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Induction of Human Dendritic Cell Maturation Using Transfection with RNA Encoding a Dominant Positive Toll-Like Receptor 4¹

Robin M. Cisco^*, Zeinab Abdel-Wahab^*, Jens Dannull^*, Smita Nair^*, Douglas S. Tyler^*,, Eli Gilboa^*, Johannes Vieweg^*,, Yehia Daaka^* and Scott K. Pruitt^2,*,

Departments of Surgery, ^* Duke University and Durham Veterans Affairs Medical Centers, Durham, NC 27710

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Maturation of dendritic cells (DC) is critical for the induction of Ag-specific immunity. Ag-loaded DC matured with LPS, which mediates its effects by binding to Toll-like receptor 4 (TLR4), induce Ag-specific CTL in vitro and in vivo in animal models. However, clinical use of LPS is limited due to potential toxicity. Therefore, we sought to mimic the maturation-inducing effects of LPS on DC by stimulating TLR4-mediated signaling in the absence of exogenous LPS. We developed a constitutively active TLR4 (caTLR4) and demonstrated that transfection of human DC with RNA encoding caTLR4 led to IL-12 and TNF- secretion. Transfection with caTLR4 RNA also induced a mature DC phenotype. Functionally, transfection of DC with caTLR4 RNA enhanced allostimulation of CD4⁺ T cells. DC transfected with RNA encoding the MART (Melan-A/MART-1) melanoma Ag were then used to stimulate T cells in vitro. Cotransfection of these DC with caTLR4 RNA enhanced the generation of MART-specific CTL. This CTL activity was superior to that seen when DC maturation was induced using either LPS or a standard mixture of cytokines (TNF-, IL-6, IL-1, and PGE₂). We conclude that transfection of DC with RNA encoding a functional signaling protein, such as caTLR4, may provide a new tool for studying TLR signaling in DC and may be a promising approach for the induction of DC maturation for tumor immunotherapy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The maturation state of dendritic cells (DC)³ is critical for the induction of effector T cell function (1). Although human DC express a variety of receptors that participate in the induction of maturation, members of the Toll-like receptor (TLR) family are central to this process. Of the 10 described human TLRs, monocyte-derived DC predominantly express TLRs 2 and 4 (2, 3). LPS, an agent commonly used to induce DC maturation experimentally, mediates its effects by binding (along with soluble CD14 and MD-2) to cell surface TLR4 (4, 5). Intracellular signaling pathways, including NF-B and p38 mitogen-activated protein kinase (MAPK), are then activated and DC maturation is induced (6, 7, 8, 9). LPS-induced maturation has been shown to markedly enhance the ability of Ag-loaded DC to stimulate Ag-specific T cell responses in vitro (1, 10, 11). In addition, Pasare and Medzhitov (12) recently reported that stimulation of TLR4 on DC using LPS blocks the T cell inhibitory activity of regulatory CD4⁺CD25⁺ T cells. Blockade of such regulatory T cell (T_reg) activity may be critically important for effective DC-based tumor immunotherapy. However, the clinical use of LPS to induce DC maturation for immunotherapy is prohibited by concerns over LPS-mediated toxicity as well as batch-to-batch variability.

RNA transfection is one method that has been shown to be effective for loading DC with tumor Ags. In both human and murine systems, in vitro and in vivo, tumor Ag RNA-transfected DC have been shown to stimulate antitumor effector T cell responses (13, 14, 15, 16). We have previously demonstrated that when RNA is transfected into DC, the RNA is translated into protein that can be processed into peptides and presented in the context of HLA class I (16). Translation of the RNA into a functional protein is not necessary for Ag processing to occur and, in fact, misfolded and incompletely translated protein products may enhance antigenic peptide presentation (17). Transfection of DC with RNA encoding a functional protein might allow control of specific cellular pathways. However, the ability of transfected RNA to be translated into a functional signaling protein remains to be shown.

