- Email: [email protected]
HLA-A Locus-Restricted and Tumor-Specific CTLs in Tumor-Infiltrating Lymphocytes of Patients with Non-Small Cell Lung Cancer
CELLULAR IMMUNOLOGY ARTICLE NO.
175, 101–110 (1997)
CI961062
HLA-A Locus-Restricted and Tumor-Specific CTLs in Tumor-Infiltrating Lymphocytes of Patients with Non-Small Cell Lung Cancer1 NAOKO SEKI,* TOMOAKI HOSHINO,* MEGUMI KIKUCHI,* AKIHIRO HAYASHI,†
AND
KYOGO ITOH*,2
*Department of Immunology and †First Department of Surgery, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan Received September 3, 1996; accepted November 26, 1996
HLA class I restriction and tumor specificity of cytotoxicity in the IL-2-activated tumor-infiltrating lymphocytes from 16 patients with non-small cell lung cancer were investigated. Six HLA class I-restricted and tumor-specific CTL lines were established: (i) HLA A2restricted and adenocarcinoma-specific CTLs in three (two A0201/ and one A0206/) patients with adenocarcinoma, (ii) HLA A3101- and A3302-restricted and adenocarcinoma-specific CTLs in an HLA A3101/3302/ patient with adenocarcinoma, and (iii) HLA A3302-restricted CTLs and (iv) HLA A2402-restricted CTLs recognizing tumors with different types of histology in an HLA A3302/ patient with adenocarcinoma and an HLA A2402/ patient with squamous cell carcinoma (SCC), respectively. The three HLA A2-restricted CTL lines recognized 4, 4, or 6 of 15 HLA A2/ adenocarcinoma cell lines that originated from lung, stomach, colon, and breast with different subtypes (HLA A0201, A0206, and A0207), respectively. Furthermore, the CTLs of an HLA A0206/ patient recognized five different fractions of peptides eluted from an HLA A0201/ adenocarcinoma cell line. These results showed evidence of the existence of HLA class I-restricted and tumor-specific CTLs recognizing peptide antigens on HLA-A alleles of adenocarcinoma or SCC in tumor sites of a substantial number of patients with non-small cell lung cancer. q 1997 Academic Press
INTRODUCTION Non-small cell lung cancers are relatively resistant to currently available chemotherapy and radiotherapy 1
Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, a grant from the Science Research Promotion Fund of the Japanese Private School Promotion Foundation, the Fukuoka Cancer Society, the Mochida Memorial Foundation, and the Japanese Foundation for Multidisciplinary Treatment of Cancer. 2 To whom correspondence should be addressed at the Department of Immunology, Kurume University School of Medicine, 67 Asahimachi, Kurume 830, Japan. Fax: 81-942-31-7699. E-mail: [email protected].
regimens and one of the malignant tumors with an extremely poor prognosis. Therefore, development of specific immunotherapy using tumor-specific CTLs3 or tumor antigens is important for improving treatment modalities. HLA class I-restricted and tumor-specific CTLs have been observed in T cells of peripheral blood mononuclear cells (PBMC) stimulated with autologous tumor cells or in IL-2-activated tumor-infiltrating lymphocytes (TILs) of patients with melanomas, esophageal cancers, ovarian cancers, and breast cancers (1– 6). Genes encoding peptide antigens recognized by CTLs have been cloned from melanomas (4–12). However, the presence of these CTLs or tumor antigens has been rarely reported in human lung cancers (13). In this investigation, we have studied HLA class I restriction and tumor specificity of cytotoxicity of IL-2-activated TILs from patients with non-small cell lung cancer. The results suggest the presence of HLA class Irestricted and tumor-specific CTLs recognizing peptide antigens on HLA-A molecules of adenocarcinoma or squamous cell carcinoma (SCC) in tumor sites of a substantial number of patients with lung cancer. MATERIALS AND METHODS Subjects and tumor cell lines. Tumor specimens or carcinomatous pleural effusion and peripheral blood were obtained from 16 patients with non-small cell lung cancer at the time of major surgery at the Kurume University Hospital. The profiles of 10 patients who displayed significant TIL proliferation and a summary of CTL activity are shown in Table 1. The TILs of the other 6 patients did not proliferate well under the employed conditions. All research followed the tenets of the Declaration of Helsinki. Autologous tumor cell line, LC1 was established from pleural effusion of a patient 3 Abbreviations used: CTL, cytotoxic T lymphocyte; EBV-BCL, Epstein–Barr virus-transformed B cell line; IL-2, interleukin-2; IFNg, interferon-g; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; SCC, squamous cell carcinoma; TIL, tumor-infiltrating lymphocyte.
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0008-8749/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
102 85 41 53 72 3 40 NT 92 NT 25 15 52 41 22 97 48 NT 8 NT 11 100 96 94 96 100 83 NT 100 NT 98 0 3 0 2 0 0 NT 0 NT 64 100 93 94 94 100 83 NTe 100 NT 34 Adeno/SCC Adeno Adeno Adeno Adeno/SCC SCC/adeno — — — Non g a
TILs of the six other cases (cases 11 to 16) did not proliferate well. Adeno, adenocarcinoma. c SCC, squamous cell carcinoma. d No cytotoxicity: TILs failed to show a significant level (ú10%) of lysis against any cell lines tested. e NT, not tested. f Mix, mixed tumor of adenocarcinoma and SCC. g MHC nonrestricted cytotoxicity was observed against all cell lines tested including one normal cell line.
I/T2N0M0 VI/T2N2M1 IIIa/T3N0M0 IIIb/T4N2M0 IV/T3N2M1 I/T2N0M0 IIIa/T3N1M0 I/T1N0M0 IIIa/T3N0M0 IIIa/T3N2M0 Adenob Adeno Adeno Adeno Adeno SCCc Adeno Adeno Mix f SCC 70/F 71/M 53/M 83/M 73/M 68/F 68/M 68/F 66/M 69/M 1 2 3 4 5 6 7 8 9 10
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b
Cw1/Cw3 Cw0303/Cw1403 Cw7/ Cw1/ Cw1/ Cw7/ Cw7/ Cw7/
B44/B48 B44/B66 B59/B62 B1511/B44031 B67/B44 B46/B52 B52/54 B7/B52 B7/B52 B52/B60
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Tumor Pleural effusion Tumor Pleural effusion Pleural effusion Tumor Tumor Tumor Tumor Tumor
A0201/A3101 A0206/A3302 A0201/A2402 A3101/A3302 A1101/A3302 A2402/ A24/ A2402/ A24/ A26/A24
Cw3/
C B A T cell source Stage/TNM Histology Age/sex Casea (n Å 10/16)
AID
A2/A3101 A2 A2 A3101/A3302 A3302 A2402 —d — — Non g
CD8 CD3 TCRgd Tumor specificity
HLA-A locus restriction of CTL HLA class I
TABLE 1
Patient Profiles and Summary of IL-2-Activated TILs from Patients with Lung Cancer
TCRab
Surface phenotype (%)
CD4
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(case 4) as previously described (14). Most of the allogenic tumor cell lines and normal cell lines used in these studies and their MHC-class I alleles have been previously reported (3). The cell lines used were HST-2 and SSTW-9 gastric cancers provided by Dr. Sato (Sapporo Medical College, Sapporo, Japan); a 1-87 lung cancer provided by Dr. Kobayashi (Tohoku University, The Research Institute for Tuberculosis and Cancer, Sendai, Japan); a YT803 lung cancer provided by Dr. Nomoto (Kyushu University, Fukuoka, Japan); an SKG-II cervical cancer provided by Dr. Nozawa (Keio University School Medicine, Tokyo, Japan); and an HAK-2 hepatocellular carcinoma and a KMCH-1 hepatocholangiocarcinoma from Dr. Kojiro of our University. The other cell lines listed in Table 2 were either established in our laboratories or purchased from JCRB (Tokyo, Japan), American Type Culture Collection (ATCC, Rockville, MD), and Morinaga Co. (Tokyo, Japan). Genotypes of HLA-A, -B, and -C alleles of tumor cell lines and patients’ PBMC were determined by the polymerase chain reaction sequence-specific oligonucleotide probe (PCR-SSOP) method, as reported with a slight modification (15–17). Sequences of the HLAA allele of SW620, R27, and KE-4 cell lines were also analyzed by the reverse-transcriptase PCR method, as previously described (18). HLA-C alleles of some tumor cell lines (TE-8, TE-10, and TE-11) were determined by the PCR restriction fragment-length polymorphism (RELP) method (19). The results from serological typing of PBMCs determined by HLA Monoclonal Reagent (One Lambda, Canoga Park, CA) were shown when genotyping data were not available. TIL preparation, expansion, and functional assays. Tumors were finely minced using surgical blades. Lymphocytes and tumor cells from pleural effusions were centrifuged and isolated by Ficoll–Conray gradient separation. These pasted mixtures of tumor cells, tissue cells and TILs, or cells of pleural effusions were then extensively washed and incubated with RPMI 1640 medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (Whittaker, Walkersville, MA), 0.1 mM MEM nonessential amino acids solution (GIBCO BRL), and 100 units/ml of recombinant IL-2 (a generous gift from Shionogi Pharmaceutical Co., Osaka, Japan) at 377C and in a 5% atmosphere of CO2 for up to 11 weeks. The surface phenotypes of the proliferating TILs were studied by direct immunofluorescence assay with FITC-conjugated anti-TCRab, -TCRgd (Becton–Dickinson Inc., Mountain View, CA), -CD3, -CD4, and -CD8 (Nichirei, Tokyo) monoclonal antibodies (mAbs) and FACScan (Becton–Dickinson), as reported (20). The results at around 50 days of culture are shown in Table 1. The IL-2-activated TILs of 10 cases were serially tested for their cytotoxicity against a panel of HLA class I-identified tumor cell and normal cell lines shown
