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The Journal of Immunology, 2006, 176: 4600-4607.
Copyright © 2006 by The American Association of Immunologists

Insufficient APC Capacities of Dendritic Cells in Gene Gun-Mediated DNA Vaccination1

Henning Lauterbach2, Anton Gruber, Christine Ried, Cedric Cheminay and Thomas Brocker3

Institute for Immunology, Ludwig-Maximilians-University Munich, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Gene gun-mediated DNA immunization is a powerful mode of vaccination against infectious diseases and tumors. Many studies have identified dendritic cells (DC) as the central players in inducing immunity upon biolistic DNA vaccination; however, none of these studies directly quantify DC-mediated responses in comparison with immunity triggered by all Ag- and MHC-expressing cells. In this study we use two different approaches to decipher the relative role of DC vs other cell types in gene gun-induced immunity. First, we directly compared the immunization efficacy of different DNA constructs, which allow Ag expression ubiquitously (CMV promoter) or specifically in DC (CD11c promoter) and would encode either for soluble or membrane bound forms of Ag. Second, we immunized transgenic mice in which only DC can present MHC-restricted Ag, and directly compared the magnitudes of CTL activation with those obtained in wild-type mice. Surprisingly, our combined data suggest that, although DC-specific Ag expression is sufficient to induce humoral responses, DC alone cannot trigger optimal CD4 and CD8 T cell responses upon gene gun vaccination. Therefore, we conclude that DC alone are insufficient to mediate optimal induction of T cell immunity upon gene gun DNA vaccination and that broad Ag expression rather than DC-restricted approaches are necessary for induction of complete immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Using DNA immunization was shown to be a promising alternative to standard protein subunit or peptide vaccine formulations (for review, see Ref.1). One of its major advantages is the potential to stimulate both humoral and cellular immunity (2), a feature that is especially important in diseases in which the combination of specific Ab and T cell responses are required for protection. Despite promising data obtained in experimental animals, the benefits of DNA vaccination for human application remain elusive. Several DNA vaccine delivery methods have been developed (for review, see Ref.1), among which biolistic gene gun application of expression plasmids seems to be the most efficient (3, 4). Using this technology, DNA-coated gold particles are propelled directly into the cytosol of dermal keratinocytes and Langerhans cells (5, 6). In this context, a series of elegant studies, using either bone marrow chimeric mice (7, 8) or skin transplantations (9, 10), have demonstrated a central role for dendritic cells (DC)4 in priming immune responses after DNA vaccination. This result was confirmed by direct in vivo visualization of DC containing gold particles and expressing reporter genes in draining lymph nodes (11). Therefore, transfected somatic cells, like myocytes or keratinocytes, are considered to serve as Ag reservoirs, which may be important for the strength and longevity, but not for the induction, of immune responses (9, 12). In fact, studies using keratinocyte-specific K14 promoter-driven DNA vaccines indicated that mainly cross-presentation rather than direct presentation by DC might be responsible for induction and maintenance of CD8 T cell immunity after gene gun immunization (13). Therefore, the cellular localization of the expressed Ag might influence both magnitude as well as quality of responses (14, 15, 16, 17). As a consequence from this apparently prominent role of DC in DNA vaccination, several groups used DC-specific promoters (17, 18) to enhance DNA vaccination efficacy by specifically targeting DC. However, most DNA vaccination studies investigating these types of vaccines either lack in vivo data or were based on i.m. DNA injection, a highly variable method inferior to gene gun immunization (4, 19, 20).

To elucidate the role of DC in gene gun vaccination and to directly evaluate their actual contribution to immunity, we combined direct in vivo readouts for T cell immunity with 1) vaccine vectors encoding for different forms of Ag (secreted vs membrane bound), 2) vectors allowing Ag expression in different cells types (ubiquitous vs DC), and 3) vaccinations of transgenic mice in which only DC were able to present Ag.

Our findings demonstrate that in gene gun application a secreted antigenic form is more efficiently inducing humoral as well as CD4 T cell responses, whereas membrane-bound antigenic forms induce stronger CD8 T cell responses. However, restricting expression of either form of Ag to DC by using the murine CD11c promoter (21) induced Ab responses comparable to ubiquitously expressing CMV promoter-driven constructs, but did not induce significant T cell responses. This lack of T cell priming and expansion was not due to weaker expression properties of the DC-specific promoter, but rather to the inability of DC to activate and expand T cells, when no other additional cell types could present the DNA-encoded Ag. Thus, Ag presentation by other cells than DC is essential for complete T cell responses after DNA gene gun immunization.


