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Cross-Priming as a Predominant Mechanism for Inducing CD8⁺ T Cell Responses in Gene Gun DNA Immunization¹

National Research Laboratory of DNA Medicine, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Hyojadong, Pohang, Kyungbuk, Korea

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA immunization induces CD8⁺ CTL responses by bone marrow-derived APCs, which are directly transfected with a plasmid DNA and/or acquire Ags from DNA-transfected non-APCs. To investigate the relative contribution of DNA-transfected APCs vs non-APCs to the initiation of CD8⁺ T cell responses, we used tissue-specific promoter-directed gene expression and adoptive transfer systems in gene gun DNA immunization. In this study, we demonstrated that non-APC-specific gene expressions induced significant CD8⁺ CTL and IFN--producing cells and Ab responses, whereas APC-specific gene expressions led to moderate CTL and IFN--producers, but no Ab responses. Interestingly, mice immunized with a non-APC-specific plasmid induced more rapid, vigorous, and prolonged proliferation of adoptively transferred Ag-specific CD8⁺ T cells than APC-specific plasmid-immunized mice. In addition, the in vivo proliferative responses elicited by a non-APC-specific plasmid administration were dependent on TAP, but were independent of CD4⁺ T cell help. Collectively, our results suggest that cross-priming, in which Ags expressed in non-APCs are taken up, processed, and presented by APCs, plays an important role in the initiation, magnitude, and maintenance of CD8⁺ T cell responses in gene gun DNA immunization.

	Introduction

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It has been shown that DNA immunization induces potent CD8⁺ T cell responses that are responsible for viral clearance and host protection (1, 2). These responses have been reported to absolutely require an Ag-presenting ability of bone marrow-derived APCs that present Ags in the context of MHC class I molecules to naive CD8⁺ T cells (3, 4, 5). There are two candidate mechanisms by which APCs may acquire Ags encoded by a plasmid DNA. First, APCs may become directly transfected by a plasmid DNA to prime CD8⁺ T cells, a process termed direct priming. Alternatively, APCs may take up Ags exogenously produced by DNA-transfected non-APCs such as myocytes and keratinocytes to prime CD8⁺ T cells, a process termed cross-priming.

It was previously reported that dendritic cells (DCs)³ transfected with a plasmid DNA were detected in draining lymph nodes (LNs) after gene gun DNA immunization (6). In addition, Klinman et al. (7) showed that rapidly migratory cells such as APCs at the site of DNA injection were critical for the initiation of cellular immunity after gene gun DNA immunization. Furthermore, Porgador et al. (8) showed that a small number of DCs transfected with a plasmid DNA were isolated in draining LNs within 24 h after gene gun DNA immunization and could stimulate a CD8⁺ T cell clone in vitro. In addition, Bot et al. (9) showed that MHC class II (MHCII)-positive cells isolated from the site of intradermal DNA inoculation were more effective in CTL priming than MHCII-negative cells upon intrasplenic infusion of these cells, suggesting the predominant role of direct priming rather than cross-priming. Alternatively, some evidence has emerged that cross-priming seems to be involved in CTL priming in DNA immunization (3, 10). Particularly, Corr et al. (11), using transactivating plasmid and bone marrow chimera systems, demonstrated that induction of CTL responses in intradermal DNA immunization was predominantly mediated via cross-priming rather than via direct priming. The discrepancy between Corr et al. (11) and Bot et al. (9) implies that the difference of experimental systems may result in quite different outcomes even when the same intradermal inoculation method was used.

Therefore, the relative contribution of direct priming vs cross-priming to the induction of CD8⁺ T cell responses in cutaneous DNA immunization has been unclear to date. Particularly, many previous studies demonstrating in vivo function of directly transfected APCs in priming Ag-specific T cell responses upon cutaneous DNA immunization have been obtained through highly invasive and extensive manipulatory procedures such as skin ablation or grafting, in vitro suspension cultures, and ex vivo explant cultures (7, 8, 9, 12, 13, 14). Because an in vitro or ex vivo manipulation of APCs might affect the functions and properties of these cells in vivo (15, 16, 17), we wanted to investigate the relative contribution of direct- or cross-presented APCs upon gene gun DNA immunization without artificial interferences due to handling. To this end, we used two experimental approaches. First, we used a tissue-specific promoter-directed gene expression system in which DNA-encoded Ags can be expressed specifically to either APCs or non-APCs in vivo. This approach allows us to exclude possible artifacts resulting from an in vitro or ex vivo manipulation of APCs from DNA-immunized mice. Second, we performed adoptive transfer of Ag-specific CD8⁺ T cells to monitor the Ag presenting capacity of direct- or cross-presented APCs directly in vivo by flow cytometry. Using these approaches, we provide insight into the relative contribution of direct priming vs cross-priming to the initiation, magnitude, and maintenance of CD8⁺ T cell responses in gene gun DNA immunization.