In this study, we created a constitutively active human TLR4 (caTLR4) and used RNA encoding this caTLR4 to transfect human monocyte-derived DC. Transfection of DC with caTLR4 RNA induced DC maturation that was comparable to that achieved using the current "gold standard" cytokine mixture of TNF-, IL-6, IL-1, and PGE₂ (18). Specifically, transfection with caTLR4 RNA induced secretion of IL-12 and TNF-, up-regulated the expression of the phenotypic markers of maturation, and enhanced the allostimulatory activity of the DC. Finally, we evaluated the ability of RNA-transfected DC to stimulate Ag-specific T cell responsiveness against the human melanoma Ag MART (Melan-A/MART-1) and found that caTLR4 RNA cotransfection enhanced the ability of HLA-A2⁺ MART RNA-transfected DC to generate MART-specific effector T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines and reagents

HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 plus 10% FCS. The cell line 293-luc-B, a stable transfectant of HEK293 containing an NF-B-dependent luciferase reporter plasmid, was created by Lipofectamine-mediated DNA transfection followed by geneticin (Invitrogen, Carlsbad, CA) selection. TAP-deficient, HLA-A0201⁺ T2 cells were a gift from Dr. P. Cresswell (Yale University, New Haven, CT) and were maintained in serum-free AIM-V medium (Invitrogen). Melanoma cell lines DM6 (Melan-A/MART1⁺, HLA-A2⁺) and DM150 (Melan-A/MART1^–, HLA-A2⁺) were derived from fresh surgical melanoma specimens and were gifts from Dr. H. Seigler (Duke University Medical Center, Durham, NC). Melanoma cell line 397mel (HLA-A1/A25, MART⁺) was a gift from Dr. S. Rosenberg (National Cancer Institute, Bethesda, MD). Hybridomas BB7.2 and MA2.1 (both anti-HLA-A2), Gap3 (anti-A3), and A11.1 (anti-A11) were obtained from the American Type Culture Collection. A purified mAb against HLA-A1 was purchased from One Lambda (Canoga Park, CA).

Monocyte-derived DC

Human DC were generated from adherent monocytes as previously described (16, 19). Briefly, PBMC were isolated by Ficoll gradient centrifugation of buffy coat lymphocytes purchased from the American Red Cross. A limited HLA-A phenotype was determined by flow cytometry of PBMC using HLA-specific mAbs. PBMC not immediately used for DC production were cryopreserved in 90% human AB serum (Valley Labs, Winchester, VA)/10% DMSO. Fresh (or thawed) PBMC were plated in tissue culture flasks in AIM-V serum-free medium. After a 2-h plastic adherence step, nonadherent lymphocytes were gently washed away and adherent blood monocytes were then cultured in serum-free medium (X-VIVO-15; BioWhittaker, Walkersville, MD) supplemented with 1000 U/ml GM-CSF (Immunex, Thousand Oaks, CA) and 1000 U/ml IL-4 (R&D Systems, Minneapolis, MN). After 5–6 days in culture, the immature DC were evaluated to confirm their DC phenotype and purity using two-color flow cytometry and a panel of mAbs specific for CD3 (T cells), CD14 (monocytes), CD19 (B cells), CD40 (DC), CD56 (NK cells), CD80 (monocytes and DC), CD86 (monocytes and DC), HLA-class I, HLA-class II, as well as isotype-matched control mAbs. Only DC cultures free of significant monocyte, B, T, or NK cell contamination were used for further studies.

DNA constructs

A chimeric caTLR4 construct consisting of the nerve growth factor receptor leader sequence fused to a FLAG epitope tag followed by a truncated TLR4 molecule was created using PCR cloning and standard molecular biology techniques. The truncation of human TLR4 was located between aa M620 and P621, creating a caTLR4 as described by Medzhitov et al. (5), based on a deletional analysis of the Drosophila Toll (20). The chimeric caTLR4 was inserted into a modified pcDNA3.1 plasmid (Invitrogen) (which contains a T7 promoter 5' to the insert site) into which a 64-adenosine synthetic poly(A) tail segment had been inserted at the 3' end of the multiple cloning site. The insert was confirmed by DNA sequencing. Western blotting of lysates from HEK293 cells transfected with the caTLR4 plasmid confirmed the generation of the chimeric caTLR4 protein. The plasmid used for the in vitro transcription of green fluorescent protein (GFP) RNA has been previously described (15, 16, 21). The plasmid encoding MART with an A27L aa mutation was created by overlapping PCR of cloned wild-type MART using primers containing the desired DNA mutation and confirmed by sequencing, Western blotting, and functional studies (22).

In vitro transcription of RNA

For the production of RNA, plasmid DNA was digested with the restriction enzyme SpeI at a site present at the 3' end of the 64-adenosine segment of the modified pcDNA3.1 plasmid. After ethanol precipitation and resuspension in water, the digested DNA was used as template for in vitro RNA transcription using a commercial kit (mMessage machine; Ambion, Austin, TX). After DNase treatment, the resultant RNA was purified, quantitated, evaluated by MOPS-formaldehyde agarose gel electrophoresis to confirm production of full-length RNA, and then stored at –80°C until use. LPS levels of the in vitro-transcribed caTLR4 RNA were below detection level (<0.5 endotoxin units/ml), as determined by the Limulus amebocyte lysate assay (Associates of Cape Cod, Falmouth, MA).

