Evidence for in situ expansion of diverse antitumor-specific cytotoxic T lymphocyte clones in a human large cell carcinoma of the lung

Hamid Echchakir, Isabelle Vergnon, Guillaume Dorothée, Dominique Grunenwald1, Salem Chouaib and Fathia Mami-Chouaib

Laboratoire Cytokines et Immunologie des tumeurs Humaines, U487 INSERM, Institut Gustave Roussy, 94805 Villejuif Cedex, France
1 Département Thoracique, Institut Montsouris, 75013 Paris, France

Correspondence to: F. Mami-Chouaib


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have isolated several cytotoxic T lymphocyte (CTL) clones from lymphocytes infiltrating a human large cell carcinoma (LCC) of the lung. All these clones were found to express a CD3+, TCR{alpha}ß+, CD8+, CD4, CD28 phenotype. According to their TCR ß chain variable region expression, they were divided in three major groups. The first group, including the majority of the clones, expressed a unique Vß3–Jß1.2 TCR. The second group expressed a Vß22–Jß1.4 rearrangement and the third group, including only two clones, expressed a Vß8–Jß1.5 TCR. Functional studies showed that all the CTL clones mediated a high cytotoxic activity against the autologous tumor cell line. While the Vß3+ clones showed a weak lysis against few allogeneic non-small cell lung cancer (NSCLC) tumor cell lines, Vß8+ and Vß22+ T cell clones were able to kill a panel of allogeneic NSCLC tumor cell lines. Cytotoxicity-blocking experiments using specific mAb indicated that, while the Vß3+ and Vß22+ CTL clones were HLA-A2 restricted, the Vß8+ clones appeared HLA-B or -C restricted. TCR transcripts expressed in the cloned cells were determined by CDR3 size and sequence analyses, and compared to those present in fresh tumor tissue. Interestingly, our studies demonstrated that the CTL clones identified in vitro were selectively expanded in vivo at the tumor site as compared to autologous peripheral blood lymphocytes. These results further provide evidence that an immune response may take place in NSCLC and that effector T cells may contribute to tumor regression.

Keywords: cytotoxic T lymphocyte, non-small cell lung cancer, TCR, tumor-associated antigen, tumor-infiltrating lymphocyte


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lung cancer is one of the leading causes of malignancy-related death in Europe and the US because of its frequency, and its relative resistance to currently available chemotherapy and radiation therapy. Therefore, the development of a specific immunotherapy based on the use of tumor-specific cytotoxic T lymphocytes (CTL) would be important to provide novel therapeutic modalities. Tumor-specific CTL have been isolated either from peripheral blood lymphocytes (PBL) or from tumor-infiltrating lymphocytes (TIL) of patients with various cancers, i.e. melanoma, ovarian and renal cell carcinomas, as well as T cell lymphoma (16). The CTL clones, isolated from patients with melanomas, were used to identify genes encoding tumor-associated antigens (TAA) such as Mart-1, gp100 and tyrosinase (7), as well as MAGE, BAGE and GAGE (1,8). The latter group of genes, identified as cancer testis antigen (CTA), were found to be expressed in a wide spectrum of human tumors including non-small cell lung cancer (NSCLC) but not in normal tissues except testis (9). The generation of CTL clones from NSCLC patients has only rarely been reported (10,11). A cellular immune response has been documented by electron microscopy analysis of human lung cancers identifying TIL with morphological evidence of activation and neighboring tumor cells were damaged or destroyed (12).

Several groups, including ours, have previously described, using different molecular approaches, oligoclonality in T cell populations infiltrating some fresh tumor biopsies from NSCLC (1316). Among the tumors with dominant T cell clonotypes, relative expansion of particular T cell subpopulations was observed and confirmed by sequencing analysis of Vß–D–Jß junctional regions (13). These results strengthen the view that local antigen-driven selection may occur and support the hypothesis that an antitumor immune response may take place in some NSCLC. To further characterize tumor-specific T cell response in NSCLC, we have generated T cell clones derived from TIL of a large cell carcinoma (LCC) of the lung. All these clones mediated a specific HLA class I-restricted cytotoxic activity against the autologous tumor cell line. CDR3 size and sequence analyses of the TCR ß chain transcripts of the cloned T cells and their comparison to those expressed in the fresh tumor tissue provide evidence that these specific CTL were expanded in situ. These results further support the view that T cells with biased TCR are activated in vivo as a result of contact with TAA.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient
The patient under the study (Heu) is a 61-year-old man suffering from a LCC of the lung. He was not treated with radio- or chemotherapy before and after surgery. Excision biopsy specimen and blood samples were taken after informed consent was obtained. A surgical biopsy of the primary tumor was performed and tumor fragments were frozen in liquid nitrogen for subsequent RNA extraction. In parallel, biopsy samples were obtained for standard histological examination. Autologous PBL were also obtained at the time of surgery and preserved at -80°C.

