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The Wild-Type Sequence (wt) p53_25–35 Peptide Induces HLA-DR7 and HLA-DR11-Restricted CD4⁺ Th Cells Capable of Enhancing the Ex Vivo Expansion and Function of Anti-wt p53_264–272 Peptide CD8⁺ T Cells¹

Daisuke Ito^*,, Andreas Albers^*,, Yong Xiang Zhao^*, Carmen Visus^*,, Ettore Appella, Theresa L. Whiteside^*,, and Albert B. DeLeo^2,*,

^* Division of Basic Research, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15213; Department of Pathology and Department of Pathology Otolaryngology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213; and National Cancer Institute, Bethesda, MD 20839

	Abstract

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Tumor peptide-based vaccines are more effective when they include tumor-specific Th cell-defined as well as CTL-defined peptides. Presently, two overlapping wild-type sequences (wt) p53 helper peptides, p53_108–122 and p53_110–124, have been identified as HLA-DR1- and/or HLA-DR4-restricted epitopes. These HLA-DR alleles are expressed by 35% of subjects with cancer. To identify Th cell-defined wt p53 peptides suitable for use on the remaining subject population, a dendritic cell (DC)-based coculture system was developed. CD4⁺ T cells isolated from PBMC obtained from HLA-DR4^– normal donors were stimulated ex vivo with autologous DC transfected with wt p53 or mutant p53 cDNA. Reactivity of T cells was tested in ELISPOT IFN- assays against DC pulsed individually with a panel of algorithm-predicted, multiple HLA-DR-binding wt p53 peptides. The wt p53_25–35 peptide was identified as capable of inducing and being recognized by CD4⁺ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules, each of which is expressed by 15% of the population. In addition, the presence of anti-p53_25–35 CD4⁺ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53_264–272 CD8⁺ T cells, which from one donor were initially "nonresponsive" to the wt p53_264–272 peptide. The wt p53_25–35 peptide has attributes of a naturally presented Th cell-defined peptide, which could be incorporated into antitumor vaccines applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable, as well as used for monitoring anti-p53 Th cell activity in cancer subjects receiving p53-based immunotherapy.

	Introduction

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Development of tumor peptide-based vaccines that are capable of targeting a wide range of cancers is a major area of cancer immunotherapy research. Current evidence indicates that CD4⁺ Th cells (Th cell) as well as CD8⁺ CTL are required for effective antitumor immunity and that peptide-based vaccines are more effective when they include tumor-specific Th cell- as well as CTL-defined peptides. Recently, several HLA class II-restricted, Th cell-defined tumor-associated peptides have been identified that potentially can provide tumor-specific helper activity when combined with CTL-defined tumor peptides (1). However, for developing broadly applicable vaccines, p53-derived peptides are attractive candidates (1, 2, 3). The rationale for targeting p53 is that alterations in p53 structure and function are associated with a wide range of cancers, and a missense mutation in p53 is the most frequently occurring genetic event in cancer. p53 was originally identified as a transformation-related Ag by IgG Abs present in the sera of mice immunized against chemically induced tumors (4). Subsequently, anti-p53 IgG was detected in the sera of subjects with various types of cancer (5, 6, 7). Its presence in these sera indicated that anti-p53 CD4⁺ Th cells were induced in these subjects.

We recently identified the wild-type sequence (wt)³ p53_110–124 peptide as the first naturally presented, HLA-DRB1*0401(HLA-DR4)-restricted Th cell-defined p53 epitope, which could be used as a component of cancer vaccines, and for immunological monitoring of CD4⁺ T cell-mediated p53-related responses in subjects with cancer (8). This epitope was identified by "reverse immunology" and used for in vitro stimulation (IVS) of CD4⁺ T cells obtained from HLA-DR4⁺ normal donors or cancer subjects with autologous dendritic cells (DC) pulsed with recombinant p53 protein as APC. The outgrowing CD4⁺ T cells were then tested for proliferative responses against a panel of algorithm-predicted, HLA-DR4-binding wt p53 peptides (9). Furthermore, we demonstrated the ability of anti-wt p53_110–124 CD4⁺ T cells to enhance the ex vivo generation and function of tumor-specific CTL, an attribute critical to the potential use of the wt p53_110–124 peptide in cancer vaccines. More recently, the overlapping wt p53_108–122 peptide was identified using HLA-DR4 transgenic mice, as an HLA-DR1/DR4-binding epitope (10), suggesting that multiple HLA-DR-binding wt p53 peptides might be derived from this region.