	Materials and Methods

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Mice

Female BALB/c and C57BL/6 mice were obtained from Charles River Laboratories (Wilmington, MA). MHCII- and TAP-deficient C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Transgenic C57BL/6 mice for a TCR recognizing the OVA_257–264 epitope of the hen egg OVA in association with H-2K^b molecules were generously provided by Dr. W. R. Heath (The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) (18). The mice were bred and maintained in specific pathogen-free conditions. All mice used here were 6–8 wk old.

Plasmid construction

To generate the pCMV-Luc, firefly luciferase cDNA from pGL2-control vector (Promega, Madison, WI) was inserted into the pCMV vector containing the CMV promoter and the SV40 polyadenylation signal (poly(A)). The CMV promoter of the pCMV-Luc was then replaced by the 2.2-kb human keratin 14 (K14) promoter (a kind gift from Dr. E. Fuchs, University of Chicago, Chicago, IL; see Ref. 19), the 1.7-kb human CD11b promoter (a kind gift from Dr. D. G. Tenen, Harvard Medical School, Boston, MA; see Ref. 20), and the 2.1-kb murine MHCII gene promoter (a kind gift from Dr. C. Benoist and Dr. D. Mathis, Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, Strasbourg, France; see Ref. 21) to produce the pK14-, pCD11b-, and pMHCII-Luc, respectively. For investigating Ag-specific immune responses, 1.5-kb nucleoprotein (NP) cDNA of the A/PR/8/34 influenza virus or 1.2-kb OVA cDNA was used to replace the luciferase gene of the pCMV-, pK14-, pCD11b-, and pMHCII-Luc to generate pCMV-, pK14-, pCD11b-, and pMHC-NP (or -OVA), respectively. We also constructed pTV-GM-CSF and pTV-IL-4 encoding GM-CSF and IL-4, respectively, as previously described (22). These plasmids were propagated in Escherichia coli and were purified using a Endofree plasmid purification kit (Qiagen, Chatsworth, CA).

Cell culture

HaCaT (human keratinocyte cell line), RAW264.7 (mouse macrophage cell line), and COS-7 (monkey kidney cell line) cells were maintained in DMEM supplemented with 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin, 50 µM 2-ME, and 2 mM glutamine (all purchased from Life Technologies, Rockville, MD).

Cell transfection and luciferase assay

Transfection was performed by electroporation using the Gene Pulser (Bio-Rad, Hercules, CA) set at 220–280 V and 960 µF capacitance. Cells (5 x 10⁶) were resuspended in 0.4 ml of supplemented DMEM and were transfected with 45 µg of each luciferase reporter plasmid together with 5 µg of the plasmid pCH110 (Amersham Pharmacia Biotech, Piscataway, NJ), which expresses -galactosidase (-gal), as a control for differences in transfection efficiency between and within various cell lines, in 0.4-cm electrode gap cuvettes (Bio-Rad). The luciferase and -gal activities were measured 48 h after transfection using Promega assay systems as previously described (23).

Gene gun delivery of plasmid DNA

Sodium pentobarbital-anesthetized mice received two or four nonoverlapping abdominal deliveries of 0.5 mg of gold beads (1 µm) coated with various plasmid DNA using the Helios gene gun system (Bio-Rad) at a helium discharge pressure of 400–450 psi as previously described (24).

ELISA for anti-NP IgG and CTL assay

For determination of NP-specific Ab responses, microtiter plates were coated with rNP at 2 µg/ml (100 µl) in carbonate buffer, pH 9.5, and individual or pooled sera were diluted and tested by ELISA as previously described (25). For determination of NP-specific CTL responses, pooled splenocytes were cultured for 6 days at 2 x 10⁶ cells/ml in the presence of 10 µM H-2K^d-restricted NP147–155 peptide (TYQRTRALV; Alberta Peptide Institute, Alberta, Canada) and 10 U/ml murine rIL-2 (BD PharMingen, San Diego, CA). Cell-mediated cytotoxicity was determined by a standard ⁵¹Cr release assay using P815 (H-2^d) mastocytoma cells as previously described (22). Nonspecific lysis was evaluated using P815 cells pulsed with an irrelevant H-2L^d-restricted -gal peptide (TPHPARIGL; Alberta Peptide Institute). In our condition for CTL assay, lysis of P815 cells pulsed with irrelevant -gal peptide was <5% in all groups of mice (data not shown).

