Nasal Flt3 Ligand cDNA Elicits CD11c⁺CD8⁺ Dendritic Cells for Enhanced Mucosal Immunity¹

Flt3 ligand (FL),³ a type 1 transmembrane protein, binds either feral liver kinase 2 (flk2) or fms-like tyrosine kinase 3 (flt3) receptor. FL mobilizes and stimulates myeloid and lymphoid progenitor cells (1), dendritic cells (DCs) (2), and NK cells (3). In addition, FL given with IL-7 costimulates the expansion of primitive B cell progenitors (4). Thus, FL has multiple roles in early hemopoiesis and B lymphopoiesis (4). In this regard, FL activates stem cells in bone marrow and promotes their differentiation into mature B cells (4). Furthermore, FL given with IL-15 expands NK cell progenitors (3). It has been shown that daily injection of FL into mice resulted in dramatic increases in DCs in the bone marrow, the peritoneal cavity, spleen, and thymus, and subsequently enhanced Ag-specific immune responses, comparable to those supported by cholera toxin (CT) when used as mucosal adjuvant (2, 5, 6). Other studies have now shown that the FL treatment also favors the induction of immune responses when given by mucosal (5), systemic (7), or cutaneous routes (8). In each of these studies, mice were injected daily with recombinant FL on consecutive days. In recent studies, plasmid DNA encoding FL has been given with plasmids encoding protein Ag (9) or linked to the Ag itself (10). These studies confirm the adjuvant activity of FL for both Ab- and cell-mediated immunity and suggest that frequent treatment with recombinant FL can also be accomplished by injection of FL cDNA itself.

Nasal Ag delivery is the most effective way to induce mucosal secretory-IgA (S-IgA) Ab responses and plasma-specific Abs (11, 12, 13). To elicit maximal levels of Ag-specific immune responses in both mucosal and systemic lymphoid tissue compartments, it is necessary to use an appropriate mucosal adjuvant (11, 14, 15). To date, both CT produced by Vibrio cholerae and the heat-labile enterotoxin from Escherichia coli are two of the most potent mucosal adjuvants for enhancement of Ag-specific Ab responses when coadministered with protein Ag by either the oral or nasal routes (16, 17, 18). It is known that CT acts as a mucosal adjuvant by inducing CD4⁺ Th type 2 (Th2) cells and the cytokines IL-4, IL-5, IL-6, and IL-10, which are essential for induction of IgA, IgG1, and IgE Ab responses (19, 20, 21, 22, 23). In contrast, mucosal delivery of heat-labile enterotoxin results in mixed Th1 and Th2-type CD4⁺ T cells with subsequent mucosal S-IgA Ab responses (24). In addition, it has been shown that mucosal application of cytokines or chemokines with protein Ag successfully induces both mucosal and systemic CD4⁺ T cell and Ab responses. For example, nasal immunization with tetanus toxoid and IL-12 enhanced tetanus toxoid-specific Ab responses (25). Furthermore, both IL-1α and IL-1β are potent mucosal adjuvants (26). Finally, it has been shown that lymphotactin and RANTES possess mucosal adjuvant activity for enhanced Ag-specific Ab responses (27, 28).

It is well known that mucosal administration of protein Ag without appropriate adjuvant or delivery systems generally induces systemic unresponsiveness to the same Ag. This unresponsive state has been termed oral tolerance (29). In general, prolonged and frequent low doses of Ag administration or a single high dose of Ag delivery is required for the induction of mucosal tolerance. However, it has been shown that conjugation of Ag with the B subunit of CT (CT-B) facilitated induction of systemic unresponsiveness by reducing the levels and numbers of Ag doses required (30). Further, it was also reported that the expansion of DCs by parental injection of FL enhanced oral tolerance induction to OVA (31). Thus, oral tolerance was established in FL-treated mice using very low doses of OVA that generally failed to induce tolerance in normal mice (31).

To date, no reports have determined the outcome of immune responses when FL or FL cDNA is given with Ag by mucosal routes. Our study was designed to examine whether a nasal plasmid encoding FL given with OVA would simultaneously enhance mucosal immunity or alternately, induce mucosal tolerance. We show here that an expression vector containing the FL gene given with the protein OVA nasally resulted in FL protein expression in the nasal tract which enhanced OVA-specific Ab responses instead of tolerance induction through a dramatic expansion of activated, mature DCs.

