Hiroko Takedatsu

Interleukin-5 participates in the pathogenesis of ileitis in SAMP1/Yit mice.

SAMP1/Yit mice spontaneously develop ileitis resembling Crohns disease (CD) without chemical or genetic manipulations. Since the focus of studies were Th1 cytokines, only Th1‐type T cells were thought to be responsible for intestinal inflammation in these mice. To further characterize the pathogenesis of this ileitis, we investigated the implication of Th2 cytokines in ileitis of SAMP1/Yit mice. The expression of chemokine receptors (CCR) associated with both Th1 and Th2 lymphocytes, such as CCR2, CCR3, CCR4, CCR5, and CCR8, was increased. Among cytokines, IL‐5 was remarkablyincreased in Peyers patches, mesenteric lymph nodes, and mucosa involved in ileitis. Furthermore, infiltration of numerous eosinophils in ileitis was histologically evident. Severe combined immunodeficiency mice injected intraperitoneally with CD4+ cells from SAMP1/Yit mice developed colitis and ileitis, with the infiltration of eosinophils. Administration of anti‐IL‐5 antibodies significantly attenuated ileitis in these mice. We suggest that IL‐5 participates in the pathogenesis of ileitis and that anti‐IL‐5 antibodies are potentially useful for immunotherapy in CD patients. This is the first demonstration that IL‐5 is crucial for the development of ileitis in this mouse model of CD.

Phase I clinical study of a peptide vaccination for hepatitis C virus‐infected patients with different human leukocyte antigen‐class I‐A alleles

Hepatitis C virus (HCV) infection has a high risk of liver cirrhosis and hepatocellular carcinoma at later stages. We recently identified a peptide derived from the HCV core protein capable of inducing both cellular and humoral responses to nearly all HCV‐positive patients in Japan with different human leukocyte antigen (HLA)‐class I‐A alleles. To assess the safety and immune responses to this novel peptide, we conducted a phase I dose‐escalation study of the vaccination for 26 HCV‐positive patients who were either non‐responders to the interferon‐based therapy (n = 23) or refused it (n = 3). The regimen was well tolerated, with no severe vaccine‐related toxicity. Twenty‐five and 22 patients completed the first and second cycle vaccination (6 and 12 vaccine injections), respectively. After a series of six vaccine injections, peptide‐specific CTL activity was augmented in peripheral blood mononuclear cells from 15 of 25 patient samples, with an expected optimal dose of 1 mg peptide. After 12 vaccine injections, peptide‐specific IgG production was augmented in plasma from the majority of patients (15 of 22 patients) tested, but not in a dose‐dependent fashion. There were two HCV RNA responders with >1 log declines. Among patients whose pre‐vaccination levels of alanine aminotransferase and alpha feto‐protein exceeded the normal ranges, a <30% decrease was found in 7 of 24 and three of six patients, respectively. Because of its tolerability and higher rate of immune boosting, this protocol is recommended for a phase II study to investigate its clinical efficacy. (Cancer Sci 2009; 100: 1935–1942)

Identification of Peptide Vaccine Candidates for Prostate Cancer Patients with HLA-A3 Supertype Alleles

