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Immunology |
Division of Basic Research, University of Pittsburgh Cancer Institute and the Departments of Pathology [K. C., A. A., J. S., T. L. W., A. B. D.] and Otolaryngology [T. L. W.], School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213; Virginia Mason Research Institute and University of Washington School of Medicine, Seattle, Washington 98101 [W. W. K.] and National Cancer Institute, Bethesda, Maryland 20892 [E. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although several class II HLA-restricted tumor-associated peptides have been identified that potentially could be used in vaccines against several frequent types of carcinomas, none is derived from a tumor antigen as widely expressed as p53 (12) . p53 was originally identified as a transformation-related antigen by IgG antibodies present in the sera of mice immunized against chemically induced tumors (13) . Subsequently, anti-p53 IgG was detected in the sera of some patients with various types of cancer (14 , 15) . The IgG nature of the anti-p53 humoral responses in cancer patients mandates that anti-p53 responses involve CD4+ T cells and suggests that p53-specific precursor cells are present in these individuals. Although anti-p53-specific proliferative responses of PBMC-derived T cells obtained from normal donors as well as cancer patients have been reported, no definitive identification of naturally presented class II-HLA-restricted wt p53 peptides has been made (16, 17, 18) . Therefore, in this study, we sought to identify a naturally presented HLA-DRB1*0401-restricted wt p53 peptide that could be used to probe patients’ CD4+ T cell-mediated responses to p53 and also have the potential of being a critical component of broadly applicable cancer vaccines. To achieve this, we stimulated CD4+ T cells with autologous DCs in the presence of rhp53 and tested the outgrowing lymphocytes for responses against a panel of HLA-DRB1*0401-binding wt p53 peptides predicted by a computer-based algorithm to give optimal reactivity (19 , 20) . This approach identified the wt p53110–124 peptide as a CD4+ Th cell-defined and naturally presented HLA-DRB1*0401-restricted epitope. HLA-DRB1*0401/p53 peptide tetramer complexes (tetramers) confirmed the presence of T cells recognizing this epitope in T-cell lines and peripheral circulation of normal donors and patients with cancer. Importantly, we demonstrate the ability of these CD4+ T cells to enhance the ex vivo generation and function of antitumor CD8+ T-cell effectors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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rhp53 and p53 Peptides.
rhp53 was purified by metal ion chromatography from insect cell extracts expressing a rhp53 construct (20)
. The eight p53-derived peptides, wt p5322–36, 47–61, 94–108, 106–120, 110–124, 123–137, 127–141, and 192–206, were algorithm predicted to bind to HLA-DRBI*0401 molecules(19)
. They were synthesized using standard methodologies, purified, and stored as lyophilized preparations. Peptides containing cysteine residues were dissolved in PBS just before use.
Induction of Anti-wt p53 CD4+ T Cells by IVS Using rhp53 or p53 Peptides.
PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation of blood samples obtained from HLA-DRB1*0401+ normal donors and patients in accordance with institutional guidelines, using an Institutional Review Board-approved protocol and consent forms. DCs were generated from PBMCs, as described previously (6)
. CD4+ T cells were positively isolated from nonadherent PBMCs with immunomagnetic beads (Miltenyi Biotech, Auburn, CA). CD4+ T cells (1 x 106) and DCs (1 x 105) were cocultured in the presence of rhp53 protein (1 µg/ml) in wells of a 48-well plate in a final volume of 1 ml/well CM supplemented with 1000 units/ml IL-6 and 10 ng/ml IL-12. On days 7, 14, and 21, the responder cells were restimulated with autologous DCs in the presence of rhp53 and grown in media supplemented with 20 IU/ml IL-2 and 2 ng/ml IL-7. The responding cells were assayed on day 21 for proliferative activity against the p53 peptides. Cloned T-cell lines were obtained from the bulk line by limiting dilution at 1 cell/well in the wells of 96-well plates containing 1 x 105 irradiated PBMCs from two different normal donors and 1 x 104 peptide-pulsed allogeneic HLA-DR4+ EBV-B cells in 0.2 ml/well culture medium. Peptide-specific CD4+ T-cell clones were then restimulated every week and expanded using irradiated allogeneic PBMCs and peptide-pulsed HLA-DR4+ EBV-B cells as feeder and stimulator cells, respectively.