Female C57BL/6 mice (6- to 8-wk-old) were purchased from the Frederick Cancer Research Facility (Frederick, MD). These mice were transferred to microisolators and maintained in horizontal laminar flow cabinets, and provided sterile food and water as part of a specific pathogen-free facility. The health of the mice was monitored by both serology for bacterial and viral pathogens and immunohistology. All of the mice used in these experiments were free of bacterial and viral pathogens.

The plasmid pORF9-mFLt3L (pFL) consists of the pORF9-mcs vector (pORF) plus the full-length murine FL cDNA gene (InvivoGen, San Diego, CA). The pORF is an expression vector containing the hybrid elongation factor (EF)1-α/human T cell leukemia virus promotor and the ampicillin resistance gene (858 bp) which allowed selection of bacteria carrying the plasmid. This plasmid DNA was purified using the GeneElute Endotoxin-Free Plasmid kit (Sigma-Aldrich, St. Louis, MO). The Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) resulted in <0.1 endotoxin unit of LPS per 1 μg of plasmid.

Mice were immunized three times at weekly intervals nasally with 6 μl/nostril PBS containing 50 μg of pFL and 100 μg of OVA (fraction V; Sigma-Aldrich). As controls, mice were immunized nasally with 50 μg of pORF (empty plasmid) and 100 μg of OVA under anesthesia. Plasma, saliva, and fecal extracts (FEs) were collected on days 0 and 21. Stimulated saliva and FE samples were obtained as described previously (18, 32, 33). Mice were sacrificed 7 days after the last immunization (day 21) and nasal washes were obtained by instillation of 1 ml of PBS on three occasions into the posterior opening of the nasopharynx with a 30-gauge hypodermic needle (34, 35).

OVA-specific Ab levels in plasma and mucosal secretions were determined by ELISA as previously described (18, 33, 36, 37). Briefly, 96-well Falcon microtest assay plates (BD Biosciences, Oxnard, CA) were coated with 1 mg/ml OVA in PBS. After blocking with 1% BSA in PBS, 2-fold serial dilutions of samples were added to each well. Following incubation overnight at 4°C, HRP-labeled goat anti-mouse μ, γ, or α H chain-specific Abs (Southern Biotechnology Associates, Birmingham, AL) were added to wells. For IgG Ab subclass analysis, biotinylated mAbs specific for IgG1, IgG2a, IgG2b, and IgG3 (BD PharMingen, San Diego, CA) and peroxidase-conjugated goat anti-biotin Ab (Vector Laboratories, Burlingame, CA) were used for detection. The color reaction was developed for 15 min at room temperature with 100 μl of 1.1 mM 2,2′-azino bis (3-ethylbenz-thiazoline-6-sulfonic acid) in 0.1 M citrate phosphate buffer (pH 4.2) containing 0.01% H₂O₂. Endpoint titers were expressed as the reciprocal log₂ of the last dilution that gave an OD₄₁₅ of 0.1 greater than background.

Mononuclear cells from the spleen and cervical lymph nodes (CLNs) were isolated aseptically by a mechanical dissociation method using gentle teasing through stainless steel screens as described previously (18, 33, 37, 38). For isolation of mononuclear cells from nasal passages (NP) and nasopharyngeal-associated lymphoreticular tissues (NALT), a modified dissociation method was used based upon a previously described protocol (39, 40, 41). Mononuclear cells from submandibular glands (SMG), Peyer’s patches, and lamina propria (LP) were isolated by a combination of an enzymatic dissociation procedure with collagenase type IV (0.5 mg/ml; Sigma-Aldrich) followed by discontinuous Percoll (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation (32). Mononuclear cells obtained from mucosal and systemic lymphoid tissues were subjected to ELISPOT assay to detect numbers of OVA-specific AFCs (33, 37, 39, 41). Briefly, 96-well nitrocellulose plates (Millititer HA; Millipore, Bedford, MA) were coated with 1 mg/ml for analysis of anti-OVA-specific AFCs. The numbers of OVA-specific AFCs were quantified with the aid of a stereomicroscope as described elsewhere (18, 32, 38).