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HLA-A LOCUS-RESTRICTED CTL IN LUNG CANCER
in Table 2. Except for an LC1 tumor that was established from case 4, all were allogenic tumor cell lines. Autologous tumor cell lines were not available from the other 9 cases due to the lack of tumor cell proliferation in vitro in culture. A cytotoxic assay against each of the target cells was repeated at least three times in most tumor cell lines in separate experiments. Representative results at around 7 weeks of culture are shown in Table 2. A standard 6-hr 51Cr-release assay was used to measure CTL activity, as reported (21). If the mean of triplicate determinants of percentage specific lysis exceeded 10%, it was evaluated as a significant level of cytotoxicity in this study. Inhibition experiments by anti-CD3 (OKT3) (ATCC), -CD4 (NUTh/i), -CD8 (NU-Ts/c), -CD14 (H14), anti-HLA class I (W6/32) (ATCC), and anti-HLA DR (H-DR1) mAb were performed as described previously (3, 21). Interferon-g (IFN-g) production by IL-2-activated TILs in response to various tumor cells was also measured. Briefly, IL2-activated TILs were incubated with target cells at an effector to target cell (E/T) ratio of 2:1 for 18 hr. The amounts of IFN-g in cell-free supernatants were measured using an IFN-g ELISA kit (Ohtsuka Assay, Tokushima, Japan; limit of sensitivity, 5 pg/ml). Peptide extraction and synthesis. Peptides were extracted and purified from an HLA A0201/ colon adenocarcinoma cell line (SW620) using the pH 3.3 acid elution technique, as reported (22). Briefly, 5 1 108 cell equivalents of extracted peptide were pretreated with C18 Sep Pak (Waters, Milford, MA), prefractionated with Centricon-3 (Amicon, Beverly, MA) (consisting of peptides £3000 Mr , É30 amino acids in length), and fractionated with reverse-phase HPLC. HPLC solvents were A Å 99.92% water/0.08% trifluoroacetic acid and B Å 99.94% acetonitrile/0.06% trifluoroacetic acid. Gradients consisted of the following linear step intervals: isocratic A solvent for 0 to 5 min; 0 to 10% B (in A) from 5 to 10 min; and 10 to 35% B (in A) from 10 to 60 min. The flow rate was 0.8 ml/min. Individual HPLC fractions were lyophilized and reconstituted in 200 ml of HBSS. HLA-A2 binding HER2/neuderived peptide (IISAVVGIL, GP2) (7) and MAGE-3-derived peptide (FLWGPRALV) were synthesized as reported (23). Peptides were dissolved in aliquots of HBSS at a concentration of 1 mg/ml and stored at 0807C. These peptides were used at a concentration of 20 mg/ml in the peptide-pulsed cytotoxicity assay using an HLA A0201/ T2 cell line (7) where 20 ml of individual HPLC fractions or synthesized peptides was added to a well containing 100 ml of 51Cr-labeled 1 1 104 T2 cells, followed by incubation for 1 hr at 377C to allow the binding of peptides to target cells. Then, 1 1 105 CTLs were added to the well followed by harvesting 6 hr later. RESULTS HLA-A2-restricted CTL lines. The CTL lines from three HLA-A2 patients with lung adenocarcinoma
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(case 1, A0201/3101; case 2, A0206/3302; and case 3, A0201/2402) displayed a similar pattern of cytotoxicity. They showed a significant level (ú10%) of lysis against three lung adenocarcinomas (A0207/ 1-87, A0201/ 1118, and A0206/ PC-9 cell lines) and a colon adenocarcinoma (A0201/ SW620) (Table 2). Recognition of these lung adenocarcinomas by the CTL line of case 1 was confirmed by the assay of IFN-g production. Namely, it produced 18, 235, or 40 pg/ml of IFN-g by recognition of 1-87, 11-18, or PC-9 tumor, respectively, but did not produce detectable levels (ú5 g/ml) of IFN-g by recognition of any of the other 15 solid tumors tested that were not susceptible to lysis (Table 3). The CTL line of case 1 (A0201/3101) also lysed three histologically different but all A3101/ tumors (LC1 lung adenocarcinoma, HSC-4 oral SCC, and KMCH-1 hepatocholangiocarcinoma), while that of case 2 (A0206/3302) lysed A3302/ LC1 lung, A2/ KATO-III stomach, and A0201/ R27 breast adenocarcinomas. In contrast, none of the other cell lines including nine HLA A2/ adenocarcinoma cell lines (established from stomach, colon, pancreas, kidney, or ovarium) and three A2/ normal cell lines, autologous B cell lines, and K562 tumor cells were susceptible to lysis by any of these CTL lines (Table 2). Cytotoxicity of all three cases against most of the cell lines was confirmed at three different E/T ratios, and the representative results of case 1 are shown in Fig. 1A. The levels of cytotoxicity increased depending upon the increased numbers of effector cells. Kinetic study showed that the IL-2-activated TILs of case 1 consistently lysed an HLA A0201/ 11-18 lung adenocarcinoma cell line between 45 and 65 days of culture, although they showed nonspecific lysis around 20 to 30 days of culture probably due to the presence of lymphokine-activated killer cells in the culture (Fig. 2). Similar kinetics were obtained in the IL-2-activated TILs of cases 2 and 3 (data not shown). Cytotoxicity of the CTL line of case 1 was inhibited by anti-CD3, antiCD8, or anti-HLA class I, but not by anti-CD4 or antiHLA class II (DR) mAb. Namely, the percentage specific lysis of an HLA A0201/ lung adenocarcinoma cell line (11-18) by this CTL was 1, 21, 14, 31, or 33% lysis in the presence of each mAb, respectively, versus 36% lysis with an irrelevant mouse mAb (CD14) at an E/T ratio of 40. All these results indicate that the IL-2-activated TILs of cases 1, 2, and 3 commonly exhibited HLA A2restricted lysis against three lung adenocarcinomas with A0201, 0206, and 0207 subtypes and one HLA A0201/ colon adenocarcinoma. In addition, the unique pattern of cytotoxicity was observed in the CTL lines of cases 1 and 2. CTL lines restricted to HLA-A other than A2 allele. IL-2-activated TILs of case 4 (HLA A3101/3302) showed a higher level of lysis against the autologous tumor cell
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RERF-LC-MS 1-87 A549 11-18 PC-9 LC1b YT803 KWS KATO-III MKN-45 MKN-28 SSTW-9 HST-2 SW620 HCT116 KM12LM COLO 201 COLO 205 COLO 320 R27 Panc-1 KMG-A KUR-11 KUR-20 KOC-7C LC-99A 86-2 LC-65A LK79 Sq-1 RERF-LC-AI QG-56 LC-lsq KE-3 KE-4 TE-8 TE-9 TE-10 TE-11 KUMA-1 Ca9-22 HSC-2 HSC-3 HSC-4 SAS SKG-II HCS
Cell line
Target cells
Squamous cell carcinoma
Small cell carcinoma
Large cell carcinoma
Adenocarcinoma
Histology
TABLE 2
Lung Lung Lung Lung Lung Lung Lung Stomach Stomach Stomach Stomach Stomach Stomach Colon Colon Colon Colon Colon Colon Breast Pancreas Gall bladder Kidney Kidney Ovarium Lung Lung Lung Lung Lung Lung Lung Lung Esophagus Esophagus Esophagus Esophagus Esophagus Esophagus Head and neck Oral Oral Oral Oral Oral Uterus Uterus
Origin
3 6 5 3 0 1 0 2 0 7 6 2 2 5 12 2 0 0
1 0 6 5
0 3 1 2 0 8 15 0 6 4 0 0 6 4 5
6 18 3 32 24 30
Case 1 A0201/3101
0
0 2 1 0 0 0 0 0 0 6 4 0 4 0 0
2 11 0 0 0 0 23 0 0 0 0 0 16 0 0 0 0 4 0 7 1
0 31 0 11 16 21
Case 2 A0206/3302
5
0 0
8
0
1 6 0 0
26
5 5
0
3
0 3
15
2 0 0 0 1 4
6
0 0
25 15
0 3
5
Case 5 A1101/3302
0
2 8 3 4 1 8 2 2
0 0 1 14 1 0 6 0 5 0 0 0 1 4 5 11
3 5 0 4 0 29
Case 4 A3101/3302
0 0
1
0 2
2 0
0 0
16 0 0 0 0 0
0 0
0 4
0 22 4 10 16
Case 3 A2402/0201
Adeno
% cytotoxicitya
7 8 3 17 18 5 8 0 4 8 6 3 4 16 3 6 26 3 5 12 8
0
5 0 10 4 4 5
6 6 6 0
1 5 4 11 6
SCC Case 6 A2402/ A1101/ A0207/A1101 A2603/A3001 A0201/A2402 A0206/A2402 A3101/A3302 A3101/A3302 A0206/ A2/A2402 A2402/ A3101/ A2402/A2601 A0206/A3101 A0201/A2402 A0101/A0201 A2/A2402 A0101/A0201 A0101/A0201 A2402/ A0201/ A0201/A1101 A2601/ A2402/A3302 A0201/A0210 A0201/A3101 A2402/ A1101/ A1101/A2402 A2402/ A1101/A2402 A2402/ A2601/ A11/A2402 A0206/A2402 A2402/A2601 A2402/A2601 A3302/ A2/A2402 A2402/A2601 A2603/A3302 A0207/A2402 A2402/A3302 A0201/A2402 A2402/A3101 A2402/ A2402/ A2402/
A
B5201/
B1501/B44031 B35/B4601 B44031/B5401 B4801/B5201 B0702/B5101 B5201/
B55/ B1503/
B51/B52 B5201/B5401 B5101/ B4002/ B4002/ B1501/5201 B5201/ B4601/ B44031/
B5101/ B1501/B4601 B3802/B5502 B0702/B1518 B4501/ B0702/ B0702/0801 B0702/0801 B1402/ B4402/ B3801/ B4002/
B5401/ B4601/B5401 B1801/B44031 B5201/B5401 B0702/B5502 B1511/B44031 B44031/B5101 B1301/ B1501/B4601
B
C
Cw1202/
Cw0302/0102 Cw1401/ Cw0303/Cw1403 Cw0102/Cw1402 Cw0102/Cw1403 Cw0803/Cw1202 Cw0702/Cw1402 Cw1202/
Cw0102/Cw1202 Cw0401/Cw1502 Cw0304/ Cw0303/ Cw0401/Cw1202 Cw1202/ Cw0102/ Cw1203/Cw1601 Cw0401/Cw1502 Cw0101/Cw0302 Cw0102/
Cw0304/ Cw0102/Cw0303 Cw0102/Cw0702 Cw0702/Cw0704 Cw0501/Cw0701 Cw0702/ Cw0701/Cw0702 Cw0701/Cw0702 Cw0802/ Cw0501/ Cw1203/ Cw0304/
Cw0102/ Cw0102/ Cw1203/Cw1601 Cw0102/Cw1201 Cw0303/Cw0702 Cw0303/Cw1403 Cw1402/Cw1403 Cw0303/ Cw0102/Cw0303
HLA class I of target cells
HLA-A Locus-Restricted Cytotoxicity of CTL Lines Derived from TILs of Patients with Lung Cancer
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a Cytotoxicity of the TILs from six patients with lung cancer against different types of cell lines was measured in a 6-hr 51Cr-release assay at an E/T ratio of 20 for cases 2, 3, 4, and 5 and of 40 for cases 1 and 6. Representative results of the repeated experiments on IL-2-activated TIL at around 7 weeks of culture are shown. b Autologous tumor cell line of case 4. c ne, no expression of HLA A alleles. d, e, f Autologous EBV-transformed B cell lines of cases 1, 2, and 6, respectively.
