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The Journal of Immunology, 2008, 180, 8126 -8134
Copyright © 2008 by The American Association of Immunologists, Inc.
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A Novel Adenovirus Expressing Flt3 Ligand Enhances Mucosal Immunity by Inducing Mature Nasopharyngeal-Associated Lymphoreticular Tissue Dendritic Cell Migration1

Shinichi Sekine*, Kosuke Kataoka*,{ddagger}, Yoshiko Fukuyama*, Yasuo Adachi{dagger}, Julia Davydova§, Masato Yamamoto§, Ryoki Kobayashi*, Keiko Fujihashi*, Hideaki Suzuki*, David T. Curiel{dagger}, Satoshi Shizukuishi{ddagger}, Jerry R. McGhee* and Kohtaro Fujihashi2,*

* Departments of Pediatric Dentistry and Microbiology, Immunobiology Vaccine Center and {dagger} Division of Human Gene Therapy, Department of Surgery, Medicine and Pathology, Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294; {ddagger} Department of Preventive Dentistry, Graduate School of Dentistry, Osaka University, Osaka, Japan; and § Department of Surgery, University of Minnesota, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4+ Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4+ T cell proliferative responses and OVA-induced IFN-{gamma} and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b+CD11c+ DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b+CD11c+ DCs that migrate to effector sites for subsequent CD4+ Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nasal delivery of Ag plus mucosal adjuvant has emerged as perhaps the most effective way to induce both peripheral and mucosal immunity, including salivary secretory IgA (S-IgA)3 Ab responses (1). Most studies can be divided into those that use soluble vaccine components together with mucosal adjuvants, such as native cholera toxin (nCT), and those that use attenuated bacterial or viral vectors, such as the adenovirus (Ad) vector (1). In this regard, it has been shown that nasal immunization with the weak protein Ag OVA plus mutant cholera toxin elicited OVA-specific S-IgA Ab responses in the submandibular glands (SMGs) in addition to other mucosal effector lymphoid tissues (2, 3). Further, our previous studies have shown that nasal vaccines containing tetanus toxoid and mutant cholera toxin induced protective immunity and generated tetanus toxin-specific neutralizing Abs (4, 5). In the case of Ad vectors, mucosal administration of the E1/E3-deleted Ad5 vector expressing the transgene β-galactosidase-induced mucosal S-IgA Ab responses (6). Most recently it was shown that nasal immunization with the heavy chain of a β-trefoil structure of botulinum toxin A genetically fused to the Ad2 fiber protein plus nCT rapidly induced protective S-IgA and plasma IgG Ab responses (7). These findings show that an appropriate nasal vaccine can provide effective mucosal and systemic immunity against mucosal pathogens.

The human homologue of the murine flt3 ligand (FL) has been cloned and shown to play a central role in the proliferation and differentiation of early hematopoietic precursor stem cells (8, 9, 10, 11, 12). It has also been shown to mobilize and stimulate dendritic cells (DCs) (13), NK cells, and B cells (14). Further, recent studies have indicated that FL treatment favors the induction of immune responses when given by mucosal (15), systemic (16), or cutaneous routes (17) to adult mice. Others have also shown that FL treatment of newborn mice leads to an increased resistance against the intracellular pathogens HSV-1 and Listeria monocytogenes by increasing the numbers of DCs and the levels of IL-12, a DC-derived cytokine (18). In addition, the systemic administration of recombinant FL protein resulted in a marked expansion of myeloid- and lymphoid-type DCs in various tissues (10, 13, 19), as well as inducing impressive antitumor effects in several murine models (20, 21).

DCs are regulators of the immune response and provide a link between the innate and adaptive immune systems. They take up pathogens (22, 23), produce the appropriate cytokines for innate defense (22, 24, 25), and migrate to lymph nodes where they present the processed Ags to T cells and thereby initiate an adaptive immune response (22, 25). Several distinct subpopulations of DCs, differing in surface phenotype and function, are found in adult mice. Conventional DCs, which display surface MHC class II and costimulatory molecules, can be subdivided based upon surface expression of other molecules such as CD8, CD4, CD11b, and DEC-205 (24, 26, 27, 28, 29).

In our previous study, we showed that nasal coadministration of DNA plasmid FL (pFL) activates CD11C+CD8+ DCs, thereby inducing mucosal S-IgA and systemic IgG Ab responses (30). We also showed that nasal pFL exerts its adjuvant effect by expanding the production of activated lymphoid-type DCs and Th2-type cytokines (i.e., IL-4) (30). Based upon these findings, we thought it important to determine whether other types of FL-based immune modulators could induce mucosal S-IgA Ab responses and whether they could elicit Th1-type responses as well as Th2-type responses, thereby providing cell-mediated immunity and facilitating vaccine development. In pursuit of these goals, we have developed an FL expressing replication-defective recombinant Ad serotype 5 vector and have assessed its adjuvanticity for the induction of coadministered protein Ag-specific mucosal and systemic humoral as well as CTL responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Six- to 8-wk-old female C57BL/6 mice were purchased from the Frederick Cancer Research Facility (National Cancer Institute, National Institutes of Health, Frederick, MD). Upon arrival, these mice were transferred to microisolators, maintained in horizontal laminar flow cabinets, and provided sterile food and water in a specific pathogen-free facility. All mice used in these experiments were free of bacterial and viral pathogens.