Tumor cell line
IGR-Heu tumor cell line was established in vitro from Heu patient biopsy after primary engraftment into nude mice as described previously (17). Briefly, small (1–2 mm3) tumor pieces were implanted s.c. into nude mice flank. After growth of engrafted tumor, mice were sacrificed, and tumor samples were mechanically dissociated and maintained in culture for tumor cell line generation in complete DMEM/F12 media supplemented with 10% FCS (Seromed, Berlin, Germany). Human origin of the cells was checked with an anti-MHC class I mAb (W6/32). Intra-cytoplasmic and surface immunofluorescence analyses with respectively anti-cytokeratin (KL1; Immunotech, Marseille, France) and Ber-EP4 (Dako, Glostrup, Denmark) mAb confirmed the epithelial origin of the tumor cell line.

Generation of TIL cell line and clones
Fresh tumor biopsy was mechanically and enzymatically dissociated in DMEM medium, 1 mM HEPES, 0.3 U/ml DNase, 0.5 U/ml collagenase and 0.28 U/ml hyaluronidase (Gibco/BRL, Life Technologies, Cergy Pontoise, France), and then frozen. Viable cells were isolated using Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) density- gradient centrifugation. Resulting single-cell suspensions were cultured at 37°C (5% CO2) in complete media supplemented with 10% human AB serum (Jacques Boy Institute, Reims, France) and rIL-2 (20 U/ml, Roussel-Uclaf, Romainville, France) in the presence of irradiated autologous tumor (3x103/well) and lymphoblastoid (4x104/well) cell lines. Cells were fed every 3 days with complete media supplemented with rIL-2 and stimulated with irradiated cell lines every 2 weeks.

TIL cell line was then cloned by limiting dilution (0.5 cell/well) in 96-well V-shaped microtiter plates (12 plates used; Nunc, Roskilde, Denmark) in the presence of irradiated autologous tumor (3x103/well) and lymphoblastoid (4x104/well) cell lines, IL-2 (100 U/ml), and 3% conditioned medium from phytohemagglutinin-activated lymphocytes. After 2 weeks of culture, the resulting T cell clones were expanded in the same conditions.

mAb and immunofluorescence analysis
OKT3, OKT4 and OKT8 mAb (Ortho Diagnostics, Westwood, MA) recognize CD3, CD4 and CD8 molecules respectively. BMA031 mAb (Behring, Marburg, Germany) is directed against a monomorphic determinant of the TCR{alpha}ß. Anti-NKH-1 (N901) mAb is directed against the CD56 molecule. Anti-CD28 mAb was purchased from Becton Dickinson (San Jose, CA). W6/32 (anti-HLA-A/-B/-C) and 9-49 (18) mAb recognize non-polymorphic determinants of HLA class I and class II gene products respectively. MA2.1 (anti-HLA A2, -B17), B1.23.2 (anti-HLA-B and -C) and B9.12.1 (anti-HLA-A/-B/-C) were kindly provided by Dr F. Lemonnier (Paris, France). Anti-TCR mAb MPB2D5, CH92, IMMU157, 36213, 3D11, ZOE, 56C5, FJN9, C21, VER2.32.1, IMMU222, JU-74, CAS1.1.3, TAMAYA 1.2, E17.5F3, BA62, ELL1.4, IG125, IMMU546 and AF23 (Immunotech, Marseille, France) are directed against the products of Vß2, Vß3, Vß5.1, Vß5.2, Vß5.3, Vß7.1, Vß8.1 and 8.2, Vß9, Vß11, Vß12, Vß13.1, Vß13.6, Vß14, Vß16, Vß17, Vß18, Vß20, Vß21.3, Vß22 and Vß23 respectively.

Phenotypic analysis of the TIL cell line and clones was performed by indirect immunofluorescence using a FACScan flow cytometer, and data were processed using the CellQuest program (Becton Dickinson).

Cytotoxicity assays and cytokine release
The cytotoxic activity of the TIL cell line and clones was measured by a conventional 4 h 51Cr-release assay using triplicate cultures in round-bottomed 96-well plates. E:T ratios were 30:1, 10:1, 3:1 and 1:1 on 3000 target cells/well. Percent specific cytotoxicity was calculated conventionally; SD was <5%. Determination of tumor necrosis factor (TNF)-{alpha} and IFN-{gamma} concentration in culture supernatants was performed using ELISA kits from Immunotech (Marseille, France).

