Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells

Abstract

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help.

Introduction

Dendritic cells (DCs) loaded with tumor-associated antigen (TAA) are frequently applied as vaccines for cancer patients.1 These DCs should express high levels of co-stimulatory molecules2 and be able to migrate towards T-cell areas in the lymph nodes.3 Furthermore, the DCs should secrete high amounts of interleukin-12 (IL-12p70), which is a crucial third signal for induction of functional T helper (Th) cell 1 and cytotoxic T lymphocytes (CTL) responses.4 In addition, for clinical application DCs have to be generated under conditions free of animal sera. The current golden standard maturation cocktail (monocyte-conditioned medium (MCM) mimic) consists of IL-1β, tumor necrosis factor-α (TNFα), IL-6 and prostaglandin E2 (PGE2). This cocktail induces mature DC (mDC) expressing high levels of co-stimulatory molecules and CCR7.5 These DC migrate efficiently towards CCR7 ligands.6 However, whereas PGE2 is important for induction of migratory capacity of DCs, it significantly impairs IL-12p70 production.7 Recently an alternative maturation cocktail was proposed that includes polyinosinic:polycytidylic acid (p-I:C), interferon (IFN)-α and IFN-γ. This cocktail generates mature DC (αDC) capable of producing IL-12p70 whereas retaining migratory capacity.8 Vaccination with DCs loaded with single CTL epitopes has lead to some therapeutic effects.9 However, it has become clear that the induction of both Th cell and CTL responses are necessary for an effective anti-tumor response. Delivery systems allowing DCs to present multiple epitopes presented by both major histocompatibility complex (MHC) class I and II are currently explored. Transfection of messenger RNA (mRNA) encoding tumor antigens is a safe and relatively easy method to accomplish this, and has already lead to clinical results.10 Here we show that in contrast to mDC, αDC do not express transgenes after mRNA transfection. In order to compensate for the reduced IL-12p70 production, PGE2-matured mDC were co-transfected with mRNA encoding IL-12 ensuring constitutive IL-12p70 production. CTL were induced with TAA mRNA-transfected DCs co-transfected with either green fluorescent protein (GFP) or IL-12 mRNA. In comparative experiments, higher numbers of melanoma antigen recognized by autologous T-cells (MART-1)-specific CTL with higher functional avidity were induced after stimulation with mDC transfected with MART-1 in combination with IL-12 mRNA. Thus, in contrast to αDC, MCM-mimic matured DC can be efficiently electroporated with mRNA. These DC express high levels of co-stimulatory molecules and are capable of migrating towards lymph node derived CCR7 ligands. Co-electroporation with IL-12 mRNA ensures constitutive IL-12p70 production at high levels.

Results

Modifications of mRNA sequences and in vitro transcription

Previously it has been described that proteosomal targeting of antigens transfected into antigen presenting cells (APC) using ubiquitin improves cell-surface MHC class I presentation.11 Antigen presentation after electroporation with in vitro transcribed (IVT) mRNA from the MART4 construct (a minigene encoding four repeats of the human leukocyte antigen (HLA)-A2.1 restricted MART1-derived altered peptide ligand MART126–35L), was compared with mRNA from a construct of the same minigene but preceded by ubiquitin (ubi(MART)4). Figure 1 shows that antigen presentation by DCs transfected with mRNA from the ubi(MART)4 construct to a specific CTL clone is not only increased but also prolonged compared to MART4 mRNA-transfected DCs. Over 75% of the CTL were positive for intracellular IFNγ after stimulation with the peptide-loaded positive control APC, in comparison to 72% after stimulation with ubi(MART)4-transfected DC and 17% after stimulation with MART4-transfected DC (Figure 1a, top panel). In addition, possibly owing to this increased MART epitope expression, the antigen presentation was also prolonged, 24 and 72 h post-transfection. ubi(MART)4-transfected DC were still capable of inducing IFNγ production by the specific CTL clone (51 and 23% of CTL-positive, respectively) at these time points whereas hardly or no IFNγ-positive CTL were detected when stimulated with MART4-transfected DC (Figure 1a lower two panels).

Figure 1
figure1

Modification of mRNA sequences and in vitro transcription. (a) MoDC were electroporated with a minigene of four repeats of the MART126–35L-modified epitope (MART4) mRNA or with MART4 mRNA preceded by ubiquitin (ubi(MART)4). These were used as APC in an intracellular IFNγ assay, 3, 24 and 72 h after electroporation using a MART126–35-specific CTL clone (4.4TG). Peptide pulsed (2 h before the start of each IFNγ assay) JY cells were used as positive control APC. One experiment out of two performed is shown. Percentages of IFNγ-producing cells of the second experiment after 3, 24 and 72 h: MART(4) mRNA: 6, 0.6 and 0.3%; ubi(MART)4-2A mRNA: 74, 39 and 37%; MART1 peptide: 98, 95 and 90%. (b) MoDC were electroporated with IL-12, ubi(MART)4-2A or ubi(MART)4 mRNA linked with the 2A sequence to IL-12 mRNA (uMART2-A-IL12). These were used as APC in an intracellular IFNγ assay 3 h after electroporation using another MART126–35-specific CTL clone (4TG-D8b). Peptide pulsed JY cells were used as positive control APC. Dot plots of one experiment out of three performed are shown. Numbers in the upper right quadrant represent the mean (s.d.) percentage of IFNγ-positive cells of those three experiments. (c and d) MoDC were electroporated with IL-12 mRNA IVT using the conventional cap (CAP) or the modified cap (ARCA). MoDC electroporated with conventional capped GFP were used as a negative control. MoDC were resuspended to 2 × 10E5/ml and five replicate wells were set up. After 24 h supernatant was harvested, cells were washed extensively and cultured for another 24 h after which supernatant was collected. This was repeated for four consecutive days (n=3).