Preparation of the adenovirus vector

Replication-incompetent adenovirus vectors expressing firefly luciferase (Ad-Luc) and FL (Ad-FL), respectively, were constructed through homologous recombination in Escherichia coli using the AdEasy system (31). Both of the vectors used in our experiments contained transgene cassettes driven by the human CMV promoter placed in the E1-deleted region of an adenoviral vector backbone. Thus, the recombinant Ad-FL was constructed by inserting the murine FL cDNA into an early region (E1). Expression of cDNA was driven by the human CMV immediate gene promoter and terminated by the polyadenylation sequence, poly(A), of SV40. The viruses were propagated in the Ad-packaging cell line, human embryonic kidney 293 cells (Microbix Biosystems), and purified by double CsCl density gradient centrifugation followed by dialysis against PBS with 10% glycerol. The Ad vectors were titrated by plaque assay and stored at –80° C until use.

FL gene expression and in vitro FL production

A549 cells (American Type Culture Collection) were plated in triplicate to 24-well plates at a concentration of 5 x 104 cells/well 1 day before gene transfer. After overnight culture, the cells were infected with Ad-FL at a multiplicity of infection of 50 PFU in medium containing 2% FCS for 1 h. The infecting medium was then replaced with complete medium. Two days after inoculation, the infected cells and culture medium were harvested, and the supernatants from 1 x 104 ultrasonicated cells were collected (32). The culture medium and cell extracts were assessed by FL-specific Western blot analysis using anti-FL mAb (R & D Systems).

Tissue distribution of Ad vector

To determine the lymphoid tissue distribution of the Ad vector, mice were nasally administered Ad-Luc (1 x 108 PFU/mouse), including the same CMV promotor as was used for Ad-FL. Three days later, mucosal (nasopharyngeal-associated lymphoreticular tissue (NALT), SMGs, and lungs) and systemic tissues (liver and spleen) as well as external secretions (nasal washes, saliva, lung washes, and fecal extracts) were harvested (2, 4, 6, 30) and assessed for luciferase gene expression using a luciferase assay system kit (Promega).

Nasal immunization and sample collection

Mice were nasally immunized three times at weekly intervals with 3 µl per nostril of PBS containing 1 x 108 PFU of Ad-FL and 100 µg of OVA (fraction V; Sigma-Aldrich). Control mice were immunized nasally with 1 x 108 PFU of Ad-Luc and 100 µg of OVA under anesthesia. In some experiments, mice were given OVA plus pFL (50 µg/mouse) three times at weekly intervals. Plasma, saliva, and nasal washes were collected on days 0 and 21 or 28. Stimulated saliva was obtained as described previously (2, 4, 30). In some experiments, mice were sacrificed 14 days after the last immunization (day 28) and nasal washes were obtained as previously described (2, 4, 30).

OVA-specific ELISA

OVA-specific Abs in plasma and external secretions were determined by ELISA as previously described (2, 4, 30, 33). Briefly, 96-well Falcon microtest assay plates (BD Biosciences) were coated with 1 mg/ml OVA in PBS. After blocking with 1% BSA in PBS, 2-fold serial dilutions of samples were added and incubated overnight at 4°C. HRP-labeled goat anti-mouse µ, {gamma}, or {alpha} heavy chain-specific Abs (Southern Biotechnology Associates) were added to individual wells. For IgG Ab subclass analysis, biotinylated mAbs specific for IgG1, IgG2a, IgG2b, and IgG3 (BD Biosciences) and peroxidase-conjugated goat anti-biotin Ab (Vector Laboratories) were used for detection. The color reaction was developed for 15 min at room temperature with 100 µl of 1.1 mM ABTS. End point titers were expressed as the reciprocal log2 of the last dilution that gave an OD at 415 nm of 0.1 greater than background.

OVA-specific ELISPOT

Mononuclear cells from the spleen were isolated aseptically by gentle teasing through stainless steel screens as described previously (2, 4, 30, 33). Nasal passages (NPs) and NALT were isolated using a previously described protocol with some modifications (2, 4, 30). Mononuclear cells from SMGs were isolated by an enzymatic dissociation procedure with collagenase type IV (0.5 mg/ml; Sigma-Aldrich) followed by discontinuous Percoll gradient (Amersham Biosciences) centrifugation (2, 4, 30). Mononuclear cells obtained from mucosal lymphoid tissues and spleen were subjected to an ELISPOT assay to determine the numbers of OVA-specific Ab-forming cells (AFCs). Briefly, 96-well nitrocellulose plates (Millititer HA; Millipore) were coated with 1 mg/ml and OVA-specific AFCs were counted with the aid of a stereomicroscope as described elsewhere (2, 4, 30).

