supernatants every 24 h and replacing them with 10 ml of DMEM-PS. Harvested retroviruses were used immediately in experiments.

Retroviral transduction of polyclonal expanded CD4+ or CMV-specific CD8+ T cells

To allow insertion of the hCAT gene into the genome, CD4+ T cell proliferation was induced before transduction. To induce proliferation in CD4+ T cells, 108 freshly isolated human PBMC were cultured in 100 ml of AIM-V with 600 IU/ml IL-2 and 10 ng/ml OKT-3 Ab for 72 h at 37°C and 5% CO2. The use of OKT-3 as a polyclonal mitogen favored the expansion of CD4+ T cells (>90% CD4+ T cells after stimulation and expansion in the presence of G418). CMV-specific CD8+ T cells were not subject to additional stimulation since they were already activated following coculture with peptide-pulsed DC. T cells were resuspended in retroviral supernatants at a concentration of 1 × 106 cells/ml and supplemented with 600 IU/ml IL-2 and 4 μg/ml polybrene. The cell suspension was added to a 24-well tissue culture plate (1 ml/well) and the plates were centrifuged at 1000 × g for 90 min at 32°C. Following incubation for 4 h, 1 ml of fresh DMEM-PS with 600 IU/ml IL-2 was added to each well. Transduced cells were incubated overnight, and this transduction procedure was repeated the next day with fresh supernatants.

Rapid expansion protocol and selection of transduced cells

A previously established rapid expansion protocol (REP) was used with minor modifications (38). Cryopreserved and thawed allogeneic PBMC from healthy donors were pooled and used as feeder cells after irradiation (50 Gy). In brief, 0.5 × 106 polyclonal CD4+ cells or CMV-specific CD8+ T cells were cocultured for 48 h with 40 × 106 feeder cells in 25 ml of AIM-V medium containing 2% human AB serum and 30 ng/ml OKT-3. Subsequently, 300 IU/ml IL-2 and 0.8 mg/ml G418 were added and cells were incubated for an additional 48 h. Half of the medium was then aspirated and replaced with fresh AIM-V supplemented with 2% human AB serum, 0.8 mg/ml G418, and 300 IU/ml IL-2. rIL-2 (300 IU/ml) was added every third day and cells were spitted if the cell density reached >1.5 × 106/ml. On day 15 of the REP, cells were harvested and analyzed. Expansion of polyclonal T cells generated cell cultures containing >90% CD4+ T cells and expansion of CMV-specific CD8+ T cells produced cell cultures containing >85% CD8+ T cells (data not shown).

ELISPOT

CMV-specific response was determined by IFN-γ ELISPOT analysis using a commercial kit in accordance with the manufacturer’s protocol. Briefly, 96-well plates with nitrocellulose membrane (Millipore) were coated with an anti-IFN-γ capture Ab for 24 h and washed. Target cells (T2), pulsed with CMV or control peptide, and hCAT- or control-transduced CMV-specific CD8+ T cells were coincubated 1:1 in the ELISPOT plate in 200 μl of X-Vivo 15 for 24 h. Thereafter, biotinylated secondary anti-IFN- γ Ab was added for 2 h and the plates were incubated with streptavidin-alkaline phosphatase reagent and stained with NBT and 5-bromo-4-chloro-3-indolyl phosphate. The number of spots was quantified in an ELISPOT reader (AID ELISPOT Reader; AID).

Viability assay

hCAT and control-transduced T cells were seeded in AIM-V medium in a 48-well tissue culture plate (1 × 106 cells/well in 1 ml). Oxidative stress was induced either by addition of H2O2 or the oxidized lipid HNE at different concentrations or by coculture with autologous activated (PMA: 50 ng/ml) granulocytes in different lymphocyte:granulocyte ratios. Cells were incubated at 37°C overnight and oxidative stress-induced cell death was analyzed by flow cytometry as described below.

Cell concentration assay

Control- or hCAT-transduced cells (1 × 106 cells/well in 1 ml in a 48-well tissue culture plate) were exposed to different concentrations of H2O2. After overnight incubation, 30 ng/ml OKT-3 was added to cells and the cells were incubated for an additional 5 days. Numbers of viable cells were measured by trypan blue exclusion in a hemocytometer. Counting was done in a blinded fashion by two investigators.

