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. 1998 May;72(5):3812–3818. doi: 10.1128/jvi.72.5.3812-3818.1998

Dendritic Cells Efficiently Induce Protective Antiviral Immunity

Burkhard Ludewig 1,*, Stephan Ehl 1, Urs Karrer 1, Bernhard Odermatt 1, Hans Hengartner 1, Rolf M Zinkernagel 1
PMCID: PMC109604  PMID: 9557664

Abstract

Cytotoxic T lymphocytes (CTL) are essential for effective immunity to various viral infections. Because of the high speed of viral replication, control of viral infections imposes demanding functional and qualitative requirements on protective T-cell responses. Dendritic cells (DC) have been shown to efficiently acquire, transport, and present antigens to naive CTL in vitro and in vivo. In this study, we assessed the potential of DC, either pulsed with the lymphocytic choriomeningitis virus (LCMV)-specific peptide GP33-41 or constitutively expressing the respective epitope, to induce LCMV-specific antiviral immunity in vivo. Comparing different application routes, we found that only 100 to 1,000 DC had to reach the spleen to achieve protective levels of CTL activation. The DC-induced antiviral immune response developed rapidly and was long lasting. Already at day 2 after a single intravenous immunization with high doses of DC (1 × 105 to 5 × 105), mice were fully protected against LCMV challenge infection, and direct ex vivo cytotoxicity was detectable at day 4 after DC immunization. At day 60, mice were still protected against LCMV challenge infection. Importantly, priming with DC also conferred protection against infections in which the homing of CTL into peripheral organs is essential: DC-immunized mice rapidly cleared an infection with recombinant vaccinia virus-LCMV from the ovaries and eliminated LCMV from the brain, thereby avoiding lethal choriomeningitis. A comparison of DC constitutively expressing the GP33-41 epitope with exogenously peptide-pulsed DC showed that in vivo CTL priming with peptide-loaded DC is not limited by turnover of peptide-major histocompatibility complex class I complexes. We conclude that the priming of antiviral CTL responses with DC is highly efficient, rapid, and long lasting. Therefore, the use of DC should be considered as an efficient means of immunization for antiviral vaccination strategies.

Dendritic cells (DC) derived from bone marrow (bmDC) are found as antigen-presenting cells (APC) scattered throughout nonlymphoid tissues. After antigen acquisition and processing, DC migrate via lymph vessels or blood to the T-cell areas of regional secondary lymphoid organs, where they present major histocompatibility complex (MHC) class I- and II-restricted peptides to naive T cells (42). The combination of these properties, i.e., antigen uptake, processing, transport, and presentation, makes DC particularly suitable as vehicles for antigens in immunotherapy. DC pulsed with tumor-derived peptides (29, 39, 46), tumor-associated proteins (16), or tumor cell RNA (9) have been shown to activate tumor-specific cytotoxic T lymphocytes (CTL) and to reduce tumor load.

The high efficiency of DC in eliciting T-cell responses against infectious agents has been demonstrated with different experimental systems; e.g., DC can prime immune responses against mycobacteria (19), Borrelia burgdorferi (30), and Leishmania major (31). Induction of virus-specific CTL by DC in vitro with virus peptides or virus proteins has been shown for influenza virus (27), Sendai virus (22), and human immunodeficiency virus (26). In vivo, adoptive transfer of human immunodeficiency virus peptide-pulsed DC (43) or hepatitis B virus protein-pulsed DC (10) elicits virus-specific CTL responses. Whether these CTL responses reflect the ability to provide antiviral protection in vivo, however, has not been addressed in these studies. This aspect is particularly important in viral infections, since the high speed of replication and amplification imposes demanding quantitative and functional requirements on protective CTL responses (6, 15, 24).

