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Identification of Natural Antigenic Peptides of a Human Gastric Signet Ring Cell Carcinoma Recognized by HLA-A31-Restricted Cytotoxic T Lymphocytes¹

Kazuhiro Suzuki^*,, Hiroeki Sahara^*,, Yohjiro Okada^*,, Takahiro Yasoshima, Yoshihiko Hirohashi^*,§, Yuki Nabeta^*, Itaru Hirai^*, Toshihiko Torigoe^*, Shuji Takahashi^*, Akihiro Matsuura^*, Nobuaki Takahashi^*,, Aya Sasaki^*, Manabu Suzuki^¶, Junji Hamuro^¶, Hideyuki Ikeda^||, Yoshimasa Wada, Koichi Hirata, Kokichi Kikuchi^*,# and Noriyuki Sato^2,*

Departments of ^* Pathology and Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan; Marine Biomedical Institute, Sapporo Medical University School of Medicine, Rishirifuji-cho, Hokkaido, Japan; ^§ Department of Otolaryngology, Wakayama Medical College, Wakayama, Japan; ^¶ Central Research Laboratories, Ajinomoto Co. Inc., Kawasaki, Japan; ^|| Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; and ^# Sapporo Immunodiagnostic Laboratory, Sapporo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Peptides of human melanomas recognized by CD8⁺ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
An increasing amount of evidence has indicated that a patient’s immune system can respond against his or her own neoplastic cells (1, 2, 3, 4, 5). This is particularly true for melanoma patients. CD8⁺ CTLs from these patients can recognize MHC class I-bound tumor antigenic peptides (6, 7, 8, 9, 10, 11, 12, 13, 14). There is the possibility that the elucidation of human tumor Ags may directly lead to a drastic improvement in tumor immunotherapy and establish a new modality of cancer therapy. Indeed, it is very interesting that therapeutic attempts using the antigenic peptides of these tumor Ags frequently resulted in the regression of primary melanoma tumors as well as metastatic tumor foci (15, 16, 17, 18).

Meanwhile, it is also obviously important to determine the tumor antigenic peptides in tumors of epithelial origins such as colon, breast, lung, stomach, and liver cancers. As the incidence of tumors derived from these tissues is obviously much higher than that of melanomas, determination of the antigenic peptides that are effective against these tumors will be of great therapeutic significance. However, the nature of these tumors is not known except for a few reports (19, 20).

We previously reported that autologous CD8⁺ CTLs that were cytotoxic against tumors of epithelial origins could be generated and expanded in vitro from patients’ tumor-infiltrating lymphocytes and PBL (1, 2, 21, 22, 23, 24). In one of these systems, gastric signet ring cell carcinoma HST-2 cells were lysed by CD8⁺ CTL clone TcHST-2 in the context of HLA-A31 restriction (22).

In the current study, by using acid elution and biochemical analyses we determined the structure of natural antigenic peptide of HST-2, designated as F4.2. To our knowledge, this is the first report indicating the primary amino acid sequence of human gastric tumor antigenic peptide. F4.2 is composed of 10 aa. Because our preliminary data showed that TcHST-2 could be cytotoxic to allogeneic gastric tumor cells upon HLA-A31 gene transfection, this antigenic peptide may be expressed among certain human gastric tumors and be presented by HLA-A31 molecules to CTLs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
CTLs and human tumor lines

The procedure for establishing CTL against tumor lines was previously described (21). In the current investigation, we used a gastric signet ring cell carcinoma line, HST-2, and CD8⁺ CTL clone, TcHST-2 (HLA haplotype: HLA-A2, A31, B38, B54, C1, C7, DR4.1, DRw53, DQ3, DQ4). TcHST-2 is specifically cytotoxic to autologous HST-2 cells in the context of HLA-A31 restriction, because this cytotoxicity was completely blocked by anti-HLA-A31-specific mAb as previously reported (22). rIL-2 was kindly provided by Takeda Pharmaceutical (Osaka, Japan) and Shionogi Pharmaceutical (Osaka, Japan). We also employed HLA-A31(-) allogeneic lines, such as M-EB (EB virus-transformed B cell line), C1R (B cell line), MKN28 (gastric tumor line), and HOBC8 (HeLa cells transfected with SV40 large T). HOBC8 was kindly provided by Dr. P. Coulie (Ludwig Institute for Cancer Research, Brussels, Belgium).

