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The Journal of Immunology, 2003, 171: 4121-4130.
Copyright © 2003 by The American Association of Immunologists

Enhanced Effector and Memory CTL Responses Generated by Incorporation of Receptor Activator of NF-{kappa}B (RANK)/RANK Ligand Costimulatory Molecules into Dendritic Cell Immunogens Expressing a Human Tumor-Specific Antigen 1,2

Carsten Wiethe3,*,{dagger},{ddagger}, Kurt Dittmar*, Tracy Doan{dagger},{ddagger}, Werner Lindenmaier* and Robert Tindle4,{dagger},{ddagger}

* Gesellschaft für Biotechnologische Forschung, Department of Molecular Biotechnology, Braunschweig, Germany; and {dagger} Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, and {ddagger} Clinical Medical Virology Centre, University of Queensland, Brisbane, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-{kappa}B (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8+ T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-{gamma}-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Initiation of T cell immunity depends on the Ag-presenting capacity of mature dendritic cells (DCs). 5 DCs occur in tissues as immature cells and are specialized to capture and process foreign or tumor Ag. After Ag capture, they mature in response to inflammatory stimuli characterized by up-regulation of MHC and costimulatory molecules (1), and migrate to the T cell areas of draining lymph nodes where processed Ag is presented to T cells. DC-induced stimulation of CD8+ CTL directed to tumor epitopes is a vital component of the immune response to tumors (2).

DCs present Ags to T cells via the TCR/MHC/epitope synapse and, in so doing, signal T cells for activation. This event requires a second signal, also known as a costimulatory signal, which is classically provided by ligation of CD28 on T cells with CD80 (B7-1) or CD86 (B7-2) on DCs, but is now known to be provided by a family of B7 molecules, by the TNF superfamily, and by cytokines (3, 4).

The interaction between DCs and T cells results in up-regulation of receptor-ligand pairs of the TNF superfamily, including 4-1BB ligand (4-1BBL) (CD137L), FasL (CD95L), CD27, CD30, CD154 (CD40L), receptor activator of NF{kappa}B (RANK)L (or TNF-related activation-induced cytokine (TRANCE)), lymphotoxin, TNF-related apoptosis-inducing ligand, and members of the TNFR superfamily including 4-1BB (CD137), RANK, and CD40 (5, 6). Coligation of these receptor-ligand pairs induces downstream signaling events via TNFR-associated factor adaptor molecules. This signaling up-regulates adhesion and costimulatory molecules (7), enhances stable cellular interactions between DCs and T cells, regulates survival of either the APC or the T cell (8), and leads to production of T cell-stimulatory cytokines (e.g., IL-12, IL-1, and IL-6) (5, 8, 9, 10) and/or down-regulation of T cell-inhibitory cytokines (e.g., IL-10) (9). A result is amplification and sustenance of the ensuing immune response.

Manipulation of DCs ex vivo to display tumor epitopes before reinfusion is an attractive approach to tumor immunotherapy, but the approach is limited by the short life span of fully differentiated or mature DCs (11). This short life span is related to their rapid apoptosis, but this can be relieved at least in tissue culture by treatment of DCs with several costimulatory molecules, including RANKL and CD40L (12). Furthermore, pretreatment of Ag-pulsed mature DCs with soluble RANKL in vitro enhances the number and persistence of Ag-presenting DCs in draining lymph nodes in vivo (13). In addition, RANKL treatment increased Ag-specific T cell responses (13).

In the present study, we used novel adenovirus (Ad) vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus type 16 (HPV16)) and costimulatory molecules in DCs. We investigated whether the immune response induced by immunization with DCs expressing the tumor Ag would be enhanced by coprovision of RANK/RANKL or CD40/CD40L DC/T cell receptor-ligand pairs to the DCs. We reasoned that ligation of these costimulatory molecules and their respective receptors within DCs may effect an autocrine activation of individual DCs and/or allow reciprocal activation of interacting DCs. This would augment the activating signals received by DCs from T cells that themselves express RANKL and CD40L. We demonstrate that coexpression RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs increased E7-directed effector and memory CTL responses. Similarly, we demonstrate that coexpression of the T cell costimulatory molecules (CD40L and RANKL) or DC costimulatory molecules (CD40 and RANK) in E7-expressing DCs also increased E7-directed effector and memory CTL responses. We show that augmentation of T cell responses correlated with up-regulation of CD80 and CD86 in DCs by these costimulatory molecule combinations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell culture

293LP cells, an Ad-transformed human embryonic kidney cell line that provides phenotypic complementation of the E1 genes, were purchased from Microbix Biosystems (Toronto, Ontario, Canada). A549 cells were a gift from T. Adrian (Medizinischen Hochschule Hannover, Hannover, Germany). The E7-expressing EL4.E7 cell line was derived from EL4 cells (H-2b thymoma) transfected to stably express the full-length HPV16 E7 gene (14). The EL4.A2 cell line has been described (13). Cells were grown in RPMI 1640 (Life Technologies, Grand Island, NY) or in high-glucose DMEM (Life Technologies) with 10% FBS supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), HEPES buffer (20 mM), 2-ME (50 µM), penicillin (100 IU/ml), and streptomycin (100 µg/ml).

Peptides and epitopes

Peptide E7/Db (44QAEPDRAHYNIVTFCCKCD62) of HPV16 E7 was synthesized and used as described (15). E7/Db contains the H-2Db-restricted CTL epitope 49RAHYNIVTF57 (16).

