Dendritic cells (DC) play a key role in the initiation of T cell-mediated immune responses and may therefore be successfully used in antiviral and antitumor vaccination strategies. Because both strength and duration of an immune response determines the outcome of a vaccination protocol, we evaluated the life span of DC-induced antiviral CTL memory against systemic and peripheral challenge infections with lymphocytic choriomeningitis virus (LCMV). We found that expansion and activation of CTL by DC was transient. Protection against systemic LCMV infection after DC immunization was relatively long-lived (>60 days), whereas complete protection against peripheral infection via intracerebral infection or infection into the footpad with LCMV, where rapid recruitment of effector T cells to the site of infection and elimination of viral pathogen plays a major role, was short-lived (<30 days). Protective immunity was most efficiently restored by administration of antigenic peptides via DC, rather than in combination with IFA or in liposomes. These results suggest that Ag presentation by DC may be crucial for both initiation and maintenance of protective CTL-mediated immunity against viruses infecting solid organs or against peripheral mesenchymal or epithelial tumors.

Dendritic cells form a network of sentinel cells in the periphery to facilitate Ag delivery to secondary lymphoid organs (1). Transport of microbial pathogens by DC4 is probably of key importance to initiate an immune response. In addition, because of their excellent T cell-stimulatory properties, small numbers of DC are sufficient to induce an immune response in vivo (2, 3, 4). Hence, DC presenting microbial Ags efficiently induce specific immune responses against mycobacteria (5), Leishmaniamajor (6), and influenza virus (7). Furthermore, DC rapidly expand and activate antiviral CTL, leading to protective immunity against systemic and peripheral challenge with LCMV (8). Thus, DC may be utilized as an immunotherapeutic tool for vaccination against infectious diseases. DNA vaccination represents an elegant way to take advantage of the special properties of DC to induce immune responses against infectious pathogens (9, 10, 11).

A prerequisite for successful vaccination is the establishment of a long-lasting protective immune response. Although Abs control and protect against reinfection via mucosae or blood, T cells are essential to protect against viruses or intracellular bacteria infecting cells in peripheral solid organs (12). T cell memory is characterized by elevated precursor frequencies that are proportional to the initial clonal burst size (13, 14). However, antiviral CTL memory able to protect against infection of cells in peripheral solid organs, where noncytopathic infection may cause immunopathological disease, requires not only increased precursor frequency but also antigenic restimulation for effector cells to confer protection against secondary infections (15). The aim of the current study was to examine the duration and protective capacity of DC-induced antiviral CTL memory responses. Using bone marrow-derived DC from transgenic mice ubiquitously expressing the immunodominant epitope (GP33–41; hereafter referred to as GP33) of LCMV WE strain (16), we analyzed the kinetics of CTL induction and correlated this with antiviral protection against systemic and peripheral virus challenge infections. In addition, various methods of peptide delivery were compared to determine the conditions to maintain long-term antiviral protection against peripheral infection.

All mice were obtained from the Institut für Labortierkunde (University of Zurich, Switzerland) at the age of 8–16 wk and were sex matched. Transgenic mice expressing the LCMV GP33 epitope in all tissues (H8 mice) have been previously described (16). Mice transgenic for a Vα2/Vβ8 TCR specific for H2-Db and the major LCMV-GP epitope, GP33–41 (GP33), were used as donors of transgenic T cells (17). All animals were kept under specific pathogen-free conditions.

LCMV WE strain, originally obtained from Dr. F. Lehmann-Grube (Hamburg, Germany), was propagated on L929 cells and titrated as described previously with MC-57 cells (18). EL-4 (H-2b) thymoma cells were used as target cells.

Supernatants from the following mAb-producing hybridomas were used: rat anti-mouse CD4 (YTS191.1) (19); rat anti-mouse CD8 (YTS169.4.2) (19); rat anti-mouse CD45R (RA3-3A1/6.1) (American Type Culture Collection (ATCC), Manassas, VA); and rat anti-mouse I-Ab (B21-2) (ATCC). LCMV GP33 (KAVYNFAT) (17) was synthesized by the solid-phase method and purchased from Neosystem Laboratoire (Strasbourg, France).

