T-cell receptor (TCR) gene transfer is an attractive strategy to generate antigen-specific T-cells for adoptive immunotherapy of cancer and chronic viral infection. However, current TCR gene transfer protocols trigger T-cell differentiation into terminally differentiated effector cells, which likely have reduced ability to mediate disease protection in vivo. We have developed a lentiviral gene transfer strategy to generate TCR-transduced human T-cells without promoting T-cell differentiation. We found that a combination of interleukin-15 (IL15) and IL21 facilitated lentiviral TCR gene transfer into non-proliferating T-cells. The transduced T-cells showed redirection of antigen specificity and produced IL2, IFNγ and TNFα in a peptide-dependent manner. A significantly higher proportion of the IL15/IL21-stimulated T-cells were multi-functional and able to simultaneously produce all three cytokines (P<0.01), compared with TCR-transduced T-cells generated by conventional anti-CD3 plus IL2 stimulation, which primarily secreted only one cytokine. Similarly, IL15/IL21 maintained high levels of CD62L and CD28 expression in transduced T-cells, whereas anti-CD3 plus IL2 accelerated the loss of CD62L/CD28 expression. The data demonstrate that the combination of lentiviral TCR gene transfer together with IL15/IL21 stimulation can efficiently redirect the antigen specificity of resting primary human T-cells and generate multi-functional T-cells.
Adoptive T-cell transfer is one of the most effective forms of immunotherapy of cancer and infection.1, 2, 3, 4, 5, 6, 7, 8 The Lymphocyte populations of desired specificity for transformed or infected cells can be isolated and expanded in vitro, followed by adoptive transfer into patients. In some studies cloned T-cells were used to deliver a highly specific cell population, although in vivo survival of these cells was often short lived.9, 10 More recently, transfer of polyclonal T-cell populations into lymphodepleted patients has improved T-cell survival in vivo, which was associated with improved disease control.4, 7 The efficacy of adoptive cellular immunotherapy depends on several factors including T-cell specificity and the ability of transferred cells to engraft efficiently, migrate to disease sites and persist long-term in the host. In the setting of melanoma, there is clear evidence that clinical responses correlated with the long-term persistence of the transferred T-cells.11
The parameters that determine the persistence and in vivo function of adoptively transferred T-cells are currently not fully understood. However, recent experiments using primates showed poor persistence of adoptively transferred T-cells derived from the effector memory pool, whereas T-cells derived from the central memory pool showed long-term persistence.12 Similarly, in a murine infection model, adoptively transferred central memory T-cells had a greater capacity than effector memory cells to proliferate in vivo and provide protective immunity.13 Experiments using a murine T-cell receptor (TCR) transgenic melanoma model showed that continued in vitro antigen activation of transgenic T-cells triggered progressive differentiation into effector T-cells. Surprisingly, T-cell differentiation led to reduced ability of adoptively transferred cells to proliferate in vivo and protect animals against the growth of melanoma tumours.14 Together, these observations support the notion that in vitro T-cell differentiation is likely to impair their functional activity in the adoptive immunotherapy setting.15
The demonstration that cloned TCR genes can be used to produce T-lymphocyte populations of desired specificity offers new opportunities to widen adoptive T-cell therapy to include malignancies and infections that do not naturally stimulate detectable T-cell responses. TCR gene transfer using retroviral vectors provides a strategy to readily redirect the specificity of T-cells against poorly immunogenic antigens of transformed or infected cells.16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Further, it has recently been shown that transfer of affinity-matured TCRs can generate T-cells with enhanced functional activity.30
The first clinical trial using adoptive transfer of TCR gene-modified T-cells in melanoma patients has been published.31 Two out of 15 patients showed persistent high-level T-cell engraftment, which correlated with melanoma regression. Inefficient T-cell persistence was associated with lack of clinical responses in 13 patients. This proof-of-principle trial illustrated the feasibility and potential benefit of TCR gene therapy, while at the same time suggesting that strategies to enhance the in vivo persistence of engineered T-cells may improve clinical response rates.
To date, most TCR gene transfer protocols have used γ-retroviral vectors. Stable genomic integration of retroviral vectors requires full T-cell activation and proliferation during the transduction process. This process requires stimulation through the TCR complex using anti-CD3 antibodies, with or without anti-CD28, to stimulate progression through the cell cycle, followed by a period of in vitro expansion in the presence of interleukin-2 (IL2). During this in vitro activation process, T-cell differentiation occurs and cell-surface molecules important for homing to secondary lymphoid organs (that is, CD62L) or co-stimulation (that is, CD28) are downregulated.