Catalase activity assay

Catalase activity in lysates of T cells transduced with hCAT or control vector was measured using a fluorescence- based method, in which the inhibition of an H2O2-dependent dimerization of a fluorescent product is quantitated (39). The method measures the accumulation of fluorescent HVA dimers. Transduced T cells (5 × 106) were lysed by freeze thawing in 300 μl of lysis buffer (49.5 ml of 50 mM phosphate buffer, 14.6 mg of 1 mM EDTA, and 0.5 ml of 1% Triton X-100). The lysates were frozen at −20°C until analysis. A reaction mix consisting of 30 mM HVA and 1 IU/ml HRP was prepared in PBS. Eighty microliters of the cell lysates, diluted 1/3 in RPMI 1640 was added to 15 μl of reaction mix and 5 μl of H2O2 (final concentration 300 μM) in a black 96-well flat-bottom plate (Labsystems Cliniplate; Thermo Scientific). Samples were analyzed in duplicates and a standard curve was plotted using serial dilutions of human catalase. HVA dimers were quantitated fluorometrically at 355-nm excitation and 420-nm emission using a Victor multilabel counter (Wallac-PerkinElmer) within 35 min.

ELISA

IFN-γ release by T cells was measured by ELISA using a commercial kit as per the manufacturer’s instructions. Forty-eight hours after stimulation with OKT-3/IL-2, 50 μl of supernatant was taken from hCAT- or control-transduced T cell cultures and diluted 1/30 in PBS. Samples were assayed colorimetrically, in duplicates, at 405 nm using a Versamax microplate reader (Molecular Devices). A standard curve was plotted using recombinant human IFN-γ.

Cytotoxicity assay

The ability of T cells to lyse peptide-loaded C1RA2 cells was measured in a standard 4-h 51Cr release assay. C1RA2 cells were collected and labeled with 51Cr (Amersham Biosciences) for 1 h at 37°C. The cells were then washed and resuspended in X-Vivo 15 medium and incubated with control or CMV peptide at 37°C for 1 h. In a 96-well V-bottom plate, 3000 C1RA2 cells/well were added, followed by T cells at a 50:1 E:T ratio. The experiments were conducted in triplicates. The release of 51Cr was measured 4 h later by quantification of gamma radiation in the supernatant by a gamma counter (Wallac). Specific lysis was calculated according to the formula: percent specific lysis = [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100.

FACS

Cells were immunofluorescently labeled in a V-bottom 96-well plate according to a standard FACS staining protocol. Each sample was stained with 30 μl of Ab mixture (containing, e.g., 7-AAD, anti-CD3-allophycocyanin, and anti-CD4-FITC) at 4°C for 15 min. The cells were washed, resuspended in 200 μl of PBS/paraformaldehyde, and transferred to FACS tubes for analysis. Cells were analyzed using a four-color (FACSCalibur; BD Biosciences) FACS machine. The data analysis was performed using CellQuest Pro (BD Biosciences) and FlowJo software (Tree Star).

Statistical analyses

Results from the different groups were compared by a two-tailed Student t test. A single and double asterisk corresponds to p < 0.05 and p < 0.01, respectively. Correlative significance was determined using the Pearson analysis.

Results

Efficient catalase expression and function after retroviral-mediated catalase gene transfer into primary T cells

In vitro studies have implicated H2O2 as one of the major effector molecules involved in tumor-induced immune suppression. NK cell and T cell function can be protected from the harmful effects of activated monocytes, granulocytes, and myeloid-derived suppressor cells (MDSCs) by exogenously added catalase (5, 30, 40). Catalase consists of a homotetramer which, with heme as a cofactor, catabolizes H2O2 into O2 and H2O (41). Thus, the catalase protein can very efficiently decompose H2O2 and therefore could be used to counteract oxidative stress. To this end, we have developed a retroviral gene delivery system containing the human catalase gene (cDNA, Fig. 1⇑), which enables catalase gene transfer into primary T cells. Anti-CD3 Ab-stimulated polyclonal T cells were transduced with control or hCAT retrovirus and expanded in a REP in the presence of the antibiotic G418 to select for stably transduced cells. This procedure, which mainly produced CD4+ T cells, resulted in eight times higher intracellular catalase activity in hCAT-transduced cells compared with control-transduced T cells (Fig. 2⇓). In addition, CMV-specific CD8+ T cells were generated through stimulation of PBMC-derived T cells by autologous DC pulsed with CMV peptide and transduced with control or hCAT retrovirus before expansion by REP in the presence of G418. When analyzing their intracellular hCAT activity, a significant increase in hCAT activity of hCAT-transduced compared with control-transduced T cells was observed (Fig. 2⇓). However, hCAT activity was lower in these CMV-specific CD8+ T cells as compared with the polyclonally activated CD4+ T cells. Thus, we were able to produce hCAT-transduced CD4+ and CD8+ T cells, which expressed increased levels of functional catalase enzyme.