CD4⁺ T cells from spleen and CLNs were purified by use of an automated magnetic activated cell sorter (AutoMACS) system (Miltenyi Biotec, Auburn, CA) as described previously (18, 37). Briefly, a nylon wool column enriched T cell fraction was incubated with biotinylated anti-CD4 mAb (GK 1.5) (BD PharMingen) followed by streptavidin-conjugated microbeads and sorted to purity with the AutoMACS. This purified T cell fraction was >97% CD4⁺ and the cells were >99% viable. This purified CD4⁺ T cell fraction was resuspended in RPMI 1640 (Cellgro; Mediatech, Washington, DC) 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 × 10⁶ cells/ml), and cultured in the presence of 1 mg/ml OVA of cultures of T cell-depleted, irradiated (3000 rad) splenic APCs taken from nonimmunized, normal mice. To assess OVA-specific T cell proliferative responses, an aliquot of 0.5 μCi of tritiated [³H]TdR (Amersham Biosciences, Arlington Heights, IL) was added during the final 18 h of incubation, and the amount of [³H]TdR incorporation was determined by scintillation counting. The supernatants of identically treated T cell cultures not incubated with [³H]TdR were then subjected to a cytokine-specific ELISA as described below.

The levels of cytokines in CD4⁺ T cell culture supernatants from spleen or CLNs were determined by cytokine-specific ELISA as described previously (36, 42, 43, 44). The culture supernatants were collected on day two for IFN-γ, IL-2, IL-4, IL-5, IL-6, and IL-10 analysis. The immunoplates (Nalge Nunc International, Rochester, NY) were coated with anti-cytokine capturing mAb. After blocking with 3% BSA in PBS, serial 2-fold diluted samples and standards were added, and incubated overnight at 4°C. The plates were washed and respective detection mAb was added. After incubation overnight at 4°C, HRP-labeled goat anti-biotin Ab (Vector Laboratories) was added and incubated for 4 h at room temperature. The color reaction was developed at room temperature with 100 μl of 1.1 mM 2, 2′-azino bis (3-ethylbenz-thiazoline-6-sulfonic acid) in 0.1 M citrate phosphate buffer (pH 4.2) containing 0.01% H₂O₂. The detection limits for each cytokine were: 106.3 pg/ml for IFN-γ, 15.6 pg/ml for IL-2, 4.66 pg/ml for IL-4, 156 pg/ml for IL-5, 156 pg/ml for IL-6, and 391 pg/ml for IL-10.

For evaluation of cytokine-specific mRNA levels in OVA-stimulated CD4⁺ T cells, real-time PCR was used with a LightCycler (Roche Applied Science, Indianapolis, IN) (45, 46). The CD4⁺ T cells were harvested after 2 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 RT reaction using Superscript II Reverse Transcriptase (Invitrogen/Life Technologies, Carlsbad, CA). Samples were treated with 1 μl of 10 μg/ml RNase H (Invitrogen/Life Technologies) and incubated at 37°C for 30 min. The levels of synthesized cDNA were measured by 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.

Aliquots of mononuclear cells (0.2–1.0 × 10⁶ cells) were isolated from tissues of mice immunized with OVA plus pFL or mice given OVA plus pORF (empty plasmid). Cells were stained with FITC-conjugated anti-mouse CD11b, CD8, or B220 mAbs, PE-labeled anti-mouse I-A^b, CD11c, CD40, CD80, or CD86 mAbs (BD PharMingen) and biotinylated anti-mouse CD11c mAbs (BD PharMingen) followed by CyChrome-streptavidin. The samples were then subjected to FACS analysis (FACSCalibur; BD Biosciences).

NALT were taken from mice immunized nasally with OVA plus pFL or OVA plus pORF (empty plasmid) and were snap-frozen at −160°C. Cryostat sections of 4 μm were then fixed with 4% formaldehyde before staining using conjugated Abs. Sections were stained with PE-conjugated biotin coupled anti-CD40, -CD80, -CD86, and anti-CD11c mAbs. Biotin-labeled anti-CD11c mAb was followed by HRP-conjugated streptavidin-Alexa Fluor 488 (Molecular Probes, Eugene, OR). Sections were examined with a fluorescence microscope (BX50/BXFLA; Olympus, Tokyo, Japan) equipped with a digital image capture system (Olympus).

To determine the tissues which take up the plasmid, DNA was extracted from the NPs, NALT, and SMG and amplified with ampicillin resistance gene-specific primers by PCR. Tissues were digested by proteinase K (100 μg/ml), and the DNA was extracted twice with phenol equilibrated with Tris buffer (pH 8.0) (47). For detection of the ampicillin resistance gene, extracted DNA was amplified by oligonucleotide primers specific for the ampicillin resistance gene (sense, 5′-CCAATGCTTAATCAGTGAGGC-3′; antisense, 5′-ATGAGTATTCAACATTTCCGTGTCG-3′). The PCR products were separated by electrophoresis in 2% agarose gels and visualized by UV light illumination following ethidium bromide (0.5 mg/ml) staining.