Purpose: The peptide vaccine candidates identified to date have been focused on the HLA-A2 and HLA-A24 alleles. The HLA-A11, HLA-A31, and HLA-A33 alleles share binding motifs and belong to an HLA-A3 supertype family. In this study, we attempted to identify CTL-directed peptide candidates, derived from prostate-related antigens and shared by HLA-A11+, HLA-A31+, and HLA-A33+ prostate cancer patients. Experimental Design: Based on the binding motif to the HLA-A3 supertype alleles, 42 peptides were prepared from prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and prostatic acid phosphatase (PAP). These peptides were first screened for their ability to be recognized by immunoglobulin G (IgG) of prostate cancer patients and subsequently for the potential to induce peptide-specific and prostate cancer–reactive CTLs from peripheral blood mononuclear cells (PBMC) of cancer patients with the HLA-A11, HLA-A31, and HLA-A33 alleles. Results: Five peptide candidates, including the PSA16-24, PAP155-163, PAP248-257, PSMA207-215, and PSMA431-440 peptides, were frequently recognized by IgGs of prostate cancer patients. These peptides efficiently induced peptide-specific and prostate cancer–reactive CTLs from PBMCs of cancer patients with the HLA-A11, HLA-A31, and HLA-A33 alleles. Antibody blocking and cold inhibition experiments revealed that the HLA-A3 supertype–restricted cytotoxicity against prostate cancer cells could be ascribed to peptide-specific and CD8+ T cells. Conclusions: We identified prostate-related antigen-derived new peptide candidates for HLA-A11-, HLA-A31-, and HLA-A33-positive prostate cancer patients. This information could facilitate the development of a peptide-based anticancer vaccine for patients with alleles other than HLA-A2 and HLA-A24.

Ran, a Small GTPase Gene, Encodes Cytotoxic T Lymphocyte (CTL) Epitopes Capable of Inducing HLA-A33–restricted and Tumor-Reactive CTLs in Cancer Patients

Purpose: The purpose is to identify a gene coding for tumor-associated antigen and peptide capable of inducing CTLs reactive to tumor cells with a HLA-A33–restricted fashion to provide scientific basis for specific immunotherapy to HLA-A33+ cancer patients. Experimental Design: An expression gene-cloning method was used to identify the tumor-associated antigen gene. Northern blot analysis and immunohistochemistry were used to examine the mRNA and protein expression levels in various cells and tissues, respectively. Synthetic peptides were examined for their ability to induce HLA-A33+ tumor-reactive CTLs in peripheral blood mononuclear cells from cancer patients. Result: A gene of small GTPase, Ran, which controls the cell cycle through the regulation of nucleocytoplasmic transport, mitotic spindle organization, and nuclear envelope formation, was found to encode epitopes recognized by the HLA-A33–restricted CTLs established from T cells infiltrating into gastric adenocarcinoma. The expression of the Ran gene was increased in most cancer cell lines and cancer tissues at both the mRNA and protein levels. However, it was not enhanced in the surrounding normal cells or tissues. It was also undetectable in normal tissues as far as tested. Ran-derived peptides at positions 48–56 and 87–95 could induce CD8+ peptide-specific CTLs reactive to tumor cells from HLA-A33+ epithelial cancer patients in a HLA class I-restricted manner. Conclusions: Because of its increased expression in cancer cells and involvement in malignant transformation and/or the enhanced proliferation of cancer cells, the two Ran-directed peptides could be potent candidates in use for specific immunotherapy against HLA-A33+ epithelial cancers.

Identification of Peptide Vaccine Candidates Sharing among HLA-A3+, -A11+, -A31+, and -A33+ Cancer Patients

Purpose: Only a few studies have been reported on CTL epitope peptides restricted with alleles other than HLA-A2 and -A24. The HLA-A11, -A31, and -A33 alleles share similar binding motifs with HLA-A3 and -A68 alleles, and, thus, are classified as an HLA-A3 supertype. This study tried to identify CTL epitope peptides as vaccine candidates sharing by HLA-A3+, -A11+, -A31+, and -A33+ cancer patients. Experimental Design: Seven peptides possessing the ability to induce HLA-A31-restricted and tumor-reactive CTLs were examined for their ability to induce HLA-A3-, -A11-, and -A33-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells (PBMCs) of 18 epithelial cancer patients. The five reference peptides all have the ability to induce CTL activity restricted with one of the HLA-A3 supertypes, and, thus, were also examined as positive controls. Results: Three peptides (2 from β-tublin5- and 1 from CGI37-derived peptides) induced tumor-reactive CTLs in PBMCs of HLA-A3+, -A11+, and -A33+ cancer patients with various frequencies (17–50%). One RLI- or KIAA0036-derived peptide induced tumor-reactive CTLs in PBMCs of HLA-A3+ and -A11+ or HLA-A11+ and -A33+ cancer patients also with various frequencies (22–67%), respectively, whereas the other peptide induced CTL activity in only HLA-A33+ patients. Among the five reference peptides tested, one peptide, TRP2–197, induced CTL activity in both HLA-A11+- and -A33+-restricted manners. Conclusions: We identified new peptide vaccine candidates for HLA-A3, -A11, -A31, and -A33 positive cancer patients. This study may facilitate the development of both basic and clinical studies of peptide-based immunotherapy for cancer patients with other alleles of HLA-A2 and -A24.