FIGURE 2.
FIGURE 2.

Lysate from hCAT-transduced T cells has higher ability to decompose H2O2. After 3 days of anti-CD3 Ab stimulation, PBMC were transduced either with hCAT or control retrovirus. Also, CMV-specific CD8+ T (CD8+ TCMV) cells were transduced with hCAT or control retrovirus. The cells were thereafter subjected to REP including G418 as selection agent (see Materials and Methods for details). This procedure favored an accumulation of CD4+ T cells (90% CD4+, 8% CD8+, 2% non-T cells) in the culture using anti-CD3 Ab stimulation and CD8+ T cells (87% CD8+, 13% CD4+) using CMV peptide as initial stimulation. After 15 days of REP, T cells were harvested and 5 × 106 cells were spun at 2000 × g for 5 min. Supernatant was discarded and the cell pellet was resuspended in cell lysis buffer and subjected to three rounds of freezing and thawing in liquid N2. The cell lysate from hCAT- or control-transduced CD4+ or CD8+ T cells was diluted in PBS and the ability of the lysate of CD4+ or CD8+ T cells to decompose H2O2 was determined. This figure shows one representative experiment of three and the bars show the SD.

Catalase gene transfer into CD4+ T cells improves T cell function and viability after H2O2 exposure

To test whether transduction of the catalase gene into primary polyclonal (CD4+) T cells could improve the ability of the cells to resist oxidative stress, the function of the expanded hCAT- or control -transduced CD4+ T cells after exposure to H2O2 was determined. This was performed by exposing CD4+ T cells to increasing concentrations of H2O2, followed by their stimulation with anti-CD3 Abs 1day later. The results showed a significant difference in the production of IFN-γ between hCAT CD4+ and control CD4+ T cells after H2O2 exposure (Fig. 3⇓A) following 3 days of anti-CD3 stimulation. A severe (81%) decrease of IFN-γ secretion was apparent in the H2O2-exposed control CD4+ T cells at 200 μM, as compared with unexposed control CD4+ T cells. In contrast, the hCAT CD4+ T cells only showed a 37% decrease in IFN-γ secretion. This difference in the ability of control and hCAT CD4+ T cells to withstand H2O2, although most prominent at 200 μM H2O2, was statistically significant at all doses of H2O2. Moreover, on exposure to increasing concentrations of H2O2, significantly higher relative cell numbers were recovered from 6-day cultures of anti-CD3-stimulated hCAT CD4+ T cells as compared with control CD4+ T cells (Fig. 3⇓B).

FIGURE 3.
FIGURE 3.

hCAT transduction improves CD4+ T cell function after H2O2 exposure. T cells were transduced with hCAT or control retrovirus. After 15 days of REP, cells were harvested and resuspended to 1 × 106 cells/ml in fresh X-Vivo 15 with 2% AB serum medium containing increasing concentrations of H2O2. A, T cells were left untreated or exposed to 0, 100, 200, or 300 μM H2O2 for 24 h (day 0) before anti-CD3 Ab activation (day 1). After 2 days (day 3), supernatants from the cultures were collected and the concentration of IFN-γ was measured in ELISA. The relative concentration of IFN-γ produced by H2O2-exposed T cells compared with untreated cells is shown. B, T cells were left untreated or exposed to 0, 25, 50, 100, 150, or 200 μM H2O2 for 24 h before anti-CD3 Ab activation as in A. After 5 days (day 6), the cell concentration of viable T cells was determined by manual cell counting. B, The relative cell concentration of viable cells compared with untreated cells. A and B, One representative experiment of three and the bars show the SD.