HLA-A31 DNA typing of HST-2 cells, and introduction of HLA-A31 gene to allogeneic tumor cells

A cDNA library of HST-2 cells was made by a poly(A) Tract mRNA purification kit (Promega, Madison, WI) and cDNA synthesis kit (Pharmacia, Uppsala, Sweden) according to the manufacturer’s manuals. PCR was employed for HLA-A DNA typing of HST-2 cells by using HLA-A-specific primer set including SalI site and HindIII site (forward primer, 5'-GCG CGT CGA CCC CAG ACG CCG AGG ATG GCC-3'; backward primer, 5'-CCG CAA GCT TTT GGG GAG GGA GCA CAG GTC AGC GTG GGA AG-3'). The reagents and condition of the PCR were the same as previously described except that we used Vent DNA polymerase (New England Biolabs, Beverly, MA) (25). The 1.3-kb PCR products were inserted into the T-vector derived from pBluescript SK⁺, and 20 subclones were sequenced by using M13 primers and other HLA primers (ABC2SF, ABC3SF, and unpublished sequence data), which were kindly provided by Dr. A. Kimura (Tokyo Medical and Dental University School of Medicine, Tokyo, Japan). The sequence data was identical with that of HLA-A*31012 (26).

For the expression of HLA-A*31012 cDNA in allogeneic tumor lines, the 1.3-kb full-length HLA-A*31012 cDNA was inserted into the XhoI/XbaI site of the pBJ mammalian expression vector containing neomycin resistant gene (27). Several tumor lines (MKN28 and HOBC8) were transfected with pBJ-A*31012 by the electroporation method (Gene pulser II; Bio-Rad, Richmond, CA) or Lipofectin Reagent (Life Technologies, Gaithersburg, MD), and selected with 100–400 µg/ml G418 (Life Technologies) containing medium. Cells were subjected to a single cell cloning, and MKN28-A31-2 and HOBC8-A31-12 clones were obtained. All of these clones were positive for the expression of HLA-A31 molecules on the cell surface as assessed by FACS with anti-HLA-A31-specific mAb, 2D12, as described below.

Establishment of anti-HLA-A31-specific mAb

BALB/c mice were immunized i.p. with C1R-A*31012 cells at weekly interval for 4 wk, and hybridoma cells were established as previously described (28). A screening of mAb production was done with a positive reaction to C1R-A*31012, but negative to C1R and C1R-A*3303 cells. C1R-A*31012 and C1R-A*3303 cells were C1R transfectants of genomic HLA-A*31012 and HLA-A*3303 DNA, respectively, and were kindly provided by Dr. M. Takiguchi (Kumamoto University School of Medicine, Kumamoto, Japan). Each mAb was also analyzed for its immunoprecipitating capability. We only selected mAbs that could make immunoprecipitates corresponding to MHC class I heavy and light chains, and thus representatively obtained one of such anti-HLA-A31-specific mAbs, namely mAb2D12, which reacted only with HLA-A31(+) lines, but not with HLA-A31(-) lines in which other HLA class I Ags such as HLA-A1, A2, A3, A11, A24, A26, A33, B7, B18, B35, B44, B51, B55, B60, B61, C1, C7, and C9 are expressed (data not shown).

Acid extraction and separation of the antigenic peptides, amino acid sequencing, and synthesis of the antigenic peptides

The isolation procedure for the natural antigenic peptides of cells was previously described by Falk et al. (29) and York and Rock (30). Approximately 4–5 x 10¹⁰ HST-2 cells were harvested from in vitro cultivation with RPMI 1640 medium plus 10–15% FCS in the presence of 50 U/ml of IFN- (Chugai Pharmaceutical, Tokyo, Japan). Cells were washed with PBS three times, homogenized, 0.1% trifluoroacetic acid (TFA)³ was added, and the mixture was incubated for 30 min at room temperature. After centrifuging at 10,000 x g, the supernatant was applied to Sephadex G-25 column (20 x 300 mm) chromatography to recover samples of less than 5 kDa in molecular size. Then, antigenic peptides were purified from these samples by reverse-phase HPLC (RP-HPLC) in three steps. In the first and the second step, these samples were loaded to the preparative C₁₈ column (µBondasphere, 5µ C₁₈, 300 Å, 19 x 150 mm, Nihon Waters, Tokyo, Japan) and applied with a linear gradient of buffer A (0.1% TFA in H₂O) and B (0.1% TFA in acetonitrile) as described below; a linear gradient from 0 to 80% buffer B for 30 min and from 40 to 50% buffer B for 30 min were employed in the first and second step of RP-HPLC, respectively. In the final (third) step, samples including antigenic peptides were loaded to a ZORBAX-ODS column (4.6 x 250 mm; Rockland Technologies, Gilbersville, PA) and applied with a linear gradient from 0 to 60% buffer B for 60 min. In all steps of RP-HPLC, the flow rate was 1 ml/min.

