Spontaneous antibody, and CD4 and CD8 T-cell responses against XAGE-1b (GAGED2a) in non-small cell lung cancer patients

Authors


Abstract

The spontaneous immune responses against XAGE-1b (GAGED2a) were analyzed in non-small cell lung cancer (NSCLC) patients. An antibody response against XAGE-1b (GAGED2a) was observed in 10% (20/200) of NSCLC patients and in 19% (13/69) of stage IIIB/IV lung adenocarcinoma patients. A CD4 T-cell response was detected in 88% (14/16) and a CD8 T-cell response in 67% (6/9) in the XAGE-1b (GAGED2a) antibody-positive patients examined. Frequent antibody responses and CD4 and CD8 T-cell responses in XAGE-1b (GAGED2a) antibody-positive patients indicate the strong immunogenicity of the XAGE-1b (GAGED2a) antigen in NSCLC patients. We established T-cell clones from PBMCs of antibody-positive patients and determined the DRB1*04:05-restricted XAGE-1b (GAGED2a) 18–31 peptide (14-mer) as a CD4 T cell epitope and the A*02:06-restricted XAGE-1b (GAGED2a) 21-29 peptide (9-mer) as a CD8 T cell epitope. As for peptide recognition, CD4 and CD8 T-cell clones responded to naturally processed antigen. The CD4 T-cell clone recognized DCs pulsed with the synthetic protein or a lysate from XAGE-1b-transfected 293T cells. The CD8 T-cell clone showed cytotoxicity against a tumor expressing XAGE-1b (GAGED2a) and the appropriate HLA class I allele. These findings establish XAGE-1b (GAGED2a) as a promising target for a lung cancer vaccine.

More than 70 cancer/testis (CT) antigen gene families have been identified by immunological or genetic approaches.1–3 Several CT antigens such as the NY-ESO-1 antigen etc. have been shown to elicit humoral and cellular immune responses in cancer patients.4, 5 Because of their restricted expression in normal tissues and high immunogenicity, CT antigens are considered attractive targets for cancer vaccines.6–9

XAGE-1 was originally identified by the search for PAGE/GAGE-related genes using an expression sequence tag database10 and was shown to exhibit CT antigen characteristics.11, 12 Five identical genes XAGE1A to E have now been identified, located in dispersed fashion in different orientations in a region of approximately 350 kilobases on chromosome Xp11.22.13 They belong to X antigen family genes. The associated protein is designated as G antigen family D member 2 (GAGED2), and GAGED2a and d isoforms have been identified.10, 13 Four transcript variants XAGE-1a, b, c and d have been extensively studied and were shown to be expressed in metastatic melanoma, Ewing sarcoma, and various epithelial tumors such as breast, lung and prostate cancers.14–17 In a serologic search for antigens using recombinant expression cloning (SEREX), we identified XAGE-1b as a dominant antigen recognized by serum from a lung adenocarcinoma patient using an autologous tumor cell line established from malignant pleural effusion as a source of the cDNA library.18 From the analysis with transfected 293T cells using a USO 9–13 mAb specific for XAGE-1b (GAGED2a) protein, we showed that the XAGE-1a and b transcripts code for the 81 amino acid XAGE-1b (GAGED2a) protein.19 The XAGE-1c transcript codes for 9- and 17-a.a. peptides from an alternative reading frame. The XAGE-1d transcript codes for a protein consisting of 69 amino acids (GAGED2d).

In relation to clinical relevance, we showed that XAGE-1b (GAGED2a) expression by itself had no correlation with overall survival in non-small cell lung cancer (NSCLC) patients.20 However, both XAGE-1b (GAGED2a) and HLA class I expression correlated with prolonged survival. Moreover, expression of XAGE-1b combined with down-regulated HLA class I expression correlated with even worse survival. These findings suggested that XAGE-1b (GAGED2a) and HLA expression elicited a T-cell response against tumors and resulted in prolonged survival.