Ideally, a broadly applicable p53-based vaccine should incorporate a Th cell-defined wt p53 peptide as well as CTL-defined wt p53 peptides. Although a number of well-characterized HLA-A*0201 (HLA-A2)-restricted, CTL-defined wt p53 peptides are available for incorporation into vaccines, only two p53 helper peptides, p53_108–122 and p53_110–124, are identified. Both peptides are HLA-DR4 restricted, and the average frequency of expression of this allele is 24.4% (11). As part of a program designed to develop p53-based cancer vaccines, we set out to identify a multiple HLA-DR-binding wt p53 helper peptide, using an in vitro strategy applied to enriched populations of CD4⁺ T cells isolated from the PBMC of HLA-DR4^– normal donors. Following IVS with autologous DC transfected with either wt or mutant p53 cDNA, the outgrowing lymphocytes were tested for reactivity against a panel of peptides, which were algorithm-predicted to bind to multiple HLA-DR allelic molecules (12). These experiments resulted in the identification of the wt p53_25–35 peptide as a naturally presented, Th cell-defined peptide that can induce/expand CD4⁺ Th cells that are restricted, at minimum, by HLA-DR7, -11 alleles. The average frequencies of expression of these alleles are 14 and 15.5%, respectively, and comparable to that of the HLA-DR4 allele, which is 24.4% (11). Consequently, the identification of the wt p53_25–35 and p53_110–124 peptides increases the percentage of HLA-A2⁺ subjects eligible to receive a multiepitope p53 peptide-based vaccine incorporating a p53 helper peptide to >50%. Furthermore, the ability of anti-wt p53_25–35 Th cells to enhance ex vivo the generation of HLA-A2-restricted, anti-wt p53_264–272 CD8⁺ T cells is demonstrated, thereby providing the proof-of-principle that the helper activity of the wt p53_25–35 peptide is likely to benefit p53-targeted immune responses and thus should be considered as a component of p53-based immunotherapy in the future.

	Materials and Methods

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Blood, cell lines, and Abs

This study was approved by the University of Pittsburgh Institutional Review Board, and written informed consent was obtained from each participating individual. The HLA genotypes of PBMC obtained from normal donors were determined using PCR with sequence-specific primers (13). PBMC were either used fresh or were cryopreserved (5 x 10⁷ cells/ml) in human AB serum (Pel-Freeze) plus 10% DMSO (Fisher Scientific). The HLA-DR-transfected mouse L cells, L.DR1, L.DR7, and L.DR11 (14) were obtained from Dr. H. Zarour (University of Pittsburgh Cancer Institute, Pittsburgh, PA). The HB95 and HB55 hybridomas, which produce anti-HLA class I (W6/32) and anti-HLA-DR (L243) mAb, respectively, were obtained from the American Type Culture Collection. The mAbs were purified from tissue culture supernatants by protein G-Sepharose immunoaffinity chromatography.

p53 plasmids

The pCMV-p53 and pCMV-p53mt135 plasmids, which encode wt p53 and p53 codon 135 missense mutation, respectively, were purchased from Clontech Laboratories, and the HindIII/EcoR1 restriction sites were used to excise and ligate the p53 cDNA inserts into the pVAX1 vector (Invitrogen Life Technologies). A 509-bp cDNA fragment encoding wt p53 codons 1–169, designated p53 F1, was constructed using the forward p53-F1 (nucleotides (n)-5–12) 5'-AGC TTA TGG AGG AGC CG-3' and reverse p53-R1 (n485–505) 5'-TGT GCT GTG ACT GCT TGT AGA-3' primers and pCMV-p53 cDNA as template. The F1 509-bp product was then amplified using the forward p53-c-F1 (n1–19) 5'-CCC AAG CTT GGG ACC ATG GAG GAG CCG CAG TCA G-3' and reverse p53-c-S1 (n485–506) 5'-GC TCT AGA GCA TGT GCT GTG ACT GCT TGT AGA-3' primers. The CCC AAG CTT GGG ACC ATG at the 5' end of p53-c-F1 encodes an EcoR1 restriction site, a Kozak translation initiation sequence, and an ATG initiation codon. The p53-c-S1 primer contained an XbaI restriction site and a stop codon (GCT CTA GAG C). The resulting p53 F1 cDNA fragment contained the appropriate restriction sites for ligation into pVAC-1 using the TA-cloning-kit (Invitrogen Life Technologies). All PCR products and plasmids were purified using the appropriate Qiagen kits (Qiagen). The sequences of PCR products and plasmids were determined at the University of Pittsburgh Cancer Institute DNA Sequencing Facility.

Peptides

Fourteen wt p53 peptides (11 to 16 mer), which were predicted to bind to multiple HLA-DR allelic molecules using the Tepitope-2000 β version algorithm obtained from Dr. J. Hammer (Roche Pharmaceutical, Nutley, NJ), as well as p53_25–25, the modified HLA-DR-binding tetanus toxoid peptide, AQYIKANSKFIGITEL (15), and PADRE peptide (16) were synthesized by standard N-(9-fluorenyl) methoxycarbonyl methodology. The peptides were purified by reverse-phase HPLC, and sequences were confirmed by mass spectrometry analysis. All peptides, with the exception of those containing cysteine, were dissolved in DMSO (Fisher Scientific) at 1 mg/ml and diluted with PBS just before use. Cysteine-containing peptides were weighed and dissolved in PBS immediately before their use.

