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The Journal of Immunology, 2003, 171: 6344-6348.
Copyright © 2003 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: A Potent Adjuvant Effect of Ligand to Receptor Activator of NF-{kappa}B Gene for Inducing Antigen-Specific CD8+ T Cell Response by DNA and Viral Vector Vaccination 1

Yasushi Miyahira2,*, Hisaya Akiba{dagger}, Masaharu Katae*,{ddagger}, Kaori Kubota§, Seiki Kobayashi, Tsutomu Takeuchi, Adolfo García-Sastre||, Yoshinosuke Fukuchi{ddagger}, Ko Okumura{dagger}, Hideo Yagita{dagger} and Takashi Aoki*

Departments of * Molecular and Cellular Parasitology, {dagger} Immunology, and {ddagger} Respiratory Medicine, Juntendo University School of Medicine, § Periodontology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, and Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo, Japan; and || Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ligand to receptor activator of NF-{kappa}B (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8+ T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8+ T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8+ T cell-mediated immunity against infectious agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ligand to receptor activator of NF-{kappa}B (RANK-L) 3 is a member of the TNF family, which has been implicated in immune regulation and bone homeostasis (1, 2). RANK-L is expressed on activated T cells (3), and a major target for RANK-L in the immune system appears to be dendritic cells (DCs) that express a high level of RANK (4, 5). The RANK-L/RANK interaction is critical for CD40 ligand (CD40L)-independent Th cell activation (2), and the treatment of Ag-pulsed DCs with soluble RANK-L before immunization enhances T cell priming in vivo (6). RANK-L has functional similarity to CD40L, which is one of the pivotal costimulatory molecules for T cell activation by DCs (1). Considering the potent immunomodulatory capacity of the CD40L/CD40 costimulation (7), it was not surprising that the coadministration of plasmid DNA expressing CD40L with plasmid DNA expressing a specific Ag significantly enhanced cellular and/or humoral immune responses, and improved vaccine efficacy against infectious agents and tumors (8, 9, 10). Although RANK-L shares DC-activating properties with CD40L, the immunostimulatory effect of the RANK-L gene on DNA vaccination has not yet been determined.

We recently identified a major epitope of trans-sialidase surface Ag (TSSA) recognized by CD8+ T cells in Trypanosoma cruzi-infected C57BL/6 mice (11, 12, 13). T. cruzi is the etiological agent of Chagas’ disease in Central and South America (14), and we demonstrated that vaccination with plasmid DNA encoding TSSA can induce CD8+ T cell-mediated protective immunity against lethal T. cruzi infection (11, 12, 13). In this report, we demonstrated that the coadministration of the RANK-L gene with the TSSA gene markedly enhanced the induction of TSSA-specific CD8+ T cells and improved DNA vaccine efficacy. The RANK-L gene also potentiated the induction of CD8+ T cell responses by vaccination with an influenza virus vector. This is the first study demonstrating the potent adjuvant effect of the RANK-L gene for the induction of protective immunity against infectious agents by DNA and viral vector vaccination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and parasites

Female C57BL/6 (B6) (H-2b) and BALB/c (H-2d) mice, 5–8 wk of age, were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan). Blood-form trypomastigotes of the T. cruzi Tulahuen strain (15) were maintained by infecting mice (11, 12, 13).

Cells and culture

The B6-derived thymoma cell line EL-4 or the DBA/2 (H-2d)-derived mastocytoma cell line P815 was used as APC for CD8+ T cell cultures and assays. These cells were cultured in high-glucose DMEM (Life Technologies, Rockville, MD) supplemented with ingredients as described previously (11, 12, 13) (complete DMEM). The medium used for ELISPOT assays and the culture of lymphocytes was supplemented with PMA-stimulated EL-4 cell culture supernatant as a source of 30 U/ml IL-2 (complete DMEM-IL-2).

Plasmid DNA

pCMV-Tag epitope-tagging mammalian expression vector (pCMV) (Stratagene, La Jolla, CA) was used to construct the T. cruzi TSSA gene-expressing plasmid DNA (pTSSA) (11, 12, 13). Murine CD40L (16) and RANK-L (17) cDNAs were amplified by PCR and cloned into pcDNA3.1- (Invitrogen, Carlsbad, CA) (pcDNA3) at XhoI and NotI sites (pCD40L and pRANK-L, respectively).