Infection of mice with lymphocytic choriomeningitis virus (LCMV) is a well-characterized model system for studying CTL responses in vivo. Control of an acute virus infection is almost exclusively achieved by CD8+ T-cell-mediated, perforin-dependent cytotoxicity (21). Various in vivo and in vitro assays with graded sensitivities allow assessment of the efficiency and biological relevance of an immunization strategy (5, 12). In addition, several CTL immunization strategies, including peptide vaccination (1, 2, 41) and priming with recombinant vaccinia virus (17), have been well characterized for this experimental system. Therefore, the study of DC-induced immunity against LCMV infection allows an interesting qualitative comparison with other vaccination approaches.

In the present study, we evaluated the potential of DC to induce CTL-mediated antiviral immunity in vivo using bone marrow-derived DC either pulsed with peptides or obtained from transgenic mice constitutively expressing the immunodominant epitope (GP33-41; hereafter referred to as GP33) of the LCMV (WE strain) glycoprotein (14a). Immunization with these cells allowed us to address the following questions. (i) How many DC are needed to induce protection against a viral infection? (ii) What are the in vivo kinetics of DC-induced CTL activation? (iii) Do DC-induced antiviral CTL emigrate to peripheral tissues to resolve viral infections, and are they protective against virus-induced immunopathology? (iv) Does turnover of peptide-MHC class I complexes influence DC immunogenicity?

MATERIALS AND METHODS

Mice.

C57BL/6 mice were obtained from the Institut für Labortierkunde (University of Zürich, Zürich, Switzerland). Transgenic mice expressing the LCMV GP33 epitope ubiquitously in all tissues were generated by use of a construct containing the H-2Kb regulatory elements and the coding sequence for amino acids 1 to 60 of the LCMV glycoprotein (H8 mice) (14a). The transgene construct was injected into fertilized C57BL/6 oocytes. All animals were kept under specific-pathogen-free conditions.

Viruses and cell lines.

LCMV strain WE was originally obtained from F. Lehmann-Grube (Hamburg, Germany) and propagated on L929 cells. Recombinant vaccinia virus expressing LCMV glycoprotein (Vacc-G2) was obtained from D. H. Bishop (Oxford, United Kingdom) and grown on BSC40 cells. Viruses were titrated as described previously with MC-57 cells for LCMV (8) and BSC40 cells for Vacc-G2 (17) cells. EL-4 (H-2b), a thymoma cell line, was used as the target cell line.

Antibodies and peptides.

Supernatants from the following monoclonal antibody-producing hybridomas (American Type Culture Collection) were used: rat anti-mouse CD4 (YTS191.1), rat anti-mouse CD8 (YTS169.4.2), rat anti-mouse CD45R (RA3-3A1/6.1), and rat anti-mouse I-Abd (B21-2).

LCMV GP33 (KAVYNFATM) (37) was synthesized by the solid-phase method and purchased from Neosystem Laboratoire (Strasbourg, France). To prevent dimer formation, the original cysteine at anchor position 41 in LCMV GP33 was replaced by methionine.

Preparation of DC.

For generation of DC from C57BL/6 and H8 mouse bone marrow cultures (H8-bmDC and B6-bmDC, respectively), the procedure of Inaba et al. (20) was used, with minor modifications. Briefly, bone marrow was flushed from femurs and tibias and subsequently depleted of erythrocytes with ammonium chloride. Bone marrow cells were depleted of lymphocytes, B cells, and I-Abd+ cells by use of a cocktail of monoclonal antibodies (YTS191.1, YTS169.4.2, RA3-3A1/6.1, and B21-2) and goat anti-rat immunoglobulin G-coated Dynabeads (Dynal, Oslo, Norway). Cells were plated at 0.5 × 106/ml in RPMI 1640 supplemented with 5% fetal calf serum (FCS), penicillin-streptomycin, 10 ng of recombinant murine granulocyte-macrophage colony-stimulating factor (kindly supplied by Sandoz, Vienna, Austria) per ml, and interleukin 4-containing supernatant from cell line X63-IL4 (kindly provided by M. Kopf, Basel, Switzerland) at a final concentration of 100 ng/ml. At days 2 and 4 of culturing, 50% of the supernatant was removed and replenished with fresh medium, and fresh cytokines were added. At day 6, nonadherent cells were collected and further purified over metrizamide (14.5% in RPMI 1640 containing 5% FCS) (Sigma) to remove cell debris and high-density cells.