Plasmids and construction of expression cassettes

The expression cassette for enhanced green fluorescent protein (eGFP), including the CMV immediate-early (CMVie) promoter and the 3' regulatory sequences, was amplified by PCR from pEGFP-C1 (Clontech, Palo Alto, CA) using primers with added SwaI and Psp1406I sites as well as ClaI and XbaI sites to the 5' and 3' ends, respectively. The PCR fragment was cloned into pGEM-T (Invitrogen, San Diego, CA) to yield pGEMegfp. For the construction of the expression cassette encoding HPV16 E7 oncoprotein, we used an HPV16 E7 variant with two mutations in the Cys-X-X-Cys repeats (position 58, Cys->Gly; position 91, Cys->Gly) provided by L. Gissmann (Deutsches Krebsforschungszentrum, Heidelberg, Germany) (17). The HPV16 E7 double-mutation variation (E7mut) was amplified by PCR, and the PCR fragment was cloned into pGEM-IRESegfp (W. Lindenmaier, unpublished data) to yield the bicistronic pGEME7mutIRESegfp. To generate the bicistronic expression cassette encoding the murine RANK and RANKL, respectively, we obtained the cDNAs encoding murine RANK or RANKL from Immunex (Seattle, WA). The cDNA encoding either RANK or RANKL was released from the pBluSK plasmids. Each of these DNA fragments was cloned into the bicistronic construct pGEMRec-1IRESegfp (W. Lindenmaier, unpublished data) by replacing the mouse ecotropic retrovirus receptor gene Rec-1 with either RANK or RANKL, yielding pGEMRANKIRESegfp and pGEMRANKLIRESegfp, respectively. To generate the plasmid encoding the murine CD40 or CD40L, we isolated total RNA using the RNeasy mini-kit (Qiagen, Valencia, CA) from 107 C57BL/6 mouse splenocytes. The cDNAs were synthesized with Superscript II (Life Technologies) using oligo(dT) primers. The genes encoding either the murine CD40 or CD40L were amplified by PCR using primers designed according GenBank sequences M83319 (CD40) and X65453 (CD40L). After cloning the PCR fragment into the pCR-Blunt-II-TOPO (Invitrogen) resulting in pCRII-CD40 and pCRII-CD40L, respectively, we sequenced the inserts. In comparison to GenBank sequences, we found in all our CD40 clones 1 aa substitution at position 227 (Met->Ile), and in the CD40L clones 1 aa substitution at position 198 (Ile->Ser). This mutation in CD40L has been described (GenBank sequence S21738) and does not affect biological function (18). The mutation in CD40 has also been described (GenBank sequence X65453). To create the bicistronic expression cassette for either the murine CD40 or CD40L, the mouse ecotropic retrovirus receptor gene Rec-1 from the bicistronic construct pGEMRec-1IRESegfp was replaced by the murine CD40 or CD40L gene from pCRII-CD40 or pCRII-CD40L, resulting in pGEMCD40IRESegfp and pGEMCD40LIRESegfp, respectively.

Construction of Ad cosmids

rAds derived from human Ad type 5 were constructed using a cosmid cloning procedure that allows direct assembly of rAds by cloning in Escherichia coli (W. Lindenmaier, unpublished data). Briefly, Ad cosmid pAdcos45 was digested by XbaI and ClaI or SwaI and ClaI for CD40L, dephosphorylated, and ligated to the Psp1406 I/XbaI- or SwaI/XbaI-excised mono- or bicistronic expression cassettes including CMVie promoter and 3' regulatory sequences from pGEMegfp, pGEME7mutIRESegfp, pGEMRANKIRESegfp, pGEMRANKLIRESegfp, pGEMCD40IRESegfp, and pGEMCD40LIRESegfp, respectively. Packaging in vitro and transduction into E. coli DH5{alpha} yielded Ad cosmids with the corresponding expression cassettes integrated into the E1 region.

rAds

rAds were propagated, purified, and titrated as described (19) with minor modifications. For production of rAd, cosmid DNA was transfected into 293LP cells. The formation of rAds was confirmed by monitoring eGFP+ adenoviral plaques, and virus particles were harvested. Restriction analysis of viral DNA was performed using DNA extracted from benzonase (Merck, West Point, PA)-digested lysates of infected cells. Purified virus was isolated by two rounds of CsCl density gradient centrifugation, and extensively dialyzed. The titers of the dialyzed stocks were determined by plaque assay on the 293LP cells.

Western blot analysis

For detection of E7 protein expression, A549 cells were infected with rAds (multiplicity of infection (MOI) of 100). Two days later, cells were lysed and subject to SDS-PAGE and Western blotting with anti-E7 mAbs 6D and 8F (20), followed by peroxidase-coupled goat anti-mouse Ab (Dianova, Hamburg, Germany). After washing, E7 protein was visualized using an ECL detection kit (Amersham, Arlington Heights, IL).

Immunofluorescence and immunochemistry

To detect costimulatory molecule proteins by indirect immunofluorescence and E7 protein by indirect immunochemistry, A549 cells or DCs were infected with rAds (MOI of 100). Two days later, the cells were fixed, permeabilized, and blocked, and then incubated with manufacturer-recommended dilutions of the following Abs: anti-mouse CD40 (clone 3/23; BD PharMingen, San Diego, CA), anti-mouse CD40L (clone MR1; BD PharMingen), anti-mouse RANK (R&D Systems, Minneapolis, MN), and anti-mouse TRANCE/RANKL (R&D Systems), or with a 1:1 mix of mAbs 6D and 8F, and then with biotinylated secondary Ab (Immunotech, Westbrook, ME). The cells were then incubated with Cy3-coupled streptavidin (Dianova) or alkaline phosphatase-coupled streptavidin (Immunotech), and color development and counterstaining with hematoxylin were performed according to the manufacturer’s guidelines.