In several experiments, caTLR4 RNA was digested with RNaseI_f (New England Biolabs, Beverly, MA) according to the manufacturer’s instructions and then boiled. This RNase-treated caTLR4 RNA was then used for electroporation of DC as described below.

DC transfection

Immature human DC were electroporated with RNA as previously described (16). Briefly, the immature DC were harvested and washed three times in cold PBS, then resuspended in cold Opti-MEM (Invitrogen) at 1–2 x 10⁶/100 µl. Two hundred-microliter aliquots of cells were mixed in a 2-mm gap cuvette along with a total of 20 µg RNA. When a combination of two RNAs was used, 10 µg of each RNA was used. The cells and RNA were then subjected to 300 V for 500 µs in a square wave electroporator (BTX, San Diego, CA). The electroporated DC were immediately transferred to tissue culture wells with fresh X-VIVO-15 medium containing IL-4 and GM-CSF. Control DC were electroporated with GFP RNA, which allowed for assessment of electroporation transfection efficiency using fluorescence microscopy.

Induction of DC maturation

Maturation agents were added to the DC culture medium 2–4 h after DC were electroporated with RNA. The maturation agents used included poly(I:C) (12.5 µg/ml; Sigma-Aldrich, St. Louis, MO), LPS (5 µg/ml; Sigma-Aldrich), or cytokine mixture (18) consisting of TNF- (5 ng/ml), rhIL-1 (5 ng/ml), rhIL-6 (150 ng/ml; all from R&D Systems), and PGE₂ (1 µg/ml; Sigma-Aldrich).

Cytokine secretion

DC culture supernatants were harvested 24 and 48 h after transfection and stored at –20°C until use. IL-12 p70 levels and TNF- concentrations were assessed using commercially available ELISA kits (R&D Systems).

Allostimulatory capacity of RNA-transfected DC

To evaluate the ability of DC electroporated with caTLR4 RNA to stimulate CD4⁺ T cells, we performed allogeneic MLRs using RNA-electroporated DC as stimulators. In these experiments, DC were electroporated with RNA, and after 1 h in culture, maturation stimuli were added as indicated. After an additional 3 h of incubation, the DC were washed and resuspended in medium and irradiated (3000 rad). Allogeneic CD4⁺ T cells (1 x 10⁵), isolated by magnetic bead separation (Miltenyi Biotec, Auburn, CA) from PBMC, were then stimulated at a 10:1 (responder:stimulator) ratio with the RNA-electroporated DC. After 4 days in culture, 1 µCi of [³H]thymidine (NEN, Boston, MA) was added to each well and incorporated radioactivity was measured after 16 h using liquid scintillation counting. GFP RNA-electroporated DC were used to establish the baseline proliferation induced by immature electroporated DC. GFP RNA-electroporated DC treated with various maturation stimuli served as a positive control for the stimulation of T cell proliferation by mature DC. DC alone and T cells alone were used as negative controls.

In vitro T cell cultures

For T cell cultures, only DC from HLA-A2⁺ donors were used, as determined by flow cytometry of donor PBL using the anti-HLA mAbs. Approximately 4 h after transfection, DC were added to autologous nonadherent lymphocytes (as a source of T cells) at a 1:10 (stimulator:responder) ratio in tissue culture medium consisting of RPMI 1640 plus 10%FCS supplemented with antibiotics and 10 µM 2-ME. IL-2 at 20 U/ml was added beginning on day 7 and then twice per week thereafter. Each T cell culture was restimulated with RNA-transfected DC at 7- to 10-day intervals for a total of three to four stimulations. As a positive control for maturation, we used our current standard regimen consisting of exposure of RNA-electroporated DC to cytokine mixture beginning 1 h after transfection. After 4 h in culture, DC were washed and added to the T cell cultures. As a negative control for maturation, DC were left untreated after MART RNA transfection and added to T cell cultures in the same fashion. Beginning 7 days after the final stimulation, Ag specificity and T cell effector function were evaluated.