GP33 was labeled with BODIPY FL,SE (4,4 difluoro-5,7 dimethyl-4-bora-3a, 4a-diazasindacene-3-propionic acid, succinimidyl ester) (Molecular Probes, Eugene, OR) for the determination of encapsulated peptide in the liposomes. GP33, 1 mg dissolved in 0.3 ml bicarbonate (pH 8.0) and 2.5 mg BODIPY FL,SE (50 mg/ml), was reacted at room temperature for 2 h. The reaction was stopped by addition of 30 μl hydroxylamine (1.5 M, pH 8.5). After 1 h, the mixture was eluted with phosphate buffer (PB, 67 mM, pH 7.4) on a Bio-Gel P6-DG (30 × 1 cm, Bio-Rad, Richmond, CA) to remove unreacted dye. Labeled GP33 was used as tracer in mixtures with known amounts of unlabeled peptide. Small unilamellar liposomes were prepared by freeze-thawing followed by sequential filter extrusion as described previously (20). The basic composition of the liposomes used was 80 mg/ml soy phosphatidylcholine, 10 mg/ml cholesterol and 0.45 mg/ml dl-α-tocopherol. The dried lipid mixture was solubilized with GP33 (4 mg/ml) and repetitively extruded through Nuclepore (Sterico, Dietikon, Switzerland) filters (0.8, 0.4 and 0.2 μm). Unencapsulated GP33 was removed by dialysis, and the amount of encapsulated peptide was determined by fluorescence measurement at 513 nm. Liposome size and homogeneity were determined by laser light scattering (Submicron Particle Sizer Model 370, Nicomp, Santa Barbara, CA) resulting in homogeneous populations of unilamellar liposomes with a size range of 80–180 nm.

Dendritic cells were prepared from bone marrow cultures as previously described (8). Briefly, bone marrow was flushed from femurs and tibias and subsequently depleted of erythrocytes with ammonium chloride. Bone marrow cells were depleted of T cells, B cells and I-Ab-positive cells using a mixture of mAbs (CD4, YTS191.1; CD8, YTS169.4.2; CD45R, RA3-3A1/6.1; I-Ab, B21-2) and goat anti-rat IgG-coated Dynabeads (Dynal, Oslo, Norway). Cells were cultured in RPMI 1640 supplemented with 5% FCS, penicillin/streptomycin, 10 ng/ml recombinant murine GM-CSF (kindly supplied by Novartis, Vienna, Austria), and 5 ng/ml IL-4 obtained by adding IL-4-containing supernatant from the cell line X63-IL4 (kindly provided by Dr. M. Kopf, Basel, Switzerland). At day 8, nonadherent cells were collected and further purified over metrizamide (14.5% in RPMI 1640, 5% FCS) (Sigma, St. Louis, MO). Cells were washed three times with balanced salt solution, and injected in a volume of 0.5 ml balanced salt solution i.v. H8-DC present solely the GP33 CTL epitope. Virus-specific Th cells, e.g., TCR-transgenic Th cells from Smarta mice (21), were not specifically stimulated by H8-DC, unless the respective peptide was provided.

Specific cytotoxicity was determined in a standard 51Cr release assay as described (22). Briefly, spleen cell suspensions were prepared from spleens of immunized mice at the indicated time point after priming. EL-4 cells were labeled with GP33 (10−6 M) and 250 μCi 51Cr for 1.5 h at 37°C. Target cells, 104 per well, were incubated for 15 h in 96-well round-bottom plates with 3-fold serial dilutions of spleen effector cells, starting at an E:T ratio of 90:1. EL-4 cells without peptide served as controls. The supernatant of the cytotoxicity cultures was counted in a Cobra II γ Counter (Canberra Packard, Downers Grove, IL). The percentage of specific lysis was calculated as [(experimental release − spontaneous release)/(total release − spontaneous release)] × 100. Spontaneous release was always below 29%.