Prostatic acid phosphatase as a target molecule in specific immunotherapy for patients with nonprostate adenocarcinoma.

Prostatic acid phosphatase (PAP) is one of the prostate-related antigens that are applicable to specific immunotherapy for patients with prostate cancer. In this study, we determined whether or not PAP could be a target molecule in specific immunotherapy for patients with nonprostate cancer. A variety of adenocarcinoma cell lines were examined for their PAP expression at the mRNA and protein levels by reverse transcription polymerase chain reaction and western blot analysis, respectively. Considerable percentages of colon, gastric, and breast cancer cell lines were found to be positive for PAP at both the mRNA and the protein levels. The PAP expression in cancer tissues was also confirmed by immunohistochemical staining. In addition, we examined whether cancer-reactive cytotoxic T lymphocytes (CTLs) could be induced from peripheral blood mononuclear cells (PBMCs) of human leukocyte antigen (HLA) A24+ nonprostate cancer patients by in vitro stimulation with a PAP peptide. As a result, tumor-specific CTLs could be induced from the PBMCs of HLA-A24+ colon and gastric cancer patients. Their cytotoxicity against HLA-A24+ cancer cells was dependent on PAP peptide-specific and CD8+ T cells. These findings indicate that PAP could be a target molecule in specific immunotherapy for patients with nonprostate adenocarcinomas including colon and gastric cancers.

Identification of SART3-derived peptides having the potential to induce cancer-reactive cytotoxic T lymphocytes from prostate cancer patients with HLA-A3 supertype alleles

SART3-derived peptides applicable to prostate cancer patients with HLA-A3 supertype alleles were identified in order to expand the possibility of an anti-cancer vaccine, because the peptide vaccine candidates receiving the most attention thus far have been the HLA-A2 and HLA-A24 alleles. Twenty-nine SART3-derived peptides that were prepared based on the binding motif to the HLA-A3 supertype alleles (HLA-A11, -A31, and -A33) were first screened for their recognizability by immunoglobulin G (IgG) of prostate cancer patients and subsequently for the potential to induce peptide-specific cytotoxic T lymphocytes (CTLs) from HLA-A3 supertype+ prostate cancer patients. As a result, five SART3 peptides were frequently recognized by IgG, and two of them—SART3 511-19 and SART3 734-42—efficiently induced peptide-specific and cancer-reactive CTLs. Their cytotoxicity toward prostate cancer cells was ascribed to peptide-specific and CD8+ T cells. These results indicate that these two SART3 peptides could be promising candidates for peptide-based immunotherapy for HLA-A3 supertype+ prostate cancer patients.

Diminished cytokine signalling against bacterial components in mononuclear leucocytes from ulcerative colitis patients after leukocytapheresis