Mice

H-2b (A2.1Kb) mice have been described (15). Mice were housed under specific pathogen-free conditions, and genetic authenticity was tested at intervals. Mice were used at 6–15 wk of age, but within a given experiment, mice were littermates or closely age- and sex-matched.

Generation of DCs, infection with rAds, and immunization

DCs were prepared from mouse bone marrow (femur and tibia) as previously described (21). Briefly, erythrocytes were removed by lysis with ammonium chloride, and total bone marrow leukocytes were cultured in RPMI 1640 supplemented with 10% FCS, recombinant murine GM-CSF (1 ng/ml; PeproTech, Rocky Hill, NJ), and IL-4 (2 ng/ml; PeproTech) at 1–2 x 106/ml, with medium changes every 2 days. On day 4 of culture, flasks of DCs were pooled and then divided according to the number of experimental groups in a given experiment. This approach normalizes for any variation in DC purity that might occur between groups. The DCs were infected with rAds at the indicated MOI in PBS with 2% FCS (1–2 ml) for 1 h at room temperature. At day 6 of culture, nonadherent and loosely adherent cells (DC) were used for flow cytometry analysis or immunization. Transduction efficiencies were in the range of 20–30%.

Flow cytometry analysis

DC were harvested and washed three times with RPMI 1640 with 10% FCS. Cells were then incubated with the primary Abs for 30 min on ice. A second incubation with Cy5-coupled streptavidin (Dianova) was performed when necessary. The following Abs were used: hamster IgG monoclonal anti-mouse CD11c (clone HL3; BD PharMingen), rat IgG monoclonal anti-mouse I-Ad/I-Ed MHC II (clone 2G9; BD PharMingen), hamster IgG monoclonal anti-mouse CD80 (clone 16-10A1; BD PharMingen), and rat IgG monoclonal anti-mouse CD86 (clone GL1; BD PharMingen). Cells were fixed with 4% paraformaldehyde. Samples were analyzed on a FACSVantage (BD Biosciences, Mountain View, CA) with CellQuest, version 3.3, software.

Immunization

Experimental mice (four per group) were immunized either with DC or peptide s.c. at the base of the tail. Before injection, DC were washed three times with PBS and resuspended in PBS at 5 x 106/ml, and each mouse was immunized with 5 x 105 DC. For peptide immunization, the mice were immunized with 30 µg of peptide, 10 µg of Quil A adjuvant (CSL, Parkville, Victoria, Australia), and 2.5 µg of tetanus toxoid (CSL, Melbourne, Australia) in 100 µl of PBS. Ten days later, splenocytes were harvested and either used in ELISPOT assays or depleted of APC as described (22). APC-depleted splenocytes (1 x 107/well) were restimulated in vitro with irradiated E7-expressing EL4.E7 cells (1 x 106/well) for 6 days in 24-well tissue culture plates and then used in CTL assay or IFN-{gamma} ELISPOT assay.

CTL assays

Chromium-51 release CTL assays were conducted as previously described (15). In summary, target cells (104 per well) sensitized at 37°C for 1 h with 1 µg/ml cognate peptide, or medium alone, and labeled with 100 µCi of 51Cr, were incubated with effector cells at various E:T ratios in triplicate in 96-well microtiter plates. Negative controls included wells containing target cells but no effector cells (background). Supernatants were harvested from CTL assays at 4 h, and 51Cr release was quantified by gamma counting. Results are expressed as percent cytotoxicity ± SD ((51Cr release in experimental wells - background/detergent-mediated total release - background) x 100%).

IFN-{gamma} ELISPOT

Cells secreting IFN-{gamma} were detected using an ELISPOT assay (23). In brief, serially diluted ex vivo or restimulated splenocytes (2 x 104 to 5 x 105 cells/well) were incubated overnight with or without cognate peptide (1 µg/ml) in 96-well nitrocellulose plates (Multiscreen HA; Millipore, Bedford, MA) coated with capture anti-mouse IFN-{gamma} mAb R4-6A2 (BD PharMingen) per well in medium supplemented with human rIL-2 (50 U/ml; Sigma-Aldrich, St. Louis, MO). Controls included spleen cells from irrelevantly immunized mice. After washing, plates were incubated with biotinylated anti-mouse IFN-{gamma} mAb XMG1.2 (BD PharMingen) per well for 3 h at room temperature, washed, and incubated with avidin-alkaline phosphatase conjugate (BD PharMingen). Spots were developed by adding an alkaline phosphatase substrate (Sigma-Aldrich). The number of spots corresponding to IFN-{gamma}-secreting cells was determined by using an automatic AID ELISPOT reader (Autoimmun-Diagnostika, Strassberg, Germany). The E7/Db-specific T cell response, monitored as IFN-{gamma}-secreting T cells per 106 splenocytes, was determined as follows: (number of spots with peptide - number of spots without peptide)/(number of cells per well) x 106.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
rAds

We constructed four rAds containing bicistronic expression cassettes encoding murine CD40, CD40L, RANK, or RANKL, respectively, driven from a human CMVie promoter, and eGFP driven from an internal entry ribosome site (IRES) (Fig. 1A). The bicistronic rAd encoding a mutated E7 protein of HPV16 with reduced transforming activity (17) and eGFP (AdE7), and a monocistronic rAd encoding eGFP (Adegfp), were constructed using an identical strategy, and have already been described (24). We used the rAds to infect A549 cells and murine bone marrow-derived DCs. Expression of the costimulatory molecules in A549 cells and in DCs infected with the respective rAds was confirmed by immunofluorescence staining with specific Abs (Fig. 1, B and C). eGFP fluorescence of infected cells was a convenient marker for recombinant gene expression (Fig. 1B). Among DCs, we recorded similar percentages of cells immunostaining with the various costimulatory molecules within a given experiment.



