[CANCER RESEARCH 62, 5664 –5667, October 15, 2002] Advances in Brief Identical T-cell Receptor Transcripts in Multiple Melanoma Metastases 1 David Schrama, Eva Fuchs, Eva-Bettina Bro ¨cker, Per thor Straten, and Ju ¨ rgen C. Becker 2 Department of Dermatology, Julius Maximilians-University, D-97080 Wu ¨rzburg, Germany [D. S., E. F., E-B. B., J. C. B.], and Tumor Cell Biology, Division of Cancer Biology, Danish Cancer Society, DK-2100 Copenhagen, Denmark [P. t. S.] Abstract Immune therapy for melanoma largely relies on preexisting T-cell responses. However, recent reports demonstrated the localized nature of such responses. Thus, we characterized the effect of immune therapy on the distribution of clonotypic T cells. To this end, we analyzed the T-cell receptor repertoire of multiple metastases of differentially treated mela- noma patients revealing oligoclonal T-cell responses and the occurrence of identical T-cell clones in several metastases. However, these findings were not limited to immune therapy but were also observed after chemother- apy, suggesting its similar impact on the distribution of T cells. Introduction The mainstay of immune surveillance is the circulation of cells, e.g., dendritic cells scan the body for the presence of new antigens and experienced T cells circulate to discover and eliminate appropriate antigen-bearing cells (1). In melanoma, however, the latter does not seem to be the case because clonotypic analysis of multiple metastases from untreated melanoma patients displayed a predominance of strictly localized T-cell clonotypes (2). Accordingly, it was demon- strated in a murine melanoma model that the reoccurrence of identical T cells in different metastatic lesions of the same mouse was virtually restricted to animals receiving IL-2 3 (3, 4). Hence, these findings raise the question of whether therapies modulating immune responses would also enhance the distribution of clonotypic T cells. Although the clinical and immunological effects of immunotherapy have been established (5), prior studies failed to address this question directly. Consequently, we analyzed and compared the TCR usage of TILs in multiple metastases from melanoma under therapy by a RT-PCR/ DGGE-based method. Patients and Methods Patients. A total of 31 metastases used for the experiments were collected from six patients (two females and four males) suffering from stage IV melanoma. These patients received immune and/or cytostatic therapies. The details of the therapies applied to each patient are provided in Table 1. The patients gave informed consent for parts of their surgically removed metastases to be used for immunological monitoring. TCR Clonotype Mapping by DGGE. The DGGE analysis used for clono- type mapping of the human TCR BV regions 1–24 has been described previously (6). Briefly, RNA was extracted using the “absolutely RNA RT- PCR” Kit (Stratagene, La Jolla, CA), and synthesis of cDNA was done with 1–3 g of total RNA, oligo(dT), and SuperScript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). cDNA was amplified by primers specific for BV families 1–24 and a constant region primer that contains a 50-bp GC-rich sequence at the 5'-end. DGGE analyses were performed in 6% polyacrylamide gels containing a gradient of urea and formamide ranging from 20% to 80% separating the amplicons due to their melting properties, which are based on their nucleotide sequence. To prove the identity of clonotypic T cells in different metastases, we applied comparable clonotype mapping. Amplicons of the same BV family of different metastatic lesions were ana- lyzed next to each other in a denaturing gradient gel, and transcripts resolving at similar positions in the gel were subjected to sequence analysis. Quantification of the TCR Repertoire. Quantification of the TCR reper- toire of TILs was performed by real-time PCR with Taqman technology. The previously given BV primers served as forward primers, 5'-GGTGTGGGAGA- TCTCTGCTTC-3' served as reverse primer, and 5'-carboxyfluorescein-ATG- GCTCAAACACAGCGACCTCGG-6-carboxytetramethylrhodamine-3' served as a probe for quantification of the TCR BV families. Primers and probe for the constant region of the TCR chain were designed with Primer Express software (Applied Biosystems, Weiterstadt, Germany) and are 5'-GTCAC- CCAGATCGTCAGCG-3' (forward primer), 5'-CACTGACCAGCACG- GCATAC-3' (reverse primer), and 5'-carboxyfluorescein-AGCAGACTG- TGGCTTCACCTCCGAGT-6-carboxytetramethylrhodamine-3' (probe). The primer efficiencies were determined as 78  15%. The relative expression of each TCR BV family was calculated in relation to the expression of mRNA coding for the constant part of the TCR. Results and Discussion Specific T-cell responses to melanoma, albeit not sufficient to control this tumor, have been repeatedly reported to occur spontane- ously (7, 8). Therefore, a major branch of therapies to treat melanoma is based on improving and/or eliciting immune responses to fight the tumor (9). Despite some encouraging successes, the overall response rate to these therapeutic interventions remained generally low. Several mechanisms have been proposed to explain the lack of efficiency of cellular immune responses, e.g., loss of target antigens on tumor cells, signal transduction defects in effector cells, or impaired tumor homing of specific T cells. The latter hypothesis could be readily addressed by analysis of the T-cell repertoire usage of TILs. Nevertheless, only a few research groups have investigated the T-cell clonality of TILs, and most of them focused on overexpressed TCR variable regions (10). Only recently, our research group analyzed the complete T-cell repertoire of multiple metastases of two untreated stage IV melanoma patients, revealing the predominance of strictly localized T-cell clono- types within the different metastases (2). This finding suggested a limited capacity of tumor-specific T cells to recirculate and infiltrate different tumor manifestations. Thus, we asked ourselves whether this limitation could be improved by therapies stimulating the immune response. To study the effect of immune modulation on the distribu- tion of identical T cells, we analyzed multiple metastases by the same highly sensitive technique based on RT-PCR and DGGE that was used by us previously (6). This technique allows us to distinguish between oligoclonal infiltrates, represented by distinct bands in the denaturing gel, and polyclonal infiltrates, indicated by a smear. To this end, a total of 25 metastatic lesions from four patients receiving different forms of immune therapy were investigated. The summary of results from these analyses and details of the therapies administered are provided in Table 1. However, to allow a clear presentation of the Received 8/9/02; accepted 8/28/02. 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 Supported in part by Deutsche Krebshilfe Grant 10-1845-BeI (to D. S.) and Wilhelm- Sander-Stiftung Grant 2000.056.1. 2 To whom requests for reprints should be addressed, at Department of Dermatology, University of Wu ¨rzburg, Josef-Schneider-Strasse 2, Building 13, D-97080 Wu ¨rzburg, Germany. Fax: 49-931-201-26700; E-mail: becker-jc.derma@mail.uni-wuerzburg.de. 3 The abbreviations used are: IL-2, interleukin 2; BV,  variable; DGGE, denaturing gradient gel electrophoresis; TCR, T-cell receptor; TIL, tumor-infiltrating lymphocyte; RT-PCR, reverse transcription-PCR. 5664 Research. on March 15, 2016. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from