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The Journal of Immunology, 2001, 166: 3440-3450.
Copyright © 2001 by The American Association of Immunologists

Prophylactic Tumor Vaccination: Comparison of Effector Mechanisms Initiated by Protein Versus DNA Vaccination1

Margot Zöller2,*,{dagger} and Oliver Christ*

* Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg, Germany; and {dagger} Department of Applied Genetics, University of Karlsruhe, Karlsruhe, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Clinical success in tumor vaccination frequently does not reach expectation. Since vaccination protocols are quite variable, we used the murine renal cell carcinoma line RENCA transfected with the lacZ gene (RENCA-{beta}-gal) to compare the efficacy of two different vaccination strategies or their combination and to elaborate on the underlying mechanisms. BALB/c mice were vaccinated either with naked lacZ DNA or with attenuated Salmonella typhimurium transformed with lacZ DNA or with dendritic cells (DC) loaded with the {beta}-galactosidase protein or mice were vaccinated with both DNA and protein. Although all regimens led to a prolongation of survival time, oral vaccination with transfected S. typhimurium followed by i.v. transfer of protein-loaded DC provided the optimal schedule. In this setting, >50% of mice remained tumor free after challenge with 10 times the lethal tumor dose of RENCA-{beta}-gal. As explored in transfer experiments, the superior efficacy of combining DNA and protein vaccination is due to the facts that 1) optimal protection depends on both activated CD4+ and CD8+ cells and 2) CD8+ CTL are most strongly activated by vaccination with transformed Salmonella, whereas vaccination with protein-loaded DC is superior for the activation of Th. The latter induced sustained activation of CTL and recruitment of nonadaptive defense mechanisms. The data demonstrate the strength of DNA vaccination, particularly by the oral route, and provide evidence that a combined treatment with protein-loaded DC can significantly increase the therapeutic efficacy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The identification of tumor rejection Ags has first been demonstrated by the group of Boon (1). Starting from this seminal work, advances in genomic technology led to new strategies which significantly accelerate the identification of gene products that are selectively overexpressed in the tumor (2, 3). An alternative approach is based on the isolation of peptides being presented by MHC class I and class II molecules (4). Furthermore, by a better crystallographic resolution of the peptide-binding groove of MHC molecules, rather reliable predictions of binding peptides can be done in silico (5, 6). Thus, it soon will be feasible to provide a comprehensive map of tumor-associated Ags to set the ground for therapeutic vaccination of the major tumor types. Such an extrinsic stimulus, however, is mandatory because patients with cancer obviously do not mount an antitumor response spontaneously even in case tumor cells do express immunogenic Ags.

Different approaches of active immunization are currently explored, i.e., vaccination with cDNA (7, 8), RNA (9), proteins, or peptides themselves (10, 11). DNA vaccines induce immune responses by direct expression of the antigenic proteins in host cells. Delivery of naked DNA has mostly been following either the i.m. or the intradermal route. It is supposed that after i.m. application both monocytes and DCs may express the Ag. Transcription can be observed rather rapidly and may last for about 1 mo. Both Th1 and Th2 cell responses have been recorded, depending on whether the Ag is a secretory product (Th2) or an intracellular or membrane-anchored molecule (Th1) (12, 13). In addition, certain DNA motifs (CpG) are immunostimulatory and strongly augment the amplitude of immune response (13, 14). Packaging of DNA in attenuated Salmonella or Listeria has opened the way of oral application (15, 16). These bacteria can cross the epithelial barrier of the gut and will be taken up by macrophages in the peritoneal cavity, where they survive for a limited number of cell cycles. It has been described that the DNA introduced into the bacteria will be transcribed in the host cells, which either present the Ag directly or eventually may die such that dendritic cells (DC)3 will take up the cell debris and present resulting peptides preferentially (although not exclusively) on MHC class II molecules. Monocytes which have ingested the bacteria will present the molecules in the context of MHC class I molecules. These features explain why after DNA vaccination via a bacterial transporter Th as well as CTL responses and Ab production can be observed (17, 18).

Vaccination with tumor extracts, tumor-associated proteins, and peptides derived thereof has become an important tool after succeeding with the in vitro generation of DC from bone marrow cells or from peripheral blood leukocytes (19, 20, 21, 22, 23, 24, 25, 26). Ex vivo-loaded DC have been shown to most efficiently activate T cells (25, 26, 27). Notably, in clinical studies reported so far, responses without side effects have been observed (28, 29, 30, 31, 32). The efficacy of vaccination with protein-loaded DC possibly could be due to the preferential activation of Th cells, which accounts for a number of well-known advantages in initiating an immune response (21, 33, 34, 35, 36, 37, 38). Thus, it is known that full activation of CTL depends on the right cytokines being provided by Th cells (21, 36). Furthermore, recruitment and activation of nonadaptive defense mechanisms, presented mainly by NK cells, monocytes/macrophages, and granulocytes, can be achieved by activation of Th cells (39, 40).

