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The Journal of Immunology, 2007, 179, 5033-5040
Copyright © 2007 by The American Association of Immunologists, Inc.
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CD8+ CTL Priming by Exact Peptide Epitopes in Incomplete Freund’s Adjuvant Induces a Vanishing CTL Response, whereas Long Peptides Induce Sustained CTL Reactivity1

Martijn S. Bijker*, Susan J. F. van den Eeden*, Kees L. Franken*, Cornelis J. M. Melief*, Rienk Offringa* and Sjoerd H. van der Burg2,{dagger}

* Department of Immunohematology and Blood Transfusion and {dagger} Department of Clinical Oncology, Leiden University Medical Centre, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Therapeutic vaccination trials, in which patients with cancer were vaccinated with minimal CTL peptide in oil-in-water formulations, have met with limited success. Many of these studies were based on the promising data of mice studies, showing that vaccination with a short synthetic peptide in IFA results in protective CD8+ T cell immunity. By use of the highly immunogenic OVA CTL peptide in IFA as a model peptide-based vaccine, we investigated why minimal CTL peptide vaccines in IFA performed so inadequately to allow full optimization of peptide vaccination. Injection of the minimal MHC class I-binding OVA257–264 peptide in IFA transiently activated CD8+ effector T cells, which eventually failed to undergo secondary expansion or to kill target cells, as a result of a sustained and systemic presentation of the CTL peptides gradually leaking out of the IFA depot without systemic danger signals. Complementation of this vaccine with the MHC class II-binding Th peptide (OVA323–339) restored both secondary expansion and in vivo effector functions of CD8+ T cells. Simply extending the CTL peptide to a length of 30 aa also preserved these CD8+ T cell functions, independent of T cell help, because the longer CTL peptide was predominantly presented in the locally inflamed draining lymph node. Importantly, these functional differences were reproduced in two additional model Ag systems. Our data clearly show why priming of CTL with minimal peptide epitopes in IFA is suboptimal, and demonstrate that the use of longer versions of these CTL peptide epitopes ensures the induction of sustained effector CD8+ T cell reactivity in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tumor cells express tumor-associated Ags on their cell surface in the context of MHC class I molecules. These tumor-associated Ags can be recognized by CD8+ T cells, which are the essential players in the clearance of tumor cells in vivo (1, 2). In recent years, many epitopes have been identified (3, 4), providing the option to apply these minimal MHC class I-restricted CD8+ T cell peptide epitopes in vaccination strategies for the immunotherapy of solid tumors (3, 4). Unfortunately, these minimal CTL peptide-based vaccines in oil-in-water formulations (e.g., IFA or Montanide) have in general met with limited immunological and clinical success (4, 5).

The use of minimal CTL peptide-based vaccines was based on murine studies demonstrating that prophylactic vaccinations with minimal CTL peptides in IFA induced protective CD8+ T cell (antitumor) immunity (6, 7, 8, 9). However, other reports show that vaccination with the minimal CD8+ T cell epitopes can also result in the induction of CD8+ T cell tolerance (8, 10, 11, 12, 13, 14). Although in some of these studies tolerance was the result of repetitive vaccination with high dose of Ag (8, 13, 14), others reported that even after a single s.c. vaccination, tolerance could be induced (11, 12). In these studies, the injection of mice with low amounts of adenovirus E1A (11) or E1B (12) minimal CTL peptide emulsified in IFA resulted in functional impairment of activated CD8+ T cells, as was evident from the enhanced outgrowth of tumors (11, 12). Intravenous coinjection of agonistic CD40 Ab (FGK), to activate and to mature dendritic cells (15, 16), resulted in a transient adenovirus-specific CD8+ effector T cell response, detectable 10 days after vaccination, but not at 30 days after vaccination, and did not protect mice against a tumor challenge (17). These data indicate that an initial proper activation of the CD8+ T cell response by minimal CTL peptide vaccines in IFA does not ensure long-term effectiveness of these CD8+ T cells. Such long-term effectiveness is particularly important to control chronic diseases such as cancer.

To date, it is unclear what the common long-term result is with respect to immunological outcome of injection with minimal CTL peptides. Therefore, we have thoroughly studied different peptide vaccination strategies using the highly immunogenic model Ag OVA (18, 19, 20, 21, 22), containing the MHC class I-restricted CD8+ T cell epitope OVA257–264 (OVA8) and the CD4+ Th cell epitope OVA323–339 (ThOVA17). Peptides were mixed in IFA, because oil-in-water formulations are standard vehicles for peptide vaccination in clinical trials. The efficacy of the different peptide vaccine formulations was tested by the analysis of the following three important parameters: 1) the magnitude of the CD8+ T cell response; 2) the ability of CD8+ T cells to undergo secondary expansion upon Ag challenge in vitro (23); and 3) the in vivo killing capacity of the CD8+ T cells. These parameters were tested at either day 10 (peak of the response) or day 30 after vaccination, the time point in which adenovirus-specific CD8+ T cells were tolerized after injection with E1A peptide in IFA (17). Furthermore, we investigated how to formulate a peptide-based vaccine that triggers CTL immunity without the risk for exhaustion/tolerance.

In this study, we report that vaccination with immunogenic OVA8 peptide in IFA also results in the activation of effector CD8+ T cells, which eventually ceased to expand or to kill target cells. This cessation of T cell function was associated with long-term systemic presentation of the minimal CTL peptide in vivo, but could be prevented by either complementation with a minimal Th peptide or extension of the minimal CTL peptide to a 30-aa-long peptide. These data clearly show the potential hazard of using minimal CTL peptide vaccines, and the benefits of long peptide vaccines for the induction of an effective CD8+ T cell response.