W6/32 (anti-class I), 9-49 (anti-class II), MA2.1 (anti-HLA-A2), B1.23.2 (anti-HLA-B, -C) and B9.12.1 (anti-HLA-A/-B/-C) mAb were used in functional assays. Functional effects of the mAb on target cells were tested by incubating each of them for 2 h at 37°C before the assay at the predetermined saturating concentration.

Target cell lines
K562 (derived from a patient with chronic myelogenous leukemia) and Daudi (B cell line) were used in NK and LAK assays respectively. Allogeneic NSCLC cell lines, A549 [adenocarcinoma (ADC) HLA-A26/30], Ludlu [squamous cell carcinoma (SCC)] and SK-MES (SCC; HLA-A3/30, B7/27), were purchased from ATCC (Rockville, MD); H1155 (LCC, HLA-A2-), H1355 (ADC, HLA-A2+), H460 (LCC; HLA-A2) and H820 (ADC; HLA-A2) were kindly provided by S. Rogers (Boston MA; 11); IGR-Pub (ADC; HLA-A2, B7/Bw6, Cw7) and IGR-Bla (LCC; HLA-A2/68, B35/38; Cw4/Cw12; 17) were used as targets in cytotoxicity assays (Table 1Go). GE renal cell carcinoma (HLA-A9/32, -B7/21, CW7; 19) as well as Fon (A2/A29, B44, CW802/CW1601; 20) and M10 melanomas (HLA-A3/-A10, -B14/-B35, -CW4/-CW8; 21) were included.


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Table 1. NSCLC tumor cell line HLA typing and characteristics
 
CDR3 size analysis of TCR Vß transcripts
To study the TCR Vß transcripts expressed by TIL and PBL, a PCR-based method that determines V–D–J junction (CDR3) size distribution patterns was used (2224). The Vß and Cß primers, including the Cß-Fam and 13 Jß-Fam primer sequences, have been described previously (2526). Briefly, tumoral samples (0.2–0.5 g tissue squeezed by a Spex 6700 pulverizer; Spex Industries, Edison, NJ) were resuspended in 6 M guanidium thiocyanate buffer. Total RNA was then purified by cesium chloride gradient centrifugation. For PBL (5x106 cells), total RNA was extracted using a modified guanidium thiocyanate–phenol–chloroform method (RNABle; Eurobio, Les Ulis, France). cDNA was prepared by standard method using reverse transcriptase and an oligo-dT primer (Invitrogen, Groningen, Netherlands). cDNA, synthesized from 5 µg of RNA, were amplified by 40 cycles of PCR using Vß/Cß primer pairs in 50 µl final volume and aliquots (2 µl) were copied in one to five cycles of run-off reactions primed with fluorescent (ABI fluorophore Fam)-labeled oligonucleotides specific for Cß or Jß fluorophores. Run-off products were then subjected to electrophoresis on an ABI (Applied Biosystem, Foster City, CA) sequencer in the presence of fluorescent size markers, and analyzed by automatic fluorescence quantification and size determination using the computer program Genescan 672 (Applied Biosystem).

TCR Vß repertoire analysis
TCR Vß gene segment usage was determined using a semi-quantitative PCR analysis as described previously (26). cDNA amplification was performed over 30 cycles with the fluorescent Cß primer and the same panel of Vß primers as for the CDR3 size analysis. The intensities of the different peaks present in all Vß subfamilies were added, and the percentages of each Vß subfamily were calculated and represented as histograms. Although the PCR efficiency may vary from one oligonucleotide pair to another, this method was previously described to allow intersample comparison (26).

Cloning and sequencing of TCR Vß transcripts
The procedure used for cloning and sequencing of TCR Vß transcripts has been described previously (27). A Vß3 primer was used for cloning (25). Briefly, cDNAs were submitted to 40 cycles of PCR and amplified products were purified using Qiagen columns (Qiaquick PCR purification kit; Hilden, Germany). The purified material was ligated into pGEM-T Easy Vector (Promega, Paris, France) and used to transform XL1-blue supercompetent cells (Stratagene, La Jolla, CA). White colonies were screened by a dot-blot technique using a 32P-labeled Jß1.2 oligonucleotide probe. Plasmid DNA was extracted from positive colonies and sequenced with thermosequenase fluorescent labeled primer cycle sequencing kit (Amersham, Little Chalfont, UK) and a 373A DNA sequencer (Applied Biosystems).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Derivation and characterization of autologous tumor and cytotoxic TIL cell lines
The IGR-Heu tumor cell line was generated from a human LCC of the lung (pT2 N0) as described in Methods. Intracytoplasmic and surface immunofluorescence analyses indicated respectively that cytokeratin and epithelial membrane antigen (Ber-EP4) were strongly expressed by virtually all the cells, consistent with an epithelial lineage (data not shown). The IGR-Heu cell line was found to express surface MHC class I but not class II molecules, the B7-1 B7-2 and CD54 (ICAM-1) accessory molecules were not expressed, and the expression of CD58 (LFA-3) was moderate (data not shown).