For induction of anti-p53 peptide CD4+ T cells, DCs were incubated with peptide (20 µg/ml) for 4 h at 37°C and irradiated, and 1 x 104 DCs were cultured with autologous CD4+ T cells (1 x 105) per well in 96-well, round-bottomed plates in 0.2 ml of CM containing 1000 units/ml IL-6 and 10 ng/ml IL-12. The CD4+ T cells were restimulated on days 7, 14, and 21 with autologous DCs pulsed with the peptide and grown in CM containing 20 IU/ml IL-2 and 2 ng/ml IL-7. On day 21, microcultures were tested for proliferative responses to peptide-pulsed T2.DR4 cells. Selected cells were transferred to wells of a 24-well plate and restimulated with peptide-pulsed autologous PBMCs or HLA-DR4--matched allogeneic EBV-B cells.
Proliferation Assays.
T cells (2 x 104 cells/well) were mixed with irradiated autologous PBMCs (1 x 105 cells/well) or T2.DR4 (2 x 104 cells/well) in the presence of peptides in 96-well, round-bottomed plates. The cultures were incubated at 37°C for 72 h and pulsed with 1 µCi/well 3H-thymidine for the last 16 h, and the incorporated radioactivity was measured by liquid scintillation counting. In mAb blocking experiments, 10 µg/ml anti-class I HLA mAb, w6/32, or anti-HLA-DR mAb, L243, were added.
ELISPOT IFN
Assay.
The ELISPOT IFN assay was performed in 96-well, flat-bottomed nitrocellulose plates (MAHAS4510; Millipore, Bedford, MA) 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 mAbs were obtained from Mabtech, Nacka, Sweden), as described previously (6)
. Plates were developed with avidin-peroxidase (Vectastain Elite kit; Vector, Burlingame, CA) followed by 3-amino-9-ethyl-carbazole (Sigma Chemical Co., St. Louis, MO). The spots were automatically counted by computer-assisted video image analysis (ELISPOT 4.14.3; Zeiss, Jena, Germany). For antibody blocking experiments, target T cells were preincubated with anti-HLA-class I or anti-HLA-DR mAb for 30 min. Cryopreserved aliquots of PBMCs obtained from a normal donor were thawed and, after stimulation with PMA (1 ng/ml) and ionomycin (1 µM; both from Sigma), were used as a positive control for each assay. The interassay reproducibility of the assay was acceptable with a coefficient of variation = 15% (n = 30).
Flow Cytometry Analyses.
The streptavidin-phycoerythrin-labeled HLA-DRB1*0401/peptide tetramers were prepared for this study as described previously (23)
. Three- and four-color flow cytometry assays of PBMCs and T-cell lines were performed using FITC-anti-CD3, CyChrome anti-CD8, allophycocynanin (APC) anti-CD4, and PE-tetramer. The fluorochrome-conjugated mAbs and appropriate isotype controls were obtained from BD PharMigen (San Diego, CA). Cells were stained at 10 µg/ml for 2 h at 37°C. In general, 3 x 105 events were collected progressively after live gating on lymphocytes by forward and side scatter. Detection of intracellular IFN was determined using cells fixed with 0.5% paraformaldehye (Fisher Scientific Co., Pittsburgh, PA) followed by permeabilization with 0.1% saponin (Sigma). The permeabilized cells were then stained with FITC-anti-IFN mAb (BD PharMingen).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The response to the p53110–124 peptide was blocked by anti-HLA-DR mAb, L243, but not anti-class I HLA mAb, w6/32 (Fig. 1B)
. We further confirmed the HLA-DRB1*0401 restriction of the reactivity of the bulk population of effectors for the wt p53110–124 peptide in the ELISPOT IFN assay using peptide-pulsed T2.DR4 target T cells (Fig. 2A)
, a result which also indicated that these effectors were Th1 biased as well. A weaker but noticeable response using these effectors was also detected in this assay system against the overlapping wt p53106–120 (SYGFRLGFLHSGTA) peptide.