We determined the fraction of the first and second step RP-HPLC that enhanced the cytotoxicity of TcHST-2 clone against adequate target cells in a ⁵¹Cr release cytotoxicity assay. In these experiments, 3 µl of each fraction was added to a 100 µl medium/well containing ⁵¹Cr-labeled target cells, such as HST-2 and HLA-A31(-) M-EB cells, and incubated for 60 min in a CO₂ incubator. A 100-µl volume of medium containing TcHST-2 was then added and cultured for 6–10 h. We applied the antigenic fraction to the third step of RP-HPLC. The amino acid sequencing of each peak of the third step RP-HPLC was then performed by Edman’s degradation method (477A Protein Sequencer, Applied Biosystems, Foster City, CA).

Based upon these sequences, peptides were synthesized (431A Peptide Synthesizer, Applied Biosystems) and purified with a ZORBAX-ODS column. We also utilized synthetic C-terminal-deleted peptides, peptides substituted with Ala or Arg at certain positions, and HLA-A31-binding hepatitis B antigenic peptide. These peptides were purified similarly, resolved in PBS containing 1% DMSO, and were tested in cytotoxicity and TNF production bioassays by TcHST-2 CTL clone as described below.

Treatment of cells with peptides, and cytotoxicity and TNF assays

The cytotoxicity assay has been described previously (21, 22). In the current investigation, the fractions of RP-HPLC and synthetic peptides were assessed for their ability to stimulate and enhance the TcHST-2 reactivity in the cytotoxicity and/or TNF production assays. For the cytotoxicity assay, ⁵¹Cr-labeled target cells, such as HST-2 cells, were pulsed with the samples for 60 min in a CO₂ incubator, washed with PBS, and mixed with TcHST-2 at certain E:T ratios. Thereafter, we followed the procedure as previously noted (21, 22). The TNF assay used was as described by Espevik and Nissen-Meyer (31). HST-2 cells were treated with certain amounts of the peptides for 60 min in a CO₂ incubator. TcHST-2 CTLs were then admixed and the cells were cultured for various times in a 5% CO₂ incubator. The culture supernatant was harvested, and its TNF production was assessed using W13 (WEHI-164 clone 13) cells as described previously (31). Briefly, W13 cells were plated in the presence of 2 µg/ml of actinomycin D and 40 mM LiCl, and the experimental supernatant was distributed onto the plate. After incubation for 20 h, viability of the W13 cells was analyzed by MTT assay. W13 was kindly provided by Dr. P. Coulie. The TNF content of the supernatant was determined from the standard TNF solution.

Minigene construction, transfection, and assays

To express peptide Ags in endogenous form, we constructed an expression minigene vector, pF4.2ss. pcDSR-E3, which contains an adenovirus E3, 19-kDa protein signal sequence (32) under the control of SR promotor, was kindly gifted by Drs. E. De Plaen and P. Chomez (Ludwig Institute for Cancer Research, Brussels, Belgium) and Dr. J. R. Miller (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA). pF4.2ss was constructed by insertion of oligonucleotides corresponding to F4.2 peptide into the PstI and XbaI site of pcDSR-E3 expression vector. pF4.2ss and pcDSR-E3 were transfected to HST-2, MKN28-A31-2 and HOBC8-A31-12 cells by lipofectin reagent. At first, the reactivity of TcHST-2 against HST-2, HOBC8-A31-12, and K562 cells, which were introduced with pF4.2ss and pcDSR-E3, was assessed in transient assays for 48-h transfection of genes using TNF production from TcHST-2. Then, we also obtained stable transfectant lines of HOBC8-A31-12 and MKN28-A31-2, which were cotransfected with pF4.2ss and puromycin resistant gene, pBabe Puro (33, 34), at a 20:1 molar ratio of DNA. The cells were selected with 1.0 µg/ml of puromycin, and the cell lines were obtained. The insertion of oligonucleotide sequence coding F4.2 in these lines was confirmed by using PCR analysis. These lines were used in the cytotoxicity experiment by TcHST-2. We also constructed an expression minigene vector, pF4.2ssAla9. This minigene codes peptides substituted with Ala at the ninth position Trp of F4.2 wild peptide.