TIL, previously isolated from the tumor, were cultured in the presence of low doses of IL-2 (20 U/ml), irradiated tumor and lymphoblastoid cell lines. The cells were then expanded with high doses (100 U/ml) of IL-2 and irradiated autologous feeder cells. This TIL-derived T cell line (TIL-Heu) displayed a high cytotoxic activity against the autologous tumor cells (Fig. 1Go). In contrast, no lysis of allogeneic melanoma, renal cell carcinoma, autologous and allogeneic Epstein–Barr virus-transformed B (EBV-B) cell lines, Daudi or K562 was observed. No killing was also observed toward any allogeneic NSCLC except A549 ADC which was lysed, although at a lower efficiency than the autologous tumor (Fig. 1Go). Immunofluorescence analysis of the TIL-Heu cell line revealed a CD3+, TCR{alpha}ß+, CD8+, CD2+, LFA-1+ (CD11a), CD4 and CD28 phenotype (data not shown). It should also be noted that NK cells were not detected in any of the TIL cultures.



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Fig. 1. Cytotoxic activity of the TIL-Heu cell line against the IGR-Heu autologous tumor cell line, IGR-Pub, IGR-Bla and A549 allogeneic NSCLC tumor cell lines, the EBV-Heu autologous lymphoblastoid cell line, and K562 and DAUDI targets. E:T ratios were 30:1, 10:1, 3:1 and 1:1.

 
TCR Vß repertoire analysis in the TIL cell line
TCR Vß gene segment usage in the TIL-Heu cell line, generated following three in vitro stimulations with irradiated feeder cells, was assessed by semi-quantitative PCR analysis with a series of previously described oligonucleotides (13), and compared to that in uncultured fresh tumor and autologous PBMC. As shown in Fig. 2, GoVß1, Vß3, Vß7, Vß8 and Vß22 were over-expressed in TIL-Heu. Note that Vß22 gene segment expression increased in the TIL cell line as compared to fresh tumor. Conversely, Vß13, the most in situ overexpressed Vß gene segment [4 times more expressed in fresh tumor than in autologous peripheral blood mononuclear cells (PBMC)], decreased during in vitro cell culture.



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Fig. 2. TCR Vß gene segment usage in tumor, PBL and cultured TIL cell line of Heu patient. Relative gene segment usage was determined by semi-quantitative PCR and expressed as percentage (histogram bars) of the sum of the fluorescence intensities present in all detected peaks obtained after PCR amplification with a fluorescent Cß primer.

 
The expression of TCR Vß regions in the cultured cell line was then investigated by immunofluorescence analysis using a panel of 23 anti-human TCR Vß mAb. An additional in vitro stimulation of the TIL cell line with IL-2 and autologous lymphoblastoid and tumor cells was, however, required for this study. Our results indicate that 73% of TIL-Heu cells reacted with anti-Vß3 mAb and that 17% reacted with anti-Vß22 mAb. Vß5, Vß6, Vß7, Vß8 and Vß13 represented 3% only (data not shown). In contrast, autologous PBL showed a similar TCR Vß repertoire as compared to normal individuals (28) without a major amplification of a particular T cell subpopulation (data not shown). A discrepancy between results obtained by semi-quantitative PCR and cytometry analyses could be observed, and may result from the fourth performed in vitro stimulation and/or the restricted specificity of the mAb used.

Generation and molecular characterization of autologous LCC-reactive TIL clones
The TIL-Heu cell line was cloned in the presence of irradiated autologous tumor and lymphoblastoid cell lines as well as IL-2. Among the 184 obtained clones, 54 displayed a cytotoxic activity against the IGR-Heu autologous tumor cell line. Fourteen clones were selected for their ability to proliferate and to display a high cytotoxic activity toward the autologous tumor cells. Flow cytometry analysis showed that all the clones had a CD3+, TCR{alpha}ß+, CD8+, CD4, CD28 phenotype similar to that of the TIL cell line (data not shown). The CD56 molecule was present only on a fraction of the cells. According to their TCR Vß gene segment expression determined by immunofluorescence analysis using appropriate anti-Vß mAb, these clones were divided in three major groups. The first group, including the majority of the clones (seven clones), expressed a Vß3 variable region. The second group, including five clones, expressed a Vß22 gene segment, while the third group including only two clones (Heu171 and Heu115) expressed a Vß8 gene product.