In this study, we investigated spontaneous antibody response, and CD4 and CD8 T-cell responses, against XAGE-1b (GAGED2a) in NSCLC patients. We showed a high frequency of antibody responses in this patient population. CD4 and CD8 T-cell responses were detected in most of the antibody-positive patients. Furthermore, we determined CD4 and CD8 T-cell epitopes using cloned T-cell lines. A CD4 T-cell clone recognized naturally processed antigen on DCs pulsed with the synthetic protein or a lysate from XAGE-1b-transfected 293T cells, and a CD8 T-cell clone showed cytotoxicity against a tumor cell line expressing XAGE-1b (GAGED2a) and the appropriate HLA class I allele.

Abbreviations

CLSM: confocal laser scanning microscopy; CT: cancer/testis; DAPI: 4′,6-diamidino-2-phenylindole; HRP: horseradish peroxidase; IHC: immunohistochemistry; NSCLC: non-small cell lung cancer; OLP: overlapping peptides; PHA: phytohemagglutinin

Material and Methods

Blood samples

Peripheral blood was drawn from lung cancer patients and healthy donors after obtaining written informed consent at Kawasaki Medical School Hospital from 2005 to 2010. Sera were obtained from 200 patients with NSCLC including 118 adenocarcinomas, 44 squamous cell carcinomas, six pleiomorphic carcinomas, four adenosquamous carcinomas, one large cell carcinoma and 27 unclassified. Sera were also obtained from 50 healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation using a Histo-paque 1077 (Sigma–Aldrich, St. Louis, MO). CD4-, CD8- and CD19-positive cells were purified by magnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany). The residual cells were kept for use as antigen presenting cells (APCs). The cells were stored in liquid N2 until use. HLA typing was done with PBMCs by a sequence-specific oligo-nucleotide probe and sequence-specific priming of genomic cDNA using a standard procedure.

Cell lines

OU–LC–KI, OU–LC–SK, OU–LC–ON, PC–9 and 1–87 were lung adenocarcinoma cell lines.21 RERF–LC–AI was a lung squamous cell carcinoma cell line. HEK293T was a human embryonic kidney cell line. OU–LC–KI, OU–LC–SK and OU–LC–ON were established in our laboratory. RERF–LC–AI, 1–87 and HEK293T were obtained from RIKEN (Riken Bioresource Center, Ibaragi, Japan). These cell lines were kept in tissue culture by serial passage. Epstein-Barr virus (EBV)-B cells were generated from CD19-positive peripheral blood B cells using a culture supernatant from EBV-producing B95-8 cells. The medium used to maintain these cell lines was RPMI1640 supplemented with 2 mmol/L Glutamax, antibiotics, 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Invitrogen, Carlsbad, CA) and 10% fetal calf serum (FCS) (JRM, Bioscience, Lenexa, KA). An HEK293T transfectant was generated by introducing pcDNA 3.1/Zeo (+) containing XAGE-1b cDNA using LipofectAMINE 2000 (Invitrogen).

Antibodies

Anti-human CD4, anti-human CD8, anti-HLA pan class I and anti-HLA pan class II mAbs were purchased from BD Bioscience (San Jose, CA).

Overlapping peptides

The following series of 17 16-mer overlapping XAGE-1b (GAGED2a) peptides spanning the entire protein 1-16, 5-20, 9-24, 13-28, 17-32, 21-36, 25-40, 29-44, 33-48, 37-52, 41-56, 45-60, 49-64, 53-68, 57-72, 61-76 and 65-81 were synthesized using Fmoc chemistry on a Multiple Peptide Synthesizer (AMS422, ABIMED, Langenfeld, Germany) at Okayama University.

Synthetic XAGE-1b (GAGED2a) protein

XAGE-1b (GAGED2a) protein (81 amino acids) was synthesized using a peptide synthesizer by GL Biochemistry (Shanghai, China).

Reverse transcription (RT)-PCR

Total RNA was obtained from cells using an RNeasy Mini kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. Two micrograms of each sample was subjected to cDNA synthesis using Ready-To-Go first strand beads kit (GE Healthcare, Tokyo, Japan). Sequences of primer pairs for XAGE-1b were X-1, 5′-TTTCTCCGCTACTGAGACAC-3′ and X-2, 5′-CAGCTTGCGTTGTTTCAGCT-3′, and sequences for G3PDH were G3PDH-S, 5′-ACCACAGTCCATGCCATCAC-3′, G3PDH-AS, 5′-TCCACCACCCTGTTGCTGTA-3′. The amplification was performed using 30 cycles as described.22