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

C57BL/6 (B6; H-2b) and MHC class II knockout (class II–/–) mice were purchased from Charles River Laboratories. CD90.1 mice were bred at TNO-PG. CD45.1 OT-1 (24) and OT-2 (25) mice are CD8+ T cell and CD4+ T cell TCR transgenic (Tg)3 mice expressing the TCR {alpha}-chain and beta-chain recognizing OVA257–264 in H2-Kb and OVA323–339 in I-Ab, respectively, and were bred at the Leiden University Medical Centre animal facility. All mice were kept at the Leiden University Medical Centre animal facility and used at 8–14 wk of age in accordance with national legislation and under supervision of the animal experimental committee of the University of Leiden.

Tumor cells

EG7 (EL4-OVA) (26) tumor cells were cultured in IMDM (Invitrogen Life Technologies) supplemented with 8% (v/v) FCS (Greiner Bioscience), 50 µM 2-ME, 2 mM glutamine, 100 IU/ml penicillin (complete medium), and 400 µg/ml Geneticin (Invitrogen Life Technologies).

Peptides and peptide vaccination

Peptides were generated as described before (6). The following dominant minimal CTL peptides were used: OVA257–264 SIINFEKL (OVA8); human papillomavirus (HPV)16E7 peptide E749–57 RAHYNIVTF (HPV9) (6); and the adenovirus protein E1A234–243 SGPSNTPPEI (E1A10) (11). The minimal Th peptide sequence of OVA was the following: OVA323–339 ISQAVHAAHAEINEAGR (ThOVA17). In addition, the following long peptides deduced from the natural sequence of each protein were used: CTL peptide OVA241–270 SMLVLLPDEVSGLEQLESIINFEKLTEWTS (OVA30) (note that this peptide does not contain the C-terminal Th epitope OVA265–280 (27)); Th peptide OVA317–347 SSAESLKISQAVHAAHAEINEAGREVVGSAE (ThOVA31); CTL peptide of HPV protein E743–77 GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (HPV35) (28); and CTL peptide of protein E1A223–252 RECNSSTDSCDSGPSNTPPEIHPVVRLCKK (E1A30). Mice were s.c. vaccinated with 40 nmol of (each) peptide admixed in PBS or in PBS and IFA (Difco Laboratories) (50% v/v) in a total volume of 200 µl.

Ab treatment

Agonistic CD40 Ab (FGK45) (50 µg) was provided i.v. in the tail vein on days 0, 1, and 2 in 200 µl of PBS. Complete CD4+ T cell depletion was obtained by i.p. injection of 25 µg of anti-CD4 (clone GK1.5) in PBS 3 days and 1 day before vaccination and once per week throughout the experiment. CD4 depletion was regularly checked by FACS analysis and showed that the mean percentage of CD4+ T cells was ≤0.003 ± 0.005% in the mice receiving GK1.5, whereas naive mice displayed 13.8 ± 2% of CD4+ T cells.

CFSE labeling and adoptive transfer of Tg T cells

Single-cell suspension was made from spleen and peripheral lymph nodes (LN) of CD45.1 OT-1 mice or OT-2 mice. Erythrocytes were lysed by ammonium chloride treatment, and 10 x 106 cells/ml were incubated at 5 µM CFSE end concentration in 0.1% PBS/BSA at 37°C for 15 min. The reaction was blocked with 10% v/v of pure FCS. The cells were washed twice with PBS, and 1–2 x 106 Tg T cells were injected into the tail vein in 200 µl of PBS.

Ex vivo detection of Ag

Mice were vaccinated with either OVA8, OVA30, ThOVA17, or ThOVA31 mixed in IFA. Two days later, the draining LN (dLN; inguinal and axillary) and the nondraining LN (ndLN; mesenteric) were isolated. A single-cell suspension was made using a 70-µm cell strainer. To detect the CTL epitope ex vivo, 0.5 x 106 LN cells were plated in a 96-well plate, and to detect the Th epitope, 1 x 106 LN cells were plated in a 96-well plate. Purified and CFSE-labeled OT-1 (1 x 105) or OT-2 (2 x 105) Tg cells were added to these wells, respectively. Three days later, division of OT-1 Tg T cells was measured by flow cytometry by gating on CD45.1+ and CD8+ lymphocytes. In the case of OT-2 cells, 4 days later, division was determined by gating on Va2+ and CD4+ lymphocytes. Ag presentation was determined by the dilution of the CFSE of the Tg T cells.

In vivo cytotoxicity assay

Erythrocytes of B6/CD45.2 splenocytes were lysed, and the splenocytes were split into two equal fractions. Cells were differentially labeled with CFSE to either 5 (target) or 0.5 (control) µM end concentration (see CFSE labeling). The target cell population was pulsed with 1.0 µg/ml OVA257–264, and the control population with 1.0 µg/ml p53 peptide (H2-Kb, p53158–166) at 37°C in complete medium for 60 min. The cells were washed four times with PBS before the two populations were mixed in a 1:1 ratio, and a total of 8 x 106 cells was injected i.v. Either 1 or 2 days after injection of the target cells (day 10 or 30 after vaccination, respectively), spleens were removed and analyzed for specific killing. The ratio of CFSElow/CFSEhigh cells was determined by flow cytometry by gating on CD45.1+ lymphocytes. Specific killing of OVA257–264-pulsed (CFSEhigh) target cells was calculated as follows: (1 – ((CFSEhigh/CFSElow)vaccinated x (CFSElow/CFSEhigh)naive)) x 100%.

