p53 Specific (auto)immunity in mice
Lauwen, M.M.
Citation
Lauwen, M. M. (2008, October 16). p53 Specific (auto)immunity in mice. Retrieved from https://hdl.handle.net/1887/13147
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden Downloaded from: https://hdl.handle.net/1887/13147
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Chapter 2
Self-tolerance does not restrict the CD4+ T-helper response against the p53 tumor antigen
Marjolein M. Lauwen, Sander Zwaveling, Linda de Quartel, S. Carmela Ferreira Mota, Janine A.C. Grashorn, Cornelis J.M. Melief, Sjoerd H. van der Burg and Rienk Offringa
Cancer Research, 2008, 68 (3), 893-900
Chapter 2
2
Abstract
Tumorigenesis is frequently associated with mutation and over-expression 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 (Th) cell responses in effective anti-tumor 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 fully available for targeting of cancer.
Chapter 2
2
Introduction
The pivotal role of p53 as tumor suppressor is illustrated by the fact that this protein is found mutated in approximately 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-γ-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 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 self antigen results in deletion of the high avidity T-cell repertoire against this antigen, while permitting T cells with low avidity and/or specificity for sub- dominant 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 immunotherapy of cancer, we have systematically investigated the impact 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
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 LUMC (Leiden, Netherlands). Genotyping was performed as described previously (10). The experiments were approved by the animal experimental committee of Leiden University.
Chapter 2
Peptides and protein. Recombinant wt.p53 murine protein was produced in E.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 reverse- phase HPLC 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 subcutaneously (s.c.) with 100 μg p53 protein emulsified in 50% incomplete Freund’s adjuvant (IFA) at a total volume of 200 μl. After two weeks mice were boosted with 1 x 10⁷ plaque forming units (PFU) ALVAC-mt.p53 (Sanofi Pasteur, Toronto, Ontario, Canada; 22) injected intravenously (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 3 times with an interval of 2 weeks with a total of 150 μg peptide, comprising 1 or 2 overlapping 30-mer peptides, depending on the epitope (see Figure 1). Splenocytes were harvested for ex vivo analysis fourteen days after the final vaccination.
In vitro culture and restimulation. All cells were cultured in complete medium consisting of Iscove’s Modified Dulbecco’s Medium (IMDM; BioWhittaker, Walkersville, MD, USA) supplemented with 8% FCS, penicillin (100 IU/ml), 2 mM glutamine (ICN, Aurora, Ohio, USA) and 30 µM 2-ME (Merck, Darmstadt, Germany). T cells were grown from splenocyte cultures of immunized mice by culturing spleen cells (4 x 10⁶ cells/well of a 24 wells plate) in complete medium in the presence of D1 cells (1 x 10⁴ cells/well) (23,24). Before use, the D1 cells were incubated for 24 hours with p53 protein (5 μg/ml), subsequently activated by adding LPS (10 μg/ml) for 6 hours, and then thoroughly washed. After seven days of in vitro restimulation, dead cells were removed from the splenocyte cultures by centrifugation on a density gradient (Lympholyte-M, Cedarlane, Ontario, Canada). For obtaining long- term cultures, T cells were subsequently cultured in the presence of 5 IU/ml IL-2 (Chiron BV, Amsterdam, the Netherlands) and 2% supernatant from Concanavalin A stimulated rat spleen cells. Cells were restimulated once every 2 weeks with 3000 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 peptide without 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 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 minutes on ice. Cells and Dynabeads (M450, Dynal) were washed separately three times with PBS/2% FCS. Cells and beads were incubated for 30 minutes 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 5-10x10⁴ cells/well in a 96-well U-bottom plate (Costar). Irradiated C57BL/6 spleen cells were pulsed with 2-3 μM of wt.p53-derived peptides and added as stimulator cells at a concentration of 5-10x10⁴ cells/well. For avidity analysis, cells were stimulated with escalating doses up to 5 μM of wt.p53-derived peptides or p53 protein. For testing the recognition of naturally processed antigen, T cell clones were restimulated with 2 μM wt.p53-derived peptides or p53 protein or 6000 rad irradiated 4J tumor cells (p53 and H-
2
Ras transformed B6 mouse embryonic cells; 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, that was prepared by means of the same procedures as p53, was used as control. No exogenous IL-2 was added. After 18-24 hours at 37°C, supernatant was harvested and IFN-γ or IL-2 production was measured by sandwich ELISA in maxisorp plates (Nunc, Roskilde, Denmark) using anti-mouse-IFN-γ- 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, San Diego, CA) streptavidin conjugated poly-HRP (CLB, Amsterdam, The Netherlands), and ABTS ((2,2’-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid); Sigma, St.Louis, MO) as a substrate.