When co-transfecting two separate mRNA transcripts, most of the cells become double positive for both genes (not shown). However, for clinical application it is preferable to be able to transfect with a single mRNA transcript if two or more genes need to be transfected. Therefore we employed the self-cleaving 2A peptide derived from the insect virus Thosea asigna.12 Through a ribosomal skip mechanism the 2A peptide impairs normal peptide bond formation between the last glycine of the 2A peptide and the proline which precedes the second protein, without negatively affecting translation of the second protein.13 Here we tested whether it is possible to generate a single IVT mRNA strain from a construct in which a TAA sequence is linked to the IL-12 sequence via a 2A peptide. Antigen presentation was comparable after DC transfection with IVT mRNA from the ubi(MART)4-2A-IL12 and ubi(MART)4-2A constructs (Figure 1b). IL-12p70 production over the first 6 h after transfection was comparable after transfection with either IL-12 or ubi(MART)4-2A-IL12 mRNA (204 and 352 pg/ml, respectively).

Because IL-12p70 is secreted, constitutive expression mediated by mRNA transfection is expected to be rapidly lost in time. Therefore we investigated whether ARCA-capped mRNA results in prolonged IL-12p70 secretion by transfected DCs. Figure 1c shows that by using ARCA-capped mRNA, IL-12p70 production over the first 24 h is about twofold higher compared to conventionally capped mRNA. Over the next 5 days (extensively washed) DCs transfected with ARCA mRNA continued to produce higher levels of IL-12p70, but IL-12p70 production from DC transfected with conventionally capped mRNA produced much lower levels which had become undetectable by day 5. Although the IL-12p70 production wanes over 5 days after transfection, the IL-12p70 produced was stable during this period (Figure 1d).

mRNA-transfected αDC fail to express the transfected gene

Recently published data suggested that α-type-1 polarized DC (αDC) are superior to DCs matured with a cytokine mix that mimics MCM (mDC) owing to higher levels of IL-12p70 production by αDC.8 We set out to compare mDC and αDC for antigen-specific CD8 T-cell induction after mRNA electroporation. DC electroporated after maturation migrate more efficiently in vitro14, 15 and are more effective in antigen presentation15 compared to DC electroporated before maturation. Furthermore, during the 48 h of in vitro maturation antigen presentation will be lost owing to loss of antigen expression (see Figure 1a). Electroporation of DC was therefore performed at the mature state. As previously described, both mDC and αDC are negative for CD14, but do express CD1a, CD83 and CCR7 as well as high levels of co-stimulatory molecules (data not shown).8 However, αDC did not express nerve growth factor receptor (NGFR) or GFP after transfection with the corresponding mRNA (not shown and Figure 2b), whereas mDC strongly express the transgene after mRNA electroporation (Figure 2a). Therefore, with the current protocols, mRNA transfection cannot be used for TAA loading of αDC.

Figure 2
figure2

Transgene expression and migration of mature DC after electroporation. (a) mDC and (one experiment out of five performed is shown, mean % GFP (s.d.): 76% (4.3%) (b) αDC (one experiment out of three performed is shown, mean % GFP (s.d.): 1% (0.45%) were electroporated with mRNA encoding GFP. DCs were harvested 18 h after GFP electroporation and GFP expression was analyzed and (c) cells were used in an overnight migration assay (n=3); NEP: not electroporated (open bars), GFP: electroporated with GFP mRNA (black bars) migration towards 6Ckine, and migration of NEP DC towards medium (gray bars). (d) IL-12p70 production was measured over 24 h after electroporation with GFP or IL-12 mRNA with (+J558) or without (−J558) CD40 ligation, by mDC or αDC (n=3).

For optimal induction of T-cell responses, DCs need, next to a fully mature status, respond to lymph node-derived chemokines. It was shown that αDC migrate towards CCR7 ligands, be it with a lower efficiency compared to mDC.8 Here we confirm these results with DCs that were not electroporated (Figure 2c). Electroporation with GFP mRNA did not affect the migratory capacity of either mDC or αDC (Figure 2c). In contrast to a better migration by mDC, αDC are better equipped for IL-12p70 production,7, 8 which is crucial for the induction of a strong type 1 cell-mediated immune response.4 This is also confirmed here, αDC already produce considerable amounts of IL-12p70 without CD40 ligation (−J558), which is highly increased by stimulation with the CD40L-transfected cell line J558 (Figure 2d). Electroporation with IL-12 mRNA does not decrease, nor increase the IL-12p70 production by αDC, the latter is to be expected as mRNA-transfected αDC do not express the transgene (Figure 2b). As expected, mDC produced no IL-12p70 without CD40 ligation and relatively low levels after stimulation with J558. However, this lack of IL-12p70 production could be remedied by transfection with IL-12 mRNA (Figure 2d). Figure 1c shows that this IL-12p70 production could even be boosted further by using ARCA-capped mRNA.

DCs cultured without FCS have a fully mature phenotype and can be transfected efficiently with mRNA

For clinical application it is necessary to generate DC under fetal calf serum (FCS)-free conditions. Previously it was shown that DC generated in X-vivo supplemented with 2% human AB serum (hAB) serum and matured in MCM were fully mature, migrated both in vivo and in vitro and induced T-cell activation.16 Here, we compared X-vivo cultured DC with DCs generated in Iscove’s modified Dulbecco’s medium (IMDM)/FCS and the medium specifically developed for the FCS-free generation of DC (CellGro-DC), with respect to phenotype, capacity to be transfected with mRNA and capability to migrate to 6Ckine after mRNA transfection. DCs generated in these specialized media supplemented with human serum were negative for CD14, but expressed lower levels of CD1a compared DCs generated in the presence of FCS. After maturation with MCM, mDC generated in all three media expressed high levels of CD40, CD80, CD83 and CCR7 (Figure 3a). FCS-free DC were efficiently transfected with mRNA (Figure 3b) and migrated equally well towards 6Ckine as DC generated in the presence of FCS (Figure 3c). Thus, mDC generated under FCS-free conditions can be efficiently transfected with mRNA, were fully mature and migrate towards CCR7 ligands.

