Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4+CD25+ regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8+, CD4+ T cells and antibody response. The appearance of a CD4+ T-cell response correlated with a stronger memory response. The combined CD25+ inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.
Immunomodulation has emerged as an important component in inducing a strong and effective immune response against tumor-associated antigens (TAA).1 In this respect, inactivation of regulatory T cells (TR) has proved efficacious against tumor cell lines, although permanent TR depletion is embedded with autoimmune-related side effects such as colitis and gastritis.2
The induction of a long lasting and effective CD8+ response is key to most cancer vaccine treatments against TAA. To achieve this result it is also important to induce a CD4+ helper response that can sustain an effective CD8+ response over time.3 However, CD4+cells also play a role in regulating the amplitude and duration of the immunological response raised against TAA, mainly through the action of TR. They express high levels of interleukin (IL)-2 receptor (CD25), GITR, 41BB and the transcription factor forkhead FOXP3.2 T-cell transfer upon TR depletion leads to autoimmune diseases that are suppressed by complementation with TR cells.4 Thus, an ideal setting in cancer vaccine could be the induction of a transient autoimmune state (TR depletion or inactivation) where antigen-specific immune response could be induced by a genetic vaccination. Many reports have shown depletion of TR in tumor cell vaccination has a positive impact, but only a few have explored the impact of TR on a specific peptide vaccine or xenogeneic vaccine.5, 6 Apparently, the impact of anti-CD25 antibodies on TR is more complex than simple depletion as recently shown by Kohm et al.7 Anti-CD25 antibody induces down expression of CD25 receptor, which is associated with functional inactivation, but they remain present, as indicated by FOXP3 expression in CD4+ cells.
The impact of TR depletion or inactivation on self-TAAs vaccination has not been explored extensively. To verify the efficacy of this strategy we explored a transient TR inactivation as a sort of ‘immunization window’ where genetic vaccination against TAA may provoke a stronger immune response that in turn can protect mice against tumor challenge when TR subsequently return to normal level. To test this hypothesis we used a CEA transgenic mouse model where we have recently shown that an adenovirus (Ad) vector expressing the carcinoembryonic antigen (Ad-CEA) succeeded in breaking immunological tolerance.8
Materials and methods
An Ad serotype 5 expressing a codon optimized gene encoding for CEA, here named Ad-CEA, has been previously described with the name Ad-CEAopt.8 The same CEA codon optimized expression cassette was inserted in an Ad serotype 6 generating Ad6-CEA.
Lyophilized CEA peptides were purchased from Bio-Synthesis, Lewisville, TX, USA and resuspended in dimethylsulfoxide at 40 mg/ml. Pools of peptides of 15 aa overlapping by 11 residues were assembled as described.9 Peptide pool C-D's final concentration was 0.8 mg/ml. Peptides were stored at −80°C.
Cell lines and mice
MC38-CEA cells10 were cultured in Dulbecco's modified Eagle's medium (DMEM) 10% fetal bovine serum (FBS), supplemented with Penicillin and Streptomycin (10 000 units Penicillin and 10 mg Streptomycin/ml), Glutamine (2 mM) and Zeocyn (400 μg/ml). PC61 hybridoma was purchased from American Type culture Collection (ATCC, Manassas, VA) and cultured in DMEM 10% FBS. All animal studies described in this report were approved by the IRBM institutional animal care Committee, CEA transgenic mice (CEA.Tg) are in a C57BL/6 background and were provided by J Primus (Vanderbilt University).10 Animals were kept in standard conditions.
Mice immunization and tumor challenge
For immunization protocol, CEA.Tg were subjected to two intramuscular injections of Ad vectors (1 × 109 viral particles/injection) as described previously.9 For tumor challenge experiments, spleens of CEA.tg mice were injected with 5 × 104 MC38-CEA cells and monitored for survivors.
To develop a non-invasive bioluminescence detection of tumor growth in vivo, MC38-CEA cells were transfected with a plasmid encoding firefly luciferase and several stable clones selected in vitro. A cell line, named MC38-CEA-L, was selected on the basis of high bioluminescence detected in vitro. The tumor growth rate of the luciferase expressing cell lines was compared with the non-glowing parental MC38-CEA cells in a subcutaneous (s.c.) xenograft experiment. Mean external caliper measurements indicated that the two cell lines grew at a similar rate. The mean photons emitted from the tumors over time in each cohort were compared with the corresponding tumor volumes of the same animals. Bioluminescent data correlated with tumor volume, with overall R2 values of 0.95–0.98 (data not shown). Having validated the new tumor cell line, mice were injected in the spleen to follow hepatic metastases over time. Liver tumor bioluminescence started to increase about 3–4 weeks after cell injection in Ad-CEA-vaccinated mice.