Comparative animal studies to explore the efficacy of DNA, RNA, or protein/peptide vaccination are largely missing, but are required to eventually give an idea which strategy is best suited for clinical application. In this study, we addressed the basic alternative of tumor vaccination by DNA vs protein vaccination.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and tumors

BALB/c (H-2d) and SCID (H-2 d) mice were bred at the central animal facilities of the German Cancer Research Center. Animals were kept under specific pathogen-free conditions. They were fed sterilized food and water at libitum. Animals were used for experiments at the age of 8–10 wk. The BALB/c-derived renal cell carcinoma line (RENCA) (41) was used throughout. RENCA cells were cotransfected with the lacZ cDNA-containing plasmid pVAX (Invitrogen, San Diego, CA) and the pcDNA3 vector containing the neomycin resistance gene (RENCA-{beta}-gal) by electroporation (1050 µF, 260 V). Transfected cells were selected by growth in G418-containing medium (500 µg/ml). Surviving cells were cloned by limiting dilution. Expression was tested by 5-bromo-4-chloro-3-indolyl {beta}-D-galactoside (X-Gal) staining. Cells were cultured in RPMI 1640 supplemented with 10% FCS.

Monoclonal antibodies

The following mAb were used: YTA 3.1.2 (anti-CD4), YTS169.4.2.1 (anti-CD8), YBM 6.1.10 (anti-Mac-1), and 33.1 (anti-DC). An I-Ad-specific mAb (K24-199) (42) was kindly provided by G. Hämmerling (German Cancer Research Center, Heidelberg, Germany). Anti-CD28, anti-CD40, anti-CD40 ligand (CD40L), anti-CD80, anti-CD86, anti-CD152, anti-IL-2, anti-IL-4, anti-IL6, anti-IL10, anti-IL12, anti-IFN-{gamma}, anti-TNF-{alpha}, and secondary dye-labeled (FITC or PE) Abs were obtained commercially (PharMingen, Hamburg, Germany).

For flow cytometry, 3–5 x 105 cells were stained according to routine procedures. When assaying cytokine-producing cells, lymphocytes were fixed with formaldehyde and permeabilized with PBS/1% Tween 20. Fluorescence was determined using a FACStar (Becton Dickinson, Heidelberg).

Transformation of Salmonella typhimurium aroA (SL7207)

The attenuated S. typhimurium aroA strain SL7207 (43) (SL) was grown in Luria-Bertani (LB) medium. Competent bacteria were transformed with the plasmid pVAX containing the lacZ gene cDNA (SL-lacZ; Invitrogen) by electroporation 2500 V, 25 µF). Transformation was controlled by selection with kanamycin. After 1 h at 37°C in LB medium, cells were cultured overnight on agar plates containing 50 µg/ml kanamycin. For vaccination via the oral route, transformed SL were cultured overnight in LB medium containing 2% NaCl and 50 µg/ml kanamycin at 37°C. After determination of OD600 (OD600 1 roughly corresponds to 1 x 108 SL/ml), cells were centrifuged at 6000 rpm and washed twice with PBS. Cells were resuspended in PBS (containing 10% bicarbonate, pH 9.6), pH 8.3–8.5, to neutralize the acid pH of the stomach. Mice received 1 x 108 bacteria in 100 µl via probang.

DNA preparation

The K12/DH5a Escherichia coli strain was transformed with the pVAX or the pVAXlacZ plasmid as described above. Cells were cultured in LB medium with kanamycin (50 µg/ml). Plasmid DNA was isolated using the mega-prep kit (Quiagen, Chatsworth, CA).

Generation of DC

A modification of the protocol of Inaba et al. (44) has been used. In brief, freshly harvested bone marrow cells (1.5–2 x 106/ml) were suspended in IMEM containing 5% FCS and were seeded in 50-ml culture flasks. After an incubation period of 2 days, nonadherent cells were removed by washing and the plastic-adherent cells were cultured in IMEM containing 5% FCS and 8 ng/ml murine GM-CSF, 2.5 ng/ml IL-4, and 50 U/ml IFN-{gamma} (Stratmann, Hannover, Germany). The medium was exchanged every third day. After 7 days of culture, DC were loaded with 20 µg/ml recombinant {beta}-galactosidase ({beta}-gal). Loading was performed for 2 days. The protein-containing medium was sucked off and DC were carefully washed with IMEM/5% FCS. DC were allowed to mature for an additional 2 days in the medium described above. The maturity of DC was verified by FACS analysis (high expression of MHC class II and a DC marker) and the microscopic appearance of veiled cells.

In vitro assays

Proliferative activity of spleen cells (SC) and lymph node cells (LNC) was evaluated by culturing titrated numbers of cells (2 x 105-2.5 x 104/well) in the presence or absence of 10 µg/ml {beta}-gal in 96-well round-bottom plates. Cells were cultured for 3 days, adding [3H]thymidine (10 µCi/ml) during the last 16 h of culture. Plates were harvested using an automatic harvester and [3H]thymidine incorporation was evaluated in a beta counter.

Ab production was determined by ELISA. Serum (1:20 dilution) or culture supernatants were placed in 96-well ELISA plates that had been coated with 10 µg/ml {beta}-gal. After overnight incubation at 4°C, plates were washed and alkaline phosphatase-conjugated anti-mouse IgG was added for 4 h. Substrate was added after washing and the OD of the enzyme reaction was measured after 20–30 min at 405 nm.