The resulting bmDC populations showed a high purity, with 80 to 90% of the cells showing the distinct stellate DC morphology. More than 85% of the cells were CD11b+ and showed strong expression of MHC class I and II antigens (5 to 10 times higher than that of naive B cells). A total of 70 to 85% of the cells were positive for CD80, CD86, and the DC marker NLDC-145, as determined by flow cytometry (data not shown). The differentiation stage of these DC was immature, as judged by the ability to take up, process, and present native viral antigens to transgenic T-helper cells (data not shown). Furthermore, the activation of bmDC with tumor necrosis factor induced significant upregulation of MHC and costimulatory antigens (data not shown).

To enrich for splenic DC (sDC), spleens were digested with collagenase and ground through a stainless steel screen with a sterile syringe plunger. Cells were resuspended in RPMI 1640 supplemented with 10% FCS and antibiotics at 2 × 107/ml and cultured on 10-cm tissue culture dishes for 90 min. Nonadherent cells were removed by washing with phosphate-buffered saline, and adherent cells were cultured overnight in RPMI 1640 supplemented with 10% FCS, 10 ng of granulocyte-macrophage colony-stimulating factor per ml, and 100 ng of interleukin 4 per ml. Nonadherent, low-density cells were separated with metrizamide. Cell suspensions prepared in this way contained 30 to 60% DC, as determined by morphology and flow cytometry.

Peptide treatment and immunization of mice.

Purified C57BL/6 bmDC and sDC (1 × 106 to 5 × 106) were resuspended in 0.5 ml of medium containing GP33 at a concentration of 10−6 M and incubated for 60 min at 37°C on a rocking platform. Cells were washed three times with balanced salt solution (BSS) and resuspended at 2 × 106/ml in BSS, and serial 10-fold dilutions were made. DC were injected in a volume of 0.5 ml intravenously (i.v.), 0.05 ml subcutaneously (s.c.) at the base of the tail, or 0.02 ml directly into the spleen.

Cytotoxicity assays.

Spleen cells (4 × 106/well) from primed mice were restimulated for 5 days in 24-well tissue culture plates with 2 × 106 GP33-labeled, irradiated (3,000 rads) spleen cells or with 2 × 105 LCMV-infected, irradiated peritoneal macrophages in Iscove’s modification of Dulbecco’s medium (IMDM) supplemented with 10% FCS, penicillin- streptomycin, and 0.001 M 2-mercaptoethanol. Restimulated spleen effector cells from one well were resuspended in 1 ml of minimal essential medium containing 2% FCS, and serial threefold dilutions were made (indicated in figures as dilution of culture). For detection of primary ex vivo cytotoxicity, effector cell suspensions were prepared from spleens of immunized mice at various times after priming. EL-4 cells were pulsed with LCMV GP33 (10−6 M, 1.5 h, 37°C) and used in a standard 5-h 51Cr release assay or in an overnight (15-h) assay. 51Cr-labeled nonpulsed EL-4 cells served as controls. Spontaneous release was always below 19% for 5-h assays and below 29% for overnight assays.

CTLp assay.

Quantification of GP33-specific precursor CTL (CTLp) per spleen was performed by limiting-dilution analysis as described previously (32). Responder spleen cells were titrated and cultured with 104 LCMV-infected, irradiated peritoneal macrophages and 105 irradiated feeder spleen cells in IMDM–10% FCS and 10% concanavalin A supernatant in 16 wells per dilution step. After 6 days of culturing, cytotoxicity was tested on GP33 containing loaded or unloaded EL-4 cells in a 51Cr release assay, and CTLp frequencies were calculated (44).

CFSE labeling of DC and immunohistochemistry.