Optical Density at 415 nm was measured using kineticalc 2.12 software in an EL312e Biokinetics ELISA plate reader (Biotek Instruments, Winooski, VT). The avidity of a specific T-cell population was determined in escalating dose-response experiments by comparing the peptide concentration resulting in the ½ Max IFN-γ secretion. ELIspot analyses were performed by seeding freshly isolated splenocytes from immunized mice (2 x 10⁵/well) into IFN-γ capture antibody coated 96-well Multiscreen ELIspot plates and overnight incubation in the presence of 2μg/ml of the relevant peptide antigen. The used antibodies and reagents were the same as for our IFN-γ 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, Frankfurt, Germany).
Adoptive transfer. Female, p53+/+ T-cell deficient (nude) C57BL/6 mice were challenged with 10*10⁶ p53 over-expressing 4J tumor cells. At day 7, when small (10 mm²) palpable tumors were present, mice were infused with 10*10⁶ p53 specific CD4+ T cells and/or 10*10⁶ p53- specific CD8+ CTL 1H11 (26), where indicated in combination with a s.c. depot of 6*10⁵ IU IL-2 in 50% IFA at day 7 and day 14. Our previous experiments have demonstrated that outgrowth of 4J tumor cells is not significantly affected by either the IL-2 depot alone, the combination of IL-2 depot with a non-relevant CTL clone, or the combination of CTL clone 1H11 and a non-relevant 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 (Figure 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 pro- apoptotic and cell cycle regulatory functions of p53 in these cells. Tumor size was measured every 3-4 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.
Results
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 anti-tumor immunity (13), we examined the impact 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 dendritic cells (DCs) for one week,
Chapter 2
followed by measurement of their IFN-γ 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 (Figure 1), in that comparable levels of IFN-γ secretion were observed in the presence of the same 30-mer peptides. The fingerprints in figure 1 point at the presence of at least three immunodominant T-cell epitopes in the p53 sequence, which are located between aa 62- 107 (referred to as epitope 1), aa 190-235 (epitope 2) and 334-363 (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-γ 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 figure 1. On basis to the modest strength of these responses, we deem it unlikely that over-stimulation 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
Figure 1. Comparison of p53-specific CD4+ T-cell responses in p53 -/- and p53 +/+ mice.
p53-/- (left panel) and p53+/+ mice (right panel) received subsequent vaccinations with recombinant p53 protein and ALVAC-p53. After one 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 μM of the indicated 30-mer peptides. Numbers on the horizontal axis represent the amino acid positions of the peptides that together cover the entire p53 sequence.
Supernatants were analyzed for IFN-γ content by ELISA. Each graph depicts mean values and SEM of two mice.
1-30 4-33 20-49 35-64 46-75 62-91
78-107 94-123 110-139 126-155 142-171 158-187 174-203 190-219 206-235 222-251 238-267 254-283 270-299 286-315 302-331 318-347 334-363 350-379 366-390
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78-107 94-123 110-139 126-155 142-171 158-187 174-203 190-219 206-235 222-251 238-267 254-283 270-299 286-315 302-331 318-347 334-363 350-379 366-390
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2
78-107 62-81 67-86 72-91 77-96 82-101 87-106 -
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IFN-J (ng/ml) 78-107 62-81 67-86 72-91 77-96 82-101 87-106 -0
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IFN-J (ng/ml) 334-363 334-353 339-358 344-363 -0.0
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206-235 - 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
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334-363 - 0.0 0.2 0.4 0.6
IL-2 ng/ml 334-363 -0.0
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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 one 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 μM of the indicated 30-mer peptide (IFN-γ and IL-2) as well as against overlapping sets of 20-mer peptides (IFN-γ). Numbers on the horizontal axis represent the amino acid positions of the peptides. Supernatants were analyzed for IFN-γ (filled bars) and IL-2 content (open bars) by ELISA. Each graph depicts mean values and SEM of three mice.