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  		FIGURE 1. A, Schematic overview of rAds showing bicistronic construction. Expression of costimulatory molecules and their ligands is driven from a human CMVie promoter located upstream from an IRES which drives expression of egfp gene. Immunofluorescence of A549 cells (B) and DCs (C) infected with rAds as indicated and reacted with Abs specific for CD40, CD40L, RANK, or RANKL, respectively, followed by Cy3-labeled second Ab. egfp was detected by autofluorescence microscopy. A549 cells transduced with the bicistronic rAds and staining positive for costimulatory molecules were invariably positive for egfp, whereas A549 cells transduced with Adegfp were never positive for CD40, CD40L, RANK, or RANKL, confirming the specificity of costimulatory molecule staining.

 
Expression of E7 was confirmed in AdE7-infected A549 cells by E7-specific Western blotting and immunohistochemistry (24).

DCs infected with AdE7 elicit an E7-directed CTL response

To determine whether E7-expressing bone marrow-derived DCs would elicit an E7-directed CD8+ T cell response, we immunized mice once with DCs that had been infected with AdE7 at MOIs of 125 or 250. Control mice were immunized with DCs infected with Adegfp at an MOI of 300 or unimmunized. Ten days later, splenocytes were harvested and cells secreting IFN-{gamma} in the presence of E7/Db peptide (containing the major H-2Db-restricted CTL epitope) were quantified in IFN-{gamma} ELISPOT assay, either directly (to measure an effector response) or after a further 6-day restimulation with E7-expressing EL4.E7 cells (to measure a memory response). Splenocytes from mice immunized with DCs infected with AdE7 at an MOI of 250 showed significantly elevated numbers of E7/Db-specific IFN-{gamma}-secreting effector splenocytes (p = 0.0003) (Fig. 2A) and memory splenocytes (p = 0.0016) (B), when compared with splenocytes from mice immunized with DCs infected with Adegfp or unimmunized mice. Immunization of mice with DCs infected with AdE7 at an MOI of 125 also elicited significantly higher numbers of E7/Db-specific IFN-{gamma}-secreting effector (p = 0.0003) (Fig. 2A) and memory (p = 0.0022) (B) splenocytes, but fewer spots than DCs infected at an MOI of 250, indicating a suboptimal response at the lower MOI. The response at MOI of 250 exceeded that elicited by immunization with E7/Db peptide (Fig. 2, A and B).



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  		FIGURE 2. Effector and memory T cell responses induced by immunization with rAd encoding E7 protein. A and B, IFN-{gamma}-secreting splenocytes from mice (four per group) immunized with bone marrow-derived DCs infected with rAds, with uninfected DCs, or with E7/Db peptide as indicated, were measured by ELISPOT assay either ex vivo (A) (*, p = 0.0003) or after restimulation for 6 days in vitro with EL4.E7 cells (B) (*, p = 0.0022; **, p = 0.0016). ELISPOTs were developed after 20-h incubation in vitro with E7/Db peptide. C, CTL assay of restimulated splenocytes from mice immunized with DCs infected with rAds, with uninfected DCs, or with E7/Db peptide as indicated. Splenocyte effector cells were reacted with E7/Db peptide-pulsed or unpulsed EL4.A2 target cells in a 51Cr release at the E:T ratios indicated (*, p = 0.0001; **, p < 0.0001). Data are pooled from three independent experiments; significant differences were determined with the Student’s t test.

 
We wished to confirm the induction of E7-directed CTL by immunization with AdE7-infected DCs in a functional assay. We examined restimulated splenocytes for cytotoxic activity against syngeneic target cells presenting the E7/Db CTL epitope in a chromium-51 release assay. Splenocytes from mice immunized with DCs infected with AdE7 specifically killed EL4.A2 target cells pulsed with E7/Db peptide. DCs infected with AdE7 at an MOI of 250 were more effective than DCs infected at an MOI of 125 (Fig. 2C).

Together, these data indicate that immunization with DCs infected with AdE7 elicits an E7-directed, IFN-{gamma}-secreting CTL response. Immunization with DCs infected with AdE7 at an MOI of 125 was suboptimal to immunization with DCs infected at an MOI of 250, suggesting that, at the lower MOI, the amount of E7 presented to the immune system by DCs may have been a limiting factor in E7-specific CD8+ T cell activation.