Evaluation of MART peptide/HLA-A2 tetramer-positive T cells

A PE-labeled class I HLA-A2 tetramer loaded with the Melan-A_26–35 A27L immunodominant peptide analog ELAGIGILTV, in combination with a FITC-labeled anti-CD8 was purchased from Beckman Coulter (San Diego, CA) and was used according to the manufacturer’s instructions. Staining was evaluated using flow cytometry.

Ag-specific T cell IFN- release

In vitro-stimulated T cells were evaluated for Ag-specific effector function using an IFN- ELISPOT assay as previously described (16). This assay was performed at a stimulator:responder ratio of 1:10 in triplicate. T2 cells loaded exogenously with either MART A27L peptide or M1 peptide (influenza matrix protein 1 HLA-A2-restricted immunodominant peptide GILGFVFTL, as a negative control) were used as targets. In addition, melanoma cell lines, previously HLA-typed and evaluated for MART expression, were used as targets. Plates were scanned and images were counted by an ImmunoSpot Series Analyzer (Cellular Technology, Cleveland, OH).

Determination of cytotoxicity

⁵¹Cr release assays were performed in triplicate using established methods as previously described (23). Percent specific lysis was calculated from the equation: ((experimental release – spontaneous release)/(maximum release – spontaneous release (targets in medium))) x 100. Spontaneous release was <15% of maximum release. SDs for triplicate wells were <5%.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction and evaluation of caTLR4

We wanted to demonstrate the functional consequences of transfecting cells with constitutively active chimeric TLR4. To generate a constitutively active molecule, we deleted the extracellular portion of TLR4 (aa 1–620) (5). This deletion was based on a mutational analysis of Drosophila Toll in which the deletion of the homologous portion of the molecule created a constitutively active Toll (20). After confirming the identity of our construct by DNA sequencing, we demonstrated that HEK293 cells transfected with this construct expressed a FLAG-tagged protein of appropriate molecular mass by Western blotting of cell lysates (data not shown). Transfection of HEK293 cells with the plasmid containing this caTLR4 construct led to the activation of NF-B, demonstrating that our chimeric caTLR4 DNA encoded a functionally active protein (data not shown).

Although these transfection experiments using plasmid DNA showed that the caTLR4 plasmid DNA had constitutive NF-B-activating activity, our main goal was to determine whether RNA transcribed from this plasmid DNA could be transfected into cells and be translated into a functionally active signaling protein. We therefore used the caTLR4 plasmid DNA as a template for in vitro RNA transcription and evaluated whether transfection with this caTLR4 RNA would activate NF-B. For these experiments, we created a stable transfectant of HEK293 cells containing a NF-B dependent luciferase reporter (293-luc-B). When 293-luc-B cells were transfected with caTLR4 RNA, NF-B activation was induced (Fig. 1), thus confirming that transfected caTLR4 RNA was translated into a functional signaling protein.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. RNA encoding caTLR4, when transfected into 293-luc-B cells, activates NF-B. 293-luc-B cells (HEK293 cells stably transfected with a NF-B inducible luciferase reporter gene) were transfected with either caTLR4 or GFP (negative control) RNA. After 6 h of serum starvation, luciferase activity was measured in cell lysates. TNF- was added as indicated as a positive control for NF-B activation. Data are shown as fold increase in luciferase activity above basal.

View larger version (66K): [in this window] [in a new window]   FIGURE 2. Transfection of DC with caTLR4 RNA activates p38 MAPK. Human DC were transfected with the indicated RNA and left untreated or were exposed to poly(I:C) (PIC) to induce maturation. Forty-eight hours later, cell lysates were prepared, subjected to SDS-PAGE, and analyzed for expression of phospho-p38 (P-p38) and total p38 by Western blotting. Equal amounts of total protein were loaded in each lane. Positive staining is seen for DC samples treated with poly(I:C) as well as for DC transfected with caTLR4 RNA. The results are representative of two experiments.

DC maturation was evaluated by phenotypic analysis of cell surface markers after transfection with caTLR4 RNA. Fig. 3 shows that transfection of DC with caTLR4 RNA induced surface expression of the maturation marker CD83 and stimulated increased surface expression of CD40 and of costimulatory molecules CD80 and CD86. Expression of these phenotypic markers of maturation in caTLR4 RNA-transfected DC was comparable, or superior, to that induced by cytokine mixture or LPS in DC transfected with control RNA. In addition, Fig. 3 also demonstrates that treatment of caTLR4 RNA with RNase abolished its maturation-inducing effects on the DC phenotype, indicating that the effects observed after caTLR4 RNA transfection were not due to LPS contamination. These results confirmed that transfection of DC with caTLR4 RNA induced phenotypic maturation of DC. The next step was to measure the functional consequences of caTLR4 RNA-induced DC maturation. Various parameters of DC function were examined, starting with cytokine secretion.