Cw1/ 5
0 0 0 0
0 0
0 0 0
2
0
0 2 0 3 11 4 0
6 5
0 3
Hepatocarcinoma
HAK-1B HAK-2 Hep-G2 PLC/PRF/5 SK-HEP-1 KMCH-1 K562 VA-13 100394 SOM 052594 ONI T2 TK EBV-BCLd NM EBV-BCLe OS EBV-BCL f Ban B1 COS-7
Liver Liver Liver Liver Liver Liver Erythroblast Fibroblast Keratinocyte Keratinocyte T/B cell B cell B cell B cell B cell
0
0 0 0 0
0
2
0
18 0
1 0
0
0 6 6
nec A0207/A2402 A2402/A3101 A0201/ A0201/A3101 A0206/A3302 A2402/ A3101/A3302
A0201/A3302 A0201/A1101 A0201/A2402 A3302/ A0201/A2402 A1101/A3101
B5101/ B44/48 B44/66 B46/52 B44/B51
B44031/B5401 B3801/ B3501/B5101 B4201/B5301 B44031/B3504 B5101/
Cw0102/ Cw3/
Cw0102/Cw1403 Cw1203/ Cw0401/Cw1602 Cw1701/ Cw0401/ Cw1402/
HLA-A LOCUS-RESTRICTED CTL IN LUNG CANCER
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line (LC1) and modestly lysed two allogenic tumor cell lines of HLA A3101/ stomach adenocarcinoma (MKN-28) and HLA A3302/ renal cell adenocarcinoma (KUR-11) (Table 2). LC1 and KUR-11 tumors were also lysed by TILs of case 5 (HLA A1101/3302). Case 5 showed cytotoxicity against the three histologically distinct, but all HLA A3302/ tumors (YT803, TE-9, and HAK-1B). None of the other cell lines were susceptible to lysis by these CTL lines. The levels of cytotoxicity against these susceptible tumors increased depending upon the increased numbers of added effector cells (Figs. 1B and 1C). The CTLs of case 5 produced 343, 870, 561, 1864, or 151 pg/ml of IFNg by recognition of LC1, YT803, KUR-11, TE-9, or HAK1B tumor, respectively, but did not produced detectable levels of IFN-g by recognition of any of the other 11 solid tumors tested that were not susceptible to lysis (Table 3). The percentage specific lysis of LC1 tumor cells by the case 4 CTL line in the presence of anti-CD3, -CD8, -class I, -CD4, -DR mAb, or an irrelevant mAb was 12, 9, 12, 22, 23, or 22% at an E/T ratio of 20, respectively. These results suggest that the IL-2-activated TILs of case 4 contained both HLA A3101- and A3302-restricted CTLs recognizing antigens on adenocarcinomas, whereas those of case 5 were HLA A3302-restricted CTLs recognizing antigen(s) expressed on histologicaly different tumors. IL-2-activated TILs of a patient with lung SCC (case 6, HLA A2402/) showed ú10% lysis against 7 HLA A2402/ tumor cell lines with different types of histology (two adenocarcinomas, four SCCs, and one small cell lung cancer). None of the other cell lines, including 20 HLA A2402/ tumor cell lines, were susceptible to lysis by this CTL line. The levels of cytotoxicity against all four susceptible tumors tested increased depending upon the increased numbers of effector cells (Fig. 1D). These results suggest that the IL-2-activated TILs of case 6 contained HLA A2402-restricted CTLs recognizing antigen(s) expressed on histologically different tumors. In contrast to clear HLA-A locus restriction in these six cases of CTL lines, no apparant HLA-B or -C locus restriction was observed in these cases. HLA-B and -C loci are not yet genotyped in all the patients except for case 4 and a part of the tumor cell lines in Table 2. Therefore, HLA-B or -C locus restriction needs further investigation after genotyping. IL-2-activated TILs of cases 7–9 failed to show a significant level (ú10%) of lysis to any of the tumor and normal cell lines tested at several different times at around 30 to 60 days of culture. Those of case 10, which contained large numbers of TCRgd/ cells, showed MHC-nonrestricted cytotoxicity during all culture periods from 30 to 60 days. For example, the IL2-activated TILs at 53 days of culture (34% TCRab/ and 64% TCRgd/) showed higher levels of lysis (13 to 86%, mean Å 41%) at an E/T ratio of 20:1 against all 24 tumor cell lines tested with different HLA-A alleles and 1 normal cell line (VA13). TILs of the remaining
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TABLE 3 Specificity of Cytokine Release IFN-g a (pg/ml) (% lysisb) Target cells Histology Adenocarcinoma
Squamous cell carcinoma
Hepatocarcinoma a b
Cell line
Origin
HLA A2/ A3302 expression
1-87 A549 11-18 PC-9 LC1 YT803 MKN-45 SSTW-9 SW620 HCT116 KM12LM COLO 201 COLO 320 R27 KUR-11 Sq-1 QG-56 TE-8 TE-9 TE-10 TE-11 KUMA-1 Ca9-22 HSC-2 HSC-3 HAK-1B HAK-2
Lung Lung Lung Lung Lung Lung Stomach Stomach Colon Colon Colon Colon Colon Breast Kidney Lung Lung Esophagus Esophagus Esophagus Esophagus Head and neck Oral Oral Oral Liver Liver
//0 0/0 //0 //0 0// 0// 0/0 0/0 //0 //0 //0 //0 0/0 //0 0// 0/0 0/0 0/0 0// //0 0/0 0// //0 0// //0 /// //0
Case 1 A0201/3101 18 õ5 235 40
(18) (3) (32) (24)
Case 5 A1101/3302 õ5 õ5 (3) 343 (25) 870 (15)
õ5 (1) õ5 (0) õ5 (0) õ5 (4) õ5 (0)
õ5 õ5 õ5 õ5 õ5 õ5
(3) (5) (0) (2) (0) (7)
õ5 õ5 õ5 õ5 õ5 õ5 õ5 561
(0) (6) (2) (0) (0) (1) (15)
1864 (26)
õ5 (0) õ5 (2) õ5 (2) õ5 (5) 151 (18) õ5 (0)
The TILs were incubated with target cells at an effector to target ratio of 2:1 for 18 hr, and IFN-g release was measured by ELISA. Cytotoxicity of the TILs from case 1 and case 5 was measured in a 6-hr 51Cr-release assay at an E/T ratio of 40 and 20, respectively.
six cases did not proliferate well under the employed conditions. Peptide fractions recognized by A2-restricted CTLs. Peptides binding to HLA class I molecules were eluted from an HLA A0201/ colon adenocarcinoma cell line (SW620) by acid treatment and were size-fractionated by Centricon-3 followed by partial purification with reverse-phase HPLC (Fig. 3A). Each of these fractions was pulsed to a T2 cell line, followed by measurement of their susceptibility to lysis by the CTL line of case 2. Experiments were repeated twice with consistent results. Five distinct HPLC fractions (fractions 13, 23, 27, 30, and 37) displayed modest but significant levels of the biological activity (Fig. 3B). In contrast, this CTL line failed to lyse T2 cells pulsed with MAGE-3-derived peptide (1% lysis), HER2/neu-derived peptide (0% lysis), or without pulsing any peptide (õ1% lysis) at an E/T ratio of 10. The CTL line of case 1 also failed to lyse T2 cells when pulsed with these two peptides. DISCUSSION We demonstrated in this study evidence of the existence of HLA-A locus-restricted and tumor-specific
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CTLs in IL-2-activated TILs in 6 of 16 patients with non-small cell lung cancer. Lung cancer is one of the most commonly observed cancers worldwide. However, there is no previous full report in the literature on the studies of HLA-A restriction and tumor specificity of the cytotoxicity of IL-2-activated TILs in lung cancer. This may be in part due to few available HLA class Igenotyped lung cancers and the other solid tumor cell lines that are needed to evaluate HLA-A restriction and tumor specificity of cytotoxicity of IL-2-activated TILs. To remove this obstacle, we provided 54 different solid tumor cell lines, including 15 lung cancers and 9 normal cell lines with which HLA-A loci are genotypically identified in most cases. HLA-B and -C loci of many tumor cell lines were also identified. The majority of these cell lines express HLA class I molecules on their surfaces as determined by staining with anti-HLA class I (W6/32) mAb (data not shown). The other reason for this obscurity may be the small numbers of CTL precursors in TILs of lung cancer. CTL precursors in melanoma TILs might be greater than those of lung cancer TILs, since the appearance in vitro of HLA-restricted and melanoma-specific CTLs in IL2-activated melanoma TILs is much faster than that
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FIG. 1. Cytotoxicity of the HLA A-restricted CTL lines. (A) HLA A2-restricted cytotoxity against HLA A0201/, A0206/, or A0207/ adenocarcinoma cell lines in the IL-2-activated TILs of case 1 (HLA A0201/A3101); (B) HLA A3101- and A3302-restricted cytotoxicity against HLA A3101/ and/or A3302/ adenocarcinoma cell lines in case 4 (HLA A3101/A3302); (C) HLA A3302-restricted cytotoxicity against HLA A3302/ tumors in case 5 (HLA A1101/A3302); and (D) HLA A2402-restricted cytotoxicity against HLA A2402/ tumors in case 6 (HLA A2402/). Representative results of a 6-hr 51Cr-release assay at three different E/T ratios (10, 20, and 40) at around 50 days of culture are shown.
in IL-2-activated lung cancer TILs. Namely, HLA-restricted CTL activity of the former or the latter was detectable at around 2 to 4 weeks, as reported (1) or 6 to 8 weeks shown herein in the culture with IL-2, respectively. The levels of lysis are also generally
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higher in the former than in the latter. Furthermore, HLA class I-restricted and tumor-specific cytotoxicity was observed in IL-2-activated TILs in a majority of melanoma patients (1, 2, 9, 10), but only in about onethird of lung cancer patients in our investigation. It is
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FIG. 2. Kinetics of cytotoxicity in IL-2-activated TILs of case 1 (HLA A0201/A3101). HLA A2-restricted CTL activity against 11–18 lung adenocarcinoma cell line was observed between 45 and 65 days of culture. Representative results of a 6-hr 51Cr-release assay at an E/T ratio of 40 are shown.
of note that the IL-2-activated TILs of one case with a high level of MHC-nonrestricted cytotoxicity possessed 64% TCRgd/ T cells, suggesting the existence of MHC-
nonrestricted immuno-responses at the tumor site in a few cases. In addition to an LC1 tumor of case 4, HLA-A-geno-
FIG. 3. Peptide fractions recognized by the HLA A2-restricted CTL line. (A) HPLC fractionation of SW620 peptides. Peptides were eluted from the SW620 tumor cell line (HLA A0201/ colon adenocarcinoma cell line) by pH 3.3 acid treatment followed by fractionation on reverse-phase HPLC in a 0–35% acetonitrile gradient. (B) T2 cell line was pulsed with peptides eluted from the SW620 tumor cell line, followed by testing their susceptibility to lysys by the HLA A2-restricted CTL line of case 2 (HLA A0206/A3302). Representative results of a 6-hr 51Cr-release assay at an E/T ratio of 10:1 are shown. Lysis of a negative control [peptide (—)] was less than 1%.