Naive C57BL/6 mice or C57BL/6 mice transfused i.v. with 5 × 105 syngeneic spleen cells from 318 mice on day −1 (B6/318) were immunized i.v. with 2 × 105 H8-DC. At the indicated times after DC immunization, mice were challenged i.v. with 200 PFU of LCMV or i.c. with 30 PFU LCMV or s.c. into one hind footpad with 5 × 104 PFU LCMV. Virus titers in the spleens were determined 4 days after i.v. challenge and in the footpad at the indicated times in a LCMV infectious focus assay as previously described (18). The incidence for convulsive LCM disease, scored as severe wasting and convulsions upon tail spinning, was registered during 3 wk. The LCMV-induced footpad swelling reaction (22) was monitored daily with a spring-loaded caliper.

Naive C57BL/6 mice were transfused i.v. with 5 × 105 syngeneic spleen cells from 318 mice (containing ∼5 × 104 TCR-transgenic CTL) on day −1 (B6/318). To detect expansion of transgenic TCR-expressing T cells (23) after immunization with H8-DC or infection with LCMV, peripheral blood cells were stained for CD8, transgenic Vα2 and Vβ8.1, using FITC-conjugated rat anti-mouse CD8, PE-conjugated rat anti-mouse Vα2, and biotinylated rat anti-mouse Vβ8.1 (all from PharMingen, San Diego, CA) followed by streptavidin-Tricolor (Caltag, South San Francisco, CA). Activation of GP33-specific CTL was detected by staining with biotinylated anti-CD62L (PharMingen) followed by incubation with streptavidin-Tricolor (Caltag). Erythrocytes were lysed with FACS lysis solution (Becton Dickinson, Mountain View, CA), and the cell suspensions were analyzed on a FACScan flow cytometer (Becton Dickinson) after gating on viable lymphocytes.

In a first set of experiments, we compared the expansion and activation of virus-specific CTL by LCMV and H8-DC. To this end, 5 × 105 spleen cells from 318 TCR-transgenic mice expressing a GP33-specific TCR (17) on 50–60% of their CD8+ T cells were adoptively transferred into naive C57BL/6 recipient mice (B6/318). After 24 h, mice were immunized i.v. with 200 PFU LCMV (Fig. 1, A–C) or 2 × 105 H8-DC (Fig. 1, D–F). After LCMV infection, the CTL response peaked on day 8 when 70–80% of the CD8+ lymphocyte population and 30–40% of the PBL were transgenic T cells (Fig. 1,A). Maximal expansion of virus-specific CTL occurred between days 4 and 8 and was followed by a slow but continuous decrease until day 30 (Fig. 1,B); then levels remained constant until at least 120 days after infection (not shown and 23). Highly activated CTL with immediate cytototxic effector function are CD62L negative (24); therefore, the activation status of CTL can be directly measured by monitoring the expression levels of the lymph node homing receptor CD62L. More than 95% of the Vα2+CD8+ CTL had down-regulated CD62L during the phase of maximal expansion, and on day 30 still more than 70% of these cells were CD62L negative (Fig. 1,C). In comparison with the vigorous expansion of TCR-transgenic CTL after LCMV infection, these cells expanded only modestly after H8-DC priming. The peak of the response (35–45% Vα2+Vβ8.1+CD8+ lymphocytes and 4–7% of the PBL) was reached on day 4 (Fig. 1, D and E). Down-regulation of CD62L on Vα2+CD8+ lymphocytes in DC-primed mice was transient and reached significantly lower levels than after LCMV infection (Fig. 1 F).

FIGURE 1.