It has been previously reported that H2O2, in relatively low concentrations, can induce cell death in T cells (42, 43). To test whether hCAT CD4+ T cells would be more resistant to H2O2-induced cell death, control- or hCAT-transduced CD4+ T cells were exposed for a short term (24 h) to increasing concentrations of H2O2 and their viability was assessed by flow cytometry (Fig. 4⇓A). Control-transduced CD4+ T cells exhibited an increased cell death (7-AAD+, shift in forward scatter/side scatter) starting at 100 μM H2O2 and at 200 μM H2O2 only 50% of the cells were viable (7-AAD−, no shift in forward scatter/side scatter) compared with 91% viability in untreated cells. In marked contrast, catalase expression by CD4+ T cells greatly improved their resilience to H2O2-induced cell death, with only a marginal loss in viability when cells were treated with 200 μM H2O2 (90% viability) compared with untreated cells (95% viability) (Fig. 4⇓A). Next, control- and hCAT-transduced CD4+ T cells were cultured in the presence of H2O2 (150 μM) for 1, 2, 3, and 4 days to determine for how long a time catalase transduction would protect T cells from H2O2-induced cell death. As shown in Fig. 4⇓B, after 3 days of exposure to H2O2, catalase could still provide resistance to H2O2-induced cell death (70% viable cells). At day 4, cell death increased markedly and catalase was no longer protective. To confirm that the improved ability of the CD4+ T cells to resist cell death was due to their increased catalase activity, T cells from the same donor were transduced with different amounts of hCAT or control retrovirus and the different cultures were analyzed for both their intracellular catalase activity and their sensitivity to H2O2 exposure. The results showed a strong correlation between intracellular catalase activity and the ability to withstand H2O2-induced cell death (Fig. 4⇓C). These experiments indicate that transduction of the catalase gene into CD4+ T cells partially abrogated the H2O2-induced decrease in IFN-γ production, sustained the ability of T cells to proliferate in the presence of H2O2, and rescued cells from H2O2-induced cell death.

FIGURE 4.
FIGURE 4.

hCAT transduction rescues cells from H2O2-induced cell death. T cells were transduced with hCAT or control retrovirus. After 15 days of REP, cells were harvested and resuspended to 1 × 106 cells/ml in fresh AIM-V medium containing different concentrations of H2O2. A, T cells were left untreated or exposed to 50, 100, 150, or 200 μM H2O2 for 24 h. This figure shows one representative experiment of three. B, T cells were exposed to 150 μM H2O2 for 1–4 days. The cells were then stained with 7-AAD and subsequently analyzed in FACS. This figure shows one representative experiment of three. C, Anti-CD3-activated PBMC from the same donor were transduced with control (•) or different amounts of hCAT (▴) retrovirus and propagated in a REP. After 15 days of REP, the cells were harvested and the ability of the lysate to decompose H2O2 was determined. This rendered four different CD4+ T cell sublines with different intracellular hCAT activity. The T cells were then exposed to 200 μM H2O2 for 24 h and further analyzed in FACS for cell viability. The correlation of intracellular catalase activity (x-axis) and the percent viable cell of total lymphocytes (y-axis) are shown.

Catalase-transduced T cells are protected against oxidized lipid-induced apoptosis

Oxidized lipids such as HNE are present during oxidative stress and can induce apoptosis (34, 35). Therefore, we examined whether catalase could protect T cells from HNE-induced apoptosis. Control- and hCAT-transduced polyclonally expanded PBL were incubated for 24 h at different concentrations of HNE and subsequently cells were subjected to flow cytometry analysis and apoptosis was examined on gated CD4+ and CD8+ T cell populations. HNE induced apoptosis in a concentration-dependent manner in both T cell subsets and. importantly, catalase could fully protect both CD4+ (Fig. 5⇓A) and CD8+ (Fig. 5⇓B) T cells from HNE-induced apoptosis as compared with control T cells. Additionally, we observed that CD8+ T cells were more sensitive to HNE-induced apoptosis as compared with CD4+ T cells.

FIGURE 5.
FIGURE 5.

hCAT-ransduced T cells are protected from apoptosis induced by oxidized lipid. Polyclonally expanded PBL were transduced with hCAT or control retrovirus. After 15 days of REP, cells were harvested and resuspended to 1 × 106 cells/ml in fresh AIM-V medium containing different concentrations (0, 5, 10, or 20 μM) of the oxidized lipid HNE and cultured for 24 h. PBL were then stained with annexin V and 7-AAD and subsequently analyzed by FACS and gated on CD4+ (A) or CD8+ (B) T cells. This figure shows one representative experiment of three.