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typed allogenic tumors were used in these studies as target tumors since there were no available autologous tumor cells. Therefore, the method employed in this study does not provide direct evidence that the IL-2activated TILs, except for case 4, are cytotoxic against autologous tumor cells, even though they showed HLAA locus-restricted and tumor-specific cytotoxicity. The level of cytotoxicity of these CTLs was relatively low. This was possibly due to the fact that only a portion of the CTL lines (15 to 52% except for 97% in case 5) were CD8/ T cells. In addition, this may also be in part due to the low affinity of the peptide antigens binding to the HLA-A alleles recognized by these CTL lines (12, 24). Four, 6, or 4 of 15 HLA A2/ adenocarcinoma cell lines established from different organs (lung, stomach, colon, and breast), but none of the HLA A2/ SCC, hepatocellular carcinomas, or normal cell lines tested, were susceptible to lysis by the HLA A2-restricted CTL lines of cases 1, 2, or 3, respectively. The 4 of A2/ adenocarcinoma cell lines (three from lung and one from colon) were commonly recognized by all 3 CTL lines. These results suggest that the CTL lines of these three cases recognize antigens binding to the HLA A2 molecules of adenocarcinomas from distinct organs. The results also suggest the presence of tumor antigens shared by adenocarcinomas like MART-1, gp100, or the other melanoma shared antigens (4). Furthermore, the CTLs from cases 1, 5, and 6 lysed histologically distinct tumors in an HLA-A locus-restricted manner, suggesting the presence of tumor antigens shared by distinct tumors, like MAGE, GAGE, and BAGE (4). The subtypes of the HLA A2 molecules of adenocarcinoma cell lines susceptible to lysis by the CTL lines of cases 1 (A0201), 2 (A0206), and 3 (A0201) were HLA A0201, A0206, and A0207. The CTL of case 2 recognized five different fractions of antigenic peptides eluted from an HLA A0201/ SW620 adenocarcinoma cell line. Binding motifs of peptides to these three HLA A2 subtypes were closely related, and some of them were identical (25, 26). Storkus et al. reported that HLA A0201 molecules presented at least six shared antigenic peptides recognized by HLA A0201-restricted and melanoma-specific CTL lines (22). HLA A2-restricted CTL lines from HLA A2/ patients with ovarian cancers or breast cancers recognized HER2/neu-derived peptide (6, 7), whereas the HLA A2-restricted CTL lines of case 1 and case 2 did not recognize either HER2/neuor MAGE-3-derived peptide on an HLA A0201/ T2 cell line. Our results, along with these observations, indicate that at least several different peptides, except for HER2/neu- and MAGE-3-derived peptides, capable of binding to HLA A2 molecules of adenocarcinomas, may be recognized by these IL-2-activated TILs. Antigen frequencies of HLA-A2 in Yugoslavian (Caucasians), North American (Amerinds), South African (Blacks in Cape Town), and Japanese are 45.2, 44.5,
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30.3, and 42.8%, respectively, while those of HLA-A24 (A31/A33) are 20.3% (3.0%/3.0%), 35.4% (47.4%/2.0%), 12.0% (2.8%/11.6%), and 57.9% (15.4%/14.8%), respectively (27). HLA A0201, A2402, A3101, and A3302 are the most frequently expressed in Japanese. HLA A0201 along with A0206 and A0207 share more than 95% of serologically defined HLA A2-positive individuals. HLA A2402, A3101, and 3302 share more than 95% of A24, A31, and A33, respectively (15). This study showed evidence for the existence of HLA-A locus (A2, A2402, A3101, and A3302)-restricted and tumor-specific CTLs in IL-2-activated TILs of a substantial number of patients with non-small cell lung cancer. These findings may be important for understanding immunorecognition of lung cancer cells at tumor sites. Large numbers of patients with non-small cell lung cancer could be treated via specific immunotherapy with CTLs and/or tumor antigens. REFERENCES 1. Itoh, K., Platsoucas, C. D., and Balch, C. M., J. Exp. Med. 168, 1419, 1988. 2. Rosenberg, S. A., Packard, B. S., Aebersold, P. M., Solomon, D., Topalian, S. L., Toy, S. T., Simon, P., Lotze, M. T., Yang, J. C., Seipp, C. A., Simpson, C., Carter, C., Bock, S., Schwartzentruber, D., Wei, J. P., and White, D. E., N. Engl. J. Med. 319, 1676, 1988. 3. Nakao, M., Yamana, H., Imai, Y., Toh, Y., Toh, U., Kimura, A., Yamana, S., Kakegawa, T., and Itoh, K., Cancer Res. 55, 4248, 1995. 4. Boon, T., Gajewski, T. F., and Coulie, P. G., Immunol. Today 16, 334, 1995. 5. Ioannides, C. G., Fisk, B., Jerome, K. R., Irimura, T., Wharton, J. T., and Finn, O. J., J. Immunol. 151, 3793, 1993. 6. Yoshino, I., Peoples, G. E., Goedegebuure, P. S., Maziarz, R., and Eberlein, T. J., J. Immunol. 152, 2393, 1994. 7. Linehan, D. C., Goedegebuure, P. S., Peoples, G. E., Rogers, S. O., and Eberlein, T. J., J. Immunol. 155, 4486, 1995. 8. van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., de Plaen, E., van der Eynde, B., Knuth, A., and Boon, T., Science 254, 1643, 1991. 9. Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Rivoltini, L., Topalian, S. L., Miki, T., and Rosenberg, S. A., Proc. Natl. Acad. Sci. USA 91, 3515, 1994. 10. Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Sakaguchi, K., Appella, E., Yannelli, J. R., Adema, G. J., Miki, T., and Rosenberg, S. A., Proc. Natl. Acad. Sci. USA 91, 6458, 1994. 11. Coulie, P. G., Brichard, V., Van Pel, A., Wolfel, T., Schneider, J., Traversari, C., Mattei, S., de Plaen, E., Lurquin, C., Szikora, J. P., Renauld, J. C., and Boon, T., J. Exp. Med. 180, 35, 1994. 12. Rivoltini, L., Kawakami, Y., Sakaguchi, K., Southwood, S., Sette, A., Robbins, P. F., Marincola, F. M., Salgaller, M. L., Yannelli, J. R., Appella, E., and Rosenberg, S. A., J. Immunol. 154, 2257, 1995. 13. Slingluff, C. L., Jr., Cox, A. L., Stover, J. M., Jr., Moore, M. M., Hunt, D. F., and Englehard, V. H., Cancer Res. 54, 2731, 1994. 14. Itoh, K., Tilden, A. B., and Balch, C. M., Cancer Res. 45, 3173, 1986. 15. Date, Y., Kimura, A., Kato, H., and Sasazuki, T., Tissue Antigens 47, 93, 1996.
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16. Fernandez-Vina, M., Lazalo, A. M., Sun, Y., Miller, S., Ferero, L., and Stastny, P., Tissue Antigens 45, 153, 1995. 17. Bunce, M., O’neill, C. M., Barnardo, M. C. N. M., Kurausa, P., Browning, M. J., Morris, P. J., and Welsh, K. I., Tissue Antigens 46, 355, 1995. 18. Hoshino, T., Seki, N., Kikuchi, M., Kuramoto, T., Iwamoto, O., Kodama, I., Koufuji, K., Takeda, J., and Itoh, K., Int. J. Cancer, in press, 1997. 19. Tatari, Z., Fortier, C., Bobrynina, V., Loiseau, P., Charron, D., and Raffoux, C., Proc. Natl. Acad. Sci. USA 92, 8803, 1995. 20. Hoshino, T., Yamada, A., Honda, J., Imai, Y., Nakao, M., Inoue, M., Sagawa, K., Yokoyama, M. M., Oizumi, K., and Itoh, K., J. Immunol. 151, 2237, 1993. 21. Kharkevitch, D. D., Seito, D., Balch, G. C., Maeda, T., Balch, C. M., and Itoh, K., Int. J. Cancer 58, 317, 1994.
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22. Storkus, W. J., Zeh, H. J., III, Maeurer, M. J., Salter, R. D., and Lotze, M. T., J. Immunol. 151, 3719, 1993. 23. van der Bruggen, P., Bastin, J., Gajewski, T., Coulie, P. G., Boe¨l, P., De Smet, C., Traversari, C., Townsend, A., and Boon, T., Eur. J. Immunol. 24, 3038, 1994. 24. Fairchild, P. J., and Wraith, D. C., Immunol. Today 17, 80, 1996. 25. del Guercio, M. F., Sidney, J., Hermanson, G., Perez, C., Grey, H. M., Kubo, R. T., and Sette, A., J. Immunol. 154, 685, 1995. 26. Sudo, T., Kamikawaji, N., Kimura, A., Date, Y., Savoie, C. J., Nakashima, H., Furuichi, E., Kuhara, S., and Sasazuki, T., J. Immunol. 155, 4749, 1995. 27. Imanishi, T., Akaza, T., Kimura, A., Tokunaga, K., and Gojobori, T., in ‘‘HLA,’’ 1991 Vol. 1, Oxford Scientific Publications, Oxford, 1992.