Peptides

The H-2Kb-restricted antigenic peptide, ANYNFTLV, derived from TSSA (11, 12, 13) and the H-2Kd-restricted antigenic peptide, SYVPSAEQI, derived from the Plasmodium yoelii circumsporozoite protein (18) were synthesized and purified on HPLC.

Transient expression of murine RANK-L and CD40L and FACS analysis

Human embryonic kidney (HEK) 293 cells (1 x 106) were plated in 60-mm dishes. On the following day, the cells were transfected with 3 µg of pRANK-L, pCD40L, or pcDNA3 using lipofectamine (Invitrogen) according to the manufacturer’s instruction. After 48 h, the cells were harvested and incubated with biotin-conjugated anti-CD40L mAb (MR1; BD PharMingen, San Diego, CA), anti-RANK-L mAb (IK22-5, described below), or hamster or rat IgG isotype control (BD PharMingen). After washing with PBS twice, the cells were incubated with PE-labeled streptavidin (BD PharMingen), washed with PBS twice, and then analyzed on a FACSCalibur (BD Biosciences, San Jose, CA), and data were processed using the CellQuest program (BD Biosciences). The anti-murine RANK-L mAb (IK22-5) was generated in our laboratory (detailed characterization of this mAb will be described elsewhere). Briefly, an SD rat was immunized with murine RANK-L-transfected baby hamster kidney cells into the hind footpads three times at 7-day intervals. Three days after the final immunization, the popliteal lymph node cells were fused with murine myeloma P3U1 cells as described previously (19). After hypoxanthine/aminopterin/thymidine selection, one hybridoma-producing mAb IK22-5 (rat IgG2a, {kappa}) was identified by its strong reactivity with RANK-L-transfected cells, but not with mock-transfected cells.

DNA vaccination and challenge infection

The protocols for DNA vaccination and T. cruzi challenge infection were described in detail in each figure legend.

Quantification of Ag-specific T cells by ELISPOT assay

The frequency of Ag-specific CD8+ T cells was determined by ELISPOT assay for IFN-{gamma}-secreting cells essentially as described previously (11, 12, 13, 18).

Immunization of mice with recombinant influenza virus expressing a P. yoelii Ag

Recombinant influenza virus expressing an H-2Kd-restricted NEDSYVPSAEQI epitope, derived from the P. yoelii circumsporozoite protein, in the hemagglutinin molecule (FluME) was described in detail previously (20). The wild-type WSN influenza virus (FluWSN) was used as the control. The protocols for the immunization of mice with recombinant or wild-type virus and the immunological assay were described in detail in figure legend.

Statistical analyses

Statistical analyses were performed by the Dunnett’s two-tailed t test for the ELISPOT assays and the counts of parasitemia. The unpaired Mann-Whitney U test was used for the survival data. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of pCD40L and pRANK-L

We first verified the expression of pRANK-L and pCD40L by transient transfection into HEK293 cells. As shown in Fig. 1, high expression of RANK-L and CD40L on the surface of HEK293 cells was observed after the transient transfection of pRANK-L and pCD40L as estimated by staining with anti-RANK-L and anti-CD40L mAbs, respectively.



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  		FIGURE 1. Transient expression of RANK-L and CD40L on HEK293 cells. HEK293 cells were transfected with pcDNA3, pRANK-L, or pCD40L, and then stained with biotinylated anti-RANK-L or anti-CD40L mAb followed by PE-labeled streptavidin. The bold line indicates staining with the indicated mAb and the broken line indicates background staining with control IgG.

