Dendritic cells at a DNA vaccination site express the encoded influenza nucleoprotein and prime MHC class I-restricted cytolytic lymphocytes upon adoptive transfer

Adrian Bot1,4, Alexandru C. Stan1,5, Kayo Inaba2, Ralph Steinman3 and Constantin Bona1

1 Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA
2 Laboratory of Immunology, Department of Zoology, Faculty of Science, Kyoto University, Kyoto, Japan
3 Laboratory of Cell Biology and Immunology, Rockefeller University, New York, NY 10021, USA

Correspondence to: A. Bot, Department of Exploratory Biological Research, Alliance Pharmaceutical Corp., 6175 Lusk Boulevard, San Diego, CA 92121, USA


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Intradermal inoculation of plasmids expressing antigens that contain MHC class I-restricted epitopes leads to the induction of specific CD8+ cytotoxic T lymphocytes (CTL). The role of in situ transfected antigen-presenting cells (APC) in the priming of specific CTL subsequent to intradermal DNA immunization was investigated using a plasmid (NPV1) expressing the nucleoprotein (NP) of influenza virus that contains a nuclear targeting signal and a dominant class I/Kd-restricted epitope. Inoculation of NPV1 leads to in situ transfection of MHC class II+ and class II cells, as revealed by the nuclear localization of NP. Between 2 and 3% of MHC class II+ and class II cells with the ability to migrate out of the epidermis expressed NP. Upon adoptive transfer into naive recipients, class II+ migratory cells recovered from the area inoculated with NP-expressing plasmid were significantly superior regarding the ability to prime virus-specific CTL as compared to MHC class II cells. Together, these results are consistent with the role of local dendritic cells loaded with antigen in the priming of CTL by intradermal DNA immunization.

Keywords: adoptive transfer, cytotoxic cells, dendritic cells, DNA vaccination, nucleoprotein


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mechanisms responsible for the generation of MHC class I-restricted immune responses by DNA vaccination are still poorly understood. Since earlier studies found that primarily non-professional antigen-presenting cells (APC) in the muscle (1) or skin (2) express inoculated DNA, it was not clear how the naive T cells were turned on. More recently, it was shown that the cytotoxic T lymphocyte (CTL) immune response generated by DNA vaccines is restricted to the MHC molecules on bone marrow-derived professional APC (35). At this point, direct transfection of professional APC (6) or antigen transfer between in situ transfected non-professional APC and professional APC (7) were suggested as mechanisms for CTL induction upon DNA vaccination. Direct transfection of professional APC may occur in situ or at remote sites from injection. Whereas direct transfection of professional APC facilitates the use of the conventional Tap-dependent MHC class I presentation pathway, the antigen-transfer mechanism implies non-conventional pathways of antigen processing and presentation in the context of class I molecules.

Among the professional APC, the DC are thought to play a critical role in the priming of MHC class I- and II-restricted T cells (8). The Langerhans cells, that are immature dendritic cells (DC), predominantly populate the dermis. They have the ability to rapidly pick up and process antigens as well as to produce soluble pro-inflammatory mediators subsequent to stimulation by a broad range of factors. However, they have reduced ability to prime specific T cells due to decreased expression of MHC molecules and co-stimulatory factors. Stimulation of Langerhans cells induces the migration to local lymph nodes simultaneously with maturation and up-regulation of MHC and co-stimulatory molecules. In the lymph nodes, maturated DC have the ability to activate specific T cells into effector and memory cells (8).

It is not clear to what extent DC that migrate from the area of DNA inoculation are responsible for the induction of MHC class I-restricted immunity upon intradermal DNA vaccination. A possibility would be that the plasmid is rapidly disseminated throughout the body via lymph and blood (9), and reaches remote areas like lymph nodes were it is picked up and expressed by resident APC. Another possibility is that non-DC rather than DC in situ transfected or loaded with antigen either carry the antigen to or effectively prime immune responses in the lymph nodes. We provide evidence that upon intradermal plasmid injection, both DC and non-DC are in situ transfected and migrate out of the inoculation area. However, the DC loaded with antigen are significantly more effective as compared to non-DC relative to the priming of naive MHC class I-restricted T cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
BALB/c H-2d mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed at the Mount Sinai Animal Core Facility according to federal and local regulations.