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HLA-A Locus-Restricted and Tumor-Specific CTLs in Tumor-Infiltrating Lymphocytes of Patients with Non-Small Cell Lung Cancer1 NAOKO SEKI,* TOMOAKI HOSHINO,* MEGUMI KIKUCHI,* AKIHIRO HAYASHI,†
AND
KYOGO ITOH*,2
*Department of Immunology and †First Department of Surgery, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan Received September 3, 1996; accepted November 26, 1996
HLA class I restriction and tumor specificity of cytotoxicity in the IL-2-activated tumor-infiltrating lymphocytes from 16 patients with non-small cell lung cancer were investigated. Six HLA class I-restricted and tumor-specific CTL lines were established: (i) HLA A2restricted and adenocarcinoma-specific CTLs in three (two A0201/ and one A0206/) patients with adenocarcinoma, (ii) HLA A3101- and A3302-restricted and adenocarcinoma-specific CTLs in an HLA A3101/3302/ patient with adenocarcinoma, and (iii) HLA A3302-restricted CTLs and (iv) HLA A2402-restricted CTLs recognizing tumors with different types of histology in an HLA A3302/ patient with adenocarcinoma and an HLA A2402/ patient with squamous cell carcinoma (SCC), respectively. The three HLA A2-restricted CTL lines recognized 4, 4, or 6 of 15 HLA A2/ adenocarcinoma cell lines that originated from lung, stomach, colon, and breast with different subtypes (HLA A0201, A0206, and A0207), respectively. Furthermore, the CTLs of an HLA A0206/ patient recognized five different fractions of peptides eluted from an HLA A0201/ adenocarcinoma cell line. These results showed evidence of the existence of HLA class I-restricted and tumor-specific CTLs recognizing peptide antigens on HLA-A alleles of adenocarcinoma or SCC in tumor sites of a substantial number of patients with non-small cell lung cancer. q 1997 Academic Press
INTRODUCTION Non-small cell lung cancers are relatively resistant to currently available chemotherapy and radiotherapy 1
Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan, a grant from the Science Research Promotion Fund of the Japanese Private School Promotion Foundation, the Fukuoka Cancer Society, the Mochida Memorial Foundation, and the Japanese Foundation for Multidisciplinary Treatment of Cancer. 2 To whom correspondence should be addressed at the Department of Immunology, Kurume University School of Medicine, 67 Asahimachi, Kurume 830, Japan. Fax: 81-942-31-7699. E-mail: [email protected].
regimens and one of the malignant tumors with an extremely poor prognosis. Therefore, development of specific immunotherapy using tumor-specific CTLs3 or tumor antigens is important for improving treatment modalities. HLA class I-restricted and tumor-specific CTLs have been observed in T cells of peripheral blood mononuclear cells (PBMC) stimulated with autologous tumor cells or in IL-2-activated tumor-infiltrating lymphocytes (TILs) of patients with melanomas, esophageal cancers, ovarian cancers, and breast cancers (1– 6). Genes encoding peptide antigens recognized by CTLs have been cloned from melanomas (4–12). However, the presence of these CTLs or tumor antigens has been rarely reported in human lung cancers (13). In this investigation, we have studied HLA class I restriction and tumor specificity of cytotoxicity of IL-2-activated TILs from patients with non-small cell lung cancer. The results suggest the presence of HLA class Irestricted and tumor-specific CTLs recognizing peptide antigens on HLA-A molecules of adenocarcinoma or squamous cell carcinoma (SCC) in tumor sites of a substantial number of patients with lung cancer. MATERIALS AND METHODS Subjects and tumor cell lines. Tumor specimens or carcinomatous pleural effusion and peripheral blood were obtained from 16 patients with non-small cell lung cancer at the time of major surgery at the Kurume University Hospital. The profiles of 10 patients who displayed significant TIL proliferation and a summary of CTL activity are shown in Table 1. The TILs of the other 6 patients did not proliferate well under the employed conditions. All research followed the tenets of the Declaration of Helsinki. Autologous tumor cell line, LC1 was established from pleural effusion of a patient 3 Abbreviations used: CTL, cytotoxic T lymphocyte; EBV-BCL, Epstein–Barr virus-transformed B cell line; IL-2, interleukin-2; IFNg, interferon-g; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cell; SCC, squamous cell carcinoma; TIL, tumor-infiltrating lymphocyte.
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0008-8749/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
102 85 41 53 72 3 40 NT 92 NT 25 15 52 41 22 97 48 NT 8 NT 11 100 96 94 96 100 83 NT 100 NT 98 0 3 0 2 0 0 NT 0 NT 64 100 93 94 94 100 83 NTe 100 NT 34 Adeno/SCC Adeno Adeno Adeno Adeno/SCC SCC/adeno — — — Non g a
TILs of the six other cases (cases 11 to 16) did not proliferate well. Adeno, adenocarcinoma. c SCC, squamous cell carcinoma. d No cytotoxicity: TILs failed to show a significant level (ú10%) of lysis against any cell lines tested. e NT, not tested. f Mix, mixed tumor of adenocarcinoma and SCC. g MHC nonrestricted cytotoxicity was observed against all cell lines tested including one normal cell line.
I/T2N0M0 VI/T2N2M1 IIIa/T3N0M0 IIIb/T4N2M0 IV/T3N2M1 I/T2N0M0 IIIa/T3N1M0 I/T1N0M0 IIIa/T3N0M0 IIIa/T3N2M0 Adenob Adeno Adeno Adeno Adeno SCCc Adeno Adeno Mix f SCC 70/F 71/M 53/M 83/M 73/M 68/F 68/M 68/F 66/M 69/M 1 2 3 4 5 6 7 8 9 10
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b
Cw1/Cw3 Cw0303/Cw1403 Cw7/ Cw1/ Cw1/ Cw7/ Cw7/ Cw7/
B44/B48 B44/B66 B59/B62 B1511/B44031 B67/B44 B46/B52 B52/54 B7/B52 B7/B52 B52/B60
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Tumor Pleural effusion Tumor Pleural effusion Pleural effusion Tumor Tumor Tumor Tumor Tumor
A0201/A3101 A0206/A3302 A0201/A2402 A3101/A3302 A1101/A3302 A2402/ A24/ A2402/ A24/ A26/A24
Cw3/
C B A T cell source Stage/TNM Histology Age/sex Casea (n Å 10/16)
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A2/A3101 A2 A2 A3101/A3302 A3302 A2402 —d — — Non g
CD8 CD3 TCRgd Tumor specificity
HLA-A locus restriction of CTL HLA class I
TABLE 1
Patient Profiles and Summary of IL-2-Activated TILs from Patients with Lung Cancer
TCRab
Surface phenotype (%)
CD4
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(case 4) as previously described (14). Most of the allogenic tumor cell lines and normal cell lines used in these studies and their MHC-class I alleles have been previously reported (3). The cell lines used were HST-2 and SSTW-9 gastric cancers provided by Dr. Sato (Sapporo Medical College, Sapporo, Japan); a 1-87 lung cancer provided by Dr. Kobayashi (Tohoku University, The Research Institute for Tuberculosis and Cancer, Sendai, Japan); a YT803 lung cancer provided by Dr. Nomoto (Kyushu University, Fukuoka, Japan); an SKG-II cervical cancer provided by Dr. Nozawa (Keio University School Medicine, Tokyo, Japan); and an HAK-2 hepatocellular carcinoma and a KMCH-1 hepatocholangiocarcinoma from Dr. Kojiro of our University. The other cell lines listed in Table 2 were either established in our laboratories or purchased from JCRB (Tokyo, Japan), American Type Culture Collection (ATCC, Rockville, MD), and Morinaga Co. (Tokyo, Japan). Genotypes of HLA-A, -B, and -C alleles of tumor cell lines and patients’ PBMC were determined by the polymerase chain reaction sequence-specific oligonucleotide probe (PCR-SSOP) method, as reported with a slight modification (15–17). Sequences of the HLAA allele of SW620, R27, and KE-4 cell lines were also analyzed by the reverse-transcriptase PCR method, as previously described (18). HLA-C alleles of some tumor cell lines (TE-8, TE-10, and TE-11) were determined by the PCR restriction fragment-length polymorphism (RELP) method (19). The results from serological typing of PBMCs determined by HLA Monoclonal Reagent (One Lambda, Canoga Park, CA) were shown when genotyping data were not available. TIL preparation, expansion, and functional assays. Tumors were finely minced using surgical blades. Lymphocytes and tumor cells from pleural effusions were centrifuged and isolated by Ficoll–Conray gradient separation. These pasted mixtures of tumor cells, tissue cells and TILs, or cells of pleural effusions were then extensively washed and incubated with RPMI 1640 medium (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (Whittaker, Walkersville, MA), 0.1 mM MEM nonessential amino acids solution (GIBCO BRL), and 100 units/ml of recombinant IL-2 (a generous gift from Shionogi Pharmaceutical Co., Osaka, Japan) at 377C and in a 5% atmosphere of CO2 for up to 11 weeks. The surface phenotypes of the proliferating TILs were studied by direct immunofluorescence assay with FITC-conjugated anti-TCRab, -TCRgd (Becton–Dickinson Inc., Mountain View, CA), -CD3, -CD4, and -CD8 (Nichirei, Tokyo) monoclonal antibodies (mAbs) and FACScan (Becton–Dickinson), as reported (20). The results at around 50 days of culture are shown in Table 1. The IL-2-activated TILs of 10 cases were serially tested for their cytotoxicity against a panel of HLA class I-identified tumor cell and normal cell lines shown