To determine the levels of FL protein present in nasal washes (NW), saliva, and plasma, samples were collected from mice given nasal Ag plus plasmid. These samples were then analyzed with a Quantikine M mouse Flt3 Ligand ELISA kit (R&D Systems, Minneapolis, MN).

The results are expressed as the mean ± SEM, and mouse groups were compared with control mice using an unpaired Mann-Whitney U test with Statview software (Abacus Concepts, Berkley, CA) designed for Macintosh computers. Values of p of <0.05 or <0.01 were considered significant.

We initially examined whether nasal administration of FL plasmid (pFL) would enhance OVA-specific immune responses or alternatively would result in tolerance in both mucosal and systemic lymphoid tissues. Mice given nasal OVA plus pFL exhibited OVA-specific IgA Ab responses in NW, FEs, and saliva (Fig. 1,a). In contrast, OVA-specific S-IgA Ab levels of mice immunized with either OVA alone or OVA with empty plasmid gene (pORF) did not produce S-IgA anti-OVA Ab responses (Fig. 1,a). To further support these findings, mice given the nasal pFL plus OVA displayed high numbers of OVA-specific IgA AFCs in NPs and SMG. In addition, a high frequency of OVA-specific IgA AFCs were seen in small intestinal LP (Fig. 1,b). Interestingly, our results showed that OVA-specific IgA AFCs were also effectively induced in mucosal inductive tissues themselves, i.e., in NALT and Peyer’s patches following nasal immunization with pFL and OVA (Fig. 1,c). These findings clearly show that nasal immunization with OVA and pFL is an effective way to induce Ag-specific IgA Abs in mucosal secretions. Further, this result clearly shows that nasal pFL does not induce mucosal tolerance. Elevated levels of OVA-specific plasma IgG Ab responses were seen in mice given nasal OVA plus pFL when compared with mice immunized with OVA plus empty plasmid (pORF) (Fig. 2,a). The plasma from mice nasally immunized with OVA alone did not contain detectable Ab levels (data not shown). Both OVA-specific IgG1 and IgG2b Ab levels were markedly elevated when compared with control mouse groups (Fig. 2,b). Relatively low IgG2a and no IgG3 Ab responses were seen (Fig. 2,b). These findings suggest that a nasal vaccine containing FL plasmid induces a Th2-type cytokine-mediated OVA-specific immune response. To support this, the numbers of OVA-specific IgM, IgG, and IgA AFCs in spleen and CLNs were significantly increased when compared with control mice (Fig. 2 c). Taken together, these results indicate that nasal immunization with pFL as mucosal adjuvant effectively induces OVA-specific Ab responses in both mucosal and systemic immune compartments. Further, pFL did not induce mucosal tolerance.

We next assessed OVA-specific CD4⁺ T cell responses induced by the expression vector containing the FL gene as nasal adjuvant. Both splenic and CLN CD4⁺ T cells from mice given nasal pFL plus OVA showed higher proliferative responses than did mice immunized with pORF plus OVA (Table I). We next examined levels of Ag-specific Th1- and Th2-type cytokine produced by OVA-specific CD4⁺ T cells from spleen and CLNs of mice given nasal pFL as mucosal adjuvant. OVA-stimulated CD4⁺ T cells isolated from spleen or CLNs of mice given nasal OVA plus pFL exhibited significantly higher levels of IL-2 and IL-4 production when compared with mice given OVA plus pORF (empty plasmid). In contrast, no significant increases in other Th1- or Th2-type (IL-5, IL-6, and IL-10) cytokines were seen (Table I). These results were further confirmed by quantitative real-time PCR. Thus, OVA-stimulated CD4⁺ T cells from the spleen and CLNs of mice given nasal pFL contained significantly increased levels of IL-4-specific mRNA when compared with mice immunized nasally with OVA plus pORF (Table I). In addition, higher levels of IL-2-specific mRNA were detected in CD4⁺ T cells from spleen and CLNs of mice given pFL as nasal adjuvant when compared with mice given pORF (Table I). In contrast, IFN-γ-specific mRNA levels were essentially identical between mice given pFL and empty plasmid (Table I). These results show that both IL-2 and IL-4 are essential cytokines for the induction of the adjuvant effects of pFL.