Coexpression of RANK/RANKL, but not CD40/CD40L, in DC immunogens enhances E7-directed effector and memory responses

We wanted to ask whether coexpression of costimulatory molecule receptor-ligand pairs in addition to E7 in DCs used for immunization would enhance E7-directed effector and memory CD8+ T cell responses. DCs infected with AdE7 at the suboptimal MOI of 125 were coinfected with AdRANK and AdRANKL, or with AdCD40 and AdCD40L, or with all four of these rAds, and used to immunize mice. Control mice were immunized with DCs infected with AdE7 and Adegfp, or with DCs without AdE7 but infected with rAds encoding the costimulatory molecules (Fig. 3). IFN-{gamma} ELISPOT assays were conducted on ex vivo splenocytes harvested 10 days after immunization (effector response) or after a further 6-day restimulation in vitro with EL4.E7 cells (memory response). Mice immunized with DCs expressing E7 and the RANK/RANKL receptor-ligand pair, but not mice immunized with DCs expressing E7 and the CD40/CD40L pair, showed significantly enhanced numbers of E7-specific effector (p = 0.0103) (Fig. 3A) and memory (p = 0.0014) (B) IFN-{gamma}-secreting cells, compared with mice immunized with DCs expressing E7 and irrelevant protein (eGFP), or mice immunized with DCs expressing receptor-ligand pairs in the absence of E7. Splenocytes from mice immunized with DCs expressing E7 and both receptor-ligand pairs (RANK/RANKL and CD40/CD40L) showed significantly enhanced numbers of E7-specific effector (p = 0.0192) (Fig. 3A) and memory (p = 0.0075) (B) IFN-{gamma}-secreting cells compared with these controls, but did not show increased numbers of IFN-{gamma}-secreting cells when compared with splenocytes from mice immunized with DCs expressing E7 and RANK/RANKL alone (Fig. 3, A and B).



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  		FIGURE 3. Coexpression of RANK and RANKL together (RANK/RANKL) with E7 in DCs enhances T cell responses in DC-immunized mice. A and B, IFN-{gamma}-secreting splenocytes from mice (four per group) immunized with DCs transduced with AdE7 (MOI, 125) alone or cotransduced with AdE7 (MOI, 125) plus AdRANK and Ad RANKL, or AdCD40 and AdCD40L, or all four of these rAds, or Adegfp, as indicated, were measured by ELISPOT assay, either ex vivo (A) (*, p = 0.0103; **, p = 0.0192) or after specific restimulation with EL4.E7 cells for 6 days (B) (*, p = 0.0014; **, p = 0.0075). ELISPOTs were quantified after overnight incubation with E7/Db peptide. C, 51Cr release CTL assay of restimulated splenocytes from mice immunized with DCs infected with AdE7 alone or with rAds encoding costimulatory molecules or ligands or eGFP as indicated. Effector cells were reacted with E7/Db-pulsed or unpulsed EL4.A2 target cells at the E:T ratios indicated (*, p = 0.0021; **, p = 0.0150). rAds encoding costimulatory molecules were used to infect DCs at a MOI of 100. Adegfp was used at a MOI of 300. Pooled data from three independent experiments are shown; significant differences were determined with the Student’s t test.

 
We also measured E7-directed memory CTL responses in chromium-51 release cytotoxicity assays. In accord with the above findings, splenocytes from mice immunized with DCs expressing E7 and RANK/RANKL, but not E7 and CD40/CD40L, showed significantly enhanced killing of EL4.A2 target cells pulsed with E7/Db peptide compared with splenocytes from mice immunized with DCs infected with AdE7 and irrelevant protein Adegfp (p = 0.0021), or with DCs infected with Ads encoding receptor-ligand pairs in the absence of E7 (p = 0.0009) (Fig. 3C).

Together, these data indicate that immunization with DCs expressing E7 and RANK/RANKL receptor-ligand pair, but not DCs expressing E7 and CD40/CD40L receptor-ligand pair, enhances E7-directed effector and memory CD8+ CTL responses when compared with immunization with DCs expressing E7 alone. They further demonstrate that inclusion of CD40/CD40L does not further enhance the response elicited by DCs expressing E7 and RANK/RANKL.

Coexpression of T cell costimulatory molecule or DC costimulatory molecules in DC immunogens up-regulates the E7-directed CTL response

Within the immune system, CD40 and RANK costimulatory molecules are predominantly expressed on DCs, and CD40L and RANKL costimulatory molecules are predominantly expressed on activated T cells (25). We examined the effect of transduction of E7-expressing DCs with DC-associated costimulatory molecules or with T cell-associated costimulatory molecules, on E7-directed T cell responses elicited by DC immunization. Groups of mice were immunized with DCs infected with AdE7 at an MOI of 125, and coinfected with rAds encoding CD40L and RANKL, or coinfected with rAds encoding CD40 and RANK. Control mice were immunized with DCs infected with rAds encoding costimulatory molecules alone, or DCs infected with AdE7 and irrelevant Adegfp. Mice immunized with DCs coexpressing E7, CD40L, and RANKL showed significantly enhanced effector responses (p = 0.0486) (Fig. 4A) and memory responses (p = 0.0025) (B) compared with splenocytes from mice immunized with DCs expressing E7 alone. Similarly, mice immunized with DCs coexpressing E7, CD40, and RANK showed enhanced effector responses (p = 0.0036) (Fig. 4A) and memory responses (p = 0.0017) (B) compared with mice immunized with DCs expressing E7 alone.