Figure 3
figure3

High expression levels of co-stimulatory molecules and efficient transfection with mRNA of mDCs generated without animal serum. (a) Phenotype of MoDC generated different media (as indicated) were matured with MCM mimic for 48 h. The open histograms indicate the fluorescence intensity of cells stained with isotype controls, the closed histograms indicate the fluorescence intensity of the indicated markers. Plots of one experiment out of three performed are shown. Numbers in dot plots indicate the mean (s.d.) percentage of CD1a and CD14-positive cells, the numbers in the histograms indicate the mean fluorescence index (s.d.) which was calculated as described in the Materials and methods, of the three experiments performed. (b) GFP expression 18 h after transfection with GFP mRNA, of MCM mimic matured MoDC generated different media (as indicated). One experiment out of three performed is shown, mean % GFP (s.d.) of those three experiments: IMDM/FCS: 78(16); X-vivo/hAB: 76(4) and CellGro/hAB: 95(2). (c) MoDC generated different media (as indicated) were matured with MCM mimic for 48 h and used in an overnight migration assay towards medium or 6Ckine. Two out of two experiments performed are shown.

IL-12 and MART1 mRNA DC co-transfection leads to the efficient induction of CTL with high functional avidity

It was investigated whether constitutive expression of IL-12p70 by mDC, achieved by transfection with IL-12 mRNA, could enhance the induction of antigen-specific CD8-positive T cells using the MART126–35L altered peptide ligand as a model antigen. Isolated CD8β-positive T cells were stimulated with mDC electroporated with ubi(MART)4 mRNA in combination with either GFP or IL-12 mRNA in multiple small-scale bulk cultures. After each stimulation, individual cultures were analyzed for the presence of specific T cells by Tm staining. Direct ex vivo analysis showed no MART-specific CTL. After in vitro stimulation MART-specific CTL could be detected in all (8/8) bulk cultures (Figure 4a). Already after the first stimulation a significantly higher number of Tm-positive T cells could be detected in the bulk cultures stimulated with IL-12 mRNA co-transfected DC. This difference was sustained after a second stimulation, clearly indicating a quantitative difference.

Figure 4
figure4

IL-12 mRNA co-transfection enhances peptide-specific CD8-positive T-cell proliferation and effector function. (a) Isolated CD8β were stimulated with mDC electroporated with ubi(MART)4 mRNA in combination with either GFP or IL-12 mRNA. After the second stimulation the number of MART1-specific CTL was determined using Tm staining. The line indicates the mean. One representative donor of five tested is shown (2/5 experiments were performed with ubi(MART)4-2A-IL-12 mRNA. Differences between stimulation conditions were compared using a two-sided student's t-test. (b) Intracellular IFNγ expression after stimulation with Mel-JKO and Mel-AKR. Percentage of IFNγ-positive cells within the TmMART126–35L-positive fraction is given. One representative donor of three tested is shown. (c and d) Chromium release assay. The number of effector cells was corrected for the percentage of TmMART126–35L-positive cells. If sufficient cells were available, effector to target (E:T) ratio's started at 80 TmMART126–35L-positive T cells to one target cell, otherwise E:T ratio's started at 10:1. T cells stimulated with ubi(MART)4 plus GFP mRNA-transfected DC: squares (□, ▪); T cells stimulated with ubi(MART)4 plus IL-12 mRNA-transfected DC: circles (, •). (c) JY target cells: open symbols (□, ); MART126–35L peptide-loaded JY target cells: closed symbols (▪, •). (d) Mel-JKO target cells: open symbols (□, ); MEL-AKR target cells: closed symbols (▪, •). One representative donor of three tested is shown. (e) Functional avidity analysis of pooled bulk cultures as determined by intracellular IFNγ/TmMART126–35L staining using HLA-A2.1-positive JY cells as target, loaded with serial 10-fold dilutions of the MART126–35L peptide. The percentage of IFNγ-positive cells within the CD8+ TmMART126–35L+ population is plotted. Open squares (□), T cells stimulated with ubi(MART)4 plus GFP mRNA-transfected DC; closed squares (▪) T cells stimulated with ubi(MART)4 plus IL-12 mRNA-transfected DC.

The generated T cells were functional as they specifically recognize target cells loaded with the altered peptide ligand in a cytotoxicity assay (Figure 4c). Furthermore, these CTL recognize wild type, endogenously processed antigen, as shown by specific IFNγ production (Figure 4b) and cytolytic activity (Figure 4d) in response to the MART1 and HLA-A2.1-positive melanoma cell line Mel-AKR, with only low back-ground reactivity in response to the MART1-positive but HLA-A2.1-negative melanoma cell line Mel-JKO.

Apart from the quantitative difference between CTL induced with DCs transfected with or without IL-12 mRNA, these data also demonstrate a qualitative difference; the proportion of IFNγ-producing CTL increased massively within the Tm-positive cell population stimulated with IL-12 mRNA-transfected DCs (Figure 4b). In the cytotoxicity assays the number of effector cells used, was corrected for the percentage of Tm-positive T cells within the pooled bulk cultures, leading to equal Tm-positive specific T cell to target-cell ratio's. Peptide pulsed JY and in particular Mel-AKR were more efficiently killed by T cells induced with IL-12 mRNA-transfected DC (Figure 3c and d). This qualitative difference was even more evident in peptide titration assays which showed an increased functional avidity of the CTL which were induced by DCs co-transfected with IL-12 mRNA. Intracellular IFNγ production was analyzed in response to JY pulsed with 10-fold serial dilutions of the MART126–35L peptide. Only a maximum of 50% of the specific Tm-positive T cells induced with DC co-transfected with GFP mRNA responded to JY pulsed with 1 μ M peptide. On the other-hand already 60% of the specific Tm-positive T cells induced by DC co-transfected with IL-12 mRNA responded to JY pulsed with a million fold lower peptide concentration (1 pM), whereas a maximum of 89% of the Tm-positive T-cells responding cells was reached after stimulation with JY loaded with 100 nM peptide (Figure 4e). These results clearly show that DCs overexpressing IL-12p70 not only induce higher numbers of specific T cells, but induce T cells with a higher functional avidity as well.