Flow cytometric analysis

To characterize the phenotype of DCs, mononuclear cells (2 x 105 cells) were stained with FITC-conjugated anti-CD8, PE-labeled anti-CD11b, allophycocyanin-tagged anti-B220, and biotinylated anti-CD11c mAbs (BD Biosciences) followed by PerCP-Cy5.5-streptavidin. Before flow cytometric analysis (FACSCalibur; BD Biosciences), in some experiments mononuclear cells were incubated with FITC-conjugated anti-CD8, anti-CD11b, or anti-B220 PE-labeled anti-mouse I-Ab, CD40, CD80, or CD86 (BD Biosciences) and biotinylated anti-CD11c mAbs followed by PerCP-Cy5.5-streptavidin. For intracellular IFN-{gamma} and IL-4 analyses, cells were incubated with ionomycin (1 µg/ml; Sigma-Aldrich) and PMA (25 ng/ml; Sigma-Aldrich) for 3 h in the presence of monensin and then stained with FITC-labeled anti-CD4 before being stained intracellularly with PE-labeled anti-IFN-{gamma} or anti-IL-4 mAbs (BD Biosciences).

OVA-specific CD4+ T cell responses

CD4+ T cells from spleen, NALT, and cervical lymph nodes (CLNs) were purified using an autoMACS system (Miltenyi Biotec), as described previously (2, 4, 30, 33). This purified CD4+ T cell fraction (>97% CD4+; >99% cell viability) was resuspended in RPMI 1640 (Cellgro Mediatech) supplemented with HEPES buffer (10 mM), L-glutamine (2 mM), nonessential amino acid solution (10 ml/L), sodium pyruvate (10 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), gentamicin (80 µg/ml) and 10% FCS (complete RPMI 1640) (4 x 106 cells/ml), before being cultured in the presence of 1 mg/ml OVA and T cell-depleted, irradiated (3000 rad) splenic APCs for 2 or 5 days. The supernatants of T cell cultures were subjected to a cytokine-specific ELISA whereas cells were subjected to cytokine-specific quantitative RT-PCR analyses as described below.

Cytokine-specific ELISA

Levels of cytokines in culture supernatants were measured by an ELISA. The details of the ELISA for IFN-{gamma} and IL-4 have been described previously (2, 4, 30, 33). The detection limits for each cytokine were 106.3 pg/ml for IFN-{gamma} and 4.66 pg/ml for IL-4.

Quantitative analysis of cytokine-specific mRNA

The CD4+ T cells were harvested after two days of incubation, and total RNA was isolated by the acid guanidium thiocyanate-phenol-chloroform extraction procedure. Aliquots of extracted RNA (25 µg/ml) were subjected to reverse transcriptase reaction and were treated with one µl of 10 µg/ml RNase H (Invitrogen). The levels of synthesized cDNA were measured using a GeneQuant RNA/DNA calculator (Amersham Biosciences). The sample cDNA and the external standards were amplified with cytokine-specific primers and SYBR Green I by using the LightCycler (Roche Applied Science). The specificity of PCR products was confirmed by a melting curve as well as by agarose gel electrophoresis. The concentration of sample cDNA was determined using linear, diluted external standards obtained by an identical PCR protocol with the LightCycler (30).

CTL assay

CD8+ T cells (2 x 106) from spleen and CLNs were incubated with splenic DCs (5 x 105) prepulsed with OVA for 5 days in the presence of recombinant mouse IL-2 (50 U/ml). Serially diluted CD8+ T cells were added to 96-well U-bottom culture plates containing OVA peptide expressing EG7 target cells (2 x 103) at different E:T ratios. Plates were incubated for 4 h and the supernatants recovered. Using the CytoTox 96 kit (Promega) according to the manufacturer’s instructions, we determined the specific lysis by measuring the amount of lactate dehydrogenase released after target cell lysis.

