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Cancer Research 68, 893, February 1, 2008. doi: 10.1158/0008-5472.CAN-07-3166
© 2008 American Association for Cancer Research
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Immunology

Self-Tolerance Does Not Restrict the CD4+ T-Helper Response against the p53 Tumor Antigen

Marjolein M. Lauwen1, Sander Zwaveling1, Linda de Quartel1, S. Carmela Ferreira Mota1, Janine A.C. Grashorn1, Cornelis J.M. Melief1, Sjoerd H. van der Burg2 and Rienk Offringa1

Departments of 1 Immunohematology and Blood Transfusion and 2 Clinical Oncology, Leiden University Medical Center, Leiden, the Netherlands

Requests for reprints: Rienk Offringa, Leiden University Medical Center, Leiden, the Netherlands. E-mail: R.Offringa{at}LUMC.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumorigenesis is frequently associated with mutation and overexpression of p53, which makes it an attractive target antigen for T cell–mediated immunotherapy of cancer. However, the magnitude and breadth of the p53-specific T-cell repertoire may be restricted due to the ubiquitous expression of wild-type p53 in normal somatic tissues. In view of the importance of the CD4+ T-helper cell responses in effective antitumor immunity, we have analyzed and compared the p53-specific reactivity of this T cell subset in p53+/+ and p53–/– C57Bl/6 mice. This response was found to be directed against the same three immunodominant epitopes in both mouse types. Fine-specificity, magnitude, and avidity were not affected by self-tolerance. Immunization of p53–/– and p53+/+ mice with synthetic peptide vaccines comprising the identified epitopes induced equal levels of Th1 immunity. Our findings imply that the p53-specific CD4+ T-cell repertoire is not restricted by self-tolerance and is fully available for the targeting of cancer. [Cancer Res 2008;68(3):893–900]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The pivotal role of p53 as a tumor suppressor is illustrated by the fact that this protein is found mutated in ~50% of human cancers. In most cases, mutations in p53 greatly increase the otherwise short half-life of this protein and cause it to accumulate in tumor cells. The aberrant p53 expression in many malignancies offers an attractive opportunity for antigen-specific immunotherapy of cancer. Although immune targeting could potentially be directed against the mutated sequences within p53, the great diversity of point mutations found in cancers (1) dictates that widely applicable intervention strategies should target tumors on the basis of the high expression level of p53 rather than its mutation. Furthermore, the intracellular localization of both wild-type and mutated p53 cause anti-p53 antibodies to be ineffective against tumors. Immune targeting therefore relies on the T cell–mediated recognition of p53-derived peptides in the context of surface-expressed MHC molecules.

Wild-type p53 expression in normal tissues is very low, but it does extend to all tissues including the thymus (2), suggesting that the T-cell repertoire against this protein may be restricted by self-tolerance. Indeed, strong indications exist that this is true for the p53-specific CD8+ T-cell repertoire (3–5). Multiple reports of CD4+ T cell and IgG-type humoral responses against this antigen in humans and mice bearing p53-overexpressing tumors suggest that the p53-specific CD4+ T-cell repertoire may be less affected by tolerance (6–10). The availability of a potent p53-specific CD4+ T-cell response is of great interest for cancer immunotherapy, even in the case of MHC class II–negative cancers, because IFN-{gamma}–secreting CD4+ Th1 cells play an important role in orchestrating and sustaining the immune attack by CD8+ CTL and innate immune effector cells (11–13). Once activated in the lymph nodes draining the vaccination site(s), such Th1 cells can travel to the tumor site, recognize cross-presented p53 epitopes at the surface of dendritic cells (DC) that have taken up tumor cell debris, and thereby provide local help to tumoricidal effector cells.