Cytotoxicity of lymphokine-activated killer (LAK) cells and CTL was measured according to the protocol described by Matzinger (45). In brief, target cells were labeled overnight with [3H]thymidine (10 µCi/ml) and were seeded (104 cells/well) after washing on titrated numbers (5 x 105-6 x 104) of effector cells in round-bottom microtiter plates. After an incubation for 6 h at 37°C, plates were harvested and the remaining radioactivity was determined in a beta counter. LAK activity was determined by using nontransfected RENCA cells as target cells. CTL activity was defined by the lysis of lacZ-transfected RENCA cells. To differentiate between LAK and CTL activity, cultures contained a 10-fold excess of cold target RENCA cells. Activity of LAK and CTL was determined using SC and LNC of vaccinated mice that had been cultured for 2 days in the presence of IL-2 (10 U/ml, LAK) or for 10 days in the presence of {beta}-gal (10 µg/ml) in medium supplemented with 10% TCGF (supernatant of Con A-stimulated rat SC) (CTL). Cytotoxicity is presented as percent cytotoxicity = 100 x (counts in control wells - counts in test wells)/(counts in control wells). The spontaneous release of RENCA/RENCA-{beta}-gal cells was in the range of 6–12%; SD of triplicate cultures were in the range of 3–5%.

The frequencies of proliferating and cytotoxic cells were evaluated under limiting dilution (LD) conditions. Cells were titrated using 24 replicates of 200–25,600 cells/well. Irradiated BALB/c thymocytes were added as feeder cells (1 x 105/well) in medium containing 20 µg/ml {beta}-gal. Proliferative activity was determined after 5 days of culture and cytotoxic activity after 10 days of culture. In the latter case, 3–5 x 103 [3H]thymidine-labeled target cells were added during the last 6 h of culture. The frequencies of proliferating CTL were calculated according to the formula: F0 (fraction of nonresponding cultures) = e-u, where u = c/w (number of cells (c) distributed in wells (w)) (46).

In vivo studies

BALB/c mice received a s.c. injection of 5 x 104 RENCA-{beta}-gal cells. Tumor growth was controlled by measuring the mean diameter twice per week. Mice were killed when the mean tumor diameter reached 2.5 cm or when animals became anemic, cachetic, or short of breath. These time points were defined as survival time.

Mice were vaccinated at 21-day intervals. They received either 1 x 108 SL-lacZ orally or 100 µg of lacZ DNA i.m., or 5 x 105 {beta}-gal-loaded DC i.v. Tumors were inoculated after the third vaccination. The vaccination schedule was maintained during tumor growth. In some experiments, mice were vaccinated with lacZ cDNA or SL-lacZ before tumor cell inoculation and with {beta}-gal-loaded DC after tumor cell inoculation. In selected experiments, LNC were collected after three rounds of vaccination and were transferred into SCID mice, which had been conditioned by irradiation with 300 rad. SCID mice were "reconstituted" i.v. either with 4 x 107 LNC, 2.5 x 107 T cells, 2.5 x 107 CD4+ cells, or 2.5 x 107 CD8+ cells. T cells were enriched by depletion of monocytes by plastic adherence and by depletion of B cells by panning for 90 min at 4°C on anti-mouse IgM-coated petri dishes. CD4+ and CD8+ cells were separated from these T cell preparations by incubating 108 cells with 1 mg anti-CD4 or anti-CD8, respectively, for 20 min at room temperature. Cells were washed and panned on anti-rat IgG-coated petri dishes for 2 h at room temperature, collecting the adherent cells. Mice received concomitantly the i.v. injection of lymphocytes and the s.c. injection of tumor cells.

Statistical analysis

Significance of differences was calculated according to Student’s t test (in vitro assays) or the Wilcoxon rank sum test (in vivo assays).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retarded tumor growth after vaccination with naked DNA, transformed Salmonella, and protein-loaded dendritic cells

BALB/c mice (six per group) were vaccinated three times at 21-day intervals with either 100 µg lacZ cDNA (i.m.), 108 SL-lacZ (orally), or 5 x 105 {beta}-gal-loaded DC (i.v.). Three days after the last vaccination, mice received a s.c. injection of 5 x 104 RENCA-{beta}-gal cells. Tumor growth was controlled twice per week (Fig. 1GoA). Tumor growth was retarded by all three vaccination schemes. Vaccination with {beta}-gal-loaded DC was most efficient with respect to the survival time and survival rate (Fig. 1GoB). With respect to DNA vaccination, the efficacy of vaccination with SL-lacZ exceeded the one of vaccination with lacZ cDNA. In this experiment, two and one of six mice remained tumor free after vaccination with loaded DC and transformed SL, respectively. Additional experiments confirmed the result; i.e., of 30 mice/group, none of the nonvaccinated mice survived, whereas two, six, and nine mice survived after vaccination with DNA, SL, and DC, respectively.



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  		FIGURE 1. Retardation of tumor growth by vaccination: BALB/c mice (six per group) were vaccinated three times with lacZ cDNA i.m., with SL-lacZ orally, or with {beta}-gal DC i.v. and received thereafter a single s.c. injection of 5 x 105 RENCA-{beta}-gal cells. Mean tumor volume (A, only of mice that developed a tumor) and survival time/survival rate (B) were monitored. Significant differences in the survival times are indicated.

 
Vaccination-induced retardation of tumor growth depends on an unimpaired immune system

Whether the therapeutic effect of vaccination with DNA or protein was based on activation of alike or different immune response mechanisms was evaluated in a transfer experiment, where SCID mice, which had not been depleted of NK cells, received either CD4+ cells or CD8+ cells or T cells or unseparated LNC concomitantly with a tumor challenge. Transferred cells were derived either from untreated mice or from mice that had been vaccinated three times with SL-lacZ or {beta}-gal-loaded DC (Fig. 2Go). After the transfer of lymphocytes from DC-vaccinated mice, a strong protective effect was only observed when unseparated LNC were transferred. Notably, the transfer of CD8+ cells was ineffective. A quite different result was obtained when transferring lymphocytes from SL-vaccinated mice. Tumor growth was retarded irrespective of whether unseparated LNC, T cells, or CD4+ T cells were transferred. The transfer of CD8+ cells was most efficient, i.e., three of eight mice remained tumor free. It should, however, be noted that the survival time of the remaining five mice that developed tumors was not prolonged as compared with mice receiving CD8+ cells from nonvaccinated mice.