Western blot analysis

The XAGE-1b (GAGED2a) antigen in the cell lysate was immunoprecipitated using USO 9-13 mAb and the antigen/antibody complex was purified using protein G Sepharose beads. The beads were washed with a lysis buffer [500 nM HEPES (pH7.9), 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid, 1% Triton X-100, 100 μM 4-(2-aminoethyl)-benzensulfonyl fluoride, 1 μg/mL leupeptin and pepstatin A], and the immune complex was dissolved in a sample loading buffer [250 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 20% glycerol and 5% β-mercaptoethanol]. The sample was heated at 95°C for 5 min and then separated in a 10–20% gradient gel by SDS-PAGE. The materials in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with a membrane-blocking reagent (GE Healthcare) and then incubated with USO 9-13 mAb at room temperature for 2 hr. After washing, the membrane was incubated with a peroxidase-conjugated second antibody (MBL, Nagoya, Japan) and the bands were visualized using an ECL plus Western Blotting Detection System (GE Healthcare).

Confocal laser scanning microscopy

Cells were fixed in ethanol (Wako Pure Chemical Industries, Osaka, Japan) and permeabilized with a 0.1% Tween 20/5% FCS/phosphate buffered saline (PBS) buffer. The cells were then stained with USO 9-13 mAb and FITC-conjugated goat anti-mouse IgG (Sigma–Aldrich). For intracellular localization, 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) was used. The stained cells were visualized under a confocal laser scanning microscope (CLSM) (Model Fluoview FV10i for the magnification of 60×, Olympus, Tokyo, Japan).

ELISA to detect the XAGE-1b (GAGED2a) antibody

Synthetic XAGE-1b (GAGED2a) protein (1 μg/mL) in a coating buffer was adsorbed onto a 96-well ELISA plate (Nunc, Roskilde, Denmark) and incubated overnight at 4°C. Plates were washed with PBS and blocked with 5% FCS/PBS (200 μL/well) for 1 hr at 37°C. After washing, 100 μL of serially diluted serum was added to each well and incubated for 2 hr at 4°C. After washing, horseradish peroxidase (HRP)-conjugated goat anti-human IgG (MBL) was added to the wells, and the plates were incubated for 1 hr at 37°C. After washing and development, absorbance was read at 490 nm.

In vitro stimulation of CD4 and CD8 T-cells

CD4 (2 × 106/well) and CD8 (1 × 104/well) T-cells were cultured on a micro-culture plate and a 96-well culture plate (BD Bioscience), respectively, with an equal number of irradiated (40 Gy), autologous CD4- and CD8-depleted cells as APCs in the presence of a mixture of 17 16-mer overlapping peptides (10−6 M) for 10–14 days at 37°C in a 5% CO2 atmosphere. The medium was AIM-V (Invitrogen) supplemented with 5% heat-inactivated pooled human serum, 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 units/mL recombinant IL-2 (Takeda Chemical Industries, Osaka, Japan) and 10 ng/mL recombinant IL-7 (Peprotech, London, UK).

Establishment of CD4 and CD8 T-cell clones

CD4 and CD8 T-cells were cloned by limiting dilution after one or two in vitro stimulations in round-bottomed 96-well plates in the presence of irradiated (40 Gy), allogeneic PBMCs as feeder cells. The medium used was AIM-V (Invitrogen) supplemented with 5% heat-inactivated pooled human serum, 2 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 units/mL recombinant IL-2 (Takeda Chemical Industries), 10 ng/mL recombinant IL-7 (Peprotech) and 1 μg/mL phytohemagglutinin-L (PHA) (Sigma–Aldrich).

Preparation of dendritic cells (DCs)

Monocytes were isolated from PBMCs using anti-CD14 mAb-coated magnetic beads (Miltenyi Biotec) and cultured in AIM-V medium supplemented with 5% heat-inactivated pooled human serum, 10 ng/mL recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) (Kyowa Hakko Kirin, Tokyo, Japan) and 10 ng/mL rhIL-4 (PeproTech) for 10 days at 37°C in a 5% CO2 atmosphere.