Analysis of the T-cell repertoire in a number of transgenic mouse models has revealed that recognition of the self-antigen results in deletion of the high-avidity T-cell repertoire against this antigen, although permitting T cells with low avidity and/or specificity for subdominant epitopes to escape from central tolerance (3, 14–18). Even though p53-specific CD4+ T cell immunity has been observed in several studies, currently available data do not exclude that most of these responses may reflect the second-tier CD4+ T-cell repertoire against this antigen. In view of the potential value of p53-specific CD4+ T-cell response in the immunotherapy of cancer, we have systematically investigated the effect of normal wt.p53 expression on the p53-specific CD4+ T-cell repertoire. Our results show that the magnitude and specificity of the p53-specific CD4+ T-cell response are indistinguishable between p53–/– and p53+/+ C57Bl/6 mice, and therefore argue that this T-cell repertoire is fully available for p53-specific targeting of cancers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. C57BL/6 nu/nu mice were purchased from Taconic Europe (Denmark). C57BL/6 (p53+/+) and p53 knockout (p53–/–) mice (19) were bred at our own facilities at the Leiden University Medical Center (Leiden, Netherlands). Genotyping was performed as described previously (10). The experiments were approved by the animal experimental committee of Leiden University.

Peptides and protein. Recombinant wt.p53 murine protein was produced in Escherichia coli and purified as described elsewhere (20). Different sets of 30-mer and 20-mer wt.p53 peptides were generated in our own facilities as described previously (21). The 30-mer peptides were designed to overlap 14 amino acids. The purity of the peptides was determined by reversed phase high-performance liquid chromatography and was found to be routinely >90% pure. Peptides were dissolved in 0.5% DMSO in PBS and, if not used immediately, stored in aliquots at –20°C.

Immunization of mice. For whole antigen vaccination, p53+/+ and p53–/– mice were injected s.c. with 100 µg of p53 protein emulsified in 50% incomplete Freund's adjuvant at a total volume of 200 µL. After 2 weeks, mice were boosted with 1 x 107 plaque-forming units of ALVAC-mt.p53 (Sanofi Pasteur; ref. 22) injected i.v. in PBS in a total volume of 200 µL. Mice that were immunized for chicken ovalbumin (OVA) received two subsequent doses of ALVAC-OVA. For peptide vaccination, mice were injected thrice with an interval of 2 weeks with a total of 150 µg of peptide, comprising one or two overlapping 30-mer peptides, depending on the epitope (see Fig. 1 ). Splenocytes were harvested for ex vivo analysis 14 days after the final vaccination.


Figure 1
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  		Figure 1. Comparison of p53-specific CD4+ T-cell responses in p53–/– and p53 +/+ mice. p53–/– (left) and p53+/+ mice (right) received subsequent vaccinations with recombinant p53 protein and ALVAC-p53. After 1 week of in vitro restimulation in the presence of p53 protein–pulsed DC, viable T cells were tested for their reactivity against splenocytes pulsed with 2 µmol/L of the indicated 30-mer peptides. Numbers on the horizontal axis, the amino acid positions of the peptides that together cover the entire p53 sequence. Supernatants were analyzed for IFN-{gamma} content by ELISA. Columns, mean from two mice; bars, SE.

 
In vitro culture and restimulation. All cells were cultured in complete medium consisting of Iscove's modified Dulbecco's medium (Bio Whittaker) supplemented with 8% FCS, penicillin (100 IU/mL), 2 mmol/L of glutamine (ICN), and 30 µmol/L of 2-ME (Merck).