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

All mice were bred and maintained under standard conditions in the animal facilities of the Institute for Immunology (Ludwig-Maximilians-University, Munich, Germany). DO11.10 mice (expressing transgenic TCR specific for OVA323–339-MHC class II I-Ad) were obtained from The Jackson Laboratory. OT-1 mice (expressing transgenic TCR specific for OVA257–263-MHC class I I-Kb) (22) and rat insulin promoter (RIP)-OVAlow mice have been previously described (23). Glucosuria in RIP-OVAlow mice was determined daily using test stripes (Diabur 5000; Roche Diagnostics). Mice were considered diabetic at levels ≥5.5 mM glucose. CD11c-MHC class I (CD11c-MHCI) transgenic mice, expressing beta2-microglobulin under control of the CD11c promoter, have been previously described (24, 25). All mice were bred for over 10 generations to the C57BL/6 background and maintained at the animal facility of the Institute for Immunology (Ludwig-Maximilians-University, Munich, Germany).

Plasmid construction

The construction of CMV-OVA was previously described. Briefly, a BamHI/XhoI fragment of rabbit beta-globin was cloned into a BamHI/XhoI-opened pcDNA3 vector (Invitrogen Life Technologies) to create pcDNA3-beta-globin. The CMV-OVA vector encoding the secreted form of chicken OVA was constructed by cloning a 1.9-kb EcoRI fragment, which contained the entire coding sequence of OVA, into the EcoRI site of pcDNA3-beta-globin. Similarly, to obtain CMV-mOVA encoding the membrane bound form of OVA (mOVA), a 1.8-kb EcoRI fragment from the plasmid pBlueRIP/Tfr-OVA (provided by F. Carbone, Walter and Eliza Hall Institute, Melbourne, Australia), which contained the first 118 amino acids of the human transferrin receptor fused to amino acids 139–386 of OVA, was cloned into an EcoRI-opened pcDNA3-beta-globin vector. To create CD11c-OVA and CD11c-mOVA, OVA-cDNA and mOVA-cDNA were cloned into an EcoRI-opened CD11c-beta-globin vector, which was previously described (21). Plasmids were prepared from Escherichia coli with Qiagen Mega kits.

Adoptive transfer

DO11.10 and OT-1 T cells were prepared from lymph nodes and spleens of transgenic mice. Briefly, spleen and lymph nodes were taken out, and single cell suspensions were prepared. Spleen RBC were removed using ACK buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2 EDTA, (pH 7.4)) for 4 min at room temperature and OT-1 T cells from OT-1 transgenic mice were isolated by negative selection (>96% purity, CD8 T cell enrichment columns; R&D Systems). After determining the percentage of DO.11.10 or OT-1 TCR transgenic T cells by flow cytometry, 2.5 x 106 transgenic DO11.10 or 5 x 106 OT-1 T cells were injected into the lateral tail veins of age- and sex-matched recipient mice.

Vaccination

DNA immunization was performed by gene gun administration (Bio-Rad). Cartridges of DNA-coated gold particles were prepared according to the manufacturer’s instructions. For each preparation, 25 mg of gold particles (diameter, 1 µm) were coated with 200 µg of DNA. Mice were anesthetized before vaccination with a mixture of Ketavet/Rompun in PBS. A total of 8 µg of plasmid DNA was delivered to the shaved abdominal skin of adult mice with a discharge pressure of 400 psi.

ELISA

For the detection of OVA-specific Abs, 96-well microtiter plates (MaxiSorp; Nunc) were coated with 150 µg/ml OVA (Sigma-Aldrich) at room temperature overnight. Plates were blocked (PBS, 0.5% milk powder and 0.05% NaN3), and immune sera (diluted 1/150 in blocking buffer) were incubated for 2 h at room temperature. After washing five times with PBS, HRP-labeled second-step goat sera specific for mouse IgM or IgG (Serotec), IgG1 or IgG2a (Southern Biotechnology Associates) in PBS (0.5% milk powder and 0.05% Tween 20) were added and incubated for 2 h. After five washing steps, the amount of bound Ab was determined by addition of substrate solution (1 mM 3,3',5,5' tetramethylbenzidine, 0.3 µl/ml 30% H2O2 in 0.2 M potassium acetate). The reaction was stopped by addition of 2 N H2SO4, and the absorbance at 450 nm was determined with a Vmax-microplate reader (Molecular Devices).