Others have previously shown that lentiviral vectors can successfully transduce minimally proliferating T-cells in the presence of cytokines alone.32, 33 At present it is not clear whether cytokine-mediated lentiviral TCR gene transfer is a suitable strategy to generate fully functional human T-cells of desired antigen specificity.
In this study, lentiviral vector constructs have been generated containing both TCR α- and β-chain genes. We have explored whether the homeostatic cytokines IL2, IL7, IL15 and IL21 can promote lentiviral TCR gene transfer without triggering progressive T-cell differentiation. We have shown that cytokine-mediated TCR gene transfer can produce antigen-specific, poly-functional T-cells that were less differentiated than T-cells produced by the conventional transduction process.
Validation of lentiviral WT1 TCR construct in anti-CD3+IL2-activated primary human T-cells
The pSIN lentiviral vector used encodes the α- and β-chain genes of a TCR specific for a peptide epitope of the Wilms' tumour antigen-1 (WT1) presented by human leukocyte antigen (HLA)-A*0201 class-I molecules. To improve expression in human T-cells, we used hybrid TCR genes containing the human TCR α and β variable regions and murine constant regions.34 The hybrid α and β gene sequences were codon-optimized and linked by a self-cleaving 2A sequence. A schematic diagram of the lentiviral vector is shown in Figure 1a.
In the initial set of experiments with primary human CD8+ bead-sorted T-cells, we used a well-established retroviral transduction protocol, involving full T-cell activation by anti-CD3 antibodies (OKT3) and IL2 to determine the TCR transfer efficiency of the lentiviral construct described above.
Immediately after transduction 29% of CD8+ T-cells expressed the introduced WT1-specific TCR as determined by Vβ2.1 staining of transduced cells, compared with mock-transduced human T-cells with 6.3% endogenous Vβ2.1 expression (Figure 1b). The expression of functional α/β TCRs was confirmed using peptide stimulation followed by intracellular cytokine staining experiments. The TCR-transduced T-cells produced IL2, interferon-γ (IFNγ) and tumour necrosis factor-α (TNFα) in response to the TCR-recognized pWT126 peptide but not an HLA-A2-binding control peptide (Figure 1b).
These experiments confirmed that the lentiviral vector redirected the peptide specificity of primary human T-cells after ‘conventional’ polyclonal activation that is generally used for retroviral transduction.
Cytokine stimulation of T-cells is sufficient for lentiviral TCR gene transfer
We then examined whether cytokines that share the receptors containing the common γ-chain were able to render primary T-cells susceptible to lentiviral TCR gene transfer. Previous studies have shown that IL2 and IL7 were able to facilitate lentiviral transfer of GFP33 and HSV-TK35 into human T-cells. In this study, we explored which of the common γ-chain cytokines IL2, IL7, IL15, IL21 alone, or in combination, was most effective for lentiviral TCR gene transfer.
Purified human CD8+ T-cells were exposed to cytokines before lentiviral transduction and fluorescence-activated cell sorting (FACS) analysis to measure expression of the introduced TCR. Successful TCR gene transfer and surface expression were seen after purified CD8+ T-cells were exposed to the single cytokines IL2, IL7 and IL15 (Figure 2a). The highest transduction efficiency was achieved with IL15 (22% of CD8+ T-cells), which was approximately half of the efficiency seen with conventional activation with anti-CD3 antibodies OKT3 plus IL2 (41% of CD8+ T-cells). Although IL21 alone did not support T-cell survival or lentiviral TCR gene transfer (data not shown), we explored whether it was possible to combine it with the other cytokines. Figure 2a shows that the transduction efficiencies with IL2+IL21, IL7+IL21 or IL15+IL21 were similar to those seen with IL2, IL7 or IL15 alone, indicating that IL21 did not impair lentiviral TCR gene transfer. The TCR expression analysis in five independent experiments showed that the highest percentage of TCR-expressing cells was achieved with IL15 in combination with IL21, followed by IL15 combined with IL7 (Figure 2b). The advantage of these two cytokine combinations was even more apparent when the total number of TCR-expressing T-cells after transduction of a fixed number of primary human T-cells was assessed. Transduction in the presence of IL15+IL21 and IL15+IL7 generated substantially more TCR-expressing cells than any of the other cytokines (Figure 2c). Again IL15+IL21 was superior to IL15+IL7, although this was not statistically significant.