T cells were grown from splenocyte cultures of immunized mice by culturing spleen cells (4 x 106 cells/well of a 24-well plate) in complete medium in the presence of D1 cells (1 x 104 cells/well; refs. 23, 24). Before use, the D1 cells were incubated for 24 h with p53 protein (5 µg/mL), subsequently activated by adding lipopolysaccharide (10 µg/mL) for 6 h, and then thoroughly washed. After 7 days of in vitro restimulation, dead cells were removed from the splenocyte cultures by centrifugation on a density gradient (Lympholyte-M; Cedarlane). For obtaining long-term cultures, T cells were subsequently cultured in the presence of 5 IU/mL of interleukin 2 (IL-2; Chiron BV) and 2% supernatant from concanavalin A–stimulated rat spleen cells. Cells were restimulated once every 2 weeks with 3,000 rad irradiated naïve spleen cells at a ratio 1:1 in the presence of a mixture of 30-mer peptides to a total of 5 µg/mL of peptide without the addition of exogenous cytokines. For in vitro depletion of CD4+ or CD8+ T cell subsets, the restimulated splenocytes were taken up in 1.5 mL of cold PBS/2% FCS. Depleting antibodies for CD4+ T cells (clone GK1.5) or CD8+ T cells (clone 2.43) were added at 20 µg/mL and incubated for 30 min on ice. Cells and Dynabeads (M450; Dynal) were washed separately thrice with PBS/2% FCS. Cells and beads were incubated for 30 min on ice before magnetically sorting bound cells twice. The depletion efficacy was verified by standard staining and analysis on FACScan (Becton Dickinson).

ELISA and ELIspot assays. In vitro restimulated splenocyte cultures (see above) were seeded at a concentration of 5 to 10 x 104 cells/well in a 96-well U-bottomed plate (Costar). Irradiated C57BL/6 spleen cells were pulsed with 2 to 3 µmol/L of wt.p53-derived peptides and added as stimulator cells at a concentration of 5 to 10 x 104 cells/well. For avidity analysis, cells were stimulated with escalating doses up to 5 µmol/L of wt.p53-derived peptides or p53 protein. For testing the recognition of naturally processed antigen, T-cell clones were restimulated with 2 µmol/L of wt.p53-derived peptides or p53 protein or 6,000 rad irradiated 4J tumor cells (p53 and H-Ras transformed B6 mouse embryonic cells; ref. 25) at a ratio 1:1 in the presence of D1 cells at a ratio 1:10. The human papilloma virus 16 E7 (HPV16E7) protein, which was prepared by means of the same procedures as p53, was used as a control. No exogenous IL-2 was added. After 18 to 24 h at 37°C, supernatant was harvested and IFN-{gamma} or IL-2 production was measured by sandwich ELISA in maxisorp plates (Nunc) using anti-mouse IFN-{gamma}–specific antibodies [clones R4-6A2 (capture) and XMG1.2 (detection)], or anti-mouse IL-2–specific antibodies [clones JES6-5H4 (capture) and JES6-1A12 (detection); PharMingen] streptavidin-conjugated poly-HRP (CLB), and ABTS [(2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid); Sigma] as a substrate. Absorbance was measured at 415 nm using kineticalc 2.12 software in an EL312e Biokinetics ELISA plate reader (Biotek Instruments). The avidity of a specific T-cell population was determined in escalating dose-response experiments by comparing the peptide concentration resulting in the 1/2 max IFN-{gamma} secretion. ELIspot analyses were performed by seeding freshly isolated splenocytes from immunized mice (2 x 105/well) into IFN-{gamma} capture antibody–coated 96-well Multiscreen ELIspot plates and overnight incubation in the presence of 2 µg/mL of the relevant peptide antigen. The used antibodies and reagents were the same as for our IFN-{gamma} ELISA. The ELIspot plates were developed according to standard procedures and analyzed with a Bioreader 2000 ELIspot reader using Bioreader v 8.3 software (Biosys).