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in Table 2. Except for an LC1 tumor that was established from case 4, all were allogenic tumor cell lines. Autologous tumor cell lines were not available from the other 9 cases due to the lack of tumor cell proliferation in vitro in culture. A cytotoxic assay against each of the target cells was repeated at least three times in most tumor cell lines in separate experiments. Representative results at around 7 weeks of culture are shown in Table 2. A standard 6-hr 51Cr-release assay was used to measure CTL activity, as reported (21). If the mean of triplicate determinants of percentage specific lysis exceeded 10%, it was evaluated as a significant level of cytotoxicity in this study. Inhibition experiments by anti-CD3 (OKT3) (ATCC), -CD4 (NUTh/i), -CD8 (NU-Ts/c), -CD14 (H14), anti-HLA class I (W6/32) (ATCC), and anti-HLA DR (H-DR1) mAb were performed as described previously (3, 21). Interferon-g (IFN-g) production by IL-2-activated TILs in response to various tumor cells was also measured. Briefly, IL2-activated TILs were incubated with target cells at an effector to target cell (E/T) ratio of 2:1 for 18 hr. The amounts of IFN-g in cell-free supernatants were measured using an IFN-g ELISA kit (Ohtsuka Assay, Tokushima, Japan; limit of sensitivity, 5 pg/ml). Peptide extraction and synthesis. Peptides were extracted and purified from an HLA A0201/ colon adenocarcinoma cell line (SW620) using the pH 3.3 acid elution technique, as reported (22). Briefly, 5 1 108 cell equivalents of extracted peptide were pretreated with C18 Sep Pak (Waters, Milford, MA), prefractionated with Centricon-3 (Amicon, Beverly, MA) (consisting of peptides £3000 Mr , É30 amino acids in length), and fractionated with reverse-phase HPLC. HPLC solvents were A Å 99.92% water/0.08% trifluoroacetic acid and B Å 99.94% acetonitrile/0.06% trifluoroacetic acid. Gradients consisted of the following linear step intervals: isocratic A solvent for 0 to 5 min; 0 to 10% B (in A) from 5 to 10 min; and 10 to 35% B (in A) from 10 to 60 min. The flow rate was 0.8 ml/min. Individual HPLC fractions were lyophilized and reconstituted in 200 ml of HBSS. HLA-A2 binding HER2/neuderived peptide (IISAVVGIL, GP2) (7) and MAGE-3-derived peptide (FLWGPRALV) were synthesized as reported (23). Peptides were dissolved in aliquots of HBSS at a concentration of 1 mg/ml and stored at 0807C. These peptides were used at a concentration of 20 mg/ml in the peptide-pulsed cytotoxicity assay using an HLA A0201/ T2 cell line (7) where 20 ml of individual HPLC fractions or synthesized peptides was added to a well containing 100 ml of 51Cr-labeled 1 1 104 T2 cells, followed by incubation for 1 hr at 377C to allow the binding of peptides to target cells. Then, 1 1 105 CTLs were added to the well followed by harvesting 6 hr later. RESULTS HLA-A2-restricted CTL lines. The CTL lines from three HLA-A2 patients with lung adenocarcinoma
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(case 1, A0201/3101; case 2, A0206/3302; and case 3, A0201/2402) displayed a similar pattern of cytotoxicity. They showed a significant level (ú10%) of lysis against three lung adenocarcinomas (A0207/ 1-87, A0201/ 1118, and A0206/ PC-9 cell lines) and a colon adenocarcinoma (A0201/ SW620) (Table 2). Recognition of these lung adenocarcinomas by the CTL line of case 1 was confirmed by the assay of IFN-g production. Namely, it produced 18, 235, or 40 pg/ml of IFN-g by recognition of 1-87, 11-18, or PC-9 tumor, respectively, but did not produce detectable levels (ú5 g/ml) of IFN-g by recognition of any of the other 15 solid tumors tested that were not susceptible to lysis (Table 3). The CTL line of case 1 (A0201/3101) also lysed three histologically different but all A3101/ tumors (LC1 lung adenocarcinoma, HSC-4 oral SCC, and KMCH-1 hepatocholangiocarcinoma), while that of case 2 (A0206/3302) lysed A3302/ LC1 lung, A2/ KATO-III stomach, and A0201/ R27 breast adenocarcinomas. In contrast, none of the other cell lines including nine HLA A2/ adenocarcinoma cell lines (established from stomach, colon, pancreas, kidney, or ovarium) and three A2/ normal cell lines, autologous B cell lines, and K562 tumor cells were susceptible to lysis by any of these CTL lines (Table 2). Cytotoxicity of all three cases against most of the cell lines was confirmed at three different E/T ratios, and the representative results of case 1 are shown in Fig. 1A. The levels of cytotoxicity increased depending upon the increased numbers of effector cells. Kinetic study showed that the IL-2-activated TILs of case 1 consistently lysed an HLA A0201/ 11-18 lung adenocarcinoma cell line between 45 and 65 days of culture, although they showed nonspecific lysis around 20 to 30 days of culture probably due to the presence of lymphokine-activated killer cells in the culture (Fig. 2). Similar kinetics were obtained in the IL-2-activated TILs of cases 2 and 3 (data not shown). Cytotoxicity of the CTL line of case 1 was inhibited by anti-CD3, antiCD8, or anti-HLA class I, but not by anti-CD4 or antiHLA class II (DR) mAb. Namely, the percentage specific lysis of an HLA A0201/ lung adenocarcinoma cell line (11-18) by this CTL was 1, 21, 14, 31, or 33% lysis in the presence of each mAb, respectively, versus 36% lysis with an irrelevant mouse mAb (CD14) at an E/T ratio of 40. All these results indicate that the IL-2-activated TILs of cases 1, 2, and 3 commonly exhibited HLA A2restricted lysis against three lung adenocarcinomas with A0201, 0206, and 0207 subtypes and one HLA A0201/ colon adenocarcinoma. In addition, the unique pattern of cytotoxicity was observed in the CTL lines of cases 1 and 2. CTL lines restricted to HLA-A other than A2 allele. IL-2-activated TILs of case 4 (HLA A3101/3302) showed a higher level of lysis against the autologous tumor cell
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RERF-LC-MS 1-87 A549 11-18 PC-9 LC1b YT803 KWS KATO-III MKN-45 MKN-28 SSTW-9 HST-2 SW620 HCT116 KM12LM COLO 201 COLO 205 COLO 320 R27 Panc-1 KMG-A KUR-11 KUR-20 KOC-7C LC-99A 86-2 LC-65A LK79 Sq-1 RERF-LC-AI QG-56 LC-lsq KE-3 KE-4 TE-8 TE-9 TE-10 TE-11 KUMA-1 Ca9-22 HSC-2 HSC-3 HSC-4 SAS SKG-II HCS
Cell line
Target cells
Squamous cell carcinoma
Small cell carcinoma
Large cell carcinoma
Adenocarcinoma
Histology
TABLE 2
Lung Lung Lung Lung Lung Lung Lung Stomach Stomach Stomach Stomach Stomach Stomach Colon Colon Colon Colon Colon Colon Breast Pancreas Gall bladder Kidney Kidney Ovarium Lung Lung Lung Lung Lung Lung Lung Lung Esophagus Esophagus Esophagus Esophagus Esophagus Esophagus Head and neck Oral Oral Oral Oral Oral Uterus Uterus
Origin
3 6 5 3 0 1 0 2 0 7 6 2 2 5 12 2 0 0
1 0 6 5
0 3 1 2 0 8 15 0 6 4 0 0 6 4 5
6 18 3 32 24 30
Case 1 A0201/3101
0
0 2 1 0 0 0 0 0 0 6 4 0 4 0 0
2 11 0 0 0 0 23 0 0 0 0 0 16 0 0 0 0 4 0 7 1
0 31 0 11 16 21
Case 2 A0206/3302
5
0 0
8
0
1 6 0 0
26
5 5
0
3
0 3
15
2 0 0 0 1 4
6
0 0
25 15
0 3
5
Case 5 A1101/3302
0
2 8 3 4 1 8 2 2
0 0 1 14 1 0 6 0 5 0 0 0 1 4 5 11
3 5 0 4 0 29
Case 4 A3101/3302
0 0
1
0 2
2 0
0 0
16 0 0 0 0 0
0 0
0 4
0 22 4 10 16
Case 3 A2402/0201
Adeno
% cytotoxicitya
7 8 3 17 18 5 8 0 4 8 6 3 4 16 3 6 26 3 5 12 8
0
5 0 10 4 4 5
6 6 6 0
1 5 4 11 6
SCC Case 6 A2402/ A1101/ A0207/A1101 A2603/A3001 A0201/A2402 A0206/A2402 A3101/A3302 A3101/A3302 A0206/ A2/A2402 A2402/ A3101/ A2402/A2601 A0206/A3101 A0201/A2402 A0101/A0201 A2/A2402 A0101/A0201 A0101/A0201 A2402/ A0201/ A0201/A1101 A2601/ A2402/A3302 A0201/A0210 A0201/A3101 A2402/ A1101/ A1101/A2402 A2402/ A1101/A2402 A2402/ A2601/ A11/A2402 A0206/A2402 A2402/A2601 A2402/A2601 A3302/ A2/A2402 A2402/A2601 A2603/A3302 A0207/A2402 A2402/A3302 A0201/A2402 A2402/A3101 A2402/ A2402/ A2402/
A
B5201/
B1501/B44031 B35/B4601 B44031/B5401 B4801/B5201 B0702/B5101 B5201/
B55/ B1503/
B51/B52 B5201/B5401 B5101/ B4002/ B4002/ B1501/5201 B5201/ B4601/ B44031/
B5101/ B1501/B4601 B3802/B5502 B0702/B1518 B4501/ B0702/ B0702/0801 B0702/0801 B1402/ B4402/ B3801/ B4002/
B5401/ B4601/B5401 B1801/B44031 B5201/B5401 B0702/B5502 B1511/B44031 B44031/B5101 B1301/ B1501/B4601
B
C
Cw1202/
Cw0302/0102 Cw1401/ Cw0303/Cw1403 Cw0102/Cw1402 Cw0102/Cw1403 Cw0803/Cw1202 Cw0702/Cw1402 Cw1202/
Cw0102/Cw1202 Cw0401/Cw1502 Cw0304/ Cw0303/ Cw0401/Cw1202 Cw1202/ Cw0102/ Cw1203/Cw1601 Cw0401/Cw1502 Cw0101/Cw0302 Cw0102/
Cw0304/ Cw0102/Cw0303 Cw0102/Cw0702 Cw0702/Cw0704 Cw0501/Cw0701 Cw0702/ Cw0701/Cw0702 Cw0701/Cw0702 Cw0802/ Cw0501/ Cw1203/ Cw0304/
Cw0102/ Cw0102/ Cw1203/Cw1601 Cw0102/Cw1201 Cw0303/Cw0702 Cw0303/Cw1403 Cw1402/Cw1403 Cw0303/ Cw0102/Cw0303
HLA class I of target cells
HLA-A Locus-Restricted Cytotoxicity of CTL Lines Derived from TILs of Patients with Lung Cancer
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a Cytotoxicity of the TILs from six patients with lung cancer against different types of cell lines was measured in a 6-hr 51Cr-release assay at an E/T ratio of 20 for cases 2, 3, 4, and 5 and of 40 for cases 1 and 6. Representative results of the repeated experiments on IL-2-activated TIL at around 7 weeks of culture are shown. b Autologous tumor cell line of case 4. c ne, no expression of HLA A alleles. d, e, f Autologous EBV-transformed B cell lines of cases 1, 2, and 6, respectively.