Monoclonal Abs and flow cytometry

Lymphocytes were analyzed using the following mAbs: anti-CD4 allophycocyanin (L3T4), anti-V{alpha}2-TCR biotin and anti-Vbeta5.1/5.2 FITC from BD Pharmingen, and KJ1-26 FITC specific for DO11.10 TCR, anti-CD8{alpha} allophycocyanin, and anti-CD44 PE from Caltag Laboratories. Biotinylated mAbs were detected with streptavidin-CyChrome (Caltag Laboratories). Analytic flow cytometry was performed on a FACSCalibur (BD Biosciences), and the data were analyzed using CellQuest software (BD Biosciences).

In vivo CTL assay

This assay was performed as described before (26). Syngeneic C57BL/6 spleen and lymph node cells were depleted of erythrocytes by osmotic lysis. Cells were washed and split into two populations. One population was pulsed with 10–6 M OVA257–264 peptide for 1 h at 37°C, washed and labeled with a high concentration of CFSE (2.5 µM, CFSEhigh cells). The second control population was labeled with a low concentration of CFSE (0.25 µM, CFSElow cells). For i.v. injection, an equal number of cells from each population (CFSEhigh and CFSElow) was mixed, such that each mouse received a total of 20 x 106 cells. Cells were injected into mice immunized 13 days before. Twenty hours later, mice were sacrificed and spleen and lymph nodes removed. Cell suspensions were analyzed by flow cytometry; ~5 x 105 CFSE+ cells were collected for analysis. Peptide-pulsed and unpulsed target cells were recognized according to their different CFSE intensities. To calculate specific lysis, the following formulas were used: ratio = ((percentage of CFSElow/percentage of CFSEhigh)); percentage of specific lysis = (1 – (ratio unprimed/ratio primed) x 100)).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To investigate whether different expression specificities (DC vs all transfected skin cells) as well as different Ag locations (secreted vs membrane-bound Ag) have an influence on the immune response, we constructed four different expression plasmids: CMV-OVA, CMV-mOVA, CD11c-OVA, and CD11c-mOVA (Fig. 1). The mOVA Ag consists of the first 118 amino acids of the human transferrin receptor fused to amino acids 139–385 of OVA (27). The CD11c promoter was already shown to provide DC-specific transgene expression in several transgenic mouse lines (21, 28, 29).


Figure 1
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  		FIGURE 1. Schematic representation of expression plasmids. A, cDNAs from full-length OVA or membrane-bound OVA (mOVA) truncated OVA139–386 ({blacksquare}) fused to the first 118 amino acid of the human transferrin receptor ({cjs2108}) were cloned into EcoRI sites of pcDNA3-beta-globin and CD11c-beta-globin, respectively (B, BamHI; E, EcoRI; N, NotI; P, PstI; S, SalI; and X, XhoI). BALB/c mice were immunized at days 1 and 14 with CMV-OVA (B), CMV-mOVA (C), CD11c-OVA (D), and CD11c-mOVA (E). OVA-specific Ab serum levels of the isotypes IgG (B and C) and IgG1 (D and E) were determined by ELISA. Mice immunized with OVA-encoding constructs (B and D) and mice immunized with mOVA-encoding constructs (C and E) are shown. Sera were obtained at days 0, 13, and 29 postimmunization and diluted 1/150. Results are expressed as the mean OD at 450 nm (OD450) ± SD (error bars) from five individual mice per group. Immunization with irrelevant vaccines yielded background levels of ≤0.05 (OD450) in this OVA-specific ELISA (data not shown). One of three experiments with similar outcome is shown.

 
Effect of cellular localization and transcriptional targeting on the Ab response

First, we immunized mice by two consecutive administrations of CMV-OVA, CMV-mOVA, CD11c-OVA, or CD11c-mOVA via gene gun. Whereas no OVA-specific IgM titers could be detected in any case (data not shown), all mice showed a significant increase in OVA-specific IgG titers (Fig. 1, B and C). A comparison of kinetics and strengths of the anti-OVA response in the different experimental groups indicates a clear superiority of the secreted form of OVA (Fig. 1B). Irrespective of the promoter, the OVA-specific IgG titers induced by cDNA encoding the secreted form of OVA (Fig. 1B) were between 13- and 20-fold stronger than the titers induced by mOVA encoding cDNA constructs (Fig. 1C) after one single immunization (IgG titer day 13, p < 0.05, using t test). After the second immunization at day 14, this difference decreased to ~1.5-fold (CMV) to 2-fold (CD11c) and was statistically not any more significant (IgG titer day 29, p > 0.05).