T-cell depletion and passive transfer
In vivo inactivation of TR was conducted by intraperitoneal (i.p.) injection of 0.5 mg of mAb anti-CD25 (PC61) produced in nude mice and purified using a protein G column (Amersham, Buckinghamshire, UK). Efficiency of TR inactivation was assessed in peripheral blood by flow cytometry using mAbs: phycoerythrin-conjugated (PE) anti-CD4, peridinin-chlorophyll-protein (PercP) anti-CD8, allophycocyanin-conjugated (APC) anti-CD25 (BD Pharmingen, San Diego, CA); PE anti-mouse/rat FOXP3 Staining Set (eBioscience, San Diego, CA). The CD4+ T cells or CD8+ T cells of immunized animals were depleted, by injecting anti-CD4 (GK1.5 hybridoma, ATCC Manassas, VA) or anti-CD8 (2.43 hybridoma, ATCC Manassas, VA). 0.5 mg of antibodies (100 μl diluted acites/dose) were injected on day −7 relative to the tumor challenge and injected every week for 3 weeks after injection of 5 × 104 MC38-CEA cells or MC38-CEA-L cells. Serum transfer studies were conducted as follows: 200 μl of serum from mice that had been treated with PC61 antibody and vaccinated with Ad-CEA were injected i.p. into CEA.tg mice three times: 24 h before the tumor challenge, the same day of the tumor challenge with MC38-CEA-L cells, and 120 h after the tumor challenge. Tumor growth was followed by the xenogen system.
IFNγ intracellular staining
The assay was carried out as described previously from splenocytes.9 and was adapted to peripheral mononuclear cells (PBMC) with minor modifications. Briefly, One to two million mouse PBMC or splenocytes were incubated with the indicated pool of peptides (5–6 μg/ml for each peptide) and treated subsequently with brefeldin A (1 μg/ml; BD Pharmingen) at 37°C for 12–16 h. Cells were washed, stained with surface antibodies, fixed, permeabilized and incubated with the isothiocyanate-conjugated anti-mouse (FITC) IFNγ antibodies (BD Pharmingen). Cells were fixed with 1% formaldehyde solution in PBS and analyzed on a FACS-Calibur flow cytometer, using CellQuest software (Becton Dickinson, Franklin Lakes, TX, USA).
Memory cells analysis
PBMC were prepared as described above and stained with: tetramer anti-CEA-PE (Proimmune, Oxford, UK); Ab anti-CD8-PercP (BD Pharmingen); Ab anti-CD62L-APC (BD Pharmingen). Memory cells were identified by sequential gating using the following criteria: morphological, CD8+, Tetramer+, CD62L high expression.11, 12, 13
Antibody detection and titration
Sera for antibody titration were obtained by retro-orbital bleeding. Enzyme-linked immunosorbant assay plates (Nunc maxisorp) were coated with 100 ng/well of CEA protein (Fitzgerald, Concorde, MA, highly pure CEA), diluted in coating buffer (50 mM NaHCO3, pH 9.4) and incubated O/N at 4°C. Plates were then blocked with PBS containing 5% bovine serum albumin (BSA) for 1 h at 37°C. Mouse sera were diluted in PBS 5% BSA (dilution 1/50 to evaluate seroconversion rate; dilutions from 1:10 to 1:31 250 to evaluate titer). Pre-immune sera were used as background. Diluted sera were incubated O/N at 4°C. Washes were carried out with PBS 1% BSA, 0.05% Tween 20. Secondary antibody (goat anti-mouse, IgG Peroxidase, Sigma, St Louis, MO) was diluted 1/2000 in PBS, 5% BSA and incubated 2–3 h at RT on a shaker. Plates were developed with 100 μl/well of tetramethylbenzidine (TMB) substrate (Pierce, Rockford, IL) and were read at 450/620 nm. Anti-CEA serum titers were calculated as the reciprocal limiting dilution of serum, producing an absorbance at least threefold greater than the absorbance of autologous preimmune serum at the same dilution.
Where indicated, results were analyzed by the long rank or two-tailed Student's t-test. P<0.05 was considered significant.