Statistical analysis

The results are presented as the mean ± 1 SEM. All mouse groups were compared with control mice using an unpaired Mann-Whitney U test with StatView software (Abacus Concepts) designed for Macintosh computers. p < 0.05 or p <0.01 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Tracking FL expression in vivo

To assess FL production in vitro, culture supernatants and cell extracts from Ad-FL-transfected A549 cells (50 pfu/cell) were examined using Western blot analysis and ELISA. At 48 h after Ad-FL transfection, significantly increased levels of FL protein (multiple bands from 18 to 36 kDa due to heterogenous glycosylation) were detected in both exogenous and endogenous cells (Fig. 1A). The levels of FL expression were stable for up to 5 days after transfection (data not shown). To track Ad in vivo, mice were given Ad-Luc (1 x 108 PFU/mouse) by the nasal route. Three days later, luciferase activity in mucosal and peripheral lymphoid tissues as well as in external secretions was examined. Among lymphoid tissues, only NALT showed significantly increased luciferase activity (p < 0.01; Fig. 1B). Increased levels of luciferase were also detected in nasal washes (p < 0.01; Fig. 1C). These findings suggest that Ad effectively delivers the expressed molecule into the mucosal inductive tissues and their external secretions.


Figure 1
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  		FIGURE 1. Expression of FL or luciferase protein with recombinant Ad in vitro and in vivo. A, Western blot assay for FL protein expression. For each virus, A549 cells (5 x 104 cells/well) were added in triplicate to 24-well plates and transfected with the constructed recombinant Ad-Luc (lanes 1 and 3) or Ad-FL (lanes 2 and 4) at 50 PFU/cell. The expression of FL was examined in cell extracts (1 x 104 cells, lanes 1 and 2) or in the medium (10 µl; lanes 3 and 4) by Western blot analysis using FL-specific mAb. The recombinant mouse FL protein was used as a positive control (20 pg; lane 5). B and C, Distribution of Ad in vivo. Three days after the nasal administration of Ad-Luc, luciferase activity in various tissues (B) and external secretions (C) was determined. The readings in relative light units (RLU) were normalized to the amount of protein in tissues or volume in secretions as determined by a bicinchoninic acid assay. Samples from nonimmunized mice were used as negative controls. The values shown are the mean ± SEM taken from 20 mice in each experimental group. *, p < 0.05; **, p < 0.01 compared with nonimmunized mice.

 
Nasal Ad-FL administration enhances mucosal and systemic immunity

To assess Ag-specific mucosal and systemic IgA and IgG Ab responses, nasal washes, saliva, and plasma were collected 1 wk after the last nasal immunization and subjected to OVA-specific ELISA. The nasal washes and saliva of mice given nasal OVA plus Ad-FL exhibited higher levels of OVA-specific S-IgA and IgG Ab responses than did those of mice immunized with OVA plus Ad-Luc or OVA alone (p < 0.05; Fig. 2A). The high numbers of OVA-specific IgA and IgG AFCs observed in the NPs, SMGs, and NALT of mice given nasal Ad-FL as a mucosal adjuvant support these findings at the cellular level (Fig. 2B; *, p < 0.05; **, p < 0.01). However, the numbers of total IgA and IgG AFCs in these lymphoid tissues were essentially unchanged among the groups (NPs: ~480/106 IgG and ~2400/106 IgA; SMGs: ~400/106 IgG and ~400/106 IgA; NALT: ~100/106 IgG and ~130/106 IgA). Similarly, levels of OVA-specific IgA and IgG Ab responses increased significantly more in the plasma of mice given nasal OVA plus Ad-FL than in mice given Ad-Luc as a control (Fig. 3A; *, p < 0.05; **, p < 0.01). In this regard, higher numbers of anti-OVA IgM, IgG, and IgA AFCs were seen in the spleens of mice given Ad-FL than in those of mice receiving Ad-Luc (Fig. 3C; p < 0.05), although the numbers of total IgM, IgG, and IgA AFCs were essentially the same between two groups (~500/106 IgM, ~850/106 IgG, and ~300/106 IgA).


Figure 2
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  		FIGURE 2. OVA-specific Ab responses in the external secretions and mucosa-associated lymphoid tissues. Each mouse group was nasally immunized weekly for three consecutive weeks with 100 µg of OVA plus 1 x 108 PFU of Ad-FL or Ad-Luc as mucosal adjuvants. A, Fourteen days after the last immunization, levels of anti-OVA S-IgA and IgG Abs in nasal washes and saliva were determined by OVA-specific ELISA. B, Mononuclear cells from NPs, SMGs, and NALT were isolated 14 days after the last immunization and subjected to OVA-specific ELISPOT assay to determine the numbers of IgG and IgA AFCs. Mice nasally immunized with OVA alone as a control group did not exhibit any anti-OVA AFCs. The values shown are the mean ± SEM taken from 25 mice in each experimental group. *, p < 0.05; **, p < 0.01 when compared with mice immunized with OVA plus Ad-Luc.