Binding assays of peptides to purified HLA-A31 molecules

Purified HLA-A31 molecules were obtained from the lysates of C1R-A*31012 cells by using immunoaffinity chromatography with anti-HLA-A31-specific mAb 2D12. Namely, C1R-A*31012 cells were homogenized with the lysate buffer (10 mM Tris-HCl (pH 7.0) 150 mM NaCl, and 0.02% NaN₃) and protease inhibitor cocktail tablet (complete; Boehringer Manheim, Manheim, Germany) in the presence of 0.1% octyl-glycoside (Sigma, St. Louis, MO), centrifuged at 15,000 rpm for 30 min, and supernatant was obtained as the cell lysate. The lysate was incubated with mAb 2D12 immobilized Affi-Gel 10 beads (Bio-Rad) at 4°C for 12 h. After washing with the buffer (0.1 M Tris-HCl (pH 8.0) and 0.2 M NaCl), purified HLA-A31 molecules were eluted with 0.1 M glycine buffer (pH 2.5) and immediately neutralized with 1.0 M Tris. A small sample of eluate was run on 10% SDS-PAGE and, purification was confirmed by silver staining of gel.

The binding of peptides to purified HLA-A31 molecules was determined by the method described by Tsai et al. (35). This assay was based on the competitive inhibition of radiolabeled standard peptide binding to HLA-A31 molecules with test peptides. We used the standard peptide Lys-Ile-Met-Lys-Trp-Asn-Tyr-Glu-Arg (KIMKWNYER), which was shown to bind strongly to HLA-A31 by Falk et al. (36) and Rammensee et al. (37). This peptide was radiolabeled with ¹²⁵I by the Iodo-Beads method (Pierce, Rockford, IL) under the manufacturer’s protocol. Approximately 14 pM HLA-A31 molecules were admixed with 125 pM standard radiolabeled peptide in 0.1 ml PBS containing 1mM ß₂-microglobulin and protease inhibitor cocktail tablet (complete; Boehringer Mannheim). Approximately 10 mM to 1 nM of the test (competiter) peptides was added to the above reaction mixture and incubated at room temperature for 48 h. Peptide-bound HLA-A31 molecules were recovered by a PD-10 column (Pharmacia) to remove nonbound free peptides. Then, the radioactivity of peptide-bound HLA-A31 molecules was determined by a scintillation gamma counter. The percent inhibition was calculated as "radioactivity (CPM) of test peptides/radioactivity (CPM) of standard peptide alone", and the concentration of test peptides necessary to inhibit the binding of radiolabeled standard peptide by 50% (IC₅₀) was also calculated.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Separation of TFA-extracted antigenic peptides of HST-2 tumor cells recognized by TcHST-2

To determine the natural antigenic peptides that are recognized by TcHST-2, 4–5 x 10¹⁰ HST-2 cells were harvested and treated with 0.1% TFA solution. The supernatant was applied to a Sephadex G-25 column, and molecules less than 5 kDa in molecular size were obtained. They were then applied to the first (Fig. 1A) and second (Fig. 2A) step of preparative RP-HPLC. We examined whether the samples of each fraction could enhance the cytotoxicity by TcHST-2 CTL clone against ⁵¹Cr-labeled autologous HST-2. As a negative control, we used HLA-A31(-) target cells such as M-EB cells. In this experiment, we employed an E:T ratio of 10:1. Because this condition confers a base line of specific cytotoxicity of only <10% against HST-2 target cells in the absence of exogenously added peptide treatment, it allowed determination of whether peptide fractions have antigenic activity.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Elution profile (A) of the first step RP-HPLC of TFA-extracted HST-2 Ags and determination of antigenicity (B) of fractions. Approximately 4–5 x 1010 HST-2 cells were harvested from in vitro culture. Ags were extracted with 0.1% TFA as indicated in Materials and Methods. After harvesting molecules <5 kDa in molecular size, a sample of 500 µg in protein content was loaded to a C-18 column (19 x 150 mm) with a linear gradient of 0–80% buffer B for 30 min. A total of 3 µl of each fraction was added to 100 µl medium containing 51Cr-labeled HST-2 () and M-EB (•) cells and incubated for 60 min in a CO2 incubator. A 100 µl of medium containing TcHST-2 clone was added at an E:T ratio of 10:1, and the cytotoxicity culture was done for 6–10 h. An arrow indicates an active fraction.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Elution profile (A) and antigenicity (B) of the second step RP-HPLC of fraction no. 12 obtained from the first step of preparative RP-HPLC. Samples of fraction no. 12 in the first step of RP-HPLC were harvested and concentrated, and 50 µg was loaded to a C18 column (19 x 150 mm) with a linear gradient of 40–50% buffer B for 30 min (thick arrows). The antigenic activity of each fraction was also determined similarly as described in Fig. 1 using HST-2 () and M-EB (•) cells. A thin arrow indicates the active fraction.