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 co-administration of CpG ODN, was chosen because we found it highly effective in inducing antigen-specific T-cell responses in other experimental systems (29, our additional unpublished data). 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 (Figure 2). We therefore conclude that
A
B
C
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Figure 3. Location of T-cell epitopes in the murine p53 sequence.
Boxed sequences represent the three newly defined immunodominant CD4+ T- cell epitopes. Epitope 1 maps between amino acid positions 78-96, epitope 2 between positions 205-219 and epitope 3 between positions 344- 358. Underlined sequences represent a previously described MHC class I (H-2Kb) restricted CD8+ T-cell epitope (double line, (26) and a subdominant MHC class II (I-Ab) restricted CD4+ T-cell epitope (single line, 10).
1 MTAMEESQSD.ISLELPLSQE.TFSGLWKLLP.PEDILPSPHC.MDDLLLPQDV.EEFFEGPSEA
61 LRVSGAPAAQ.DPVTETPGPV.APAPATPWPL.SSFVPSQKTY.QGNYGFHLGF.LQSGTAKSVM
121 CTYSPPLNKL.FCQLAKTCPV.QLWVSATPPA.GSRVRAMAIY.KKSQHMTEVV.RRCPHHERCS
181 DGDGLAPPQH.LIRVEGNLYP.EYLEDRQTFR.HSVVVPYEPP.EAGSEYTTIH.YKYMCNSSCM
241 GGMNRRPILT.IITLEDSSGN.LLGRDSFEVR.VCACPGRDRR.TEEENFRKKE.VLCPELPPGS
301 AKRALPTCTS.ASPPQKKKPL.DGEYFTLKIR.GRKRFEMFRE.LNEALELKDA.HATEESGDSR
361 AHSSYLKTKK.GQSTSRHKKT.MVKKVGPDSD
the T-cell repertoires of these mice target the same three immunodominant p53 epitopes, the core sequences of which are located between aa 78-96, aa 205-219 and aa 344-358 respectively (Figure 3). In addition, all T-cell cultures specific for these epitopes produce IL-2 upon in vitro restimulation (Figure 2, open bars). Our experimental data do not rule out subtle differences in the p53-specific T-cell repertoires, such as in TCR-chain or CDR3 usage.
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 as shown in figures 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-γ response in the presence of the three identified T-cell epitopes, although abrogated by CD4+ depletion, was not significantly affected by CD8+ depletion (Figure 4A-B).
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 can result in elimination of high avidity T cells from the repertoire, while permitting T cells with low avidity and/or specificity for sub-dominant epitopes to escape from central tolerance (3,14-18). Notably, the experiments shown in figures 1 and 2 do not discriminate between high and low avidity T cells, because saturating amounts of peptides
2
were used as target antigen in the IFN-γ 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 figures 4C and 4D, 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 (Figures 2 and 4A-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-γ and IL-2.
Figure 4. Newly identified epitopes are recognized by CD4+ T cells.
Panels 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 legend to figure 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. Each graph depicts mean values and SEM of three mice.
Short term, polyclonal T cell cultures from p53-/- and p53 +/+ mice display equal avidity for their epitopes Panels 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 legend to figure 2. In vitro reactivity was measured against splenocytes pulsed with different doses of the relevant 30-mer peptides. Graphs depict in vitro responses of T cells from p53-/- (open symbols) and p53+/+ (closed symbols) mice immunized with epitope 1 (panel C) or epitope 2 (panel D). The dotted lines indicate the peptide dose at which 50% of the maximal IFN-γ secretion was reached.
Epitope 1: p53-/- 0.19 µM, p53+/+ 0.17 µM. Epitope 2: p53-/- 0.27 µM, p53+/+ 0.26 µM.