Catalase rescues T cells from granulocyte-mediated cell death

Next, we explored whether catalase could overcome the immunosuppressive activity mediated by autologous activated granulocytes and protect T cells from cell death. Control- and hCAT-transduced polyclonally expanded PBL were cocultured with an increasing number of PMA-activated granulocytes for 24 h and, subsequently, cell death was analyzed in gated CD4+ and CD8+ T cells by FACS. Catalase expression protected both CD4+ (Fig. 6⇓A) and CD8+ (Fig. 6⇓B) T cells from granulocyte-induced cell death as compared with cells transduced with control vector.

FIGURE 6.
FIGURE 6.

hCAT-transduced T cells are protected from granulocyte-mediated cell death. Polyclonally expanded PBL were transduced with hCAT or control retrovirus. After 15 days of REP, cells were harvested and resuspended at 1 × 106 cells/ml in fresh AIM-V medium and cocultured with fresh autologous activated (PMA: 50 ng/ml) granulocytes in different PBL:granulocyte ratios (1:0, 1:0.25, 1:0.5, and 1:1 ratio) for 24 h. PBL were then stained with 7-AAD and analyzed by FACS and gated on CD4+ (A) or CD8+ (B) T cells. This figure shows one representative experiment of three.

Catalase-transduced Ag-specific CD8+ T cells are more resistant to oxidative stress

Previous studies have demonstrated that adoptively transferred CD8+ CTLs can eradicate tumors (44) and chronic virus infections (45, 46). Because advanced cancer and chronic virus infections are associated with oxidative stress (5, 29, 30, 47, 48), we examined whether catalase gene transfer can improve the ability of CD8+ T cells to resist an H2O2 -induced decrease of function. CMV-specific HLA-A2-restricted CD8+ T (CD8+ TCMV) cells were transduced with hCAT or control retrovirus and expanded in a rapid expansion protocol. In a series of experiments, control- and hCAT-transduced CD8+ TCMV cells were exposed to increasing concentrations of H2O2 for 24 h. The following day, the capacity of these CD8+ TCMV cells to recognize CMV peptide-pulsed target cells, as measured by IFN-γ ELISPOT assay (Fig. 7⇓A) and to lyse CMV peptide-loaded target cells, as measured in a standard 51Cr release assay (Fig. 7⇓B), was analyzed. At concentrations of 100 μM or higher, H2O2 significantly reduced the number of Ag-specific, IFN-γ- secreting, control CD8+ TCMV cells (Fig. 7⇓A). This loss-of-function was not noted in hCAT CD8+ TCMV cells even when exposed to the highest concentration of H2O2 (150 μM), demonstrating that catalase transduction in CD8+ T cells conferred complete protection to high-dose H2O2. Furthermore, hCAT expression significantly improved the ability of hCAT CD8+ TCMV cells to lyse target cells at all H2O2 concentrations analyzed compared with control CD8+ TCMV cells. In conclusion, only minor loss-of-function was noted in hCAT CD8+ TCMV cells even when exposed to high levels of H2O2, demonstrating that catalase transduction in CD8+ T cells mediated protection to H2O2.

FIGURE 7.
FIGURE 7.

hCAT-transduced CMV-specific CD8+ T cells withstand H2O2-induced loss-of-function and cell death. CMV-specific CD8+ T (CD8+ TCMV) cells were transduced with hCAT or control retrovirus. After 15 days of REP, cells were harvested and resuspended to 1 × 106 cells/ml in fresh X-Vivo 15 with 2% AB serum medium containing different concentrations of H2O2. A, CD8+ TCMV were left untreated or exposed to 75, 100, 125, or 150 μM H2O2 for 24 h. Cells were then carefully resuspended and a set volume was transferred to an anti-IFN-γ mAb-coated ELISPOT plate containing T2 cells prelabeled with CMV or control peptide and cultured overnight. A, The relative number of spots compared with untreated cells according to the formula (no. of spotsT cell + T2 + CMV-peptide at X μM H2O2 − no. of spotsT cell + T2 + control-peptide at X μM H2O2)/(no. of spotsT cell + T2 + CMV-peptide at 0 μM H2O2 − no. of spotsT cell + T2 + control-peptide at 0 μM H2O2). One representative experiment of three is shown and the bars show the SD. B, CD8+ TCMV were left untreated or exposed to 100, 150, or 200 H2O2 for 24 h. Cells were then carefully resuspended and a set volume was then transferred to a 96-well plate containing 3000 51Cr-labeled target cells pulsed with control or CMV peptide. Specific lysis of target cells was assessed in a standard 4-h 51Cr release assay. B, One representative experiment of three and the bars show the SD. C, CD8+ TCMV were left untreated or exposed to 50, 100, 125, or 150 μM H2O2 for 24 h. The cells were then stained with 7-AAD and subsequently analyzed in FACS. C, Dot plots of total lymphocytes. The percent viable cells (7-AAD−/no shift in forward scatter) are indicated in each dot plot.