Determination of amino acid sequence

Fraction no. 17 of the second step of preparative RP-HPLC (Fig. 2A) was further purified by a ZORBAX-ODS RP-HPLC column. As shown in Fig. 3, several peaks were demonstrated, and we characterized each of these as to whether they contained peptides. It was shown by using Edman’s degradation method for protein sequencing that only fractions 4, 5, and 6 contained amino acids, and the other fractions did not. Both fraction 4 (fr. 4) and fraction 6 (fr. 6) (arrows in Fig. 3) were shown to be composed of 10 aa, as demonstrated in Table I; fraction 4 demonstrated the primary amino acid sequence of Tyr-Ser-Trp-Met-Asp-Tyr-Ser-Cys-Trp-Ile (designated F4.1) or Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile (F4.2). They appeared to differ from one another only in N-terminal sixth amino acid, because two clear peaks corresponding to Tyr and Ile were detected with almost the same quantity in cycle 6 of the HPLC chart of Edman’s degradation method. Meanwhile, fraction 6 showed Ile-Ala-Pro-Cys-Pro-Leu-Arg-Arg-Pro-Ala (designated F6). It was shown that the third amino acid Trp of F4.1 and F4.2, and the sixth Ile of F4.2, corresponded to the anchor motif of HLA-A31-bound peptides (36, 37). This was also true for the sixth amino acid Leu of F6. Fraction 5 was shown to be composed of only 4 aa. Because peptides that bind to HLA-class I molecules are usually 8–13 aa (29, 30), we omitted fraction 5 as a candidate of antigenic peptides.

View larger version (10K): [in this window] [in a new window]   FIGURE 3. Elution profile of the final (third) step RP-HPLC using a ZORBAX-ODS column of fraction no. 17 obtained from the second step RP-HPLC. A linear gradient of 0–60% buffer B for 60 min was used. Flow rate was at 1 ml/min. Two fractions, fr. 4 and fr. 6 (arrows), were further analyzed for the protein sequence. See text for details.

Three peptides (F4.1, F4.2, and F6), whose primary sequences were obtained from analyses of fraction 4 and fraction 6 in the third (final) step of RP-HPLC, were synthesized and purified with RP-HPLC. To determine the antigenicity of these peptides, we used the cytokine production assay by CTLs, because this method seems to be highly sensitive in determining the antigenicity of peptides (11, 38). Namely, we determined if TcHST-2 could enhance the TNF production when HST-2 cells were pulsed with exogeneously added synthetic peptides. In the experiments, certain amounts of each peptide were pulsed to HST-2 cells. TcHST-2 was added at an E:T ratio of 10:1 following in vitro cultivation for 6 h, and the TNF production was assessed in each culture supernatant of wells with the MTT assay using W13 cells (31). As shown in Table II, at an E:T ratio of 10:1, base line TNF production was indicated at 200–300 pg/ml in response to HST-2 cells without peptide treatment. Meanwhile, when peptides were pulsed to HST-2 cells, it appeared that TcHST-2 enhanced the production of TNF most preferentially with F4.2 peptides. Other peptides seemed to fail to enhance the TNF production by TcHST-2. Furthermore, none of these synthetic peptides at any concentration stimulated TcHST-2 when they were pulsed to M-EB cells. We also determined the specificity of TcHST-2 response against peptides. As shown in Fig. 4, F4.2 peptide could enhance the TNF production in a dose-dependent manner, whereas HBA31 (STLPETTVVRR), which is the antigenic peptide of CTL specific for the HLA-A31-restricted hepatitis B Ag (39), could not affect TcHST-2 reactivity.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. Specificity of TcHST-2 response against F4.2 (YSWMDISCWI) and HBA31 (STLPETTVVRR) peptides. HST-2 cells were pulsed with 0.1, 1.0, and 10 µM of these synthetic peptides or without peptide, and mixed for 6 h with TcHST-2 at an E:T ratio of 3:1. The TNF production by TcHST-2 was determined as described in Materials and Methods.