Treatment with an anti-CD25 antibody enhances immune response against a self-TAA
A previous study has shown that CEA-specific cellular immune responses were enhanced by vaccination with Ad-CEA in the CEA.Tg mouse model.8 Here, we evaluated whether the administration of anti-CD25 mAb could enhance the CEA-specific immune responses.
As first control we checked whether the anti-CD25 antibody, PC61, could deplete mice of CD25+ cells or whether the presence of the antibody led to a functional inactivation of TR cells as recently shown.7 Analysis of FOXP3 expression in CD4+ cells showed that also in C57BL/6 mice, anti-CD25 treatment results in complete disappearance of CD4+CD25+ cells but does not affect the number of CD4+FOXP3+ cells (Figure 1a). This observation indicates that anti-CD25 monoclonal antibodies induce the functional inactivation, but not depletion of TR cells. To characterize the impact of TR on Ad-CEA vaccination, CEA.Tg mice were treated with PC61, and vaccinated 4 days later with two injections of Ad-CEA vaccine 2 weeks apart. This schedule was chosen in order to perform vaccination treatment in the absence of active TR cells. The CEA-specific immune response was evaluated when the CD25 expression on TR cells returned to normal levels at day 35 (Figure 1). To measure the cellular-mediated immune response, INFγ+ intracellular staining (ICS) analysis was carried out with splenocytes restimulated in vitro with a pool of peptides covering the entire CEA protein.
Mice that have been treated with PC61 and vaccinated with Ad-CEA showed a statistically significant increase (P=0.0016) in the CD8+ T-cell response against the C-terminal region of CEA (Figure 2a). The average response was more than fivefold higher than Ad-CEA-treated mice. No CD8+ response was observed using peptide pools covering the N-terminus or the central region of CEA protein (data not shown). In agreement with the predominant C-terminal immunogenicity of CEA8, 9 a low but statistically significant increase in the IFNγ-positive CD4+ T cells was observed in seven out of eight mice inactivation of TR, whereas only one mouse injected with Ad-CEA showed a detectable response (Figure 2b). Finally, an antibody response was detected in most mice inactivation of TR (6/8) and only in one of the control group (P=0.028) (Figure 2c). Anti-CEA-specific cellular response and antibody response were not observed in mice treated only with anti-CD25 antibody (data not shown).
These results indicate that TR inactivation enhanced the Ad-CEA induced CD8+, CD4+ T cells and antibody response in a tolerant animal model.
Ad-CEA vaccination in association with TR inactivation resulted in an increased CEA-specific memory response
In view of the higher immune response observed in mice vaccinated upon TR cells inactivation, we asked whether this vaccination protocol could affect persistence of the immune response as well as the induction of a memory response against CEA. To verify the persistence of immune response, CEA.Tg mice were treated with or without anti-CD25 antibody and vaccinated with Ad-CEA as described in Figure 1a. CEA-specific immune response was analyzed over time in the same group of animals by IFNγ ICS analysis performed with a pool of PBMC.
As expected from the immune response measured with splenocytes, higher frequency of CEA-specific IFNγ+CD8+ T cells was observed in the pool of PBMC of mice inactivation of TR. This observation was confirmed at different time points showing a similar persistence in the two groups of vaccinated mice. The signal returned to basal level at week 10 (Figure 3a).
At this time point, to verify whether TR inactivation had induced a higher level of memory CD8+ T cells, PBMC were analyzed for the presence of memory CD8+ T cells using a tetramer staining with peptide CGIQNSVSA and analyzed for the expression of CD62L, considered a marker of central memory.11, 12, 13 Anti-CEA CD8+ cells were slightly higher in the pool of PBMC of TR inactivation mice (178 events) than in the group of mice treated only with Ad-CEA (124 events), whereas a low background was observed in the untreated control pool (20 events). More interesting is the higher expression of CD62L that was observed in TR inactivation mice as indicated by the higher mean value (Figure 3b).
To further support this finding, both groups of mice were boosted with Ad6-CEA, a different Ad serotype, which expresses the same CEA transgene. Using a different Ad serotype is justified by the presence of neutralizing antibodies against Ad5, which impede effective vaccination with the same serotype.14 Importantly, both groups were boosted in the presence of TR cells. A higher CD8+ response was registered in the group originally TR-inactivated, and this response followed similar kinetics and became undetectable 3 months later (Figure 3a).
These results showed that TR inactivation not only increased cellular-mediated immune response, but also enhanced the central memory response.