Interferon (IFN) γ ELISA

Responder CD4 or CD8 T-cells (1 × 104) from a stimulation culture were cultured with EBV-B cells (1 × 104) pulsed with overlapping peptide (OLPs) in a 96-well round-bottomed culture plate for 24 hr at 37°C in a 5% CO2 atmosphere. Culture supernatants were then collected and the amount of INFγ was measured by sandwich ELISA. For antibody blocking experiments, each mAb (5 μg/mL) was added to the assay culture.

IFNγ capture assay

Responder CD4 or CD8 T-cells (5 × 104) from the stimulation culture were cultured with autologous EBV-B cells (5 × 104) pulsed with OLPs for 4 or 8 hr, respectively. The cells were then treated with a bi-specific CD45 and IFNγ antibody (IFNγ catch reagent) (2 μL) for 5 min on ice. The cells were diluted in AIM-V medium (3 mL) and placed on a slow rotating device (Miltenyi Biotec) to allow IFNγ secretion at 37°C in a 5% CO2 atmosphere. After incubation for 1 hr, the cells were washed with cold buffer and treated with PE-conjugated anti-IFNγ (detection reagent), and FITC-conjugated anti-CD4 or anti-CD8 mAb. After incubation for 10 min at 4°C, the cells were washed and analyzed by FACS Calibur or Canto II (BD Bioscience).

Cytotoxicity assay

Cytotoxicity was assayed by a luminescent method using a aCella-Tox kit (Cell Technology, Mountain View, CA). Effector cells were incubated with 5,000 target cells at various ratios in 96-well round-bottomed culture plates for 12 hr at 37°C in a 5% CO2 atmosphere. The plate was read by a luminometer (multi-detection microplate reader, DS Pharma, Osaka, Japan).

Statistical analysis

All values are expressed as the mean ± S.D. of individual samples. Samples were analyzed using the Student's t-test for two groups and ANOVA for multiple groups using IBM SPSS Statistics 19 for Windows (IBM, New York, NY). Values of p < 0.05 were considered significant.

Results

XAGE-1b (GAGED2a) expression in lung cancer cell lines

We previously showed that XAGE-1b (GAGED2a) was expressed predominantly in lung adenocarcinoma and rarely observed in esophagus, stomach, liver, colon or breast cancer.18, 19, 22 In normal adult tissues, XAGE-1b (GAGED2a) was expressed only in the testes. In this study, we investigated XAGE-1b (GAGED2a) expression in lung cancer cell lines by Western blot and CLSM. In a conventional Western blot, we found that XAGE-1b (GAGED2a) mAb (clone USO 9-13) gave a 9 kDa band in cell lysates from XAGE-1b transfectants,22 but not in XAGE-1b-mRNA expressing tumor cell lines. For this reason, we used a modified method in which cell lysates were first incubated with the mAb and the antigen/antibody complex was purified using protein G Sepharose beads. Then, the eluate of the XAGE-1b (GAGED2a)-enriched solution was analyzed by Western blot. As shown in Figure 1, XAGE-1b-mRNA-positive cell lines gave a 9 kDa band. CLSM showed nuclear localization of the protein with a granular or diffuse pattern in XAGE-1b (GAGED2a)-positive lung cancer cell lines, consistent with the immunohistochemistry (IHC) results with lung cancer tissues.22

Figure 1.

RT-PCR and Western blot analyses (a) and CLSM (b) for XAGE-1b (GAGED2a) expression in lung cancer cell lines. OU–LC–KI, OU–LC–SK and 1–87 are lung adenocarcinoma (Ad) cell lines. RERF-LC-AI is a lung squamous cell carcinoma (Sq) cell line. In RT-PCR, G3PDH was used as the internal control. In Western blot, the cell lysate was first incubated with USO 9-13 mAb and the antigen/antibody complex was purified using protein G Sepharose beads. The eluate was run on the gel. The pcDNA3.1/XAGE-1b-transfected HEK293T cell lysate was used as a control. In CLSM, the cells were stained with USO 9–13 mAb and FITC-conjugated goat anti-mouse IgG (green). Nuclei are stained with DAPI (blue). XAGE-1b (GAGED2a) derived fluorescence is merged with differential interference contrast (Merge 1) and DAPI (Merge 2). Scale bar indicates 20 μm. HLA expression of the cell lines was A*02:01, *02:06; B*39:01, *51:01; C*03:04, *14:02 for OU–LC–KI: A*24:02, -; B*51:01, -; C*14:02, - for OU–LC–SK: A*24:02, -; B*52:01, -; C*12:02, - for RERF-LC-AI: A*02:07, *11:01; B*46:01, *54:01; C*01:02, - for 1–87. No expression of HLA class II antigens was observed in these cell lines.