Although it appeared that TcHST-2 could respond to pulsed F4.2 peptide in the highly sensitive TNF production assay, we attempted to express F4.2 peptide in an endogenous form (32) by constructing an expression vector pF4.2ss. Because it is uncertain whether F4.2 peptide could enter into the endoplasmic reticulum via TAP, we used the expression vector encoding the signal sequence plus F4.2 peptide. pF4.2ss minigene encodes adenovirus E3, 19-kDa protein signal sequence plus F4.2 peptide, as shown in Fig. 5.

View larger version (11K): [in this window] [in a new window]   FIGURE 5. The construction of a minigene, pF4.2ss, encoding the adenovirus E3 signal sequence plus F4.2 peptide. The oligonucleotides encoding F4.2 peptide were ligated at PstI and XbaI sites in PcDSR-E3, and a pF4.2ss minigene was obtained. An arrow A indicates a site cleaved by signal peptidase, and an underline B shows a Kozak consensus sequence.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. TcHST-2 reactivity against HST-2, HOBC8-A31-12, and K562 cells in which F4.2 peptide was expressed transiently with the introduction of pF4.2ss minigene. HST-2 (A), HOBC8-A31-12 (B), and K562 (C) cells were transfected for 48 h with various concentrations of pF4.2ss or control pcDSR-E3, which encodes the adenovirus E3 signal sequence alone. Then the cells were cultured with or without TcHST-2 for 6 h at an E:T ratio of 3:1. The TNF production was determined as shown in Fig. 4.

View larger version (13K): [in this window] [in a new window]   FIGURE 7. Cytotoxicity by TcHST-2 against stable transfectants of HOBC8-A31-12 and MKN28-A31-2 cells with pF4.2ss minigene. HOBC8-A31-12 (A) and MKN28-A31-2 (B) cells were transfected with pF4.2ss and pBabe Puro at 20:1 of DNA molar ratio, or with pBabe Puro alone. The cells were selected in the presence of 1.0 µg/ml of puromycin, and the cell lines were obtained. Then the cells were labeled with 51Cr and mixed with TcHST-2 at various E:T ratios. Bars represent mean ± SE.

We further studied the characteristics of F4.2 peptide. As shown in an upper panel of Fig. 8A, we synthesized and purified peptides such as F4.2-9, F4.2-8, and F4.2-7, which were deleted with C-terminal 1, 2, and 3 aa residues, respectively, from F4.2 decamer wild peptide. Then these peptides were assessed for antigenicity to TcHST-2 in the TNF production assay. HST-2 cells were pulsed with 0.1, 1.0, and 10.0 µM peptides, and mixed with TcHST-2 at an E:T ratio of 3:1. As shown in a lower panel of Fig. 8A, F4.2 and F4.2-9 peptides could enhance TcHST-2 reactivity in a dose-dependent manner, whereas F4.2-8 and F4.2-7 lost almost completely the antigenicity to TcHST-2. Because these data suggested that the N-terminal ninth Trp may be important in the interaction with TcHST-2, we studied the interaction by using synthetic peptides which are substituted with Ala (F4.2-Ala⁹) or Arg (F4.2-Arg⁹) at this ninth position (upper panel of Fig. 8B). Although F4.2-Ala⁹ may show very weak reactivity with higher concentrations of peptides, it may be concluded that neither of these peptides could enhance TcHST-2 reactivity (lower panel of Fig. 8B). It appears that F4.2-Arg⁹ has a strong anchor motif for the binding to HLA-A31 at the ninth position (36, 37), but it almost completely failed to stimulate TcHST-2. Although the content of TNF production by TcHST-2 was different in Fig. 8A and Fig. 8B, this seemed due to the different level of activation state of TcHST-2 by recombinant IL-2.

View larger version (14K): [in this window] [in a new window]   FIGURE 8. The antigenicity of F4.2 C-terminal-deleted synthetic peptides (A) and variant F4.2 peptides substituted at the ninth position (B). Identical amino acid as F4.2 amino acid sequence. HST-2 cells were pulsed with 0.1, 1.0, and 10.0 µM of peptides or without peptides, and mixed for 6 h with TcHST-2 at an E:T ratio of 3:1. The TNF production by TcHST-2 was determined as in Fig. 4.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. A, Construction and nucleotide sequence (underline) of pF4.2ss minigene variant, pF4.2ss-Ala9. DNA fragments coding F4.2 variant in which the ninth amino acid is substituted with Ala (ssF4.2-Ala9) instead of Trp of ssF4.2 wild form were inserted into the PstI and XbaI site of PcDSR-E3 expression vector, and pF4.2ss-Ala9 vector was obtained. B, Reactivity of pF4.2ss-Ala9. HOBC8-A31-12 cells were transfected for 48 h with 100 and 33 ng/ml of pF4.2ss and pF4.2ss-Ala9, and TcHST-2 was added at an E:T ratio of 3:1. Then, TNF production was determined.