Sequence analysis of the TCRß transcripts of all the Vß3+ clones and the TIL cell line indicated that they expressed a unique Vß3–Jß1.2 rearrangement with a CCACGGGTGCCC encoding junctional sequence (Table 2Go). This result indicated that all the Vß3+ clones derived from a unique T cell, which was greatly amplified. Similarly, Vß8 (Heu171 and Heu115) and all Vß22 T cell clones were identical, and expressed Vß8–Jß1.5 rearranged gene segments with a TGGGACGGAC junctional region and Vß22–Jß1.4 rearrangement with a GAAGAGCCCC junctional sequence respectively (Table 2Go). Thus it became evident that unique T cells, in particular Vß3+ and Vß22+ lymphocytes, have been greatly amplified in culture; one clone of each, i.e. Heu161 (Vß3) and Heu118 (Vß22), was selected for functional studies.


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Table 2. V–D–J sequences of CTL clones
 
Functional activity of the CTL clones
Heu161, Heu118 and Heu171 T cell clones were assessed for their cytotoxic activity against IGR-Heu autologous tumor cells, autologous and allogeneic EBV-B cell lines as well as a panel of allogeneic tumor cell lines including K562 and Daudi in a conventional 51Cr-release assay. The three clones displayed a high cytotoxic activity against the autologous LCC cell line but failed to lyse autologous and allogeneic EBV-B cells, allogeneic melanoma and renal cell carcinoma as well as Daudi tumor cells (Fig. 3Go and data not shown). Although the Heu161 clone failed to kill all NSCLC targets tested, including IGR-Bla (Table 1Go), it was able to mediate a weak lysis against A549 and IGR-Pub ADC. In contrast, Heu171 and Heu118 clones displayed a high level of cytotoxicity against A549, IGR-Pub, H1155 and H820 tumor cell lines (Fig. 3Go and data not shown). These results suggest that Heu171 and Heu118 T cell clones possess a larger spectrum of reactivity than Heu161 T cells.



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Fig. 3. Cytotoxic activity of Heu161 (A), Heu171 (B) and Heu118 (C) T cell clones against the IGR-Heu autologous tumor cell line, IGR-Pub, LUDLU and A549 selected allogeneic NSCLC tumor cell lines, the EBV-Heu autologous lymphoblastoid cell line, and K562 and DAUDI targets. E:T ratios were 30:1, 10:1, 3:1 and 1:1.

 
Heu118 and Heu171 were found to mediate a killing against K562 target cells. Such lysis, varying from one experiment to another, depended on the effector cell status subsequent to IL-2-induced activation. Cytotoxic experiments performed against IGR-Heu in the presence of K562 cold target (30,000–50,000 cells/well for 1000 NSCLC tumor cells/well), showed a partial inhibition of Heu171 and Heu118 lytic activity varying from 20 to 50%. These results indicate that TCR Vß8+ and TCR Vß22+ clones mediate both a specific and a non-MHC-required cytotoxicity toward the autologous tumor cell line. The cytotoxic activity of both clones toward allogeneic target cells may therefore correspond to `NK-like' activity.

HLA restriction of tumor-specific CTL clones
To determine the implication of the TCR and the MHC molecules in the interaction of the CTL clones with their specific target, we tested a panel of mAb for their ability to inhibit the specific lysis. Anti-CD3, anti-TCR{alpha}ß and anti-CD2 mAb were able to inhibit the CTL function. Anti-CD8 was also inhibitory, while anti-CD4 mAb had no effect (Fig. 4Go and data not shown). We then tested anti-HLA class I (W6/32) and anti-HLA class II (9–49) mAb for their inhibitory potential on the interaction of Heu161, Heu118 and Heu171 with IGR-Heu autologous tumor cell line. As shown in Fig. 5Go, pre-incubation of autologous LCC with W6/32 mAb inhibited the lytic activity of the three clones, while 9-49 had no effect. A similar inhibitory effect was observed with B9.12.1 mAb (data not shown).



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Fig. 4. Cytolytic activity of Heu161 (A), Heu171 (B) and Heu118 (C) T cell clones against the IGR-Heu autologous tumor cell line. E:T ratio was 30:1. Cytolytic experiments were performed either in medium or in the presence of the indicated mAb. CTL clones were preincubated for 2 h with saturating concentrations of anti-TCR{alpha}ß (BMA031), anti-CD8 (OKT8) or anti-CD4 (OKT4) mAb and then 51Cr-labeled IGR-Heu target cells were added.