Adoptive transfer. Female, p53+/+ T cell–deficient (nude) C57BL/6 mice were challenged with 10 x 106 p53-overexpressing 4J tumor cells. At day 7, when small (10 mm2) palpable tumors were present, mice were infused with 10 x 106 p53-specific CD4+ T cells and/or 10 x 106 p53-specific CD8+ CTL 1H11 (26), where indicated in combination with a s.c. depot of 6 x 105 IU IL-2 in 50% incomplete Freund's adjuvant at days 7 and 14. Our previous experiments have shown that the outgrowth of 4J tumor cells was not significantly affected by either the IL-2 depot alone, the combination of IL-2 depot with a nonrelevant CTL clone, or the combination of CTL clone 1H11 and a nonrelevant helper T-cell clone (10, 26–28). The p53-specific CD4+ and CD8+ T cells used constituted clonal T-cell lines obtained from p53–/– mice. Even though p53-specific CD4+ T-cell clones of p53+/+ origin were available (Fig. 5), these cultures could not be expanded to sufficient numbers to permit adoptive transfer experiments. The better growth characteristics of T-cell clones of p53–/– origin is most likely due to the absence of the proapoptotic and cell cycle regulatory functions of p53 in these cells. Tumor size was measured every 3 to 4 days, and mice were killed when tumor size reached 100 mm2. Statistical analysis of the resulting data was performed by means of a log-rank test using GraphPad software.


Figure 5
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  		Figure 5. T-helper clones from p53–/– and p53+/+ origin recognize naturally processed antigen with equal avidity. A and B, T-cell clones from p53–/– ({circ}) and p53+/+ origin (bullet), isolated from mice immunized with epitope 1–containing peptides, were tested for their reactivity against different doses of the relevant 30-mer peptide (A) or full-length recombinant p53 protein (B). Dotted lines, the peptide dose at which 50% of the maximal IFN-{gamma} secretion was reached. Peptide: p53–/– (0.04 µmol/L), p53+/+ (0.03 µmol/L). Protein: p53–/– (0.22 µmol/L), p53+/+ (0.17 µmol/L). C and D, p53-specific T helper cells were tested for secretion of IFN-{gamma} (filled columns) and IL-2 ELISA (open columns) in the presence of DC pulsed with 2 µmol/L of the relevant 30-mer peptide, 2 µmol/L of the full-length recombinant p53 protein, control protein (HPV16E7; D) or a cell lysate from p53-overexpressing tumor cells at a ratio of 1:1 (C). As a control, reactivity was also measured against DCs with medium (C and D) and tumor cells in the absence of DCs (C).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specificity of the p53-specific CD4+ T-cell response in p53–/– and p53+/+ mice. In view of the importance of tumor-specific CD4+ T helper responses in sustained and effective antitumor immunity (13), we examined the effect of self-tolerance on the specificity and magnitude of the p53-specific CD4+ T-cell response. Immunization of p53+/+ and p53–/– C57Bl/6 mice was performed with a heterologous prime boost protocol involving subsequent administration of recombinant p53 protein and recombinant canarypoxvirus (ALVAC) encoding p53 that we found to work optimally for this antigen in our pilot studies. Analysis of the p53-specific CD4+ T-cell response involved in vitro restimulation of the splenocytes with protein-pulsed DC for 1 week, followed by measurement of their IFN-{gamma} secretion in the presence of an array of overlapping 30-mer peptides encompassing the entire sequence of wild-type murine p53. We have previously employed this methodology for comparison of the CD4+ T-cell responses against carcinoembryonic antigen (CEA) in CEA-transgenic and control mice (18). Interestingly, T-cell cultures from p53-immunized p53–/– and p53+/+ mice showed a similar reactivity pattern (Fig. 1), in that comparable levels of IFN-{gamma} secretion were observed in the presence of the same 30-mer peptides. The fingerprints in Fig. 1 point at the presence of at least three immunodominant T-cell epitopes in the p53 sequence, which are located between amino acids 62 and 107 (referred to as epitope 1), amino acids 190 and 235 (epitope 2), and amino acids 334 and 363 (epitope 3), respectively. Because adjacent peptides have an overlap of 14 amino acid residues, it is likely that the detection of responses against two such peptides reflects a T-cell response against a single T-cell epitope shared by these peptides.