In vitro stimulation

EG7 cells were incubated with 50 µg/ml mitomycin C (Kyowa) in complete medium at 37°C for 1 h. Subsequently, the cells were washed four times and then irradiated (4000 rad). A total of 1 x 106 EG7 cells was incubated with 10 x 106 splenocytes; total volume was 2 ml. After 7 days, viable splenocytes were isolated over a Ficoll gradient and stained for H-2Kb tetramer (TM)-OVA257–264, CD8b2, and propidium iodide (to exclude dead cells).

Overnight intracellular cytokine staining

Splenocytes were incubated for 1 h with 1 µM ThOVA17 or ThOVA31 peptide or no peptide (background) before 1 µg/ml Golgiplug (containing Brefeldin A; BD Pharmingen) was added. The next day, cells were permeabilized and stained using the Cytofix/Cytoperm Plus kit (BD PharMingen), according to manufacturer’s instructions, and stained for CD4 and intracellular IFN-{gamma}.

Flow cytometry

Single-cell suspensions of spleens or LN were stained in PBS/0.1% BSA. The Abs that were used were the following: directly allophycocyanin conjugated; TM-OVA257–264; CD45.1 (A20; eBioscience); IFN-{gamma} (XMG1.2; BD Pharmingen); CD90.2 (53-2.1; BD Pharmingen); CD8a (53-6.7; BD Pharmingen) and PE-conjugated; and CD8b2 (53-5.8; BD Pharmingen), CD4 (RM4-5; BD Pharmingen). Data acquisition and analysis were done on a BD Biosciences FACScan with CellQuest software.

Statistical analysis

Statistical analysis was done using GraphPad InStat software (version 3.0) and GraphPad Prism 4 (GraphPad). Student’s two-tailed t test with Welch correction was applied for the statistical analysis of the samples, except for the data analyzed in Fig. 1A, OVA8 (IFA) day = 10 vs day = 30 (nonparametric two-tailed Mann-Whitney U test), and Figs. 7 and 8 (Kruskal Wallis test nonparametric ANOVA) because Student’s t test was not applicable.


Figure 1
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  		FIGURE 1. Minimal CTL peptide in IFA induces only transient CTL immunity. Mice were vaccinated s.c. with the minimal OVA8 CTL peptide mixed in IFA ({blacksquare}) or in PBS ({square}) in the presence or absence of 50 µg of FGK administered i.v. on days 0, 1, and 2. A, Enumeration of TM-OVA+ CD8+ T cells by flow cytometry. Ten or 30 days after vaccination, splenocytes were harvested and stimulated in vitro with OVA-expressing tumor cells for 7 days before analyzing the presence of TM-OVA+ CD8+ T cells (OVA8 (IFA) day = 10 vs day = 30, p < 0.01; OVA8 + FGK (IFA) day = 10 vs day 30, p < 0.001). The data are depicted as mean ± SEM (n = 5–7). B, In vivo cytotoxicity assay. Nine or 28 days after vaccination, CFSE-labeled CD45.1+ target cells were injected and cytotoxicity was measured in the spleen at day 10 or 30, respectively, by FACS analysis (OVA8 (IFA) day 10 vs day 30, p < 0.01; OVA8 + FGK (IFA) day 10 vs day 30, p < 0.01). Cells were gated on CD45.1+ lymphocytes. The data are depicted as mean ± SEM (n = 6–10). Representative FACS plots of the experiment are shown below.

 

Figure 7
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  		FIGURE 7. Dominant-negative effect of the minimal OVA CTL peptide on immunogenic vaccination with the long OVA30 CTL peptide (p < 0.01, Kruskal Wallis test nonparametric ANOVA). Mice were vaccinated s.c. with the minimal CTL peptide ({square}), the long CTL peptide ({blacksquare}), or both peptides (Figure 7) mixed in IFA. Thirty days later, mice were subjected to an in vivo cytotoxicity assay (n = 4–6).

 

Figure 8
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  		FIGURE 8. Failure to induce long-lasting CTL immunity is a general feature for minimal CTL peptides. Mice were vaccinated s.c. with peptides mixed in IFA with either the minimal CTL peptide ({square}), the long CTL peptide ({blacksquare}), or the long CTL peptide in which mice were depleted for CD4+ T cells with GK1.5 Ab (Figure 8). Mice that were depleted for CD4+ T cells received 3 and 1 days before vaccination 25 µg of CD4-depleting Ab GK1.5 (i.p.), and thereafter once every week 25 µg to maintain complete CD4+ T cell depletion. Thirty days later, mice were subjected to an in vivo cytotoxicity assay. A, HPV peptides (n = 10) (HPV9 vs HPV35 with or without GK1.5, p < 0.001; Kruskal Wallis test nonparametric ANOVA); B, E1A peptides (n = 5) (E1A10 vs E1A30 with or without GK1.5, p < 0.001; Kruskal Wallis test nonparametric ANOVA). The data are depicted as mean ± SEM.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Vaccination with the minimal CTL peptide OVA257–264 in IFA induces a transient and then functionally impaired CTL response

To explore the short- and long-term outcome of the CD8+ T cell response after peptide vaccination with the minimal MHC class I-binding peptides, mice were vaccinated with OVA8 in IFA, and subsequently, the secondary expansion potential of the CD8+ T cells in vitro as well as the in vivo effector function were determined, either 10 days (short-term) or 30 days after vaccination (long-term). In addition, a strong DC stimulus was provided by i.v. injection of FGK to systemically activate APC in vivo (15, 16, 29).