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Transfection of DC with caTLR4 induces phenotypic maturation. DC were transfected with the indicated RNA and then either left untreated or exposed to LPS or the cytokine mixture (CC) to induce maturation. Forty-eight hours later, DC were harvested and surface phenotype was evaluated by flow cytometry. These results are representative of four experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Transfection of DC with RNA encoding caTLR4 results in IL-12 and TNF- secretion. Immature monocyte-derived human DC were transfected with the indicated RNA and placed in culture. In some cultures, either the cytokine mixture (CC) or LPS was added as a maturation inducer. Forty-eight hours later, supernatants were harvested and levels of secreted TNF- (A) and IL-12 p70 (B) were determined by ELISA. (GFP RNA = RNA encoding GFP used as a negative control). The results shown (A and B) are representative of eight experiments, each with similar results. The effect of RNase digestion on the ability of caTLR4 RNA transfection to induce DC cytokine secretion was also evaluated for TNF- (C) and IL-12 p70 (D). The results shown (C and D) are representative of three experiments, each with similar results.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Transfection with caTLR4 RNA enhances the allostimulatory activity of DC. Human DC were transfected with either caTLR4 RNA or with GFP RNA as a control. The GFP RNA-transfected DC were then either left untreated (negative control) or exposed to a maturation stimulus (M, which for MLR-A was LPS and for B through E was the cytokine mixture). After a 4-h incubation, these DC were irradiated (3000 rad) and cultured with allogeneic CD4+ T cells. Proliferation was assessed 4 days later using [3H]thymidine incorporation.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Enhanced generation of MART HLA-A2 tetramer-positive CD8+ T cells after stimulation with DC transfected with MART RNA plus caTLR4 RNA. The effector T cell populations generated after serial stimulation with RNA-transfected DC were stained with a MART peptide-loaded HLA-A2 tetramer. MART peptide-specific CD8+ T cells were then identified using flow cytometry. The percentages indicate the fraction of tetramer-positive cells within the entire population of T cells. The results are representative of five experiments.

View larger version (37K): [in this window] [in a new window]   FIGURE 7. IFN- ELISPOT of T cells stimulated with DC transfected with MART and TLR4 RNA. Autologous T cells were serially stimulated with DC transfected with the indicated combinations of RNA. As a positive control for maturation induction, RNA-transfected DC were treated with cytokine mixture (CC) before being added to T cell cultures. After three serial stimulations, IFN- ELISPOT was performed using melanoma cell line targets DM6 (MART+, A2+) and 397mel (A3+, A2–, MART+) as well as T2 cells loaded with either the HLA-A2-restricted immunodominant MART A27L peptide or influenza M1 peptide (as a negative control). These results are representative of four experiments.

View larger version (25K): [in this window] [in a new window]   FIGURE 8. MART Ag-specific lytic activity of T cells stimulated with caTLR4-transfected DC. After three serial stimulations with DC transfected with the indicated RNA, T cells were evaluated for Ag-specific lytic activity using a 51Cr release assay. Targets included melanoma cell lines DM6 (MART+, A2+), DM150 (MART–, A2+), and DM150 loaded with MART A27L peptide, as well as T2 cells loaded with either MART A27L peptide or influenza M1 peptide (as a negative control). These results are representative of three separate experiments, each with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The use of LPS for the induction of DC maturation in clinical immunotherapy is unlikely because of toxicity (potentially leading to toxic shock) and batch-to-batch variability. However, using in vitro and in vivo animal models, DC induced to mature using LPS and then loaded with MHC-restricted antigenic peptides are highly effective in stimulating Ag-specific T cell responses (1, 10, 11). By using caTLR4 RNA to induce DC maturation, we sought to stimulate the pathways that mediate the effects of LPS on DC without using this exogenous potentially toxic agent.

When LPS binds to TLR4, the adaptor molecules myeloid differentiation factor 88, Toll-IL-1R domain-containing adapter protein/myeloid differentiation protein 88 adapter-like, and Toll-IL-1R domain-containing adapter inducing IFN- are recruited to the intracellular Toll-IL-1R domain of TLR4, leading to activation of multiple signaling pathways, including NF-B and p38 MAPK (30). Using inhibitors of these signaling pathways, Ardeshna et al. (6) showed that both are critically important for optimal DC maturation by LPS. As demonstrated in Figs. 1 and 2, transfection of cells with caTLR4 RNA activates both NF-B and p38 MAPK, similar to the known effects of LPS on intracellular signaling.