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  		FIGURE 4. Coexpression of CD40L and RANKL together with E7, and coexpression of CD40 and RANK together with E7 in DCs, enhance T cell responses in DC-immunized mice. IFN-{gamma}-secreting splenocytes from mice (four per group) immunized with DCs transduced with AdE7 (MOI, 125) alone or cotransduced with AdE7 (MOI, 125) plus AdCD40L and AdRANKL, or with AdCD40 and AdRANK, or all four of these rAds, or Adegfp, as indicated, were measured by ELISPOT assay, both ex vivo (A) (*, p = 0.0486; **, p = 0.0036) and following specific restimulation with EL4.E7 cells for 6 days (B) (*, p = 0.0025; **, p = 0.0017). ELISPOTs were measured after overnight incubation with E7/Db peptide. C, 51Cr release CTL assay of restimulated splenocytes from mice immunized with DCs infected with AdE7 alone or with rAds encoding costimulatory molecules or egfp as indicated. Effector cells were reacted with E7/Db-pulsed or unpulsed EL4.A2 target cells at the E:T ratios indicated (*, p = 0.0036; **, p = 0.0023). Ads encoding costimulatory molecules were used to infect DCs at a MOI of 100; Adegfp was used at an MOI of 300. Data are pooled from three independent experiments; significant differences were determined with the Student’s t test.

 
When E7-directed memory CTL responses were measured in chromium-51 release cytotoxicity assays, an increase in E7-expressing DC immunogen efficacy related to CD40L and RANKL coexpression, and to CD40 and RANK coexpression was also recorded. Thus, splenocytes from mice immunized with DCs transduced with E7 and CD40L and RANKL, or with DCs transduced with E7 and CD40 and RANK showed significantly enhanced killing of EL4.A2 target cells pulsed with E7/Db peptide compared with splenocytes from mice immunized with DCs transduced with E7 alone (p = 0.0036 and 0.0023) or with irrelevant Adegfp, or with DCs transduced with costimulatory molecules in the absence of E7 (Fig. 4C).

Together, these data are compatible with the notion that immunization with DCs transduced with E7 and DC-associated costimulatory molecules, or with DCs transduced with E7 and T cell-associated costimulatory molecules, enhances E7-directed effector and memory CD8+ CTL responses when compared with immunization with DCs transduced with E7 alone.

Transduction with costimulatory molecules augments the proportion of DCs expressing CD80 and CD86

One possible mechanism for the enhanced immune response observed when the costimulatory molecules were introduced into E7-expressing DC immunogens is up-regulation of the expression of second-signal molecules and MHC class II. Therefore, using cell surface marker staining, we asked whether transduction with rAds encoding costimulatory molecules would enhance the CD80, CD86, and MHC class II expression in DCs. Transduction of cells with AdRANK and AdRANKL augmented CD80 and CD86 expression compared with untransduced DCs or DCs transduced with irrelevant Adegfp. Thus, 84.4% of AdRANK/AdRANKL-transduced DCs expressed CD80, and 89.2% expressed CD86, compared with 32.4 and 55.9% for Adegfp-transduced DCs, respectively. Lesser augmentation of CD80 and CD86 was seen in DCs transduced with CD40 and CD40L (49.3 and 74.2%, respectively) (Fig. 5, A and B). Transduction with AdRANKL and AdCD40L caused an up-regulation of CD80 (79.9%) and CD86 (83.2%) (i.e., less than RANK/RANKL). Compared with the T cell-associated RANKL and CD40L, coexpression of DC-associated CD40 and RANK induced a lesser augmentation of CD80 and CD86 (45.3 and 67.3%, respectively), similar to that observed with CD40/CD40L.



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  		FIGURE 5. Effect of expression of costimulatory molecules on second-signal molecules and MHC class II in transduced DCs. DCs (pooled from two mice) were infected with Adegfp, AdCD40/AdCD40L, AdRANK/AdRANKL, AdCD40 and AdRANK, or AdCD40L and AdRANKL, stained with Abs to CD11c, and to MHC II or CD80 or CD86 as indicated, and subjected to FACS analysis. The number of DCs staining positive for MHC II, CD80, or CD86 is expressed as a percentage of CD11chigh plus CD11clow cells. In a series of preliminary experiments using isotype control primary Abs, no staining of DCs was recorded using anti-B220 (IgG2a{kappa}) for CD86 and MHC II, anti-CD3 (hamster IgG2) for CD80, and anti-TNP (hamster IgG1{lambda}) for CD11c (data not shown).

 
These results are consistent with the notion that expression of an exogenous source of the costimulatory molecules up-regulates expression of CD80 and CD86 signaling molecules in DCs.

Similarly, DCs expressing exogenous costimulatory molecules displayed higher levels of MHC II (AdRANK/AdRANKL, 73.2%; AdCD40L/AdRANKL, 73.8%), although these differed only marginally from DCs transduced with Adegfp (52.4%) or untransduced (69.4%) (Fig. 5C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present studies, we investigated whether provision of an exogenous source of certain costimulatory TNF and TNFR molecules to DC immunogens expressing a tumor-associated Ag (TAA) would enhance TAA-directed effector and memory CTL responses. Several costimulatory molecule receptor-ligand pathways come into play when an Ag-presenting DC encounters a cognate T cell. The outcome is bidirectional signaling that enhances the maturation of DCs and the activation of T cells. We generated Ad vectors expressing the E7 tumor Ag of HPV16, or one of the costimulatory molecules RANK, RANKL, CD40, or CD40L. We confirmed expression of these transgenes in A549 tissue culture cells (Fig. 1B and Ref.24). Although autofluorescence of eGFP confirmed the expression of the rAd-encoded bicistronic constructs in DCs used for immunization, we also confirmed expression of some costimulatory molecules by indirect immunofluorescence (Fig. 1C). We demonstrate that splenocytes from mice immunized with DCs coexpressing RANK/RANKL (but not CD40/CD40L) and E7 TAA showed enhanced IFN-{gamma} production when stimulated with cognate peptide, either ex vivo (effector response) or after a period of restimulation in vitro (memory response). In addition, restimulated splenocytes showed enhanced killing of E7 peptide-sensitized target cells (memory response). Splenocytes from mice immunized with DCs coexpressing CD40L and RANKL or with DCs expressing CD40 and RANK, plus E7, also showed enhanced E7-directed effector and memory CTL responses. In these experiments, we elected to use DCs expressing suboptimal levels of E7 for specific immune response induction as vehicles for cotransduced costimulatory molecules. We have previously reported that, under conditions of high amounts of E7 expression in DCs, coexpression of RANK/RANKL (or 4-1BBL) did not augment E7-directed T cell responses in DC immunized mice (24). In this context, others (26, 27) have shown that costimulation plays a more important role where Ag is limiting, probably relating to strength of signaling through the TCR (28).