 

Figure 3
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  		FIGURE 3. OVA-specific Ab responses in plasma and peripheral lymphoid tissues of mice immunized with OVA plus Ad-FL or OVA plus Ad-Luc. A and B, Each group of mice was nasally immunized weekly for three consecutive weeks with 100 µg of OVA plus 1 x 108 PFU of Ad-FL or Ad-Luc as a mucosal adjuvant. Seven days after the last immunization the levels of anti-OVA IgM, IgG and IgA (A) or IgG subclass Abs (B) in plasma were determined by OVA-specific ELISA. Mice immunized with OVA alone as a control group did not exhibit detectable plasma anti-OVA Abs. C, Seven days after the last immunization, mononuclear cells isolated from spleen were subjected to OVA-specific ELISPOT assay to determine the numbers of IgM, IgG, and IgA AFCs. D, Levels of IgG and IgA anti-OVA Abs in plasma of mice given OVA plus Ad-FL or pFL only as a nasal adjuvant were monitored weekly by OVA-specific ELISA. The values shown are the mean ± SEM taken from 25 mice in each experimental group. *, p < 0.05; **, p < 0.01 when compared with mice immunized with OVA plus Ad-Luc.

 
OVA-specific IgG2a Ab responses were the highest of the IgG subclasses (Fig. 3B; p < 0.05). However, both IgG1 and IgG2b Ab responses were significantly higher than those seen in the control group (Fig. 3B; p < 0.05). Taken together, these results clearly indicate that nasal immunization with Ad-FL as a mucosal adjuvant effectively induces OVA-specific Ab responses in both mucosal and systemic immune compartments. Having shown in our previous study that pFL had significant adjuvant activity, we set out in this study to determine the efficacy of Ad-FL as a nasal adjuvant. We found that OVA-specific IgA and IgG Abs in the plasma taken from mice given OVA plus Ad-FL were maintained until at least 8 wk after the final immunization, whereas those responses in mice given nasal pFL showed a sharp decline at 4 wk after the final immunization (Fig. 3D; p < 0.05).

Ad-FL as nasal adjuvant increases CD11b+ DC numbers in mucosal lymphoid tissues

We next determined the frequency and phenotype of DCs in mucosal and peripheral lymphoid tissues of mice given nasal Ad-FL as a mucosal adjuvant. Seven days after the final immunization, much higher CD11c+ DC numbers were noted in the NPs and SMGs of mice given nasal Ad-FL than in mice given Ad-Luc (Fig. 4A; p < 0.01). Among these induced DCs, CD11b+CD11c+ DCs were selectively increased in NPs and SMGs (Table I and Fig. 4B; p < 0.01). Although the numbers of CD11c+ DCs were essentially unchanged, a higher frequency of CD11b+CD11c+ DCs was noted in lymphoid tissues such as NALT and spleen (Table I). Further, DCs from NALT, NPs, and CLNs of mice given nasal Ad-FL as adjuvant expressed higher levels of costimulatory molecules (CD40, CD80, and CD86) and MHC class II than did those of mice given Ad-Luc alone (Table II; p < 0.05). In addition, DCs from SMGs and spleen showed significantly increased levels of CD80 and CD86 expression when mice were immunized with OVA plus Ad-FL (Table II; p < 0.05).


Figure 4
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  		FIGURE 4. Comparison of the frequency of CD11b+ DCs in the mucosal and peripheral tissues of mice given nasal OVA plus Ad-FL or Ad-Luc. Mononuclear cells were isolated from NPs, NALT, SMGs, lung, spleen, and CLNs 1 wk after the last immunization and stained with FITC-conjugated anti-CD11b, PE-labeled anti-CD8, biotinylated anti-CD11c, and allophycocyanin-tagged anti-B220 mAbs followed by PerCP-Cy5.5-streptavidin. All samples were subjected to flow cytometric analysis by FACSCalibur. A, The frequencies of CD11c+ DCs were determined in various lymphoid tissues. B, The CD11c and CD11b expression of SMG and NP samples in the CD8- and B220-negative population was determined. C, Cells from NALT and CLNs were isolated at different time points and the frequency of CD11b+ CD11c+ DCs was determined. The values shown are the mean ± SEM taken from 25 mice in each experimental group. *, p < 0.05; **, p < 0.01 when compared with mice immunized with OVA plus Ad-Luc.

 

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  		Table I. Comparison of the subpopulation of CD11c+ DCs in mucosal and peripheral lymphoid tissues of mice given nasal OVA plus Ad-FL or Ad-Luca

 

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  		Table II. Comparison of costimulatory molecules and MHC class II expression by CD11c+ DCs in mucosal and peripheral lymphoid tissues of mice given nasal OVA plus Ad-FL or Ad-Luca

 
Because the NALT is a primary lymphoid tissue exposed to nasally delivered Ag and adjuvant, a kinetic analysis of CD11b+ DC subsets was performed. Early expansion of this DC population was seen in the NALT of mice given Ad-FL as nasal adjuvant (Fig. 4C; p < 0.05). Thus, the highest numbers of CD11b+ DCs were noted in NALT 3 days after the second immunization with OVA plus Ad-FL (day 10). When an identical kinetic analysis was performed in CLNs, the number of CD11b+ DCs peaked 7 days after the second immunization (day 14) (Fig. 4C; p < 0.05. Taken together, these results indicate that nasal administration of Ad-FL preferentially increases mature-type CD11b+CD11c+ DCs in NALT before these DCs migrate into the effector lymphoid tissues (i.e., NPs and SMGs) through CLNs.