Expansion and activation of virus-specific cytotoxic T cells by LCMV and H8-DC. C57BL/6 mice that had been adoptively transfused with 5 × 104 TCR transgenic CTL (B6/318) were immunized i.v. 1 day after transfusion with 200 pfu LCMV (A–C) or with 2 × 105 H8-DC (D--F). PBL were analyzed at the indicated time points for CD8 and TCR Vα2 and Vβ8.1 expression by flow cytometry (A, B, D, and E). TCR-transgenic CTL expanded maximally on day 8 after LCMV infection (A) and on day 4 after DC immunization (D); the numbers in bold represent the percentage of TCR Vα2+Vβ8.1+ cells within the CD8+ T cell population and the numbers in brackets indicate the percentage of transgenic T cells of total PBL. The kinetics of virus-specific T cell expansion after LCMV infection (B) and H8-DC (E) immunization was followed for 30 days. Activation of TCR transgenic CTL after LCMV infection (C) and H8-DC immunization (F) was monitored by three-color FACS analysis using down-regulation of CD62L on CD8+Vα2+ T cells as a marker. Values in B–C and E–F represent the mean values ± SD of two to three mice per group. One of two experiments is shown.

FIGURE 1.

Expansion and activation of virus-specific cytotoxic T cells by LCMV and H8-DC. C57BL/6 mice that had been adoptively transfused with 5 × 104 TCR transgenic CTL (B6/318) were immunized i.v. 1 day after transfusion with 200 pfu LCMV (A–C) or with 2 × 105 H8-DC (D--F). PBL were analyzed at the indicated time points for CD8 and TCR Vα2 and Vβ8.1 expression by flow cytometry (A, B, D, and E). TCR-transgenic CTL expanded maximally on day 8 after LCMV infection (A) and on day 4 after DC immunization (D); the numbers in bold represent the percentage of TCR Vα2+Vβ8.1+ cells within the CD8+ T cell population and the numbers in brackets indicate the percentage of transgenic T cells of total PBL. The kinetics of virus-specific T cell expansion after LCMV infection (B) and H8-DC (E) immunization was followed for 30 days. Activation of TCR transgenic CTL after LCMV infection (C) and H8-DC immunization (F) was monitored by three-color FACS analysis using down-regulation of CD62L on CD8+Vα2+ T cells as a marker. Values in B–C and E–F represent the mean values ± SD of two to three mice per group. One of two experiments is shown.

Close modal

The phenotypic analysis revealing that priming with DC resulted in less expansion and fewer activated CTL correlated with the GP33-specific CTL activity in cytotoxicity assays (Fig. 2). B6/318 mice were immunized with either 200 PFU LCMV (Fig. 2,A) or H8-DC (Fig. 2,B). To control for the contribution of initially elevated CTLp frequencies due to the transfer of naive 318 TCR transgenic CTL, untreated C57BL/6 mice were also immunized with H8-DC (Fig. 2,C). On days 8, 16, and 30 after immunization, splenic CTL activity was measured in a 15 h 51Cr release assay which permits the detection of relatively weak CTL activities (8, 24, 25). Direct ex vivo CTL activity after LCMV infection was high for 30 days in B6/318 mice (Fig. 2,A), comparable with LCMV infection of untreated mice (not shown). In contrast, DC-induced CTL activity was ∼10–20 times lower and had completely waned by day 30 in both B6/318 and control B6 mice (Fig. 2, B and C). Taken together, these results show that priming of CTL by DC was rapid, but less efficient compared with infection with LCMV. Expansion and activation of Ag-specific CTL after DC priming was less extensive and direct ex vivo CTL activity was short-lived.

FIGURE 2.

Comparison of long-term CTL activity after LCMV infection and DC priming. B6/318 mice were immunized i.v. on day 0 with 200 PFU LCMV (A) or 2 × 105 H8-DC (B). Untreated C57BL/6 mice were immunized i.v. on day 0 with 2 × 105 H8-DC to control for the contribution of elevated CTLp frequencies (C). At different time points after immunization, direct ex vivo CTL activity was tested in a 15-h 51Cr release assay on GP33-labeled (filled symbols) or on unpulsed (open symbols) EL-4 target cells at the indicated E:T ratios. Spontaneous release after 15 h was <26%. Data from two individual mice per group are shown. One of two experiments is shown.