We determined amino acid residue(s) of F4.2 peptide which acts as the agretope in the binding to HLA-A31 molecules. To this end, we developed anti-HLA-A31 mAb 2D12. This mAb reacts with C1R-A*31012, but not with C1R or C1R-A*3303. 2D12 also does not react with HLA-A1, A2, A3, A11, A24, A26, B7, B18, B35, B44, B51, B55, B60, B61, C1, C7, and C9-positive cells, indicating that 2D12 is highly specific for HLA-A31. Therefore, HLA-A31 molecules were purified with mAb 2D12 from the lysates of C1R-A*31012 cells. As shown in Fig. 10, it is indicated that mAb 2D12 could precipitate molecules corresponding to HLA class I heavy and light chains. Then, the binding of synthetic peptides to HLA-A31 molecules was assessed in a competitive inhibition with HLA-A31-restricted radiolabeled standard peptide, Lys-Ile-Met-Lys-Trp-Asn-Tyr-Glu-Arg (KIMKWNYER) (36, 37). The radioactivity of peptide-bound HLA-A31 molecules was determined, and the concentration of test peptides that could inhibit the radiolabeled standard peptide by 50% (IC₅₀) was calculated. As shown in Table III, F4.2 wild peptide indicated the strongest binding affinity to HLA-A31 molecules among all the peptides. Although TcHST-2 CTL responded to F4.2-9, but not to F4.2-8, both of these C-terminal-deleted peptides showed almost similar binding capability. As compared with F4.2, F4.2-Arg⁹ showed almost the same binding capability, and F4.2-Ala⁹ showed a minimally reduced binding capability. However, these peptides could not stimulate TcHST-2 reactivity, confirming our notion that the N-terminal ninth Trp may act as the T cell epitope residue. F4.2-7 showed much lower binding capability, but it seemed that this peptide could still bind to HLA-A31 molecules, since the N-terminal sixth Ile appeared to be the most important residue as the agretope. Indeed, when we substituted F4.2 with Ala (F4.2-Ala⁶) at the N-terminal sixth Ile, the binding capability was drastically impaired. Meanwhile, F4.2-Ala³ minimally lost its binding affinity to HLA-A31 molecules. Thus, in F4.2 peptide it appeared that the most critical residue to the binding to HLA-A31 was the sixth Ile.

View larger version (17K): [in this window] [in a new window]   FIGURE 10. Photograph of silver staining of immunoprecipitates made by 2D12 mAb with the lysates of C1R-A*31012 cells in 10% SDS-PAGE. The thickest band corresponded to MHC class I heavy () chain (HLA-A31 molecule).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our previous report indicated that a gastric signet ring cell carcinoma, HST-2 cells, was lysed by autologous PBL-derived CD8⁺ CTL clone, TcHST-2, in the context of HLA-A31 (22). The TCR gene structure analysis of CTLs in this system has suggested that HST-2 cells may express the tumor Ag on the cell surface, because MLTC of patient’s PBL frequently resulted in the clonal expansion of CTLs which have the same DNA sequence in the complimentarity-determining region-3 (CDR-3) as TcHST-2 (23, 24). Therefore, using an autologous pair of HST-2 tumor cells and TcHST-2 CTLs, we have attempted to determine the primary structure of the antigenic peptide of HST-2 cells. In the current investigation, we used TFA elution of Ags and purified the antigenic peptide with several steps of biochemical procedures, including RP-HPLC. This is the first report demonstrating the primary amino acid sequence of human gastric signet ring cell tumor Ag recognized by CD8⁺ CTLs. Our data suggested that TcHST-2 could preferentially recognize 10 aa composed of Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile. We designated this peptide as F4.2.

The immunogenic activity of fractions no. 12 and no. 17 from the first and second step, respectively, of preparative RP-HPLC could be detected in the cytotoxicity assays. However, although that of synthetic peptides was detected in TNF production assays, it was rather obscure in the conventional cytotoxicity assays. Perhaps these observations were due to the sensitivity of the assay system. TNF assays using W13 cells are more sensitive than the cytotoxicity assays, and can detect even <10 pg/ml of TNF. These facts may imply that F4.2 peptide has a relatively weak immunogenicity. Meanwhile, one more possible explanation is that there may exist a conformational difference between natural antigenic peptides and synthetic ones. It is also possible that the peptide exchange was relatively ineffective between exogenously added F4.2 peptides and already-bound endogenous, i.e. resident, peptides on the cell surface of HLA-A31 molecules.