 


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Fig. 5. Cytotoxic activity of Heu161 (A), Heu171 (B) and Heu118 (C) T cell clones against the IGR-Heu autologous tumor cell line. E:T ratio was 30:1. Cytolytic experiments were performed either in medium or in the presence of the indicated mAb. IGR-Heu target cells were preincubated for 2 h with saturating concentrations of anti-class I (W6/32, B1.23.2 and MA2.1) or anti-class II (9.49) mAb and then effector cells were added. Represents one experiment of five.

 
HLA typing of Heu is HLA-A2, -A68, -B7, -B35, -Cw4, -Cw7. To determine more precisely the restriction element recognized by the CTL clones, we used mAb specific either for HLA-A2 (MA2.1) or HLA-B -C (B1.23.2) in cytotoxicity blocking experiments. While B1.23.2 mAb had no effect on the cytotoxic activity of Heu161 and Heu118, MA2.1 inhibited the interaction of both CTL clones with IGR-Heu autologous LCC. In contrast, B1.23.2 mAb partially inhibited the lytic activity of Heu171 toward the IGR-Heu target. These results indicated that tumor recognition by Heu161 and Heu118 was HLA-A2 restricted, while that of Heu171 clone seemed to be HLA-B or -C restricted (Fig. 5Go). Note that the inhibitory activity of anti-class I mAb of IGR-Heu lysis by Heu171 and Heu118 was partial; the remaining cytotoxicity may in part correspond to MHC-unrestricted, IL-2-induced LAK-like activity.

Cytokine response of the CTL clones to autologous LCC
Specific cytokine release was assayed after stimulation of the CTL clones with autologous or allogeneic NSCLC tumor cells. Significant secretion of TNF-{alpha} (Fig. 6Go) and IFN-{gamma} (data not shown) was observed when the three clones were incubated with autologous but not with allogeneic (IGR-Bla) tumor cells. As shown in Fig. 6Go, TNF-{alpha} production was inhibited by anti-class I and anti-CD8 mAb but not with irrelevant antibodies, confirming that Heu161, Heu118 and Heu171 T cell clones specifically recognize their target in an HLA class I-restricted manner. Results presented in Fig. 6Go also confirm that Heu161 and Heu118 are HLA-A2 restricted.



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Fig. 6. TNF release of Heu161 (A), Heu171 (B) and Heu118 (C) T cell clones co-cultured with the IGR-Heu autologous tumor cell line or IGR-Bla allogeneic NSCLC tumor cell line. Experiments were performed either in media or in the presence of indicated mAb. Target or effector cells were preincubated for 2 h with saturating concentrations of anti-class I (W6/32, B9.12.1, B1.23.2 and MA2.1), anti-class II (9.49) or anti-CD8 (OKT8) mAb respectively, and then effector or target cells were added. Determination of TNF-{alpha} concentrations was performed on 24 h supernatants using ELISA.

 
Evidence for in situ clonal expansion of the CTL
To determine whether Vß3–Jß1.2, Vß8–Jß1.5 and Vß22–Jß1.4 CTL were present and potentially overexpressed at the tumor site, we analyzed the corresponding transcripts after PCR amplification in the fresh tumor, autologous PBMC as well as in the TIL cell line. CDR3 size analysis was performed using the corresponding Vß oligonucleotides in combination with Cß or the appropriate Jß primers. Monoclonal peaks of 118, 223 and 287 nucleotides corresponding to Vß3–Jß1.2, Vß8–Jß1.5 and Vß22–Jß1.4 combinations were observed in Heu161, Heu171 and Heu118 T cell clones respectively. These peaks were also observed in the TIL cell line and were maintained in culture throughout T cell stimulation, thus confirming sequence analysis. Interestingly, oligoclonal expansion of cloned cell TCR transcripts were also observed in fresh tumor (Fig. 7Go). Indeed, a dominant peak corresponding to the Heu161 Vß3–Jß1.2 combination was detected in a tumor biopsy but was much weaker in the patient's PBMC. Similarly, a single peak corresponding to Heu171 Vß8–Vß1.5 rearrangement was observed in fresh tumor; a polyclonal pattern was, in contrast, observed in autologous PBMC. A monoclonal peak of 287 nucleic acids corresponding to Heu118 Vß22–Jß1.4 was also detected in uncultured tumor and was less dominant in the periphery.