Splenocytes were collected 10 days after vaccination and stimulated with OVA-expressing APC in vitro. The OVA-specific CD8+ T cells in these cultures were capable of undergoing secondary expansion in vitro, as measured by high numbers of TM-OVA+ CD8+ T cells (Fig. 1A, left). To determine whether vaccination resulted in effector CD8+ T cells, an in vivo cytotoxicity assay was performed (30). Both groups of mice vaccinated with OVA8 peptide in IFA (±FGK) displayed a similar capacity to kill target cells in vivo 10 days after vaccination (Fig. 1B, left).

When splenocytes were isolated 30 days after vaccination, OVA-specific CD8+ T cells displayed a strongly decreased capacity to undergo secondary expansion because only low numbers of OVA8-specific (TM-OVA+) CD8+ T cells could be observed after stimulation in vitro (Fig. 1A). Accordingly, mice showed a markedly reduced capacity to kill target cells in vivo at day 30 (Fig. 1B), indicating that the ability to undergo secondary expansion in vitro correlated with the ability to lyse target cells in vivo. Provision of a DC-stimulatory signal by FGK, although enhancing CTL levels at day 10, was unable to preserve the CTL response at day 30 (Fig. 1). Interestingly, when the OVA8 peptide was applied in PBS in combination with systemic administration of FGK, the CD8+ T cell response was not lost over 30 days, as indicated by their ability to expand after in vitro stimulation (Fig. 1A) and their capacity, albeit at lower levels, to kill target cells in vivo (Fig. 1B). Thus, vaccination with the minimal OVA8 CTL peptide in IFA induces a transient effector CD8+ T cell response, followed by functional impairment of these activated CD8+ T cells.

Peptide vaccination in IFA induces long-term presentation of the minimal CTL peptide

The major difference between PBS and IFA is the capacity of the latter formulation to function as a depot for the peptides. To test whether the OVA8 peptide in IFA was presented long-term in vivo, mice were vaccinated with the OVA8 peptide in IFA and, after either 30 or 60 days, CFSE-labeled OT-1 CD8+ T cells were adoptively transferred into C57BL/6 mice as probes to detect Ag presentation in vivo. Extensive proliferation of OT-1 T cells was observed at both days 30 and 60 (Fig. 2A) in the dLN of vaccinated mice, indicating that the minimal OVA8 CTL peptide was still presented 60 days after vaccination. In contrast, when the peptide was applied in PBS, no proliferation of OT-1 CD8+ T cells was observed at day 30 (Fig. 2A). This indicated that vaccination with the OVA8 peptide in PBS was associated with relatively short duration of Ag presentation, whereas vaccination in IFA induced long-term Ag presentation in vivo.


Figure 2
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  		FIGURE 2. IFA promotes extended duration of minimal CTL and minimal Th peptide presentation in vivo. Mice were vaccinated s.c. with the minimal OVA8 CTL (A) or minimal ThOVA17 (B) peptide mixed in IFA. A, Either 30 or 60 days later, 1–2 x 106 CFSE-labeled CD8+CD45.1+ OT-1 T cells were i.v. injected into C57BL/6 mice. Three days later, the dLN (inguinal) was harvested and proliferation was determined by flow cytometry analysis. Cells were gated on CD45.1+CD8+ cells. B, Either at day 30 or 60, 1 x 106 CFSE-labeled CD4+CD90.2+ OT-2 T cells were i.v. injected into CD90.1+ recipient mice. Four days later, the dLN (inguinal) was harvested and proliferation was determined by FACS analysis. Cells were gated on CD90.2+CD4+ T cells. As a control, peptides were administered in PBS and Ag presentation was analyzed on day 30. The data are representative of four mice per time point and per vaccine.

 
Addition of Th OVA323–339 peptide to the CTL/IFA vaccine retains CD8+ T cell function

Provision of FGK for APC activation (15, 16) was unable to prevent CD8+ T cell tolerance induction after vaccination with OVA8 peptide in IFA. Because a dose of mAb is generally rapidly cleared from the system (31, 32), long-term presentation of OVA8 peptide beyond this period (Fig. 2) occurs in the absence of a potent APC-activating agent. Because CD4+ T cells can also activate APC via CD40-CD40L interaction (33), the minimal Th peptide ThOVA17 was mixed into the same IFA depot as the OVA8 peptide. Adoptively transferred CFSE-labeled OT-2 cells into ThOVA17-vaccinated mice showed also that the ThOVA17 peptide in IFA is presented for at least day 60 in vivo in the dLN (Fig. 2B).

We therefore tested whether vaccination with ThOVA17 peptide in IFA was able to induce long-lasting OVA323–339-specific CD4+ T cell responses in C57BL/6 mice. Indeed, OVA323–339-specific IFN-{gamma}+ CD4+ T cells could be detected in all mice when tested directly ex vivo, 30 days after vaccination with the ThOVA17 peptide in IFA (Fig. 3A). Subsequently, we examined whether addition of the ThOVA17 peptide to the IFA depot could rescue the function of OVA8-induced CD8+ T cells. As shown in Fig. 3B, s.c. vaccination of mice with a combination of OVA8 and ThOVA17 peptide in IFA resulted in the detection of similar percentages of TM-OVA+ CD8+ T cells following in vitro stimulation, at both days 10 and 30 after vaccination. Moreover, the combination with the ThOVA17 peptide retained the cytolytic capacity of these CD8+ T cells at day 30 (compare Figs. 3C and 1B, respectively). The addition of an extra DC-stimulatory signal, FGK, slightly enhanced the expansion of the OVA-specific CD8+ T cell response (Fig. 3, B and C).