The fluorescent dye CFSE (5- and 6-carboxyfluorescein diacetate succinimidyl ester) was purchased from Molecular Probes (Eugene, Oreg.). DC were washed with BSS, resuspended at 106/ml in BSS containing 0.5 μM CFSE, and incubated for 10 min at 37°C. After the cells were labeled, FCS was added to a final concentration of 5%, and the cells were washed twice. DC viability after CFSE labeling was >95%, as determined by trypan blue exclusion. CFSE-labeled DC (3 × 106) were adoptively transferred into recipient mice. Organs were removed at days 1 and 2, immersed in Hanks BSS, and snap frozen in liquid nitrogen. Histological procedures were performed as described previously (33) with rabbit antifluorescein antibody (Dako).

RESULTS

Migration pattern for bmDC.

Apart from their high stimulatory capacity, the ability of DC to migrate to secondary lymphoid organs and to home to T-cell areas is a prerequisite for efficient CTL priming. To assess the homing pattern for bmDC, cells were labeled with the fluorescent dye CFSE and adoptively transferred into C57BL/6 recipient mice. One to 2 days later, several organs were analyzed for the presence of the donor DC by immunohistochemistry. After i.v. injection, the majority of the CFSE-labeled bmDC homed to the T-cell areas of the spleen (Fig. 1A). In the T-cell areas, DC displayed the typical stellate morphology, thereby facilitating interaction with a large number of T cells (Fig. 1B). Homing of bmDC to paracortical areas of regional hepatic lymph nodes was also observed, as previously described for rat blood DC (23). Transferred DC were hardly detectable in other lymph nodes (mediastinal, inguinal, and axillary) or other tissues (liver, lung, and kidney) (data not shown). Thus, after i.v. injection, bmDC preferentially home to the T-cell areas of the spleen.

FIG. 1.

FIG. 1

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Homing of bmDC to splenic T-cell areas. bmDC (3 × 106) were CFSE labeled, injected i.v., and visualized in cryostat sections with an antifluorescein antibody. (A) bmDC were found in the interfollicular areas (follicles are marked with asterisks). Magnification, ×40. (B) High magnification of boxed area in panel A showing dense follicular area (asterisk), DC with typical stellate morphology (arrow), and central artery (arrowhead). Magnification, ×350.

How many DC are needed to induce antiviral immunity?

To evaluate the antiviral priming capacity of DC, we used transgenic mice expressing the first 60 amino acids of the LCMV glycoprotein under the control of the H-2Kb promoter (H8 mice). This characteristic results in the ubiquitous expression of the immunodominant LCMV epitope GP33 (14a). When H8-bmDC were used, external loading of DC with either peptide or protein was not needed.

To determine the minimal number of DC required for the induction of antiviral immunity and to assess the influence of DC migration on the efficiency of CTL induction, C57BL/6 mice (H-2b) were immunized with different doses of H8-bmDC by direct injection into the spleen (Fig. 2A), i.v. (Fig. 2B), or s.c. (Fig. 2C). Eight days later, spleens were harvested, and cytotoxicity was determined after restimulation for 5 days with GP33-pulsed, irradiated spleen cells. The minimal dose needed to elicit a measurable CTL response after intrasplenic injection was approximately 100 H8-bmDC (Fig. 2A); injection of lower numbers of DC did not induce a CTL response (data not shown). The minimal dose needed to induce a CTL response in the spleen after i.v. injection was 103 DC (Fig. 2B). In contrast, approximately 10 times more H8-bmDC had to be injected s.c. to reach similar levels of CTL induction in the spleen (Fig. 2C).

FIG. 2.

