Mechanisms underlying the effects of leukocyte apheresis with a fiber filter in a rat model of dextran sulfate sodium-induced colitis.

While several clinical trials have suggested that leukocytapheresis (LCAP) by filtration can benefit patients with active ulcerative colitis, the mechanisms underlying these benefits are largely unknown. The aim of this study was to address the mechanisms that may underlie the therapeutic effects of LCAP using a dextran sulfate sodium-induced colitis model in rats. Treatment with the active column, but not the sham column, improved disease severity by down-regulating pro-inflammatory events, including the cell-proliferative responses and inflammatory cytokine and reactive oxygen production, as well as by up-regulating protective events, including hepatocyte growth factor production, bone marrow-derived endothelial progenitor cell induction, and colonic blood flow levels, which were mediated predominantly by calcitonin gene-related peptide. The improvement was also associated with the increase of Ki-67 labeling in the colonic epithelium. In conclusion, the LCAP procedure was used in a dextran sulfate sodium-induced colitis model in rats under extracorporeal circulation conditions. This approach down-regulated pro-inflammatory events and up-regulated protective events in association with disease improvement. These data suggest that LCAP is feasible in animals and should shed light on the mechanisms of LCAP in clinical settings.

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.

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in non-small cell lung cancer.

BACKGROUND: Our previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer. METHODS: In the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein. RESULTS: CCL21 protein production from transduced DC was detected in supernatants (24-72 hours, 3.5-6.7 ng/4-5 x 10(6) cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro. CONCLUSION: Viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

New peptide vaccine candidates for epithelial cancer patients with HLA-A3 supertype alleles.

We previously identified 2 cancer-associated antigens, immediate early response gene X-1 (IEX) and small GTPase (Ran), and their 5 epitopes using human leukocyte antigen (HLA)-A33-restricted and tumor-infiltrating T cells from a colon cancer patient. In this study, we examined whether or not these peptides can induce cytotoxic T lymphocytes (CTLs) in HLA-A11+ or HLA-A31+ epithelial cancer patients because the HLA-A11, HLA-A31, and HLA-A33 alleles share binding motifs as an HLA-A3 supertype family, which is widely distributed in many ethnic populations. Among them, the 2 peptides, IEX 47-56 and IEX 61-69, induced peptide-specific CTLs from peripheral blood mononuclear cells of cancer patients with the HLA-A11 and HLA-A31 alleles more efficiently than the other 3 peptides. Antibody blocking and cold inhibition experiments revealed that the cytotoxicity of peptide-induced CTLs against cancer cells was attributable to peptide-specific and CD8+ T cells. Together with our previous findings, these results indicate that the 2 IEX peptides could be appropriate vaccine candidates for HLA-A11, HLA-A31, and HLA-A33 positive epithelial cancer patients. This information could expand the chance of a peptide-based cancer vaccine for epithelial cancer patients of many ethnic populations.

Identification of new immunogenic peptides in conserved regions of hepatitis C virus (HCV) 1b with the potentiality to generate cytotoxic T lymphocytes in HCV1b(+) HLA-A24(+) patients.

AIM: Hepatitis C virus (HCV) 1b is resistant to standard interferon therapy and has a high risk of developing into hepatocellular carcinoma at the late stage of infection. Therefore, new therapeutic modalities for HCV1b infection must be developed. One approach would be active specific immunotherapy with highly immunogenic HCV1b peptides. METHODS: HCV1b-derived 44 synthetic peptides were selected based on their binding scores to HLA-A24. Peptide-specific IgG were measured by ELISA. Peptide-specific cytotoxic T-lymphocytes (CTLs) were induced in vitro by repeated peptide-stimulation. RESULTS: We identified three novel candidate peptides of HCV1b proteins containing HLA-A24 binding motifs. Each of them had the ability to induce HLA-A24-restricted and peptide-specific CTL activity, and IgGs specific to each of them were detected in the plasma of HCV1b patients. Among these three peptides, a peptide NS5A 2132-2142 was recognized by both cellular and humoral immunities in the majority of blood samples of patients tested. More importantly, the peptide-stimulated peripheral blood mononuclear cells (PBMCs) showed cytotoxicity against cells cotransfected with NS5A and HLA-A2402 genes in an HLA-restricted manner. This is an additional report to our previous study. CONCLUSION: These findings may provide a new insight into the development of a peptide-based specific immunotherapy for HCV1b-infected patients.

Depletion of endothelial progenitor cells in the peripheral blood of patients with ulcerative colitis.

There is strong evidence to suggest that endothelial progenitor cells (EPCs) play a significant role in re-endothelialization and subsequent tissue repair. This study examined the role of EPCs in inflammatory bowel disease, a disease in which impairment of mucosal healing has been implicated. Peripheral blood mononuclear cells obtained from 50 patients with ulcerative colitis (UC), 29 patients with Crohn's disease (CD), 14 patients with infectious colitis, and 35 normal control subjects were cultured in EPC medium, harvested after 7 days, and characterized by immunocytochemistry and flow cytometry. Colony assay for hematopoietic progenitor cells was also performed. Patients with active UC had a significantly decreased number of circulating EPCs as compared with healthy controls (p=0.0013), patients with inactive UC (p=0.0099), patients with active CD (p=0.0235) and patients with infectious colitis (p=0.0002). On the other hand, patients with infectious colitis had a significantly increased number of circulating EPCs as compared with healthy controls (p=0.0406), patients with active UC (p=0.0002), and patients with active CD (p=0.0316). In patients with UC, the number of circulating EPCs was correlated with the serum hemoglobin levels (r=0.485, p=0.007) and inversely with the platelet count (r=-0.372, p=0.0382). The number of hematopoietic progenitor cell colonies was comparable among patients with UC, patients with CD, patients with infectious colitis, and healthy controls. Our observations indicate that the number of circulating EPCs in patients with UC is significantly reduced. Further studies are needed to define the mechanisms that underlie the reduction in the number of circulating EPCs and to better understand the pathophysiological consequences of this event in patients with UC.

