Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.


Introduction
The use of genetically modified lymphocytes in basic and translational research has increased dramatically in recent years [1,2]. By engineering CD8 + T cells to express TCRs derived from patients' tumor-specific cytotoxic T cells (CTLs), they can be converted from a population of polyclonal CD8 + T cells to CTL of monoclonal TCR specificity [1,2]. Furthermore, T cells engineered to express MHC-unrestricted chimeric antigen receptors (CARs) have demonstrated efficacy in human trials [1,2]. These approaches are attractive because CTLs with high affinity and specificity are increasingly easy to generate and can be adapted to treat a number of different tumor types. Almost all of the studies using the TCR gene transfer approach showed T cell response to tumor peptide antigen. However, the innate lymphocyte's TCR transfer has not been studied. Among innate lymphocytes, invariant natural killer T (NKT) cells have several unique features that differentiate them from T cells and NK cells. NKT cells express a nearly invariant T cell receptor encoded by Vα14Jα18 in mice and Vα24Jα18 in humans and can rapidly produce both IFN-γ and IL-4 after ligand stimulation [3,4]. An exogenous glycolipid, αgalactosylceramide (α-GalCer), is widely used as a synthetic ligand for activating iNKT cells and is presented to them by the monomorphic, HLA-class I-like molecule, CD1d.
Receptor-transfer strategies using viral vectors, such as retroviral and lentiviral vectors, are often utilized in experiments that require significant transgene expression in primary T cells [1]. The use of viral vector-based gene delivery systems results in stable genomic integration of the transgene and constitutive expression of the transgenic TCR. However, integration of the provirus into the genome may bear the risk of insertional mutagenesis and theoretically, malignant transformation of T cells. Because of this, RNA molecules have recently received attention as a potentially safer delivery system of genomic material to primary lymphocytes. The expression of RNA-derived CAR [5] or RNA-derived TCR [6] in T cells is transient and disappears after short period and a possible toxicity is thought to rapidly abate [5,7,8].
γδT cells are innate lymphocytes that comprise 3% to 5% of peripheral blood T cells [9][10][11]. γδT cells recognize 'phospho-antigens', such as isopentenyl pyrophosphate (IPP) and 1-adenosin-5-ylester 3-(3-methylbut-3-enyl) ester (ApppI) via their TCR [9]. The predominant Vγ9Vδ2T subset can be expanded in vitro using bisphosphonate zolendronic acid (Zol), which blocks the mevalonate pathway, leading to intracellular accumulation of endogenous γδT cell ligand, IPP and ApppI mevalonate metabolite [9]. Although γδT cell ligands are expressed on some cancer cells and Zol-or bisphosphonate-treated cells, γδT cells in PBMCs of cancer patients often demonstrate impaired activation and proliferation [12]. Different from CTL, γδT cells attack MHC-low expressing tumor cells. Therefore, augmenting γδT cell function in cancer patients could improve patient responses to a broad range of malignancies. To alter the antigen-specificity of γδT cells, an approach to transfect antigen-specific αβTCR genes into γδT cells was recently reported [13,14]. In the current study, we transfected γδT cells with NKT derived-TCR αand β-chains and evaluated their anti-tumor effects. This approach resulted in potent bi-functional γδT cells, which recognized both the NKT cell and the γδT cell ligands.

Human PBMCs and cell lines
Human PBMCs were obtained from healthy volunteers and separated by Ficoll-Hypaque (Amersham Pharmacia Biotech) density centrifugation. All studies were approved by the RIKEN institutional review board and the approval number was [H26-18]. All participants gave written informed consent. Jurkat and K562 cell lines were purchased from RIKEN BRC. HEK293 cell line was purchased from ATCC.

In vitro generation of human NKT cell lines and Vγ9Vδ2 T cells lines
To prepare NKT cell lines, PBMCs were cultured in RPMI 1640 containing 10% FCS the presence of α-GalCer (100 ng/ml) and IL-2 (100 U/ml). After 10-14 days, human NKT cells were sorted using anti-Vα24 mAb and were maintained as NKT cell lines in the presence of IL-2 (100 U/ml), IL-7 (5 ng/ml), and IL-15 (10 ng/ml) in complete medium and restimulated with irradiated PBMCs pulsed with α-GalCer for at least one month as previously described [15].
To prepare Vγ9Vδ2T cell lines, PBMCs were cultured in the presence of Zol (5 μM) and IL-2 (300 U/ml). After sorting using anti-Vγ9 mAb, Vγ9Vδ2T cell cells were maintained as γδT cell line in the presence of IL-2 (300 U/ml).
Electroporation of in vitro-transcribed mRNA RNA electroporation of T cells was performed as described previously [7]. In brief, PBMCs were stimulated in vitro at 10 6 cells/ml with 50 ng/ml anti-CD3 mAb OKT3 (Janssen pharmaceutical, Inc.) and 300 U/ml IL-2. Three days later, T cells were washed and resuspended in OptiMEM. γδT cells were established as previously described and resuspended in OptiMEM. In transfection by electroporation, 10 μg each RNA was pulsed in square-wave pulse, 500V, 3 msec with an ECM 830 square were electroporation system (BTX). Immediately after electroporation, the cells were cultured with 300 U/ml IL-2 containing culture medium until use in the assay.