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Fig. 7. CDR3 size distribution patterns of Vß3, Vß8 and Vß22 transcripts in Heu T cell clones, the TIL-Heu cell line, uncultured tumor and autologous PBL analyzed with fluorescent Cß, and indicated Jß primers. RNA was extracted from CTL clones, the TIL cell line, fresh tumor and PBL, and cDNA were synthesized and amplified in a PCR reaction primed by Vß3-, Vß8-, Vß22- and Cß-specific primers. The unlabeled amplification products were elongated with nested fluorescent Cß, Jß1.2, Jß1.4 and Jß1.5 as indicated. Aliquots were subjected to electrophoresis and analysis on an automated sequencer. The profiles obtained show size in nucleotides (x-axis) and fluorescence intensity of different amplified products (y-axis).

 
The presence of recurrent TCR Vß3 transcripts in Heu fresh tumor was further investigated by cloning and sequencing PCR-amplified Vß3 cDNA products from the corresponding tumor lesion. Sequence analysis revealed that the cDNA clone encoding the same junctional sequence as Heu161 CTL represented 89% (eight of nine sequences) of the Vß3–Jß1.2 transcripts. These results are in line with CDR3 size distribution analysis indicating in situ clonal expansion of several CTL clones mediating a cytotoxic response against autologous tumor cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have previously demonstrated through direct sequencing of TCRß transcripts of NSCLC TIL that particular T cell populations were selected and amplified at the lesion site, and that these cells may contribute to the antitumor response (13). To further assess the immune response in NSCLC, we have generated CTL clones derived from TIL of a human LCC. Analysis of the TCR gene segment expression in the cultured cell lines revealed a profile substantially different from that detected in situ. Indeed, multiple Vß gene segments present in vivo were no longer evidenced in vitro, while others, i.e. Vß1, Vß3, Vß7, Vß8 and Vß22, were overexpressed. Further studies based on immunofluorescence analysis indicated that the major cell subpopulations expressing Vß3 and Vß22 gene segments had become predominant in culture, and represented 73 and 18% of the cells respectively. Stimulation by autologous tumor cells may be responsible of such strong selection in vitro since functional studies indicated that both Vß3 and Vß22 subpopulations were tumor specific. Alternatively, it may be related to a growth advantage dependent upon cytokine susceptibility or particular T lymphocyte activation state. Similar clonal T cell expansion was previously reported in a T cell line derived from lymphocytes infiltrating a spontaneously regressing melanoma (21).

Furthermore, comparison of the TCR repertoire in fresh tumor to that of the TIL cell line indicated that Vß13+ cells, which were found to be amplified in vivo as compared to autologous PBMC (4-fold more in tumor than in PBMC), were less predominant in vitro. These cells may react with fresh tumor and not with the in vitro established autologous tumor cell line. Indeed, in vitro selection of tumor cells was previously described in melanoma. CTL clones selectively recognizing autologous fresh tumor cells and not tumor cell lines were identified (20). Alternatively, Vß13+ lymphocytes may have reached a final stage of differentiation and proliferation in situ, preventing further amplification in vitro. These findings confirm previous observations demonstrating that TCR repertoire modifications frequently occur upon subsequent in vitro culture in the presence of autologous tumor cells (13,19). Additional studies of the underlying causes of this selection and determination of the specificity and function of these cells will be required to clarify the biology of tumor–lymphocyte interactions. Our experimental system based on in vitro and in vivo analysis of TIL may therefore more adequately reflect the effective antitumor immune response, and constitutes a powerful tool for evaluating the oligoclonality of in vitro-expanded TIL potentially useful in clinical trials.

Several CTL clones were derived from the in vitro established TIL-Heu cell line. These CTL clones were able to produce TNF-{alpha} and IFN-{gamma} in the presence of autologous tumor cells. The majority of these clones expressed a Vß3–Jß1.2 and mediated a specific cytotoxic activity against the autologous LCC cell line. Indeed, cytolysis was found to be CD3–TCR, CD8 dependent and HLA-A2 restricted with low cross-reactivity with allogeneic target cells. T cell clones with an apparent larger spectrum of reactivity (i.e. cytotoxicity toward the autologous tumor cell line and some allogeneic NSCLC cell lines) were also identified, and were found to express distinct TCR ß chains encoded by Vß22–Jß1.4 and Vß8–Jß1.5 rearrangements. No obvious homology was observed at the TCRß CDR3 region sequences, thought to play an important role in peptide antigen recognition. Blocking experiments, performed with mAb directed against relevant HLA class I molecules, indicated that Vß22+ clones recognized their specific target in a HLA-A2-restricted manner, while Vß8+ clones seemed to be HLA-B or -C restricted. These results indicate that in vivo antitumor response is complex and probably involves different HLA loci for peptide presentation of either the same or distinct TAA.

