Kazuhiko Nakagawa

XAGE-1 expression in non : Small cell lung cancer and antibody response in patients

Purpose:XAGE-1 was originally identified by the search for PAGE/GAGE-related genes using expressed sequence tag database and was shown to exhibit characteristics of cancer/testis-like antigens. Four transcript variants XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d have been identified thus far. We recently identified XAGE-1b as a dominant antigen recognized by sera from lung adenocarcinoma patients. We here investigated the mRNA expression of four XAGE-1 variants and XAGE-1 protein expression in non–small cell lung cancer (NSCLC). Humoral immune response to XAGE-1b was also evaluated in patients. Experimental Design: Forty-nine NSCLC specimens were analyzed for the expression of four XAGE-1 transcript variants by conventional 30-cycle and real-time reverse transcription-PCR and XAGE-1 protein expression by immunohistochemistry. Sera from 74 patients were analyzed for XAGE-1b antibody production by ELISA and Western blot. Results:XAGE-1b and XAGE-1d mRNA were detected in 15 and 6 of 49 lung cancer specimens, respectively. No XAGE-1a or XAGE-1c mRNA expression was observed. XAGE-1b mRNA expression was observed in 14 of 31 (45%) adenocarcinoma and 1 of 18 (6%) lung cancer with other histologic types. Immunohistochemical analysis using a XAGE-1 monoclonal antibody showed that 14 of 15 XAGE-1b mRNA-positive and 3 of 34 XAGE-1b mRNA-negative specimens expressed XAGE-1 protein. Seropositivity was observed in 5 of 56 patients with adenocarcinoma, whereas none of 18 patients with other histologic types produced XAGE-1b antibody. Conclusion: XAGE-1b is highly and strongly expressed in lung adenocarcinoma and immunogenic in patients, suggesting that XAGE-1b is a promising antigen for immunotherapy against lung adenocarcinoma.

Immunoscreening of a cDNA library from a lung cancer cell line using autologous patient serum: Identification of XAGE-1b as a dominant antigen and its immunogenicity in lung adenocarcinoma.

By serologic identification of antigens by recombinant expression cloning (SEREX) analysis using an autologous lung adenocarcinoma cell line, OU‐LU‐6, as a cDNA library source, we demonstrated that XAGE‐1 was the dominant antigen recognized by serum from a patient. By immunoscreening, we obtained 38 positive cDNA clones consisting of 16 genes designated as OY‐LC‐1 to ‐OY‐LC‐16. OY‐LC‐1, represented by 18 clones, was identical to XAGE‐1. OY‐LC‐2 to ‐16, represented by either a single or 2 clones, were identical to known genes shown to be ubiquitously expressed in various normal tissues. RT‐PCR analysis showed that of 4 XAGE‐1 transcripts—XAGE‐1a, b, c and d—XAGE‐1b was expressed in OU‐LU‐6 dominantly. Furthermore, XAGE‐1b mRNA was expressed in 4 of 10 lung cancer tissues, whereas no expression was observed in normal tissues. Of 4 XAGE‐1b mRNA positive cancer tissues, 3 were adenocarcinoma and one was poorly differentiated squamous cell carcinoma. Of 32 sera from lung cancer patients, 8 sera were reactive with the XAGE‐1b product. Those 8 sera were from patients with adenocarcinoma. These findings indicated strong immunogenicity of XAGE‐1b in lung adenocarcinoma and suggested its potential use as a target for vaccine‐based immunotherapies.

Induction of donor-specific hyporesponsiveness and prolongation of cardiac allograft survival by jejunal administration of donor splenocytes.

BACKGROUNDnDonor-specific immunosuppression is important in transplantation surgery. We examined the immunosuppressive effects of donor splenocytes administered postoperatively into the jejunum and the effect of such treatment on the survival of heterotopic vascularized cardiac allograft in rats.nnnMETHODSnLewis (LEW, RT-1l) recipient rats were treated with 5x10(7) Brown Norway (BN, RT-1n) donor splenocytes for 5 days orally, intrajejunally, or subcutaneously. The immune responses of LEW treated with either donor BN or irrelevant Wistar King A (WKA, RT-1k) were examined by mixed lymphocyte reaction (MLR) and delayed type hypersensitivity (DTH). The effect of postoperative enteral treatment for 6 days with suboptimal dose of cyclosporine (CsA) on heterotopic cardiac allotransplantation was investigated. We measured the production of cytokines (interleukin [IL]-2, IL-4, IL-10, and interferon-gamma [IFN-gamma]) in the supernatant of MLR by ELISA. The effect of intravenous dose of GdCls to block Kupffer cell function was also investigated before the administration of splenocytes.nnnRESULTSnMLR and DTH responses were strongly inhibited in a BN-restricted manner after jejunal or oral feeding of donor BN splenocytes but not by subcutaneous injection or injections by any routs of WKA splenocytes. The effect was more prominent in jejunal than oral feeding. Immunosuppression was associated with a significant inhibition of IL-2 and IFN-gamma production and increased concentrations of IL-4 and IL-10 in MLR supernatants. Immunosuppression was abrogated by pretreatment with GdCl3. Postoperative intrajejunal feeding of donor splenocytes with CsA significantly prolonged cardiac allograft survival time (18.7+/-7.3 vs. 9.9+/-1.7 days for control animals).nnnCONCLUSIONnJejunal administration of splenocytes produces donor-specific immunosuppression and prolongs cardiac allograft survival. Our results suggest the involvement of T helper (Th) 2 cytokines and Kupffer cells in the induction of immune hyporesponsiveness, and indicate that this method represents a unique approach for induction of donor-specific immunosuppression.