Tumor protection is induced by Ad-CEA vaccination only in TR-inactivated mice
An open question was whether Ad-CEA vaccination in TR-inactivated mice could prevent tumor growth in CEA.tg mice when the CD25+ population returned to normal level. To answer this question, MC38 cells expressing CEA were injected in the spleen. This tumor model is characterized by formation of metastasis in the liver.15 Tumor challenge was started at day 45 after TR inactivation and Ad-CEA vaccination as described in Figure 1. As control we used mice that were untreated, treated with a control IgG and vaccinated with Ad-CEA or only injected with PC61 antibody.
As shown in Figure 4 a statistically significant increase in the survival rate was observed in TR-inactivated/Ad-CEA-vaccinated mice with respect to either TR inactivation alone (P=0.0006) or untreated mice (P<0.00005). Thirteen out of 15 TR-inactivated mice were still alive 300 days after tumor challenge. No statistically significant increase in survivals was observed in mice only injected with PC61 antibody (3/15) or in the group treated with Ad-CEA alone (1/15) compared to control mice (0/15).
As this is a metastatic model, we decided to verify whether this vaccination protocol was able to restrict tumor growth within the spleen or whether it could reverse metastases established in other organs such as liver. To discriminate between these two hypotheses, a non-invasive monitoring system was adopted based on the chemioluminescence luciferase activity. MC38-CEA cells were engineered to express luciferase (MC38-CEA-L) and tumor growth monitored over time by luciferase activity. To validate this system, metastases formation was confirmed by examining tissue ex vivo in parallel with the original cell line (data not shown). Mice injected with MC38-CEA-L showed a clear signal in the liver with occasional signs of luminescence in the lung. Thus, to follow metastases formation in the liver, mice were subjected to different vaccination protocols and challenged with MC38-CEA-L. Mice were imaged immediately after injection of cells and once a week thereafter. Ventral images of two representative groups of mice at different time points are shown in Figure 5a. It is worth noting that a much lower signal was observed in TR-inactivated and Ad-CEA-vaccinated mice than in control mice (Max=6.3 × 103 vs Max=4.2 × 106). Within 2 weeks, localized chemioluminescence signals appeared in the spleen of the control group, whereas TR-inactivated and Ad-CEA-vaccinated mice clearly showed a reduced abdomen signal which always remained lower than the control group (Figure 5b). Of interest was mouse n.1, which showed a weak and transient signal on the left side corresponding to the liver. From this observation we can speculate that liver metastases could have been reversed, at least in this case.
To determine the possible prophylactic mechanisms associated with this combined therapy, immunological analysis was conducted using CEA.Tg mice implanted intrasplenic (i.s.) with MC38-CEA-L tumors. We examined which immunological component was required for antitumor activity by depleting either CD4+ or CD8+ cells in vaccinated mice before tumor challenge. CD8+ depletion but not CD4+ depletion strongly affected tumor growth (Figure 6a). Tumor protection was not observed in naïve mice that have received serum transfer before tumor challenge (Figure 6b).
Here, we have shown that TR inactivation in association with Ad-CEA vaccination resulted in the induction of a greater CD8+ response, the appearance of CD4+ response and increased antibody titers. The enhanced immune response was associated with tumor protection in a CEA immunological tolerant model.
In our experimental model the antitumor activity of Ad-CEA vaccination was efficacious only in the presence of TR inactivation (Figures 4 and 5). This observation is not related to antitumor activity observed immediately after injection of the anti-CD25 monoclonal antibody as reported in different experimental settings.16 Here, we used transient TR inactivation and thus tumor challenge was performed when TR cells had already returned to normal levels. In line with published literature, at this time point tumor growth did not show a statistically significant difference from control groups, although a trend in tumor protection was shown (Figure 4). This observation may be explained by marginal antitumor activity of inactivated TR cells and thus be worthwhile investigating. In contrast, a clear and statistically significant difference was observed between the deaths of almost all mice vaccinated with Ad-CEA alone and the number of survivals in the group vaccinated with the same Ad-CEA vector upon TR inactivation. One potential explanation is the significant increase of CEA-specific IFNγ+CD8+ cells and the appearance of a CD4+ response. These observations are consistent with other reports where the magnitude of immune response was correlated with the biological effect under investigation, as shown by protection from viral challenges afforded by vaccination in mice inactivation of TR cells. In agreement with published data, we observed a 2 to 5-fold increase in the CD8+ response. This CEA-specific IFNγ+ CD8+ response is likely to play a crucial role in tumor protection, as indicated by the loss of protection upon CD8+ cells depletion before tumor challenge. Similar depletion of CD4+ cells or serum transfer from immunized mice had no effect on tumor take and progression over time (Figure 6). The direct role of CEA-specific antitumor CD8+ response is in line with previous reports regardless of the CEA.Tg model utilized.17, 18 By contrast, CEA immune response induced by poxvirus-derived vectors with or without costimulatory molecules showed an antitumor activity associated with CD4+ response.19, 20 This discrepancy may be explained by the use of different vaccination protocols in similar but not identical transgenic mouse models.