Antigen specificity of non-proliferating T-cells can be re-directed by lentiviral TCR gene transfer
Next, we performed experiments to explore whether lentiviral TCR gene transfer and cell-surface TCR expression required T-cell proliferation. Carboxyfluorescein succinimidyl ester (CFSE)-labeled human T-cells were stimulated with IL2, IL7 or IL15 alone or in combination with IL21 and infected with lentiviral TCR vectors. The CFSE dilution profile was analysed after 72 h, the time point of lentiviral infection, and after 120 h, the time point when TCR expression was measured by FACS. Figure 3a shows that after 72 h of cytokine exposure the vast majority of cells were undivided, whereas some of the cells activated by anti-CD3 plus IL2 had undergone two divisions. After 120 h, the majority of the CD3-stimulated T-cells had divided extensively, whereas only a small number of cytokine-stimulated cells had undergone two divisions.
To test directly whether transduction of undivided cells occurred, WT1-specific TCR expression (as determined by Vβ2.1 staining) was measured on gated CFSE-high cells (undivided cells) and the CFSE-low cells that had undergone two divisions. Figure 3b shows the analysis of IL15+IL21-stimulated T-cells, indicating that 27% of the CD8+ population expressed Vβ2.1. The Vβ2.1 expression in non-divided cells was 17% and in the divided T-cell population it was 38%. Subtracting the level of endogenous Vβ2.1 expression seen in the mock-transduced T-cells (Figure 3b) indicated that the introduced TCR was expressed in 16% of all CD8+ T-cells, in 7% of non-divided and in 25% of the divided T-cells. Although cytokine stimulation reproducibly promoted lentiviral TCR gene transfer in non-divided T-cells (Figure 3c), the efficiency appeared to be substantially greater in the divided T-cell population (Figure 3b). In the experiments described lentiviral infection was performed 72 h after cytokine stimulation, a time point when cell division was undetectable (Figure 3a). Nevertheless, flow-cytometric analysis performed 48 h later (120 h after cytokine stimulation) showed preferential TCR expression in the divided T-cell population (Figure 3b). This suggests that at 72 h the lentiviral vectors preferentially infected human T-cells that were committed to subsequent proliferation. This is consistent with a previous study showing that lentiviral GFP vectors preferentially transduced human T-cells that had progressed from the G0 into the G1 phase of the cell cycle.33
Cytokine-stimulated, TCR-transduced T-cells are less differentiated than CD3-activated, TCR-transduced T cells
To determine the effect of the common γ-chain cytokines on the phenotype of TCR-transduced T-cells, we focused on the expression of CD62L and CD28. CD62L was used because its loss was associated with decreased tumour protection by adoptively transferred T-cells, and in several murine and primate models it was used to select T-cell subpopulations that survived and persisted after cell transfer.12, 13, 14 The CD28 co-receptor was used because its loss is a feature of fully differentiated CD8+ T-cells with poor proliferative potential.36, 37
As expected, only subtle differences in CD62L and CD28 expression were seen in freshly transduced T-cells. However, following antigen stimulation in the presence of cytokines striking differences were observed (Figure 4a). A substantial loss of CD62L and CD28 was seen in 68% of T-cells that were transduced by CD3 stimulation followed by antigen stimulation in the presence of IL2. In contrast, loss of CD62L and CD28 was less pronounced (42%) when cells were transduced and antigen-stimulated with IL2. Transduction and antigen stimulation in the presence of IL7 maintained CD62L and CD28 expression in 51% of the cells, whereas only 21% of T-cells were CD62L/CD28-positive after transduction and stimulation with IL15. Interestingly, IL7 was unable to prevent the IL15-induced loss of CD62L/CD28 expression. In contrast, IL21 was able to prevent the IL15-driven loss of CD62L/CD28, as 62% of cells retained expression of these molecules. Similarly, IL21 increased the percentage of CD62L/CD28-expressing cells when used in combination with IL2 or IL7. The fold increase in the expression of CD28 (as measured by mean fluorescence intensity) was significantly greater after transduction in the presence of IL15 and IL21 as shown in Figure 4b. Together, these data indicate IL21 in combination with other common γ-chain cytokines was most effective in maintaining high levels of CD62L and CD28 expression in TCR-transduced human T-cells.