To assess in more detail whether p53 responses against these epitopes are indeed of the same magnitude in p53–/– and p53+/+ mice, the p53-reactive T cells in freshly isolated splenocytes of p53-immunized mice were enumerated by IFN-{gamma} ELIspot analysis. However, no significant p53-specific T-cell reactivity was detected in these assays, even though parallel analysis of splenocytes from ovalbumin-immunized mice readily revealed ovalbumin-specific T cells (data not shown; see Materials and Methods). In view of these results, we conclude that the CD4+ T-cell responses against p53 are relatively weak, both in p53–/– and p53+/+ mice, and that ex vivo detection requires the brief expansion phase employed in the experiments shown in Fig. 1. On the basis of the modest strength of these responses, we deem it unlikely that overstimulation as a result of the immunization scheme used could have overshadowed differences in the p53-specific T-cell repertoire between p53–/– and p53+/+ mice.

To further exclude differences in T-cell reactivity between p53+/+ and p53–/– mice, a more detailed analysis of the T-cell responses against the three identified immunogenic regions in p53 was performed. For these experiments, mice received two subsequent immunizations with the 30-mer peptide(s) covering either of these sequences. Immunization with peptides, rather than vaccines comprising or encoding full-length p53, allowed us to more accurately distinguish between the T-cell repertoires directed against the three immunogenic regions. Furthermore, this vaccination regime, which involved coadministration of CpG ODN, was chosen because we found it highly effective in inducing antigen-specific T-cell responses in other experimental systems (29).3 The reactivity of the splenocytes isolated from the immunized mice was evaluated not only against 30-mer peptides, but also against overlapping sets of 20-mer peptides spanning the regions of interest. Results from these experiments revealed that the fine specificity of the responses is indistinguishable between p53–/– and p53+/+ mice (Fig. 2 ). We therefore conclude that the T-cell repertoires of these mice target the same three immunodominant p53 epitopes, the core sequences of which are located between amino acids 77 and 91, 205 and 219, and 344 and 358, respectively (Fig. 3 ). In addition, all T-cell cultures specific for these epitopes produce IL-2 upon in vitro restimulation (Fig. 2, open columns). Our experimental data do not rule out subtle differences in the p53-specific T-cell repertoires, such as in TCR-chain or CDR3 usage.


Figure 2
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  		Figure 2. Fine specificity of p53-specific CD4+ T-cell responses in p53–/– and p53+/+ mice. p53–/– (left) and p53+/+ mice (right) were immunized with 30-mer peptide(s) comprising epitope 1 (A), epitope 2 (B), or epitope 3 (C). After 1 wk of in vitro restimulation in the presence of p53 protein–pulsed DC, viable T cells were tested for their reactivity against splenocytes pulsed with 2 µmol/L of the indicated 30-mer peptide (IFN-{gamma} and IL-2) as well as against overlapping sets of 20-mer peptides (IFN-{gamma}). Numbers on the horizontal axis, the amino acid positions of the peptides. Supernatants were analyzed for IFN-{gamma} (filled columns) and IL-2 content (open columns) by ELISA. Columns, mean from three mice; bars, SE.

 

Figure 3
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  		Figure 3. Location of T-cell epitopes in the murine p53 sequence. Boxed sequences, the three newly defined immunodominant CD4+ T-cell epitopes. Epitope 1 maps between amino acid positions 77 and 91; epitope 2 between positions 205 and 219; and epitope 3 between positions 344 and 358. Underlined sequences, a previously described MHC class I (H-2Kb)–restricted CD8+ T-cell epitope (double line; ref. 26) and a subdominant MHC class II (I-Ab)–restricted CD4+ T-cell epitope (single line; ref. 10).

 
As a result of the experimental design, in particular, the in vitro restimulation with protein-pulsed DC and the use of long peptide antigens as target antigens, our analyses shown in Figs. 1 and 2 are geared towards the detection of CD4+ T-cell responses (18). To formally exclude that CD8+ T cells would contribute to the reactivity patterns observed, splenocyte cultures were depleted of either CD8+ or CD4+ cells before functional analysis. Importantly, the IFN-{gamma} response in the presence of the three identified T cell epitopes, although abrogated by CD4+ depletion, was not significantly affected by CD8+ depletion (Fig. 4A and B ).


