Functional and Molecular Characterization of a KIR3DL2/P140 Expressing Tumor-Specific Cytotoxic T Lymphocyte Clone Infiltrating a Human Lung Carcinoma
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Oncogene (2003) 22, 7192–7198 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Functional and molecular characterization of a KIR3DL2/p140 expressing tumor-specific cytotoxic T lymphocyte clone infiltrating a human lung carcinoma Guillaume Dorothe´ e1, Hamid Echchakir1,Be´ atrice Le Maux Chansac1, Isabelle Vergnon1, Faten El Hage1, Alessandro Moretta2, Armand Bensussan3, Salem Chouaib1 and Fathia Mami-Chouaib*,1 1Laboratoire Cytokines et Immunologie des tumeurs Humaines, INSERM U487, Institut Gustave Roussy, F-94805 Villejuif, Cedex, France; 2Dipartimento di Medicina Sperimentale, Sezione di Istologia, and Centro di Eccellenza per la Ricerca Biomedica, Universita` di Genova, 16132 Genova, Italy; 3INSERM U448, Faculte´ de Me´decine de Cre´teil, 94010 Cre´teil, Cedex, France T lymphocytes infiltrating a human lung carcinoma receptors, which recognize ligands on target cells, and stimulated in vitro with autologous tumor cell line showed inhibitory receptors specific for MHC class I molecules. a TCRVb13.6 þ T-cell expansion. This subset was isolated Ag/MHC complexes serve as activating signals for T using TCRVb-specific antibody and several T-cell clones cells and are recognized by TCR/CD3 complex (Zin- were generated. All these clones expressed a unique Vb13.6- kernagel and Doherty, 1974; Townsend et al., 1986). Jb2.7 TCR with the same junctional region strongly Reciprocally, inhibitory signals can be provided by suggesting that they derived from the same cell. They were engagement of a variety of cell surface inhibitory CD8 þ /CD28À and expressed the MHC class I binding receptors. These include the Ig-like killer inhibitory killer cell Ig-like receptor (KIR)3DL2/p140, but not receptors (KIRs) specific for HLA-A, -B, -C, the CD94/ KIR3DL1/p70, KIR2DL1/p58.1 and KIR2DL2/3/p58.2. NKG2A heterodimer, specific for HLA-E and the Sequence analysis indicated that KIR3DL2/p140 cDNA leukocyte Ig-like receptor 1/Ig-like transcript 2 (LIR1/ was identical to the previously reported 3DL2*002 allele ILT2), characterized by a broad specificity for different except for two nucleic acid substitutions. Functional HLA class I molecules (Moretta et al., 1994; Moretta studies showed that KIR3DL2/p140 þ CTL secrete a et al., 1995; Colonna et al., 1997). KIRs are expressed on significant level of IFNc and mediate an HLA-A2- a subset of human CD8 þ T cells and their interaction restricted cytotoxicity against the autologous and some with their specific ligands induces inhibition of cell- allogeneic tumor cells but not towards the autologous mediated lysis of target cells bearing the appropriate EBV-B cells. Strikingly, both the lytic and the cytokine HLA class I allotype (Ferrini et al., 1994; Phillips et al., secretion activities induced upon specific cell interactions 1995; Ikeda et al., 1997; Bakker et al., 1998). Ligation of were unaffected by anti-KIR3DL2/p140 antibody. In KIRs results in tyrosine phosphorylation of the im- addition, crosslinking KIR3DL2/p140 molecules on CTL munoreceptor tyrosine-based inhibition motifs (ITIM) did not result into the modification of cytotoxicity and in their cytoplasmic domain, leading to the recruitment cytokine production triggered by anti-CD3 antibody. of SH2 domain-containing tyrosine phosphatase 1 These results strongly suggest that, as opposed to distinct (SHP-1) and inhibition of cell-mediated cytotoxicity KIR expressed by CTL, the in vitro KIR3DL2/p140 and cytokine production (Burshtyn et al., 1996; Fry engagement does not result into inhibitory (nor activatory) et al., 1996). effects on tumor-specific CTL. It has been previously reported that CD94/NKG2A Oncogene (2003) 22, 7192–7198. doi:10.1038/sj.onc.1206627 heterodimer (Le Drean et al., 1998; Noppen et al., 1998; Speiser et al., 1999) and KIRs, in particular KIR2DL1/ Keywords: TIL; KIR3DL2; CTL; TCR p58.1 and KIR2DL2/3/p58.2 inhibitory receptors for HLA-C (Ikeda et al., 1997; Guerra et al., 2000; Gati et al., 2001), were expressed by tumor-specific CTL clones and were able to inhibit their lytic activity Introduction towards the autologous tumor cells. Regarding KIR3DL2/p140 inhibitory receptor for HLA-A, no The function of effector lymphocytes is regulated by a data related to CTLs are available and its expression balance between signals transmitted by activating and function on tumor infiltrating T lymphocytes (TILs) has never been reported. Solid tumors, including *Correspondence: FMami-Chouaib, U487 INSERM, Institut non-small cell lung carcinomas (NSCLC), are often þ þ Gustave Roussy, 39 rue Camille-Desmoulins, F-94805 Villejuif, infiltrated by TILs, most of which are TCRa/b ,CD8 , France; E-mail: [email protected]. CD28À (Echchakir et al., 2000 and unpublished data). Expression and function of KIR3DL2/p140 on TIL G Dorothe´e et al 7193 We have