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(Fig. 2B)
. The genotype of PCI-13 was determined to be HLA-DRB1*0401,-DRB1*0701. The bulk population of anti-wt p53110–124 CD4+ T cells produced and secreted IFN in response to cytokine-treated PCI-13 tumor cells. This response was blocked by anti-HLA-DR mAb, L243, but not anti-class I HLA mAb, w6/32, and was consistent with wt p53110–124 peptide being a naturally presented Th-defined epitope (Fig. 2C)
. As this bulk population of CD4+ T cells was induced with rhp53, its recognition of PCI-13 cells could have involved a p53-derived epitope(s) other than, or in addition to, the wt p53110–124 peptide. Accordingly, we isolated several anti-p53110–124 peptide-specific T-cell lines from the bulk population by limiting dilution and selected one of the more potent lines, clone #21, for additional characterization. Clone #21 was highly reactive in ELISPOT IFN assays against p53110–124 peptide-pulsed T2.DR4 cells but not against nonpulsed, p53106–120 or p53 22–36 peptide-pulsed T2.DR4 cells (Fig. 3A)
. As expected, clone #21 cells also responded in ELISPOT assays to PCI-13 cells pretreated with IFN alone or in combination with TNF
. This reactivity was blocked by the L234 mAb, but not the w6/32 mAb, confirming that wt p53110–124 peptide as representing a naturally presented HLA-DRB1*0401-restricted, Th-defined wt p53 epitope (Fig. 3B)
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. No significant recognition was noted. In a subsequent experiment, the p53110–120 and p53110–121 peptides were also found not to be recognized (data not shown). These results imply that the binding/recognition of the p53110–124 peptide is critically dependent on the leucine at codon 111 and the COOH-terminal amino acid residues, in particular, the valine at codon 122.
Induction of Anti-wt p53110–124 CD4+ T Cells from PBMCs in HLA-DR4+ OSCC Patients.
The potential utility of the wt p53110–124 peptide in cancer vaccination protocols was demonstrated by generation of anti-wt p53110–124 CD4+ Th cells from enriched populations of CD4+ T cells isolated from PBMCs obtained from two HLA-DR4+ OSCC patients, including the patient whose tumor gave rise to the PCI-13 cell line. After 4 x IVS, both bulk populations showed specificity against the relevant peptide in proliferation and IFN ELISPOT assays. The antipeptide reactivity of the effectors derived from the PCI-13 patient, designated F3, was demonstrated in ELISPOT IFN assays (Fig. 4A)
. The response was blocked by L243 mAb but not w6/32 mAb. In addition, we found that the F3 cells were responsive to the autologous tumor cell line, PCI-13, which had been pretreated with IFN alone or in combination with TNF (Fig. 4B)
. This reactivity was blocked by the anti-HLA-DR mAb but not the anti-class I HLA mAb. The anti-p53110–124 CD4+ T cells derived from another HLA-DRB1*0401+ OSCC patient were also reactive against PCI-13 target T cells (data not shown).
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. In contrast, the frequency of flu tetramer+ cells in this population was 1%. The comparable analysis of the anti-PCI-13 CD4+ T cell 13.6 cell line yielded a frequency for the p53 tetramer+ cells of 0.4%, whereas the flu tetramer+ cell frequency was 0.1%. As a baseline for this analysis, we also analyzed unstimulated PBMCs obtained from the PCI-13 patient with both tetramers. The frequency of the flu tetramer+ cells in the CD3+CD4+ PBMC-gated population was 0.13%, whereas that of the p53 tetramer+ cells was slightly lower at 0.1% (Fig. 5)
. The F3 cell population, therefore, represented 
170-fold expansion of anti-p53110–124 precursor Th cells present in PBMCs, whereas only a 4-fold expansion of these cells occurred during the in vitro generation of the autologous but antigenically unrelated 13.6 cell line. These results confirm the specificity of the HLA-DRB1*0401/p53 peptide tetramer for CD4+ T cells recognizing the wt p53110–124 peptide.