Cytokine-stimulated T-cells are multi-functional
Finally, we analysed the ability of freshly transduced human T-cells to produce IL2, IFNγ and TNFα upon stimulation with the TCR-recognized peptides. In particular, we tested the ability of T-cells to simultaneously produce two or three cytokines, as this has been associated with protective T-cell immunity against infections in humans and mice.38, 39
Purified CD8+ T-cells were transduced using cytokine exposure, or CD3 activation with OKT3, followed by stimulation with the TCR-recognized pWT126 peptides. Intracellular staining was used to measure IL2, IFNγ and TNFα production in individual cells. Figure 5 contains representative FACS plots showing the specificity of TCR-transduced T-cells producing either IL2 and IFNγ (Figure 5a) or IL2 and TNFα (Figure 5b) in response to either pWT126-relevant peptide or pWT235-irrelevant peptide. The FACS plots are gated on viable CD8+ T-cells. The transduction conditions shown are OKT3+IL2 and cytokine alone (IL2). The TCR expression level in each T-cell population was similar to that shown in Figure 2. Despite lower levels of Vβ2.1 TCR expression in the IL2-stimulated cells (17 versus 41%) the total number of peptide-responsive T-cells was equivalent. Of note, the T-cells transduced in the presence of OKT3 and IL2 (upper row) contained increased numbers of monofunctional (IL2 only secreting) T-cells (3.19–4.27%) as compared with T-cells stimulated with only IL2 (0.77–0.84%).
Figure 6 shows the cytokine production profile of the T-cells that responded to peptide stimulation. Following transduction by CD3 activation, a substantial proportion of the responding T-cells (36%) produced only one cytokine. In contrast, following cytokine-mediated transduction, only 3–12% of T cells produced one cytokine, whereas 88–97% of cells produced two or three cytokines. Interestingly, all common γ-chain cytokines individually were able to generate a large proportion of multi-functional T-cells, which was unaltered when they were used in combination with IL21 (Figure 6).
There is increasing evidence that ex vivo expansion resulting in differentiation of T-cells is associated with loss of in vivo protection against tumour growth and virus infection. In a murine antigen-specific adoptive transfer model, several phenotypic markers have been used to distinguish central memory T-cells from more differentiated effector T-cells. The loss of CD62L expression on adoptively transferred murine T-cells observed with differentiation towards the effector T-cell phenotype, was associated with a relative loss of protection against growth of melanoma tumours.14 Conversely, in a recent primate study CD62L served as a marker for isolated central memory T-cells that showed long-term persistence following adoptive cell transfer.12
To date, the vast majority of TCR transfer protocols in the human system have involved direct TCR stimulation through CD3 to fully activate T-cells, followed by retroviral transduction in the presence of IL2. Such transduction protocols efficiently generate antigen-specific T-cells expressing the introduced TCR α- and β-chains, but also result in marked effector differentiation, which can limit subsequent in vivo persistence.31 There have been a number of studies, which have used lentiviral vectors for TCR (or chimaeric antigen receptor) gene transfer, but the T-cell activation protocol used in these studies involved CD3 stimulation.40, 41, 42 Similarly, studies, which have demonstrated lentivirus-mediated gene transfer into cytokine-stimulated resting human T-cells, have examined the cell-surface phenotype after transduction, but have not explored the antigen-specific T-cell function.43
In this study we have shown that the CD3-mediated stimulation protocol accelerated T-cell differentiation resulting in loss of CD62L and CD28 expression. In contrast, lentiviral TCR gene transfer after cytokine stimulation was able to prevent differentiation in a large proportion of transduced T-cells. Of the common γ-chain cytokines tested in this study, IL15 triggered more pronounced T-cell differentiation than IL2 and IL7. Although IL21 alone was unable to support lentiviral T-cell transduction, when combined with other common γ-chain cytokines, it prevented differentiation as measured by continued expression of CD62L and CD28 in the majority of transduced T-cells. This is consistent with recent studies showing that IL21 was able to sustain CD28 expression in human T-cells, which was associated with improved antigen-specific functional avidity.44, 45 Similarly, murine experiments showed that in vivo administration of IL21 in combination with IL15 resulted in a cooperative effect on tumour protection.46 Exposure of murine T-cells to IL21 triggered a characteristic gene expression profile that included upregulation of CD62L and was associated with enhanced tumour immunity following adoptive T-cell transfer.47 Importantly, in addition to preservation of a more naïve phenotype, use of IL21 and IL15 in combination supported the greatest proliferation of TCR-transduced cells as compared with other minimally activating conditions, which is essential for translation of novel transduction protocols for early-phase clinical trials.
