[CANCER RESEARCH 64, 2865–2873, April 15, 2004] Effector Function of Human Tumor-Specific CD8 T Cells in Melanoma Lesions: A State of Local Functional Tolerance Alfred Zippelius, 1 Pascal Batard, 1 Verena Rubio-Godoy, 1 Gilles Bioley, 1 Danielle Lie ´nard, 1,2 Ferdy Lejeune, 2 Donata Rimoldi, 3 Philippe Guillaume, 3 Norbert Meidenbauer, 4 Andreas Mackensen, 4 Nathalie Rufer, 5,6 Norbert Lubenow, 7 Daniel Speiser, 1 Jean-Charles Cerottini, 3,6 Pedro Romero, 1,6 and Mikae ¨l J. Pittet 1 1 Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research and 2 Multidisciplinary Oncology Center, University Hospital (Centre Hospitalier Universitaire Vaudois), Lausanne, Switzerland; 3 Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; 4 Department of Hematology/Oncology, University of Regensburg, Regensburg, Germany; 5 Swiss Institute for Experimental Cancer Research and 6 National Center of Competence in Research Program on Molecular Oncology, Epalinges, Switzerland; 7 Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt University, Greifswald, Germany ABSTRACT Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differen- tiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART- 1-specific CD8 T cells that circulate in the blood display robust inflam- matory and cytotoxic functions, those that reside in tumor lesions (par- ticularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression. INTRODUCTION Naive CD8 T cells constantly travel from the blood to secondary lymphoid organs whereas dendritic cells capture antigens in nonlym- phoid peripheral tissues, migrate via afferent lymphatics to lymphoid organs, and present processed antigenic peptides to the naive T cells. After appropriate activation, antigen-primed T cells undergo prolifer- ation and differentiation (1, 2). Their progeny includes effector T cells, which gain the ability to migrate to peripheral tissues and display immediate effector function to contain invasive pathogens or cancer cells, as well as memory cells, which travel through secondary lymphoid organs and can generate a new wave of effector cells after re-encounter with antigen. At the site of immune attack, the functions exerted by effector CD8 T cells include the release of cytokines to mediate local inflammation (3) and deposition of cytotoxic granules at the vicinity of target-cell membranes to induce target-cell apoptosis (4, 5). In human cancer, a state of immune tolerance has been documented for both T-cell activation and function (6 – 8). However, current knowledge of the in vivo functions of human tumor antigen- specific T cells is mainly restricted to peripheral blood T cells and is largely inferred from the expression of phenotypic markers (9 –13). Studies on the functional activities of tumor antigen-specific T cells derived from human tumor lesions have been rarely performed and are in their infancy. Intriguingly, despite the coexistence of potentially tumor-reactive T cells and growing tumor cells, previous work on the efficacy of antitumor responses suggests adequate effector functions of T cells ex vivo and after in vitro stimulation in metastatic lymph nodes (LNs; Refs. 11, 14, 15). In this study, we present a comprehensive analysis of the function of CD8 T cells directed against the tumor differentiation antigen Melan-A/MART-1 (hereafter, Melan-A) in the following three dis- tinct body compartments: peripheral blood, metastatic LNs, and soft tissue/visceral metastases (referred to as nonlymphoid tissue metas- tases). Tissue samples were collected from a series of 61 stage III/IV HLA-A2 melanoma patients. We have chosen Melan-A as a model antigen because (a) Melan-A is expressed in the majority of HLA-A2 melanoma patients (16, 17), (b) immunological ignorance to Melan-A is overcome in the majority of these patients (14), and (c) high frequencies of Melan-A-specific T cells are readily detectable ex vivo (18, 19). We analyzed inflammatory and cytotoxic responses of Melan-A-specific CD8 T cells and compared them with those of T cells that control spreading of cytomegalovirus (CMV) infection in immune competent individuals. We provide evidence that Melan-A- specific CD8 T cells with high T-cell receptor (TCR) avidity are triggered in vivo and that they efficiently accumulate in tumor lesions (both within the lymphoid and nonlymphoid compartments) in the majority of melanoma patients. We show, however, that the tumor antigen-specific T cells in tumor lesions lack major T-cell effector functions (i.e., are functionally tolerant). Our findings highlight the importance of the microenvironment in shielding tumor cells from T-cell immune attack. MATERIALS AND METHODS Tissues and Cells. Peripheral blood, metastatic LNs, control LNs as diag- nosed tumor free by pathological examination, and nonlymphoid tissue me- tastases were obtained from a total of 61 HLA-A2 melanoma patients; clinical characteristics appear in Table 1. Informed consent was obtained from all patients. The study and treatment of patients were approved by the ethical committee of the Medical Faculty, University of Lausanne, and the Ludwig Institute for Cancer Research. Thirty-eight HLA-A2 healthy subjects were blood donors at the blood transfusion center in Lausanne, Switzerland, or in Greifswald, Germany. Mononuclear cells were purified and immediately fro- zen as described previously (14). Major Histocompatibility Complex/Peptide Multimers and Monoclonal Antibodies (mAbs). Phycoerythrin- or allophycocyanin-labeled HLA-A2/ peptide multimers (14) were synthesized around Melan-A 26 –35 A27L analog Received 9/30/03; revised 1/28/04; accepted 2/10/04. Grant support: V. Rubio-Godoy was supported in part by Grant SLK 782-2-1999 from the Swiss Cancer League, N. Rufer by a grant from the National Center of Competence in Research Program on Molecular Oncology, and A. Zippelius in part by the Emmy-Noether Program of the Deutsche Forschungsgemeinschaft. 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. Note: M. J. Pittet is currently at the Center for Molecular Imaging Research, Massa- chusetts General Hospital, Harvard Medical School, Building 149, 13th Street, Room 5029, Charlestown, MA 02129. A. Zippelius is currently at the Department of Oncology, University Hospital Zurich, 8091 Zurich, Switzerland. Requests for reprints: Mikae ¨l J. Pittet, Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Building 149, 13th Street, Room 5029, Charlestown, MA 02129. Phone: 1-617-726-5788; Fax: 1-617-726-5708; E-mail: mpittet@hms.harvard.edu or Pedro Romero, Division of Clinical Onco- Immunology, Ludwig Institute for Cancer Research, Ho ˆpital Orthope ´dique, Niveau 5, Aile est, Avenue Pierre Decker 4, 1005 Lausanne, Switzerland. E-mail: pedro.romero@ isrec.unil.ch. 2865 Research. on December 20, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from