Further analyses of the IgG subtypes showed an IgG1-dominated response for all plasmids (Fig. 1, D and E), whereas IgG2a was not detectable (data not shown). Also in this experiment, the differences in IgG1 Ab titers induced by soluble vs membrane bound forms of Ag were statistically significant day 14 postimmunization (p < 0.05, CD11c-OVA vs CD11c-mOVA and p < 0.05, CMV-OVA vs CMV-mOVA) using a t test (Fig. 1, D and E). However, statistical analysis of Ab titers induced by CMV promoter-driven as compared with CD11c promoter-driven plasmids, expressing the same form of Ag (Fig. 1, B and D, soluble or Fig. 1, C and E, membrane-bound), showed no significant difference (p > 0.05 using a t test). Therefore DC-specific expression of Ag was sufficient to elicit Ab titers comparable to plasmids that confer ubiquitous Ag expression. These data indicate that the kinetics and strengths of the humoral immune response are mainly dependent on the cellular localization of Ag (OVA vs mOVA) after gene gun immunization, and only marginally on the expressing cell type (ubiquitous vs DC-specific). In addition, our results confirm previous findings suggesting that the amount of Ag provided by DC transfected with the CD11c constructs was sufficient to allow similar levels of Ab being produced as compared with mice immunized with the ubiquitous CMV promoter (30).

DC-specific DNA vaccines are insufficient to induce complete CD4 T cell responses

To visualize the capacity of the different DNA vaccines to elicit T cell responses, we used adoptive transfer models of CD4 or CD8 T cells derived from mice transgenic for an OVA-specific TCR as previously described (31). First we monitored the expansion TCR transgenic, OVA323–339-specific CD4 T cells (DO11.10 cells) after immunization with the different OVA-encoding vaccines in vivo by flow cytometry (Fig. 2A). Although nonimmunized mice or mice immunized with control vaccines encoding for irrelevant Ag hemagglutinin (HA) contained very few DO11.10 T cells (Fig. 2A and data not shown), the percentage in draining lymph nodes increased significantly upon gene gun vaccination with CMV-OVA (Fig. 2A). This increase in relative percentage (Fig. 2A) corresponded also to elevated total numbers of DO11.10 T cells, which were ~16 times (Fig. 2B, CMV-OVA) or ~8 times (Fig. 2B, CMV-mOVA) higher than those found in mice immunized with a control vaccine (Fig. 2B). In contrast, neither vaccination with CD11c-OVA nor CD11c-mOVA did augment the DO11.10 frequencies (data not shown) or cell numbers (Fig. 2B) significantly above those found in control (CMV-HA)-immunized animals. Simultaneously, we monitored the activation status of DO11.10 T cells with a mAb specific for CD44, a cell surface marker modulated from low levels on naive T cells to high expression on activated T cells (Fig. 2C). Although the CMV-driven construct induced significant up-regulation of CD44 expression on DO11.10 cells (CMV-OVA 77.3%) (Fig. 2C), immunization with CD11c-OVA could not increase CD44 levels on DO11.10 T cells significantly higher than those found in CMV-HA control vaccinated mice (15.8 vs 19.7%) (Fig. 2C). As previously described, the peak of expansion was always observed between days 5 and 7 and then followed by a decline of Ag-specific T cells thereafter (data not shown and Ref.31). However, CD11c-OVA or CD11c–mOVA constructs did neither elicit measurable expansion at this expected time point nor at earlier or later time points (data not shown). These findings indicate that CD11c-driven DNA vaccines are not sufficient to induce Ag-specific activation of DO11.10 T cells.


Figure 2
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  		FIGURE 2. Monitoring of OVA-specific CD4 T cell response in vivo. BALB/c mice received 2.5 x 106 naive DO11.10 cells at day –1 and were immunized with CMV-OVA, CMV-mOVA, CD11c-OVA, and CD11c-mOVA at day 0. A, The frequency of DO11.10 T cells present in the inguinal lymph nodes at day 5 postimmunization was measured by flow cytometry. Cell suspensions were stained with anti-CD4 and KJ1-26 (DO11.10 TCR-specific) as shown in representative dot plots. The mean percentage (± SD) of CD4+ KJ1-26+ DO11.10 T cells among lymphocytes is indicated in each dot plot. B, Graphs show mean total numbers ± SD (error bars) in inguinal lymph nodes of mice immunized with the indicated vaccines. C, Activation status of transgenic DO11.10 T cells was determined 5 days after immunization by staining for CD44 surface expression. Histograms show the mean percentage ± SD (error bars) of DO11.10 cells with a high CD44 expression (n = 3 mice per group). Data from one of three experiments with similar outcome are shown.