We are in the process of establishing cytokine-mediated lentiviral gene transfer into murine T-cells to test directly whether continued CD62L and/or CD28 expression results in enhanced antitumour immunity of TCR-transduced T-cells in vivo.
In addition to phenotypic analysis of lentivirus-transduced human T-cells, we also performed functional studies. We focused on the ability of transduced T-cells to produce three cytokines in an antigen-specific manner. There is increasing evidence that multi-functional T-cells are less differentiated than mono-functional T-cells, and that the former provide protective immunity more effectively than the latter.38, 39, 48 Following cytokine-mediated TCR transduction a significantly greater proportion of antigen-specific T-cells were multi-functional compared with CD3-mediated TCR transduction.
Together, these data indicate that cytokine-mediated TCR gene transfer can generate antigen-specific human T-cells that are functionally and phenotypically less differentiated than T-cells produced by conventional gene transfer into CD3-activated cells. Although there is currently no direct evidence that this will improve the function of TCR-transduced human T-cells in patients, existing evidence using murine and primate models strongly suggest that this will be the case. The data presented in this study support the testing of lentiviral vectors and cytokine stimulation in future clinical TCR gene therapy trials.
Materials and methods
Media, cells, antibodies, cytokines, peptides and FACS analysis
Unless otherwise stated, all culture media were RPMI-1640 (Cambrex, Charles City, IA, USA) supplemented with 10% heat-inactivated foetal calf serum (FCS) (Sigma-Aldrich, Gillingham, Dorset, USA), 1% L-glutamine (Invitrogen Life Technologies, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen Life Technologies). The cell lines used were the human TCR-negative Jurkat 76 cell line and the HLA-A2 positive T2 cell line, which is deficient in TAP (transporter associated with Ag processing) and can be efficiently loaded with exogenous peptides. Peripheral blood mononuclear cells were obtained from volunteer donors from the National Blood Service (Colindale, London, UK). CD8+ T-lymphocytes were isolated by positive selection using anti-CD8 magnetic beads as per manufacturer's protocol (MACS system; Miltenyi Biotech, Bergish Gladbach, Germany).
The concentration of common γ-chain cytokines used either alone or in combination as stated was as follows: 20 U ml−1 of human recombinant IL2 (Roche, Basel, Switzerland), 5 ng ml−1 human recombinant IL7 (R&D Systems, Abingdon, Oxfordshire, UK), 10 ng ml−1 human recombinant IL15 (R&D Systems) and 30 ng ml−1 mouse recombinant IL21 (R&D Systems).
The peptides used in this study were the HLA-A2-binding peptides pWT126 (RMFPNAPYL) and pWT235 (CMTWNQMNL), which were synthesized by ProImmune as described previously.29
Antibodies for flow cytometry were anti-human CD3 PerCP, CD28 fluorescein isothiocyanate, CD62L APC, CD45RO fluorescein isothiocyanate and CD8 APC (all from BD Biosciences, Cowley, Oxfordshire, UK) and anti-human Vβ2.1-phycoerythrin (Immunotech, Quebec, Canada). The antibodies used for intracellular cytokine staining were anti-human IFNγ, anti-human TNFα and anti-human IL2 (all from BD Biosciences). FACS analysis was performed using a LSR II flow cytometer and the data were analysed using FACSDiva software (BD Biosciences).