The ability of LPS-matured DC to generate effector T cells may in part be mediated by LPS-induced IL-12 secretion. IL-12 has been demonstrated to stimulate Th cell proliferation and is vital for the generation of Th1 responses (31, 32). As seen in Fig. 4, DC transfected with caTLR4 RNA secreted IL-12 at levels comparable to those achieved after exposure to LPS, demonstrating that our RNA construct is equal in potency with respect to this LPS-induced effect. When compared with the cytokine mixture used clinically to induce DC maturation, caTLR4 RNA transfection was superior to the cytokine mixture in inducing secretion of both IL-12 and TNF-.

An additional mechanism by which LPS-treated DC may function to more effectively induce Ag-specific effector T cell responses is by promoting expansion of Th cells and biasing the Th response toward Th1. As assessed by the ability to stimulate proliferation of allogeneic CD4⁺ T cells, DC transfected with caTLR4 were comparable, or even superior, to DC transfected with control RNA and then matured using either LPS or cytokine mixture (Fig. 5).

LPS may also enhance the ability of DC to stimulate Ag-specific T cells through the inhibition of T_reg. Pasare and Medzhitov (12) recently demonstrated that ligation of TLR4 (using LPS) or TLR9 (using CpG oligonucleotides) in a murine model reversed the ability of CD4⁺CD25⁺ T_reg to inhibit CD4⁺ T cell proliferation. We are currently investigating the ability of caTLR4 RNA-transfected human DC to overcome the effects of T_reg in vitro.

Although DC transfection with caTLR4 RNA clearly induces maturation, one of our most important findings is that MART RNA-transfected DC, when matured using caTLR4 RNA transfection, are superior inducers of anti-MART effector T cells. When compared with the use of cytokine mixture as a maturation inducer, caTLR4 RNA-transfected DC stimulated the generation of higher numbers of MART-specific effector T cells as assessed by both tetramer binding (Fig. 6 and Table I) and IFN- secretion (Fig. 7). In addition, T cells stimulated with DC transfected with MART RNA and induced to mature using caTLR4 RNA transfection had the highest lytic activity against melanoma cells expressing MART (Fig. 8). Thus, the induction of DC maturation using caTLR4 transfection might have potential clinical applications for the stimulation of antitumor effector T cell responses during immunotherapy.

The use of caTLR4 RNA transfection to induce DC maturation has several additional practical advantages with respect to clinical applications for DC-based tumor immunotherapy. First, RNA transfection using electroporation is a simple procedure that eliminates the need for potentially toxic transfection reagents (such as lipid modifiers) that have not been approved for clinical use. Second, RNA encoding tumor Ags can be cotransfected, along with caTLR4 RNA, into DC for tumor Ag loading. Third, RNAs encoding additional signaling molecules involved in specific signaling pathways could potentially be combined to enhance certain aspects of DC maturation. We are currently developing constitutively active mutants of other TLRs as well as of downstream signaling molecules involved in TLR-mediated signaling for this purpose. Finally, induction of DC maturation using caTLR4 RNA transfection avoids the significant expense of purchasing clinical grade recombinant human cytokines.

The use of constitutively active signal molecule RNA also has potential applications for more fully elucidating the role of specific TLRs in the induction of DC maturation. Constitutively active forms of several other TLRs that we are currently developing will be ideal for this purpose. As an example, dsRNA, the ligand for TLR3, has many additional effects on cellular signaling that are not mediated directly through binding to TLR3 (33). An RNA encoding a constitutively active TLR3 will allow us to specifically evaluate and manipulate dsRNA-mediated maturation-inducing effects in DC that are TLR3 mediated.

In summary, we show that transfection of human monocyte-derived DC with caTLR4 RNA induces DC maturation. Both phenotypically and based on cytokine secretion, as well as ability to stimulate proliferation of allogenic CD4⁺ T cells, the DC maturation induced with caTLR4 RNA transfection is comparable or even superior to that induced using either LPS or the clinically utilized cytokine mixture of TNF-, IL-1, IL-6, and PGE₂. Most importantly, DC transfected with RNA encoding the MART melanoma Ag most effectively induced MART-specific effector T cells when maturation was induced by cotransfection with caTLR4 RNA. The induction of DC maturation using caTLR4 RNA transfection may therefore be a promising approach for DC-based clinical tumor immunotherapy.