previously demonstrated a T-cell response in including 99% TCRVb13.6 þ was generated. This cell NSCLC and isolated several tumor-specific CTL clones line was cloned by limiting dilution and a panel of from TILs infiltrating a human lung carcinoma where T-cell clones, expressing a unique Vb13.6-GGA-Jb2.7 they appeared clonally expanded (Echchakir et al., 2000, sequence, was isolated (data not shown). This result 2001). In the present study we have isolated from the strongly suggests that these clones are derived from the same patient TILs, several tumor-specific CTLs expres- same cell. Immunofluorescence analysis of one selected sing KIR3DL2/p140 receptor and a unique Vb13.6- clone, Heu33, indicated that it expressed CD3 complex Jb2.7 TCR rearrangement. The functional effects of the (data not shown) associated to a TCRa/b (Figure 1). engagement of KIR3DL2/p140 on cytokine release and Furthermore, this clone was TCRb13.6 þ /CD8 þ / cytotoxic functions of these effector T cells were CD56 þ /CD28À and consistently expressed significant investigated. level of p140-KIR3DL2 determined with both Q66 and AZ158 mAbs (Figure 1). In contrast, expression of CD94 and NKG2A was low and KIR3DL1/p70, KIR2DL1 (p58.1/CD158a) and KIR2DL2/3 (p58.2/ Results CD158b) inhibitory receptors were not expressed (Figure 1). The same phenotype was obtained with the Isolation and characterization of Heu33 CTL clone parental H4C TIL cell line. In contrast, the TCRb13.6À The IGR-Heu tumor cell line was derived from a fraction did not express KIR3DL2/p140 (data not NSCLC tumor biopsy (Asselin-Paturel et al., 1998). shown). Mononuclear cells that infiltrated the primary tumor were isolated by Ficoll–Hypaque density gradient Molecular characterization of KIR3DL2/p140 receptor centrifugation and stimulated with irradiated IGR-Heu expressed by Heu33 CTL clone cells, irradiated autologous EBV-transformed B cells and IL-2. On day 15, the responding lymphocytes were To determine whether the cDNA encoding KIR3DL2/ analysed by flow cytometry and were shown to include p140 molecule expressed by Heu33 CTL clone corre- 97% CD3 þ and 49% TCRVb13.6 þ cells. The sponds to one of the 12 already described alleles TCRVb13.6 þ TILs were immunoselected using specific (Shilling et al., 2002), reverse transcription–polymerase monoclonal antibody (mAb), and a cell line (H4C) chain reaction (RT–PCR) was performed and the Figure 1 Flow cytometry analysis of Heu33 CTL clone. Briefly, 3 Â 105 cells were stained with the indicated receptor-specific mAbs (gray fill) or isotype-matched control (without fill). For KIR3DL2/p140 staining, two mAbs Q66 (anti-KIR3DL2/p140) and AZ158 (recognizing both KIR3DL2/p140 and KIR3DL1/p70) were used and were indicated between parentheses Oncogene Expression and function of KIR3DL2/p140 on TIL G Dorothe´e et al 7194 resulting product was cloned into pcDNA3.1 plasmid and sequenced. RT–PCR performed on RNA derived from the T-cell clone, using a set of primers able to amplify the entire cDNA encoding KIR3DL2 (Bagot et al., 2001), showed a 1.4 kb size product (data not shown), similar to that previously described (Pende et al., 1996; Bagot et al., 2001; Gardiner et al., 2001). Furthermore, nucleic acid sequence analysis indicated that it was identical to one of the previously reported alleles, the 3DL2*002 allele (Pende et al., 1996; Shilling et al., 2002), except for two nucleotide replacements in exon 1 which may represent an additional polymorph- ism in the KIR3DL2/p140 gene. Figure 2 shows the amino-acid sequence alignment of Heu33 KIR3DL2 together with the 3DL2*001 (Colonna and Samaridis, 1995) and the 3DL2*002 (Pende et al., 1996) encoded proteins. The two amino-acid substitutions in the leader peptide sequence are shown (Figure 2). Heu33 CTL clone recognizes autologous and allogeneic lung tumor cell lines Heu33 T-cell clone was assessed for its cytotoxic activity against IGR-Heu autologous tumor and EBV-B cells as well as a panel of allogeneic tumor cell lines including K562 and Daudi in conventional 51Cr release assay. This clone displayed a high cytotoxic activity towards the autologous IGR-Heu tumor cell line as well as some allogeneic cells, including IGR-Pub and Ludlu HLA- Figure 3 Analysis of specificity and HLA restriction element of A2 þ NSCLC cell lines, but not against the autologous Heu33 CTL clone (a) Cytotoxic activity of Heu33 T-cell clone against IGR-Heu autologous tumor cell line, IGR-Pub, IGR-B1, EBV-B cells, Daudi (data not shown) and K562 Ludlu, H1155 and SK-MES selected allogeneic NSCLC tumor cell (Figure 3a). It should however be mentioned that the lines, Heu-EBV autologous lymphoblastoid cell line and K562 cytotoxicity towards K562 varied from one experiment targets. E/T ratios were as indicated. One representative experi- to another (0–30% of lysis at 30 : 1 E/T ratio) and ment out of three is shown (b) Cytotoxic activity of Heu33 T-cell depended upon the effector cell status for IL-2-induced clone against Ludlu allogeneic tumor cell line.