Spontaneous CD8 T Cell Responses against the Melanocyte Differentiation Antigen RAB38/NY-MEL-1 in Melanoma Patients This information is current as Senta M. Walton, Marco Gerlinger, Olga de la Rosa, Natko of October 2, 2021. Nuber, Ashley Knights, Asma Gati, Monika Laumer, Laura Strauss, Carolin Exner, Niklaus Schäfer, Mirjana Urosevic, Reinhard Dummer, Jean-Marie Tiercy, Andreas Mackensen, Elke Jaeger, Frédéric Lévy, Alexander Knuth, Dirk Jäger and Alfred Zippelius J Immunol 2006; 177:8212-8218; ; Downloaded from doi: 10.4049/jimmunol.177.11.8212 http://www.jimmunol.org/content/177/11/8212 http://www.jimmunol.org/ References This article cites 42 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/177/11/8212.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Spontaneous CD8 T Cell Responses against the Melanocyte Differentiation Antigen RAB38/NY-MEL-1 in Melanoma Patients1 Senta M. Walton,2* Marco Gerlinger,2* Olga de la Rosa,* Natko Nuber,* Ashley Knights,* Asma Gati,* Monika Laumer,† Laura Strauss,* Carolin Exner,* Niklaus Scha¨fer,* Mirjana Urosevic,‡ Reinhard Dummer,‡ Jean-Marie Tiercy,§ Andreas Mackensen,† Elke Jaeger,¶ Fre´de´ric Le´vy,ʈ Alexander Knuth,* Dirk Ja¨ger,3# and Alfred Zippelius3,4* The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro Downloaded from sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-150–58 exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 0201؉ melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of naturalءT cells from HLA-A processing of RAB38/NY-MEL-150–58 peptide. Accordingly, monoclonal RAB38/NY-MEL-150–58-specific T cell populations were /capable of specifically recognizing HLA-A2؉ melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2 RAB38/NY-MEL-150–58 multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell http://www.jimmunol.org/ response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY- MEL-150–58 is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials. The Journal of Immunology, 2006, 177: 8212–8218. ritical advances have been made toward understanding nogenic epitopes derived from the two major groups of nonmu- the magnitude and characteristics of cellular immune re- tated shared Ags, namely cancer-testis (i.e., NY-ESO-1, MAGE, sponses in cancer patients (for review, see Refs. 1 and 2). and SSX) and differentiation Ags (i.e., Melan-A, tyrosinase, and C by guest on October 2, 2021 In particular, the discovery of tumor Ags and the precise charac- NY-BR-1). Melanocyte differentiation Ags are expressed in nor- terization of their immunogenic sequences involved in T cell me- mal melanocytes and melanomas, and their function in melano- diated anti-tumor immunity (3) have allowed, for the first time, to genesis is frequently associated with pigment production. systematically analyze anti-tumor T cell responses and have sub- There is considerable evidence that melanocyte differentiation stantially progressed the development of specific cancer vaccines Ags presented to the immune system of melanoma patients can (4–6). Vaccination trials are focused mainly on the use of immu- overcome tolerance and induce T cell-based immunity (7–9). The relative immunogenicity of Melan-A/MART-1 is thought to be the highest of this group of Ags. We could demonstrate previously that *Medical Oncology, Department of Internal Medicine, University Hospital Zurich, Zurich, Switzerland; †Department of Hematology/Oncology, University of Regens- Melan-A/MART-1-specific CD8 T cells with potential tumoricidal burg, Regensburg, Germany; ‡Dermatologische Klinik, University Hospital Zurich, capacity are selectively primed in vivo and are directly detectable Zurich, Switzerland; §Transplantation Immunology Unit, University Hospital, Geneva, Switzerland; ¶II Medizinische Klinik, Krankenhaus Nordwest, Frankfurt, by fluorescent HLA-A2/peptide multimeric constructs (thereafter Germany; ʈLudwig Institute for Cancer Research, Lausanne Branch, University of multimers) even in circulating lymphocytes from melanoma pa- Lausanne, Epalinges, Switzerland; and #Medizinische Onkologie, National Center for tients (10). Apart from the Melan-A/MART-1-specific CD8 T cell Tumor Diseases, University Hospital Heidelberg, Germany repertoire, but similarly to the apparent paucity of ex vivo detect- Received for publication December 15, 2005. Accepted for publication September 22, 2006. able CD8 T cell responses to cancer testis Ags, multimers can only rarely be used to readily identify CD8 T cells specific for other 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 differentiation Ags, e.g., tyrosinase (11, 12). Notably, the applica- with 18 U.S.C. Section 1734 solely to indicate this fact. tion of CD8 T cell epitopes derived from melanocyte differentia- 1 This work was supported in part by a Swiss National Science Foundation special tion Ags in cancer vaccination trials has demonstrated favorable program and a grant from the Cancer Research Institute/Ludwig Institute for Cancer effects on disease progression in individual patients with only very Research Cancer Vaccine Collaborative; the Terry-Fox, Hanne-Liebermann, and Claudia-von-Schilling Foundation; and the UBS Wealth Management. A.Z., A.G., limited toxicity (13–15). Recently, we reported on the melanocyte and S.W. were supported in part by the Emmy-Noether Program (Zi685-2/3) of the differentiation Ag RAB38/NY-MEL-1 (thereafter RAB38) (16), Deutsche Forschungsgemeinschaft. F.L. was supported by the Swiss National Foun- dation and the Cancer Research Institute. that is highly expressed in normal melanocytes and melanoma tis- sues but not in other normal tissues or cancer types. Importantly, 2 S.M.W. and M.G. contributed equally to this work. RAB38 is immunogenic leading to spontaneous Ab responses in a 3 D.J. and A.Z. contributed equally to this work. large proportion of melanoma patients (17). In this study, we dem- 4 Address correspondence and reprint requests to Dr. Alfred Zippelius, Medical On- cology, Department of Internal Medicine, University Hospital Zurich, Raemistrasse onstrate for the first time the identification of a naturally occurring 100, CH-8091 Zurich, Switzerland. E-mail address: [email protected] RAB38-specific CD8 T cell response through the association of in Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 8213 vitro sensitization with overlapping peptides and in silico predic- cells were then cocultured overnight with the target cells in the presence of tion of potential HLA class I ligands. Tumor cell recognition and 10 ␮g/ml brefeldin A (GolgiPlug; BD Pharmingen) and then analyzed ␥ in vitro proteasomal digestion assays document natural processing using the intracellular IFN- assay. and presentation of the HLA-A2 restricted RAB3850–58 peptide. RNA extraction, quantitative real-time PCR, and Western blot We show that, in the T cell repertoire of melanoma patients, analysis RAB38 -specific CD8 T cells exist that can even be detected 50–58 RNA extraction, RAB38-specific quantitative real-time PCR, and Western spontaneously applying multimer technology and are endowed blot assays to detect circulating RAB38-specific Ab responses were per- with sufficient avidity to lyse RAB38-expressing tumor cells. Its in formed as described previously (17). To analyze the proteasome subunits, vivo immunogenicity reflected by the presence of cellular and hu- rabbit anti-hLMP-2 (b1i; PW8345), rabbit anti-hLMP-7 (b5i; PW8355), moral anti-RAB38 immune responses in cancer patients opens the rabbit anti-hMECL-2 (b2i; PW8350), rabbit anti-hb5 (PW8895), mouse avenue for developing Ag-specific immunotherapy targeting anti-hb1 (Y; PW8140), and mouse anti-hb2 (Z; PW8145) were purchased from Affiniti