Our results support a notion that provision to DCs of a DC/T cell costimulatory molecule receptor-ligand pair (RANK/RANKL) serves to up-regulate the effector and memory T cell responses to a coexpressed TAA. Similarly, provision of DC-associated costimulatory molecules (CD40 and RANK) or T cell-associated costimulatory molecules (CD40L and RANKL) serves to up-regulate the effector and memory T cell responses. We did not dissect out the contribution of individual costimulatory molecules. Because DCs express CD40 and RANK endogenously, the enhancement observed by provision of an exogenous source of these suggests that the amount of these molecules in DCs may be limiting in the generation of an immune response, at least in our system, and that the enhanced expression of CD40 and RANK may improve the capacity of the modified DCs to interact with and activate T cells. The enhancement observed by provision of the T cell costimulatory molecules or the DC/T cell costimulatory molecule receptor-ligand pair suggests that an autocrine activation of the transduced DCs, and/or reciprocal activation of interacting DCs, may play a role in the enhanced effector and memory T cell responses we observe. This autocrine activation would presumably occur in addition to the DCs receiving activating signals during the interaction with T cells via the ligation of TNF/TNFR pairs. The results extend our recent report that RANK/RANKL acts additively with 4-1BBL to enhance the specific immunogenicity of DCs expressing E7 (24). Our findings are congruent with those of Josien et al. (13), who showed that pulsing of DCs with soluble TRANCE (RANKL) protein before immunization enhanced their adjuvant capacity, resulting in better T cell priming in vivo, probably due at least in part to the enhanced survival of RANKL-treated DCs as measured by recovery in greater numbers from draining lymph nodes.

The seminal role of DCs in initiating adaptive immune responses to infectious organisms and tumors underlies the approach of reinfusing ex vivo-modified DCs as vaccines (1). At least in some cases, Ag presentation by DCs is the limiting factor in antitumor immunity (29). Strategies that use DCs to deliver TAAs have shown considerable promise in inducing tumor Ag-directed CD8+ CTL responses and curtailment of tumor growth (1). Thus, DCs pulsed with TAA-derived peptides, recombinant tumor proteins, virus-like particles containing TAA, and apoptotic tumor cells have proved effective in generating tumor protection in animal models (30, 31, 32) and in generation of tumor Ag-specific T cell responses in vitro in humans (33, 34). DC immunogens genetically modified to express TAA improve on the approach of pulsing DCs with peptide before infusion, by providing more prolonged antigenic stimulation (35, 36). Rapid peptide turnover and inefficient presentation of exogenous Ag critically limit the activation of CTL by DCs (37). Therefore, a major aim has been to optimize transduction and expression of TAA within DCs and to maximize TAA-directed T cell responses when genetically modified DCs are used for immunization. Gene transfer using Ad vectors has helped circumvent difficulties in introduction of TAA-expressing foreign genetic material into DCs using traditional methodologies, e.g., lipofection and electroporation. Ad vectors also have the advantage of allowing high expression of transgene, inducing some DC activation, and not grossly interfering with DC maturation (38).