LDA for CTL

Limiting dilution analysis (LDA) for cytotoxicity was performed as previously reported (26). Pooled splenocytes were 3-fold serially diluted in 96-well U-bottom plates (24 replicates/dilution). The 10 µM NP147–155 peptide and 10 U/ml murine rIL-2 were added to each well. On day 6, 100 µl of cultured cells from each well was transferred to 96-well plates containing 10 µM NP147–155 peptide-pulsed ⁵¹Cr-labeled P815 cells for a standard ⁵¹Cr release assay. Wells were scored as positive for CTL recognition if the specific lysis exceeded 3 SD above the mean control release from the target cells pulsed with irrelevant -gal peptide. The CTL precursor (CTLp) frequency was estimated at which 37% of the wells were negative from the slope of a regression plot of the log percentage of negative vs input cell numbers.

ELISPOT assay

An ELISPOT assay was performed as previously described (27). Nitrocellulose plates (96-well; Millipore, Bedford, MA) were coated with the anti-mouse IFN- mAb (5 µg/ml; BD PharMingen). Pooled splenocytes were 3-fold serially diluted and incubated for 24 h with 10 µM NP147–155 peptide or irrelevant -gal peptide in the presence of 10 U/ml murine rIL-2. Each dilution was seeded in quadruplicate. The plates were washed six times with PBS containing 0.05% Tween 20. To detect IFN--specific spots, 2.5 µg/ml biotinylated anti-mouse IFN- mAb (BD PharMingen) was added and incubated at room temperature for 2 h, followed by alkaline phosphatase-coupled streptavidin (BD PharMingen) for 1 h. Spots of IFN--secreting cells were visualized by adding 5-bromo-4-chloro-3-indolyl phosphate/tetranitroblue tetrazolium (TNBT) substrate solution (Calbiochem, San Diego, CA). The reaction was stopped after 15–20 min at room temperature by several washes with distilled water. After drying, spots were counted under a dissecting microscope. The frequency of peptide-specific T cells is expressed as the number of IFN--secreting cells per 10⁶ splenocytes.

Preparation of OT-I cells for adoptive transfer

Transgenic OVA-specific MHC class I-restricted CD8⁺ T (OT-I) cells were purified from the spleen and LNs of OT-I mice as previously described (28). Cell suspensions were treated with J11d (anti-HSA), RL172 (anti-CD4), and M5/114.15.2 (anti-MHCII) for 30 min on ice, and then depleted by treatment with rabbit complement (Calbiochem) for 30 min at 37°C. The purified OT-I cells were resuspended at 5 x 10⁷ cells/ml in PBS containing 0.1% BSA and then incubated with 10 µM CFSE (Molecular Probes, Eugene, OR) for 10 min at 37°C. Cells were washed twice with cold RPMI 1640 containing 10% FBS followed by two washes in PBS. CD8⁺V⁺CFSE⁺ OT-I cells (2 x 10⁶) in 200 µl of PBS were injected into the tail vein of mice.

FACS analysis

Inguinal LN cells were isolated and stained with PE-conjugated anti-CD8 (53-6.7) mAb, PE- or biotin-conjugated anti-V2 TCR (B20.1), and anti-V5.1/2 TCR (MR9-4) mAb (BD PharMingen). Biotin-labeled mAbs were detected with streptavidin-conjugated PerCP (BD Biosciences, Palo Alto, CA). Two- or three-color flow cytometric analysis was performed on a FACSCalibur (BD Biosciences). Live gates were set on lymphocytes by forward and side scatter profiles. Live lymphocytes (100,000–150,000) were collected and then analyzed using CellQuest software (BD Biosciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid vectors with the K14, CD11b, or MHCII gene promoter can drive the cell type-specific expression of a reporter gene in various cell lines