CTL induced with IL-12 mRNA-transfected DC have a reduced expression of CD27 (P=0.06) and in particular reduced CD28 expression (P=0.03, Figure 5), which indicates an advanced differentiation into effector cells compared to CTL induced with control DCs. Other markers which are lost upon differentiation into effector cells such as CD62L and CCR7 were not expressed on any of the MART1-specific CTL whereas expression levels of CD45RA(+/−) and CD45RO (+/−) were similar on CTL induced with or without IL-12p70 (not shown).

Figure 5
figure5

Induced MART26–35-specific CTL have an effector T-cell phenotype. Expression of CD27 and CD28 on TmMART126–35L-positive CD8+ T cells was measured on CTL induced with DC co-transfected with either GFP or IL-12 mRNA (n=5). Mean fluorescence index is shown, line indicates the mean. MF indices were compared using a Wilcoxon-signed rank test. An example of a CD27 and a CD28 staining is shown: thin lines, CTL induced with DC co-electroporated with GFP mRNA; bold lines, CTL induced with DC co-electroporated with IL-12 mRNA. The horizontal bars represent the isotype histograms.

CD4+ T-cell help increases numbers but not functional avidity of specific CTL

CD4-positive T cells provide help during the induction of antigen-specific CD8+ T cells by DCs, also by inducing IL-12p70 production by DCs. To investigate whether CD4+ T cells could still further enhance the induction of antigen-specific CTL by mature DCs transfected with IL-12 mRNA, MART-specific CTL were induced in the presence or absence or irradiated autologous CD4+ T cells with ubi(MART)4-transfected DC co-transfected with either GFP or IL-12 mRNA. Indeed, despite the constitutive high expression of IL-12p70, addition of irradiated CD4+ T cells further increased the number of antigen-specific CTL (Figure 6a), but CD4 help did not support an increase in functional avidity (Figure 6b).

Figure 6
figure6

CD4+ T-cell help induces higher frequencies of specific CTL without affecting the functional avidity. (a) Isolated CD8β were stimulated with mDC electroporated with ubi(MART)4 mRNA in combination with GFP or with ubi(MART)4-2A-IL-12 mRNA, either in the absence or presence of irradiated autologous CD4+ T cells. After the second stimulation the number of MART1-specific CTL was determined using Tm staining. The lines indicate the mean. One representative donor of three tested is shown. Differences between stimulation conditions were compared using a two-sided student's t-test. (b) Functional avidity analysis of pooled bulk cultures as determined by intracellular IFNγ/TmMART126–35L staining using HLA-A2.1-positive JY cells as target, loaded with serial 10-fold dilutions of the MART126–35L peptide. The percentage of IFNγ-positive cells within the CD8+ TmMART126–35L+ population is plotted. Open squares (□), T cells stimulated with ubi(MART)4 plus IL-12 mRNA-transfected DC in the absence of CD4+ T cells; closed squares (▪) T cells stimulated with ubi(MART)4 plus IL-12 mRNA-transfected DC in the presence of CD4+ T cells. One representative experiment of three performed is shown.

Discussion

DCs are the most powerful APC able to induce primary T cells and maintain previously induced immune responses. Methodologies to generate DCs from circulating precursors in vitro have boosted the adoptive transfer of antigen-loaded DCs as adjuvant therapy for various types of cancers. Multiple clinical trials have shown induction of limited vaccine-specific immune responses. As then numerous studies have addressed optimizing maturation and antigen loading procedures of DCs. Transfection with mRNA coding for TAA has multiple advantages above peptide loading, DNA-vector transfection or use of recombinant viruses. mRNA transfection is safe, highly efficient, leads to high levels of protein expression of which a broad spectrum of epitopes over various HLA restrictions can be presented to T cells. Clinical studies have shown that adoptive transfer of mRNA-transfected DCs is safe and induces TAA-specific T-cell responses10, 17, 18, 19 However, the in vitro generation of DC possessing all the features crucial for the induction of a type 1 immune response, such as mature state, migratory potential and IL-12p70 production is complicated. In particular PGE2, which is essential for efficient DC migration,20 inhibits IL-12p70 production. An alternative cytokine cocktail without PGE2 but including IFNα and poly I:C was proposed by Mailliard et al.8 These so-called αDC were still able to migrate towards CCR7 ligands, be it with lower efficiency than PGE2 matured DC, whereas still producing high levels of IL-12p70 after CD40 ligation. Here, we have confirmed these findings. However, we found that after transfection with mRNA, αDC do not express the transgene, αDC are therefore not suitable for antigen loading with mRNA.