Cytotoxicity assay
The cytotoxic activity against adherent cells was analyzed using an LDH assay kit according to the manufacturer's instructions (Takara Bio Company). CD1d gene-transfected HEK293 (CD1d-HEK293) were treated with or without 500 ng/ml α-GalCer or 5 μM Zol for 24 h to use them as target cells. Ten thousands target cells were cultured with 1x10 5 effector cells for 12 h. After subtracting the background control value, cytotoxicity values (%) were calculated as previously described [17].

Statistical analysis
Differences in in vitro data were analyzed using the Student T-test. P<0.05 was considered statistically significant.

Results
Cloning of human Vα24 + Vβ11 + NKT-TCRα and β DNAs and construction for mRNA synthesis We established an NKT cell line from a healthy donor ( Fig 1A). After sorting, TCR cDNAs of Vα24 + Vβ11 + NKT cells were cloned using RT-PCR. The resultant NKT-TCR α and β pairs, i.e., Vα24 and Vβ11 chains were separately inserted into a pGEM-3Z vector ( Fig 1B). We then generated TCR mRNA of Vα24 and Vβ11 using an in vitro transcription approach and evaluated that the mRNAs before polyadenylation (Fig 1C (-)) were compatible in size with their template cDNAs whereas the poly-A tails were more than 200 bases long after the reaction (Fig 1C  (+)). The expression of NKT-TCR mRNA was tested in the Jurkat T cell leukemic cell line. After transfecting Jurkat cells with both TCR Vα24 and Vβ11 mRNA, the expression of NKT derived-Vα24 and Vβ11 TCR (denoted as NKT-TCR) was confirmed by flow cytometry using anti-Vα24 Ab and Vβ11 Ab (Fig 1D right). In some experiments, we used anti-6B11 Ab which reacts with an epitope of the CDR3 formed by the germ-line configuration of the Vα24 and Jα18 of the TCRα locus of an invariant NKT-TCR (Fig 1D, left). The level of NKT-TCRs was up-regulated in transfected cells after 6 h, and a high expression of Vα24 and Vβ11 TCRs was detected on approximately 90% of Jurkat cells (Fig 1D).

NKT-TCR mRNA-transfected T cells respond to the NKT ligand
Vα24 and Vβ11 TCR mRNAs were transfected into bulk PBMCs. Expression of NKT-TCR, as measured by flow cytometry started at 2 h, was present on 60-70% of T cells at 12 h, and was still detectable until 48 h (Fig 2A and 2B), confirming that both chains were expressed on the T cells for 48 h. Vα24 + Vβ11 + TCR-transfected cells and Vα24 -Vβ11 -TCR non-transfected cells were sorted using CD3-FITC, Vα24-PE and Vβ11-APC by FACS Aria (Fig 3A). We then investigated whether ERK signaling was induced in a ligand specific way when Vα24 + Vβ11 + TCR--transfected cells were stimulated by the NKT cell ligand. For this purpose, ERK phosphorylation was assessed after stimulation on solid phase of α-GalCer-loaded CD1d-dimer protein. As shown in Fig 3B, Vα24 + Vβ11 + NKT-TCR mRNA-transfected T cells showed the ERK kinase phosphorylation in response to α-GalCer, but not Vα24 -Vβ11non-transfected T cells. The MAPK participates in activation of some transcription factors [18,19]. Indeed, MAPK phosphorylation was detected in NKT-TCR mRNA-transfected T cells when activated by NKT cell ligand, indicating that the transfected TCR indeed recognized the NKT cell ligand (Fig 3B).