To separately investigate the in vivo function of DNA-transfected APCs and non-APCs in gene gun DNA immunization, we used a specialized plasmid DNA with well-characterized tissue- and cell type-specific promoters that selectively produce DNA-encoded Ags in either APCs or non-APCs in vivo. Gene gun administration of a plasmid DNA is known to primarily transfect non-APCs such as keratinocytes, which play a role as an Ag reservoir in the epidermis of the bombarded skin (29, 30, 31, 32). In contrast, the gene gun immunization has been shown that a small number of APCs such as DCs were transfected (7, 8, 31). Although Langerhans cells and dermal DCs are known to be potent dendritic APCs in the bombarded skin, macrophages are also likely candidates for APC function. Thus, we used two APC-specific promoters, the CD11b and the MHCII gene promoter, which are specific for all skin APCs, including macrophages, Langerhans cells, and dermal DCs (20, 21, 33, 34, 35). The K14 promoter was also selected for specific gene expression in squamous epithelial cells such as keratinocytes (19, 36, 37).

The tissue and cell-type specificities of these promoters have been well known in various transgenic mice systems (19, 20, 21, 33, 34, 36). To further confirm the strength and tissue specificities of these promoters in cultured cell lines, we constructed various plasmid DNA encoding the luciferase gene under the control of the K14, the CD11b, the MHCII, or the CMV promoter as a positive control (Fig. 1A). These plasmids were transiently transfected into various cell lines and their relative luciferase activities were analyzed (Fig. 1B). Significant luciferase activity from pK14-Luc was observed only in the keratinocyte-derived cell line (HaCaT), but pCD11b- and pMHCII-Luc showed luciferase activity exclusively in the macrophage-derived cell line (RAW264.7). In contrast, there was no luciferase activity in the nonspecific COS-7 cell line. We also could not detect any significant luciferase activity in other cell lines including the muscle, kidney, melanoma, ovary, colon, cervix, liver, and fibroblast cell lines (data not shown). Similar results were also obtained when the green fluorescent protein (GFP) was used as the reporter gene (data not shown). It was notable that the relative luciferase activity driven by the K14 promoter in HaCaT cells was similar to that driven by the CD11b or the MHCII promoter in RAW264.7 cells, although their activities were approximately two to three times lower than those driven by the CMV promoter. These results indicate that a plasmid DNA containing the CD11b, the MHCII, or the K14 promoter would allow us to separately investigate the in vivo function of DNA-transfected APCs and non-APCs after gene gun DNA immunization.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. A, Schematic diagram of expression plasmids used in the transient transfection assays. B, The relative luciferase activity driven by the K14, the CD11b, or the MHCII promoter in different cell lines. The plasmids were transiently transfected by electroporation into various cell lines, and luciferase activity was determined 48 h after transfection. The -gal-expressing plasmid was used for normalization of the transfection efficiencies. The relative luciferase activity was calculated by dividing the luciferase activity obtained from pK14-, pCD11b-, or pMHCII-Luc transfection by that obtained from pCMV-Luc transfection for each cell line. Transfections with pCMV-Luc resulted in the luciferase light unit of 1138998 in COS-7 cells, 136089 in RAW 264.7 cells, and 22592 in HaCaT cells. Data represent the mean values of the relative luciferase activities ± SD of three independent experiments.

Using these promoters described above, we investigated whether DNA-transfected APCs or non-APCs are capable of producing a sufficient level of Ags for inducing Ab responses after gene gun DNA immunization. Female BALB/c mice were immunized at 0 and 3 wk by two nonoverlapping gene gun deliveries with gold beads coated with pCMV-, pK14-, pCD11b-, or pMHCII-NP plasmid encoding influenza virus NP or with a mock plasmid DNA. At 5 wk after the first DNA immunization, pooled sera from the immunized mice were analyzed for NP-specific Ab responses.

As expected, mice immunized with pK14-NP that directs NP expression in keratinocytes produced significant levels of anti-NP IgG responses, suggesting that keratinocytes are the major cells that take up the injected plasmid DNA and produce enough Ags to generate Ab responses (Fig. 2A). These data support a previous study in which nonmigratory cells such as keratinocytes influenced the magnitude of Ab responses after gene gun DNA immunization (7). It is of interest that there were no detectable anti-NP IgG responses in mice immunized with pCD11b- or pMHCII-NP that directs NP expression in APCs. However, we could detect a few GFP-positive DCs in draining LNs of pCD11b- or pMHCII-GFP-immunized mice (data not shown). Given that the relative strength of the CD11b or the MHCII promoter was similar to that of the K14 promoter (Fig. 1B), the lack of Ab responses in pCD11b- or pMHCII-NP-immunized mice is not likely to result from the weak promoter activities or no in vivo transfection of APCs. Alternatively, this may be caused by the insufficient amount of Ags produced by a small number of DNA-transfected APCs in vivo. As a positive control, pCMV-NP-immunized mice induced slightly higher anti-NP IgG responses than pK14-NP-immunized mice (Fig. 2A), presumably due to an increased amount of Ags resulting from a stronger and broader specificity into various cell types of the CMV promoter in comparison with the K14 promoter (Fig. 1B). In contrast, there were no anti-NP IgG responses in mock plasmid DNA-immunized mice as a negative control (Fig. 2A).