Figure 3
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  		FIGURE 3. Long-term immunity after minimal CTL peptide plus minimal Th peptide vaccination in IFA. Mice were vaccinated s.c. with the minimal OVA8 CTL peptide and minimal ThOVA17 peptide mixed in IFA ({blacksquare}) or in PBS ({square}) in the presence or absence of 50 µg of FGK (i.v. days 0, 1, and 2). A, Enumeration of CD4+ IFN-{gamma}+ T cells by intracellular cytokine staining. Thirty days after vaccination, mice were sacrificed and the splenocytes were stimulated overnight in the presence or absence of the minimal ThOVA17 peptide and Golgiplug. The next day, the cells were stained for the surface marker CD4 and for intracellular IFN-{gamma} (n = 5; naive vs vaccinated mice, p < 0.01). B, Enumeration of TM-OVA+ CD8+ T cells by FACS. Ten or 30 days after vaccination, splenocytes were harvested and stimulated in vitro with OVA-expressing tumor cells (EL4-OVA) for 7 days and then analyzed for the presence of CD8+ T cells capable of binding TM-OVA (n = 5–8). C, In vivo cytotoxicity assay. Nine or 28 days after vaccination, target cells were injected and cytotoxicity was measured in the spleen at day 10 or 30, respectively. Cells were gated on CD45.1+ lymphocytes. The data are depicted as mean ± SEM (n = 6).

 
Injection of OVA8 and ThOVA17 in PBS, in combination with systemic injection of FGK, resulted in an OVA8-specific CD8+ T cell response with a strong capacity to undergo secondary expansion, but with only a moderate to kill target cells in vivo (Fig. 3, B and C). Direct ex vivo analysis shows that at this time point the CD8+ T cell response has contracted (percentage of TM+ CD8+ cells, mean ± SD, 0.13 ± 0.03 vs naive 0.12 ± 0.04; data not shown), with too low numbers of circulating T cells to exert a direct strong in vivo measurable cytotoxic response. In contrast to the T cells that were formed after vaccination with OVA8 in IFA, delivery of OVA8 + FGK ± ThOVA17 in PBS did not affect their potential to expand in vitro following one round of stimulation (Figs. 1 and 3).

In conclusion, simultaneous activation of Th cells for the sustained deliverance of license to kill signals (34) prevented premature termination of the CD8+ T cell response that is observed otherwise after vaccination with OVA8 in IFA.

Long CTL peptide induces long-lasting CD8+ T cell immunity independent of CD4+ T cell help

We have recently shown that long peptide vaccination (35-aa-long peptide) was superior to a minimal CTL peptide, with respect to the induction of the magnitude and functionality of the CD8+ T cell response when vaccinated in PBS in combination with CpG-oligodeoxynucleotides (28). To study whether the functional impairment of the CD8+ T cell response induced by the minimal OVA8 CTL peptide in IFA can be prevented by the use of a longer peptide in IFA, a long CTL peptide OVA30 and the long ThOVA31 peptide were designed, using the natural OVA protein-flanking residues to extend the minimal CTL and Th peptides. By use of TCR Tg detector T cells OT-1 (CD8) and OT-2 (CD4), we confirmed also that the extended CTL (OVA30) and Th (ThOVA31) peptides were presented at least for 60 days in vivo in the dLN, when administered in IFA (Fig. 4).


Figure 4
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  		FIGURE 4. IFA promotes extended duration of long CTL peptide and long Th peptide presentation in vivo. Mice were vaccinated s.c. with either the long CTL peptide OVA30 or the long Th peptide ThOVA31 mixed in either IFA or PBS. A, Either 30 or 60 days later, 1–2 x 106 CFSE-labeled CD8+CD45.1+ OT-1 T cells were i.v. injected into C57BL/6 recipient mice. Three days later, the dLN (inguinal) was harvested and proliferation was determined by FACS analysis gating on CD45.1+ and CD8a+ lymphocytes. B, Either 30 or 60 days later, 1 x 106 CFSE-labeled CD4+CD90.2+ OT-2 T cells were injected i.v. into CD90.1+ recipient mice. Four days later, the dLN (inguinal) was harvested and proliferation was determined by FACS analysis. Cells were gated on CD90.2+ and CD4+ lymphocytes. As a control, peptides were administered in PBS and Ag presentation was analyzed on day 30. Representative figure is shown of four mice per time point and per vaccine.

 
To ensure that vaccination with the ThOVA31 peptide also resulted in the induction of OVA-specific CD4+ T cells, we enumerated the number of IFN-{gamma}-producing CD4+ T cells 30 days after vaccination by FACS analysis. The magnitude of the OVA-specific CD4+ T cell response induced by the ThOVA31 peptide was comparable to what was observed after vaccination with the ThOVA17 peptide (Fig. 5A). To assess whether the use of long peptides in IFA prevented the induction of CD8+ T cell tolerance at day 30, mice were vaccinated with the OVA30 peptide in the absence or the presence of the ThOVA31peptide with or without i.v. injection of FGK (Fig. 5). In contrast to what we observed with OVA8, all the vaccine combinations with OVA30 resulted in high numbers of TM-OVA+ CD8+ T cells after stimulation in vitro at day 30, indicating that the CD8+ T cells were capable of undergoing extensive secondary expansion in vitro (compare Fig. 5B with 1A, right-hand side). Addition of the ThOVA31 peptide to the OVA30 peptide/IFA bolus enhanced the CD8+ T cell response (Fig. 5B) compared with OVA30 peptide alone (mean TM-OVA+, 28 vs 13%, respectively). An additional, albeit small increase in the number of CD8+ T cells was observed in mice that received the OVA30, ThOVA31 peptide in IFA in combination with systemic FGK (Fig. 5B).