Generation of the lentiviral WT1 TCR construct
A pSIN second-generation lentiviral vector, containing a spleen focus-forming virus long terminal repeat promoter and the HIV-1 central polypurine tract cis-active element, was modified for this study. The codon-optimized, hybrid, HLA-A*0201-restricted, WT1-specific TCR genes, previously cloned into the pMP71 retroviral vector, have been described before.35 The hybrid TCR sequences contain murine constant human variable region sequences. An additional disulphide bond between the α- and β-chains was created by the introduction of two additional cysteine residues in the α- and β-chain constant regions. The genes for the TCR α- and β-chains cloned into the lentiviral vector were separated by a porcine tsechovirus self-cleaving 2A sequence to optimize expression of both TCR α- and β-chain genes. The leader sequence was derived from the pMP71 retroviral vector34 and the full-length woodchuck hepatitis virus post-transcriptional regulatory element was truncated to prevent encoding of the oncogenic protein-X.49
Lentiviral vector production and transduction of primary human T-cells
For lentiviral vector production, 2 × 106 293T packaging cells were cultured in 10-cm tissue culture plates for 24 h at 37 °C under 10% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 1% penicillin/streptomycin and 1% L-glutamine. The packaging cells were co-transfected with pMD.G, a plasmid encoding the vesicular stomatitis virus glycoprotein envelope, and pCMVΔ8.91, a plasmid encoding genes required for generation of functional viral particles, together with the pSIN vector encoding the TCR genes using the Fugene transfection kit as per manufacturer's instructions (Roche). The lentiviral supernatant was harvested at 48 and 72 h and concentrated 100 × times by ultracentrifugation and stored at −80 °C until required for transduction. The lentiviral supernatant was added to the primary human T-cells for 24 h in the presence of the relevant cytokine combination at the concentrations stated above. After 24 h of co-culture the medium was changed. After 48 h cell-surface TCR expression was determined by FACS analysis. The multiplicity of infection of lentiviral supernatant was calculated using serial dilutions of the supernatant to transduce Jurkat cells, which were cultured in RPMI supplemented with 10% FCS, 1% penicillin/streptomycin and 1% L-glutamine. Control transductions using anti-CD3 and IL2 required peripheral blood mononuclear cells to be activated for 48 h using the anti-CD3 antibody OKT3 at 30 ng ml−1 and IL2 20 U ml−1 before lentiviral transduction.
Peptide stimulation of TCR-transduced T-cells
Transduced primary T-cells were stimulated and expanded every 9 days. The stimulations were performed in 24-well plates in 2 ml of culture medium containing 10% non-heat-inactivated FCS and 10 U ml−1 IL2 (Roche) or other cytokine combinations as stated, at 37 °C with 5% CO2. Each well contained 5 × 105 transduced cells, 2 × 105 irradiated T2 cells loaded for 2 h with 100 μM of the pWT126 (stimulator cells) and 2 × 106 irradiated peripheral blood mononuclear cells as feeder cells.
CFSE proliferation assays
For CSFE labelling, 107 T-cells were incubated with 5 μM CFSE (Molecular Probes, Invitrogen, Carlsbad, CA, USA) for 5 min at 37 °C in 1 ml of phosphate-buffered saline. After incubation, the cells were quenched with 10 × volume of 5% FCS-phosphate-buffered saline at room temperature (RT) and centrifuged for 5 min at 300 g at 20 °C. The CFSE-stained T-cells were then washed twice in FCS-phosphate-buffered saline RT before cytokine culture in a 96-well plate. A total of 106 CFSE-stained T-cells were cultured in the presence of no cytokines; OKT3 (30 ng ml−1) and IL2 (600 U ml−1); IL2 (20 U ml−1); IL7 (5 ng ml−1); IL15 (10 ng ml−1); IL2 and IL21 (30 ng ml−1); IL15 and IL21; IL7 and IL21; or IL7 and IL15. The cells were harvested at 72 or 120 h and analysed by FACS following staining for CD3, TCR and CD8 expression.
Intracellular cytokine secretion assays
These assays were performed using 96-well round-bottom plates. TCR-transduced T-cells and T2 stimulator cells loaded with relevant (pWT126) or irrelevant (pWT235) peptide were added at 4 × 105 per well in 200 μl of culture medium containing brefeldin-A (Sigma-Aldrich) at 2 μg ml−1. After incubation for 16 h at 37 °C with 5% CO2, the cells were then fixed, permeabilized (BD cytofix kit) and stained for intracellular IFNγ, TNFα and IL2. Data were analysed using FACSDiva (BD Biosciences).
Statistical analysis was performed using the GraphPad Prism software (version 5.0) and Microsoft Excel.
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ECM, MP and HJS designed the research, analysed the data and wrote the paper. MP, JT, SX, LG and DE performed the research. MCG, JT and CP contributed new reagents. MC and DH analysed data. Grant Support: Leukaemia Research, Medical Research Council.
The authors declare no conflict of interest.
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