Plasmid, peptides and viruses
NPV1 plasmid was constructed at the Merck Laboratories (West Point, PA) by inserting the open reading frame of the nucleoprotein (NP) gene of a A/PR8/34 virus into the BglII site of a mutant pBR322 plasmid, containing 1.96 kb of enhancer, promoter and intron A of the immediate early gene of cytomegalovirus. The control plasmid (CP) was obtained by excising the NP open reading frame from NPV1 plasmid. The plasmids were propagated in Escherichia coli and purified by the alkaline lysis method on resin columns (Qiagen, Valencia, CA). The DNA was ethanol precipitated and resuspended in 0.9% saline to a concentration of 30 µg/100 µl for immunization.

The synthetic peptide NP 147–155 with the sequence TYQRTRALV, the dominant MHC class I-restricted epitope for H-2d, was synthesized in the Protein Core Facility of Mount Sinai School of Medicine.

Influenza virus strains A/PR8/34 (H1N1) and B/Lee/40 were grown in the allantoic cavity of 10 day embryonated hen eggs (Spafas, Norwich, CT). Allantoic fluids were harvested 48 h later and stored at –80°C.

Recovery and separation of DC
DC were obtained from mice injected intradermally 4 times at 12 h interval into the dorsal side of each ear with 30 µg NPV1 plasmid DNA or CP, or with 5 µg purified influenza virus. One hour after the last injection, the mice were sacrificed and sheets of ear skin placed into tissue culture for 1–4 days. The RPMI/10% FCS culture medium was supplemented with Amphotericin B according to the manufacturer's instructions (Gibco, BRL, Grand Island, NY). DC are the major cell type of cells that migrate into the culture medium from the skin explants, displaying intense staining for MHC class II antigens, CD11/CD18, ICAM-1, B7/BB1, CD40, and lacking macrophage, lymphocyte and endothelium markers (10,11). The emigrants are termed `crawl-out' cells, and consist of MHC II+ and MHC II cells. In some experiments, the cells that migrated out during the first 24 h were discarded since the percentage of MHC class II+ cells was around or below 20%, due to the presence of blood-derived lymphocytes. At later time points (24–72 h), the percentage of class II+ cells was ~50–70% but the total yields were lower. Since the yields were significantly higher, sorting experiments were carried out with all cell subsets (0–72 h). Using FACS, we sorted MHC II+ and MHC II crawl-out cells after staining with 1:10 rat anti-MHC II (I-Ad) antibody conjugated with FITC (Biosource International, Camarillo, CA) for 30 min on ice, in the presence of PBS/1% BSA sterile buffer. The cells were washed twice and resuspended in PBS for adoptive transfer.

Immunostaining of DC
Ears from immunized animals were obtained as above and cryoembeded in HistoPrep (Fisher Scientific, Springfield, NJ). Serial 7 µm sections were fixed in 1:1 methanol:acetone (Fisher Scientific) for 10 min at –20°C and air dried. Before staining, sections were rehydrated in 1% BSA/PBS (Sigma, St Louis, MO) for 15 min, and incubated for 1 h at room temperature in a moist chamber with 1:600 polyclonal rabbit anti-NP (kindly donated by Dr Peter Palese from Mount Sinai School of Medicine, New York) and 1:5 rat anti-MHC II mAb (Biosource International). Controls were non-immune rabbit serum and rat IgG. Samples from non-immunized mice were used as additional negative controls for the expression of NP. The sections were washed in 1% BSA/PBS and incubated for 30 min at room temperature with labeled secondary antibody, i.e. DTAF–(Fab')2 goat anti-rabbit IgG and Cy3–(Fab')2 donkey anti-rat IgG, according to the manufacturer's instructions (Jackson ImmunoResearch, West Grove, PA). After washing, the nuclei were counterstained for 30 min at room temperature with 1 µg/ml DAPI (Sigma). Sections were examined with a Zeiss Axiophot microscope.