Figure 5
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  		FIGURE 5. Long CTL peptide induces long-lasting CTL immunity independent of CD4+ T cell help. Mice were vaccinated s.c. with the long CTL peptide OVA30 with or without the long Th peptide ThOVA31 mixed in IFA ({blacksquare}) or in PBS ({square}) in the presence or absence of 50 µg of FGK (i.v. on days 0, 1, and 2). A, Enumeration of CD4+ IFN-{gamma}+ T cells by intracellular cytokine staining. Thirty days after vaccination, mice were sacrificed and the splenocytes were stimulated overnight in the presence or absence of the ThOVA31 peptide and Golgiplug. The next day, the cells were stained for the surface marker CD4 and for intracellular IFN-{gamma} (n = 5; naive vs vaccinated mice, p < 0.001). B, After 30 days, the spleen was harvested and cells were stimulated in vitro with OVA-expressing tumor cells (EL4-OVA) for 7 days and then analyzed for the presence of CD8+ T cells capable of binding TM-OVA (n = 4; OVA30 (IFA) vs OVA30 + ThOVA31 (IFA), p = 0.06; OVA30 + FGK (IFA) vs OVA30 + ThOVA31 + FGK (IFA), p = 0.09). In vivo cytotoxicity assay at day 30 (C) and day 90 (D). Target cells were injected at day 28 (C) or day 88 (D), and 2 days later cytotoxicity was measured in the spleen by FACS analysis. Cells were gated on CD45.1+ lymphocytes. The data are depicted as mean ± SEM (n = 5–10; C and D, IFA vaccination vs PBS vaccination, p < 0.01). Mice that were depleted for CD4+ T cells (GK1.5, light gray bar) received 25 µg i.p. of depleting anti-CD4 at 3 days and 1 day before vaccination and once every week during the experiment. The dark gray bar indicates MHC class II knockout mice (class II–/–; n = 6).

 
Subsequently, the effector function of the CD8+ T cells was tested in an in vivo cytotoxicity assay at day 30. Vaccination with OVA30 in IFA resulted in strong in vivo lytic activity (Fig. 5C). To warrant that the effect seen by OVA30 does not rely on CD4+ T cell help, by unknown cryptic helper sequences that may have been present in the long OVA30 peptide, the mice were depleted for CD4+ T cells before vaccination and once every week throughout the experiment to maintain complete CD4+ T cell depletion (≤0.01% CD4+ T cells) or MHC class II–/– mice were used. The absence of CD4+ T cells during the priming and effector phase did not demonstrably affect the OVA30-induced CD8+ T cell killing of target cells. Nor did the absence of MHC class II strongly influence the killing capacity of OVA30-induced CTL (Fig. 5C). Although the numbers of OVA-specific CD8+ T cells were higher when the OVA30 peptide was complemented with either systemic FGK, the ThOVA31 peptide, or both, it was not possible to measure a potential increase in effector function because the percentage of in vivo killed target cells in mice vaccinated with OVA30 in IFA was high already (Fig. 5C). The cytolytic activity of these CD8+ T cells was preserved over time, because a comparable killing capacity was observed at day 90 (Fig. 5D).

Similar to what was observed with the injection of OVA8 and ThOVA17 in PBS, the injection of the long peptide(s) in PBS in combination with systemic FGK resulted in an OVA-specific CD8+ T cell response that was contracted at day 30 (percentage of TM+ CD8+ cells, mean ± SD, OVA30 + FGK 0.13 ± 0.05; OVA30 + ThOVA31 + FGK 0.13 ± 0.03, vs naive 0.12 ± 0.04; data not shown) and of which the number of circulating T cells was too low to measure direct in vivo effector function (Fig. 5C). However, these vaccine-induced T cells were still capable of undergoing secondary expansion in vitro (Fig. 5B) to a similar extent as seen with OVA30 in IFA. In contrast, higher numbers of circulating OVA-specific cytotoxic CD8+ T cells were detected in mice vaccinated with the long OVA30 CTL peptide in IFA (percentage of TM+CD8+ cells, mean ± SD, 0.83 ± 0.61; data not shown).

In conclusion, despite the fact that vaccination with the minimal OVA8 peptide or the longer OVA30 peptide in IFA resulted in a comparable long Ag presentation in vivo, extension of the minimal OVA8 peptide to the OVA30 peptide prevented the functional impairment of activated CD8+ T cells and instead induced a CTL response that was sustained over a long period.

Dominant-negative effect of the OVA8 CTL peptide on induction of effector CD8+ T cells by an immunogenic vaccine

In addition to the vaccine dLN, the ndLN of mice injected with either OVA8, OVA30, ThOVA17, or ThOVA31 also were analyzed for the presentation of these peptides. In contrast to the long peptides, which were presented predominantly in the dLN, presentation of the OVA8 peptide was also observed in ndLN, indicating that this peptide spreads systemically (Fig. 6). Despite the fact that sustained presentation of the ThOVA17 peptide is readily detected in vivo (Fig. 2B), we were not able to detect presentation of the ThOVA17 peptide ex vivo (Fig. 6B), suggesting that the MHC class II off-rate of this peptide is too high to allow direct ex vivo detection. This must be related to the affinity of this peptide for MHC class II I-Ab, which is probably lower than for the MHC class II molecule I-Ad, which was first found to present this peptide (35).