 
DC-specific DNA vaccines are insufficient to induce efficient CD8 T cell responses

One of the most important advantages of DNA as compared with protein immunization is its strong induction of CTL responses. To investigate the capacity of the different vaccines to elicit OVA-specific CD8 T cell expansion, we adoptively transferred OVA257–264/MHC class I-Kb-specific, TCR-transgenic CD8 T cells (OT-1 cells) into syngeneic C57BL/6 mice before immunization. The frequency of OT-1 T cells in blood of naive (Fig. 3, A and B) or control vaccinated (Fig. 3b, CMV-HA) animals was, on average, 0.4% of all PBL (Fig. 3A) and did not increase upon immunization with CD11c-OVA or CD11c-mOVA (Fig. 3, A and B). In contrast, gene gun immunization with CMV-OVA or CMV-mOVA led to a 13- and 19-fold increase of OT-1 frequency, respectively (Fig. 3, A and B). As shown before for CD4 T cells (Fig. 2C), we also measured CD44 expression on OT-1 T cells as a marker for activation. Whereas >90% of OT-1 T cells after CMV-OVA and CMV-mOVA immunization showed high CD44 expression, only ~50% of the OT-1 cells in CD11c-cDNA immunized mice up-regulated their CD44 expression (Fig. 3C). However, this modulation of CD44 upon immunization with CD11c-OVA or CD11c–mOVA was not considered to be Ag-specific because similar CD44 modulation was observed in mice immunized with the irrelevant CMV-HA vaccine, indicating a nonspecific reaction induced by DNA vaccines. These results show that one single immunization with a CD11c-controlled expression plasmid is not sufficient to induce an Ag specific CD8 T cell expansion.


Figure 3
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  		FIGURE 3. Monitoring of OVA-specific CD8 T cell response in vivo. C57BL/6 mice received 5 x 106 naive OT-1 T cells at day –1 and were immunized with CMV-HA, CMV-OVA, CMV-mOVA, CD11c-OVA, and CD11c-mOVA at day 0. At the indicated time points the frequency of OT-1 T cells in blood was determined by flow cytometry. PBL were stained with anti-CD8, anti-V{alpha}2, anti-Vbeta5.1/5.2, and anti-CD44. A, Representative dot plots show V{alpha}2 and Vbeta5.1/5.2 stainings of CD8+ PBL. The mean percentage ± SD of OT-1 cells in PBL (gated) 5 days after immunization are indicated. B, The kinetics of OT-1 T cell expansion is shown as percentage ± SD (error bars) of OT-1 cells in total PBL. C, The activation status is shown as mean percentage ± SD of OT-1 cells with a high CD44 surface expression (n = 3 mice per group). Symbols same as shown in B. D, RIP-OVAlow mice received 1 x 106 naive OT-1 T cells at day –1 and were immunized with CMV-OVA, CMV-mOVA, CD11c-OVA, or CD11c-mOVA or used as a control with CMV-HA 1 day later. Mice were monitored for induction of diabetes by measuring glucosuria. The graph shows the average glucose concentration (mM) per time point from three mice per group. One of two experiments with similar outcome is shown. Symbols are the same as shown in B.

 
To test whether the observed CD8 T cell expansion is accompanied by acquisition of CTL effector functions in vivo, we used the RIP-OVAlow mouse model as a functional in vivo readout system. RIP-OVAlow mice express low amounts of OVA in the beta-islet cells of the pancreas, which is ignored by endogenous CD8 T cells (23). Therefore, OT-1 T cells have been transferred into these mice before immunization with CMV-OVA, CMV-mOVA, CD11c-OVA, CD11c-mOVA, or CMV-HA (Fig. 3D). Activated OT-1 cells lyse OVA-expressing islet cells and mice become diabetic due to insulin deficiency. Six days after one single CMV-OVA or CMV-mOVA immunization, all mice showed high glucose concentrations in urine and thus were considered diabetic (Fig. 3D). In contrast, no increased glucose levels could be detected after immunization with CD11c-OVA, CD11c-mOVA, or CMV-HA. These data indicate that gene gun DNA immunization with DC-specific expression constructs is not sufficient to induce effector functions in CD8 T cells.

DC as exclusive APC are insufficient to trigger full CTL responses, when Ag is broadly expressed