Skin crawl-out cells were prepared for immunolabeling after 1 day of culture. The ear sheets were transferred to 12-well plates (Fisher Scientific) containing 12 mm cross-diameter round glass coverslips previously coated with 1% gelatin/PBS (Sigma), to which the crawl-out cells attached. The latter were removed after another day, washed, fixed and stained with antibodies to NP and MHC II, and counterstained with DAPI as described above.

PCR analysis
PCR was used as previously described (12) to detect the plasmid DNA in crawl-out cells. We have used primers that amplify the NP insert (12). The crawl-out cells were washed 3 times in large excess medium before the lysis. The PCR products were identified by the restriction digestion patterns with AcsI and MlvII enzymes (Boehringer Mannheim, Indianapolis, IN).

Adoptive transfer protocol
Due to the limited number of cells that can be isolated from mouse ears, crawl-out cells (unsorted or sorted MHC II+ and MHC II cells) were injected directly in graded doses into spleens of naive BALB/c mice. The mice were anesthetized with a mixture of Rompun (Bayer, Pittsburg, PA) and Ketanest (Fort Dodge Laboratories, Fort Dodge, IO), the upper abdominal skin was incised and cells resuspended in 100 µl of saline were injected into the spleen through the peritoneum. The spleen was immobilized during injection by gentle compression toward the posterior wall of the abdominal cavity. The mice were sacrificed 7 days later, and single-cell suspensions were prepared and used for precursor CTL (pCTL) frequency estimation and secondary cytotoxic assays.

CTL assay and estimation of pCTL frequency
The CTL assay and pCTL estimation were carried out according to published protocols (13). Briefly, the secondary cultures were carried out by incubating various numbers of splenocytes from test animals with irradiated, PR8 virus-infected splenocytes from naive BALB/c mice for 5 days in RPMI supplemented with 10% FCS and 50 µM 2-mercaptoethanol. Different ratios of responder:stimulator (R:S) cells were employed and the protocol was modified so that the secondary expansion of specific CTL would be part of the read-out. Namely, a constant number (104 cells/well) of 51Cr-labeled, virusinfected or non-infected P815 target cells (MHC I+/MHC II) was added to the responder cells cultured with stimulator cells in 96-well flat-bottom plates. The plates were centrifuged at 5 h and the radioactivity in the supernatant was measured using a {gamma}-counter. The results were expressed as percentage of specific lysis after the subtraction of the background, at different R:S ratios. We have used three animals per group. Statistical analysis to ascertain the significance of the difference in cytotoxicity among various groups was carried out by Student's t-test. The stimulator or target cells were infected with live PR8 virus in DMEM/1% BSA at 37°C for 1 h, at a m.o.i. = 10.

A variant of the assay described above was used for the estimation of pCTL frequency. Responder cells were incubated in 96-well flat-bottom plates with stimulator cells at various R:S ratios. For a given R:S ratio, 20 replicates comprising virus-infected stimulator cells and four replicates comprising non-infected stimulator cells were employed. After 5 days, the effector cells were transferred into plates containing 5x103 virus-infected, 51Cr-loaded P815 target cells per well. After 5 h of incubation, the percent release was measured for all wells. The wells exhibiting 51Cr release exceeding background + 3 SD, were considered positive. The pCTL frequency was estimated by linear regression of ln(% negative cultures at certain dilution of responders) versus the number of responder cells per well, as previously described (13). The total numbers of splenic pCTL per mouse were estimated by taking into consideration the numbers of splenocytes recovered (the splenocytes were pooled from three mice per group).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In vivo transfection of dermal cells after DNA immunization
The uptake of plasmid by dermal cells was studied by PCR and immunohistochemistry. First, we investigated if cells with the ability to migrate from the inoculation site took up the plasmid. PCR analysis of extensively washed crawl-out cells demonstrated that dermal cells were transfected with NPV1 plasmid (Fig. 1Go). The identity of the plasmid was documented by the size of the amplified insert (711 bp; Fig. 1AGo) as well as by digestion of the PCR products with AcsI and MlvII which resulted in fragments with the expected size (Fig. 1BGo). Interestingly, the data shown in Fig. 1Go(A, lane 7) suggest that >1 in 8x102 crawl-out cells picked up the plasmid.