View larger version (20K): [in this window] [in a new window]   FIGURE 2. NP-specific Ab responses after gene gun immunization with various promoter-driven NP-expressing plasmids. Groups of BALB/c mice were immunized on wk 0 and 3 by two nonoverlapping gene gun deliveries with gold particles coated with 0.5 µg of the indicated plasmids. A, At 5 wk after the first DNA immunization, pooled sera from the immunized mice were 2-fold serially diluted for determining anti-NP total IgG by ELISA. B, For isotype analysis, individual sera from the immunized mice were diluted 1/100, and anti-NP IgG1 and IgG2a was determined by ELISA. The circles show data points of individual mice reading at OD 405 nm and the bars indicate the arithmetic means. The plasmids injected are indicated at the bottom of the figure and the number of mice in each group is shown in parentheses.

Immunization with pK14-NP induces higher NP-specific CTL and IFN--producing T cells than pCD11b- or pMHCII-NP immunization

Although directly transfected APCs are generally accepted to be essential for CTL priming in gene gun DNA immunization (6, 7, 8), the role of DNA-transfected non-APCs has been unclear. Thus, we wanted to determine the relative contribution of DNA-transfected APCs and non-APCs to the induction of CTL responses in gene gun DNA immunization.

At 3 wk after the first DNA immunization, we determined NP-specific CTL responses in pooled splenocytes from the immunized mice described in Fig. 2. As shown in Fig. 3A, mice immunized with pCMV-NP induced robust levels of NP-specific CTL responses, whereas mock vector-immunized mice as a negative control did not. Interestingly, pK14-NP-immunized mice elicited higher levels of NP-specific CTL responses than pCD11b- or pMHCII-NP-immunized mice, but induced slightly lower responses than pCMV-NP-immunized mice. We also observed the similar pattern of NP-specific CTL responses at 2 and 5 wk after the booster DNA immunization (data not shown).

View larger version (17K): [in this window] [in a new window]   FIGURE 3. NP-specific CTL and IFN--producing CD8+ T cells after gene gun immunization with various promoter-driven NP-expressing plasmids. Groups of BALB/c mice were immunized as described in Fig. 2. At 3 wk after the first DNA immunization, pooled splenocytes from three mice per group were assayed for determining the MHC class I-restricted NP147–155 peptide-specific CTL responses (A) and IFN--secreting CD8+ T cells (B) by performing CTL and ELISPOT assay, respectively. The data were represented as the mean ± SD of three independent experiments.

We further confirmed the above results by performing LDA of NP-specific CTLp frequency (Table I). Mice immunized with pK14-NP induced one CTLp in every 81,400–87,500 splenocytes compared with one CTLp in every 134,000–151,000 and 136,000–166,000 splenocytes in pCD11b- and pMHCII-NP-immunized mice, respectively. Mice immunized with pCMV-NP induced the highest CTLp frequency (one CTLp in every 72,000–77,000 splenocytes), whereas mock vector-immunized mice showed less than one CTLp in every 1,200,000 splenocytes.

Immunization with pK14-OVA induces more rapid, vigorous, and prolonged proliferation of adoptively transferred OT-I cells than pCD11b- or pMHCII-OVA immunization

To investigate the efficacy and kinetics for the induction of CD8⁺ T cell responses directly in vivo after gene gun DNA immunization, naive OT-I cells were labeled with CFSE, which allows us to monitor cell division in vivo, and they were then adoptively transferred into female C57BL/6 mice. One day after adoptive transfer, the mice were immunized once by two nonoverlapping gene gun deliveries with gold beads coated with pCMV-, pK14-, pCD11b-, or pMHCII-OVA plasmid encoding OVA or with a mock plasmid DNA. At various time points after DNA immunization, cells isolated from draining LNs of the immunized mice were analyzed for detecting in vivo proliferation of CFSE-labeled OT-I cells by flow cytometry.