The previous data indicate that gene gun-mediated Ag expression by DC is not leading to full T cell responses (Figs. 1–3). Another explanation for why immunizations with the CD11c promoter-driven constructs would not lead to efficient T cell priming could be weak cDNA expression levels and/or different transfection efficiency of DC as compared with CMV promoter-driven constructs. To compare these properties side-by-side, transfection studies of CD11c+ DC would be appropriate because the CD11c promoter is neither active in CD11c– primary cells nor in CD11c– cell lines (Ref.21 and T. Brocker, unpublished observation). However, transfection of GM-CSF-cultured bone marrow-derived CD11c+ DC with plasmid DNA using available nonviral reagents has been unsuccessful (Refs.32, 33, 34 and our unpublished observations). Also plasmid DNA transfection by electroporation in cultured DC showed a low efficiency (<2%) (Ref.34 and our unpublished observation). For these reasons we compared transfection with expression efficacies in primary DC in vivo. Mice were immunized with CD11c- or CMV-driven constructs expressing enhanced GFP (data not shown) or LacZ (30) and the percentage of GFP- or LacZ-expressing DC in draining lymph nodes was determined (30). In both sets of experiments, similar frequencies and expression levels could be determined (30 and data not shown). These results indicated that immunization with CD11c promoter-driven as well as CMV promoter-driven constructs yielded similar levels of transfected DC in vivo, which expressed comparable amounts of Ag.

To exclude more subtle differences such as differential inflammatory capacities or CpG contents being responsible for the observed discrepancies between CMV promoter- and CD11c promoter-driven vaccines, we next used CD11c-MHCI transgenic mice. This transgenic mouse model enables the measurement of the actual contribution of DC to the immune response because only DC are able to present Ag via MHC class I. In this model, the DC-specific CD11c promoter was used in a transgenic approach to reconstitute wild-type levels of MHC class I expression selectively in DC in an otherwise MHC class I-negative environment (24, 25). In these mice, in which only DC could present MHC class I-restricted Ag, peptide immunizations resulted in identical priming and induction of effector functions of adoptively transferred OT-1 T cells in vivo (25). We therefore compared now the expansion and effector functions of OT-1 T cells in these CD11c-MHCI mice with those transferred to wild-type animals upon DNA vaccination with the CMV-OVA constructs. In this experimental setting transfection and Ag production efficacies of the CMV-OVA vaccine are supposedly identical in both types of mice. In immunized wild-type mice all transfected cell types are able to produce OVA and present it via MHC class I. In contrast, although immunization of CD11c-MHCI mice with CMV-OVA will also lead to OVA production in all gene gun-transfected cells, only DC will be able to present the OVASIINFEKL epitope via MHC class I due to DC-selective reconstitution of MHC class I by the MHC class I transgene (24, 25). As described (Fig. 3), we adoptively transferred purified CD8+ OT-1 T cells at day –1 into CD11c-MHCI as well as into a wild-type C57BL/6 control group, immunized the cohorts of mice with the ubiquitously active CMV-OVA vaccine, and monitored expansion of OT-1 T cells in peripheral blood via the CD45.1 marker as shown in Fig. 4A. This analysis revealed a peak of expansion on day 5 in C57BL/6-MHCI and CD11c-MHCI mice, whereas in control-immunized animals no expansion was detected (Fig. 4A). However, the magnitude of expansion was approximately two to three times higher in wild-type mice as compared with CD11c-MHCI mice (Fig. 4A). This result corresponded to a 2- to 3-fold lower absolute OT-1 T cell number found in draining lymph nodes and spleens as compared with the number found in C57BL/6 mice, where all cells could present the DNA vaccine-encoded Ag via MHC class I (Fig. 4B). Also the activation markers CD62 ligand (Fig. 4A) and CD44 (data not shown) on the expanding OT-1 cells were stronger modulated when all cells could present Ag (C57BL/6) as compared with OT-1 cells in the "DC-only" environment (CD11c-MHCI) (Fig. 4A). The percentage of activated CD62Llow OT-1 cells in CD11c-MHCI mice was three to four times lower as compared with wild-type mice (Fig. 4A). In addition, also the quality of the fewer effector cells seemed to be inferior in CD11c-MHCI mice because only 40% of all OT-1 cells were found to produce the effector cytokine IFN-{gamma} as compared with ~70% in C57BL/6 mice (Fig. 4C). Furthermore, the ability of OT-1 cell to lyse target cells was reduced, when they were activated by DC-only; an in vivo killer assay (Fig. 4D, left) revealed an ~30% reduced capacity to specifically lyse target cells presenting the OT-1-specific peptide-MHC combination. This reduced capacity to activate OT-1 T cells into effector cells was not due to intrinsic properties of the transgenic CD11c-MHCI mouse model because immunization with SIINFEKL peptide in CFA (s.c.) revealed identical capacities to activate OT-1 cells as compared with wild-type mice (25 and data not shown). Therefore, DC alone are insufficient to activate a full blown CTL response upon gene gun DNA vaccination.