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  		FIGURE 2. Protective efficacy of lymphocyte subsets from vaccinated mice: BALB/c mice were vaccinated as described above. Five days after the third vaccination, mice were killed and lymph nodes were collected. T cells, CD4+ cells, and CD8+ cells were enriched from LNC by panning as described in Materials and Methods. Unseparated LNC (4 x 107), T cells (2.5 x 107), CD4+ cells (2.5 x 107), and CD8+ cells (2.5 x 107) were transferred into SCID mice. Mice concomitantly received 5 x 104 RENCA-{beta}-gal cells. Survival time and survival rate are shown. Significance of differences in the survival times is indicated.

 
The SCID mouse, particularly when reconstituted with lymphocyte subpopulations, provided a means to judge the recruitment and activation of elements of the nonadaptive defense which was explored by cytokine expression (TNF and IL-6) and by LAK activity. A very strong increase in the percentage of TNF- and IL-6-expressing cells (Fig. 3GoA) was observed after the transfer of LNC, T cells, and CD4+ T cells of DC-vaccinated mice. Expression of TNF and IL-6 was by far less impressively increased after the transfer of LNC or subpopulations of LNC from SL-vaccinated mice. Expression of IFN-{gamma}, likely in the transferred lymphocytes, was high after the transfer of LNC, T cells, and CD4+ T cells of DC-vaccinated mice as well as after the transfer of T cells or CD8+ T cells of SL-vaccinated mice. LAK activity was higher after the transfer of unseparated lymphocytes or T cells than after the transfer of CD8+ or CD4+ cells. We only noted two exceptions: LAK activity remained high after the transfer of CD8+ cells from SL-vaccinated mice as well as after the transfer of CD4+ cells of DC-vaccinated mice (Fig. 3GoB). The findings are interpreted in the sense that vaccination with protein-loaded DC supports in a strongly CD4-dependent manner the recruitment of nonadaptive defense mechanisms.



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  		FIGURE 3. Cytokine expression and cytotoxic activity of lymphocytes collected from reconstituted tumor-bearing SCID mice: "reconstituted" SCID mice were killed 3 wk after tumor cell application and lymph nodes as well as the tumor were excised. A, Cytokine expression of LNC was determined by flow cytometry. Mean values of three mice per group are shown. B, LAK and CTL activity were determined after restimulation in vitro. Percentage cytotoxicity of triplicate cultures is shown for E:T of 50:1 (LAK and CTL). Significance of differences (p < 0.01) is indicated by an asterisk.

 
A possible transfer of memory T cells was estimated by the evaluation of CTL activity (Fig. 3GoB) and by the frequencies of Th and CTL 4 wk after the transfer (Table IGo). With respect to CTL activity, three observations should be mentioned: 1) High CTL activity of LNC and tumor-infiltrating leukocytes (TIL) was only observed when transferring CD8+ cells of SL, but not of DC-vaccinated mice. 2) Transferred CD4+ cells of DC-vaccinated mice did not exhibit any significant CTL activity. 3) The recovery of CTL from TIL after the transfer of CD8+ cells from DC-vaccinated mice was very low. Yet, a good recovery was seen in mice reconstituted with CD8+ cells of SL-lacZ-vaccinated mice. Survival of transferred effector cells/memory cells as explored by LD analysis revealed high frequencies of proliferating cells after the transfer of CD4+ cell-enriched populations from mice vaccinated with SL or DC; high frequencies of CTL were only recovered when transferring CD8+ cells after SL vaccination.


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  		Table I. Frequencies of helper and cytotoxic T cells after the transfer of lymphocytes from DNA or protein-vaccinated mice

 
According to these findings, it is unlikely that CD4+ CTL have been activated by vaccination with DC and, if at all, only to a minor degree by vaccination with SL. Furthermore, vaccination with SL apparently induced CD8+ memory cells, while the antitumor responses after vaccination with DC essentially were driven by a CD4+ helper population.

Additive effect of vaccination with DNA and protein-loaded DC on tumor growth control and survival rate

If it holds true that DNA vaccination preferentially induces a (memory) CTL response and vaccination with protein-loaded DC provides an efficient means of Th activation, combining the two vaccination schemes should yield further advantage. An analysis of tumor growth rate and survival time/survival rate after prophylactic vaccination with either DC or SL, boosting both groups of mice after tumor challenge with DC, supported the hypothesis (Fig. 4Go). The SL/DC vaccination schedule significantly retarded the onset of tumor growth. Yet, the growth rate was similar to that of mice not being vaccinated or vaccinated with DC throughout. Accordingly, the mean survival time of mice developing tumors was rather independent of whether mice received only DC or SL and later on DC. However, the overall efficacy of a prophylactic vaccination by SL followed by boosting with DC was high. When mice had been vaccinated with SL and received thereafter DC, up to 60% remained tumor free. Macroscopic and histological evaluation of these mice that were killed after an overall observation period of 6 mo did not reveal any sign of micrometastases.



