Potentiation of a Tumor Cell Susceptibility to Autologous CTL Killing by Restoration of Wild-Type p53 Function 1 Je ´ro ˆ me Thiery,* Guillaume Dorothe ´e,* Hedi Haddada,* Hamid Echchakir,* Catherine Richon,* Rodica Stancou,* Isabelle Vergnon,* Jean Benard, † Fathia Mami-Chouaib,* and Salem Chouaib 2 * Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21 waf/CIP1 . Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We dem- onstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway. The Journal of Immunology, 2003, 170: 5919 –5926. T umor cells proliferate under adverse host conditions dur- ing tumor progression and use several strategies to adapt their survival by blocking the action of key regulators of the immune response and circumventing anti-tumor defenses. Con- siderable progress has been made in understanding the mecha- nisms involved in the differentiation and functional regulation of killer cells either restricted or not by the MHC. However, it has become clear that the induction of cytotoxic response is essential but not sufficient to control tumor progression (1). Indeed, two major factors have limited the effectiveness of tumor-specific ther- apy: selection and activation of a nonsignificant population of tu- mor-reactive CTL and the generation of tumor variants that are not recognized by these specific CTL (2, 3). It is becoming apparent that to fully understand and manipulate killer cells therapeutically it will be necessary to characterize the death pathways and identify the molecular events that contribute to the control of cytotoxic effector-induced tumor cell death and the protective mechanisms used by tumor cells. In addition to the several known classical strategies used by tumor cells to escape immune surveillance, the phenomenon of tumor resistance to cell death is of major concern and the mech- anisms underlying it have yet to be fully elucidated. Thus, the search for new approaches to sensitize resistant cells to killing by cytotoxic effectors remains important in immunotherapy of cancer. CTL play a key role in immunosurveillance against tumors and infected cells (4). Two major pathways, triggered following TCR recognition of target-cell MHC/Ag complex, underlie T cell-me- diated cytotoxicity. The first one is a secretory pathway involving receptor-triggered exocytosis of pre-formed secretory granules containing granzymes and perforin. The second is based on recep- tor-induced surface expression of death receptor ligands on effec- tor cells, which cross-links the corresponding receptors (Fas, TRAIL receptor (TRAIL-R), 3 TNFR I-p55 (TNFRI)) on target cells (5, 6). Indeed, stimulation of these receptors with their re- spective ligand leads to their clustering and the formation of the death-inducing signaling complex. Recruitment and oligomeriza- tion of caspase-8 to the death-inducing signaling complex results in its proteolytic activation, which initiates a cascade of caspases, leading to apoptosis (6). Many genes have been reported to regu- late these death-associated events (7–9). The p53 tumor suppressor protein plays multiple roles in cell cycle control, differentiation, genomic stability, angiogenesis, and apoptosis (10, 11). Mutations that inactivate the p53 gene product are frequently found in human cancers (12, 13). In this regard, point mutations of the p53 gene are the most frequent genetic alterations in tumor cells (14, 15). These mutations, resulting in stabilization and accumulation of high level of a defective p53 product, result in loss of the cell cycle-regulating function of the *Laboratoire “Cytokines et Immunologie des Tumeurs Humaines”, Institut National de la Sante ´ et de la Recherche Me ´dicale Unite ´ 487, Institut Fe ´de ´ratif de Recherche 54 Institut Gustave Roussy, and † Centre National de la Recherche Scientifique-Unite ´ Mixte de Recherche 1598, Institut Gustave Roussy, Villejuif, France Received for publication October 11, 2002. Accepted for publication April 14, 2003. 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 by grants from Institut National de la Sante ´ et de la Recherche Me ´dicale and the association pour la recherche sur le cancer (Grants 5253– 5129). J.T. was supported by a fellowship from the Ligue Nationale Franc ¸aise de Recherche Contre Le Cancer. 2 Address correspondence and reprint requests to Dr. Salem Chouaib, Laboratory “Cytokines et Immunologie des Tumeurs Humaines”, Institut National de la Sante ´ et de la Recherche Me ´dicale Unite ´ 487, Institut Gustave Roussy, F-94805 Villejuif Cedex, France. E-mail address: chouaib@igr.fr 3 Abbreviations used in this paper: TRAIL-R, TRAIL receptor; TNFRI, TNFR I-p55; GrB, granzyme B; CMA, concanamycin A; LU, lytic unit; wt, wild type; PI, pro- pidium iodide. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00