IL-12p70 enhances natural killer cell cytotoxicity and IFNγ production (reviewed by Trinchieri21). Furthermore, murine studies have shown that for naïve CD8+ T cells to differentiate into lytic effector cells a third signal is needed apart from T-cell receptor (TCR) engagement and co-stimulation through CD80/86 and CD28 interactions, which can be provided by IL-12p70.22, 23 Human DC, transduced with adenoviral vectors expressing the IL-12 gene, effectively stimulate specific type 1 CD4+ T cells from melanoma patients in vitro.24 Moreover, we have recently shown that IL-12p70 enhances proliferation and IFN-γ production of human antigen-specific effector memory CTL as well.25 These studies underscore the importance of IL-12p70 in the induction and maintenance of antigen-specific CTL. Therefore we co-transfected IL-12 mRNA together with TAA mRNA to compensate for the lack of IL-12p70 production by PGE2-matured DCs. For clinical application single agents are preferable, therefore we employed the 2A peptide used by various viruses to translate two separate proteins from a single mRNA strand. This system, using a retroviral vector, was previously applied by others to express all four murine CD3 proteins12 and by us to express both human TCR chains.26 Here we show, to our knowledge for the first time, that this peptide can also be used to transfect single IVT mRNA strands encoding two proteins, leading to similar protein expression as from separate transfected mRNA strands without interference with the expression and/or function of either protein.

Genetically engineered mRNA using ubiquitin11 or lysosomal-associated membrane protein (LAMP-1)27, 28 sequences targetting the HLA class I and class II pathway have been described to efficiently induce antigen-specific CD8 and CD4 T cells, respectively. Here we show that ubiquitin not only increases HLA class I restricted peptide presentation but also prolongs presentation of the TAA. ARCA-capped mRNA could not only increase protein expression as was reported previously29 but could also prolong the secretion of IL-12p70. In both cases it is not clear whether the prolonged expression is due to an initial increased protein expression or owing to an enhanced stabilization of the mRNA. This prolonged expression is of importance as it takes at least 48 h for injected DC16 to reach the lymph node, providing a rationale for the use of ubiquitin and ARCA-capped mRNA in clinical vaccination studies.

Here we show a significant increase in numbers and enhanced functional avidity of MART1-specific CTL after in vitro stimulation with DC transfected with MART1 combined with IL-12 mRNA as opposed to stimulation with DCs transfected with MART1 and GFP mRNA. This is in line with previous studies showing that functional avidity of CD8+ T cells is dependent on IL-12p70.30 Furthermore, the reduced expression of CD27 and CD28 on CTL induced with IL-12 mRNA-transfected DC indicates an advanced differentiation into effector T cells.31, 32

Initial experiments were all performed in the presence of autologous irradiated CD4+ T cells. However, as we used fully mature DC expressing high levels of IL-12p70, it may be expected that CD4 help was redundant. However, we show here that CD4+ T cells provided additional help in inducing higher numbers of CTL, but without increasing the functional avidity. This could be due to a further elevation of IL-12 levels, however Figure 2d shows that CD40 ligation only induces a minor increase in IL-12 levels of IL-12 mRNA-transfected DCs. What other factors may be involved in this extra help is currently under investigation.

Various groups have investigated co-transfection of (co-)stimulatory molecules such as, OX40L,33 4-1BBL34 and polyI:C35 or used CD40L encoding adenoviruses to transduce DC.36 Co-transfection or -transduction of these agents with tumor antigens into DCs, increased antigen-specific CTL induction, with differential effects on IL-12p70 expression and DC migration. However, none of these studies addressed the duration of the increased expression of IL-12p70 and/or the transfected co-stimulatory molecule. Furthermore, it is not clear from those studies what the quality, that is, the functional avidity of the induced CTL, is. Comparative studies are warranted to elucidate which approach or combination will be the most efficient in inducing strong tumor-specific immune responses both in vitro and in vivo.

In conclusion, we describe here a potent DC-based vaccine consisting of PGE2 matured DC transfected with ARCA-capped mRNA, encoding TAA preceded by ubiquitin and linked to IL-12 by a 2A peptide. This method provides DC with high responsiveness to secondary lymphoid organ chemokines, which secrete IL-12p70 and present antigen for at least 72 h, and which induce high avidity tumor antigen-specific CTL with, but also without, CD4 T-cell help. Our approach offers a DC-based vaccine, generated under conditions without FCS, possessing all the features considered necessary for induction of optimal tumor reactive Th1 immune responses.

Materials and methods

Media and reagents and cell lines

TNFα (50 ng/ml), IL-6 (100 ng/ml), IL-7 (5 ng/ml), IL-1β (25 ng/ml) and IL-2 (10 IU/ml) were purchased from Strathmann Biotech, Hanover, Germany. Granulocyte macrophage-colony stimulating factor (GM-CSF, Schering-Plough, Kenilworth, NJ, USA) was used at 100 ng/ml; IL-4 (R&D systems, Abingdon, UK) was used at 10 ng/ml; PGE2 (Sigma-Aldrich, St Louis, MO, USA) was used at 1 μg/ml; IFNγ (Biosource, Camarillo, CA, USA) was used at 1000 U/ml; IFNα (Peprotech, London, UK) was used at 3000 U/ml and pI:C (Sigma-Aldrich) was used at 20 μg/ml. IMDM (Cambrex, Verviers, Belgium) was supplemented with 10% FCS (Perbio, Helsingborg, Sweden). X-vivo-15 (Cambrex) and CellGro-DC (CellGenix, Freiburg, Germany) were both supplemented with 2% hAB (ICN Biomedicals, Zoetermeer, The Netherlands). All media were supplemented with 100 IU/ml sodium penicillin (Yamanouchi Pharma, Leiderdorp, The Netherlands), 100 μg/ml streptomycin sulfate (Radiumfarma-Fisiopharma, Naples, Italy), 2.0 mM L-glutamine (Invitrogen, Breda, Netherlands) and 0.01 mM 2-mercapo-ethanol (Merck, Darmstadt, Germany). The melanoma cell lines Mel-JKO and Mel-AKR and the EBV-LCL JY were cultured in IMDM/10% FCS. The HLA-A2.1 restricted melanoma-(MART-1(27–35)) specific CTL clones (4-TG-D8b and 4.4TG) were generated and propagated with feeder mix as described previously.37 Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)- or APC-labeled isotype controls and mouse mAbs to CD1a, CD86, CD40 (Pharmingen, San Diego, CA, USA), CD80 and CD14 (BD Biosciences, Heidelberg, Germany), CD83 (Immunotech, Marseille, France), CCR7 (R&D, Minneapolis, MN, USA) and NGFR (Chromaprobe, Maryland Heights, MO, USA) were used to determine the phenotype and transfection efficiency by fluorescence-activated cell sorting (FACS) analysis. Mean fluorescence index was calculated as follows: MFIndex=mean fluorescence intensity marker/fluorescence intensity isotype.