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  		FIGURE 4. Combining DNA and protein vaccination increases the therapeutic efficacy: untreated BALB/c mice and BALB/c mice that had been vaccinated (as described above) three times with SL-lacZ or {beta}-gal DC received a s.c. injection of 5 x 104 RENCA-{beta}-gal. Except for the control group, all mice were treated after tumor cell application with {beta}-gal DC every third week. The mean tumor volume (A, only of mice developing tumors) and the survival time and rate (B) are shown. Significant differences in the survival times are indicated.

 
The experiment strengthens our interpretation that vaccination with a sequence of DNA followed by protein activates different lymphocyte subpopulations, which show a small degree of overlap, only. To consolidate the view that activation of CTL by vaccination with SL can be strongly enhanced by the provision of help as initiated by protein-loaded DC, a comparative ex vivo analysis of cytokine profiles, proliferative activity, and cytotoxic activity in draining LNC and TIL was performed.

Activation and expansion of effector T cells by vaccination with DNA vs protein

Before turning to the ex vivo analysis on lymphocyte subset activation, we want to comment on the unimpaired growth rate of tumors in a subgroup of vaccinated mice. This finding was unexpected and we assumed that tumors may have lost the Ag under the pressure of the immune attack. Indeed, although the vast majority of tumor cells in nonvaccinated mice expressed {beta}-gal, tumors developing in DC-vaccinated mice were partly {beta}-gal negative, i.e., in cytospin preparations between 20 and 50% of cells were stained by X-Gal. In tumor sections, positively stained cells were found in clusters dispersed between {beta}-gal-negative cells. Only a minority of tumor cells recovered from SL- plus DC-vaccinated mice expressed the "tumor Ag," and in some tumors no clear staining was observed at all (data not shown). This is contrasted by the observation that under in vitro culture conditions, the RENCA-{beta}-gal cells stably expressed the transgene at a high level. Furthermore, tumors that were recovered from nonvaccinated mice were uniformly stained by X-Gal. This implies that in the absence of an external stimulus, the response against the foreign Ag did not suffice to provoke tumor escape. Taking into account the clustered appearance of {beta}-gal+ tumor cells and the delayed outgrowth of the tumor in vaccinated mice, we consider it as most likely that with an increasing pressure from the immune system only a few loss variants survived and expanded.

To further explore the immune mechanisms underlying the therapeutic effects of vaccination with plasmid vs SL vs DC or combinations thereof, proliferative and cytotoxic activity, cytokineexpression, Ab secretion, and frequencies of proliferative lymphocytes and CTL were evaluated. To explore whether the growing tumor interferes with T cell activation, vaccinated tumor-free and tumor-bearing mice were tested simultaneously. For the sake of clarity of presentation, the immune status of vaccinated tumor-free mice is not presented in all experiments.

We first evaluated the leukocyte subset composition. Three weeks after tumor cell inoculation, draining lymph nodes and the tumor were excised to evaluate the composition of draining LNC and TIL as well as their activation state. We noted an increased recovery of surface IgM+ cells in draining LNC and TIL after vaccination with SL-lacZ and a slight, but statistically significant increase in the percentage of CD8+ cells in draining lymph nodes after vaccination with the plasmid or SL. After vaccination with DC, the recovery of CD4+ cells and of monocytes from TIL was slightly increased (data not shown).

Expression of activation markers and of costimulatory molecules was of particular interest in TIL. CD25 and CD40L (CD154) and the costimulatory molecules CD40, CD80, and CD86 were more strongly up-regulated by vaccination with SL or DC than the plasmid, whereas CTLA-4 (CD152) was significantly up-regulated after vaccination with naked DNA. In mice vaccinated with a combination of either DNA or SL with DC, a further increase in the percentage of CD25+ and CD40L+ cells was noted. This accounted for draining LNC (data not shown) as well as TIL (Fig. 5GoA). There was no convincing evidence for a dominance of counterregulatory mechanisms, i.e., neither CTLA-4 nor CD95/CD95L (data not shown) were strongly up-regulated. A counterattack by the tumor also appeared unlikely; i.e., we did not detect CD95L expression by RENCA-{beta}-gal cells (data not shown).



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  		FIGURE 5. Lymphocyte activation in tumor-free and tumor-bearing mice by vaccination: BALB/c mice were vaccinated as described. Tumor-free BALB/c mice were killed 4–5 days after the third vaccination, tumor-bearing mice were killed 3 wk after tumor inoculation. Mice were bled via the retroorbital sinus; spleen, lymph nodes, and the tumor were excised. A, Expression of activation markers, costimulatory molecules, and their ligands on TIL was analyzed by flow cytometry. The mean percentage ± SD of stained cells from three mice per group is shown. B, Lymphocytes of tumor-free and tumor-bearing mice were titrated in triplicate and were cultured for 3 days in the absence or presence of 10 µg/ml {beta}-gal. [3H]Thymidine (10 µCi/ml) was added during the last 16 h of culture. Cells were harvested and counted in a beta counter. Mean cpm ± SD of triplicates of 1 x 105 cells/well is shown. C, Serum from tumor-bearing mice was collected and diluted 1:20, supernatants of SC, draining LNC, and TIL (tumor-bearing mice) that had been cultured for 3 days in the presence of {beta}-gal (1 x 105 cells/well) were transferred on {beta}-gal-coated plates to determine the presence of {beta}-gal-specific Abs by ELISA. Mean values ± SD of the OD405 from triplicate experiments are shown. D, Cytokine expression in draining LNC and TIL was analyzed by flow cytometry. The mean percentage ± SD of stained cells from three mice per group is shown. Significance of differences (A–D) (p < 0.01) is indicated by an asterisk.