Transfection of DC with p53 cDNA

Immature DC were generated from PBMC isolated by Ficoll-Hypaque (Amersham Biosciences) centrifugation of venous blood obtained from normal donors, as described previously (8). Six days later, immature DC were harvested and transfected with pVAX constructs expressing wtp53, mutant p53, or p53 F1 fragment. For transfection, the DC were resuspended at a cell density of 0.52 x 10⁶/0.1 ml in Human Dendritic Cell Nucleofector solution (Amaxa Biosystems), mixed with 15 µg of plasmid DNA, and transferred into an amaxa-certified curvette. The plasmid DNA transfection was performed by electroporation using a Nucleofector device, according to the manufacturer’s protocol. After transfection, the DC were harvested and resuspended into AIM-V medium and incubated at 37°C for 48 h before use.

Transfection efficiency of DC was routinely determined by monitoring DC transfected with the pEGFP-N1 vector (BD Clontech), at a concentration of 1 µg/ml per 1 x 10⁶ DC. Using fluorescent microscopy, transfection was 35% efficient (data not shown). To assess transfection of DC with p53 cDNA, quantitative RT-PCR (qRT-PCR) was performed using RNA isolated from DC transfected with either pVAX vector itself, pEGFP-N1 vector, or the pVAX/p53 cDNA constructs. The following primers were used to detect levels of p53 mRNA: forward primer, 5'-GTCCCAAGCAATGGATGATT-3'; reverse primer, 5'-GCATTCTGGGAGCTTCATCT-3'; and the probe-FAM, 5'-CAATGGTTCACTGAAGACCCAGG-3'. RNA was extracted from cell lines harvested when at 70% confluence, using TRIzol (Invitrogen Life Technologies) and the RNeasy kit (Qiagen). The purified RNA was resuspended in RNA secure solution (Ambion) and cleared of DNA. A one-step reverse transcriptase reaction was performed using 500 and 2000 ng/µl of the RNA with random hexamer primers and Superscript II (Invitrogen Life Technologies) as described previously (17). qRT-PCR was then conducted on the Applied Biosystems 7700 Sequence Detection Instrument, using a previously validated assay (17). Expression of the target gene (p53) relative to that of β-glucuronidase (GUS; an endogenous control gene) was calculated using the CT (cycle time) method described previously: relative expression = 2^–CT, where CT = CT(p53) – CT(GUS).

IVS of CD4⁺ Th cells using p53 cDNA-transfected DC as APC

wt p53-specific CD4⁺ Th cell lines were generated as follows: CD4⁺ T cells were positively isolated from nonadherent PBMC with CD4 MicroBeads (Miltenyi Biotec) and used as responder cells. On day 0, the CD4⁺ T cells and p53 cDNA-transfected autologous DC (at ratio of 1: 0.1) were cocultured in wells of 96-well round-bottom tissue culture plates in 0.2 ml/well of AIM-V medium supplemented with 5% (v/v) human AB serum and IL-6 (10 ng/ml) and IL-12 (10 ng/ml) (PeproTech). On day 7, the responder cells were restimulated with fresh p53 cDNA-transfected DC in AIM-V medium supplemented with IL-2 (20 IU/ml) and IL-7 (2 ng/ml) (PeproTech). From 5 to 7 days after the second IVS, the T cells were harvested and tested against peptide-pulsed autologous DC in ELISPOT IFN- assays.

IVS of CD4⁺ T cells using wt p53_25–35 peptide-pulsed DC as APC

wt p53_25–35 peptide-specific CD4⁺ T cell lines were generated using autologous DC that had been matured in the presence of IL-1β, IL-6, and TNF- (10 ng/ml each) (PeproTech) for 24 h, harvested, resuspended (1 x 10⁶ cells/ml) in AIM-V medium containing 20 µg/ml peptide, and incubated at 37°C for 4 h. Subsequently, the peptide-pulsed DC were irradiated (3000 rad), washed, and resuspended in AIM-V medium containing 5% (v/v) human AB serum. Autologous naive CD4⁺ T cells were isolated by positive selection from nonadherent PBMC using CD4 MicroBeads and served as responder cells. On day 0, responder cells and peptide-loaded DC acting as APC were cocultured (at a ratio of 1:0.1) in wells of 96-well round-bottom tissue culture plates in 0.2 ml of AIM-V medium supplemented with 5% (v/v) human AB serum, IL-6 (10 ng/ml), and IL-12 (10 ng/ml). On day 7 and thereafter, the responder cells were restimulated with the peptide-pulsed autologous DC in AIM-V medium supplemented with IL-2 (20 IU/ml) and IL-7 (2 ng/ml) once a week. After the fourth IVS, the responder cells were restimulated with irradiated peptide-pulsed autologous PBMC once a week. After the third or fourth IVS, the reactivity of cultured T cells was determined against autologous wt p53_25–35 peptide-pulsed DC in an ELISPOT IFN- assay.