We next investigated the frequency of CD11c⁺ DCs in various mucosal inductive and effector tissues. Our results showed major increases in numbers of CD11c⁺ DCs in NALT, NPs, SMG (Table II), and LP (data not shown) of mice given nasal FL when compared with mice given pORF. It is important to note that the numbers of CD11c⁺ DCs in the mucosal effector sites, such as NPs, NALT, and SMG were remarkably enhanced (Table II; Fig. 3, a and b). These results indicate that increased numbers of CD11c⁺ DCs were due to nasal application of the expression plasmid encoding the FL gene. To characterize the phenotype of these expanded DCs, we assessed cell surface expression by CD11c⁺ DCs in mucosal tissues of CD8, CD11b, B220, MHC class II (MHC II), CD40, CD80, and CD86. Interestingly, the majority of DCs in NPs and NALT express CD8 but not CD11b or B220 molecules. Further, high levels of MHC II, CD40, CD80, and CD86 were expressed by CD11c⁺, CD8⁺, CD11b⁻, and B220⁻ DCs (Table II; Fig. 3, c–j). Taken together, these results indicate that nasal administration of pFL preferentially expands mature DCs and also induces their activation in both mucosal inductive and effector tissues.

To assess for expression of FL protein, DNA was isolated from various tissues and amplified with ampicillin resistance gene-specific primers. The highest expression of pFL was in NALT followed by NPs. In contrast, naive mice did not express the plasmid-specific gene (Fig. 4,a). We next examined whether the levels of FL protein were increased after nasal administration of pFL. The levels of FL protein in plasma, FEs, and NWs of mice immunized given nasal pFL plus OVA were significantly elevated when compared with mice immunized with pORF plus OVA or mice given OVA only (Fig. 4 b). These results indicate that nasal application of plasmid induced higher expression of FL protein in both mucosal effector and systemic lymphoid tissues.

In our studies, we have shown that nasal delivery of the protein OVA with 50 μg of FL plasmid results in high titers of OVA-specific plasma IgG and IgA and mucosal S-IgA Ab responses. Further, elevated numbers of OVA-specific IgG and IgA AFCs were also detected in both mucosal and systemic tissues. Although it has been shown that both FL protein and encoded plasmid exhibit adjuvant activity (7, 8, 9, 10), our study is the first to show that the plasmid vector encoding FL cDNA has potent mucosal adjuvanticity instead of tolerance induction when coadministered nasally with protein Ag. Induction of Ag-specific immune responses was associated with elevated numbers of activated DCs in NALT. In this regard, we demonstrated that pFL was preferentially taken up by NALT, and FL protein levels were increased in NWs of mice given nasal OVA plus pFL. Further, our study showed that CD4⁺ T cells and IL-2 and IL-4 synthesis mediated the pFL nasal adjuvant activity.

The focus of the present study was to investigate whether nasal delivery of plasmid encoding FL would enhance mucosal immunity or alternatively would induce tolerance. It was previously reported that pretreatment with FL facilitated the induction of systemic unresponsiveness through expansion of immature DCs when protein Ag was subsequently administered via the oral route (31). In contrast, our results provide direct evidence that both mucosal and systemic immunity are enhanced when pFL is given simultaneously with Ag. The opposite outcomes between these two studies may be explained by the manner of FL treatment used. For example, we used mucosal FL plasmid administration simultaneously with OVA. Thus, significant increases in mature DCs were limited to the local nasal mucosa and SMG (Table II). In contrast, in the oral tolerance studies, consecutive FL protein delivery was performed and marked increases in DCs were seen in lymphoid tissues before oral delivery of OVA. Further, the phenotype of DCs induced by nasal pFL was totally distinct from DCs induced by consecutive injection of FL protein. Thus, increased numbers of CD11c⁺CD8⁺ DCs in NALT, NPs, and SMG with up-regulated expression of MHC II, CD40, CD80, and CD86 molecules were noted in mice given pFL when compared with DCs from mice given nasal OVA with pORF (Table II). In contrast, FL protein injection failed to induce up-regulated expression of these costimulatory molecules, although numbers of DCs in various tissues were significantly increased. Based upon these findings, it is possible that the presence of Ag with FL is an essential condition for the effective generation of activated DCs in immune inductive tissues which subsequently lead to Ag-specific immunity instead of mucosal tolerance. Indeed, DCs expanded by FL injection were capable of being activated and of expression of costimulatory molecules after LPS stimulation (5, 31). To further support this view, it has been shown that i.m. injection of a DNA vaccine encoded together with FL plasmid and Ag induced enhanced Ag-specific immune responses (10). Further, nasal application of pFL alone failed to induce costimulatory factor expression even though the DC population was markedly increased (data not shown).