 
A similar response profile was observed when evaluating proliferative activity in response to {beta}-gal (Fig. 5GoB). The strongest response consistently was observed after vaccination with SL plus DC. When mice had been vaccinated with DNA plus DC, the proliferative response also was increased as compared with vaccination with the individual components. With respect to the latter, a stronger proliferative response was observed after vaccination with DC than with DNA or SL, DNA being an equally or slightly stronger stimulus than SL. Furthermore, proliferative responses in tumor-bearing mice were not significantly decreased as compared with vaccinated tumor-free mice, and the pattern of proliferative response was quite similar in draining LNC and TIL. Finally, it should be mentioned that spontaneous proliferative activity was increased in vaccinated mice particularly after transfer of DC. This may be due to the persistence of DC loaded with {beta}-gal-derived peptides.

Activation of Th cells was indirectly confirmed by the activation of B cells as judged by the presence of {beta}-gal-specific Abs in the serum of vaccinated mice and in supernatants of SC and LNC (Fig. 5GoC). B cell activation was more efficiently achieved by vaccination with DC and the plasmid than with SL. Vaccination with both DNA plus DC or SL plus DC had no further impact on Ab secretion. This accounted for tumor-free (data not shown) and tumor-bearing mice. Notably, even from the tumor itself Ab-producing cells could be isolated, although Ab production by TIL was low.

As a further parameter of Th activation, cytokine expression was analyzed (Fig. 5GoD). The cytokine expression profile provided two additional informations: First, Th1 cytokine (IL-12 and IFN-{gamma})-expressing cells were augmented after vaccination with DNA, SL, and DC, with only minor differences between the three stimuli or combinations thereof. Expression of the counterregulatory cytokine IL-10 was increased only after SL or SL plus DC vaccination. Second, although SL or DC vaccination led to an increase in the percentage of IL-2expressing cells, a significant additional increase was seen after vaccination with SL plus DC.

Finally, it was of interest to see whether activation of Th would have sufficed for activation of nonadaptive defense. This was evaluated by testing LAK activity of vaccinated tumor-bearing mice. DNA vaccination had no impact on LAK activity at all. Yet, LAK activity was significantly increased by vaccination with SL or DC, the latter exerting the stronger effect. In tumor-bearing mice vaccinated with SL followed by DC, LAK activity was further augmented; i.e., at a LAK:RENCA ratio of 50:1, we observed 8.3% (nonvaccinated), 8.2% (DNA vaccinated), 10.3% (SL vaccinated), 11.5% (DC vaccinated), and 34.7% (SL plus DC vaccinated) lysis (data not shown).

Thus, activation of Th was induced by all three vaccines, but most efficiently by DC. Furthermore, particularly by combining the SL and DC vaccines, the overall efficacy of Th activation/expansion could be further improved. The determination of the frequencies of Th cells confirmed the assumption (Table IIGo). First to mention, Th frequencies were increased in the nonvaccinated tumor-bearing mice as compared with the tumor-free host. This implies that BALB/c mice mounted an immune response against {beta}-gal when introduced as a tumor Ag. Vaccination with DC led to a significant increase in the frequency of Th cells in spleen and lymph nodes of tumor-free and tumor-bearing mice. After vaccination with SL, higher frequencies of Th were only seen in the spleen. Yet, in mice vaccinated with SL plus DC, draining LNC and SC contained high frequencies of Th, which significantly exceeded the frequencies seen after vaccination with DC alone.


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  		Table II. Frequencies of {beta}-gal-specific helper and cytotoxic T cells after vaccination with DNA or protein-loaded DC

 
A different picture emerged when testing CTL activity (Fig. 6Go). After in vitro restimulation, the cytotoxic potential of LNC and TIL was evaluated using RENCA-{beta}-gal as target and RENCA as cold target, the latter for intra-assay subtraction of LAK activity. Activation of CTL was more efficiently achieved by vaccination with SL than by vaccination with DNA or DC. Similar to the activation of Th, activation of CTL also was strongly improved by combining the SL with the DC vaccine. This accounted for LNC of tumor-free and tumor-bearing mice as well as for TIL. The determination of CTL frequencies by LD (Table IIGo) corresponded to the results obtained in bulk cultures; i.e., the highest frequency of CTL was seen after vaccination with SL followed by DC.



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  		FIGURE 6. Pronounced activation of CTL by subsequent vaccination with SL-lacZ and {beta}-gal DC: BALB/c mice were vaccinated as described. LNC and TIL (5 x 106/ml) were cultured for 5 or 10 days in medium containing 20 µg/ml {beta}-gal and 10% TCGF. Thereafter, cells were harvested and separated from dead cells by Ficoll-Hypaque gradient centrifugation. CTL activity was evaluated by adding [3H]thymidine-labeled RENCA-{beta}-gal cells and a 10-fold excess of cold target RENCA cells for an internal subtraction of LAK activity. Cells were incubated for 6 h. Cytotoxicity of triplicate cultures is shown for E:T ratios of 80–10:1.