 
Coadministration of pRANK-L, but not pCD40L, with pTSSA enhances the induction of TSSA-specific CD8+ T cells

We then examined whether the coadministration of either pCD40L or pRANK-L could exert an immunostimulatory adjuvant effect that enhanced the DNA vaccine-induced immune responses. B6 mice were immunized twice with pTSSA or pCMV and pRANK-L, pCD40L, or pcDNA3 together. Twelve days after the second immunization, frequency of the H-2Kb-restricted TSSA antigenic peptide-specific CD8+ T cells in the freshly isolated splenocytes was assessed by the ELISPOT assay. As shown in Fig. 2A, coadministration of pRANK-L with pTSSA markedly increased the frequency of ANYNFTLV-specific IFN-{gamma}-secreting T cells as compared with the coadministration of pcDNA3 (81 ± 37 vs 1 ± 15 per 106 splenocytes, p = 0.000374). In contrast, coadministration of pCD40L did not significantly increase the frequency (6 ± 4 vs 1 ± 15, p = 0.9953). Even after the in vitro expansion of ANYNFTLV-specific T cells for 1 wk, no significant effect of pCD40L was observed as compared with pcDNA3 (3480 ± 1160 vs 5080 ± 2469, p = 0.3647), while pRANK-L markedly increased the frequency of ANYNFTLV-specific T cells (16350 ± 790 vs 5080 ± 2469, p = 0.00000007) (Fig. 2B). The administration of a sufficient amount of anti-RANK-L mAb into pTSSA/pRANK-L-vaccinated mice reversed the enhanced induction of ANYNFTLV-specific CD8+ T cells, while the treatment of pTSSA/pRANK-L-vaccinated mice with control rat IgG did not (data not shown). These results indicated that coadministration of pRANK-L, but not pCD40L, exerted a potent adjuvant effect on the induction of Ag-specific CD8+ T cells by DNA vaccination.



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  		FIGURE 2. Coadministration of pRANK-L, but not pCD40L, with pTSSA enhances the induction of TSSA-specific CD8+ T cells. B6 mice were immunized with 100 µg of pTSSA or pCMV and 100 µg of pCD40L, pRANK-L, or pcDNA3, which were all suspended in 50 µl of sterile PBS, into the right hind leg quadriceps twice at a 10-day interval. The mice were sacrificed 12 days after the second immunization, and their spleens were removed. Splenocytes from individual mice were cultured with irradiated EL-4 cells pulsed with ANYNFTLV peptide for 1 wk. The freshly isolated splenocytes (A) or the 1-wk cultured splenocytes (B) were subjected to the ELISPOT assay for IFN-{gamma}-producing cells in response to ANYNFTLV peptide-pulsed EL-4 cells. The number of IFN-{gamma}-secreting cells/1 x 106 cells was counted 24 h later. The number of IFN-{gamma}-secreting cells that appeared against peptide-unpulsed EL-4 was subtracted from the number of IFN-{gamma}-secreting cells that appeared against peptide-pulsed EL-4. Data represent the mean ± SD of four mice in each group. *, p < 0.05 by the Dunnett’s two-tailed t test. NS, not significantly different. The data are representative of one of three independent experiments.

 
Coadministration of pRANK-L, but not pCD40L, with recombinant influenza virus enhances the induction of murine malaria-specific CD8+ T cells

We next examined whether the adjuvant effect of pRANK-L could be also obtained when combined with recombinant influenza virus. We previously demonstrated that immunization with FluME-expressing H-2Kd-restricted malaria Ag (SYVPSAEQI) could induce the Ag-specific CD8+ T cells and confer protective immunity against malaria infection (20). We immunized BALB/c mice with FluME or FluWSN as a control and pRANK-L, pCD40L, or pcDNA3. Fourteen days later, the frequency of SYVPSAEQI-specific CD8+ T cells in the freshly isolated splenocytes were assessed by the ELISPOT assay. As shown in Fig. 3, coadministration of pRANK-L with FluME markedly increased the frequency of SYVPSAEQI-specific IFN-{gamma}-secreting T cells as compared with the coadministration of pcDNA3 (802 ± 225 vs 219 ± 46 per 106 splenocytes, p = 0.000462). In contrast, coadministration of pCD40L did not significantly increase the frequency (253 ± 146 vs 219 ± 46, p = 0.9312).