Generation of wt p53_264–272-specific CD8⁺ T cells in the presence of anti-wt p53_25–35 CD4⁺ T cells

First, wt p53_25–35-specific helper cells were generated as follows: matured DC, obtained from PBMC of HLA-A2⁺/DR4^– normal donors, were resuspended at the concentration 1 x 10⁶ cells/ml in AIM-V medium containing 40 µg/ml wt p53_264–272 peptide and 20 µg/ml wt p53_25–35 peptide, and incubated at 37°C for 4 h. The peptide-pulsed DC and autologous peptide-specific CD4⁺ T cells were irradiated (3000 rad), washed, and resuspended in AIM-V medium containing 5% (v/v) human AB serum. Next, autologous CD8-enriched PBMCs were negatively isolated from nonadherent PBMC using CD4 MicroBeads. On day 0, responder cells (CD8⁺ T cells), wt p53_264–272 peptide-loaded DC, and peptide-specific CD4⁺ T cells (in a ratio of 1:0.1:1, respectively) were cocultured in wells of 48 or 24-well tissue culture plates in AIM-V medium supplemented with 5% (v/v) human AB serum and IL-7 (10 ng/ml). On day 7, the responder cells were restimulated with peptide-pulsed autologous and peptidespecific CD4⁺ T cells in AIM-V medium supplemented with IL-2 (20 IU/ml) and IL-7 (5 ng/ml). One week after the second IVS, the reactivity of the cultured cells was tested against wt p53_264–272-pulsed T2 cells in an ELISPOT IFN- assay.

Flow cytometry analysis using HLA-A2/peptide tetrameric complexes (tetramer)

The PE-labeled HLA-A2/wt p53_264–272 tetramer was obtained from the Tetramer Facility of the National Institute of Allergy and Infectious Disease (Atlanta, GA). Its was previously confirmed by its staining of a CTL line specific for this peptide and lack of staining of irrelevant CTL or HLA-A2^– PBMC obtained from normal donors (18, 19). Three-color flow cytometry assays (FACScan; BD Biosciences) were performed with energy-coupled dye labeled anti-CD3 and FITC-anti-CD8 Abs (Beckman Coulter) and PE-tetramer. Generally, 60,000 events per sample were collected after gating on lymphocytes by forward- and side-scatter.

ELISPOT IFN- assay

The ELISPOT IFN- assay was performed in 96-well flat-bottom nitrocellulose plates (MAHAS4510; Millipore) using the anti-IFN- mAb, 1-D1K, as the capture mAb and the biotinylated anti-IFN- mAb, 7-B6-1, as the detection mAb (both were obtained from Mabtech), as described previously (8). Plates were developed with avidin-peroxidase (Vectastain Elite kit; Vector Laboratories) followed by 3-amino-9-ethyl-carbazole (Sigma-Aldrich). The spots were automatically counted by computer- assisted video image analysis (ELISPOT 4. 14.3; Zeiss). For mAb blocking experiments, stimulator cells were preincubated with anti-HLA-class I (W6/32) or anti-HLA-DR (L243) mAb at a concentration of 10 µg/ml for 30 min.

Statistical analysis

The results of ELISPOT IFN- assays were analyzed using Student’s t test and were considered significant when p < 0.05.

	Results

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Identification of wt p53_25–35 as a CD4⁺ Th cell-defined wt p53 epitope

In two sets of experiments, IVS of CD4⁺ T cells isolated from PBMC obtained from six HLA-DR4^– normal donors using autologous DC transfected with various p53 cDNA constructs as APC was used to identify wt p53 helper peptides for vaccine use in HLA-DR4^– subjects. A panel of 14 wt p53 peptides, 13 of which were predicted by the TERITOPE algorithm (12) to bind to multiple HLA-DR allelic molecules, were tested (Table I). The wt p53_25–35 peptide was tested also because it overlapped the algorithm-predicted wt p53_22–34 peptide and may be a HLA-A2-restricted, CTL-defined wt p53 peptide (20). It was, therefore, possible that it might be a helper as well as cytotoxic peptide. The outgrowing lymphocytes from IVS cultures were tested in ELISPOT IFN- assays for reactivity against peptide-pulsed DC in ELISPOT IFN- assays. The IVS responses to the individual peptides was the basis for ranking the peptides for further evaluation.