 
Thus, all three vaccination protocols supported the view that DNA was more efficient in CTL activation and protein-loaded DC were more efficient in Th cell activation. When mice received DC after an initial stimulation via SL, the provision of help resulted in an additional significant increase in the frequency and efficacy of CTL.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In animal models, promising results have been reported by vaccination with either DNA or protein (26, 47, 48). At the protein/peptide level, loading of DC as the professional APCs is the method of choice (25, 49). Optimization of DNA vaccination is still a matter of debate. Direct delivery, transfection of, for example, DC, or the use of transporter systems like attenuated bacteria are the most common alternatives (47). Particularly the oral route of application using attenuated Salmonella or Listeria provides a technically very attractive route that is being successfully explored in vaccination against a variety of infectious agents (15, 17, 18, 50, 51, 52, 53, 54). Since clinical success in cancer immunotherapy is still far behind expectation (55, 56), we considered it important to explore in an animal model system whether combining DNA and protein vaccination provides a means of strengthening an antitumor response. Using {beta}-gal as an artificial tumor Ag of the renal cell carcinoma line RENCA, we could show that vaccination with both lacZ-transformed attenuated S. typhimurium or with protein-loaded DC sufficed for retardation of tumor growth. When combining both vaccination approaches, roughly 50% of mice remained tumor free. This additive effect is due to the preferential, although not exclusive activation of a CTL response by oral vaccination with attenuated Salmonella and a strong Th1 response by the transfer of in vitro-generated and expanded DC.

Comparing three vaccination schemes (plasmid, SL, and DC), it became obvious that DNA vaccination was more efficient using transformed SL than naked DNA. Protein-loaded DC provided a stronger protection than both modes of DNA vaccination. Similar findings have been reported in other systems (57, 58). However, although the start of tumor growth could be retarded, the majority of mice succumbed with a progressively growing tumor. The in vitro analysis of lymphocytes from vaccinated tumor-free and tumor-bearing mice revealed that this unlikely has been due to a counterregulation of immune response by the tumor; i.e., there was no evidence for a reduction in proliferative cells of CTL or of Ab-producing cells. Expression of activation markers and their ligands as well as of cytokines was roughly unaltered or only slightly reduced in tumor-bearing mice. We also did not observe signs of tumor-mediated apoptosis of activated lymphocytes; i.e., CD95L was not up-regulated on RENCA cells when growing in the vaccinated mice, nor did those cells induce apoptosis of lymphocytes when cocultured in vitro (data not shown). These findings leave us with the option that either a less protective Th2 response has been induced, that the efficacy of response was insufficient to prevent tumor growth, or that the tumor escaped the immune attack.

Some evidence for activation of Th2 cells was only observed after vaccination with SL-lacZ, where an increased percentage of draining LNC and TIL expressed IL-10 and IL-4 (data not shown) and, accordingly, serum and culture supernatant contained more IgG1 than IgG2a Abs (data not shown). Although the oral route of vaccination with live Salmonella has been reported to mainly induce a Th1 response (59), Th2 responses, possibly in dependence on the Ag, have also been observed (60).

With respect to the efficacy of induction of an immune response, it became obvious that lacZ DNA by itself was the weakest stimulus. Although it induced a strong humoral response, it did not lead to a major up-regulation of IL-2 and its receptor CD25 nor of CD154. Furthermore, lymphocytes from lacZ-vaccinated mice exhibited a good proliferative response in the absence of nominal Ag, but the Ag-specific increase in the proliferative activity was low. Most important, hardly any cytotoxic activity could be detected in lacZ-vaccinated mice. We interpret the data in the sense that by vaccination with naked DNA we induced an immune response, probably supported by the adjuvant effect of the DNA, which only partially has been {beta}-gal specific and clearly supported activation preferentially of B cells rather than of CTL.

The situation was quite different when using SL as DNA carrier. There was no evidence for an unspecific activation of immune response; i.e., proliferation in the absence of the nominal Ag was low. It also should be noted that expansion of Th was mainly restricted to the spleen, which could have been due to the route of application (61). Furthermore, with the exception of mesenteric lymph nodes (data not shown), production of IgG Abs was not very strong. Whether this has been due to preferential induction of IgA Ab secretion with transport through the gut epithelium remains to be explored. Without any question, vaccination with SL-lacZ very efficiently supported activation and expansion of {beta}-gal-specific CTL, a phenomenon also seen after vaccination with Listeria (18). This became apparent by the level of cytotoxicity in bulk cultures, the frequency of CTL recovered from draining lymph nodes and the spleen as well as the retention of cytotoxic activity after the transfer of CD8+ cells into SCID mice and, most important, the protective effect of CD8+ cells after transfer. Based on the latter findings, it becomes tempting to speculate that a memory CTL response may have been induced.