Figure 4
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  		Figure 4. Newly identified epitopes are recognized by CD4+ T cells. A and B, immunization of p53–/– (A) and p53+/+ mice (B), in vitro restimulation of splenocytes and analysis of p53-specific T-cell responses was performed as described in Fig. 2. Cells harvested after in vitro restimulation were depleted for either CD8+ or CD4+ T-cell subsets (ND, not depleted), after which their in vitro reactivity against the indicated peptides was evaluated. Columns, mean from three mice; bars, SE. Short-term, polyclonal T-cell cultures from p53–/– and p53+/+ mice display equal avidity for their epitopes. C and D, immunization of p53–/– and p53+/+ mice, in vitro restimulation of splenocytes and analysis of p53-specific T-cell responses was performed as described in Fig. 2. In vitro reactivity was measured against splenocytes pulsed with different doses of the relevant 30-mer peptides. Points, in vitro responses of T cells from p53–/– ({circ}) and p53+/+ (bullet) mice immunized with epitope 1 (C) or epitope 2 (D). Dotted lines, the peptide dose at which 50% of the maximal IFN-{gamma} secretion was reached. Epitope 1: p53–/– (0.19 µmol/L), p53+/+ (0.17 µmol/L). Epitope 2: p53–/– (0.27 µmol/L), p53+/+ (0.26 µmol/L).

 
Avidity of the p53-specific CD4+ T-cell response in p53–/– and p53+/+ mice. Previous analyses of the T-cell response against auto-antigens in mice have shown that self-tolerance could result in the elimination of high-avidity T cells from the repertoire, whereas permitting T cells with low avidity and/or specificity for subdominant epitopes to escape from central tolerance (3, 14–18). Notably, the experiments shown in Figs. 1 and 2 do not discriminate between high- and low-avidity T cells because saturating amounts of peptides were used as target antigens in the IFN-{gamma} assays. We therefore analyzed the reactivity of short-term T-cell cultures obtained from p53-immunized mice against limiting quantities of target antigen. As shown in Fig. 4C and D, the avidity of T-cell responses directed against p53 epitopes 1 and 2 did not differ essentially between p53–/– and p53+/+ mice. Because the T-cell responses against p53 epitope 3 were consistently lower than those against epitopes 1 and 2 (Figs. 2 and 4A and B), we could not make a reliable comparison of the avidities of epitope 3–specific response between p53–/– and p53+/+ mice. We conclude that the CD4+ T-cell response against at least the two most immunogenic p53 epitopes does not differ in avidity between p53–/– and p53+/+ mice. Taken together, our data show that the specificity and magnitude of the p53-specific CD4+ T-cell response is not significantly affected by self-tolerance in p53+/+ mice. Furthermore, vaccines consisting of long synthetic peptides and CpG ODN can successfully be used for induction, in p53+/+ mice, of p53-specific CD4+ Th1 immunity that is associated with the secretion of IFN-{gamma} and IL-2.

Reactivity of p53-specific CD4+ T-helper cells against cross-presented p53 tumor antigen. On the basis of the capacity of the p53-specific CD4+ T cells from p53–/– and p53+/+ mice to respond to low concentrations of peptide antigens, one would expect p53-specific CD4+ T cells to also recognize physiologic quantities of naturally processed epitopes. This functional aspect was analyzed with p53-specific CD4+ T-cell clones specific for epitope 1. The clonality of these T-cell cultures, which were obtained by limiting dilution cloning, was confirmed by flow cytometric analysis with a panel of Vβ-specific antibodies (data not shown). Like the polyclonal short-term cultures, the CD4+ T-cell clones isolated from p53–/– and p53+/+ mice displayed indistinguishable avidity for their target antigen as determined by the reactivity against DCs pulsed with limiting amounts of peptide (Fig. 5A ). Importantly, the high avidity of the CD4+ T-cell clones as shown with peptide-pulsed DCs was also reflected by their capacity to respond to DCs pulsed with the full-length p53 protein (Fig. 5B).