Nasal Ad-FL as mucosal adjuvant elicits OVA-specific CD4+ Th1- and Th2-type cytokine responses

We next assessed OVA-specific CD4+ T cell responses induced by Ad-FL as nasal adjuvant. OVA-stimulated CD4+ T cells isolated from spleen and CLNs of mice given nasal OVA plus Ad-FL produced significantly higher levels of IFN-{gamma} and IL-4 than those from mice given OVA plus Ad-Luc (Fig. 5A; p < 0.05). These results were further confirmed by quantitative real-time PCR. Thus, OVA-stimulated CD4+ T cells from the spleen, NALT, and CLNs of mice given nasal Ad-FL contained significantly higher levels of IFN-{gamma}- and IL-4-specific mRNA than those of the control group (Fig. 5B; p < 0.05). In addition, results of intracellular cytokine analysis also revealed increased numbers of IFN-{gamma}- and IL-4-producing CD4+ T cells from spleen, NALT, and CLNs of mice given Ad-FL as a nasal adjuvant (Fig. 5C). These results show that Ad-FL as a nasal adjuvant elicits both Th1- and Th2-type cytokine responses for the induction of mucosal and peripheral OVA-specific Ab responses.


Figure 5
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  		FIGURE 5. Th1- and Th2-type cytokine production by CD4+ T cells of mice given nasal OVA plus Ad-FL or Ad-Luc. The CD4+ T cells (4 x 106 cells/ml) from each mouse group were purified from NALT, spleen, and CLNs on day 14 and cultured with 1 mg/ml of OVA in the presence of APCs (8 x 106 cells/ml). A, Culture supernatants were harvested after 5 days of incubation (or after 2 days for IL-4 by NALT) and analyzed by the respective cytokine-specific ELISA. B, The CD4+ T cells were harvested after 48 h of incubation. Total RNA was extracted from these cells and then subjected to quantitative RT-PCR analysis. C, IL-4 and IFN-{gamma} production by CD4+ T cells in NALT, CLNs, and spleen was determined by intracellular analysis. Mononuclear cells were incubated with ionomycin (1 µg/ml) and PMA (25 ng/ml) for 3 h and then stained with FITC-labeled anti-CD4. Samples were further stained intracellularly with PE-labeled anti-IL-4 or anti-IFN-{gamma} mAb. The numbers of cytokine-producing cells were determined by multiplying the percentages of cytokine-positive cells in the total lymphocyte cell count. The values shown are the mean ± SEM of 25 mice in each experimental group. The results represent the individual values from three separate experiments with five mice in each experimental group. *, p < 0.05; **, p < 0.01 when compared with mice given Ad-Luc as a nasal adjuvant.

 
Nasal Ad-FL vaccination induces high levels of Ag-specific CTLs

Because Ad-FL as a nasal adjuvant revealed significant Th1-type cytokine responses, we next examined OVA-specific CTL activity by CD8+ T cells. Splenic CD8+ T cells from naive mice and mice given OVA and Ad-Luc or OVA alone were used as controls. CD8+ T cells from CLNs and spleen of mice given nasal OVA and Ad-FL elicited remarkably higher CTL activity (CLNs: 30.4 ± 1.2%; spleen: 68.4 ± 5% specific lysis) at E:T ratios of 25:1 (Fig. 6; p < 0.05). A gradual decrease in CTL activity was seen together with reduced E:T ratios of 5:1 and 1:1 (Fig. 6), further confirming the specificity of the lysis induced by CD8+ T cells. However, at all E:T ratios studied, CTL responses were significantly higher than those of control groups (Fig. 6). These results show that nasal administration of Ad-FL induces not only Ag-specific Ab responses but also effective CTL immunity.