The effect of H2O2 on the viability of hCAT- or control-transduced CD8+ TCMV cells was examined by exposing the cells to increasing concentrations of H2O2 for 24 h (Fig. 7⇑C) followed by flow cytometric analysis. Control CD8+ TCMV cells showed a marked decrease in viability starting at 75 μM H2O2 and, at 150 μM H2O2, only 22% of the cells were viable. In contrast, hCAT CD8+ TCMV cells were more resistant to cell death upon H2O2 exposure (96% viable cells in the absence of H2O2, 82% viable cells at 150 μM H2O2). Thus, transduction of the catalase gene into Ag-specific CD8+ T cells conferred almost total resistance to H2O2-induced loss-of-function and cell death.

Discussion

Genetic engineering of T cells has had the main focus on prolonging the life span and efficacy upon adoptive transfer of CTLs in the absence of Ag-specific Th cells or cytokine infusions. Examples of this include transduction of human CTLs with chimeric GM-CSF-IL-2 receptors that deliver an IL-2 signal when they bind GM-CSF (49), engineering of T cells to express CD28 (50), the catalytic subunit of telomeras (51), or T cell receptors specific for tumor-associated Ags (52). We here show that genetic engineering of CD4+ and CD8+ T cells enabling expression of an antioxidant enzyme can render them resistant to immune suppression mediated by oxidative stress. We demonstrate that catalase transduction rendered both CD4+ and CD8+ T cells less sensitive to loss-of-function and cell death induced by coculture with activated granulocytes and addition of oxidized lipid (HNE) or H2O2. Gene transfer of catalase into CMV-specific CD8+ T cells rescued cells from cell death and improved their capacity to recognize CMV peptide-loaded target cells when exposed to H2O2. These findings are pertinent to approaches for adoptive T cell therapy in patients with advanced cancer or chronic infections. Furthermore, we believe that the doses of H2O2 used in this study are physiologically relevant at inflammatory sites, as we have recently shown that activated granulocytes release H2O2 in concentrations that would be sufficient to induce T cell death (100–200 μM/1 × 106 cells in 100-μl total volume; K. Mimura and M. G. Hanson, unpublished observation). A study from Test et al. (53) support this observation. Thus, we believe that the local concentration of H2O2 in vivo may reach or even be higher in the immediate vicinity of the T cell than the doses used in this study.

The approach to transfer antioxidative enzyme genes was previously shown to protect lung tissue against hyperoxia-induced injury (54), pancreatic islet cells against oxidant stress (55), and neural cells against ROS-induced cell damage (56). In this study, we demonstrate that by using retroviral-mediated gene transfer, T cells are efficiently protected from oxidative stress. This approach could be an effective strategy for prolonging the life of adoptively transferred T and NK cells to patients with advanced cancer or chronic viral infections. Although adoptive immunotherapy based on the transfer of ex vivo-expanded specific CTLs has been shown to be an effective treatment of viral infections (57, 58), adoptive immunotherapy of patients with advanced cancer has so far met with positive, but limited success (59, 60). This limited therapeutic effect of adoptively transferred lymphocytes may be attributable to a variety of factors, including a suboptimal ex vivo cell culture system, limited life span of the injected cells due to “trapping” in capillaries, limited replicative capacity of in vitro-cultured NK and T cells due to immunological “senescence,” or difficulties of the injected cells to traffic to the patient’s tumor (61, 62). Much evidence, however, points at the consequence of active immune suppression mediated by regulatory T cells, MDSCs, or activated granulocytes in limiting the life span of the transferred NK cells or T cells (63). A rapid elimination of the injected cells by factors produced by these immunosuppressive cell types, including H2O2, NO, and arginase, could be a difficult obstacle to overcome in this type of therapy.