FIG. 2

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Efficient priming of antiviral CTL by H8-bmDC. C57BL/6 mice were immunized by intrasplenic injection (A), i.v. (B), or s.c. (C) with graded doses of H8-bmDC. Eight days later, induction of GP33-specific CTL was tested. Spleen cells were restimulated in vitro for 5 days with peptide-labeled, irradiated spleen cells. Specific lysis was measured on GP33-labeled EL-4 target cells (closed symbols) or EL-4 cells without peptide (open symbols). Spontaneous release was <15%. Values for control mice immunized i.v. with 105 unlabeled B6-bmDC were 10, 7, 4, and 2% with labeled EL-4 cells and 12, 9, 4, and 1% with unlabeled EL-4 cells at the indicated culture dilutions (from left to right), respectively.

To test the protective capacity of H8-bmDC, mice immunized via different routes with graded doses of H8-bmDC were challenged with 200 PFU of LCMV strain WE i.v. at day 8 after DC priming. Infection of naive C57BL/6 mice caused high virus titers (>106 PFU/spleen), whereas mice immunized by intrasplenic injection with 103 H8-bmDC were able to rapidly clear the virus (Fig. 3). Mice immunized intrasplenically with 102 H8-bmDC showed a reduction of virus titers of about 2 to 4 log units but did not completely clear the virus by day 4. Immunization with 104 H8-bmDC i.v. or 105 H8-bmDC s.c. led to resistance against a low-dose LCMV challenge infection, whereas injection of 103 H8-bmDC i.v. or 104 H8-bmDC s.c. only partially protected against a low-dose challenge infection (Fig. 3).

FIG. 3.

FIG. 3

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Protection against low-dose LCMV challenge after priming with H8-bmDC. Mice were immunized intrasplenically (i.spl.), i.v. or s.c. (at the base of the tail) with the indicated number of H8-bmDC. At day 8 postimmunization, protection against LCMV strain WE was tested. Virus titers in spleens were determined 4 days after an i.v. challenge with 200 PFU of LCMV strain WE. The detection limit is represented by the broken line.

The results described above suggested that low numbers of DC are sufficient to induce an in vivo protective antiviral immune response. To assess how this response is reflected in an increase in the number of virus-specific CTLp in the spleen, mice were injected with different doses of H8-bmDC i.v. and a limiting-dilution assay was performed 8 days later. With doses of 104 to 106 DC, GP33-specific CTLp frequencies were always about 1:28,000 to 1:100,000 (Table 1). In LCMV memory mice (i.e., mice infected with 102 PFU of LCMV WE at least 50 days ago), CTLp frequencies were also about 1:10,000 (Table 1) (3). Thus, in the LCMV system, DC efficiently prime CTL to levels of protective memory responses.

TABLE 1.

Frequency of GP33-specific CTLp in spleens after immunization with different doses of H8-bmDC

Immunization Day 8
Day 60
CTLp frequencya CTLp/spleenb CTLp frequencya CTLp/spleenb
106 H8-bmDCc 1:28,000 ± 1:5,300 4,191 ND ND
105 H8-bmDCc 1:43,000 ± 1:4,100 2,241 1:96,000 ± 1:3,700 945
104 H8-bmDCc 1:110,000 ± 1:9,200 848 1:146,000 ± 1:7,600 690
Nonec <1:1,000,000 <100 <1:500,000 <100
LCMVd 1:10,000 6,000 1:11,000 5,000
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a

Splenocytes from immunized mice were tested in a limiting-dilution assay after restimulation with LCMV-infected peritoneal macrophages for 6 days. The mean number of CTLp ± standard deviation is indicated. Statistical analysis of the data with an unpaired t test revealed two-tailed significance at P < 0.05 among the groups. Data from one of two experiments are shown. 

b

The mean number of CTLp per spleen is indicated. 

c

C57BL/6 mice (three per group) were immunized i.v. with H8-bmDC or left untreated. 

d

C57BL/6 mice were infected i.v. with 200 PFU of LCMV strain WE at day 0. 

Rapid and long-lasting activation of CTL by DC.