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-treated PCI-13.B7 tumor cells (PCI-13 cells transfected to express the B7.1 costimulatory molecule) rather than the parental tumor cell line (21)
. Even under these optimized conditions, however, CD4+ T cells needed to be routinely removed by positive selection before each successive IVS of the culture to facilitate expansion of CD8+ T cells. In contrast, 2 x IVS of PBMCs or CD8+ T cells in the presence of irradiated CD4+ F3 cells and IFN-treated PCI-13.B7 tumor cells enhanced the generation of antitumor CD8+ T-cell effectors. PBMCs to F3 or CD8+ to F3 cell ratios used in these experiments ranged from 1:0.1 to 1:1 (Table 1)
. Regardless of the ratio used, the total cell and CD8+ T-cell yields, as well as numbers of antitumor effectors obtained from the PBMC cultures supplemented with irradiated F3 cells, were higher compared with the control cultures. When PBMC:F3 cells were cultured at a 1:1 ratio, the expansion of CD8+ T cells and antitumor effectors was significant. A comparable result was obtained when an enriched population of CD8+ T cells, rather than PBMCs, was cocultured with F3 cells (Table 1)
. In both types of experiments, the presence of anti-class II HLA mAb, as well as anti-class I HLA mAb, blocked the generation of antitumor effector activity (data not shown).
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+ T cells. The antigenic specificity of the anti-PCI-13 CD8+ T-cell line is presently undefined. Although PCI-13 cells are HLA-A2.1+, the anti-PCI-13 CD8+ T-cell line does not contain HLA-A2.1-restricted anti-p53 CTL reactivity, as defined by its lack of reactivity against HLA-A2.1+ p53-transfected SaOS-2 cell line, SaOS-2cl3, and p53 peptide-pulsed T2 target cells (data not shown). The percentage of CD8+ T cells expressing IFN+ increased from 40 to 82% as the ratio of CD8:CD4 cells increased from 1:0 to 1:1 (Fig. 6A)
. Furthermore, upwards of 12% of the cocultured CD8+ T cells were large lymphocytes, whereas <0.1% of these cells were detected in the control culture (Fig. 6B)
. Essentially, all of the large CD8+ T cells were IFN+ (Fig. 6C)
. This representative experiment indicates that anti-p53 CD4+ T cells have a "helper" role and are able to enhance functional activity of tumor-specific CD8+ T cells, which need not necessarily be recognizing p53-derived determinants.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic alteration in p53 is perhaps the most frequently encountered genetic event associated with human cancer. Over the past 20 years, anti-p53 IgG has been consistently detected in the sera of 15–20% of cancer patients in many studies (15)
. Despite their anti-p53 humoral responses, however, these cancer patients have a poor prognosis (27)
, which might be attributable to a predominating Th2 antitumor immune response in these patients rather than the Th1-biased response that is generally associated with tumor eradication. A recent study has shown that a majority of the proliferative responses colon cancer patients’ PBMC to p53 protein or peptides (considered Th cell mediated) was from p53 sero-negative patients (17)
. Interestingly, several of these ex vivo responses showed Th1-cytokine profiles, which would predict that p53-based immunization of these individuals might induce robust antitumor responses.
We assessed whether p53110–124-specific pCD4+ T cells were present in the peripheral circulations of cancer patients by generating peptide-specific CD4+ T cells from PBMCs obtained from two HLA-DRB1*0401+ OSCC patients, one of whom was sero-negative for p53. The p53 serotype of the other "responsive" patient is under study. Before IVS, no significant anti-p53110–124 peptide responses were detected in unstimulated PBMCs in ELISPOT IFN assays. After 3 x IVS, however, the cultures showed proliferative activity and produced IFN, but not IL-5, in response to peptide-pulsed target T cells. The fact that the patients’ responses were Th1 biased is encouraging in terms of using this peptide in developing p53-based immunotherapy. These results suggest that precursors of p53110–124-specific CD4+ T cells are present in patient’s PBMCs, although their frequencies were too low to be detected in direct ELISPOT assays.