RANKL, expressed on activated T cells (25), is a regulator of the immune system and of bone development (reviewed in Refs. 10 and 39), whose major target in the immune system is RANK expressed on mature DCs. RANKL promotes the survival of DCs via Bcl-xL induction (12, 40) and other antiapoptotic pathways (41). RANKL induces the production by DCs of proinflammatory cytokines (e.g., IL-1 and IL-6) and cytokines that stimulate and induce differentiation of T cells (e.g., IL-12 and IL-15) (42). We show that transduction of DCs with rAds encoding RANK/RANKL, or T cell-associated RANKL and CD40L augmented expression of costimulatory B7 family members CD80 and CD86 in DCs compared with untransduced or irrelevantly transduced DCs. Lesser augmentation of CD80 and CD86 expression was seen in DCs transduced with CD40/CD40L, or with DC-associated CD40 and RANK. Ligation of CD80 or CD86 with CD28 expressed on T cells during cognate TCR-peptide/MHC interaction serves to positively activate T cells. It is reported that at least some costimulatory molecules work synergistically with CD28-CD80/86 signaling to drive activation of T cells (43). The up-regulation of CD80/86 we report in the present study suggests a mechanism by which this might occur. Wong et al. (12) found that stimulation of DCs with soluble TRANCE (RANKL) did not affect expression of CD80, CD86, or MHC class II. This apparent discrepancy with our findings may relate to the prolonged stimulation afforded in our system by RANKL and/or RANK transduction. Thus, RANKL and an exogenous source of RANK in DCs positively regulates the outcome of T cell-DC interaction, predisposing to enhanced immune responses. Reciprocally, activated RANKL-expressing T cells provide a powerful positive reinforcement to Ag-presenting DCs by enhancing their survival and cytokine production via RANK. Thus, both Ag-specific T cells and Ag-presenting DCs depend on each other for activation and survival (12). We also examined MHC class I expression in DCs following transfection with rAds expressing costimulatory molecules (not shown). Although some slight up-regulation was noted (in the range of 82–89% class I-positive cells, compared with 74% for DCs transfected with irrelevant Adegfp), it is unlikely that augmentation of the class I restriction element is a major factor in the enhanced CD8+ CTL responses we report. Similar to the RANK/RANKL system, interactions between CD40L expressed on activated CD4+ T cells and CD40 expressed on DCs can mediate DC survival and activation (12, 40). However, the two systems function independently (42, 44), and RANK/RANKL signaling offers an alternative route to CD40/CD40L signaling for the activation of T cells. Therefore, our finding that coexpression of CD40/CD40L with E7 in DC immunogens failed to enhance E7-directed effector or memory responses is at first sight somewhat surprising, because it is reported that signals through CD40 can up-regulate adhesion and second signal molecules (7) and generate antiapoptotic signals through the Bcl-xL pathway that increase survival of DCs (1, 45). However, in our experiments, although DCs expressing CD40/CD40L show up-regulation in CD80 and CD86, the effect is much less than in DCs expressing RANK/RANKL (Fig. 5). One explanation may be that CD40/CD40L-activated DCs when injected in vivo may preferentially stimulate CD4+ T cells, because CD40L is expressed primarily on activated CD4+ T cells (6, 44). We have previously shown that the CD8+ CTL response to the E7/Db epitope used in the present study is CD4+ T cell independent (15, 46). In contrast to CD40L, RANKL is expressed on activated CD8+ as well as CD4+ T cells (25), and therefore, DCs expressing RANK or RANK/RANKL may be expected to directly stimulate RANKL-expressing CD8 T cells.

Although our data were obtained at the cell population level, other studies indicate simultaneous multiple costimulatory molecule expression on a single Ag-responsive (CD4+) T cell (47). These data suggest that certain costimulatory molecules may act additively or synergistically during particular immune responses, i.e., they do not have redundant functions. We have previously reported that RANK/RANKL acts additively with 4-1BBL to induce CD8+ T cell responses to E7 (24). Our present study provides no evidence for addition/synergism between RANK/RANKL and CD40/CD40L

A possibility arises that the enhanced T cell responses we report may have been caused by adenoviral transduction of DCs per se or its downstream consequences. We went to considerable lengths to exclude this possibility. We included control groups of mice immunized with DCs transduced with an irrelevant gene (egfp), or with E7 without costimulatory molecules, or with costimulatory molecules without E7. We normalized transduction conditions such that total MOIs with Ad vector per DC were similar for groups receiving DCs expressing both costimulatory molecules and E7, and for the negative control groups. Inclusion of these controls removed the possibility that the observed augmented T cell responses were due to transduction with Ad per se.

The present data show that in vivo functional capacity of DCs can be modulated in a positive way by the RANK/RANKL system. Our findings have generic implications for more effective DC cancer vaccines. Specifically, there are implications for a therapeutic vaccine for HPV-associated cervical cancer. Although a number of E7-based vaccines, e.g., E7 peptides, whole E7 protein, and vaccinia-E7 recombinants, have been used in clinical trials (48, 49), the generation of E7-specific CTLs has been disappointing. Immunization with E7-pulsed or E7-transfected DCs elicited E7-specific CD4+ and CD8+ T cells in cervical cancer patients (33), and in mice, in which E7-specific tumor protection was conferred (31). The findings of our study suggest that incorporation of RANK and/or RANKL in DCs expressing E7 may offer an improved approach. We have experiments planned to test whether immunization with DCs expressing E7 and costimulatory molecules will prevent the development of carcinomas in mice expressing an E7 transgene in squamous epithelium (50), which constitute a model for human cervical cancer (51).


   Acknowledgments
 
We thank Dr. S. Weiss for provision of CD80 and CD86 Abs. Donna West and her staff provided excellent animal husbandry. We thank Karen Herd for helpful discussion. We are grateful to Paula Hall for help with the FACS analysis.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council (Australia). C.W. was supported by a grant from the Helmholtz-Gemeinschaft Forschungszentren-Strategiefond I Infektionsabwehr und Krebsprävention and by a short-term Ph.D. fellowship from the Deutscher Akademischer Austauschdienst. Back

2 This paper is contribution number 192 of the Sir Albert Sakzewski Virus Research Center. Back

3 Current address: Dermatologische Klinik, Universitat Erlangen, Hartmannstrasse 14, D-19052 Erlangen, Germany. Back

4 Address correspondence and reprint requests to Dr. Robert Tindle, Sir Albert Sakzewski Virus Research Centre, Royal Children’s Hospital, Herston Road, Herston, QLD 4029, Australia. E-mail address: r.tindle{at}mailbox.uq.edu.au Back

5 Abbreviations used in this paper: DC, dendritic cell; RANK, receptor activator of NF-{kappa}B; L, ligand; TRANCE, TNF-related activation-induced cytokine; HPV, human papillomavirus; eGFP, enhanced green fluorescent protein; MOI, multiplicity of infection; Ad, adenovirus; CMVie, CMV immediate early; TAA, tumor-associated Ag; IRES, internal ribosome entry site. Back

Received for publication April 10, 2003. Accepted for publication August 6, 2003.


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