These possibilities led us to investigate whether the antigenicity of F4.2 could be enhanced when it was expressed in an endogeneous form within the cells by using the minigene expression vectors (32). Because we do not know yet whether F4.2 peptide enters into the endoplasmic reticulum via TAP molecules, we constructed a minigene, pF4.2ss, encoding signal sequence plus F4.2 peptide by which means F4.2 peptide could enter directly into the endoplasmic reticulum via signal recognition particles. Our data showed that the introduction of pF4.2ss into autologous HST-2 cells resulted in drastically enhanced TcHST-2 reactivity. Furthermore, the introduction of pF4.2ss to HLA-A31(+) allogeneic tumor lines was also able to stimulate TcHST-2; TcHST-2 could produce TNF in a very efficient amount as well as lysing with these targets. These facts may suggest that the antigenicity of F4.2 peptide itself was not weak, and the relatively low antigenicity observed in the experiment using F4.2 synthetic peptides may have been due to inefficient peptide exchange between already-bound endogenous, i.e. resident, peptides and exgenously added synthetic ones on the cell surface of HLA-A31 molecules. More recent experiments suggest that F4.2 peptide may be transported via TAP, because the minigene coding F4.2 without signal sequence was similarly effective to pF4.2ss in enhancing TcHST-2 response.

So far there is no homologous protein between F4.2 peptide primary sequence and known proteins when investigated with the computer database, and it appears that the parental molecule of this peptide is an as yet unknown protein. The molecular cloning of the parental protein of F4.2 is very important for understanding the whole characteristic of this Ag, and this is now being undertaken by using degenerated oligonucleotides deduced from F4.2 peptide sequence.

We are also studying the effectiveness of in vitro CTL induction from HLA-A31(+) patients’ PBL with gastric tumors. Our preliminary data indicated that 30% of patients could clearly induce peptide-specific CTL in vitro. Our data also showed that F4.2 is immunogenic in at least some gastric tumors when they express HLA-A31 molecules; TcHST-2 was cytotoxic against gastric tumor lines MKN-28, but not to MKN-74 and HGC-25, when these lines were transfected with an expression vector of HLA-A31 gene, pBJ-A*31012. Furthermore, F4.2 peptides expressed in endogenous form within the cells could obviously enhance the reactivity of TcHST-2 in HLA-A31(+) allogeneic tumor cells. If this is true for more large panels of HLA-A31(+) gastric tumor cells, it may be recommended for immediate clinical use, because gastric signet ring cell carcinomas belong to the highly malignant cancers with very poor prognosis.

	Acknowledgments

 
This work is dedicated to Dr. Masahiko Ohta who died of gastric cancer in May 1996, at the age of 33. Dr. Ohta was one of the main investigators in the biochemical isolation and analysis of F4.2 peptide. We thank Dr. P. Coulie (Ludwig Institute for Cancer Research, Brussels) for kindly providing W13 (WEHI-164 clone 13) and HOBC8 cells. We also thank Drs. E. De Plaen and Dr. P. Chomez (Ludwig Institute for Cancer Research, Brussels) and J. R. Miller (DNAX Research Institute of Molecular and Cellular Biology) for providing pcDSR-E3 expression vector. We are also grateful to Dr. N. Yamada (Central Research Laboratories, Ajinomoto Co. Inc., Kawasaki) for his fruitful suggestions regarding this study.

	Footnotes

 
¹ This work was supported by research grants of the Princess Takamatsu Cancer Research Fund, the Japanese Medical Association Research Fund, the Akiyama Foundation, the Naito Foundation, NOVARTIS Foundation (Japan) for the Promotion of Science, and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture and Science of Japan.

² Address correspondence and reprint requests to Dr. Noriyuki Sato, Department of Pathology, Sapporo Medical University School of Medicine, S. 1, W. 17, Chuo-ku, Sapporo 060-8556, Japan. E-mail address:

³ Abbreviations used in this paper: TFA, trifluoroacetic acid; RP-HPLC, reverse phase-HPLC.

Received for publication April 26, 1999. Accepted for publication June 25, 1999.

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