This result suggested that the p53-specific CD4+ T-cell clones should also react against DCs that cross-present tumor-derived p53 in the context of MHC class II. Figure 5C and D show that this is indeed the case because the CD4+ T cells specifically released IFN-{gamma} and IL-2 in response to DCs pulsed with either p53 peptide, p53 protein, or lysate from p53-overexpressing tumor cells. T-cell activation by tumor-derived p53 antigen required cross-presentation by DCs bearing the MHC class II molecule I-Ab, because incubation of the T cells with the class I MHC-positive, class II MHC-negative tumor cell did not result in cytokine production (Fig. 5C).

We have previously shown that adoptively transferred p53-specific CD8+ T cells are capable of controlling p53-overexpressing tumors, provided that the mice also receive a subcutaneous depot of IL-2 (10, 26–28) or cotransfer of p53-specific T helper cells directed against a subdominant p53 epitope (10). Figure 6 shows that adoptively cotransferred p53-specific T-helper cells specific for an immunodominant p53 epitope could similarly harness the in vivo efficacy of p53-specific CTLs. These data suggest that the CD4+ T-cell repertoire available in both p53–/– and p53+/+ mice can enhance the CTL-mediated eradication of p53-overexpressing tumors.


Figure 6
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  		Figure 6. p53-specific CD4+ T cells provide help to antitumor CTL in vivo. p53+/+ nude mice (seven per group) received 10 x 106 p53-overexpressing 4J tumor cells in PBS in the flank. After tumor size had reached 10 mm2, mice received either no treatment ({square}), 10 x 106 CTL ({triangleup}), 10 x 106 CTL and 10 x 106 T helper cells ({blacktriangleup}), or 10 x 106 CTL and 6 x 105 IU of IL-2 in a subcutaneous depot of 50% incomplete Freund's adjuvant in the flank contralateral to the tumor (bullet). *, P = 0.006, log-rank test. This experiment was performed twice with similar outcomes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Systematic comparison of the p53-specific CD4+ T-helper immunity between p53–/– and p53+/+ C57Bl/6 mice revealed that this response was not affected by self-tolerance, despite the ubiquitous expression of wild-type p53 in normal somatic tissues of p53+/+ mice. These results are in concordance with the observation of p53-specific CD4+ T-cell and IgG responses in patients and mice bearing p53-overexpressing tumors (6–9). However, the latter reports do not exclude the possibility that such immunity might reflect low-avidity T-cell responses and/or T-cell responses directed against subdominant T-cell epitopes, as was found for the T-cell response against self-antigens in several mouse models (3, 14–18). Importantly, our present data show that the p53-specific CD4+ T-cell response is not significantly affected by central tolerance, in that it is highly similar in magnitude, specificity, and avidity between p53–/– and p53+/+ mice. This lack of self-tolerance most likely relates to the fact that p53 gene expression in normal cells is cell cycle–regulated rather than constitutive, whereas the half-life of wild-type p53 is very short. As a result of this tight regulation, the protein does not accumulate, unless cells are programmed for death because of sustained DNA damage (30). Even though apoptotic cells containing elevated levels of p53 might frequently occur in the body, their p53 content is apparently not efficiently cross-presented by DCs due to the lack of concomitant proinflammatory "danger" signals (31). Together, these conditions are expected to result in immunologic ignorance rather than peripheral tolerance. Likewise, p53-derived peptides are not expected to be processed through the endogenous or exogenous pathways into the MHC class II of thymic APC, providing a plausible explanation for the absence of central tolerance for this antigen at the level of the CD4+ T-cell response. The present results strikingly differ from those of a similar comparison of CEA-specific CD4+ T-cell responses in CEA-transgenic and nontransgenic mice (18). In the case of CEA, we found that expression of this antigen in normal somatic tissues, in a manner very similar to humans, profoundly affected the specificity, breadth, and magnitude of the CD4+ T-cell response. Importantly, CEA has a long half-life and is expressed in a constitutive rather than cell cycle–dependent fashion. As a result, CEA is available for processing into MHC class II.