View larger version (8K): [in this window] [in a new window]   FIGURE 1. Characterization of the cloned anti-wt p5325–35 CD4+ Th cell. Reactivity in ELISPOT IFN- assay against peptide-pulsed autologous DC of the cloned anti-wt p5325–35 helper cell line, no. 23, which was derived from a bulk population of CD4+ T cells having anti-wt p5325–35 peptide reactivity following IVS using wt p53 F1 cDNA-transfected DC as APC. The * indicates significance (p < 0.05) relative to unpulsed DC as targets, whereas ** indicates significant blocking in the presence of anti-HLA-DR mAb, L243.

Because the results of the above experiments were consistent with the wt p53_25–35 peptide having the properties of binding to multiple HLA-DR alleles, we directly tested its ability to generate anti-p53_25–35 peptide T cells from CD4⁺ T cell obtained from two additional HLA-DR4^– normal donors 7 (HLA-DR15/16⁺) and 8 (HLA-DR7/17⁺). Both yielded anti-wt p53_25–35 peptide CD4⁺ T cells. The reactivity in ELISPOT IFN- assay of the outgrowing Th cells obtained from donor 7 for the wt p53_25–35 peptide is shown in Fig. 2A. The reactivity of these CD4⁺ T cells was also tested against autologous DC transfected with either wt p53 F1 or EGFP cDNA. As indicated in Fig. 2B, these anti-wt p53_25–35 Th cells responded to the p53 F1 fragment-transfected DC, but not EGFP-transfected DC. In addition, the response to the wt p53 F1-transfected DC target cells was blocked by anti-HLA-DR mAb, but not anti-HLA class I mAb. These results provide additional confirmation that the wt p53_25–35 peptide behaves like a naturally presented Th cell-defined epitope, which can be presented in associated with multiple HLA-DR alleles.

View larger version (14K): [in this window] [in a new window]   FIGURE 2. Characterization of the anti-wt p5325–35 CD4+ Th cell line IVS generated using peptide-pulsed DC as APC. Reactivity in ELISPOT IFN- assay of CD4+ T cells following IVS with wt p5325–35 peptide-pulsed DC as APC against peptide-pulsed autologous DC (A) and wt p53F1 or EGFP cDNA-transfected DC (B) target cells. The * indicates significant reactivity (p < 0.05) relative to unpulsed DC. The ** indicates significant blocking of reactivity in the presence of anti-HLA-DR mAb, L243.

The ability of the wt p53_25–35 to induce and be recognized by CD4⁺ T cells in association with HLA-DR7 and -DR11 molecules was directly tested using a panel of peptide-pulsed HLA-DR-transfected mouse L cells, designated L.DR cells, as targets in ELISPOT IFN- assays. Anti-wt p53_25–35 helper cells induced from CD4⁺ T cells obtained from normal donors 5 (HLA-DR7/11⁺) and 6 (HLA-DR11/15⁺), which previously had responded to wt p53 and/or wt p53 F1 cDNA-transfected DC (Table IV), were tested against peptide-pulsed L.DR1, L.DR7, and L.DR11 cells. Because none of the donors expressed HLA-DR1, peptide-pulsed L.DR1 cells served as a negative control for these assays, whereas peptide-pulsed autologous DC served as the positive control. The results demonstrate that the anti-wt p53_25–35 helper cells generated from donor 5 (HLA-DR7/11⁺), recognized peptide-pulsed L.DR7 and L.DR11 cells, as well as autologous DC, but not peptide-pulsed L.DR1 cells (Fig. 3A). Furthermore, the responses were blocked by anti-HLA-DR mAb (L243) but not anti-HLA class I (W6/32) mAb. The ability of wt p53_25–35 peptide to bind to HLA-DR11 molecules was also evident from the analysis using helpers derived from donor 6 (HLA-DR11/15⁺). In this analysis, the helpers recognized peptide-pulsed L.DR11 and autologous DC, but not L.DR1 cells (Fig. 3B). L.DR15 cells were not available for this analysis, although the lack of response of donor 4 (HLA-DR15⁺) suggests that the wt p53_25–35 peptide probably can be presented in association with this HLA-DR allele.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. The wt p5325–35 peptide is an HLA-DR7 and -DR11-restricted helper peptide. Anti-wt p5325–35 CD4+ T cells generated from PBMC obtained from donor 5 (HLA-DR7/11+) (A) and donor 6 (HLA-DR11/15+) (B) recognize peptide-pulsed L cells expressing HLA-DR7 and/or HLA-DR11 allelic molecules. The * indicates significant reactivity (p < 0.05) relative to unpulsed target cells. The ** indicates significant blocking of reactivity in the presence of anti-HLA-DR mAb, L243.