FIGURE 2.

Comparison of long-term CTL activity after LCMV infection and DC priming. B6/318 mice were immunized i.v. on day 0 with 200 PFU LCMV (A) or 2 × 105 H8-DC (B). Untreated C57BL/6 mice were immunized i.v. on day 0 with 2 × 105 H8-DC to control for the contribution of elevated CTLp frequencies (C). At different time points after immunization, direct ex vivo CTL activity was tested in a 15-h 51Cr release assay on GP33-labeled (filled symbols) or on unpulsed (open symbols) EL-4 target cells at the indicated E:T ratios. Spontaneous release after 15 h was <26%. Data from two individual mice per group are shown. One of two experiments is shown.

Close modal

An important feature of antivirally protective CTL is their potential to migrate through solid tissue. The antiviral protective capacity of CTL during the effector and the memory phase can be evaluated in an experimental setup in which antiviral protection critically depends on the migration of CTL to the site of infection, early interference with virus spread, and rapid elimination of the virus to avoid lethal immunopathology (26). Therefore, to assess the antiviral capacity of DC-primed CTL in vivo, mice were challenged with LCMV WE i.c. (or in the the footpad, see later) and protection against immunopathological CTL-mediated LCM was monitored. C57BL/6 mice immunized with high (106) (Fig. 3,A) or intermediate (1–2 × 105) (Fig. 3,B) doses of H8-DC were protected against i.c. challenge on day 8 after immunization. Interestingly, even after adoptive transfer of 106 DC, corresponding to ∼1–2-times the number of DC present in a normal spleen (0.5–1% of all spleen cells are bone marrow-derived DC), the protection against i.c. challenge waned in only 16 days (Fig. 3,A). Elevating the precursor frequency of virus-specific CTL by adoptive transfer of 318 TCR-transgenic T cells (B6/318) significantly prolonged the protection against i.c. challenge after H8-DC priming up to day 30 when 65% of the mice survived. However, by day 60 protection was lost, despite the initially elevated CTp frequencies (Fig. 3 B, filled bars).

FIGURE 3.

Protective antiviral immunity after DC priming against peripheral challenge infection with LCMV is short-lived. A, C57BL/6 mice were immunized i.v. on day 0 with 1 × 106 (▪) or with 1 × 105 H8-DC. (□) At the indicated times postimmunization, mice were challenged with 30 PFU LCMV i.c. and checked twice daily for convulsive LCM disease. Values represent percentage of protected animals in groups of 3–4 animals. B, B6/318 mice (▪) or untreated C57BL/6 mice (□) were immunized i.v. on day 0 with 2 × 105 H8-DC and challenged i.c. with 30 PFU LCMV at the indicated times (3–4 animals per group). C, C57BL/6 mice (5–9 mice per group) were immunized on day 0 with 2 × 105 H8-DC i.v. and challenged at the indicated times postimmunization of mice with 5 × 104 pfu LCMV into one hind footpad. Memory mice that had been infected i.v. with 200 PFU LCMV on day −60 before intrafootpad challenge served as controls for optimal protection. The footpad swelling reaction was monitored daily using a spring-loaded caliper.

FIGURE 3.

Protective antiviral immunity after DC priming against peripheral challenge infection with LCMV is short-lived. A, C57BL/6 mice were immunized i.v. on day 0 with 1 × 106 (▪) or with 1 × 105 H8-DC. (□) At the indicated times postimmunization, mice were challenged with 30 PFU LCMV i.c. and checked twice daily for convulsive LCM disease. Values represent percentage of protected animals in groups of 3–4 animals. B, B6/318 mice (▪) or untreated C57BL/6 mice (□) were immunized i.v. on day 0 with 2 × 105 H8-DC and challenged i.c. with 30 PFU LCMV at the indicated times (3–4 animals per group). C, C57BL/6 mice (5–9 mice per group) were immunized on day 0 with 2 × 105 H8-DC i.v. and challenged at the indicated times postimmunization of mice with 5 × 104 pfu LCMV into one hind footpad. Memory mice that had been infected i.v. with 200 PFU LCMV on day −60 before intrafootpad challenge served as controls for optimal protection. The footpad swelling reaction was monitored daily using a spring-loaded caliper.

