Melanoma Antigens Dual Recognition of a Human Cytotoxic T-Cell Clone for Updated Version

It is well known that tumor-specific CTLs have a crucial role in the elimination of tumors and that different CTL populations recognize tu mor antigens in MHC-restricted and MHC-unrestricted manners. We have established two aßCTL clones that recognize melanoma antigens in both human lymphocyte antigen (HLA)-A2-restricted and HLA-unrestricted manners. Flow cytometry analysis showed that these CTL clones carry CD3, CDS, and aßT-cell receptor (TCR) and express low levels of CD56. In contrast, these CTL clones do not express CD16, indicating that they do not contain natural killer cells. TCR analysis of these CTL clones using an anchored PCR method revealed that each clone carries a single aßTCR. Both CTL clones contained the same Va and \'ßgene segments although they carried different Ja and .Ißgene segments. Taken together, these results confirm that CTL clones that carry a single aßTCR recog nize melanoma antigens in both HLA-A2-restricted and HLA-unrestricted manners. It is strongly suggested that the dual recognition of these CTL clones for the melanoma antigens is mediated by TCRs. The novel mechanism for antitumor immunity by these CTLs may be important in the effective elimination of tumors in vivo. INTRODUCTION Tumor cells are eliminated by specific CTLs that carry aßTCRs4 and by NK cells (1, 2). Tumor-specific CD8+ CTLs recognize a peptide presented by MHC class I molecules (3-5). In contrast, the recognition of NK cells is not restricted by MHC molecules, and molecules recognized by NK cells remain unknown. CD8 + CTLs can eliminate tumor cells effectively because of their specific immune recognition. Recent studies identified HLA class I binding peptides recognized by melanoma-specific CTLs (6-8). On the other hand, tumor cells often lose surface expression of MHC class I molecules (9-11). CD8+ CTLs fail to kill tumor cells lacking MHC class I molecules although they are eliminated effectively by NK cells (12). Previous studies showed the presence of T cells that have NK activity (13, 14). These T cells also killed target cells losing MHC class I expression effectively, such as K562 cells ( 15). Thus, MHC-restricted CTLs and NK cells or MHC-unrestricted CTLs together play a com plementary role in tumor immunity. Previous studies suggested the presence of a T-cell population that recognizes tumor antigens or alloantigens in both the HLA class I-restricted and HLA-unrestricted manners (16, 17). How ever, it remains possible that T-cell clones contain more than one Received 12/12/95; accepted 3/19/96. 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 study was supported by a Grant-in-Aid for Scientific Research in Priority Areas from the Ministry of Education. Science. Sports, and Culture, and a grant for a Compre hensive New 10-Year Strategy of Cancer Control from the Ministry of Health and Welfare, the Government of Japan. 2 Present address: Department of Dermatology, School of Medicine, Shinshu Univer sity, Matsumoto, Nagano 320, Japan. 3 To whom requests for reprints should be addressed. 4 The abbreviations used are: TCR. T-cell receptor; NK, natural killer; mAb. mono clonal antibody; IL. interleukin; MFI. mean fluorescence intensity; HLA, human lym phocyte antigen. T-cell population or NK cells because the clonality of each T-cell clone was not confirmed by analyses of TCRs as well as cell surface markers in these studies. We have investigated such a T-cell population in detail and present evidence that a single T-cell clone recognizes melanoma antigens in both HLA-A2-restricted and HLA-unrestricted manners. MATERIALS AND METHODS Cells. Melanoma cell lines DM6, DM92, DM93, DM122, DM208, and DM252 were established previously (18, 19). HST2 cells were kindly provided by Dr. N. Sato (Sapporo Medical College, Sapporo, Japan). Other tumor cell lines were provided by the Japanese Cancer Research Resources Bank. These cell lines were cultured in RPMI 1640 containing 10% FCS. Monoclonal Antibodies. Hybridoma cells secreting W6/32 anti-HLA class I, L243 anti-HLA class II, BB7.2 anti-HLA-A2, MA2.1 anti-HLA-A2, OKT3 anti-CD3, OTK4 anti-CD4, and OKT8 anti-CD8 mAbs were purchased from the American Type Culture Collection. Anti-CD56, anti-CD 16. and WT31 anti-aj3 TCR mAbs were purchased from Becton Dickinson (Mountain View, CA). HO-2 anti-HLA-A2 mAb was kindly provided by Dr. S. Ferrone (New York Medical College, Valhalla, NY). Cloning of HLA-A2 Genes. HLA class I genes were cloned from DM252 cells as described previously (20). The genes encoding HLA-A2 were selected by RFLP. The coding region of HLA-A2 genes was sequenced as described previously (21). Gene Transfer of the HLA-A*0201 Gene into DM92 Cells. The HLAA*0201 gene was transfected by electroporation with neomycin-resistant genes into HLA-A2-negattve DM92 cells. Cells were added to each well of a 96-well, flat-bottomed microtiter plate (Nunc, Roskilde, Denmark). After selection by G418, resistant cells were isolated from separate wells. Surface expression of HLA-A2 was detected by flow cytometry with anti-HLA-A2 mAb. DM92 cells expressing HLA-A*020l (DM92-A*0201) were cultured in RPMI 1640 containing 10% FCS. Flow Cytometry Analysis. About 2 X IO5 cells were incubated with 50 /JLÌ of properly dilute mAb for 30 min on ice. After two washes with PBS containing 2% PCS, the cells were incubated with 50 /*! of FITC-conjugated F(ab')2 goat antimouse immunoglobulin (Silenus Laboratories Pty. Ltd., Haw thorn, Australia) at a dilution of 1:40 for 30 min on ice. Afterward, the cells were washed three times and resuspended in PBS containing 2% FCS; then the fluorescence intensity of the cells was measured by FACS. Generation of CTL Clones. Peripheral blood lymphocytes from a mela noma patient (no. 252) were stimulated with autologous melanoma cell line DM252S1 three times for 4 weeks. The cells were cultured in RPMI 1640 containing 100 units of recombinant human IL-2. After specific cytotoxicity against DM252S1 cells was detected, CTL clones were established by using a limiting-dilution technique. T-cell blasts were placed in a 96-well, roundbottomed plate (Nunc) at 0.6 cell/well with 1 X IO3 irradiated DM252S1 cells, 100 fil of RPMI 1640 supplemented with 10% FCS. and 100 units of recom binant human IL-2. Cells were fed with the medium every 2-3 days. CTL Assay. Target cells (5 x IO5) were incubated for 60 min with 100 fid of Na,5lCrO4 in PBS and washed three times with RPMI 1640 containing 10% FCS. Labeled target cells (5 X lOVwell) were added to a 96-well, round-bottomed microtiter plate (Nunc), and effector cells at an E;T ratio of 2:1 were added. After the mixtures were incubated for 4 h at 37°C, the supematants were collected and analyzed with a gamma counter. The sponta neous "Cr release (cpm spn) was determined by measuring the cpm in the