Figure 6
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  		FIGURE 6. Location of Ag presentation. Mice were vaccinated with the indicated peptide mixed in IFA. Two days later, the dLN (inguinal and axillary) and ndLN (mesenteric) were isolated. A, Ex vivo detection of the OVA8 epitope was performed by incubation of 0.5 x 106 LN cells with 100,000 OT-1 CFSE-labeled Tg CD8+ T cells for 3 days. B, Ex vivo detection of the Th epitope was performed by incubation of 1 x 106 LN cells together with 200,000 CFSE-labeled OT-2 cells for 4 days. Dilution of CFSE was measured by gating on CD45.1+CD8+ lymphocytes for OT-1 cells, or V{alpha}2+ CD4+ lymphocytes for OT-2 cells.

 
Importantly, systemic presentation of OVA8 CTL peptide is associated with the functional impairment of the CD8+ T cell response (Fig. 1B), whereas the predominant local presentation of the OVA30 peptide results in immunity. Therefore, we wondered what effect OVA8 vaccination has on a concomitant immunogenic vaccination (OVA30; Fig. 5C). To test this, mice were vaccinated with the OVA8 peptide, the OVA30 peptide, or both peptides mixed in IFA, and the in vivo killing capacity was determined at day 30. As shown in Fig. 7, vaccination with OVA8 was unable to induce effector CD8+ T cells at day 30, whereas vaccination with the OVA30 peptide resulted in a CTL response. When the OVA8 peptide was mixed with the OVA30 peptide in the same IFA depot, a significant reduction in the in vivo effector function of OVA-specific CD8+ T cell reactivity was observed (Fig. 7). These data show that vaccination with the minimal OVA8 peptide has a dominant-negative effect on the effector functions of CD8+ T cell induced by an immunogenic peptide vaccine such as OVA30 CTL peptide.

Failure to induce long-lasting CTL immunity is a more general characteristic of vaccination with minimal CTL peptide epitopes, but can be overcome by the use of long peptides.

Because injection of the dominant minimal CTL peptide OVA8 of the model Ag OVA results in the functional impairment of CD8+ T cells when injected s.c. at a low dose in IFA, and as such acts similar to the earlier described adenoviral E1A (E1A10) (11, 17) and E1B (12) minimal CTL peptides, the impact of minimal and extended CTL epitope vaccines was compared in two additional antigenic systems.

First, we tested the HPV16 E7 minimal CTL peptide (HPV9) and the extended CTL peptide (HPV35) (28). As shown in Fig. 8A, vaccination with the HPV9 peptide resulted in marginal killing (8%) of HPV peptide-loaded target cells in vivo 30 days after vaccination, whereas the use of the HPV35 peptide resulted in 45% specific killing of target cells (Fig. 8A). The enhanced effector response induced by HPV35 in IFA was independent of CD4+ T cell help (Fig. 8A).

Similarly, vaccination with the minimal CTL peptide of the adenovirus E1A protein E1A10, known to tolerize the CD8+ T cell response (11, 17), failed to induce CD8+ T cell effector function 30 days after vaccination (3% killing) (Fig. 8B). In contrast, when the extended CTL peptide E1A30 was used, 52% of the target cells were lysed 30 days after vaccination, independently of the presence of CD4+ T cell help (Fig. 8B). These results indicated that the use of extended CTL peptide vaccines may prevent T cell tolerance (E1A10 peptide) and, in general, induce a sustained in vivo CD8+ effector T cell response, when compared with their minimal CTL peptide counterparts.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we showed that vaccination with a low dose of a highly immunogenic exact MHC class I-binding peptide (OVA257–264) can result in the activation of CD8+ effector T cells, which eventually cease to expand or to kill target cells, provided that this peptide is administered in IFA. The injection of a minimal CTL peptide in IFA did not immediately impair the responding CD8+ T cells, but was preceded by induction of a complete functional CD8+ T cell response that was still present at day 10, but absent at 30 days after vaccination. APC activation by coadministration of FGK was unable to rescue the responding CD8+ T cells from their fate. The basis for this dominant-negative effect lies in the duration of Ag presentation in vivo, the location in which these peptides are presented, and the absence of sustained systemic danger signals.

When the minimal CTL peptide is administered in PBS, this Ag is presented to CD8+ T cells for <30 days. In contrast, when the peptide is mixed with IFA, the CTL peptide was still presented at 60 days after vaccination. FGK is likely to be cleared rapidly from the system, similar to other Ab (31, 32), and as such can only temporarily activate APC. When systemic presentation of the minimal CTL peptide ensues in the absence of a continuous helper/danger signal (36), it is likely that this will result in CD8+ T cell tolerance (Figs. 1 and 2) (17). To provide continuous helper signals, we included the minimal ThOVA17 peptide in the CTL/IFA bolus to activate APC via CD40-CD40L interactions (15, 16, 33), and showed that also the Th peptide was presented for >60 days (Fig. 2). Because both peptides are continuously leaking out of the IFA depot, activation of APC and CD4+ and CD8+ T cells will take place simultaneously. The induction of CD4+ T cells not only enhanced the CD8+ T cell response (Fig. 3), as was previously reported (37, 38), but our experiments also show that it prevented the impairment of CD8+ T cell reactivity, resulting in the preservation of long-term CD8+ T cell immunity (Fig. 3).