The availability of anti-wt p53_25–35 Th cells permitted us to evaluate their ability to enhance the ex vivo generation of anti-wt p53_264–272 CD8⁺ T cells from CD8⁺ T cells obtained from HLA-A2⁺/DR4^– normal donors 7 and 8. The ratio of CD4⁺:CD8⁺ T cells used in the IVS cultures was 0.5:1, similar to that determined in our previous study to augment the generation of antitumor CD8⁺ T cell effectors (8). The outgrowing lymphocytes were tested in ELISPOT IFN- assays for responses to wt p53_264–272 peptide-pulsed T2 cells as targets. As indicated in Table V, CD8⁺ T cells obtained from donor 7 were modestly IVS responsive to the wt p53_264–272 peptide (325 ± 25 spots/1 x 10⁶ cells) in the culture, in the absence of Th cells. The IVS response in the presence of autologous anti-wt p53_25–35 helper cells and the wt p53_25–35 peptide, however, increased nearly 7-fold to 2107 ± 108/1 x 10⁶ cells. In the absence of exogenously added wt p53_25–35 peptide, however, the anti-wt p53_25–35 helper cells enhanced the generation of anti-wt p53_264–272 effectors <2-fold. In general, the level of response using the wt p53 Th cells was comparable to those observed in cultures supplemented with autologous Th cells specific for the pan HLA-DR tetanus toxoid or PADRE peptides, 2222 ± 67 and 1400 ± 128, respectively. These "pan helpers" are currently being used clinically to provide "generic" help in tumor peptide-based vaccines(15, 16). The total number of responsive T cells per culture was significantly greater in all the IVS cultures supplemented with Ag-specific CD4⁺ T cells compared with those without these cells.

We have observed in our studies that nearly two of three of CD8⁺ T cells obtained from HLA-A2⁺ normal donors or subjects with squamous cell carcinoma of the head and neck are IVS nonresponsive to the wt p53_264–272 peptide (22). CD8⁺ T cells obtained from 1 of 2 of these individuals, however, can be IVS responsive to the "optimized" p53_264–272 peptide, F270W, which can induce/expand CD8⁺ T cells that are cross-reactive against the parental wt p53 peptide (18). We determined that CD8⁺ T cells obtained from donor 8 were essentially IVS nonresponsive to wt p53_264–272 peptide (<10 spots/1 x 10⁶ cells) (Table IV). The optimized F270W peptide, however, did induce a modest anti-wt p53_264–272 CD8⁺ T cell response (383 spots/1 x 10⁶ cells), which was comparable to that of the IVS-responsive CD8⁺ T cells obtained from donor 7, which was 325 ± 25 spots/1 x 10⁶ cells. Culturing the CD8⁺ T cells of donor 8 with the wt p53_264–272 in the presence of anti-p53 Th cells yielded a response of 1007 spots/1 x 10⁶ cells, which was 3-fold greater than the activity induced using the optimized peptide alone, but still only half the response of the IVS-responsive donor 7 CD8⁺ T cells cocultured with parental wt p53 cytotoxic peptide and the anti-wt p53_25–35 Th helper cells (2107 ± 108 spots/1 x 10⁶ cells). Coculturing donor 8 lymphocytes with F270W-pulsed DC and anti-wt p53_25–35 Th cells, however, increased the induction/expansion of anti-wt p53_264–272 CD8⁺ T cells to 6600 spots/1 x 10⁶ cells, which represents 17-fold relative to the response using the optimized peptide alone (6600 vs 383/1 x 10⁶ cells), and 3-fold above the IVS response of anti-wt p53_264–272 CD8⁺ T cells obtained from donor 7 using the parental wt p53 peptide and anti-wt p53_25–35 Th cells peptides (6600 vs 2107 ± 108/1–10⁶ cells).

Interestingly, the addition of CD4⁺ T cells to cultures of donor 8’s CD8⁺ T cells failed to enhance lymphocyte yields (Table V). The numbers of lymphocytes generated in the these cultures, in the presence or absence of CD4⁺ T cells (1.6 and 1.4 x 10⁶ cells/culture), were similar and comparable to the yields of the IVS cultures of donor 7 CD8⁺ T cells in the absence of CD4⁺ T cells (1.2 and 1.9 x 10⁶ cells/culture). In contrast, the addition of the Ag-specific CD4⁺ T cells to cultures of donor 7’s CD8⁺ T cells increased cell yields 2- to 3-fold. The basis for this difference is uncertain at this time, but may be reflective of the functional activities of the different anti-wt p53_25–35 Th cell lines used in these experiments.