Figure 4
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  		FIGURE 4. DC are insufficient to prime full CTL responses upon gene gun DNA vaccination. A total of 2 x 106 OT-1 Ly5.1 cells were transferred on day –1 into wild-type C57BL/6 mice or CD11c-MHCI transgenic mice expressing MHC class I selectively on DC. At day 0, mice were gene gun immunized with CMV-OVA, whereas control mice were left untreated. A, Expansion of OT-1 cells and modulation of activation marker CD62 ligand were analyzed from PBL. *, p < 0.05 for day 5 C57BL/6 or CD11c-MHCI mice vs day 5 control group using a Student’s t test; **, p < 0.05 for CD11c-MHCI mice vs C57BL/6 mice on day 5 using a Student’s t test. B, On day 5, spleens were removed. Absolute numbers of OT-1 cells in spleen were calculated and spleen cells were restimulated in vitro with SIINFEKL peptide in the presence of GolgiPlug. C, Four hours later, cells were fixed, and intracellular IFN-{gamma} staining was performed. D, Mice were treated as described in A and they received at day 13 a 1:1 mixture of SIINFEKL-pulsed (CFSEhigh) and unpulsed (CFSElow) spleen cells. At 14 h later, spleens were removed, and remaining CFSE+ cells were analyzed (left) by flow cytometry. Percentage of specific lysis of peptide-pulsed cells (right) was calculated as described in Materials and Methods. All data are displayed as an average ± SD from groups of n = 4–5 mice. One experiment from two with similar outcome is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite promising results obtained in animal models, the usefulness of DNA vaccination in human application remains uncertain and vaccination efficacy needs to be improved. Because DC play pivotal roles in inducing immunity (35), one strategy to ameliorate vaccine efficacy is targeting Ag expression to DC as potent immunostimulatory APC. The general principle of DC targeting has been proven to efficiently enhance immunity in vivo, when immunizations were performed with protein-Ag coupled to DC-specific Abs (36, 37).

However, the data generated in our study argue against the success of a genetic transcriptional DC-targeting approach. Limiting Ag expression to cells with such a rare frequency was insufficient to trigger efficient CD4 or CD8 T cell responses as measured with highly sensitive adoptive transfer methods. Although we could reproduce the previously reported (38) clonal expansion kinetics and magnitude of DO11.10 T cells upon gene gun immunization with CMV promoter-driven constructs, all CD11c-driven vaccines failed to do so. In contrast, CD11c-driven constructs could elicit Ab responses comparable to those observed with ubiquitously active CMV promoter constructs (Fig. 1). Using membrane-bound vs soluble forms of OVA expressed from the ubiquitous CMV promoter, we confirmed previous studies demonstrating that secreted Ag was more potent in inducing humoral responses and activation of CD4 T cells (Figs. 1 and 2), whereas CD8 T cell activation was more pronounced in the case of cell-associated Ag (Fig. 3) (16). However, none of those antigenic OVA forms would trigger T cells via MHC class II or class I, when expressed directly and selectively in DC (Figs. 2 and 3). The fact that humoral responses induced by transcriptional targeting of DC were comparable to those triggered by the ubiquitous CMV construct (Fig. 1) argues that a certain amount of CD4 T cell priming must have occurred and therefore a weak Th cell response might have been initiated by the DC-specific construct. In contrast, when adoptively transferred DO11.10 CD4 T cells were used, no significant Ag-specific expansion could be measured. These findings are in line with those of a previous published study, in which upon DNA vaccination via the i.m. route no significant clonal expansion was observed (16). Yet, the authors have shown that a very low frequency of DO11.10 cells underwent up to five cell divisions (16). Eventually these few dividing cells were sufficient to provide help for B cells and allow class switches.

A recent study by Itano et al. (39) suggested that after gene gun immunization, soluble unprocessed Ag drains to lymph nodes, where resident DC take it up and activate naive CD4 T cells. A second wave of Ag reaches the lymph nodes later, by an influx of transfected Ag-presenting DC from the skin. This second wave of dermal DC was required for a complete CD4 T cell differentiation to take place (39). Membrane-bound Ag, in contrast, is not flushed into lymph nodes by the lymphatic system directly after production. The first step of CD4 T cell activation by resident DC could therefore be abrogated or at least delayed. This could explain why anti-OVA Abs were detected already 2 wk after the first immunization with CMV-OVA or CD11c-OVA but not with the membrane-bound Ag constructs CMV-mOVA or CD11c-mOVA (Fig. 1).

Our observations that DC-restricted expression of Ag is insufficient to prime CD8 T cells supports a hypothesis obtained also with the keratinocyte-specific K14 promoter (13). In the study, the authors showed that an APC-specific (CD11b) promoter was weaker in inducing T cell responses as compared with keratinocyte-specific K14 promoter constructs. As a conclusion the authors postulated that in addition to DC, non-DC have to produce Ag, which subsequently is efficiently cross-presented by DC (13). Our data in CD11c-MHCI mice contradict this finding and argue rather for an active participation of non-DC in Ag presentation rather than just producing Ag (Fig. 4).