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  		FIGURE 3. Coadministration of pRANK-L, but not pCD40L, with FluME enhances the induction of malaria Ag-specific CD8+ T cells. BALB/c mice were immunized with 104 PFU FluME or FluWSN and 100 µg of pCD40L, pRANK-L, or pcDNA3, which were all suspended in 50 µl of sterile PBS, into the hind leg quadriceps. The mice were sacrificed 14 days after the immunization, and their spleens were removed. The number of SYVPSAEQI-specific CD8+ T cells was quantified by the ELISPOT assay for IFN-{gamma}-producing cells in response to SYVPSAEQI peptide-pulsed P815 cells. The number of IFN-{gamma}-secreting cells/1 x 106 cells was counted 24 h later. The number of IFN-{gamma}-secreting cells that appeared against peptide-unpulsed P815 was subtracted from the number of IFN-{gamma}-secreting cells that appeared against peptide-pulsed P815. Data represent the mean ± SD of four mice in each group. *, p < 0.05 by the Dunnett’s two-tailed t test. NS, not significantly different. The data are representative of one of two independent experiments.

 
Coadministration of pRANK-L, but not pCD40L, with pTSSA improves the DNA vaccine efficacy against T. cruzi infection

We finally examined whether the coadministration of pRANK-L could actually improve the DNA vaccine efficacy against lethal T. cruzi infection. B6 mice were immunized with pTSSA or pCMV and pRANK-L, pCD40L, or pcDNA3 twice, and then challenged with 5000 T. cruzi blood-form trypomastigotes 13 days after the second immunization. The coadministration of pRANK-L with pTSSA significantly suppressed the parasitemia at the late stage of infection (Fig. 4A), and improved survival (Fig. 4B) as compared with the coadministration of pcDNA3. In contrast, the coadministration of pCD40L showed no significant effect on either the parasitemia or survival.



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  		FIGURE 4. Coadministration of pRANK-L, but not pCD40L, with pTSSA improves the DNA vaccine efficacy against T. cruzi infection. B6 mice were immunized with 100 µg of pTSSA or pCMV and 100 µg of pCD40L, pRANK-L, or pcDNA3 into the right hind leg quadriceps twice at a 10-day interval. The mice were infected i.m. with 5000 Tulahuen strain T. cruzi blood-form trypomastigotes 13 days after the second immunization. The number of parasites in 5 µl of blood (parasitemia) was counted periodically, and the data represent the mean ± SD of four mice in each group (A). Survival was monitored daily (B). *, p < 0.05 compared with pTSSA + pcDNA3 by the Dunnett’s two-tailed t test (A). The survival of pTSSA + pRANK-L-immunized mice was significantly different (p < 0.05 by the unpaired Mann-Whitney U test) from that of pTSSA + pcDNA3-immunized mice (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we demonstrated that the coadministration of the RANK-L gene with a T. cruzi gene could enhance the Ag-specific CD8+ T cell response (Fig. 2) and could improve the vaccine efficacy against lethal T. cruzi infection (Fig. 4). The potent adjuvant effect of the RANK-L gene was also demonstrated when recombinant influenza virus expressing a malaria Ag was used as an immunogen (Fig. 3). This is the first indication that the RANK-L gene could be used as a genetic adjuvant for potentiating CD8+ T cell-mediated protective immunity against infectious agents.

Considering the previous studies that demonstrated a potent adjuvant activity of the CD40L gene for inducing protective immunity against Leishmania major infection and tumors (9, 10), our present results indicating no significant effects of pCD40L were somewhat unexpected. However, this discrepancy may be explained by a difference in the construction of CD40L used for those studies. Although we used murine CD40L cDNA encoding the full-length transmembrane protein, those studies used cDNA encoding soluble murine CD40L fused to an IL-7 leader sequence and a leucine zipper sequence to facilitate the secretion of a functional trimer. It has been reported that the membrane form of CD40L is naturally processed by some proteases to produce a soluble form with reduced activity possibly due to destruction of the trimeric structure (21). In contrast, the naturally processed soluble RANK-L has been reported to retain potent activity (22). In our DNA vaccine setting, the inoculated cDNA encoding Ag would be primarily expressed in muscle cells, and local APCs such as macrophages and DCs would acquire the Ag from these cells. Additionally, some local APCs may directly express the inoculated antigenic cDNA. In either case, the APCs would then migrate to the lymphoid organ, where they would prime the Ag-specific T cells. The coadministered CD40L and RANK-L cDNA would be also expressed by muscle cells and APCs. However, the expressed membrane forms of these proteins might be rapidly processed by some proteases in the environment, leading to loss of CD40L function but retained RANK-L function. Therefore, RANK-L might be more suitable than CD40L as a stable adjuvant.