The outgrowing CD8⁺ T cells present in bulk IVS cultures established from lymphocytes obtained from donor 8 were also analyzed by flow cytometry for the frequency of anti-wt p53_264–272 tetramer⁺CD3⁺CD8⁺ T cells (Fig. 4A). The CD8⁺ T cells cultured in the presence of the optimized peptide and anti-wt p53_25–35 CD4⁺ T cells (culture 4; Fig. 4D) showed the highest level of tetramer⁺CD8⁺ T cells (3.3%), which represents approximately a 10-fold increase in the frequency of these T cells compared with the IVS cultures established in the absence of the Th cells (0.3%) and a 3- to 4-fold increase compared with frequency generated in the presence of the optimized peptide alone (0.8% in culture 2; Fig. 4B) or the combination of the wt p53 peptide and the anti-wt p53_25–35 CD4⁺ T cells (0.9% in culture 3; Fig. 4C). The p53 tetramer⁺ T cells generally gated as large CD3⁺CD8⁺ T cells (data not shown), consistent with their activation status, as described previously (8). The frequencies of tetramer⁺ T cells in all three sets of IVS cultures were considerably higher than the level of activity detected in the ELISPOT IFN- assay performed with the same cultures. This is not surprising, because similar discrepancy is consistently noticed in immunological monitoring of antitumor peptide responses in the peripheral blood and can be partially attributed to differences in the sensitivities of the two detection methods (23).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. The addition of anti-wt p5325–35 CD4+ Th cells enhances the ex vivo generation/expansion of anti-wt p53264–272 CD8+tetramer+ T cells from nonresponsive PBMC. A representative three-color flow cytometry analysis of CD3+CD8+ T cells using PE-labeled HLA-A2/wt p53264–272 tetramer of cells following IVS of an enriched population of CD8+ T cells obtained from normal donor 8 PBMC in the presence of wt p53264–272-pulsed DC (A), F270W peptide-pulsed DC (B), wt p53264–272/p5325–35-pulsed DC, and anti-wt p5325–35 CD4+ Th cells (C), and F270W/p5325–35-pulsed DC and anti-wt p5325–35 CD4+ Th cells (D). (See Table IV for additional details.) The analysis of an average of 60,000 CD3+CD8+ T cells from each culture is shown. Values in the upper right quadrants indicate percentages of tetramer+CD8+ T cells detected in outgrowing cells harvested from each of the four IVS cultures. The analysis was performed twice, and the values obtained for tetramer+CD8+ cells in cultures 2–4 were within 0.2%. The tetramer+CD8+ T cells present in culture 1 were 3.3 and 2.2%.

	Discussion

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The wt p53_25–35 peptide has been identified as a naturally presented Th cell-defined peptide capable of inducing and being recognized by CD4⁺ T cells in association, at a minimum, with HLA-DR7 and -DR11 molecules. Ancillary results suggest it’s binding to HLA-DR13 and HLA-DR16 molecules, as well. In addition, the presence of anti-p53_25–35 CD4⁺ Th cells was shown to enhance the in vitro generation/expansion of HLA-A2-restricted, anti-wt p53_264–272 CD8⁺ T cells from CD8⁺ T cells, whether they were IVS responsive or nonresponsive to the wt p53_264–272 peptide in the absence of anti-p53_25–35 CD4⁺ Th cells. At least in vitro, it has all the attributes of a promising tumor helper peptide for vaccines. Given the fact that it will be necessary to test this epitope in vivo, vaccines incorporating this helper peptide would be applicable to a broader population of subjects for whom a wt p53 helper peptide is presently unavailable. In fact, the average frequencies of expression of the HLA-DR7, -11 alleles, which are capable of recognizing the wt p53_25–35 peptide, are 14 and 15.5% (11), respectively. These values are comparable to those of the HLA-DR1 and -DR4 alleles, 10.4 and 24.4%, respectively, which recognize the overlapping wt p53_108–122 and wt p53_110–124 helper peptides (11). Consequently, 60% of HLA-A2⁺ subjects are now potentially eligible for a p53 peptide-based vaccine combining helper and cytotoxic peptides.

The ex vivo generation/expansion of anti-p53_25–35 CD4⁺ T cells was initially obtained using DC transfected with a nonviral plasmid expressing either wt p53 or a codon 135 mutant p53 cDNA as the APC. The mutant p53 cDNA was tested because mutated p53 molecules in some tumors display an increased stability compared with wt p53 molecules and enhanced presentation of p53-derived epitopes (2, 3). In contrast, p53 missense mutations can negatively influence proteasomal processing of p53 epitopes (25). To guard against the possibility that the p53 codon 135 mutation might interfere with the antigenic processing of the sought after multiple HLA-DR-binding peptide(s), DC transfected with wt p53 cDNA were also tested as APC. In addition, wt p53 cDNA promotes apoptosis (26), and nontransfected DC might phagocytize apoptotic DC in the culture system, further promoting the processing and presentation of p53 epitopes by DC. The lymphocytes outgrowing in this in vitro system were tested against a panel of 14 algorithm-predicted, multiple HLA-DR-binding wt p53 peptides, which had been individually pulsed onto autologous DC. IVS of CD4⁺ T cells from an additional two HLA-DR4^– donors using DC transfected with either wt p53 or wt p53 F1 fragment cDNA or pulsed with the wt p53_25–35 peptide as APC were also performed. No clear distinction between the efficacies of the various APC was apparent.