Antibody response against XAGE-1b (GAGED2a) in NSCLC patients

The antibody response against XAGE-1b (GAGED2a) was investigated in NSCLC patients by ELISA using synthetic XAGE-1b (GAGED2a) protein. Figure 2a shows titration curves of sera from 200 NSCLC patients and 50 healthy control donors. The frequency of antibody-positive patients was 10% (20/200). The frequencies of antibody-positive patients with adenocarcinoma and squamous cell carcinoma were 14% (16/118) and 2% (1/44), respectively. The frequency of antibody-positive patients with Stage IIIB/IV lung adenocarcinoma was 19% (13/69). Our previous results showed that the frequencies of XAGE-1b mRNA and IHC-positives were 31% and 23% in NSCLC, and 45% and 33%, respectively, in lung adenocarcinoma. No correlation was observed between XAGE-1b expression and disease stage or histologic grade.20, 22 These findings indicate that the frequency of antibody positives was 32–43% in NSCLC patients and 42–57% in Stage IIIB/IV lung adenocarcinoma patients with XAGE-1b-mRNA and/or XAGE-1b (GAGED2a)-expressing tumors.

Figure 2.

Antibody response against XAGE-1b (GAGED2a) in non-small cell lung cancer patients. (a) ELISA against synthetic XAGE-1b (GAGED2a) protein of serially diluted sera from 200 non-small cell lung cancer patients (left) and 50 healthy donors (right). O.D. values exceeding 1.0 were considered positive. (b) ELISA against 17 16-mer XAGE-1b (GAGED2a) overlapping peptides (OLPs) (5 μg/mL) of sera from 20 antibody-positive patients in a at 1:100 serum dilution. O.D. values exceeding 0.1 were considered positive. (c) The number of antibody-positive patients for individual 17 16-mer XAGE-1b overlapping peptides.

Epitope peptides recognized by the antibody were analyzed by ELISA using 17 16-mer XAGE-1b (GAGED2a) OLPs. As shown in Figures 2b and 2c, various regions were recognized. Within these, peptides 25–40, 29–44, 33–48 and 57–72 were relatively frequently recognized. The USO 9–13 mAb used for XAGE-1b (GAGED2a) expression analysis recognized peptide 65–81.19

CD4 and CD8 T-cell responses against XAGE-1b (GAGED2a) in NSCLC patients

Purified CD4 and CD8 T-cells from PBMCs were stimulated with a mixture of 17 16-mer XAGE-1b (GAGED2a) OLPs using irradiated (40 Gy), autologous CD4- and CD8-depleted PBMCs as APCs for 10–14 days. CD4 T-cells were cultured in a micro-culture plate (2 × 106/well) and CD8 T-cells in a 96-well culture plate (1 × 104/well). After culture, responding CD4 and CD8 T-cells were collected and stimulated with a mixture of OLPs using autologous EBV-B cells as APCs for 4 and 8 hr, respectively, and examined for IFNγ secretion by FACS.

As shown in Figure 3, CD4 and CD8 T-cell responses were observed in 14 of 16 and 6 of 9, respectively, of the XAGE-1b (GAGED2a) antibody-positive patients examined. Neither response was observed in 7 antibody-negative patients or five healthy donors.

Figure 3.