NKT-TCR mRNA-transfected γδT cells respond to both γδT and NKT cell ligands
As shown in Fig 4A, the frequency of NKT cells is lower than that of Vγ9Vδ2T cells in PBMCs of healthy donors (mean±SEM of NKT vs Vγ9Vδ2T of CD3 + T cells: 0.036±0.035 vs 0.98 ±1.46). When PBMCs were stimulated with either Zol or α-GalCer, Vγ9Vδ2T cells proliferated more than NKT cells (Fig 4A). Vγ9Vδ2T cells ordinarily do not express NKT-TCR, but transfection of these cells with NKT-TCR mRNA, led to NKT-TCR expression in addition to Vγ9Vδ2T (Fig 4B). To test the functionality of Vα24 + Vβ11 + NKT-TCR mRNA-transfected Vγ9Vδ2T cells (denoted as γδT-NKT-TCR (+)), these cells were stimulated using Zol and α-GalCer. It is well known that Vγ9Vδ2T cells in PBMC can be expanded after culturing with Zol. However, there are few reports about secondary expansion of γδT cells. It was previously reported that the proliferative response of γδT cells is transient and also that repeated injection of BrHPP and IL-2 induced activation induced cell death of Vγ9Vδ2T cell and an exhaustion of the response [20][21][22]. After ex vivo expansion from PBMCs using Zol, we assessed the number and function of γδT cells in secondary challenge with antigen. Vγ9Vδ2T cells were cultured again in the presence of soluble Zol to serve as a control for the subsequent groups (Fig 4C). The number of NKT-TCR-transfected Vγ9Vδ2T cells was higher after stimulation with CD1d-HEK293/Gal cells and CD1d-HEK293/Zol than that of parental γδT cells in secondary challenge ( Fig 4C). We evaluated whether transfection of Vα24 + Vβ11 + NKT-TCR mRNA into Vγ9Vδ2T cells allowed for activation by the NKT cell ligand. Vγ9Vδ2T cells secreted IFN-γ and low level of TNF-α after culturing with CD1d-HEK293/Zol cells, but not after the culture with CD1d-HEK293/Gal cells (Fig 4D and 4E, left). NKT-TCR-transfected Vγ9Vδ2T cells, in contrast, produced more IFN-γ and TNF-α in response to CD1d-HEK293/Gal than CD1d-HEK293/Zol. These data showed that NKT-TCR transfected Vγ9Vδ2T cells responded to NKT cell and γδT cell ligands (Fig 4D and 4E, right). To evaluate whether the activation of NKT-TCR transfected γδT cells is dependent on CD1d molecule, we cultured the cells with a CD1d blocking antibody. As shown in Fig 4F, activation of NKT TCR-γδT cells by α-GalCer is dependent on CD1d. Finally, we examined whether NKT-TCR transfected γδT cells can respond synergistically to both α-GalCer and γδT ligands. As shown in Fig 4G, IFN-γ  after stimulation with both α-GalCer-loaded CD1d-HEK293 cells and Zol-treated CD1d-HEK293 cells are almost equal to that by NKT TCR-γδT cells by α-GalCer-loaded CD1d-HEK293 cells.
Next, the cytotoxicity of NKT-TCR-transfected Vγ9Vδ2T cells against tumor cells was assessed. It is known that Daudi cells (a Burkitt's lymphoma cell line) are a highly sensitive γδT cell target due to the expression of phosphoantigen while K562 cells (myelogenous leukemia cell line) are a weakly sensitive γδT cell target [10]. We initially confirmed that Daudi cells are killed by γδT cells (40-70% of cytotoxicity, E/T = 10, data not shown), but K562 are not ( Fig  5C). Therefore, we used K562 to evaluate antitumor effect in this study. Although γδT cells without stimulation did not show cytotoxicity against K562, they showed cytotoxicity against K562 after stimulation with CD1d-HEK293/Zol (Fig 5C). Then, we measured the NKT-TCRtransfected Vγ9Vδ2T cell-mediated cytotoxicity against K562. NKT TCR-transfected Vγ9Vδ2T cells without culturing did not show cytotoxicity against K562 (Fig 5D). However, after stimulation with CD1d-HEK293/Zol or CD1d-HEK293/Gal, NKT-TCR-transfected Vγ9Vδ2T cells demonstrated a strong killing activity against K562 compared to unstimulated one (Fig 5D). Since K562 did not express CD1d (data not shown), the cytotoxic activity of NKT-TCR-transfected Vγ9Vδ2T cells was not in an NKT TCR-mediated manner. NKT-TCRtransfected Vγ9Vδ2T cells activated by CD1d-HEK293/Gal demonstrated higher cytotoxicity against K562 than those were activated by CD1d-HEK293/Zol. Next, we tested whether NKT-TCR-transfected Vγ9Vδ2T cells can be activated synergistically by both CD1d-HEK293/ Zol and CD1d-HEK293/Gal. When NKT-TCR-transfected Vγ9Vδ2T cells were activated by CD1d-HEK293/Gal together with CD1d-HEK293/Zol, the cytotoxicity was similar to that by CD1d-HEK293/Gal (Fig 5D). Thus, NKT-TCR mRNA-transfected Vγ9Vδ2T cells can be primed with α-GalCer and then respond to γδT ligands in a more functional fashion than parental γδT cells.

