1. The use of transgenic mice to generate high affinity p53 specific cytolytic T cells
Z Yu, X Liu, T M McCarty, D J Diamond, J D Ellenhorn J Surg Res. 1997 May;69(2):337-43. doi: 10.1006/jsre.1997.5058.
P53 is an attractive target immunotherapy because it is overexpressed in up to one half of all malignancies, and its overexpression often correlates with a worsened prognosis. We wanted to determine the feasibility of targeting wild-type epitopes p53 on human tumor cells. HLA A2.1 transgenic mice were immunized with the immunodominant wild-type p53 peptide epitopes, p53(149-157) and p53(264-272), along with a pan-DR helper epitope peptide in incomplete Freund's adjuvant (IFA). Twelve days later, splenocytes were harvested and stimulated with syngeneic blast cells that had been acid-treated to remove endogenous peptide and p53 peptide-pulsed. The responding cells were subsequently restimulated weekly with acid washed, peptide-pulsed Jurkat cells transfected with HLA A2.1. Peptide specific activity was tested in a chromium release assay. The resulting cytotoxic T cells (CTL) were cloned by limiting dilution. Peptide specific CTL were generated against both p53(149-157) and p53(264-272. Only p53(149-157) specific CTL were able to recognize and lyse cells that overexpressed endogenous p53. CTL clones derived from the p53(149-157) cell line demonstrated high affinity and specificity for p53(149-157) when presented by HLA A2.1+ cells. The p53(149-157) specific CTL were tested for specificity against a variety of cultured human cell lines. The CTL clones only lysed cells that overexpressed p53 in the context of HLA A2.1 and did not lyse cells with normal p53 expression or cells that lacked HLA A2.1 expression. This study demonstrates the possibility of targeting tumors, which overexpress p53, and raises the possibility transferring the high affinity, p53 specific T cell receptors from the murine CTL to human T cells.
2. [Detection of specific T cells for 264-272 and 149-157 peptides of p53 protein in peripheral blood of patients with breast cancer]
Iñaki Alvarez-Busto, et al. Med Clin (Barc). 2008 Nov 22;131(18):685-8. doi: 10.1157/13129112.
Background and objective: p53 protein is overexpressed in nearly half of all human tumours. An HLA-A2.1-restricted immunological response mediated by anti-p53 CD8+ T cells directed against the wild type p53 264-272 epitope has been demonstrated in patients with head and neck squamous carcinomas. The existence of such a response in patients with other cancer types could be determinant for the development of specific antitumour vaccines targeting the p53 protein. We aimed to determine the presence of anti-p53 specific CD8+ T cells in peripheral blood of breast cancer patients in vivo. Patients and method: p53 264-272-specific CD8+ T cells were directly enumerated in the peripheral circulation of patients with breast cancer using tetrameric p53 264-272/HLA-A2.1 complexes by multicolor flow cytometry. The same procedure was used to enumerate T cells specific for another HLA-A2.1 restricted wild type p53 epitope, p53 (149-157). Results: The 99th percentile of the concentration of anti-p53 cells in 6 HLA A2- breast cancer patients was 1/5634 (cut-off point). The median counts of anti-p53264-272 and anti-p53149-157 lymphocytes in 14 HLA A2.1+ patients were 1/2383 and 1/2335 respectively. All of the HLA A2+ patients had concentrations of anti-p53 lymphocytes above the cut-off point for at least one of the epitopes: 13/14 (93%) for p53(264-272) and 11/12 (92%) for p53(149-157). Conclusions: A specific immunological response mediated by anti-p53 CD8+ T cells has been detected in patients with breast carcinoma. More studies are needed to confirm these results and to determine its usefulness for the development of p53-based vaccines.
3. Generation of anti-p53 cytotoxic T lymphocytes from human peripheral blood using autologous dendritic cells
K Chikamatsu, K Nakano, W J Storkus, E Appella, M T Lotze, T L Whiteside, A B DeLeo Clin Cancer Res. 1999 Jun;5(6):1281-8.
CTLs recognizing the HLA-A2.1-restricted, wild-type sequence p53 epitopes p53(149-157) and p53(264-272) were generated from CD8-enriched populations of nonadherent peripheral blood lymphocytes (PBLs) obtained from healthy donors. The PBLs were restimulated in vitro with peptide-pulsed granulocyte macrophage colony-stimulating factor- and interleukin (IL)-4-induced autologous dendritic cells in the presence of IL-6 and IL-12 and subsequently cultivated with IL-1alpha, IL-2, IL-4, IL-6, and IL-7. Bulk anti-p53(264-272) CTL populations were generated from PBLs obtained from two of five donors. Both CTL populations were cytotoxic against peptide-pulsed HLA-A2+ target cells, but not against untreated target cells. A CD8+ anti-p53 CTL clone designated p264#2 was isolated from one of the bulk populations. It was found to have an intermediate affinity of approximately 10(-9) M for the epitope and to mediate cytotoxicity against several human tumor cell lines, including the squamous cell carcinoma of the head and neck cell line SCC-9, which is known to present the wild-type sequence p53(264-272) epitope. In addition, CTLs reactive against p53(149-157)-pulsed targets as well as a HLA-A2+ tumor cell line were cloned from a bulk population of antitumor CTLs obtained from one of the five normal PBLs restimulated with this epitope. The results indicate that CTLs recognizing wild-type sequence epitopes can be generated from precursors present in PBLs obtained from some normal individuals using autologous dendritic cells as antigen-presenting cells and suggest that vaccine strategies targeting these epitopes can lead to antitumor CTL generation, thereby emphasizing the therapeutic potential of p53-based cancer vaccines.