CD4 and CD8 T-cell responses against XAGE-1b (GAGED2a). (a) IFNγ capture assays. Purified CD4 and CD8 T-cells from PBMCs of antibody-positive (KLU22 and KLU187) or -negative (KLU407 and KLU479) patients were cultured with an equal number of irradiated (40 Gy), autologous CD4- and CD8-depleted PBMCs as APCs in the presence of 17 16-mer OLPs (1 μM) spanning the entire XAGE-1b (GAGED2a) protein using a micro-culture plate for CD4 T-cells (2 × 106 per well) and a 96-well culture plate for CD8 T-cells (1 × 104 per well) for 10–14 days. IFNγ secretion by CD4 and CD8 T-cells (5 × 104) was assayed against autologous EBV-B cells (5 × 104) pulsed with XAGE-1b (GAGED2a) OLPs after incubation for 4 hr in CD4 and for 8 hr in CD8. HLA types of the antibody-positive patients KLU22 and KLU187 are shown in Table 1. Those of the antibody-negative patients KLU407 and KLU479 are DRB1*15:02, -; DQB1*06:01, -; DPB1*09:01, -; A*24:02, -; B*52:01, -; C*12:02, - for KLU407: DRB1*08:03, *15:01; DQB1*06:01, *06:02; DPB1*02:01, 05:01; A*02:06, 02:07; B*39:01, *46:01; C*01:02, 07:02 for KLU479. XAGE-1b mRNA expression in a tumor specimen was positive in KLU407 and negative in KLU479. (b) The peptide regions recognized by CD4 and CD8 T-cells from antibody-positive patients. The culture supernatant of T-cells (1 × 104) stimulated with autologous EBV-B cells (1 × 104) in the presence of individual OLPs (5 μg/mL) for 24 hr was assayed by ELISA for IFNγ secretion. Predominant responses are marked by a star. HLA types of the patients are shown in Table 1. (c) The number of patients with CD4 and CD8 T-cell responses for individual 17 16-mer OLPs.

XAGE-1b (GAGED2a) peptide regions recognized by CD4 and CD8 T-cells

XAGE-1b (GAGED2a) peptide regions recognized by CD4 and CD8 T-cells were determined using 17 16-mer XAGE-1b (GAGED2a) OLPs. Purified CD4 and CD8 T-cells from PBMCs were stimulated once or twice with the OLPs using irradiated (40 Gy), autologous CD4- and CD8-depleted PBMCs as APCs as described above. The responding cells were collected and stimulated with individual OLPs using autologous EBV-B cells as APCs for 24 hr and assayed for IFNγ secretion in the culture supernatant by ELISA. As shown in Figures 3b and 3c, peptide regions recognized by CD4 and CD8 T-cells from 14 and 6 patients, respectively, who were XAGE-1b (GAGED2a) antibody-positive were quite diverse. Several peptide regions were relatively frequently recognized. CD4 T-cells recognized peptide 13–28 in 5 of 14, and peptide 33–48 in 6 of 14, XAGE-1b (GAGED2a) antibody-positive patients. On the other hand, CD8 T-cells recognized peptide 9–24 and 29–44 in three of six XAGE-1b (GAGED2a) antibody-positive patients. The patients' HLA types are listed in Table 1. No specific correlation of the peptides recognized to HLA alleles was found.

Table 1. HLA class II and I in patients analyzed for CD4 and CD8 T-cell responses, respectively, in Figure 3
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Determination of restriction molecules and minimal epitopes in the recognition of XAGE-1b (GAGED2a) peptides by CD4 and CD8 T-cell clones

CD4 and CD8 T-cell clones were established from PBMCs of XAGE-1b (GAGED2a) antibody-positive patients. As shown in Figure 4a, the restriction molecule in recognition of peptide 17–32 by the KLU187 CD4 T-cell clone was determined by antibody blocking and by using various EBV-B cells as APCs to present the peptide, while the minimal epitope peptide was determined by using N- and C-termini truncated peptides. The recognition of peptide 17–32 was restricted by DRB1*04:05 and the minimal epitope was the 14-mer peptide 18–31.

Figure 4.