To evaluate the kinetics of CTL induction by DC, LCMV GP33-specific cytotoxicity was monitored at different times after adoptive transfer of H8-bmDC into C57BL/6 mice. DC-induced primary ex vivo cytotoxicity was weak, as determined by a standard 5-h 51Cr release assay (Fig. 4). When the sensitivity of the assay was increased by prolongation of the incubation time to 15 h, direct ex vivo cytotoxicity became apparent 4 days after priming with a high dose of DC (5 × 105) (Fig. 4A to D). Immunization with a low dose of DC (104) led to detectable ex vivo cytotoxicity beginning at day 6 (Fig. 4E to G).

FIG. 4.

FIG. 4

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Kinetics of CTL induction after DC priming. Mice were primed with either 5 × 105 (A to D) or 104 (E to G) H8-bmDC i.v. At different times after immunization, ex vivo CTL activity of splenocytes was tested in a 51Cr release assay after 5 h (open symbols) or after 15 h (closed symbols) on GP33-labeled EL-4 target cells at the indicated effector/target cell (E:T) ratios. Spontaneous release after 5 h was <12%; after 15 h, it was <29%. Nonspecific lysis of unlabeled EL-4 target cells was always <5%. Values (percentages) for an LCMV-infected control mouse (day 8) were 87, 74, 61, and 35% for 5 h and 99, 98, 81, and 72% for 15 h at E:T ratios of 90, 30, 10, and 3, respectively.

Challenging H8-bmDC-primed mice at different times after adoptive transfer showed the rapid development and long duration of the antiviral immune response. Immunization with 105 H8-bmDC completely protected mice against challenge with LCMV infection as early as 2 days after priming (Fig. 5). With 104 H8-bmDC, protective immunity had not developed at day 2 but was detectable by day 8 postimmunization (Fig. 5). After 60 days, mice immunized with 105 H8-bmDC were still fully protected against LCMV challenge, whereas immunization with 104 H8-bmDC was only partially protective. In addition, CTLp frequencies at day 60 after DC immunization were significantly elevated (Table 1). Taken together, these results show that high numbers of DC rapidly induce a long-lasting protective immune response. With low numbers of DC, mounting of a fully protective immune response was slightly delayed and protection waned faster.

FIG. 5.

FIG. 5

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Rapid and long-lasting induction of protective immunity by high doses of DC. Mice were immunized i.v. with different doses of H8-bmDC and challenged with 200 PFU of LCMV strain WE i.v. after 2, 8, or 60 days. Virus titers in spleens were determined 4 days after challenge infection. The broken line represents the detection limit.

DC efficiently prime against peripheral virus challenge.

In the experiments described above, antiviral CTL activity was determined mainly in the spleen. Control of virus replication outside lymphoid tissues (such as ovaries or brain) imposes much more stringent requirements on virus-specific CTL (6, 24). To assess whether DC-primed CTL exert their antiviral activity after homing to peripheral organs in vivo, virus control in the ovaries after intraperitoneal infection with LCMV glycoprotein-expressing vaccinia virus (Vacc-G2) and in the brains after intracerebral challenge with LCMV strain WE was studied. Immunization with 103 H8-bmDC only partially protected against Vacc-G2 challenge, whereas higher doses completely inhibited Vacc-G2 growth (Table 2). After intracerebral injection of LCMV strain WE, naive mice started to show symptoms of immunopathological choriomeningitis on day 7. Mice immunized with 103 H8-bmDC and three of five mice immunized with 104 DC had already developed disease by days 5 to 6, indicative of CTL preactivation leading to enhanced immunopathology (34). Mice immunized with 105 H8-bmDC and one of five mice immunized with 104 DC survived the intracerebral challenge with LCMV strain WE without developing symptoms of choriomeningitis (Table 2). Thus, DC-primed CTL rapidly eliminate virus from peripheral tissues, thereby preventing later virus-induced, CTL-mediated immunopathology.

TABLE 2.