mRNA in vitro transcription

Enhanced GFP (EGFP) was replaced by the various genes of interest in the pGEM4Z/EGFP/A64 vector, which was used for in vitro mRNA transcription (IVT). The IL-12elasti cassette containing the p35 and p40 subunits of IL-12 joined together by a flexible linker (InvivoGen, San Diego, CA, USA) was used. This construct guarantees equal expression of both subunits and prevents over-expression of p40 and the creation of p40 homodimers which behave as an antagonist of IL-12p70.38 A codon-optimized minigene was constructed containing four repeats of the altered peptide ligand MART126–35A27L39 (MART4) in a string of beads manner (GeneArt, Regensburg, Germany). For proteosomal targeting, the MART sequence is preceded by the ubiquitin open-reading frame. To facilitate the translation of two separate proteins from one mRNA message, we made use of the T. asigna virus derived 2A sequence.12 The sequence of ubi(MART)4-2A with the ubiquitin sequence (bold and italic), the four MART epitopes (bold) and the 2A sequence (bold underlined): MQIFVKTLTGKTITLEVEPSDTIENV KAKIQDKEGIPPDQQRLIFAGKQLE GRTLSDYNIQKESTLHLVLRLRGVVNSEFKHEELAGIGILTVAEFKSEELAGIGILTVAEEELAGIGILTVAEEELAGIGILTVAEEVNRAEGRGSLLTCGDVEENPGPMGSMRGCGR. Additional sequences served as spacer or cloning sites. IVT was carried out using the T7 mMessage-mMachine kit (Ambion, Huntingdon, Cambridgeshire, UK) to generate m7G(5′)pppG-capped IVT mRNA (CAP) or the T7 MessageMachine Ultra kit (Ambion) using the anti-reverse cap analog to generate 3′-O-methyl-m7G(5′)pppG-capped IVT mRNA (ARCA) as described.40 In contrast to the conventional CAP, ARCA has only one 3′-OH group because the second 3′-OH group is replaced with OCH3. Because of this substitution, the RNA polymerase can only initiate transcription with the remaining hydroxyl group thus forcing ARCA incorporation in the forward orientation. As a result, unlike transcripts synthesized with the conventional CAP analog, 100% of the transcripts synthesized with ARCA at the 5′-end are translatable, leading to a strong stimulatory effect on translation.29 0.1 U pyrophosphatase (PPase; Frementas life sciences, St Leon-Rot, Germany) per IVT reaction of 20 μl was added. As previously described,41 addition of PPase increased the yield of IVT mRNA (1557 (SD 593) versus 1033 (SD 556) ng/μl; P=0.04, paired student's t-test). mRNA quality was checked by agarose gel electrophoresis. RNA concentration was assayed by spectrophotometrical analysis at OD260. RNA was stored at −80°C in small aliquots (1 μg/μl).

DC generation and transfection

Peripheral blood mononuclear cells were isolated from buffy coats of healthy donors by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway). Monocyte-derived DC (MoDC) were generated using GM-CSF and IL-4 in IMDM, X-vivo-15 or CellGro-DC as described.42 DC maturation was induced by an additional 2-day culture with MCM mimic (IL-1β, TNFα, IL-6 and PGE2),5 25% v/v MCM (generated as described43) supplemented with TNFα or a combination of IL-1β, TNFα, IFNγ, IFNα and pI:C.8 Electroporation (300 V, 150 μF) was carried out with 10 μg of IVT mRNA as described.40 For most experiments DCs were generated in IMDM/10% FCS and matured with MCM supplemented with TNFα unless specified otherwise. DCs transfected with IL-12 or GFP mRNA were resuspended at 2 × 10E5 cells/ml and incubated with or without CD40L-transfected cells (2 × 10E5/ml J558),42 IL-12p70 concentration was measured in 24 h supernatants by enzyme-linked immunosorbent assay.44 Chemotaxis of electroporated DCs was tested 24 h after electroporation in an overnight migration assay as described,6 using 250 ng/ml 6Ckine (Biosource). Quantification of migrated cells was by flow cytometry using a FACScalibur and True-count fluorospheres (Beckman Coulter, Mijdrecht, The Netherlands) according to the manufacturer's instructions.

Induction of MART126–35A27L peptide-specific CD8+ T cells

CD8β-positive CTL precursors from buffy coats of HLA-A2.1-positive healthy donors were isolated by positive selection on an automated magnetic sorting device (autoMACS; Miltenyi Biotec, Bergisch Gladbach, Germany) as described.45 Mature DC were transfected with ubi(MART)4 mRNA in combination with either GFP or IL-12 mRNA or with ubi(MART)4-2A-IL12 mRNA. Four hours after electroporation DCs were washed and multiple bulk cultures containing 0.5–1 × 106 CD8β T cells, 0.5–1 × 105 mRNA-transfected DCs and 0.25–0.5 × 106 irradiated autologous CD4+ T cells were set up in Yssel's medium supplemented with 1% hAB.45 The next day IL-7 was added. After 10 days, T cells were analyzed for specificity using PE- and/or APC-labeled HLA-A*0201 tetramers (Tm) presenting the MART126–35A27L epitope. Tm staining was performed as described.45 On day 10 the bulk cultures were restimulated with mRNA-transfected DCs and the next day IL-2 was added. Tm staining was performed after 7 days and the bulk cultures were restimulated as on day 10.