Determination of restriction molecules and minimal epitopes in recognition of XAGE-1b (GAGED2a) OLPs by CD4 (a) and CD8 (b) T-cell clones from patient KLU-187. For the method to establish CD4 and CD8 T-cell clones, see “Material and Methods” section. In a, recognition of XAGE-1b (GAGED2a) 17–32 and 21–36 OLPs by the CD4 T-cell clone (1 × 104) was determined by ELISA examining IFNγ in the culture supernatant after stimulation. In antibody blocking, the CD4 T-cell clone (5 × 103) was stimulated with the autologous EBV-B cells (5 × 103) in the presence of the peptide 17–32 (0.1 μM) and various antibodies (5 μg/mL). In determining a restriction molecule for peptide 17–32 recognition by the CD4 T-cell clone (5 × 103), EBV-B cells (5 × 103) with various HLA types were used for APCs. The recognition was restricted to DRB1*04:05. In determining a minimal epitope, various N- and C-terminal truncated peptides (0.01 μM in the upper panel and 0.001 μM in serum free medium in the lower panel) were examined for recognition by the CD4 T-cell clone (5 × 103). The peptide 18–31 (14-mer) was the epitope. In b, recognition of XAGE-1b (GAGED2a) 17–32 and 21–36 OLPs by a CD8 T-cell clone was similarly determined by ELISA. In antibody blocking, the CD8 T-cell clone (1 × 104) was stimulated with the autologous EBV-B cells (1 × 104) in the presence of the peptide 21–36 and various antibodies. In determining a restriction molecule for peptide 21–36 recognition by the CD8 T-cell clone, EBV-B cells with various HLA types were used for APCs. The recognition was restricted to A*02:06. In determining a minimal epitope, various N- and C-terminal truncated peptides were examined for recognition. The CD8 T-cell response to different concentrations of various peptides in serum free medium revealed peptide 21–29 (9-mer) to be the epitope. The values are the mean ± S.D. from the three independent experiments.

The restriction molecule in the recognition of peptide 21–36 by the KLU187 CD8 T-cell clone and the minimal epitope peptide were similarly determined. As shown in Figure 4b, the recognition was restricted to A*02:06 and the minimal epitope was the 9-mer peptide 21–29.

Recognition of naturally processed XAGE-1b (GAGED2a) antigen by CD4 and CD8 T-cell clones

We examined the recognition of the naturally processed XAGE-1b (GAGED2a) antigen by the DRB1*04:05-restricted CD4 T-cell clone, and the A*02:06-restricted CD8 T-cell clone shown in Figures 4a and 4b, respectively. As shown in Figures 5a and 5b, the DRB1*04:05-restricted CD4 T-cell clone recognized DCs pulsed with XAGE-1b (GAGED2a) synthetic protein or the lysate from XAGE-1b-transfected 293T cells. On the other hand, the A*02:06-restricted CD8 T-cell clone recognized DCs pulsed with the 9-mer epitope peptide, but not synthetic XAGE-1b (GAGED2a) protein. As shown in Figure 5c, the A*02:06-restricted CD8 T-cell clone showed cytotoxicity against a XAGE-1b (GAGED2a)-positive, A*02:06-positive lung cancer cell line OU–LC–KI, but not a XAGE-1b (GAGED2a)-positive, A*02:06 negative lung cancer cell line OU–LC–ON, a XAGE-1b (GAGED2a)-negative, A*02:06-positive lung cancer cell line PC-9, or a XAGE-1b (GAGED2a)-negative, A*02:06 negative lung cancer cell line 1–87.

Figure 5.

Recognition of a naturally processed XAGE-1b (GAGED2a) antigen by CD4 and CD8 T-cell clones from patient KLU187. In a, a CD4 T-cell clone (5 × 104) was stimulated for 4 hr with autologous DCs (5 × 104) pulsed with XAGE-1b (GAGED2a) synthetic protein (1 μM) or the lysate from XAGE-1b-transfected 293T cells (5 × 106). The CD8 T-cell clone (5 × 104) was stimulated for 8 hr with autologous DCs (5 × 104) pulsed with XAGE-1b (GAGED2a) synthetic protein (1 μM) or 9-mer epitope peptide (RQKKIRIQL) (1 μM). Then, the response was examined by an IFNγ capture assay. In b, the amount of IFNγ in the culture supernatant of the CD4 and CD8 T-cell clones (1 × 104) stimulated for 24 hr with autologous DCs (1 × 104) treated as above was determined by ELISA. In c, cytotoxicity of the CD8 T-cell clone for an XAGE-1b (GAGED2a)-positive and A*02:06-positive tumor cell line by a luminescent method using the aCella-Tox kit (see “Material and Methods” section) is shown. The values are the mean ± S.D. from the three independent experiments.