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Fig. 1. Detection of NPV1 plasmid in crawl-out dermal cells from DNA immunized mice. DNA extracted from crawl-out cells was amplified by PCR using primers specific for the NP insert. (A) PCR of plasmid DNA extracted from 105 (lane 4), 2x104 (lane 5), 4x103 (lane 6) and 8x102 (lane 7) crawl-out cells obtained by tissue culture of epidermal layers previously inoculated in vivo with NPV1 plasmid. The specificity of the primers was tested with positive control (NPV1 plasmid, lane 2) and negative control (CP, lane 3). Lane 1: 100 bp ladder. (B) The characterization of the PCR products by digestion with restriction enzymes. The PCR product of plasmid DNA extracted from crawl-out cells and amplified with NP primers was digested with MlvII (lane 5), AcsI (lane 7) or run undigested (lane 3). As controls, we ran PCR products of NPV1 undigested (lane 2), or digested with MlvII (lane 4) or AcsI (lane 6). Lane 1: 100 bp ladder.

 
The analysis of ear sections by immunohistochemistry using reagents for the viral NP and MHC class II molecules, showed the presence of class II+ cells that express NP. We detected the presence of rare MHC class II+ dermal cells (Fig. 2b and cGo) with or without co-expression of NP (Fig. 2a and cGo). The majority of class II+ dermal cells were negative for NP expression. The ears harvested from non-immunized mice contained only MHC class II+ and MHC class II (Fig. 2d and eGo) devoid of NP expression (data not shown). Specificity controls including isotype-matched antibodies entailed negative staining (Fig. 2fGo and data not shown).



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Fig. 2. Identification of in vivo transfected cells in cryostat sections. Ear sections obtained from NPV1 plasmid immunized (a, b, c and f) or non-immunized (d and e) mice were simultaneously stained for anti-NP and anti-MHC class II primary antibodies followed by fluorescently labeled secondary antibodies (green in the case of NP and red in the case of MHC II). The nuclei were counterstained with DAPI. (a) Staining of NP. (b and d) Staining of MHC class II. (c and e) Staining of nuclei. (f) Simultaneous staining of nuclei and isotype control staining for NP and class II in an NPV1 immunized mouse; the primary antibodies were replaced in this case with rat IgG. No significant background was noted. Downwards pointing arrowheads indicate an NP+/MHC II+ cells, whereas upwards pointing arrowheads indicate an NP/MHC II+ cell. Magnification x120.

 
The expression of NP was confirmed in crawl-out cells captured on cover slips. The crawl-out cells were triple stained for MHC class II molecules (DC primarily), the NP protein using specific antibodies and for nuclei using DAPI. As shown in Fig. 3Go(a, b and d, composite of different fields), the NP (green) is most often present in class II+ cells (red) within the nucleus, or nucleus and cytoplasm. Only rarely, we have detected clear-cut cytoplasmic staining of NP (Fig. 3cGo). However, the majority of the MHC class II+ and class II crawl-out cells did not stain for NP. A rough estimation of the number of class II+ cells expressing NP indicates that ~2–3% were transfected (Table 1Go). A similar percentage of class II crawl-out cells expressed NP. Overall, ~25–50% of the crawl-out cells expressed MHC class II molecules, varying from animal to animal. Thus, the PCR signal amplified from 8x102 crawl-out cells (Fig. 1Go, lane 7) corresponded to ~30–50 transfected cells.