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Figure 5. T helper clones from p53-/- and p53+/+ origin recognize naturally processed antigen with equal avidity
Panels A and B: T cell clones from p53 -/- (open circles) and p53+/+ origin (filled circles), 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). The dotted lines indicate the peptide dose at which 50% of the maximal IFN-γ secretion was reached. Peptide: p53-/- 0.04 µM, p53+/+ 0.03 µM. Protein: p53- /- 0.22 µM, p53+/+ 0.17 µM.
Panel C and D: p53 specific T helper cells were tested for secretion of IFN-γ (filled bars) and IL-2 ELISA (open bars) in the presence of DC pulsed with 2 μM of the relevant 30-mer peptide, 2 μM of the full-length recombinant p53 protein, control protein (HPV16 E7, 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, D) and tumor cells in the absence of DCs (C).
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Pmol/L protein Pmol/L peptide
% max IFN-J
p53 peptide p53 protein tumor cell lysate - tumor cell p53 peptide p53 protein tumor cell lysate - tumor cell 0.0
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fig5and6ab.pzf:Layout 17 - Mon Mar 10 11:01:39 2008
A B
C D
Absence of p53 T-helper cell tolerance
2
Reactivity of p53-specific CD4+ T helper cells against cross-presented p53 tumor antigen On 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 physiological 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 Vbeta-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 (Figure 5A). Importantly, the high avidity of the CD4+ T-cell clones as demonstrated with peptide-pulsed DCs was also reflected by their capacity to respond to DCs pulsed with the full-length p53 protein (Figure 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 5D demonstrate that this is indeed the case, because the CD4+ T cells specifically released IFN-γ 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 dendritic cells 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 (Figure 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 co-transfer of p53-specific T helper cells directed against a subdominant p53 epitope (10). Figure 6 shows that adoptively co-transferred 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 CTL-mediated eradication of p53- overexpressing tumors.
Figure 6. p53-specific CD4+ T cells provide help to anti-tumor CTL in vivo.
p53+/+ nude mice (7 per group) received 10*10⁶ p53-overexpressing 4J tumor cells in PBS in the flank. After tumor size had reached 10 mm², mice received either no treatment (open squares), 10*10⁶ CTL (open triangles), 10*10⁶ CTL and 10*10⁶ T helper cells (filled triangles), or 10*10⁶ CTL and 6*10⁵ IU of IL-2 in a subcutaneous depot of 50%
IFA in the flank contra-lateral to the tumor (filled circles). (* Log rank test p=0.006). This experiment was performed twice with similar outcome.
0 10 20 30 40
0 10 20 30 40 50 60 70 80 90 100
- (n=7) CTL (n=7) CTL+Th (n=7) CTL+IL-2 (n=7)
*
*
* *
days
% survival
Chapter 2
Discussion
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 demonstrate 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, while 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 dendritic cells due to the lack of concomitant pro-inflammatory ‘danger’
signals (31). Together, these conditions are expected to result in immunological 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 carcinoembryonic antigen (CEA) specific CD4+ T-cell responses in CEA- transgenic and non-transgenic 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 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 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, also 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 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 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 an I-Ab-restricted, murine p53 T helper epitope spanning amino acids 108-121 that we have described previously (10, see Figure 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
2
involving an MHC class II binding motif, followed by immunization of mice with the selected peptide epitopes. On the one hand, this shows that 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 demonstrates that a search for epitopes by algorithm-driven prediction can result in the identification of subdominant epitopes that can be effectively used as targets for potent anti-tumor T-cell immunity. This is exemplified by the fact that co-transfer 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 (10, Figure 6). Even though peptide 108-121 of murine p53 acts as a subdominant epitope in the context of I-Ab, its equivalent in human p53 appears 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 prominence of this epitope in 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 (Figure 2 and 4A-B), and demonstrated that adoptively transferred CD4+ T cells against p53 can efficiently enhance in vivo eradication of p53-overexpressing tumors by p53- specific CD8+ CTL (10, Figure 6), it will be important to test whether p53-specific vaccination can be used to launch an effective anti-tumor attack.
Acknowledgements
We would like to thank Neil Berinstein and Bryan McNeil from Sanofi Pasteur Ltd. in Toronto for providing ALVAC-p53 and ALVAC-OVA, Kees Franken for providing recombinant p53 protein, and Suzanne van Duikeren for technical assistance.
Chapter 2
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