The major contribution of the present work is the demonstration for the first time that several LCC-reactive CTL clones generated in vitro were actually selected and expanded at the tumor site, even though these particular cells may not be the most represented in vivo. Indeed, CDR3 size analysis using appropriate Vß oligonucleotides in combination with specific Jß primers indicated that Vß3–Jß1.2, Vß8–Jß1.5 and Vß22–Jß1.4 specificities appeared as dominant peaks in fresh tumor as opposed to autologous PBMC. These data, confirmed by sequencing analysis of Vß3–Jß1.2 transcripts, strongly suggested that these CTL clones were expanded in situ as a consequence of tumor antigen recognition. The fact that these cells were characterized as specific CTL, while cancer regression was documented clinically and histologically in this patient (who is alive and healthy 4 years after surgery), strongly suggests that the adaptive immune response may represent, at least in part, an active mechanism of defence against tumor.

Previous studies have demonstrated that at least one Her2/neu-derived peptide is a TAA in NSCLC and that HLA-A2 serves as a restriction element in CTL recognition (11). Since at least a subset of IGR-Heu cells express Her2/neu on its surface (data not shown), we examined whether Her2/neu HLA-A2-binding peptides (peptide 369–377 and 654–662) were implicated in Heu161 and Heu118 recognition. No lysis was observed when autologous EBV-transformed or T2 cell lines were loaded with such peptides, strongly suggesting that the CTL clones recognize distinct antigen on IGR-Heu tumor cell line (data not shown). In this regard, the HLA-A2+, Her2/neu+ cell line (H1355) was resistant to Heu161 and Heu118 clone killing. Furthermore, it has been reported that 35% of NSCLC express MAGE antigen (9). RT-PCR analysis indicated that IGR-Heu cell line express MAGE 2 and MAGE 3 but not MAGE 1 melanoma-associated antigen (data not shown). The MAGE 3 HLA-A2-binding peptide (MAGE–3271–279) was not involved in Heu161 and Heu118 T cell clone recognition of autologous tumor cells since both clones were unable to kill peptide-loaded HLA-A2+ EBV or T2 cell lines. In addition, the HLA-A2+ IGR-Bla allogeneic tumor cell line expressing both MAGE 2 and MAGE 3 was resistant to T cell clone lysis. These results strongly suggest that Her2/neu and MAGE antigen are not implicated in CTL clone recognition of LCC and that a distinct TAA is expressed by the IGR-Heu cell line. Studies are in progress to determine the nature of NSCLC-related peptides.

So far few CTL clones have been derived from PBL or TIL from NSCLC patients even after stimulation with autologous tumor cells and rIL-2. CTL specific for autologous human SCC of the lung were generated by stimulation of PBL with autologous tumor cells in vitro (10). These T cell clones recognized a peptide derived from a mutated elongation factor 2 gene in an HLA-A68.2-restricted manner (29). Several mechanisms, responsible for T cell anergy and inhibition of an effective T cell response in vivo and in vitro, are now under investigation. Current studies attempting to amplify and characterize additional T cell populations, in particular those expanded in situ, may help to assess the diversity of the antitumor response in NSCLC in the context of therapeutic transfer. This approach should also lead to the identification of a large number of TAA peptides, in particular those common to several tumor cells, which may be useful for the design of new immunotherapy and vaccination protocols in lung cancer.


    Acknowledgments
 
We thank Dr F. Lemonnier for providing anti-class I mAb, D. Charron and R. Tamouza for HLA typing, F. Gay for technical assistance, and Y. Lecluse for FACS analysis. We also thank Drs A. Caignard and C. Kosmatopoulos for critically reading the manuscript. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Institut Gustave Roussy, the Association de la Recherche contre le Cancer (grant 9307) and the Ligue Nationale Franciaise de Recherche contre le Cancer. H. E. was supported by a fellowship from the Ligue Nationale Franciaise de Recherche contre le Cancer (comité Val de Marne).


    Abbreviations
 
ADC adenocarcinoma
CTL cytotoxic T lymphocytes
CTA cancer testis antigen
EBV Epstein–Barr virus
LCC large cell carcinoma
NSCLC non-small cell lung cancer
PBL peripheral blood lymphocytes
PBMC peripheral blood lymphocytes.
SCC squamous cell carcinoma
TAA tumor-associated antigen
TIL tumor-infiltrating lymphocyte
TNF tumor necrosis factor

    Notes
 
Transmitting editor: J. F. Bach

Received 4 October 1999, accepted 6 January 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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