At first glance, our findings seem to be in conflict with previous work by others demonstrating that the avidity of the p53-specific CD8+ T-cell repertoire is limited by self-tolerance (3–5). However, the answer to this paradox lies in the short half-life of p53, in particular, in the fact that rapid breakdown of p53 involves the proteasome. As a result, endogenously expressed p53 in thymic APC is expected to be efficiently routed into the MHC class I processing pathway. Because wild-type p53 fails to accumulate, insufficient levels of this antigen remain for either direct or cross-presentation into MHC class II, explaining the split tolerance by the CD4+ and CD8+ T-cell repertoires (32). Direct testing of this hypothesis awaits the availability of transgenic mouse strains that express MHC class I and II–restricted, p53-specific T-cell receptors, which are currently being generated in our laboratory. The p53-specific T cells obtained from such mice will permit the highly sensitive detection of p53-epitope presentation by thymic and peripheral APC in vitro and in vivo.

The three p53 T-helper epitopes identified in the present study are distinct from the I-Ab–restricted murine p53 T-helper epitope spanning amino acids 108 to 121 that we have described previously (ref. 10; see Fig. 3), the sequence of which largely corresponds to that of a HLA-DR4–restricted epitope identified in the human p53 sequence (33). Notably, identification of this epitope in the context of I-Ab was based on epitope prediction involving a MHC class II–binding motif, followed by immunization of mice with the selected peptide epitopes. On the one hand, this shows that the analysis of responses elicited by whole antigen immunization provides a better means for charting the natural, immunodominant T-cell response against a given antigen. On the other hand, our earlier study shows that a search for epitopes by algorithm-driven prediction could result in the identification of subdominant epitopes that can be effectively used as targets for potent antitumor T-cell immunity. This is exemplified by the fact that cotransfer of CD4+ T cells directed against either a subdominant or dominant p53 epitope proved to be equally efficient in supporting CTL-mediated tumor eradication in vivo (ref. 10; Fig. 6). Even though peptides 108 to 121 of murine p53 act as a subdominant epitope in the context of I-Ab, its equivalent in human p53 seems to play a more prominent role in the HLA-DR4–restricted CD4+ T-cell response in humans, as T cells against this epitope were isolated following in vitro stimulation with autologous DCs pulsed with whole p53 antigen (33). The difference in the prominence of this epitope in the context of the DR4- and I-Ab–restricted CD4+ T-cell responses could be explained by a plethora of factors, including differences in the restricting MHC molecules, T-cell repertoires, and peptide sequences concerned (the human and murine peptides differ at three amino acid positions).

Now that we have shown that p53-specific Th1 responses can be efficiently induced in p53+/+ C57Bl/6 mice by immunization with synthetic peptides comprising the identified epitopes (Figs. 2 and 4A and B), and showed that adoptively transferred CD4+ T cells against p53 can efficiently enhance in vivo eradication of p53-overexpressing tumors by p53-specific CD8+ CTL (ref. 10; Fig. 6), it will be important to test whether p53-specific vaccination can be used to launch an effective antitumor attack.


   Acknowledgments
 
Grant support: Dutch Cancer Society (Koningin Wilhelmina Fonds) grant no. 2002-2735.

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.

We thank Neil Berinstein and Bryan McNeil from Sanofi Pasteur, Ltd. (Toronto, Ontario, Canada) for providing ALVAC-p53 and ALVAC-OVA, Kees Franken for providing recombinant p53 protein, and Suzanne van Duikeren for technical assistance.


   Footnotes
 
3 Our additional unpublished data. Back

Received 8/16/07. Revised 10/16/07. Accepted 12/ 3/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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