The OT-I cells in pCMV-OVA-immunized mice began to proliferate on day 3 (data not shown), some of them dividing more than five times on day 4, and continued to proliferate for up to 21 days (Fig. 4A). As a negative control, there was no detectable proliferation of OT-I cells in mock vector-immunized mice (Fig. 4E). These results indicate that OT-I cells can be activated and subsequently proliferate in draining LNs in response to OVA-derived epitope presented in association with MHC class I molecules on direct- or cross-presented APCs after gene gun DNA immunization. Interestingly, the OT-I cells in pK14-OVA-immunized mice began to proliferate 2–3 days earlier than those in pCD11b- or pMHCII-OVA-immunized mice (4 vs 7 days; Fig. 4, B–D). These results indicate that cross-priming is not only involved in priming CD8⁺ T cell responses, but can also initiate more rapid responses than direct priming in vivo. In addition to earlier OT-I cell proliferation, pK14-OVA-immunized mice showed more vigorous OT-I cell proliferation than pCD11b- or pMHCII-OVA-immunized mice, as evidenced by more intense CFSE profiles of dividing OT-I cells. These results indicate that cross-priming predominantly contributes to augmenting the magnitude of CD8⁺ T cell responses compared with direct priming, and they are consistent with our in vitro data (Fig. 3 and Table I).

View larger version (42K): [in this window] [in a new window]   FIGURE 4. In vivo proliferation of adoptively transferred CFSE-labeled OT-I cells after gene gun immunization with various promoter-driven OVA-expressing plasmids. C57BL/6 mice were i.v. injected with 2 x 106 CFSE-labeled OT-I cells, and 1 day later were immunized once by two nonoverlapping gene gun deliveries of gold particles coated with 1 µg of pCMV-OVA (A), pK14-OVA (B), pCD11b-OVA (C), pMHCII-OVA (D), or mock plasmid DNA (E). At the indicated time points after DNA immunization represented on the right of this figure, pooled draining inguinal LNs from two mice per group were analyzed by flow cytometry. Profiles were gated on CD8+ cells. Data are represented on a contour plot showing CFSE (x axis) vs CD8 fluorescence (y axis). The data are representative of two independent experiments with similar results.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. The effect of delivery number and cytokine genes on in vivo OT-I cell proliferation. C57BL/6 mice were i.v. injected with 2 x 106 CFSE-labeled OT-I cells. One day later, mice were immunized once by two (A) and four (B–D) nonoverlapping gene gun deliveries of gold particles coated with 1 µg of pCD11b-OVA (B), or cocoated with 0.5 µg of each of pCD11b-OVA and pTV-GM-CSF (C), or cocoated with 0.5 µg of each of pCD11b-OVA, pTV-GM-CSF, and pTV-IL-4 (D). At 4 days after DNA immunization, pooled draining inguinal LNs from two mice per group were analyzed by flow cytometry. Profiles were gated on CD8+ cells. Data are represented on a contour plot showing CFSE (x axis) vs CD8 fluorescence (y axis). The data are representative of two independent experiments with similar results.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. The period of Ag persistence required for in vivo OT-I cell proliferation after pK14-OVA immunization. C57BL/6 mice were immunized once by two nonoverlapping gene gun deliveries of gold particles coated with 1 µg of pK14-OVA and i.v. injected with 2 x 106 CFSE-labeled OT-I cells at days 7 (A), 21 (B), 35 (C), and 49 (D) after DNA immunization. On day 3 after adoptive transfer, pooled draining inguinal LNs from two mice per group were analyzed by flow cytometry. Profiles were gated on CD8+ cells. Data are represented on a contour plot showing CFSE (x axis) vs CD8 fluorescence (y axis). The data are representative of two independent experiments with similar results.

