[CANCER RESEARCH 55. 685-690, February 1, 1995] In Vivo Local Expansion of Clonal T Cell Subpopulations in Renal Cell Carcinoma1 Catherine Gaudin, Pierre-Yves Dietrich, Sylvie Robache, Maryvonne Guillará, Bernard Escudier, Marie-JoséeTerrier Lacombe, Anil Kumar, Frédéric Triebel, and Anne Caignard2 Laboratoire d'Immunologie Cellulaire, INSERM U333 ¡C.G., S. R., M. G., A. K., F. T., A. C.¡,Unit d'Immunothérapie [P-Y. D., B. E.J, and Départementde Pathologie ¡M.J. T. LI, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France, and Division d'Oncologie, Hôpital Universitaire. 1211 Genève 14, Switzerland [P-Y. D.J ABSTRACT Renal cell carcinoma (RCC) is one human tumor to which the immune response may control the growth of tumor cells. These tumors are infil trated by a large mononuclear infiltrate mainly composed of T lympho cytes. To characterize the lymphocytes infiltrating RCC, we analyzed the molecular structure of the T cell receptor (TCR) a and ßchains in tumor and paired peripheral blood lymphocytes from a series of 6 untreated patients. We first determined Va and Vßgene segment usage by PCR using a panel of V specific oligonucleotide primers (Val-w29 and V/ÃŒI- w24). A highly diverse usage of TCR Va and Vßgeneusage was observed in 5 of 6 tumors. In addition, the few tumor overexpressed Vß specificities detected by reverse transcription-PCR were shown to contain minor T cell expansions. Strikingly, 1 of the 6 tumor studied displayed a skewed TCR repertoire with Vß4transcript representing 25% of the TCR signals. Clonality of the tumor overexpressed Vßtranscripts was analyzed by CDR3 size distribution analysis. In the particular tumor displaying a biased repertoire large expansions of T cell subpopulations were detected (particularly in Vß4)specifically at the tumor site. Such T cells may be expanded locally in response to tumor antigens. antibody-like structure. CDR regions 1, 2, and 3 have accordingly been defined for TCR molecules (11). CDR3 regions are encoded by the hypervariable V-J or V-D-J junctions and are essential for binding to the antigenic peptide. The expression of unique rearranged TCR gene products determines the specificity of a given T cell (12). Identification of recurrent TCR transcripts (same CDR3) in T cell populations indicates antigen driven expansion of the corresponding T cells. Thus, the direct analysis of the molecular structure of TCR polypeptides expressed by TIL is one way to study how tumor cells modulate the T cell repertoire. Using different PCR approaches, we analyzed in situ the fine molecular structure of TCR a and ßchains in TIL and paired PBL from 6 RCC patients. A highly diverse repertoire was found in 5 of 6 tumors, whereas it was strongly skewed in the last tumor. Dramatic clonal expansions of few T cell subsets were indeed identified in the last patient. Such data strengthen the view that antigen driven T cell expansion may occur locally and contribute to the control of the tumor evolution at least in some RCC. INTRODUCTION The recent characterization of tumor specific antigens recognized by cytolytic T lymphocytes in melanoma (1-5) is an important step to better understand how the immune system interacts with tumor cells. Although much less studied than melanoma, RCC3 is another human cancer for which the immune system is thought to play a role in the control of malignant cell growth. Indeed, an overall response rate of about 20% is achieved with systemic interleukin 2 administration, which is now the first line treatment of metastatic RCC (6). Finally, human RCC are infiltrated by numerous lymphocytes, mainly CD3+, a/ß+ T cells and few tumor specific T cell lines, have been recently described (7-9). These observations suggest that an antitumor re sponse mediated by T cells may be taking place in RCC. Mature T cells specifically recognize antigenic peptides presented by MHC molecules through their heterodimeric surface receptor (TCR) which associates the a and the ßpolypeptides. The specific recognition is dependent upon interaction between the MHC/peptide complex and the variable region of TCR molecules. During T cell differentiation, unique variable region genes are created by recombi nation of variable (V), diversity (D), and joining (J) segments for the ß locus and V and J segments for the a locus. The joining of a random combination of these segments as well as the pairing of the two chains generate combinatorial diversity which is greatly increased by impre cise V-D-J joinings and the addition of N region nucleotides (10). The three dimensional structure of the TCR backbone is presently un known, but it is likely that the TCR antigen binding site has an Received 8/22/94; accepted 11/21/94. 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. 1This work was supported in part by Association pour la Recherche contre le Cancer Grant 2038. 2 To whom requests for reprints should be addressed. 'The abbreviations used are: RCC, renal cell carcinoma; TIL, tumor infiltrating lymphocytes; PBL, peripheral blood lymphocytes; CDR, complementary determining region; nt, nucleotides. MATERIALS AND METHODS Patients and Samples. Six previously untreated patients with RCC were studied. There were 4 males and 2 females with a mean age of 55 years (range, 42 to 70 years). All patients underwent radical nephrectomy with regional lymphadenectomy. Clear cell carcinoma was the histológica! diagnosis in 5 cases (including 2 tumors with papillary structures), whereas the last tumor corresponded to a granular cell variant with eosinophilic staining cytoplasm (patient 5). Regional lymph nodes involvement by tumor cells was histologi- cally demonstrated in 2 patients. At the time of nephrectomy, 4 patients presented with métastasesto lungs (3 cases) and/or to the liver (2 cases). Patients 2 and 4 were free of métastases. For each patient, samples were collected within the tumor, in normal kidney, and in blood. Tissue biopsies were washed with 0.9% NaCl and immediately frozen in liquid nitrogen for further RNA extraction. Blood samples (30 ml) were collected at the time of nephrectomy. In addition, lymph nodes tissues were available for patients 1 and 2. Informed consent for blood and tissue samples was obtained from all patients. TCR Repertoire Analysis. TCR Va and Vßgene segment usage was determined by reverse transcription-PCR using a panel of experimentally controlled primers specific for the 29 Va and 24 Vß subfamilies (13, 14). In brief, total RNA was prepared from tumor, lymph nodes, and PBL (0.2-0.5 g tissue or 5 X IO6 cells) using RNAzol B and converted to cDNA by standard methods using reverse transcriptase and an oligodeoxythymi- dylate primer. These cDNAs were amplified using the panel of 5' sense primers as well as a 3' antisense primer for the Cet and Cßregion. The specificity of the amplified products was assessed by the length of the PCR products after Southern blotting and hybridization with a labeled Ca and Cßoligonucleotide (15). Comparative analysis of each V product between the different samples was achieved by densitometric analysis of the signals on the autoradiographies (16). Cloning and Sequencing of V Transcripts. The procedure used for clon ing and sequencing of Vß transcripts has been described previously (13, 14). Briefly, sequences of the primers for cloning were Vßl45'-GGGCTCGG- CTTAAGGCAGACCTAC-3', Vßl7 5'-CTGCTGAATTTCCCAAAGAGG- GCC-3', and Vß85'-CCATGATGCGGGGACTGGAGTTGC-3'. The ampli fications were performed in 2 rounds of 30 cycles. After ethanol precipitation the amplified products were separated on a 2% agarose gel and purified by absorption on glass beads (Gene Clean Bio 101, Inc., La Jolla, CA). The 685 Research. on December 9, 2021. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from