Cw1/ 5
0 0 0 0
0 0
0 0 0
2
0
0 2 0 3 11 4 0
6 5
0 3
Hepatocarcinoma
HAK-1B HAK-2 Hep-G2 PLC/PRF/5 SK-HEP-1 KMCH-1 K562 VA-13 100394 SOM 052594 ONI T2 TK EBV-BCLd NM EBV-BCLe OS EBV-BCL f Ban B1 COS-7
Liver Liver Liver Liver Liver Liver Erythroblast Fibroblast Keratinocyte Keratinocyte T/B cell B cell B cell B cell B cell
0
0 0 0 0
0
2
0
18 0
1 0
0
0 6 6
nec A0207/A2402 A2402/A3101 A0201/ A0201/A3101 A0206/A3302 A2402/ A3101/A3302
A0201/A3302 A0201/A1101 A0201/A2402 A3302/ A0201/A2402 A1101/A3101
B5101/ B44/48 B44/66 B46/52 B44/B51
B44031/B5401 B3801/ B3501/B5101 B4201/B5301 B44031/B3504 B5101/
Cw0102/ Cw3/
Cw0102/Cw1403 Cw1203/ Cw0401/Cw1602 Cw1701/ Cw0401/ Cw1402/
HLA-A LOCUS-RESTRICTED CTL IN LUNG CANCER
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line (LC1) and modestly lysed two allogenic tumor cell lines of HLA A3101/ stomach adenocarcinoma (MKN-28) and HLA A3302/ renal cell adenocarcinoma (KUR-11) (Table 2). LC1 and KUR-11 tumors were also lysed by TILs of case 5 (HLA A1101/3302). Case 5 showed cytotoxicity against the three histologically distinct, but all HLA A3302/ tumors (YT803, TE-9, and HAK-1B). None of the other cell lines were susceptible to lysis by these CTL lines. The levels of cytotoxicity against these susceptible tumors increased depending upon the increased numbers of added effector cells (Figs. 1B and 1C). The CTLs of case 5 produced 343, 870, 561, 1864, or 151 pg/ml of IFNg by recognition of LC1, YT803, KUR-11, TE-9, or HAK1B tumor, respectively, but did not produced detectable levels of IFN-g by recognition of any of the other 11 solid tumors tested that were not susceptible to lysis (Table 3). The percentage specific lysis of LC1 tumor cells by the case 4 CTL line in the presence of anti-CD3, -CD8, -class I, -CD4, -DR mAb, or an irrelevant mAb was 12, 9, 12, 22, 23, or 22% at an E/T ratio of 20, respectively. These results suggest that the IL-2-activated TILs of case 4 contained both HLA A3101- and A3302-restricted CTLs recognizing antigens on adenocarcinomas, whereas those of case 5 were HLA A3302-restricted CTLs recognizing antigen(s) expressed on histologicaly different tumors. IL-2-activated TILs of a patient with lung SCC (case 6, HLA A2402/) showed ú10% lysis against 7 HLA A2402/ tumor cell lines with different types of histology (two adenocarcinomas, four SCCs, and one small cell lung cancer). None of the other cell lines, including 20 HLA A2402/ tumor cell lines, were susceptible to lysis by this CTL line. The levels of cytotoxicity against all four susceptible tumors tested increased depending upon the increased numbers of effector cells (Fig. 1D). These results suggest that the IL-2-activated TILs of case 6 contained HLA A2402-restricted CTLs recognizing antigen(s) expressed on histologically different tumors. In contrast to clear HLA-A locus restriction in these six cases of CTL lines, no apparant HLA-B or -C locus restriction was observed in these cases. HLA-B and -C loci are not yet genotyped in all the patients except for case 4 and a part of the tumor cell lines in Table 2. Therefore, HLA-B or -C locus restriction needs further investigation after genotyping. IL-2-activated TILs of cases 7–9 failed to show a significant level (ú10%) of lysis to any of the tumor and normal cell lines tested at several different times at around 30 to 60 days of culture. Those of case 10, which contained large numbers of TCRgd/ cells, showed MHC-nonrestricted cytotoxicity during all culture periods from 30 to 60 days. For example, the IL2-activated TILs at 53 days of culture (34% TCRab/ and 64% TCRgd/) showed higher levels of lysis (13 to 86%, mean Å 41%) at an E/T ratio of 20:1 against all 24 tumor cell lines tested with different HLA-A alleles and 1 normal cell line (VA13). TILs of the remaining
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TABLE 3 Specificity of Cytokine Release IFN-g a (pg/ml) (% lysisb) Target cells Histology Adenocarcinoma
Squamous cell carcinoma
Hepatocarcinoma a b
Cell line
Origin
HLA A2/ A3302 expression
1-87 A549 11-18 PC-9 LC1 YT803 MKN-45 SSTW-9 SW620 HCT116 KM12LM COLO 201 COLO 320 R27 KUR-11 Sq-1 QG-56 TE-8 TE-9 TE-10 TE-11 KUMA-1 Ca9-22 HSC-2 HSC-3 HAK-1B HAK-2
Lung Lung Lung Lung Lung Lung Stomach Stomach Colon Colon Colon Colon Colon Breast Kidney Lung Lung Esophagus Esophagus Esophagus Esophagus Head and neck Oral Oral Oral Liver Liver
//0 0/0 //0 //0 0// 0// 0/0 0/0 //0 //0 //0 //0 0/0 //0 0// 0/0 0/0 0/0 0// //0 0/0 0// //0 0// //0 /// //0
Case 1 A0201/3101 18 õ5 235 40
(18) (3) (32) (24)
Case 5 A1101/3302 õ5 õ5 (3) 343 (25) 870 (15)
õ5 (1) õ5 (0) õ5 (0) õ5 (4) õ5 (0)
õ5 õ5 õ5 õ5 õ5 õ5
(3) (5) (0) (2) (0) (7)
õ5 õ5 õ5 õ5 õ5 õ5 õ5 561
(0) (6) (2) (0) (0) (1) (15)
1864 (26)
õ5 (0) õ5 (2) õ5 (2) õ5 (5) 151 (18) õ5 (0)
The TILs were incubated with target cells at an effector to target ratio of 2:1 for 18 hr, and IFN-g release was measured by ELISA. Cytotoxicity of the TILs from case 1 and case 5 was measured in a 6-hr 51Cr-release assay at an E/T ratio of 40 and 20, respectively.
six cases did not proliferate well under the employed conditions. Peptide fractions recognized by A2-restricted CTLs. Peptides binding to HLA class I molecules were eluted from an HLA A0201/ colon adenocarcinoma cell line (SW620) by acid treatment and were size-fractionated by Centricon-3 followed by partial purification with reverse-phase HPLC (Fig. 3A). Each of these fractions was pulsed to a T2 cell line, followed by measurement of their susceptibility to lysis by the CTL line of case 2. Experiments were repeated twice with consistent results. Five distinct HPLC fractions (fractions 13, 23, 27, 30, and 37) displayed modest but significant levels of the biological activity (Fig. 3B). In contrast, this CTL line failed to lyse T2 cells pulsed with MAGE-3-derived peptide (1% lysis), HER2/neu-derived peptide (0% lysis), or without pulsing any peptide (õ1% lysis) at an E/T ratio of 10. The CTL line of case 1 also failed to lyse T2 cells when pulsed with these two peptides. DISCUSSION We demonstrated in this study evidence of the existence of HLA-A locus-restricted and tumor-specific
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CTLs in IL-2-activated TILs in 6 of 16 patients with non-small cell lung cancer. Lung cancer is one of the most commonly observed cancers worldwide. However, there is no previous full report in the literature on the studies of HLA-A restriction and tumor specificity of the cytotoxicity of IL-2-activated TILs in lung cancer. This may be in part due to few available HLA class Igenotyped lung cancers and the other solid tumor cell lines that are needed to evaluate HLA-A restriction and tumor specificity of cytotoxicity of IL-2-activated TILs. To remove this obstacle, we provided 54 different solid tumor cell lines, including 15 lung cancers and 9 normal cell lines with which HLA-A loci are genotypically identified in most cases. HLA-B and -C loci of many tumor cell lines were also identified. The majority of these cell lines express HLA class I molecules on their surfaces as determined by staining with anti-HLA class I (W6/32) mAb (data not shown). The other reason for this obscurity may be the small numbers of CTL precursors in TILs of lung cancer. CTL precursors in melanoma TILs might be greater than those of lung cancer TILs, since the appearance in vitro of HLA-restricted and melanoma-specific CTLs in IL2-activated melanoma TILs is much faster than that
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FIG. 1. Cytotoxicity of the HLA A-restricted CTL lines. (A) HLA A2-restricted cytotoxity against HLA A0201/, A0206/, or A0207/ adenocarcinoma cell lines in the IL-2-activated TILs of case 1 (HLA A0201/A3101); (B) HLA A3101- and A3302-restricted cytotoxicity against HLA A3101/ and/or A3302/ adenocarcinoma cell lines in case 4 (HLA A3101/A3302); (C) HLA A3302-restricted cytotoxicity against HLA A3302/ tumors in case 5 (HLA A1101/A3302); and (D) HLA A2402-restricted cytotoxicity against HLA A2402/ tumors in case 6 (HLA A2402/). Representative results of a 6-hr 51Cr-release assay at three different E/T ratios (10, 20, and 40) at around 50 days of culture are shown.
in IL-2-activated lung cancer TILs. Namely, HLA-restricted CTL activity of the former or the latter was detectable at around 2 to 4 weeks, as reported (1) or 6 to 8 weeks shown herein in the culture with IL-2, respectively. The levels of lysis are also generally
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higher in the former than in the latter. Furthermore, HLA class I-restricted and tumor-specific cytotoxicity was observed in IL-2-activated TILs in a majority of melanoma patients (1, 2, 9, 10), but only in about onethird of lung cancer patients in our investigation. It is
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FIG. 2. Kinetics of cytotoxicity in IL-2-activated TILs of case 1 (HLA A0201/A3101). HLA A2-restricted CTL activity against 11–18 lung adenocarcinoma cell line was observed between 45 and 65 days of culture. Representative results of a 6-hr 51Cr-release assay at an E/T ratio of 40 are shown.
of note that the IL-2-activated TILs of one case with a high level of MHC-nonrestricted cytotoxicity possessed 64% TCRgd/ T cells, suggesting the existence of MHC-
nonrestricted immuno-responses at the tumor site in a few cases. In addition to an LC1 tumor of case 4, HLA-A-geno-
FIG. 3. Peptide fractions recognized by the HLA A2-restricted CTL line. (A) HPLC fractionation of SW620 peptides. Peptides were eluted from the SW620 tumor cell line (HLA A0201/ colon adenocarcinoma cell line) by pH 3.3 acid treatment followed by fractionation on reverse-phase HPLC in a 0–35% acetonitrile gradient. (B) T2 cell line was pulsed with peptides eluted from the SW620 tumor cell line, followed by testing their susceptibility to lysys by the HLA A2-restricted CTL line of case 2 (HLA A0206/A3302). Representative results of a 6-hr 51Cr-release assay at an E/T ratio of 10:1 are shown. Lysis of a negative control [peptide (—)] was less than 1%.