The exact mechanism by which the coadministration of pRANK-L could exert the potent adjuvant effect remains to be determined. We have previously demonstrated that the coadministration of the IL-12 gene with pTSSA similarly enhances the induction of TSSA-specific CD8+ T cells and improves the vaccine efficacy against lethal T. cruzi infection (11). We have also recently demonstrated that the induction of CD8+ T cell-mediated protective immunity against T. cruzi infection by DNA vaccination with pTSSA is critically dependent on the CD28/B7-mediated T cell costimulation (13). In these contexts, it is worth noting that RANK-L, as well as CD40L, cannot only enhance the survival of DCs but can also up-regulate the production of cytokines, including IL-12, and the expression of costimulatory molecules, including B7, by DCs (3, 5, 6). Therefore, it seems likely that the coadministrated pRANK-L enhanced the induction of the TSSA-specific CD8+ T cell response by supporting the survival of Ag-presenting DCs and by up-regulating the expression of IL-12 and/or B7 by them. We have also previously demonstrated that CD4+ T cells are required for the development of CD8+ T cell-mediated protective immunity against T. cruzi infection by the DNA vaccination with pTSSA (11). Thus, the pRANK-L coadministration might also enhance the activation of TSSA-specific helper CD4+ T cells. Further studies are now under way to address these possibilities. Wiethe et al. (23) recently reported that the expression of RANK/RANK-L, but not CD40/CD40L, on the recombinant adenovirus-transduced DCs is effective for enhancing tumor-specific CD8+ T cell responses. Therefore, we believe that the increased expression of RANK-L in vivo could exert a potent adjuvant effect for inducing Ag-specific CD8+ T cell responses in different experimental designs and in different vaccination protocols. Although we have not performed protection assays against P. yoelii after FluME/pRANK-L coadministration, we expect that the enhanced induction of Ag-specific CD8+ T cells will be able to suppress the proliferation of malaria in the liver to some extent.

T cell-mediated immunity is crucial for resolving infection with intracellular pathogens including T. cruzi and malaria. Our present study has indicated that RANK-L gene can be used as a potent adjuvant to enhance the induction of T. cruzi and malaria Ag-specific CD8+ T cell responses by vaccination with plasmid DNA or recombinant influenza virus expressing the parasite Ags. This finding would be clinically relevant to the development of new vaccine strategies against these pathogens and also against other infectious agents such as HIV.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid for Scientific Research Number 14570222 from the Ministry of Education, Science, Sports, and Culture of Japan and by the Ohyama Health Foundation (to Y.M.), by a National Institutes of Health grant (to A.G.-S.), and also in part by Research for the Future Program Grant JSPS-RFTF-97L00701. Back

2 Address correspondence and reprint requests to Dr. Yasushi Miyahira, Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address: miyahira{at}med.juntendo.ac.jp Back

3 Abbreviations used in this paper: RANK-L, ligand to receptor activator of NF-{kappa}B; DC, dendritic cell; CD40L, CD40 ligand; TSSA, trans-sialidase surface Ag; HEK, human embryonic kidney. Back

Received for publication September 16, 2003. Accepted for publication October 27, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Y. Miyahira, Y. Takashima, S. Kobayashi, Y. Matsumoto, T. Takeuchi, M. Ohyanagi-Hara, A. Yoshida, A. Ohwada, H. Akiba, H. Yagita, et al.
Immune Responses against a Single CD8+-T-Cell Epitope Induced by Virus Vector Vaccination Can Successfully Control Trypanosoma cruzi Infection
Infect. Immun., November 1, 2005; 73(11): 7356 - 7365.
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