Discussion
The current study evaluates the effects of transfecting human NKT cell-derived TCR α and β chain mRNA into either innate or adaptive lymphocytes. NKT-TCR transfected T cells and γδT cells secreted IFN-γ in a ligand-specific manner. These γδT cells demonstrated cytotoxicity against K562 tumor cells and proliferated in response to NKT cell and γδT cell ligands. This study demonstrated that γδT cells and primary adaptive lymphocytes transfected with the NKT-TCR retain parental T cell qualities while acquiring NKT cell functionality, thus creating a bi-potential T cell.
NKT cell receptors are universal in humans and their specificity is not HLA restricted. Therefore, the approach of NKT-TCR mRNA transfection into primary lymphocytes and γδT cells has a number of unique features that makes it attractive for use as an immunotherapeutic approach to cancer treatment. NKT-TCR mRNA is not stably integrated into the genome [5,6] and is only expressed by transfected cells for a limited period of time, thus eliminating potential safety concerns. On the other hand, Zhao et al demonstrated that CAR mRNA was useful and safer as well, but the retroviral transfer of CAR DNA to T cells was effective for a long time [5]. As a future plan, we will examine about the efficacy using T or γδT cells retrovirally transduced with the NKT-TCR DNA.
A potential disadvantage of the TCR gene transfer approach is the possible formation of mixed TCR dimers. It has been reported that chains of the introduced TCR can pair with endogenous TCR chains, resulting in a dimer composed of both endogenous TCR and introduced TCR, thus altering TCR specificity [13]. However, since γδT cells do not have αβ chains, transfection of NKT α and β chains into γδT cells will not result in mispaired receptors.
NKT cells in naïve mice have the potential to produce both IFN-γ and IL-4 in response to α-GalCer, therefore modulating whether the immune response is Th1 or Th2 [3]. However, NKT cell secretion of IFN-γ, and not IL-4 after an administration of α-GalCer-loaded DCs has been shown to have tumor protective effects [25]. This is further demonstrated in patients with advanced non-small cell lung cancer in whom increased numbers of IFN-γ-producing PBMCs correlated with prolonged median survival time upon an administration of α-GalCer pulsed IL-2/GM-cultured PBMC [26].
In the current study, NKT-TCR-transfected lymphocytes responded to NKT cell ligand with the secretion of IFN-γ, but not IL-4. We found that the transfer of mRNA encoding NKT cell receptors drives the other lymphocytes toward IFN-γ producing NKT like cells, which might be useful against tumor cells. One of the reasons is that the transfected lymphocytes might be already polarized or skewed toward Th1 type in cytokine production. Previous studies showed that PBMCs stimulated by anti-CD3 plus IL-2 led to T-bet via TCR signaling pathway for IFN-γ in most of conventional T cells [27]. T-bet is known to be important in that it regulates antigen-driven effector T cells [28]. Although Zhao et al described a minor population of CD4T cells capable of producing IL-4 or IL-10 in these anti-CD3 Ab and IL-2 stimulated PBMCs [29], major population which were polarized to Th1 might mask the response of Th2. But, further study is needed to understand the mechanism in detail.
Aminobisphosphonates, such as palindronate-or zoledronic acid-activated Vγ9Vδ2T cells to produce IFN-γ and exhibit cytotoxicity against some tumor cell types, e.g. Daudi, K562, AML cells, lymphoma cells and myeloma cells [30][31][32]. Dieli et al showed that administration of zoledronic acid induces Vγ9Vδ2T cells to mature toward an IFN-γ-producing effector phenotypes [33]. In a recent lung cancer study, adoptive transfer of ex vivo expanded Vγ9Vδ2T cells showed some clinical efficacy [11,34]. The current study demonstrated that NKT-TCRtransfected Vγ9Vδ2T cells can be stimulated and functionally activated by either of the two ligands. These results offer a potentially new treatment modality for activating poorly responding γδT cells in cancer patients with NKT cells ligand for improved tumor responses.
The current study demonstrated that the transfer of NKT-TCR αβ chains enhances the function of both innate and adaptive lymphocytes and this approach may hold the promise of a new anti-cancer immunotherapy.