Functional assays

Eight to 10 days after the last (DC) stimulation the T-cell bulk cultures or CTL clones were harvested and the bulk cultures were pooled per condition, washed and incubated with target cells. Intracellular IFNγ staining was performed using the BD cytofix/cytoperm plus kit (BD Biosciences) according to the manufacturer's instructions. One hour after the start of the stimulation, GolgiPlug was added to each well (0,1% v/v). After 5 h cells were washed, stained with APC-labeled tetramers and PE labeled CD8 followed by intracellular staining with FITC-labeled anti-IFNγ. Cytolytic activity of the pooled bulk cultures was determined using a standard chromium release assay as described45 in the presence of a 50 times excess of unlabeled K562.

References

  1. 1

    Banchereau J, Palucka AK . Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 2005; 5: 296–306.

  2. 2

    Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N . Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 2001; 193: 233–238.

  3. 3

    Ingulli E, Mondino A, Khoruts A, Jenkins MK . In vivo detection of dendritic cell antigen presentation to CD4(+) T cells. J Exp Med 1997; 185: 2133–2141.

  4. 4

    Albert ML, Jegathesan M, Darnell RB . Dendritic cell maturation is required for the cross-tolerization of CD8+ T cells. Nat Immunol 2001; 2: 1010–1017.

  5. 5

    Lee AW, Truong T, Bickham K, Fonteneau JF, Larsson M, Da S et al. A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocyte-derived dendritic cells: implications for immunotherapy. Vaccine 2002; 20 (Suppl 4): A8–A22.

  6. 6

    Scandella E, Men Y, Legler DF, Gillessen S, Prikler L, Ludewig B et al. CCL19/CCL21-triggered signal transduction and migration of dendritic cells requires prostaglandin E2. Blood 2004; 103: 1595–1601.

  7. 7

    Vieira PL, de Jong EC, Wierenga EA, Kapsenberg ML, Kalinski P . Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction. J Immunol 2000; 164: 4507–4512.

  8. 8

    Mailliard RB, Wankowicz-Kalinska A, Cai Q, Wesa A, Hilkens CM, Kapsenberg ML et al. alpha-type-1 polarized dendritic cells: a novel immunization tool with optimized CTL-inducing activity. Cancer Res 2004; 64: 5934–5937.

  9. 9

    Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998; 4: 328–332.

  10. 10

    Su Z, Dannull J, Yang BK, Dahm P, Coleman D, Yancey D et al. Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer. J Immunol 2005; 174: 3798–3807.

  11. 11

    Rasmussen AB, Zocca MB, Bonefeld CM, von Essen M, Lauritsen JPH, Tomra S et al. Proteasomal targeting and minigene repetition improve cell-surface presentation of a transfected, modified melanoma tumour antigen. Scand J Immunol 2004; 59: 220–227.

  12. 12

    Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF et al. Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 2004; 22: 589–594.

  13. 13

    Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D et al. Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol 2001; 82: 1013–1025.

  14. 14

    Michiels A, Tuyaerts S, Bonehill A, Breckpot K, Heirman C, Van Meirvenne S et al. Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines. Gene Therapy 2005; 12: 772–782.

  15. 15

    Schaft N, Dorrie J, Thumann P, Beck VE, Muller I, Schultz ES et al. Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation. J Immunol 2005; 174: 3087–3097.

  16. 16

    de Vries IJ, Krooshoop DJEB, Scharenborg NM, Lesterhuis WJ, Diepstra JH, van Muijen GNP et al. Effective Migration of Antigen-pulsed Dendritic Cells to Lymph Nodes in Melanoma Patients Is Determined by Their Maturation State. Cancer Res 2003; 63: 12–17.

  17. 17

    Gilboa E, Vieweg J . Cancer immunotherapy with mRNA-transfected dendritic cells. Immunol Rev 2004; 199: 251–263.

  18. 18

    Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas CD et al. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest 2002; 109: 409–417.

  19. 19

    Kyte JA, Gaudernack G . Immuno-gene therapy of cancer with tumour-mRNA transfected dendritic cells. Cancer Immunol Immunother [published online, 1 September 2006].

  20. 20

    Legler DF, Krause P, Scandella E, Singer E, Groettrup M . Prostaglandin E2 Is Generally Required for Human Dendritic Cell Migration and Exerts Its Effect via EP2 and EP4 Receptors. J Immunol 2006; 176: 966–973.

  21. 21

    Trinchieri G . Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev 2003; 3: 133–146.

  22. 22

    Schmidt CS, Mescher MF . Peptide antigen priming of naive, but not memory, CD8 T cells requires a third signal that can be provided by IL-12. J Immunol 2002; 168: 5521–5529.

  23. 23

    MacGregor JN, Li Q, Chang AE, Braun TM, Hughes DPM, McDonagh KT . Ex vivo culture with interleukin (IL)-12 improves CD8+ T-cell adoptive immunotherapy for murine leukemia independ. Cancer Res 2006; 66: 4913–4921.

  24. 24

    Vujanovic L, Ranieri E, Gambotto A, Olson WC, Kirkwood JM, Storkus WJ . IL-12p70 and IL-18 gene-modified dendritic cells loaded with tumor antigen-derived peptides or recombinant protein effectively stimulate specific Type-1 CD4+ T-cell responses from normal donors and melanoma patients in vitro. Cancer Gene Ther 2006; 13: 798–805.