Our data also contradict results from vaccination experiments performed with the Langerhans-specific fascin promoter (18). However, different experimental set-ups for measuring CD8 T cell responses as well as different Ag expression patterns and strengths could explain the discrepancies. In those experiments proliferation and CTL activity were determined under in vitro restimulation conditions (18). We do not exclude that DC transfected with a CD11c construct would be able to induce proliferation and/or CTL activity of cocultured T cells in vitro, but our data clearly show that this does not reflect the situation in vivo (Figs. 2 and 3). In addition, although the CD11c promoter is constitutively active on DC, the stronger fascin promoter is up-regulated on maturing Langerhans cells. Yet, Allan et al. (40) showed for viral infections that Ag-specific CD8 T cells are not primed by infected Langerhans cells directly, but rather by lymph node resident CD8+ DC, which acquire exogenous Ag and subsequently cross-present MHC class I epitopes. Because both viral infections and DNA immunization are based on intracellular Ag production, similar ways of T cell priming can be expected. Thus, even if transfected migratory DC from the skin can be found in draining lymph nodes of gene gun-immunized mice (11), they are probably not directly responsible for CD8 T cell priming, but would have to pass "their" Ag to CD8+ resident DC. In regard of the low number of transfected DC after gene gun immunization (12) and their short life span, only very little Ag would be expected to reach the lymph nodes in a DC-restricted immunization strategy. This is the case in CD11c-OVA or CD11c-mOVA immunized mice, in which Ag-specific T cells did not become activated and did not expand, proliferate, or acquire effector functions (Figs. 2 and 3). Previous studies have already postulated that a broad Ag expression is necessary for efficient CD4 and CD8 T cell responses and that Ag uptake and Ag presentation via class I or class II are important mechanisms for priming immunity. However, the use of CD11c-MHCI mice, in which only DC can present Ag via MHC class I, demonstrates that 1) broad Ag expression via the CMV promoter and 2) availability of MHC class I+ DC are not sufficient to reach full CTL responses as compared with normal mice (Fig. 4). Although our experiments show that DC are sufficient to prime CD8 T cells upon gene gun vaccination, the few primed CD8 T cells expand only to suboptimal numbers, possess blunted cytolytic efficacy (Fig. 4D), and display less production of the effector cytokine IFN-{gamma} (Fig. 4C). As a conclusion from our experiment and those of other groups showing that cross-priming is the major mechanism of CTL-induction upon gene gun vaccination (13), one would have to postulate that other cell types in addition to DC have to (cross-) present Ag for induction of optimal T cell responses. In addition to DC as potent inducers of T cell immunity, B cells have also been shown to induce Ag-specific expansion of naive T cells in vivo, when they were appropriately activated and did express costimulatory molecules such as CD86 and CD40 (41, 42). When mice were immunized with protein Ag linked to CpG-DNA, B cells were shown to cross-present and cross–prime naive OVA-specific CD8 T cells (43). Therefore, other APC in addition to DC might be necessary to trigger efficient T cell responses upon gene gun vaccination. Recent studies with influenza virus infection demonstrated that differential presentation of viral epitopes by DC as compared with those presented by DC and non-DC induced differentially efficient immune responses (44). Only when also non-DC participated in the immune response, efficient primary as well as secondary CTL responses were detectable (44). Eventually DC are responsible for the initial priming of T cells upon gene gun vaccination, but to gain sustained effector function and expand, other cells (such as B cells) must present Ag in addition. However, further studies are necessary to evaluate the roles of non-DC during DNA vaccination, to identify the "other" APC type, and to decipher whether such priming by DC plus non-DC is one synchronized event or several subsequent events.


   Acknowledgments
 
We thank A. Steinkasserer and M. Lutz for helpful discussions, and A. Bol and W. Mertl for expert animal care.


   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 the Deutsche Forschungsgemeinschaft SFB571-B5, SFB456-B12, Wilhelm-Sander-Stiftung, and ForPrion. Back

2 Current address: Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037. Back

3 Address correspondence and reprint requests to Dr. Thomas Brocker, Institute for Immunology, Ludwig-Maximilians-Universität München, Goethestrasse 31, 80331 München, Germany. E-mail address: tbrocker{at}med.uni-muenchen.de Back

4 Abbreviations used in this paper: DC, dendritic cell; RIP, rat insulin promoter; HA, hemagglutinin. Back

Received for publication March 31, 2005. Accepted for publication January 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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