Apparently, the increase of tumor antigen-specific IFNγ response mediated by TR inactivation was not limited to CEA in CEA.Tg mice, but we did observe an enhanced immune response with this protocol using a different self-antigen, that is, mouse Ep-CAM, in an outbred CD1 mouse strain (data not shown). Interestingly, use of the same TR inactivation protocol in conjunction with Ad-mEp-CAM vaccination enhanced the anti-mEp-CAM IFNγ+CD8+ response in mice where this response was already measurable, but was ineffective in those mice where the response was undetectable. We cannot exclude that additional immune effects were induced which led to the induction of other important cytokines such as TNF-α or IL-2, and which may serve as more appropriate biomarkers to reveal antigen-specific immune responses induced by this vaccination protocol. Further studies in this direction are ongoing.
The enhanced immune response we observed was associated with tumor protection in a metastatic tumor model. We did not evaluate the therapeutic efficacy of TR inactivation in conjunction with Ad-CEA vaccination in the treatment of established tumors. This is mainly due to the observation that TR inactivation blocks MC38-CEA tumor growth (data not shown) in line with previous reports that showed lack of tumor growth during the treatment with anti-CD25 antibodies in different mouse models.16, 21, 22, 23, 24 The prophylactic treatment explored in this report is more similar to a clinical setting where patients may receive CEA vaccination as means of protection against tumor relapse after surgical removal of the primary tumor. In addition, the rationale in a prophylactic setting may be different to that of a therapeutic application. In the first case a long lasting memory response should be privileged over an immediate effector immune response, which should eradicate an existing large mass of tumor cells.25 In line with a prophylactic application, we have shown that TR inactivation enabled a persistent immune response as indicated by a higher level of memory markers, and also as a stronger CD8+ response observed upon a boost with Ad6-CEA vaccine 3 months after the initial treatment (Figure 3). This observation is in line with previous observations on viral vaccines26 or more in general on the role of TR in restricting memory CD8+ response.27
TR depletion may pose the risk of increased side effects, as shown previously when it was used in conjunction with cell line vaccines.28 On the contrary, gene-specific vaccination in association with TR inactivation restricts potential side effects to those tissues that express the antigen. Indeed, we have observed that targeting CEA in the CEA.Tg mouse model was not associated with any evident signs of tissue damage or systemic alterations (data not shown). This lack of side effects seems to be in contrast with a large number of reports showing side effects associated with transfer of T lymphocyte from mice vaccinated with TAA upon depletion of TR cells. It is worth mentioning that few studies, if any, have adopted a protocol such as that described here, and to the best of our knowledge few reports used a real tolerant model such as CEA.Tg mice. We cannot exclude, however, that side effects could be induced by undesired concomitant vaccination against other antigens, as recently outlined by concurrent induction of antitumor immunity and autoimmune thyroiditis in TR-depleted mice.29 The utilization of a short period of TR inactivation and the specificity of CEA expression in the digestive track may have limited side effects more effectively than previous protocols. Interestingly, the feasibility of CD25 targeting in the clinic was recently explored in conjunction with dendritic cells transduced with tumor RNA.30 This vaccination resulted in an enhanced vaccine-mediated antitumor immunity without apparent bystander toxicity.
In conclusion, this report has shown that transient TR inactivation can create synergies with Ad vaccination and lead to a more potent immune response and tumor protection.
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We thank Fabrizio Colaceci and Walter Castaldi for animal care and Janet Clench for editorial assistance.
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Elia, L., Aurisicchio, L., Facciabene, A. et al. CD4+CD25+ regulatory T-cell-inactivation in combination with adenovirus vaccines enhances T-cell responses and protects mice from tumor challenge. Cancer Gene Ther 14, 201–210 (2007). https://doi.org/10.1038/sj.cgt.7701004
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