Priming with DC protects against peripheral infection and virus-induced immunopathology

Immunization (no. of bmDC)a Log10 PFU of Vacc-G2/ovaryb No. of mice protected against intracerebral LCMV infection/no. testedc
105 H8-bmDC <0.7 5/5
104 H8-bmDC <0.7 1/5
103 H8-bmDC 4.1 ± 3.5 0/5
None 6.5 ± 1.05 0/5
105 B6-bmDC 6.3 ± 0.7 0/3
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a

C57BL/6 mice were immunized i.v. with H8-bmDC or unlabeled B6-bmDC or left untreated. 

b

Mice were challenged with 2 × 106 PFU of Vacc-G2 intraperitoneally. Five days later, vaccinia virus titers were determined on BSC40 cells (three to five mice per group) and are reported as mean ± standard deviation. Statistical analysis of the data with an unpaired t test revealed two-tailed significance at P < 0.05 among the groups. Data from one of two experiments are shown. 

c

Mice were challenged with 30 PFU of LCMV strain WE intracerebrally and observed twice daily for the occurrence of convulsive lymphocytic choriomeningitis. Statistical analysis of the data with an unpaired t test revealed two-tailed significance of P < 0.05 among the groups. 

In vivo CTL priming with peptide-loaded DC is not influenced by turnover of peptide-MHC class I complexes.

H8-bmDC constitutively express the transgenic glycoprotein fragment and therefore the GP33 epitope. Thus, comparison of H8-bmDC with B6-bmDC and sDC from C57BL/6 mice (B6-sDC) loaded exogenously with GP33 peptide allowed us to assess whether peptide turnover and decay influence the in vivo priming capacities of DC. In preliminary experiments, peptide loading of B6-bmDC was tested with activation of GP33-specific T-cell receptor-transgenic T cells (36). Labeling of B6-bmDC with GP33 for 1 h at a concentration of 10−6 M led to an in vitro stimulatory capacity comparable to that of H8-bmDC (data not shown). In vivo, immunization with GP33-labeled B6-bmDC or B6-sDC led to induction of CTL responses (Fig. 6A) and protection against LCMV strain WE challenge (Fig. 6B) with the same efficiency as H8-bmDC immunization.

FIG. 6.

FIG. 6

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Priming efficiencies of H8-bmDC and of peptide-labeled B6-sDC or B6-bmDC. (A) Mice were injected i.v. with 104 H8-bmDC or 104 GP33-labeled B6-sDC or B6-bmDC, and specific CTL induction was determined as described in the legend to Fig. 2. (B) Mice were immunized i.v. with different doses of H8-bmDC or GP33-labeled B6-sDC or B6-bmDC and challenged with 200 PFU of LCMV strain WE i.v. at day 8. Virus titers in spleens were determined 4 days later.

DISCUSSION

In the present study, we evaluated the ability of DC to induce CTL-mediated antiviral immunity in vivo. When administered i.v., as few as 1,000 to 10,000 DC, either constitutively expressing an MHC class I-restricted epitope or exogenously loaded with peptide, led to the induction of virus-specific CTL, protected against challenge LCMV infection, and prevented viral replication in peripheral tissues.

The high efficiency of DC in priming T-cell responses against tumors (16, 29, 39, 46) and nonviral infections (19, 30, 31) in vivo has been shown. CTL are crucial for effective immune responses against various viral infections. Induction of antiviral CTL responses by DC has been studied in vivo mainly with secondary in vitro restimulation (10, 43). In the present study, we assessed the induction of protective antiviral CTL responses by DC in vivo. The infectious LCMV mouse model is particularly useful for this purpose, as DC are infected during primary LCMV infection (11) and well-established in vivo assays (5) allow investigation of the extent of CTL priming. Furthermore, the finely graded sensitivity of these assays facilitates comparison with other anti-LCMV immunization strategies. Priming with peptide GP33 in a mild adjuvant protects against low-dose i.v. LCMV challenge (1) but fails to protect against lethal choriomeningitis (2a). In contrast, immunization with recombinant vaccinia virus expressing LCMV glycoprotein induces a strong primary immune response with clearly detectable ex vivo cytotoxicity and long-lasting protection against LCMV infection (17). Thus, the induction of protective CTL by DC is more efficient than priming with peptides but less effective than immunization with recombinant vaccinia virus. However, only 10,000 to 100,000 DC given i.v. can induce an immune status resembling a memory situation after virus infection: (i) CTLp frequencies are only slightly lower than those in LCMV long-term memory mice (3), i.e., between 1 in 105 to 1 in 104; (ii) ex vivo cytotoxicity is measurable after prolonged incubation of target cells with effector cells (15); (iii) mice are protected against i.v. LCMV challenge infection by day 4; and (iv) lethal LCMV-specific immunopathology after intracerebral infection is prevented (35).