Close modal

To support the conclusion that antiviral protection against a peripheral infection is limited after DC priming, CTL memory was assessed in a second model of peripheral infection. DC-primed C57BL/6 mice were challenged with LCMV into the footpad, and the CTL-mediated footpad swelling reaction was monitored. The kinetics and degree of the swelling reaction directly correlate with the vigor and the kinetics of the antiviral (memory) CTL response (22). Fig. 3,C shows that in naive mice the swelling reaction peaked on day 7–8. LCMV-immune mice were optimally protected and failed to generate a swelling reaction. In DC-primed mice challenged 8 days after priming, the swelling reaction was accelerated by 5–6 days vs naive mice and was less severe, indicating that activated CTL mediated relatively effective protection. In contrast, mice challenged 30 days after DC priming showed an increased swelling reaction which was accelerated only by 2–3 days (Fig. 3,C), suggesting that protection was suboptimal. This suboptimal protection was not sufficient to protect against LCM disease (Fig. 3, A and B).

The requirements for protection against a systemic infection are overall different from protection against a peripheral infection because CTL do not have to recirculate through peripheral tissues to mediate effector function. Thus, measuring CTL memory by determining virus titers in the spleen after an i.v. challenge assays for memory CTL that do not have to emigrate and home to solid tissue before being able to exert effector function. In addition, such memory CTL may be reactivated in secondary lymphoid tissues by hematogenically distributed virus (24, 27). To determine the life span of such not acutely activated memory CTL, virus titers were measured in spleens of DC-immunized mice 4 days after i.v. challenge with LCMV (Fig. 4). H8-DC primed C57BL/6 mice were efficiently protected for at least 60 days after priming. However, this protection was lost by day 150 (Fig. 4), despite the fact that virus-specific CTL still could be restimulated in vitro (not shown). Similarly, no virus was detected in the spleens of mice that had been primed with DC 30 days before challenge into the footpad (data not shown). Thus, despite the fact that after 30 days DC-primed mice only suboptimally controlled peripheral infections, they were able to prevent systemic virus spread. Taken together, DC immunization induced a relatively long-lived protective memory CTL response against systemic virus spread. However, under more physiological conditions, when activated CTL were needed to rapidly eliminate virus from peripheral solid tissues to avoid immunopathological disease in the meninges or the footpad, DC-induced CTL memory was short-lived.

FIGURE 4.

Protection against a systemic LCMV infection after priming with DC is long-lived. C57BL/6 mice were immunized i.v. on day 0 with 2 × 105 H8-DC that induce CTL but cannot induce neutralizing Ab responses or virus-specific Th cells. At the indicated times, mice were challenged i.v. with 200 PFU LCMV, and the virus titers in spleen were determined 4 days later. Untreated mice served as naive controls. The broken line represents the detection limit.

FIGURE 4.

Protection against a systemic LCMV infection after priming with DC is long-lived. C57BL/6 mice were immunized i.v. on day 0 with 2 × 105 H8-DC that induce CTL but cannot induce neutralizing Ab responses or virus-specific Th cells. At the indicated times, mice were challenged i.v. with 200 PFU LCMV, and the virus titers in spleen were determined 4 days later. Untreated mice served as naive controls. The broken line represents the detection limit.