  25. 25

    Bontkes HJ, Ruizendaal JJ, Kramer D, Meijer CJ, Schreurs MW, Hooijberg E . Interleukin-12 increases proliferation and Interferon- production but not cytolytic activity of human antigen specific effector memory Cytotoxic T-lymphocytes; power of the effect depends on the functional avidity of the T-cell and antigen concentration. Hum Immunol 2006; 66: 1137–1145.

  26. 26

    Scholten KB, Kramer D, Kueter EW, Graf M, Schoedl T, Meijer CJ et al. Codon modification of T cell receptors allows enhanced functional expression in transgenic human T cells. Clin Immunol 2006; 119: 135–145.

  27. 27

    Su Z, Vieweg J, Weizer AZ, Dahm P, Yancey D, Turaga V et al. Enhanced induction of telomerase-specific cd4+ t cells using dendritic cells transfected with rna encoding a chimeric gene product. Cancer Res 2002; 62: 5041–5048.

  28. 28

    Bonehill A, Heirman C, Tuyaerts S, Michiels A, Breckpot K, Brasseur F et al. Messenger RNA-electroporated dendritic cells presenting MAGE-A3 simultaneously in HLA class I and class II molecules. J Immunol 2004; 172: 6649–6657.

  29. 29

    Mockey M, Goncalves C, Dupuy FP, Lemoine FM, Pichon C, Midoux P . mRNA transfection of dendritic cells: Synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level. Biochem Biophys Res Commun 2006; 340: 1062–1068.

  30. 30

    Xu S, Koski GK, Faries M, Bedrosian I, Mick R, Maeurer M et al. Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism. J Immunol 2003; 171: 2251–2261.

  31. 31

    Hamann D, Roos MT, van Lier RAW . Faces and phases of human CD8+ T-cell development. Immunol Today 1999; 20: 177–180.

  32. 32

    Hiura T, Kagamu H, Miura S, Ishida A, Tanaka H, Tanaka J et al. Both regulatory t cells and antitumor effector T cells are primed in the same draining lymph nodes during tumor progression. J Immunol 2005; 175: 5058–5066.

  33. 33

    Dannull J, Nair S, Su Z, Boczkowski D, DeBeck C, Yang B et al. Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand. Blood 2005; 105: 3206–3213.

  34. 34

    Grunebach F, Kayser K, Weck MM, Muller MR, Appel S, Brossart P . Cotransfection of dendritic cells with RNA coding for HER-2//neu and 4–1BBL increases the induction of tumor antigen specific cytotoxic T lymphocytes. Cancer Gene Ther 2005; 12: 749–756.

  35. 35

    Michiels A, Breckpot K, Corthals J, Tuyaerts S, Bonehill A, Heirman C et al. Induction of antigen-specific CD8(+) cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA. Gene Therapy 2006; 13: 1027–1036.

  36. 36

    Loskog A, Ninalga C, Totterman TH . Dendritic cells engineered to express CD40L continuously produce IL12 and resist negative signals from Tr1/Th3 dominated tumors. Cancer Immunol Immunother 2006; 55: 588–597.

  37. 37

    Hooijberg E, Ruizendaal JJ, Snijders PJ, Kueter EW, Walboomers JM, Spits H . Immortalization of human CD8+ T cell clones by ectopic expression of telomerase reverse transcriptase. J Immunol 2000; 165: 4239–4245.

  38. 38

    Ling P, Gately MK, Gubler U, Stern AS, Lin P, Hollfelder K et al. Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J Immunol 1995; 154: 116–127.

  39. 39

    Valmori D, Fonteneau JF, Lizana CM, Gervois N, Lienard D, Rimoldi D et al. Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J Immunol 1998; 160: 1750–1758.

  40. 40

    Van Tendeloo VFI, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 2001; 98: 49–56.

  41. 41

    Cunningham PR, Ofengand J . Use of inorganic pyrophosphatase to improve the yield of in vitro transcription reactions catalyzed by T7 RNA polymerase. Biotechniques 1990; 9: 713–714.

  42. 42

    Bontkes HJ, de Gruijl TD, Schuurhuis GJ, Scheper RJ, Meijer CJ, Hooijberg E . Expansion of dendritic cell precursors from human CD34(+) progenitor cells isolated from healthy donor blood; growth factor combination determines proliferation rate and functional outcome. J Leukoc Biol 2002; 72: 321–329.

  43. 43

    Romani N, Reider D, Heuer M, Ebner S, Kampgen E, Eibl B et al. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods 1996; 196: 137–151.

  44. 44

    Snijders A, Hilkens CM, Van der Pouw Kraan TC, Engel M, Aarden LA, Kapsenberg ML . Regulation of bioactive IL-12 production in lipopolysaccharide-stimulated human monocytes is determined by the expression of the p35 subunit. J Immunol 1996; 156: 1207–1212.

  45. 45

    Schreurs MW, Scholten KB, Kueter EW, Ruizendaal JJ, Meijer CJ, Hooijberg E . In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones. J Immunol 2003; 171: 2912–2921.

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Acknowledgements

We thank A Stam for technical assistance, and the Maurits and Anna de Kock Foundation for financial support in the purchase of an HPLC. We thank Dr ML Kapsenberg for providing IL-12 antibodies and Dr S Hallez for providing the ubiquitin open-reading frame. We are grateful to Drs KJB Scholten and Dr MWJ Schreurs for fruitful discussions. This work was financially supported by Dutch Cancer Society (KWF) Grant VU2002-2627. VFIVT is a postdoctoral fellow of the fund for Scientific Research-Flanders (FWO-Vlaanderen).

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Correspondence to H J Bontkes.

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Bontkes, H., Kramer, D., Ruizendaal, J. et al. Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells. Gene Ther 14, 366–375 (2007). https://doi.org/10.1038/sj.gt.3302874

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Keywords

  • dendritic cells
  • Interleukin-12
  • CTL
  • functional avidity
  • mRNA

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