Further support for this premise can be gathered from reports that macrophages isolated from metastatic melanoma lesions were shown to block tumor-specific CTLs in vitro and induce decreased CD3ζ expression, effects which could be inhibited by the presence of exogenously added catalase (30). Inhibition of H2O2 production by activated monocytes can also be achieved through histamine or histamine analogs, a principle that has been successfully applied in clinical trials in cancer patients (64, 65). Furthermore, previous studies demonstrated that direct interaction of macrophages from spleens of tumor-bearing mice secreted H2O2 that induced decreased CD3ζ expression and concomitant loss of T cell function (29). Elimination of NK cell and T cell activities by oxidative stress may however also occur systemically, as it was demonstrated that peripheral blood of cancer patients contain activated granulocytes and the presence of these cells was paralleled with an increased lipid peroxidation (66). The latter finding is of particular importance, because it indicates that systemically produced ROS could be the major cause of severe systemic T cell suppression.

As an alternative to the approach we have taken here, drug-induced blocking of the harmful effects on T cells and NK cells mediated by components produced by myelomonocytic cells have been successfully used in experimental and clinical settings. In MDSCs, l-arginine is metabolized mainly by arginase 1 and NO synthase 2 (67). It has been demonstrated that the combination of arginase 1 and NO synthase 2 inhibitors or peroxynitrite scavengers can block the immune suppressive activity of MDSCs and restore specific T cell responses in mouse tumor models (68, 69, 70) as well as during chronic infection with helminthes (71). Another example of drug-induced normalization of the immune systems is the increased Th1 cytokine production and enhanced NK cytotoxicity observed in peripheral blood of patients with advanced colorectal cancer as a result of short-term supplementation with high doses of dietary vitamin E, a known antioxidant (72, 73). Systemic administration of drugs is however limited by several factors, including the difficulty of drugs to penetrate into tumors, toxicity due to the relatively high drug concentration needed to reach the necessary concentration in the tumor microenvironment, and by a limited half-life of the administered drug. In contrast, T cells will have the advantage of being able to actively migrate into tumor tissues and have the potential of replicating and remaining in the host for prolonged periods of time (74).

The finding that CMV-specific CD8+ T cells can be protected if engineered to express catalase is particularly interesting. Human PBMC-derived T cells show a difference in their sensitivity to H2O2-induced cell death, where in particular the CD8+ effector memory T cell (CD3+CD8+CCR7−CD45RA−) subset was found to be most sensitive to cell death induced by low doses of exogenous H2O2 (42). The CMV-specific CD8+ T cells included in the present study had a high proportion (94 and 95% for catalase- and control-traduced T cells, respectively) of T cells with this phenotype (data not shown). Since IL-2-expanded tumor-infiltrating lymphocytes used in adoptive transfer setting of patients with advanced cancer have this phenotype (59, 75), it may be beneficial to introduce the catalase gene into these cells. Thus, we argue that catalase-transduced T cells are resistant to elimination through ROS-dependent mechanisms and thereby may increase the efficacy of adoptive T cell transfer when treating cancer or chronic virus infections and that this new approach should be exploited.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This study was supported by grants to R.K. from the Swedish Cancer Society, the Swedish Medical Research Council, the Cancer Society of Stockholm, and by a grant to M.N. from the National Institutes of Health, Grant CA102280.

  • ↵2 T.A. and K.M. contributed equally to this article.

  • ↵3 Address correspondence and reprint requests to Dr. Rolf Kiessling, Department of Oncology and Pathology, Immune and Gene Therapy Laboratory, Cancer Center Karolinska, R8:01, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail address: Rolf.Kiessling{at}ki.se

  • ↵4 Abbreviations used in this paper: ROS, reactive oxygen species; HVA, 3-methoxy-4-hydroxyphenyl acetic acid; HNE, (E)-4-hydroxynonenal; 7-AAD, 7-aminoactinomycin D; DC, dendritic cell; LTR, long terminal repeat; PS, penicillin-streptomycin; REP, rapid expansion protocol; MDSC, myeloid-derived suppressor cell.

  • Received February 28, 2008.
  • Accepted October 20, 2008.

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