Close modal

The above data suggest that DC can induce a long-lived expansion of virus-specific CTLp. However, to achieve protective immunity also against a peripheral virus challenge, CTL had to be recently activated. Therefore, we evaluated different conditions to restimulate DC-primed CTL to restore protection against LCM disease. To this end, B6/318 mice or C57BL/6 mice were immunized with 2 × 105 H8-DC and were restimulated after 69 days, when protection against LCM disease was lost, with peptide-presenting DC, peptide delivered in the mild adjuvant IFA, or peptide encapsulated in liposomes. One day later, the mice were infected i.c. with LCMV (Table I). Protection against lethal LCM could only be efficiently restored by antigenic restimulation with DC and was very limited after restimulation with peptide in IFA or liposomes (Table I). Interestingly, protection against peripheral infection was optimally recalled only by DC, irrespective of the initial CTLp frequencies or the route of DC application (Table I). Thus, antiviral CTL memory seems to be most efficiently restored and maintained by delivery of Ag via DC.

Table I.

Protection against intracerebral LCMV infection is best maintained by restimulation with DC

Primary ImmunizationaAntigenic Restimulation on Day 69bProtection Against i.c. LCMV Challenge Infection on Day 70c
Expt. 1, H8-DC → B6/318   
2× 105 H8-DC i.v. 2× 105 H8-DC i.v. 5 /5 
2× 105 H8-DC i.v. 100 μg gp33/IFA i.d. 2 /5 
2× 105 H8-DC i.v. 100 μg gp33/Lipos. i.d. 1 /5 
2× 105 H8-DC i.v. None 0 /3 
   
Expt. 2, H8-DC → B6   
2× 105 H8-DC i.v. 2× 105 H8-DC s.c. 5 /5 
2× 105 H8-DC i.v. 100 μg gp33/IFA s.c. 1 /5 
2× 105 H8-DC i.v. 100 μg gp33/Lipos. s.c. 2 /5 
2× 105 H8-DC i.v. None 0 /3 
None 2× 105 H8-DC s.c. 0 /3 
Primary ImmunizationaAntigenic Restimulation on Day 69bProtection Against i.c. LCMV Challenge Infection on Day 70c
Expt. 1, H8-DC → B6/318   
2× 105 H8-DC i.v. 2× 105 H8-DC i.v. 5 /5 
2× 105 H8-DC i.v. 100 μg gp33/IFA i.d. 2 /5 
2× 105 H8-DC i.v. 100 μg gp33/Lipos. i.d. 1 /5 
2× 105 H8-DC i.v. None 0 /3 
   
Expt. 2, H8-DC → B6   
2× 105 H8-DC i.v. 2× 105 H8-DC s.c. 5 /5 
2× 105 H8-DC i.v. 100 μg gp33/IFA s.c. 1 /5 
2× 105 H8-DC i.v. 100 μg gp33/Lipos. s.c. 2 /5 
2× 105 H8-DC i.v. None 0 /3 
None 2× 105 H8-DC s.c. 0 /3 
a

C57BL/6 mice transfused with 5 × 105 TCR transgenic 318 spleen cells (Expt. 1) or C57BL/6 mice (Expt. 2) were immunized i.v. with 2 × 105 H8-DC or left untreated.

b

Mice received either 2 × 105 H8-DC i.v., 100 μg gp33 intradermally or s.c. either in IFA or as a liposomal formulation (Lipos.) on day 69 or were left untreated.

c

Mice were challenged i.c. (30 PFU LCMV-WE) on day 70 and observed twice daily for the occurrence of convulsive LCM disease.

The present report investigated the long term kinetics of DC-induced CTL activation and the conditions to maintain protective antiviral CTL memory after DC-immunization. Using an adoptive transfer system of TCR transgenic T cells, we found that DC rapidly expand virus-specific CTL. Given that a mouse spleen contains 5–8 × 107 lymphocytes and that ∼20% of the transferred 5 × 104 GP33-specific TCR-transgenic CTL initially reached the spleen, these CTL expanded >100-fold within 4 days after DC priming. Interestingly and in contrast to the prolonged expansion and activation of virus-specific transgenic CTL by LCMV (Ref. 23 and this report), the DC-activated CTL population rapidly collapsed and CTL activity was lost. This is probably due to the relatively short half-life of DC, because DC presenting a defined immunogenic peptide disappear after 2–3 days (28). Furthermore, elimination of antigenic peptide-presenting DC may be enhanced by specific CTL. Thus, it appears that CTL activation by DC is rapid and efficient, but limited due to the rapid loss of Ag-presenting DC. In contrast, after infection with replicating LCMV, stimulatory Ag is less limiting, and it can be assumed that larger numbers of DC present specific Ag over a prolonged period of time, probably because LCMV WE persists at very low levels in a murine host below levels that can be detected by conventional methods.5