Recently, we found that the activation of CD8+ T cells by minimal CTL peptide vaccines strongly depended on the presence of nonprofessional APC (M. Bijker, manuscript in preparation). Indeed, minimal MHC class I-binding peptides are known to bind directly to their restriction elements at the cell surface of MHC class I-expressing cells, including nonprofessional APC. However, our experiments teach us that CD8+ T cells responding to minimal CTL peptides injected in PBS (and FGK) can become fully activated and as well form memory CD8+ T cells that are able to expand upon secondary Ag stimulation in vitro, to a similar extent as when the longer peptide is injected (compare Figs. 1A and 5B, day 30, {square}). Thus, even while suboptimal presentation may occur, this does not necessarily lead to the activation of CTL that subsequently fail to expand or survive. This implies that at least other factors, such as the long-term systemic presentation of the OVA8 peptide, may play a role in the detrimental outcome of minimal CTL peptide vaccines in IFA.

Despite the fact that both the long OVA30 CTL peptide and the minimal OVA8 CTL peptide were presented for at least 60 days in vivo when applied in IFA (Figs. 4A and 2A, respectively), vaccination with the OVA30 peptide did not result in an eventual impairment of CD8+ T cell reactivity and did not require CD4+ T cell help to preserve the CD8+ T cell response (Fig. 5). This was not only the case for OVA, but could be generalized for three Ag systems (Figs. 1, 5, and 8). Extension of the minimal CTL peptide of either the OVA, HPV, or E1A protein induced CD8+ T cells with potent in vivo killing function until at least 30 days after vaccination. Furthermore, increasing the length of the minimal E1A10 CTL peptide to a 30-aa-long peptide prevented the induction of CD8+ T cell tolerance that was previously observed when the minimal CTL peptide was used (11).

In contrast to the minimal CTL peptides, which spread systemically to ndLN and noninflamed LN (17, 29, 39), where Ag probably is presented in a tolerizing fashion (40), the longer peptides are predominantly presented by APC in the vaccine dLN (Fig. 6). Because IFA induces a local inflammatory response (41, 42), it will thereby provide the necessary danger signals (36, 43) that are needed to activate the local APC that have ingested the long peptide vaccine. Together, this can explain why vaccination with long peptides in IFA does not result in a functionally impaired CD8+ T cell response and why CD4+ T cell help is not required.

So, why did many papers report that minimal peptide vaccination in IFA induces an effective CD8+ T cell response (6, 7, 8, 9) instead of CD8+ T cell tolerance? On one hand, the outcome of vaccination was studied shortly after peptide vaccination when the CD8+ T cells are not expected to be tolerized, as shown in this study (Fig. 1). On the other, many of the peptides used were much longer than the minimal CTL sequence, reaching up to 27 aa in length (2, 6, 7, 44, 45, 46, 47, 48), and/or contained a Th sequence (45, 49). In view of the current results, this might explain why these so-called short synthetic peptides performed so well compared with the exact minimal CTL peptides used in this study, or in clinical trials (4).

In addition, it should be noted that vaccination with minimal CTL peptide represses the induction of CD8+ effector T cells generated in response to concomitant proper Ag presentation (Fig. 7). One can envisage that such an effect is highly undesirable in cancer patients that receive minimal CTL peptide vaccination after chemotherapy or radio therapy. In these cases, large amounts of Ag are released and cross-presented by DC (50), which may result in the activation of tumor-specific CD8+ T cells that recognize the same sequences also present in the minimal CTL peptide vaccines in IFA. As a result, such T cell responses may be functionally impaired following vaccination.

In conclusion, our data clearly answer the question why vaccines consisting of minimal CTL peptides in oil-in-water emulsions (e.g., IFA) may have only limited immunological and clinical success in cancer patients. These vaccines need to be complemented at least by Th epitopes to sustain effective CD8+ T cell reactivity. In case that specific CD4+ T cell help is not readily available, extension of CTL peptides to longer variants may form an excellent alternative. Vaccine trials should be performed to confirm our results in a human setting.


   Acknowledgments
 
We gratefully acknowledge Astrid van Halteren (PhD), Lothar Hambach (MD), and Marij Welters (PhD) for critically reviewing the manuscript; Sytse Piersma for helping with figure preparations; the group of Jan-Wouter Drijfhout for peptide synthesis; and the people in the animal facility for assistance during animal experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Dutch Cancer Society Grant UL 2003-2817. Back

2 Address correspondence and reprint requests to Dr. Sjoerd H. van der Burg, Department of Clinical Oncology, Building 1, K1-P, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: shvdburg{at}lumc.nl Back

3 Abbreviations used in this paper: Tg, transgenic; dLN, draining lymph node; HPV, human papillomavirus; LN, lymph node; ndLN, nondraining LN; TM, tetramer. Back

Received for publication January 5, 2007. Accepted for publication August 12, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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F. M. Speetjens, P. J.K. Kuppen, M. J.P. Welters, F. Essahsah, A. M. E.G. Voet van den Brink, M. G. K. Lantrua, A. R. P.M. Valentijn, J. Oostendorp, L. M. Fathers, H. W. Nijman, et al.
Induction of p53-Specific Immunity by a p53 Synthetic Long Peptide Vaccine in Patients Treated for Metastatic Colorectal Cancer
Clin. Cancer Res., February 1, 2009; 15(3): 1086 - 1095.
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G. G. Kenter, M. J.P. Welters, A.R. P.M. Valentijn, M. J.G. Lowik, D. M.A. Berends-van der Meer, A. P.G. Vloon, J. W. Drijfhout, A. R. Wafelman, J. Oostendorp, G. J. Fleuren, et al.
Phase I Immunotherapeutic Trial with Long Peptides Spanning the E6 and E7 Sequences of High-Risk Human Papillomavirus 16 in End-Stage Cervical Cancer Patients Shows Low Toxicity and Robust Immunogenicity
Clin. Cancer Res., January 1, 2008; 14(1): 169 - 177.
[Abstract] [Full Text] [PDF]

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