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Fig. 3. Identification of transfected cells in crawl-out preparations from ears of mice intradermally injected with NPV1 plasmid. The cells were simultaneously stained for nuclei (DAPI, upper row) and immunostained for NP (middle row, green) and MHC class II (bottom row, red). (a) In a group of three class II+ cells only one shows the presence of NP in the nucleus. (b) A class II+ cell exhibiting nuclear staining for NP. (c) A class II+ cell exhibiting cytoplasmic staining for NP. (d) An NP+/MHC II cell. The figure represents a composite of cells from various microscopic fields. Magnification x1600.

 

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Table 1. The frequency of crawl-out cells expressing NP in the nucleus
 
Utilization of NPV1 enabled us to precisely determine that the cells expressed viral NP rather than taking up NP secreted by neighboring cells. While the internalized protein is expected to localize in the endosomal/lysosomal compartment, the endogenously synthesized NP localizes into the nuclei and is later exported to the cytoplasm (14). The nuclear staining shown in Fig. 3Go strongly suggested endogenous expression of NP in DC and non-DC of dermal origin.

The ability of crawl-out cells to prime virus-specific CTL after intrasplenic adoptive transfer
The capacity of crawl-out cells to prime NP-specific CTL was assessed by adoptive transfer into spleens of naive BALB/c mice. This approach allowed us to circumvent the low number of crawl-out cells usually obtained from mouse ear explants. The priming of specific CTL was assessed at day 7 after transfer, by measuring secondary cytotoxicity and by estimating the pCTL frequency against target cells that express MHC class I but not class II molecules. As a positive control, crawl-out cells were obtained from mice inoculated with PR8 influenza virus. As negative controls, we have used crawl-out cells harvested from mice inoculated with B/Lee (type B) virus or control plasmid (CP). The data depicted in Fig. 4Go(a) show that while the animals that received crawl-out cells from mice inoculated with B/Lee or control plasmid did not display significant cytotoxicity, the effector cells from animals that received crawl-out cells from mice inoculated with either PR8 or NPV1 lysed the target cells. A comparable level of cytotoxicity was triggered when ~2x105 crawl-out cells from PR8- or NPV1-injected mice were transferred (Fig. 4aGo).



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Fig. 4. Secondary cytotoxic activity of splenocytes from mice injected with unsorted (A), or sorted class II+ (B) or class II (C) crawl-out cells. Seven days after adoptive transfer of crawl-out cells into spleen of naive BALB/c mice, the splenocytes harvested from host animals were in vitro stimulated with virus-infected APC. The cytotoxic activity was measured at various R:S ratios against target cells infected or not infected with influenza virus. The results were expressed as mean ± SE of specific lysis from three animals per group. (A) Recipient mice injected with unseparated crawl-out cells from animals inoculated with PR8 virus, B/Lee virus, NPV1 plasmid and CP. (B) Recipient mice injected with class II+ crawl-out cells from NPV1 immunized mice. (C) Recipient mice injected with class II crawl-out cells from NPV1 immunized mice. The values that are significantly above the negative control (B/Lee virus) in Fig. 1Go(A) are tagged with an asterisk (P < 0.05 by t-test). The number of transferred crawl-out cells is represented in the boxes.

 
In another set of experiments, the crawl-out cells from mice injected with NPV1 plasmid were sorted into class II+ and class II cells, and then adoptively transferred into naive BALB/c mice. Induction of virus-specific, class I-restricted CTL was measured after secondary stimulation. A comparison of the results in Fig. 4Go(b and c), showed that significant CTL activity was triggered by injection of 6x104 MHC class II+ cells, but not by similar numbers of class II crawl-out cells. However, significant CTL activity induced by class II crawl-out cells was observed after transfer of 8x105 cells at 1:1 and 1:2 E:T ratios, and of 1.6x105 crawl-out cells at 1:1 E:T ratio (Fig. 4cGo). Since similar percentages of class II+ and class II crawl-out cells expressed NP (Table 1Go), these data suggested that class II+ cells were more effective in priming specific CTL.