Over-Expression of the Testis-Specific Gene TSGA10 in Cancers and Its Immunogenicity

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real‐time RT‐PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over‐expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.

Identification of glioma-specific RFX4-E and -F isoforms and humoral immune response in patients

For regulatory factor X4 (RFX4), two alternatively spliced variants, RFX4‐A and ‐B, were reported in the testis. In this study, we identified transcript variants RFX4‐C, ‐D, ‐E, and ‐F, and demonstrated by reverse transcription‐polymerase chain reaction (RT‐PCR) that RFX4‐A, ‐B and ‐C mRNAs were expressed only in the testis, and RFX4‐D mRNA was expressed only in normal brain tissues. In tumors, RFX4‐E and ‐F in addition to RFX4‐D mRNA were expressed in gliomas by rapid amplification of cDNA ends and RT‐PCR analyses. Expression of RFX4 mRNA was not observed in other tumors, such as lung, esophageal, stomach, colon or liver cancers. Quantitative real‐time RT‐PCR using common primer pairs detecting all of the variant transcripts showed high expression in normal testis, low expression in the brain (1% compared to the expression in testis), and overexpression in 17 of 61 gliomas (28%). Western blot analysis using DC28 monoclonal antibody (mAb) produced against recombinant RFX4‐D C‐terminus protein showed expression of RFX4‐A and ‐C proteins, but not RFX4‐B protein, in the testis, and expression of RFX4‐D protein in the brain. Moreover, expression of RFX4‐E and ‐F proteins, but not RFX4‐D protein, was observed in gliomas. Immunohistochemistry analysis using DC28 mAb showed positive staining in the nuclei of spermatocytes in the testis and glioma cells. Antibody against RFX4 was detected in the sera of 3 of 58 (5%) glioma patients by enzyme‐linked immunosorbent assay, suggesting the immunogenicity of RFX4‐E and ‐F proteins in glioma patients. (Cancer Sci 2005; 96: 801–809)

XAGE‐1 mRNA Expression in Prostate Cancer and Antibody Response in Patients

To evaluate the feasibility of cancer vaccine targeting XAGE‐1, we investigated the expression of 4 XAGE‐1 transcript variants and the humoral immune response to XAGE‐1 in prostate cancer patients. XAGE‐1a, b, c, d mRNA expression was analyzed in 54 prostate cancer specimens and 8 specimens of benign prostate hypertrophy (BPH) by reverse transcription‐polymerase chain reaction (RT‐PCR). The humoral response to XAGE‐1 was investigated in sera obtained from 278 patients with prostate cancer and 40 healthy volunteers by enzyme‐linked immunosorbent assay (ELISA) using recombinant protein. XAGE‐1b mRNA expression was observed in 14 of 54 (26%) prostate cancer specimens, while XAGE‐1a, c, and d mRNA expressions were observed in 1, 1, and 3, respectively. None of the 4 XAGE‐1 transcript variants was observed in the 8 BPH specimens. Antibody against XAGE‐1 was detected in sera from 2 of 129 stage D2 patients, whereas none of sera from 149 patients with localized prostate cancer or lymph node metastasis had detectable XAGE‐1 antibody. No reactivity to XAGE‐1 was found in sera from the 40 healthy individuals.

Identification of an HLA-A24-restricted OY-TES-1 epitope recognized by cytotoxic T-cells.

OY‐TES‐1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY‐TES‐1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY‐TES‐1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA‐A24‐binding OY‐TES‐1 peptide, TES401–409 (KTPFVSPLL) recognized by CD8 T‐cells. Purified CD8 T‐cells from healthy donors stimulated in vitro with the peptide‐pulsed autologous DC and PBMC produced IFN7 in response to the peptide‐pulsed PBMC and showed cytotoxicity against the peptide‐pulsed autologous EBV‐B specifically. Furthermore, cytotoxicity was also observed against an OY‐TES‐1 mRNA‐expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV‐B cells in recognition by CD8 CTL was the same as that of the fraction of the TES401–409 peptide itself, suggesting that the TES401–409 was a naturally processed peptide on LK79.