The identification of the Th-defined wt p53110–124 peptide expands the repertoire of epitopes available for the vaccine development to now include Th- as well as CTL-defined wt p53 epitopes. Previously, Fujita et al. (18) identified a panel of class II HLA-restricted wt p53 epitopes, among which, p53108–122 was defined as a DP5-restricted peptide. Although immunogenic, none of the peptides was established as being naturally presented. Our results and those of Fujita et al. (18) suggest that the region of the p53 molecule encoding these peptides might be a source of multiple wt p53 peptides capable of binding to more than one type of class II HLA allelic molecule. Contrary to this hypothesis, however, are the results reported in the recent study by van der Burg et al. (17) in which the p53-specific proliferative responses of colon cancer patients against mixtures of overlapping 30-mer peptides derived from residues 1–142, 129–270, and 257–393 were investigated. Although not directly attributed to CD4+ Th cells, fewer responses were detected against the peptides overlapping residues 1–142 than against the other two pools of p53 peptides.
While most tumors do not express class II HLA antigens, optimal induction of antitumor immunity has been shown to require CD4+ T cells as well as CD8+ T cells (28)
. The potential beneficial impact that a wt p53 Th cell-defined epitope would have on immunotherapy was probed ex vivo in a series of experiments involving elements of the OSCC PCI-13 system, an autologous oral cancer system available in our laboratory. The bulk F3 anti-p53 Th cell population was used in these studies. Although this CD4+ T-cell line was characterized by tetramer analysis, as well as ELISPOT assays, as containing only 2% functionally active anti-p53 cells, its presence in autologous PCI-13 system-based cultures consistently augmented the ex vivo generation, as well as functional activity of CD8+ antitumor effectors. Generally, these responses were not dependent on the equivalent ratio between responder lymphocytes and the F3 Th cell, which implies that the functional activity of these Th cells is potent and need not require equivalency. Apparently, enhancement of the functional activity of the antitumor CD8+ T cells need not involve direct contact between CD8+/CD4+ T cells. In ancillary experiments involving the anti-PCI-13 CD4+ T cell line, 13.6, rather than the F3 cell line, autologous PBMCs cultured with irradiated IFN-treated PCI-13.B7 tumor cells and either 13.6 T cells or supernatant from a culture of 13.6 cells and IFN-treated PCI-13.B7 tumor cells showed enhancement of outgrowth of CD8+ T cells, as well as antitumor effectors (data not shown). These results are consistent with the recent demonstration of the synergy between adoptively transferred CD4+ and CD8+ T cells in inhibiting progressively growing transplanted tumors in a nude mouse model (29)
. In the absence of anti-wt p53 Th cell line, administration of exogenous IL-2 was required to maintain the adoptively transferred anti-p53 CD8+ effectors.
Although CD8+ T cells were the responders in the ex vivo experiments we performed, the value of using CD4+ Th-defined tumor peptides for vaccination is that, in addition to augmenting CTLs, they could augment other elements of the host defense, including non-MHC-restricted inflammatory cells and natural killer cells, that also can function in tumor eradication (10) . As the first fully characterized, naturally presented class II HLA-restricted wt p53 peptide, the p53110–124 is a leading candidate for inclusion in the development of broadly applicable multiepitope cancer vaccines that could target p53, as well as other widely expressed tumor antigens.
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FOOTNOTES |
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1 Supported in part by NIH Grant PO-1 DE-12321 (to T. L. W. and A. B. D.). 
2 To whom requests for reprints should be addressed, at University of Pittsburgh Cancer Institute, Research Pavilion, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh PA 15213. Phone: (412) 623-3228; Fax: (312) 623-1415; E-mail: deleo{at}imap.pitt.edu
3 The abbreviations used are: wt, wild-type sequence; CM, complete medium; DC, dendritic cell; MSD, mean standard deviation; ELISPOT, enzyme-linked immunospot assay; IVS, in vitro stimulation; mAb, monoclonal antibody; OSCC, oral squamous cell carcinoma; PBMC, peripheral blood mononuclear cell; IL, interleukin; PE, phycoerythrin; rhp53, recombinant human p53; TNF, tumor necrosis factor; APC, allophycocynanin polyposis coli; Th, T helper.
Received 11/27/02. Accepted 5/ 5/03.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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