	Footnotes

 
¹ S.K.P. is supported by a Veterans Affairs Merit Review Grant; R.M.C. was supported by a grant from the Department of Surgery, Duke University; D.S.T. is supported by a grant from the Lustgarden Foundation; and Y.D. is supported by R01-AG17952 from the National Institutes of Health.

² Address correspondence and reprint requests to Dr. Scott K. Pruitt, Box 2624, Duke University Medical Center, Durham, NC 27710. E-mail address: scott.pruitt{at}duke.edu

³ Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; caTLR4, constitutively active TLR4; MAPK, mitogen-activated protein kinase; MART, Melan-A/Mart-1 melanoma Ag; GFP, green fluorescent protein; T_reg, regulatory T cell.

Received for publication December 9, 2003. Accepted for publication March 18, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Labeur, M. S., B. Roters, B. Pers, A. Mehling, T. A. Luger, T. Schwarz, S. Grabbe. 1999. Generation of tumor immunity by bone marrow-derived dendritic cells correlates with dendritic cell maturation stage. J. Immunol. 162:168.[Abstract/Free Full Text]
2. Re, F., J. L. Strominger. 2001. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells. J. Biol. Chem. 276:37692.[Abstract/Free Full Text]
3. Muzio, M., D. Bosisio, N. Polentarutti, G. D’amico, A. Stoppacciaro, R. Mancinelli, C. van’t Veer, G. Penton-Rol, L. P. Ruco, P. Allavena, A. Mantovani. 2000. Differential expression and regulation of Toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. J. Immunol. 164:5998.[Abstract/Free Full Text]
4. da Silva Correia, J., K. Soldau, U. Christen, P. S. Tobias, R. J. Ulevitch. 2001. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex: transfer from CD14 to TLR4 and MD-2. J. Biol. Chem. 276:21129.[Abstract/Free Full Text]
5. Medzhitov, R., P. Preston-Hurlburt, C. A. Janeway, Jr. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388:394.[Medline]
6. Ardeshna, K. M., A. R. Pizzey, S. Devereux, A. Khwaja. 2000. The PI3 kinase, p38 SAP kinase, and NF-B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells. Blood 96:1039.[Abstract/Free Full Text]
7. Arrighi, J.-F., M. Rebsamen, F. Rousset, V. Kindler, C. Hauser. 2001. A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-, and contact sensitizers. J. Immunol. 166:3837.[Abstract/Free Full Text]
8. Lyakh, L. A., G. K. Koski, W. Telford, R. E. Gress, P. A. Cohen, N. R. Rice. 2000. Bacterial lipopolysaccharide, TNF-, and calcium ionophore under serum-free conditions promote rapid dendritic cell-like differentiation in CD14⁺ monocytes through distinct pathways that activate NF-B. J. Immunol. 165:3647.[Abstract/Free Full Text]
9. Yoshimura, S., J. Bondeson, B. M. Foxwell, F. M. Brennan, M. Feldmann. 2001. Effective antigen presentation by dendritic cells is NF-B dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines. Int. Immunol. 13:675.[Abstract/Free Full Text]
10. Lapointe, R., J. F. Toso, C. Butts, H. A. Young, P. Hwu. 2000. Human dendritic cells require multiple activation signals for the efficient generation of tumor antigen-specific T lymphocytes. Eur. J. Immunol. 30:3291.[Medline]
11. Salio, M., D. Shepherd, P. R. Dunbar, M. Palmowski, K. Murphy, L. Wu, V. Cerundolo. 2001. Mature dendritic cells prime functionally superior Melan-A-specific CD8⁺ lymphocytes as compared with nonprofessional APC. J. Immunol. 167:1188.[Abstract/Free Full Text]
12. Pasare, C., R. Medzhitov. 2003. Toll pathway-dependent blockade of CD4⁺CD25⁺ T cell-mediated suppression by dendritic cells. Science 299:1033.[Abstract/Free Full Text]
13. Boczkowski, D., S. K. Nair, J.-H. Nam, H. K. Lyerly, E. Gilboa. 2000. Induction of tumor immunity and cytotoxic T lymphocyte responses using dendritic cells transfected with messenger RNA amplified from tumor cells. Cancer Res. 60:1028.[Abstract/Free Full Text]