The induction of CTL observed after adoptive transfer of APC that migrated out of the inoculation site was further documented by estimating the frequency of class I-restricted, PR8-specific CTL precursors. The data in Table 2Go show no detectable pCTL in mice inoculated with crawl-out cells harvested from animals injected with CP or B/Lee. In contrast, animals adoptively transferred with crawl-out cells from BALB/c mice injected with NPV1 plasmid or PR8 virus displayed comparable pCTL frequencies. Furthermore, 3 day crawl-out cells were more effective in priming specific CTL, as compared to 1 day crawl-out cells obtained from ears inoculated with NPV1 (Table 2Go). Consistent results were obtained when the frequency of virus-specific pCTL was estimated after adoptive transfer of MHC class II+ or class II crawl-out cells (Table 3Go). Based on the total numbers of specific pCTL generated, the MHC class II+ crawl-out cells recovered from the site of NPV1 inoculation were more effective in inducing specific cytotoxicity as compared to the class II counterparts.


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Table 2. CTL response of mice injected with crawl-out cells obtained from mice inoculated with plasmids or influenza viruses
 

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Table 3. Priming of MHC class I-restricted CTL by intrasplenic injection of in vivo transfected APC from mice immunized with pNP plasmid
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have studied the role of Langerhans cells in the generation of CTL immunity by intradermal DNA immunization with a plasmid expressing NP of influenza virus. The presence of a nuclear targeting sequence on NP (14) allowed us to pinpoint in situ transfected cells by immunohistochemistry.

Most of the NP+ cells displayed a characteristic nuclear staining, that is consistent with cytoplasmic synthesis followed by nuclear targeting rather than uptake via pinocytosis, endocytosis or phagocytosis. This argues against mere carry-over of the plasmid by migratory cells, an alternative interpretation of the data presented in the Fig. 1Go. Such positive cells were MHC class II+ or class II as shown by immunohistochemistry of frozen tissue harvested from the site of inoculation, using an I-A-specific mAb. By employing tissue cultures, we show that 2–3% of either I-A+ or I-A cells that migrate out of the epidermal layer within 3 days after DNA inoculation, were positive for NP. Thus, besides Langerhans cells that differentiate to class II+ DC, other types of cells take up the plasmid and express the antigen subsequent to intradermal DNA immunization. Such cells might be fibroblasts, keratinocytes or MHC class II leukocytes, but their ability to reach the local lymph nodes in order to prime T cell responses may be limited.

Purification of MHC class II+ and class II crawl-out cells from the site of inoculation revealed significant differences in their ability to prime CTL responses upon adoptive transfer. Thus, intrasplenic infusion of 6x104 class II+ cells that migrated out of the dermal layer within 3 days after DNA immunization induced significant priming of virus-specific CTL. In contrast, the adoptive transfer of similar numbers of class II migratory cells did not result in significant induction of CTL. However, increased numbers of class II cells were able to prime CTL against NP-expressing influenza virus. According to Table 3Go, 1.6x105 class II APC induced 1.9x105 pCTL. Further, 0.6x105 class II+ APC induced 4.9x105 pCTL. Thus, for the induction of the same number of pCTL (i.e. same level of immunity) one needs an excess of [1.6/1.9]/[0.6/4.9] of class II APC over class II+ APC, which is ~7-fold. Based on this estimation and on the similar percentage of class II and class II+ cells that are transfected with NPV1, it shows that class II+ cells that migrated out of the site of DNA inoculation were more effective in priming CTL activity as compared to class II cells. Direct stimulation of host T cells by infused APC rather than antigen transfer to host APC is indirectly but strongly suggested by the discrepancy in the effectiveness of class II+ and class II APC to induce CTL immunity upon adoptive transfer. In case of antigen transfer we would have expected similar efficiencies of class II+ and class II transfected cells in inducing CTL activity. However, we cannot rule out at this point preferential in situ loading of MHC class II+ APC by antigen released from somatic cells transfected with plasmid. It would be difficult to estimate the number of such cells in the absence of a probe for MHC class I–peptide complex, since most of the internalized antigen might be in a state that precludes staining with NP-specific antibody. The observation that migrant cells harvested between 48 and 72 h after DNA inoculation are more effective in priming CTL compared to day 1 cells supports this hypothesis. Alternatively or in addition, this observation may be explained by an increased frequency of DC that emigrate after 24 h from the skin.