Immunological T cell memory, particularly CTL memory, is mediated by long-lived memory lymphocytes, if CTLp is taken as readout (13, 14). However, it has also been shown that persisting Ag capable of reactivating memory CTL or keeping CTL in an activated state is particularly important to maintain protection of solid organs against viral infections (15, 27). The presented results show that DC priming induces rather short lived CTL memory responses that are capable of rapidly responding to and protecting against a peripheral infection to confer protection. Even when virus-specific CTLp frequency was increased by adoptive transfer of TCR-transgenic CTL, protection nevertheless faded away.

Vaccination with synthetic peptides is a well-established way to induce primary T cell responses in vivo (29, 30, 31). A dose of 100 μg GP33 applied s.c. in the mild adjuvant IFA (32) induces a protective CTL response against i.v. LCMV challenge. Furthermore, it has been shown that repetitive, high dose administration of GP33 specifically tolerizes naive (32) but not memory CTL (33). Because memory T cells have lower thresholds for activation than naive T cells (34), we hypothesized that restimulation of a protective DC-induced CTL memory response could be easily accomplished by antigenic peptides. Surprisingly, only restimulation with DC completely restored protection against the highly demanding i.c. LCMV challenge, peptides failed to efficiently restore protective antiviral immunity. Whether the insufficient restimulatory capacity of the peptide formulation is the result of overstimulation of all available GP33-specific CTL leading to their subsequent elimination (35), inappropriate stimulation during cell cycle (36), or peptide-induced immunopathology (33) remains to be elucidated. Here, it is particularly interesting that DC most efficiently restimulated memory CTL. Therefore, the strong costimulatory capacity of DC in concert with their ability to specifically deliver Ags to the T cell areas of secondary lymphoid organs seems to be not only crucial for the initiation of a T cell response but also a key for the maintenance of highly active, protective CTL immune responses.

Taken together, the presented results indicate the following rules for the application of DC for vaccination purposes. 1) The half-life of Ag delivered by DC is short-lived and correlates with the short-lived activation of CTL. Hence, DC-induced CTL memory may well protect for prolonged periods in the absence of specific Ag against a systemic infection that rapidly reactivates T cells in the lymphoid organs, leading to efficient virus clearance. However, CTL immune responses protecting effectively against infections of peripheral tissues must be preactivated and therefore require Ag. 2) Optimal restimulation of protective CTL is achieved when the Ag is delivered by DC. Thus, both improvement of Ag delivery to DC and prolongation of Ag persistence, e.g., by specifically targeting vaccines to DC via special delivery vehicles, may enhance the initiation and the maintenance of protective T cell responses against peripheral virus infections and probably also against peripheral mesenchymal and peripheral tumors.

We thank Andrew MacPherson for helpful discussions and critical reading of the manuscript.

1

This work was supported by the Swiss National Science Foundation, a postdoctoral fellowship by the Deutsche Forschungsgemeinschaft to B.L., and the Kanton Zürich.

4

Abbreviations used in this paper: DC, dendritic cells; LCMV, lymphocytic choriomeningitis virus; PB, phosphate buffer; i.c., intracranially; CTLp, CTL precursor; CD62L, CD62 ligand.

5

A. Ciurea, P. Klenerman, and R. M. Zinkernagel. Persistence of LCMV at very low levels in immune mice. Submitted for publication.

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