Using the individual vaccines, the most potent immune response has been observed after vaccination with {beta}-gal-loaded DC, which induced a strong proliferative response, Ab production, activation of LAK and CTL, as well as expansion of CTL and Th. The transfer experiment revealed that it is, indeed, the concerted action of distinct elements of the immune system which accounts for the efficacy in tumor growth retardation by vaccination with protein-loaded DC, since the transfer of unseparated LNC was more efficient than the transfer of T cells and the transfer of CD8+ cells was ineffective. Furthermore, very few CTL were recovered after the transfer of CD4+ cells and cytotoxic activity in bulk cultures hardly exceeded background levels. Thus, it is unlikely that by vaccination with {beta}-gal-loaded DC a CD4-mediated CTL response has been induced. In addition, activation of CTL apparently was a secondary phenomenon initiated after/by activation of Th cells. The generation of {beta}-gal-specific CTL by protein-loaded DC has been described before (10). Yet, it also has been demonstrated that induction of a protective response requires CD4+ and CD8+ cells (62) and, importantly, that activation of CD8+ CTL by DC priming requires CD4+ cells (63). These CTL apparently were not of the memory type, because after the transfer of CD8+ cells into SCID mice hardly any cytotoxic activity was recovered in TIL. Instead, the transfer of CD4+ cells from mice vaccinated with {beta}-gal-loaded DC sufficed for the recruitment of nonadaptive defense mechanisms as apparent by the high level of TNF-{alpha} and IL-6 expression as well as the strong LAK activity. From there we would conclude that by vaccination with protein-loaded DC, a long-lasting Th1 response has been induced which supported activation of elements of the nonadaptive and the adaptive immune systems.

Taking these features into account, it could have been expected that combining the two vaccination schemes would strengthen the efficacy of antitumor defense. This has, indeed, been the case and has been most impressive with respect to CTL activation and expansion; i.e., the additional help recruited by boosting with DC led to a significant expansion and a strong increase in lytic activity of CTL in SL-vaccinated mice. The finding is in line with the observation that Ag presentation by DC is crucial not only for the initiation, but also for the maintenance of a protective CTL response against nonlymphoid tissue, including solid tumors (64).

The optimized prophylactic vaccination protocol allowed for tumor cell eradication in roughly 50% of mice. Yet, the growth rate in those mice that developed tumors has not been significantly different from the one in nonvaccinated mice. One possibility could have been the limits of the vaccination protocol; i.e., its efficacy relies on the elimination of the tumor inoculum during the starting 3–5 wk and it fails to efficiently cope with a growing tumor burden. This may well be the case and could be due to dealing with a solid tumor mass (65). Thus, it remains to be explored how activated lymphocytes can be more efficiently targeted toward the tumor. Alternatively, only those tumor cells which develop escape mechanisms survive the immune attack. In fact, the majority of tumor cells collected from SL- plus DC-vaccinated mice had lost the tumor Ag, but still expressed MHC class I molecules (data not shown). This high percentage of Ag-loss variants probably is not due to the fact that the {beta}-gal tumor Ag was artificially introduced into the genome. Though less pronounced, we also observed a loss of the natural tumor Ag gp100 in the B16 melanoma under immune pressure.4 Furthermore, no Ag loss was seen in in vitro cultures. It is difficult to define, whether a few spontaneous loss variants grew out or whether Ag loss was a more direct consequence of the immune pressure. The failure to detect Ag loss in nonvaccinated mice argues against the immunogenicity of the "foreign" tumor Ag to be strong enough to induce an efficient immune response, at least when expressed on a solid tumor line. Finally, the finding that vaccination with SL or SL plus DC was accompanied by a pronounced Ag loss suggests a particular correlation between Ag loss and the activation of CTL. The interpretation is strengthened by the observation that Ag loss was also observed after the transfer of CD8+ cells from SL vaccinated mice into SCID mice (data not shown) as well as by the clustered appearance of the remaining {beta}-gal+ RENCA cells. Unfortunately, this problem of tumor escape may become of major importance in clinical vaccination protocols. Whether the problem can be solved by consecutive vaccination with a panel of tumor Ags remains to be explored. Nonetheless, with the optimized vaccination protocols, 50% of mice remained tumor free. We clearly could demonstrate that DNA vaccination using Salmonella as transporter via the oral route efficiently induces a CTL response which is not limited to the gut-associated immune system. We also could show that the efficacy of DNA vaccination gets significantly improved by the provision of additional help via protein-loaded DC.


   Acknowledgments
 
We cordially thank Dr. B. A. Stokker (Stanford, CA) for providing us with attenuated Salmonella typhimurium. We particularly thank R. Weth for the transfection of RENCA cells, DNA preparation, and help with the animal experiments as well as S. Hummel and M. Vitacolonna for excellent technical help. We greatly appreciate helpful discussions and suggestions during preparation of this manuscript by Dr. S. Matzku.


   Footnotes
 
1 This work was supported by the Deutsche Krebshilfe (to M.Z.). Back

2 Address correspondence and reprint requests to Dr. Margot Zöller, Department of Tumor Progression and Immune Defense, German Cancer Research Center, Im Neuenheimer Feld 280, D 69120 Heidelberg, Germany. Back

3 Abbreviations used in this paper: DC, dendritic cell; LAK, lymphokine-activated killer; LD, limiting dilution; LNC, lymph node cells; RENCA-{beta}-gal, RENCA cells transfected with the lacZ gene; {beta}-gal, {beta}-galactosidase; SL, attenuated Salmonella typhimurium, strain SL7202; SL-lacZ, SL transformed with the pVAX vector containing the lacZ gene cDNA; SC, spleen cells; TIL, tumor-infiltrating leukocytes; X-Gal, 5-bromo-4-chloro-3-indolyl {beta}-D-galactoside; LB, Luria-Bertani; CD40L, CD40 ligand. Back

4 R.Weth, O. Christ, S. Stevanovic, and M. Zöller. Gene delivery by attenuated Salmonella typhimurium: comparing the efficacy of helper versus cytotoxic T cell priming in tumor vaccination. Submitted for publication. Back

Received for publication October 6, 2000. Accepted for publication December 27, 2000.


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