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
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Abstract |
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Keywords: adoptive transfer, cytotoxic cells, dendritic cells, DNA vaccination, nucleoprotein
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Introduction |
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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.
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Methods |
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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 147155 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 14 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 (2472 h), the percentage of class II+ cells was ~5070% but the total yields were lower. Since the yields were significantly higher, sorting experiments were carried out with all cell subsets (072 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 -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).
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Results |
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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. 4(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. 4a
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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 2 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 2
). 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 3
). 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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Discussion |
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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. 1. 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 23% 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 3, 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 Ipeptide 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.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cells |
CTL cytotoxic T lymphocytes |
CP control plasmid |
DC dendritic cell |
NP nucleoprotein |
R:S responder:stimulator |
pCTL CTL precursors |
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Notes |
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5 Present address: Institute of Neuropathology, Hannover Medical School, 30625 Hannover, Germany
Received 21 July 1999, accepted 14 February 2000.
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References |
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