In total, IVS of CD4⁺ T cells from 6 of 7 HLA-DR4^– normal donors tested yielded anti-wt p53_25–35 CD4⁺ T cells using peptide-pulsed DC as APC. Based on the HLA-DR genotypes of the donors, the results indicate that HLA-DR7, -11, -13, and -16 alleles, but not HLA-DR15, can present the wt p53_25–35 peptide for recognition by CD4⁺ T cells. Direct confirmation of wt p53_25–35 peptide binding to HLA-DR7 and -DR11 alleles was obtained using HLA-DR-transfected L cells as targets in ELISPOT IFN- assays. In an ancillary study, however, no IVS responses to wt p53_25–35 peptide were generated from PBMC obtained from 10 HLA-A2⁺ normal donors, strongly discounting the possibility that this peptide might act as a cytotoxic as well helper p53 peptide (K. Chikamatsu and A. B. DeLeo, unpublished results).

Although the IVS response to the wt p53_25–35 peptide was predominant in this study, several responses to other algorithm-predicted wt p53 peptides were also detected, in particular, the wt p53_193–205 and p53_381–393 peptides. Although we continue to analyze the immunoreactivities of the latter peptides, the wt p53_25–35 peptide joins the wt p53_110–124 and p53_108–122 peptides as naturally presented HLA class II-restricted Th cell-defined p53 epitopes (8, 10). All three are encoded within the N-terminal portion of the p53 molecule, which suggests that this region of the molecule might be the prevalent source of multiple HLA class II-restricted wt p53 peptides. Interestingly, anti-p53 Ab present in the sera of cancer subjects have been shown to recognize epitopes encoded in the N-terminal transactivation region of the molecule, in which this novel p53 helper peptide is encoded (27). This suggests that Ab as well as Th cell-defined epitopes are encoded in this region. Contrary to this hypothesis, however, is the report by van der Burg et al. (28) for subjects with colon cancer, which measured the p53-related proliferative responses of these subjects to pools of overlapping 30-mer peptides corresponding to wt p53 residues 1–142, 129–270, and 257–393. The p53 129–270 peptide pool did yield the most responses, whereas none were detected against the wt p53 1–142 peptide pool. Following surgery, some responses against the third peptide pool corresponding to the p53 C-terminal region were detected. The difference between our results and those of van der Burg et al. might be due to various reasons (28). These include 1) their use of peptide pools rather than individual peptides and the resulting competition between peptides for recognition, 2) their analyses were done independent of subjects’ HLA-DR genotypes, and, perhaps more like, 3) there exists a Th peptide present in their p53 129–270 peptide pool that was not part of the panel of algorithm-predicted peptides used in our study.

We have previously demonstrated the beneficial effects of incorporating the Th-defined wt p53_110–124 peptide into IVS (8). In the experiments reported in this study, we also observed similar ex vivo effects with the wt p53_25–35 peptide on the generation of anti-wt p53_264–272 CD8⁺ T cells. As noted, we consistently find that PBMC from only 1/3 HLA-A2⁺ normal donors or cancer subjects are IVS responsive to the wt p53_264–272 peptide, i.e., generate anti-wt p53_264–272 CD8⁺ T cells ex vivo (19). To overcome this unresponsiveness, we identified F270W, an optimized p53_264–272 peptide, in which tryptophan was exchanged for phenylalanine at residue 7 in the peptide. The F270W peptide is capable of enhancing the generation of effectors that cross-react with the parental peptide from 1/2 of IVS nonresponsive PBMC (18). Importantly, the addition of wt p53_25–35-specific Th cells to IVS cultures of CD8⁺ T cell-enriched PBMC yielded higher levels of anti-wt p53_264–272 effectors, even from IVS nonresponsive CD8⁺ T cells. The identification of the wt p53_25–35 peptide as a multiple HLA-DR-binding Th cell-defined epitope is likely to provide investigators with a p53-specific Th cell component for use in p53-based vaccines that could be more efficacious and certainly applicable to a much broader population of subjects with various types of cancer than was previously possible. Ag-uptake by DC and its role in the induction of an antitumor immune response by "cross-priming" CD8⁺ as well as CD4⁺ T cells are envisioned to be most effective when CTL-defined as well as Th cell-defined epitopes are derived from the same Ag are presented (29).

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported in part by National Institutes of Health Grants P01 DE-12321 and P50 CA97190, and The Stout Family Fund for Head and Neck Cancer Research at The Eye and Ear Foundation of Pittsburgh.

² Address correspondence and reprint requests to Dr. Albert B. De Leo, University of Pittsburgh Cancer Institute, Research Pavilion, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213. E-mail address: deleo{at}imap.pitt.edu

³ Abbreviations used in this paper: wt, wild-type sequence; IVS, in vitro stimulation; DC, dendritic cell; n, nucleotide; qRT-PCR, quantitative RT-PCR; GUS, β-glucuronidase; CT, cycle time; EGFP, enhanced GFP.

Received for publication December 12, 2005. Accepted for publication August 10, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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