Figure 6
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  		FIGURE 6. Ag-specific CTL activity. An aliquot of 2 x 106 CD8+ T cells was purified from total splenocytes and CLNs 7 days after the last immunization and stimulated in culture with OVA-pulsed stimulator, splenic DCs, and recombinant human IL-2 (50 U/ml). CTL activity was determined by measuring lactate dehydrogenase release from target cells (E.G7-OVA cells) loaded with MHC class I-restricted OVA peptide. The values shown are the mean ± SEM of 15 mice in each experimental group. *, p < 0.05 when compared with mice given Ad-Luc only as a nasal adjuvant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We examined cell and molecular adjuvant events following nasal delivery of an Ad expressing flt3 ligand, finding that nasal Ad-FL as a mucosal adjuvant elicited significant levels of long-lasting OVA-specific mucosal S-IgA and systemic IgG Ab responses. These results are the first to show that Ad-FL can be used as an effective nasal adjuvant. Notably, mice given nasal Ad-FL and OVA three times at weekly intervals showed increases in the numbers of CD11b+ DCs in mucosal effector lymphoid tissues, i.e., SMGs and NPs that migrate from NALT. Furthermore, Ad-FL induced mature-type DCs that expressed higher levels of costimulatory molecules and MHC class II than did those in mice given control Ad-Luc only. Importantly, OVA-stimulated CD4+ T cells from NALT, spleen, and CLNs of mice given nasal OVA plus Ad-FL produced significantly higher levels of IFN-{gamma} and IL-4 than those from mice given nasal Ad-Luc alone. Significant Th1-type cytokine responses supported the induction of OVA-specific CTLs. Taken together, our study clearly demonstrates that Ad-FL as nasal adjuvant targets mature-type CD11b+ DCs, increasing their number and thereby enhancing Th1-type and Th2-type cytokine-, CTL-, and Ag-specific Ab responses in both mucosal and systemic immune compartments.

Our study shows that a selective increase in CD11b+ DCs follows nasal delivery of OVA plus Ad-FL. Studies of DC subsets in Peyer’s patches revealed that the CD11b-expressing DCs were immature, showed high endocytic activity, exhibited low levels of MHC and B7 molecule expression, and were characterized by a dense layer of cells in the subepithelial dome (34). These CD11b+, CD11c+, and CD8– immature myeloid-type DCs expressed CCR6, directing their migration toward the subepithelial dome (35). In contrast, much higher levels of CD40, CD80, CD86, or MHC class II were noted in the NALT, SMGs, and NPs than in the DCs of mice given nasal Ad-Luc alone, indicating that the CD11b+ DCs induced by Ad-FL are mature. A previous study by others showed that i.p. injection of FL protein induced CD11b+ DCs expressing CD40, CD86, and MHC class II (36). However, our own previous study showed that nasal pFL as mucosal adjuvant preferentially expanded CD8+ DCs in NALT, SMGs, and NPs (30). Others recently showed that parenteral delivery of Ad-FL induced all subpopulations of DCs in spleen (37). These results suggest that the formulation and delivery route for FL likely influence the subsets of DC induced in different lymphoid tissues. Similarly, different immunization regimens resulted in the induction of mature-type CD11b+ DCs possessing distinct immunoregulatory functions. Our study clearly shows that CD11b+ DCs induced by nasal immunization with OVA plus Ad-FL play key roles in the induction of CD4+ Th cells, CD8+ CTLs, and OVA-specific Ab responses. Further, others have reported that natural virus infection or immunization with adjuvant elicited CD11b+ DCs that direct T cell activation (38, 39). In contrast to these findings, CD11b+ DCs were able to down-regulate T cell responses under conditions of oral tolerance (40, 41). Attempting to shed light on these contradictory functions by CD11b+ DCs, a recent study showed that the activation of β2 integrins (CD11b/CD18) by DCs significantly hampered T cell activation during Ag presentation (42).

A kinetic analysis of the frequencies of CD11b+ DCs revealed that DCs increased in NALT and CLNs before migrating into the SMGs and NPs. Higher numbers of CD11b+ DCs were observed in NALT and CLNs at 3 and 7 days after the second nasal immunization, but not in the SMGs or NPs until 7 days after the final nasal immunization with OVA and Ad-FL. It is possible that the increase in DC numbers, particularly in the CD11b+ DC subset in SMGs and NPs, was due to the presence of Ad-FL after nasal immunization. To rule out this possibility, the localization of the Ad5 vector was determined using an Ad5 expressing luciferase. High levels of Luc activity were seen in NALT but not in other lymphoid tissues, suggesting that the migration of CD11b+ DCs from NALT into the SMGs and NPs occurs via the CLNs when mice are nasally immunized with Ad-FL plus OVA. Previously, DCs in Peyer’s patches and intestinal lamina propria have been shown to traffic to the mesenteric lymph nodes (43, 44, 45). However, to our knowledge, ours is the first study to directly demonstrate the concept of a common mucosal immune system in the oral-nasopharyngeal tract, with NALT DCs induced by nasal immunization migrating into mucosal effector tissues (e.g., SMGs and NPs) through the draining lymph nodes of the mucosal inductive lymphoid tissue (e.g., NALT). It has been shown that CCR6 and CCR7 play important roles in DC relocation and migration both within and between lymphoid tissues (35, 46, 47, 48, 49, 50, 51). In addition, recent studies showed that CCR1 was expressed by GALT CD11b+ DCs and that these cells migrated toward the subepithelial dome region, where CCL9 was produced by the follicle-associated epithelium (52). Further, it was reported that CCR2 played a critical role in the activation of lung DCs (53) and that CCR5 was an important player in antimicrobial immunity (54). Thus, the obvious question would be to determine which chemokines play central roles in the regulation of CD11b+ DC migration into the SMGs and NPs. Efforts are underway in our laboratory to elucidate the mechanisms for NALT DC migration into the SMGs.