Infiltration by circulating inflammatory cells is a prominent local inflammatory feature of ulcerative colitis (UC). Several trials have suggested that leukocytapheresis by filtration can benefit patients with active UC. We investigated how this therapy might modulate the inflammatory response. Patients with active UC who were beginning repeated filtration leukocytapheresis were studied. Mononuclear cell preparations were obtained from blood before and after the first treatment, and expression of cytokine signalling components and the cell‐proliferative response were analysed in vitro. Leukocytapheresis reduced lipopolysaccharide‐induced production of proinflammatory cytokines (interleukin‐1, ‐6, ‐8 and tumour necrosis factor‐α, P < 0·05 for all) and activation of intracellular signalling components (nuclear factor‐κB, mitogen‐activated protein kinases, and signal transducer and activator of transcription‐3), as well as surface expression of toll‐like receptor‐4 (P < 0·05) in mononuclear cells. The therapy also reduced the cell‐proliferative response by mononuclear cells stimulated with sonicated bacterial preparations from autologous intestine (P < 0·05). These results indicate that activated mononuclear cells in the peripheral blood of patients with active UC are removed by leukocytapheresis and replaced by cells with a lower activation status. This replacement may partly explain the therapeutic benefit.

A form of circulating interleukin-6 receptor component soluble gp130 as a potential interleukin-6 inhibitor in inflammatory bowel disease

The presence and the role of soluble gp130, the soluble form of a component of the interleukin (IL)‐6 receptor complex, were investigated in inflammatory bowel disease. The serum concentrations of soluble gp130 were increased in ulcerative colitis (active disease, median, 93·5 ng/ml; interquartile range, 26–125 ng/ml; inactive disease, 81 ng/ml, 24·8–137·3 ng/ml) and to a lesser extent in Crohns disease (active disease, 66 ng/ml, 44·4–87·6 ng/ml; inactive disease, 63 ng/ml, 43·5–82·5 ng/ml) compared to normal controls (43 ng/ml, 27–59 ng/ml). Paired analysis of serum samples showed a decrease of IL‐6 and soluble IL‐6 receptor concentrations in both diseases and an increase of soluble gp130 concentrations, especially in ulcerative colitis, just after the resolution of disease exacerbation. Size fractionation of the serum revealed that a part of the IL‐6 co‐eluted with soluble gp130 and soluble IL‐6 receptor. The IL‐6‐induced proliferation of murine B9 hybridoma was enhanced by recombinant soluble IL‐6 receptor, whereas the proliferation was inhibited by recombinant soluble gp130. These results indicate that soluble gp130 may function as a natural inhibitor of the IL‐6 actions in inflammatory bowel disease.

Immediate Early Response Gene X-1, a Stress-Inducible Antiapoptotic Gene, Encodes Cytotoxic T-Lymphocyte (CTL) Epitopes Capable of Inducing Human Leukocyte Antigen-A33-Restricted and Tumor-Reactive CTLs in Gastric Cancer Patients

Peptide-based vaccine therapy, which is designed to elicit T-cell immunity against tumors, is an attractive approach for the treatment of cancer patients. To provide a scientific basis for peptide therapy, an increasing number of CTL-directed peptides have been identified, and some of them have been tried as antigen-specific immunotherapy in the past decade. Only a few studies, however, have been performed on such peptides restricted with alleles other than HLA-A2 and -A24. In the present study, we show that immediate early response gene X-1 (IEX-1), a stress-inducible protein associated with the regulation of cell proliferation and apoptosis, produces antigenic epitopes recognized by 850B-CTLs, HLA-A33-restricted CTLs newly established from T cells infiltrating into gastric adenocarcinoma. The IEX-1 gene was highly expressed in most cell lines and tissues from various types of cancer at both the mRNA and protein levels. However, it was not expressed at the protein level in any normal epithelium or connective tissues tested. Three IEX-1-derived peptides at positions 47-56, 61-69, and 65-73, which were recognized by the 850B-CTLs, could induce CD8(+) peptide-specific CTL reaction to tumor cells from HLA-A33(+) gastric cancer patients and other epithelial cancer patients, but not from healthy donors, in an HLA class I-restricted manner. Because increased expression of IEX-1 is suggested to be involved in the resistance to apoptosis and in the proliferation of cancer cells, these antigenic peptides could be potent candidates for peptide-based specific immunotherapy against HLA-A33(+) gastric cancer and other epithelial cancers.