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HLA-A LOCUS-RESTRICTED CTL IN LUNG CANCER
typed allogenic tumors were used in these studies as target tumors since there were no available autologous tumor cells. Therefore, the method employed in this study does not provide direct evidence that the IL-2activated TILs, except for case 4, are cytotoxic against autologous tumor cells, even though they showed HLAA locus-restricted and tumor-specific cytotoxicity. The level of cytotoxicity of these CTLs was relatively low. This was possibly due to the fact that only a portion of the CTL lines (15 to 52% except for 97% in case 5) were CD8/ T cells. In addition, this may also be in part due to the low affinity of the peptide antigens binding to the HLA-A alleles recognized by these CTL lines (12, 24). Four, 6, or 4 of 15 HLA A2/ adenocarcinoma cell lines established from different organs (lung, stomach, colon, and breast), but none of the HLA A2/ SCC, hepatocellular carcinomas, or normal cell lines tested, were susceptible to lysis by the HLA A2-restricted CTL lines of cases 1, 2, or 3, respectively. The 4 of A2/ adenocarcinoma cell lines (three from lung and one from colon) were commonly recognized by all 3 CTL lines. These results suggest that the CTL lines of these three cases recognize antigens binding to the HLA A2 molecules of adenocarcinomas from distinct organs. The results also suggest the presence of tumor antigens shared by adenocarcinomas like MART-1, gp100, or the other melanoma shared antigens (4). Furthermore, the CTLs from cases 1, 5, and 6 lysed histologically distinct tumors in an HLA-A locus-restricted manner, suggesting the presence of tumor antigens shared by distinct tumors, like MAGE, GAGE, and BAGE (4). The subtypes of the HLA A2 molecules of adenocarcinoma cell lines susceptible to lysis by the CTL lines of cases 1 (A0201), 2 (A0206), and 3 (A0201) were HLA A0201, A0206, and A0207. The CTL of case 2 recognized five different fractions of antigenic peptides eluted from an HLA A0201/ SW620 adenocarcinoma cell line. Binding motifs of peptides to these three HLA A2 subtypes were closely related, and some of them were identical (25, 26). Storkus et al. reported that HLA A0201 molecules presented at least six shared antigenic peptides recognized by HLA A0201-restricted and melanoma-specific CTL lines (22). HLA A2-restricted CTL lines from HLA A2/ patients with ovarian cancers or breast cancers recognized HER2/neu-derived peptide (6, 7), whereas the HLA A2-restricted CTL lines of case 1 and case 2 did not recognize either HER2/neuor MAGE-3-derived peptide on an HLA A0201/ T2 cell line. Our results, along with these observations, indicate that at least several different peptides, except for HER2/neu- and MAGE-3-derived peptides, capable of binding to HLA A2 molecules of adenocarcinomas, may be recognized by these IL-2-activated TILs. Antigen frequencies of HLA-A2 in Yugoslavian (Caucasians), North American (Amerinds), South African (Blacks in Cape Town), and Japanese are 45.2, 44.5,
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30.3, and 42.8%, respectively, while those of HLA-A24 (A31/A33) are 20.3% (3.0%/3.0%), 35.4% (47.4%/2.0%), 12.0% (2.8%/11.6%), and 57.9% (15.4%/14.8%), respectively (27). HLA A0201, A2402, A3101, and A3302 are the most frequently expressed in Japanese. HLA A0201 along with A0206 and A0207 share more than 95% of serologically defined HLA A2-positive individuals. HLA A2402, A3101, and 3302 share more than 95% of A24, A31, and A33, respectively (15). This study showed evidence for the existence of HLA-A locus (A2, A2402, A3101, and A3302)-restricted and tumor-specific CTLs in IL-2-activated TILs of a substantial number of patients with non-small cell lung cancer. These findings may be important for understanding immunorecognition of lung cancer cells at tumor sites. Large numbers of patients with non-small cell lung cancer could be treated via specific immunotherapy with CTLs and/or tumor antigens. REFERENCES 1. Itoh, K., Platsoucas, C. D., and Balch, C. M., J. Exp. Med. 168, 1419, 1988. 2. Rosenberg, S. A., Packard, B. S., Aebersold, P. M., Solomon, D., Topalian, S. L., Toy, S. T., Simon, P., Lotze, M. T., Yang, J. C., Seipp, C. A., Simpson, C., Carter, C., Bock, S., Schwartzentruber, D., Wei, J. P., and White, D. E., N. Engl. J. Med. 319, 1676, 1988. 3. Nakao, M., Yamana, H., Imai, Y., Toh, Y., Toh, U., Kimura, A., Yamana, S., Kakegawa, T., and Itoh, K., Cancer Res. 55, 4248, 1995. 4. Boon, T., Gajewski, T. F., and Coulie, P. G., Immunol. Today 16, 334, 1995. 5. Ioannides, C. G., Fisk, B., Jerome, K. R., Irimura, T., Wharton, J. T., and Finn, O. J., J. Immunol. 151, 3793, 1993. 6. Yoshino, I., Peoples, G. E., Goedegebuure, P. S., Maziarz, R., and Eberlein, T. J., J. Immunol. 152, 2393, 1994. 7. Linehan, D. C., Goedegebuure, P. S., Peoples, G. E., Rogers, S. O., and Eberlein, T. J., J. Immunol. 155, 4486, 1995. 8. van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., de Plaen, E., van der Eynde, B., Knuth, A., and Boon, T., Science 254, 1643, 1991. 9. Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Rivoltini, L., Topalian, S. L., Miki, T., and Rosenberg, S. A., Proc. Natl. Acad. Sci. USA 91, 3515, 1994. 10. Kawakami, Y., Eliyahu, S., Delgado, C. H., Robbins, P. F., Sakaguchi, K., Appella, E., Yannelli, J. R., Adema, G. J., Miki, T., and Rosenberg, S. A., Proc. Natl. Acad. Sci. USA 91, 6458, 1994. 11. Coulie, P. G., Brichard, V., Van Pel, A., Wolfel, T., Schneider, J., Traversari, C., Mattei, S., de Plaen, E., Lurquin, C., Szikora, J. P., Renauld, J. C., and Boon, T., J. Exp. Med. 180, 35, 1994. 12. Rivoltini, L., Kawakami, Y., Sakaguchi, K., Southwood, S., Sette, A., Robbins, P. F., Marincola, F. M., Salgaller, M. L., Yannelli, J. R., Appella, E., and Rosenberg, S. A., J. Immunol. 154, 2257, 1995. 13. Slingluff, C. L., Jr., Cox, A. L., Stover, J. M., Jr., Moore, M. M., Hunt, D. F., and Englehard, V. H., Cancer Res. 54, 2731, 1994. 14. Itoh, K., Tilden, A. B., and Balch, C. M., Cancer Res. 45, 3173, 1986. 15. Date, Y., Kimura, A., Kato, H., and Sasazuki, T., Tissue Antigens 47, 93, 1996.
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16. Fernandez-Vina, M., Lazalo, A. M., Sun, Y., Miller, S., Ferero, L., and Stastny, P., Tissue Antigens 45, 153, 1995. 17. Bunce, M., O’neill, C. M., Barnardo, M. C. N. M., Kurausa, P., Browning, M. J., Morris, P. J., and Welsh, K. I., Tissue Antigens 46, 355, 1995. 18. Hoshino, T., Seki, N., Kikuchi, M., Kuramoto, T., Iwamoto, O., Kodama, I., Koufuji, K., Takeda, J., and Itoh, K., Int. J. Cancer, in press, 1997. 19. Tatari, Z., Fortier, C., Bobrynina, V., Loiseau, P., Charron, D., and Raffoux, C., Proc. Natl. Acad. Sci. USA 92, 8803, 1995. 20. Hoshino, T., Yamada, A., Honda, J., Imai, Y., Nakao, M., Inoue, M., Sagawa, K., Yokoyama, M. M., Oizumi, K., and Itoh, K., J. Immunol. 151, 2237, 1993. 21. Kharkevitch, D. D., Seito, D., Balch, G. C., Maeda, T., Balch, C. M., and Itoh, K., Int. J. Cancer 58, 317, 1994.
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22. Storkus, W. J., Zeh, H. J., III, Maeurer, M. J., Salter, R. D., and Lotze, M. T., J. Immunol. 151, 3719, 1993. 23. van der Bruggen, P., Bastin, J., Gajewski, T., Coulie, P. G., Boe¨l, P., De Smet, C., Traversari, C., Townsend, A., and Boon, T., Eur. J. Immunol. 24, 3038, 1994. 24. Fairchild, P. J., and Wraith, D. C., Immunol. Today 17, 80, 1996. 25. del Guercio, M. F., Sidney, J., Hermanson, G., Perez, C., Grey, H. M., Kubo, R. T., and Sette, A., J. Immunol. 154, 685, 1995. 26. Sudo, T., Kamikawaji, N., Kimura, A., Date, Y., Savoie, C. J., Nakashima, H., Furuichi, E., Kuhara, S., and Sasazuki, T., J. Immunol. 155, 4749, 1995. 27. Imanishi, T., Akaza, T., Kimura, A., Tokunaga, K., and Gojobori, T., in ‘‘HLA,’’ 1991 Vol. 1, Oxford Scientific Publications, Oxford, 1992.
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