It is well known that CD4⁺ T cells and their derived Th1 and Th2 cytokine responses are essential for the induction of Ag-specific S-IgA Ab responses. In this regard, we have compared Th1- and Th2-type cytokine production by CD4⁺ T cells from mice given either nasal pFL or pORF as mucosal adjuvant. Our results clearly showed that IL-4 production was significantly enhanced in both spleen and CLNs of mice given pFL. Further, increased levels of IL-2 synthesis by OVA-stimulated CD4⁺ T cells were also noted in both spleen and CLNs of mice immunized with OVA plus pFL. Although IL-2 is considered to be a Th1-type cytokine, the enhanced IL-2 production by CD4⁺ T cells from mice given pFL may account for IL-4 synthesis but not for Th1-type cytokine responses. To support this view, our results showed limited amounts of IFN-γ production (Table I). In this regard, OVA-specific IgG2a Ab which is a typical subclass supported by Th1-type responses were quite low in mice given nasal pFL as mucosal adjuvant. It has been shown that nasal application of the unmethylated CpG motif possessed CD4⁺ Th1-type T cell-mediated adjuvant activity when coadministered with influenza Ag (48). Thus, it would be logical to suggest that pFL adjuvanticity possibly resulted from unmethylated CpG motif effects because the FL encoded plasmid was used as mucosal adjuvant. However, our results revealed only low OVA-specific IgG2a Ab responses and essentially no OVA-induced IFN-γ synthesis by CD4⁺ T cells which would be associated with CpG adjuvant activity. A minor increment of OVA-specific IgG Ab responses induced by control cDNA plasmid is due to the common results of DNA immunization. In this regard, others reported similar results that control plasmid possessed minor adjuvant activity (49, 50). The most important point of our study is that pFL successfully induced OVA-specific IgA Ab responses in mucosal secretions and plasma. In contrast, control plasmid failed to induce detectable levels of IgA Abs in mucosal secretions or plasma. Thus, the control immunization formula did not contain actual adjuvant activity. The adjuvant activity totally depends upon pFL. Taken together, we postulate that pFL mucosal adjuvant activity may be mediated by IL-4 producing Th2-type CD4⁺ T cells. In this regard, we are currently testing pFL adjuvant activity in IL-4-deficient mice.

To date, an array of nasal adjuvants have been used successfully to elicit Ag-specific immunity in both mucosal and systemic sites; however, the precise mechanisms for the induction of Ag-specific immune responses still remains to be elucidated. For example, enterotoxins, their mutants, and chimeras have shown significant adjuvanticity when coadministered nasally with protein Ag (18, 34). Although evidence has been presented that oral native CT induces up-regulation of CD86 on Peyer’s patch B cells and macrophages and expression of CD86 was required for adjuvanticity (23, 51), the primary and main target tissues or cells of nasal native CT for the initiation of adjuvant activity are currently unknown. Contrary to this, our findings clearly showed the target tissues and cells where the initiation of FL adjuvant function occurred. In this regard, our results showed that the highest expression of the plasmid-specific ampicillin resistant gene was in NALT followed by the NPs. The FL protein levels were significantly increased in NWs when compared with those from mice given OVA only or OVA plus pORF. Similarly, FL levels in plasma were also elevated. Because the spleen as well as other lymph nodes did not express the plasmid-specific gene, we postulate that high levels of FL in plasma were primarily due to transudation from the nasal mucosa. These results show that FL-encoded plasmid was mainly taken up by NALT and subsequently the FL protein was produced locally in these tissues, which resulted in the subsequent expansion and activation of DCs.

In summary, the present study has shown that plasmid encoding FL cDNA was an effective and safe mucosal adjuvant. The mechanisms of pFL adjuvanticity were clearly due to the function of FL. Thus, the numbers of DCs in nasal inductive tissues, e.g., NALT were significantly increased. Because Ag was simultaneously administered together with pFL, the expanded DCs were activated and expressed MHC II and costimulatory molecules (CD40, CD80, and CD86). Subsequently, these activated DCs induced mainly Th2-type IL-4-producing CD4⁺ T cells for the induction of mucosal S-IgA and systemic IgG Ab responses. Use of pFL as mucosal adjuvant opens a new avenue for the development of effective and safe mucosal vaccines which prevent not only ordinary infectious diseases but also the category A agents for potential use by bioterrorists.

We thank Annette M. Pitts for excellent technical assistance in the immunohistochemistry assay, and Sheila D. Turner for the final preparation of the manuscript.