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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

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.

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.