We adopted the strategy of intrasplenic adoptive transfer for two reasons: (i) it allowed us to ascertain the ability of the APC to prime naive CTL precursors and (ii) the intrasplenic infusion limited the dissemination of APC in various organs as is the case of i.v. inoculation. We observed that I-A crawl-out cells were able to prime specific CTL. The activation of specific CTL precursors by class II cells upon intrasplenic transfer can be facilitated by a bystander co-stimulation provided by resident splenic APC. Such mechanism of three-cell cooperation was previously suggested (15). However, the relevance of this observation in the case of DNA immunization may be limited due to the fact that MHC class II+ DC rather than other subsets are thought to migrate to local lymph nodes (8). In contrast, class II transfected cells may be important for re-stimulation of primed T cells that migrate in the periphery or as reservoir of antigen that is transferred to Langerhans cells. Interestingly, in spite of the lack of an ER translocation signal on NP, somatic cells transfected with NP plasmid may release antigen or peptides in an immunogenic form (7), possibly complexed with heat shock proteins (16).

Initial studies carried out with bone marrow chimeras revealed a critical role for bone marrow-derived professional APC in the generation of MHC class I-restricted immunity subsequent to DNA immunization (35). The main question that emerged was if directly transfected APC or, alternatively, APC that internalize the antigen released by in situ transfected somatic cells are responsible for CTL priming. Earlier and more recent studies supported both mechanisms. Thus, in situ transfected DC able to migrate into the local lymph nodes were previously defined (6,17,18). Such cells were able to activate specific T cells (1719). More recently, in vivo co-transfection with antigen and a membrane marker that allowed depletion of transfected cells pinpointed the role of DC in the induction of CTL immunity by taking up the plasmid after DNA immunization (20). In contrast, studies comprising transplantation of transfected myoblasts (7) together with bone marrow chimeric mice generated subsequent to DNA inoculation (4) suggested that antigen transfer between somatic and bone marrow-derived APC may be important for CTL priming.

A few factors may be responsible for understanding these discrepancies: the heterogeneity of the models employed regarding route and dose of vaccination; type of antigen, particularly the presence or absence of targeting signals; and the co-existence to a certain degree of both mechanisms.

The results of our study are consistent with the role of in situ transfected Langerhans cells as a critical factor in the induction of MHC class I-restricted CTL subsequent to DNA vaccination and extended early observations showing that DC can directly activate CD8+ T cells (21). The following findings support our conclusion: the presence of transfected cells with the ability to migrate out of the inoculation site, the expression of MHC class II by a significant fraction of in vivo transfected cells and the enhanced ability of class II+ crawl-out cells to prime CTL immunity upon adoptive transfer. Since the MHC class II marker is preferentially expressed by DC that migrate out of skin (8,10,11), our results are consistent with a predominant role for antigen-loaded DC that migrate from the site of injection to the local lymph nodes, in the generation of CTL immunity upon intradermal DNA vaccination.


    Acknowledgments
 
We are thankful for the technical help with cell sorting to Ms George Italas (Mount Sinai School of Medicine, New York). This work was supported by a contract from Alliance Pharmaceutical Corp. (San Diego, CA) to C. B., from the grant AI13013 to R. S. and by the Yamanouchi Fund to K. I.


    Abbreviations
 
APC antigen-presenting cells
CTL cytotoxic T lymphocytes
CP control plasmid
DC dendritic cell
NP nucleoprotein
R:S responder:stimulator
pCTL CTL precursors

    Notes
 
4 Present address: Alliance Pharmaceutical Corp., San Diego, CA 92121, USA Back

5 Present address: Institute of Neuropathology, Hannover Medical School, 30625 Hannover, Germany Back

Transmitting editor: Z. Ovary

Received 21 July 1999, accepted 14 February 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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