14. Heiser, A., M. A. Maurice, D. R. Yancey, N. Z. Wu, P. Dahm, S. K. Pruitt, D. Boczkowski, S. K. Nair, M. S. Ballo, E. Gilboa, J. Vieweg. 2001. Induction of polyclonal prostate cancer-specific CTL using dendritic cells transfected with amplified tumor RNA. J. Immunol. 166:2953.[Abstract/Free Full Text]
15. Kalady, M. F., M. A. Onaitis, D. S. Tyler, S. K. Pruitt. 2001. RNA electroporation of dendritic cells results in superior antigen presentation and improved generation of specific effector T cells. Surg. Forum 52:279.
16. Kalady, M. F., M. W. Onaitis, K. M. Padilla, S. Emani, D. S. Tyler, S. K. Pruitt. 2002. Enhanced dendritic cell antigen presentation in RNA-based immunotherapy. J. Surg. Res. 105:17.[Medline]
17. Yewdell, J., L. Anton, J. Bennink. 1996. Commentary: defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules?. J. Immunol. 157:1823.[Abstract]
18. Jonuleit, H., U. Kuhn, G. Muller, K. Steinbrink, L. Paragnik, E. Schmitt, J. Knop, A. H. Enk. 1997. Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur. J. Immunol. 27:3135.[Medline]
19. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser, G. Schuler. 1996. Generation of mature dendritic cells from human blood: an improved method with special regard to clinical applicability. J. Immunol. Methods 196:137.[Medline]
20. Schneider, D. S., K. L. Hudson, T. Y. Lin, K. V. Anderson. 1991. Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo. Genes Dev. 5:797.[Abstract/Free Full Text]
21. Kalady, M. F., M. W. Onaitis, S. Emani, Z. Abdel-Wahab, D. S. Tyler, S. K. Pruitt. 2003. Sequential delivery of maturation stimuli increases human dendritic cell IL-12 production and enhances tumor antigen-specific immunogenicity. J. Surg. Res. 116:24.
22. Abdel-Wahab, Z., M. F. Kalady, S. Emani, M. W. Onaitis, O. I. Abdel-Wahab, R. Cisco, L. Wheless, T.-Y. Cheng, D. S. Tyler, S. K. Pruitt. 2003. Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution. Cell. Immunol. 224:85.
23. Abdel-Wahab, Z., P. DeMatos, D. Hester, X. D. Dong, H. F. Seigler. 1998. Human dendritic cells, pulsed with either melanoma tumor cell lysates or the gp100 peptide(280–288), induce pairs of T-cell cultures with similar phenotype and lytic activity. Cell. Immunol. 186:63.[Medline]
24. Alexopoulou, L., A. C. Holt, R. Medzhitov, R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-B by Toll-like receptor 3. Nature 413:732.[Medline]
25. Reddy, A., M. Sapp, M. Feldman, M. Subklewe, N. Bhardwaj. 1997. A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells. Blood 90:3640.[Abstract/Free Full Text]
26. Hacker, H., R. M. Vabulas, O. Takeuchi, K. Hoshino, S. Akira, H. Wagner. 2000. Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J. Exp. Med. 192:595.[Abstract/Free Full Text]
27. Wagner, H.. 2002. Interactions between bacterial CpG-DNA and TLR9 bridge innate and adaptive immunity. Curr. Opin. Microbiol. 5:62.[Medline]
28. Takeshita, F., C. A. Leifer, I. Gursel, K. J. Ishii, S. Takeshita, M. Gursel, D. M. Klinman. 2001. Cutting edge: role of Toll-like receptor 9 in CpG DNA-induced activation of human cells. J. Immunol. 167:3555.[Abstract/Free Full Text]
29. Kopp, E., R. Medzhitov. 2003. Recognition of microbial infection by Toll-like receptors. Curr. Opin. Immunol. 15:396.[Medline]
30. O’Neill, L. A. J., K. A. Fitzgerald, A. G. Bowie. 2003. The Toll-IL-1 receptor adaptor family grows to five members. Trends Immunol. 24:286.[Medline]
31. Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, M. K. Gately. 1996. IL-12-deficient mice are defective in IFN production and type 1 cytokine responses. Immunity 4:471.[Medline]
32. Gately, M. K., L. M. Renzetti, J. Magram, A. S. Stern, L. Adorini, U. Gubler, D. H. Presky. 1998. The interleukin-12/interleukin-12-receptor system: role in normal and pathologic immune responses. Annu. Rev. Immunol. 16:495.[Medline]
33. von Herrath, M. G., A. Bot. 2003. Immune responsiveness, tolerance and dsRNA: implications for traditional paradigms. Trends Immunol. 24:289.[Medline]

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