It has been shown that Ag delivery using the Ad vector preferentially induces Th1-type cytokine responses to the codelivered Ag (6), leading to the logical expectation that Th1-type cytokine responses would dominate. Indeed, nasal Ad5 expressing β-galactosidase elicited β-galactosidase-specific CD4+ Th1-type (IFN-{gamma}) cell responses with IL-6 production (6). Interestingly, however, the host vector, Ad5-specific CD4+ T cells, produced both Th1- and Th2-type cytokines, i.e., IFN-{gamma} and IL-2 as well as IL-4, IL-5, and IL-6 (6, 55). Our recent study showed that nasal immunization with Ad2, which was genetically fused with botulinum toxin A protein plus nCT, rapidly induced mixed Th1-and Th2-type cytokine responses. Another study reported that an Ad vector containing an Ag epitope in the virus capsid elicited IFN-{gamma} and IL-4 production by CD4+ T cells (56). Our previous study, which used not Ad-FL but another FL-based modulator, pFL, in combination with CpG oligodeoxynucleotide (Th1-type inducer), showed that pFL as a nasal adjuvant elicited IL-2 and IL-4 production and a mixed Th1- and Th2- type cytokine response (30). These results clearly support our findings that IFN-{gamma}- and IL-4-producing CD4+ T cells contribute to CTL activity and S-IgA Ab production and that they are induced in both mucosal and peripheral lymphoid tissues of mice given nasal OVA plus Ad-FL.

An important aspect of our findings is that Ad-FL as nasal adjuvant elicits longer-lasting Ag-specific IgA and IgG Ab responses than does pFL. It has been suggested that DCs are long-lived cells capable of carrying Ag-specific information after initial Ag processing (29, 57). Because they can be used successfully to elicit long-lasting protective immunity, DC-based vaccines show considerable promise for cancer immunotherapy aimed at regulating the growth and expansion of tumor cells (58). Further, it has been shown that antitumor immunity in tumor-bearing mice can be boosted after an adoptive transfer of DCs pretreated with tumor Ags ex vivo (59). Our two separate studies showed that nasal immunization targeting NALT DCs elicited long-lasting OVA-specific immune responses. Furthermore, the deletion of DCs by anti-CD11c mAb treatment impaired the ability of a combined nasal pFL and CpG oligodeoxynucleotide adjuvant to induce sustained elevated immune responses. Taken together, these findings clearly indicate that memory-type DCs play a critical role in the induction of long-lasting Ag-specific immunity.

In summary, Ad-FL as a nasal adjuvant preferentially triggers NALT CD11b+ DCs to multiply and migrate to efferent mucosal effector lymphoid tissues. These mature-type CD11b+ DCs are key players in the induction of Th1 and Th2 cytokine-mediated, long-lasting, Ag-specific humoral and CTL responses. Balanced Th1- and Th2-type cytokine responses could be ideal for preventing exaggerated inflammatory or allergic immune responses. Furthermore, Ag-specific Ab and CTL responses could help prevent viral infections. Because Ad-FL can be easily coadministered with any type of Ag, this adjuvant delivery system could greatly facilitate the development of mucosal vaccines against a wide variety of bacterial and viral pathogens while reducing potential side effects.


   Acknowledgments
 
We thank Sheila D. Turner for the final preparation of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants DE 12242, AI 43197, AI 18958, AG 025873, and AI 076096 as well as by Grants-in-Aid (C19592403, A19209064) from the Ministry of Education, Science, Sports and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Kohtaro Fujihashi, Departments of Pediatric Dentistry and Microbiology, Immunobiology Vaccine Center, University of Alabama at Birmingham, 1919 Seventh Avenue South, School of Dentistry Building 801A1, Birmingham, AL 35294. E-mail address: kohtarof{at}uab.edu Back

3 Abbreviations used in this paper: S-IgA, secretory IgA; Ad, adenovirus; Ad-FL, adenovirus expressing flt3 ligand; Ad-Luc, adenovirus expressing luciferase; AFC, Ab-forming cell; CLN, cervical lymph node; DC, dendritic cell; FL, flt ligand; NALT, nasopharyngeal-associated lymphoreticular tissue; nCT, native cholera toxin; NP, nasal passage; pFL, plasmid expressing Flt ligand; SMG, submandibular gland. Back

Received for publication May 17, 2007. Accepted for publication April 14, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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