It has been previously reported that in vivo cross-priming occurs in either a TAP-dependent (44, 45) or a TAP-independent manner (46, 47, 48). To investigate whether TAP is required for OT-I cell proliferation after pK14-OVA immunization, TAP-deficient mice (TAP^-/-) or wild-type (TAP^+/+) mice adoptively transferred with CFSE-labeled OT-I cells were immunized once with pK14-OVA- or mock DNA-coated gold particles. At 9 days after DNA immunization, cells from draining LNs of the immunized mice were analyzed by flow cytometry. Significant OT-I cell proliferation was detected in TAP^+/+ mice, whereas there was no detectable OT-I cell proliferation in TAP^-/- mice (Fig. 7, A and B). In addition, there was no detectable OT-I cell proliferation in both TAP^+/+ and TAP^-/- mice after mock DNA immunization (data not shown). These results further support the absolute requirement of TAP for cross-priming to occur in vivo.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. The dependency of TAP or CD4+ T cell help on in vivo OT-I cell proliferation after pK14-OVA immunization. TAP+/+ (A) and TAP-/- (B) mice, as well as MHCII+/+ (C) and MHCII-/- (D) mice were i.v. injected with 2 x 106 CFSE-labeled OT-I cells, and 1 day later were immunized once by two nonoverlapping gene gun deliveries of gold particles coated with 1 µg of pK14-OVA. At 9 days after DNA immunization, pooled draining inguinal LNs from two mice per group were analyzed by flow cytometry. Profiles were gated on CD8+ cells. Data are represented on a contour plot showing CFSE (x axis) vs CD8 fluorescence (y axis). The data are representative of two independent experiments with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have shown that cross-priming contributes predominantly to the initiation, magnitude, and maintenance of CD8⁺ T cell responses in gene gun DNA immunization. Our observations are partially inconsistent with previous results reported by Porgador et al. (8). The discrepancies could be caused by differences in the experimental methods for measuring the ability of APCs to prime CD8⁺ T cells. In particular, the previous experiments were performed in the in vitro condition in which APCs isolated from draining LNs within 24 h after gene gun DNA immunization were incubated with a CD8⁺ T cell clone to measure IFN- secretion (8). It has been previously reported that CD8⁺ T cell clones have a lower activation threshold in proliferation after Ag stimulation (54, 55, 56), presumably allowing the CD8⁺ T cell clones to be activated at earlier time points and at lower doses of Ag stimulation than would be required in naive CD8⁺ T cells in vivo. These could explain why in vivo proliferation of OT-I cells in our studies was not detected until 3 days after the pCMV-OVA immunization (data not shown). Thus, DNA-transfected APCs in draining LNs within 24 h after gene gun DNA immunization appear to be sufficient to stimulate a CD8⁺ T cell clone in vitro (8), but not naive CD8⁺ T cells in vivo. In support of this hypothesis, it was previously reported that excision of the epidermal site 24 h after cutaneous DNA immunization abrogated the induction of CTL responses (57) despite DNA-transfected DCs being detected in draining LNs less than 24 h after gene gun DNA immunization (6, 8). This indicates that the Ag presentation by a very small percentage of DNA-transfected DCs in draining LNs within 24 h after gene gun DNA immunization is insufficient to induce detectable CD8⁺ T cell responses.

Interestingly, our results demonstrated that DNA-transfected APCs were shown to initiate CD8⁺ T cell responses slower than APCs that take up and present Ags produced by DNA-transfected non-APCs (Fig. 4, B–D), which might be explained by the following reasons. First, the number of DNA-transfected APCs appears to be a critical parameter in determining earlier CD8⁺ T cell responses. It is possible that the number of DNA-transfected APCs might not be enough to induce an earlier proliferation of OT-I cells after pCD11b- or pMHCII-OVA immunization. It was previously reported that the number of DNA-transfected APCs was proportionally increased by the delivery number of DNA-coated gold particles (8). However, the kinetics of initial OT-I cell proliferation was not changed even by four nonoverlapping gene gun deliveries with pCD11b-OVA (Fig. 5B), although the magnitude of proliferative responses was slightly increased at later time points (days 7 and 9; data not shown). It is likely that the very small number of DNA-transfected APCs in draining LNs appears to be an intrinsic problem in gene gun DNA immunization, because a 2-fold increase of their number was still insufficient to induce an earlier proliferation of OT-I cells after pCD11b- or pMHCII-OVA immunization. Second, it is possible that DNA-transfected APCs might transfer their antigenic materials into other APCs in vivo because short-lived immature peripheral DCs were reported to transfer their Ags to resident DCs in draining LNs (58). It was previously reported that the dose of Ags could affect the kinetics of proliferation of CD8⁺ T cells in vitro (59) and that cross-priming occurred only in situations in which the dose of Ags exogenously produced was beyond a certain threshold level (28). These previous reports allow us to postulate that a very small number of DNA-transfected APCs after gene gun DNA immunization (8) produced only the low amount of Ags that cannot mediate efficient cross-priming in vivo, thereby leading to the delayed OT-I cell proliferation in pCD11b- or pMHCII-OVA-immunized mice (Fig. 4, C and D).