Transport of antigen from the periphery to organized lymphoid tissues by APC is crucial for the initiation of an immune response (7, 25). DC are probably the main APC population contributing to this antigen transport, as they migrate and home to the T-cell areas of lymphoid tissues, subsequent to antigen uptake in the periphery (23, 31). To address the contribution of DC migration to their immunogenicity, we monitored the migration pathway of bmDC and correlated it with their ability to induce protective immune responses. As described previously for sDC (4), bmDC home preferentially to the interfollicular T-cell areas of the spleen. A smaller proportion is also found in the liver-draining lymph nodes, probably due to their ability to translocate into the hepatic lymph system (23). To prime a protective CTL response in the spleen, only 100 to 1,000 DC had to reach the organ, as determined by direct intrasplenic injection. Ten times more cells, i.e., 1,000 to 10,000, had to be injected i.v. to achieve a similar priming efficiency. After s.c. injection, the majority of the bmDC rapidly migrated to the local lymph nodes (data not shown). However, even after s.c. injection of 10,000 to 100,000 DC, a minimal number of 100 to 1,000 DC still must have reached the spleen to induce a local CTL response and to protect against LCMV challenge infection. Thus, the migratory and homing capacities of DC are of critical importance in delivering antigens to the lymphoid environment.

Knowledge about peptide turnover and its influence on priming efficiency is particularly important for peptide- and cell-based vaccination strategies. During maturation, DC acquire the ability to present MHC class II-restricted peptides over prolonged periods of time (>100 h) (13). In contrast, MHC class I-peptide complexes generally show a rapid turnover (40) on DC (13). A comparison of H8-bmDC constitutively expressing GP33 peptide with exogenously pulsed B6-sDC and B6-bmDC showed that turnover of peptide-MHC class I complexes did not influence the capacity to induce antiviral immunity in vivo. These results confirm and further emphasize previous findings (29, 38) that the transport of exogenously loaded antigenic peptides to regional lymphoid tissues by DC is highly efficient.

One aim of the current DC research is the development of DC-based vaccination strategies against tumors and infectious agents. In tumor patients, therapy with autologous DC is possible (18) and necessary because of the high variability of tumor antigens. Clearly, however, individual treatment with adoptively transferred, peptide- and/or protein-pulsed DC is too complicated for general immunization. Therefore, other application pathways for delivering antigenic determinants specifically to DC should be considered. Manickan et al. (28) have shown that targeting DNA vaccines containing herpes simplex virus DNA encoding antigenic epitopes specifically to DC offers an efficient way to induce T-helper- and B-cell responses against this infectious agent. Further promising results come from experiments with “gene gun” technology, showing that this type of immunization mediates antiviral protection (45), probably via in vivo transfection of skin DC, which subsequently express the foreign protein in regional lymph nodes (14). In conclusion, vaccination via DC appears to be an effective way to induce long-lasting protective antiviral immunity.

ACKNOWLEDGMENTS

We thank Paul Klenerman and Peter Aichele for helpful discussions and critical reading of the manuscript and Lenka Vlk for expert technical assistance.

This work was supported by the Swiss National Science Foundation, the Deutsche Forschungsgemeinschaft (grants to B.L. and S.E.), and the Kanton Zürich.

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