Discussion

In this study, we showed that an antibody response against XAGE-1b (GAGED2a) was observed in 10% of NSCLC patients and in 19% of Stage IIIB/IV lung adenocarcinoma patients. We previously showed that the frequency of XAGE-1b mRNA and IHC-positives was 31% and 23% in NSCLC, and 45% and 33%, respectively, in lung adenocarcinoma. By calculation, this indicates that 32–43% of NSCLC patients and 42–57% of Stage IIIB/IV lung adenocarcinoma patients with XAGE-1b-mRNA and/or protein-expressing tumors elicited an antibody response. This high frequency of spontaneous antibody response against XAGE-1b (GAGED2a) in NSCLC patients was comparable to that against NY-ESO-1 in melanoma patients in a Caucasian population, and it has been shown to be one of the most immunogenic tumor antigens.5, 23 However, in lung cancer patients in the Japanese population, the frequency of the antibody response against NY-ESO-1 was approximately 5% (unpublished). On the other hand, no spontaneous antibody response was observed in lung cancer patients with MAGE-A3 or SSX2-expressing tumors.7, 23–27 The antibody response against p53 was shown to be high at around 7–27% in lung cancer patients.27 These findings emphasize the strong immunogenicity of XAGE-1b (GAGED2a) in the antibody response in lung cancer patients.

In this report, we analyzed CD4 and CD8 T-cell responses in XAGE-1b (GAGED2a) antibody-positive patients. A CD4 T-cell response was detected in 14 of 16 (88%), and a CD8 T-cell response was detected in six of nine (67%) XAGE-1b (GAGED2a) antibody-positive patients examined. Occurrence of CD4 and CD8 T-cell responses in XAGE-1b (GAGED2a) antibody-positive patients showed the strong cellular immunogenicity of the XAGE-1b (GAGED2a) antigen. This is also similar to the findings with NY-ESO-1. Thus, an integrated immune response including antibody and CD4 and CD8 T-cell responses was repeatedly shown in patients with NY-ESO-1-expressing tumors.28–30 With regard to both the XAGE-1b (GAGEG2a) and NY-ESO-1 antigens, CD4 and CD8 T-cell responses were elicited in PBMCs from antibody-positive patients after a single in vitro stimulation. Ex vivo detection of such responses was rarely possible due to the low frequencies of CD4 and CD8 T-cells responding to the antigens.31 However, in XAGE-1b (GAGED2a)-antibody-positive patients, the CD8 T-cell response appeared to be somewhat weaker than the CD4 T-cell response.

In this study, we determined the DRB1*04:05-restricted XAGE-1b (GAGED2a) 18–31 peptide (14-mer) as a CD4 T-cell epitope, and the A*02:06-restricted XAGE-1b (GAGED2a) 21–29 peptide (9-mer) as a CD8 T-cell epitope. We previously determined two XAGE-1b (GAGED2a) CD4 epitope peptides restricted to DRB1*04:1032 and DRB1*09:01.33 Moreover, we are currently determining other MHC I binding peptide epitopes. These CD4 and CD8 T-cell epitope peptides will be useful for designing vaccines and producing tetramers for immune monitoring. Tetramer production is now under investigation.

Recognition by CD4 and CD8 T-cell clones of a naturally processed XAGE-1b (GAGED2a) antigen was shown in this study. The CD8 T-cell clone showed cytotoxicity against an HLA-matched, XAGE-1b (GAGED2a)-positive tumor cell line.

XAGE-1b (GAGED2a) is 81 amino acids long and is expressed in most XAGE-1b mRNA expressing NSCLC. Thus, XAGE-1b (GAGED2a) was the predominant isoform in NSCLC. However, in hepatocellular carcinoma or prostate cancer, although XAGE-1b and d mRNA expression have been frequently observed, XAGE-1b (GAGED2a) protein expression has rarely been observed by IHC. It is possible that another isoform, XAGE-1d (GAGED2d), was expressed in these tumors. Production of mAb detecting XAGE-1d (GAGED2d) is now being studied.

Acknowledgements

The authors thank Dr. Masao Nakata (Department of General Thoracic Surgery, Kawasaki Medical School) for support during this study and Dr. Hirofumi Matsumoto (Department of Translational Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences) for technical advice. They also thank Ms. Junko Mizuuchi for preparation of the article.

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