It was previously reported that naive transgenic CD4⁺ T cells were not activated when adoptively transferred into mice that had been s.c. immunized with a plasmid DNA 20 days before (13). However, Doe et al. (3) showed that significant CD8⁺ CTL responses were induced when immunocompetent spleen and bone marrow cells were transferred into histoincompatible SCID mice at 21 days after i.m. DNA immunization. These discrepancies may be due to the route of DNA administration (s.c. vs i.m.), the nature of encoded Ags, the difference of activation threshold between CD4⁺ and CD8⁺ T cells, and the methods for evaluating the induced T cell responses. In the present study, we demonstrated that adoptively transferred naive OT-I cells can proliferate when mice immunized with pK14-OVA at 35 days before adoptive transfer (Fig. 6). Considering the short half-life of both DCs (60, 61) and extracellular plasmid DNA in vivo (62), it is likely that DNA-transfected keratinocytes persistently produce Ags up to at least 5 wk after pK14-OVA immunization, thereby allowing APCs to take up and present them to naive OT-I cells in vivo. Our results agree well with the previous results that DNA-transfected keratinocytes could produce Ags for long periods after gene gun DNA immunization (32).

It has been reported that cross-priming plays an important role in inducing CD8⁺ T cell responses to peripheral self, viral, tumor, and bacterial Ags (28, 45, 46, 63). This indicates that cross-priming is a general mechanism for the induction of CD8⁺ T cell immunity and/or tolerance. However, it remains to be determined how Ags produced by DNA-transfected keratinocytes are transferred into APCs. One possibility is that heat shock protein carrying antigenic peptides might be released from transfected keratinocytes and be transferred into APCs. Recent studies have suggested that the transfer of heat shock protein 70-linked peptides into APCs may induce CD8⁺ T cell responses after DNA immunization (64). Alternatively, the transfected keratinocytes might somehow undergo either necrosis or apoptosis by which proteins could be taken up and processed by APCs to induce CD8⁺ T cell responses (65, 66, 67). It was previously reported that apoptotic but not intact keratinocytes transfected with a plasmid DNA resulted in the activation of an Ag-specific T cell line via cross-priming in vitro (13). In addition, it was recently shown that a cell-associated Ag is more efficient for inducing CD8⁺ T cell responses in vivo than a soluble Ag (68).

To our knowledge, these studies are the first to show the kinetics of in vivo proliferation of Ag-specific CD8⁺ T cells after gene gun DNA immunization, providing clear in vivo evidence that cross-priming plays an important role in the initiation, magnitude, and maintenance of CD8⁺ T cell responses in gene gun DNA immunization. Our findings further suggest that appropriate methods that facilitate Ag transfer from DNA-transfected non-APCs to APCs will be one of the critical factors for designing optimal DNA vaccines to induce faster and stronger CD8⁺ T cell responses in gene gun DNA immunization.

	Acknowledgments

 
We thank Dr. D. G. Tenen and Dr. E. Fuchs for the generous gift of a plasmid DNA containing the CD11b and the K14 promoter, respectively. We thank Dr. C. Benoist, Dr. D. Mathis, and Dr. C. H. Chang for the generous gift of a plasmid DNA containing the MHCII promoter. We are grateful to Dr. W. R. Heath for the generous gift of OT-I mice and helpful advice in our experiments. We also thank Sang Chun Lee for devoted animal care and Yun Ji Park for technical assistance.

	Footnotes

 
¹ This work was supported by a National Research Laboratory Grant from the Korea Institute of Science and Technology Evaluation and Planning (2000-N-NL-01-C-202) and by grants from the Korea Research Foundation and Superior Research Center of University of Ulsan College of Medicine.

² Address correspondence and reprint requests to Dr. Young Chul Sung, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyojadong, Pohang, 790-784, Korea. E-mail address: ycsung{at}postech.ac.kr

³ Abbreviations used in this paper: DC, dendritic cell; LN, lymph node; K14, keratin 14; MHCII, MHC class II; NP, nucleoprotein; -gal, -galactosidase; LDA, limiting dilution analysis; CTLp, CTL precursor; OT-I cells, transgenic OVA-specific MHC class I-restricted CD8⁺ T cells; GFP, green fluorescent protein.

Received for publication February 14, 2001. Accepted for publication September 4, 2001.

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