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 (511-519) and SART3 (734-742)-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.

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 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 PSA(16-24), PAP(155-163), PAP(248-257), PSMA(207-215), and PSMA(431-440) 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.

Identification of human papillomavirus 16-E6 protein-derived peptides with the potential to generate cytotoxic T-lymphocytes toward human leukocyte antigen-A24+ cervical cancer.

Human papillomavirus 16 (HPV16)-E6 and -E7 proteins are considered to be appropriate targets in specific immunotherapy for cervical cancer. In this study, we attempted to identify epitope peptides from the HPV16-E6 protein that have the potential to generate cytotoxic T-lymphocytes (CTLs) toward human leukocyte antigen (HLA)-A24+ cervical cancer. Two HPV16-E6 peptides at positions 75-83 and 91-100 effectively induced peptide-specific CTLs from peripheral blood mononuclear cells of HLA-A24+ cervical cancer patients. These HPV16-E6 peptide-induced CTLs showed cytotoxicity against HLA-A24+ and HPV16-E6 protein-expressing cervical cancer cells. Experiments with blocking antibodies and cold inhibition targets revealed that the cytotoxicity was mainly dependent on peptide-specific and CD8+ T cells. In addition, based on our observation that induction of immunoglobulin G (IgG) reactive to administered CTL-directed peptides is correlated with clinical responses, we attempted to detect IgG reactive to HPV16-E6 peptides in the plasma of cancer patients. As a result, IgGs reactive to the HPV16-E691-100 peptide were detected in 4 of 12 cervical cancer patients. These results indicate that these HPV16-E6-derived peptides are good candidates in peptide-based immunotherapy for HLA-A24+ cervical cancer patients.

Determination of thrombopoietin-derived peptides recognized by both cellular and humoral immunities in healthy donors and patients with thrombocytopenia.

Thrombopoietin (TPO) is a cytokine that promotes megakaryocytopoiesis and thrombopoiesis and is considered a drug suitable for patients with thrombocytopenia. However, unexpected severe thrombocytopenia has developed in some healthy individuals participating in phase I clinical trials with a pegylated recombinant human megakaryocyte growth factor (PEG-rHuMGDF) that contained the first 163 amino acids of endogenous TPO, which resulted in hampering the further development of clinical trials. Autoimmune responses to PEG-rHuMGDF, which cross-reacted with endogenous TPO, were suggested to be involved in this rare but severe adverse event, although the immunogenic epitopes have not yet been determined. To better understand the molecular basis of such autoimmune reactions, we investigated the reactivity of 18 TPO-derived peptides with HLA-A2-binding motifs to plasma and T cells, both from patients with thrombocytopenia (n=24) and from healthy donors (HDs) (n=24). Four peptides, including those possessing amino acids in receptor-binding sites, were preferentially reactive to plasma from at least 20% of the patients, whereas one peptide at position 101-109 was equally reactive to those of the patients and the HDs. Each of the five peptides had the ability to induce peptide-specific cytotoxic T lymphocytes (CTLs) in both groups, albeit with less frequency among the patients. More important, each of these five peptides had the ability to induce HLA-A2-restricted and peptide-specific CTL activity reactive to cells that produce TPO. These results may provide new insights to gain a better understanding of autoimmune reactions to TPO.

Immunological evaluation of vaccination of peptides derived from epithelial cancer-related antigens in two patients with hematological malignancy.

Recent advances in tumor immunology have resulted in identification of many epithelial cancer-related antigens and peptides applicable to specific immunotherapy. We and others have reported that several epithelial cancer-related antigens are also expressed in hematological malignancies. Two patients with hematological malignancy (multiple myeloma and chronic lymphocytic leukemia) were vaccinated with peptides derived from epithelial cancer-related antigens to evaluate the immune responses to peptides under a personalized peptide vaccination regimen. There was no adverse event except for local skin reaction at the injection site. The peptide vaccination augmented both peptide-specific CTLs cytotoxic to hematological malignant cells in post-vaccination peripheral blood mononuclear cells and peptide-specific IgG in post-vaccination sera. A transient but obvious decrease of malignant cells was observed at the early phase of the vaccination in both cases. Vaccines consisting of peptides derived from epithelial cancer antigens safely increased anti-tumor cell activity in patients with hematological malignancies. These results may provide a scientific rationale in use of epithelial cancer-related antigens for specific immunotherapy to patients with hematological malignancies.

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.

Expression of epithelial cancer-related antigens in hematologic malignancies applicable for peptide-based immunotherapy.

Recent advances in tumor immunology have resulted in identification of many epithelial cancer-related antigens and peptides applicable to specific immunotherapy. The authors investigated whether these peptides, which are being studied clinically, could be appropriate target molecules for treatment of patients with hematologic malignancies. The majority of hematologic malignant cells studied expressed five different epithelial cancer-related antigens. Cytotoxic T lymphocyte (CTL) precursors reactive to these antigen-derived peptides were detected in peripheral blood mononuclear cells (PBMCs) of the majority of HLA-A24 patients, and the mean number of peptides recognized by CTL precursors was 2.4 per patient, ranging from 0 to 8 among the 10 peptides tested. These peptide-stimulated PBMCs exhibited HLA-A24-restricted cytotoxic activity against hematologic malignant cells but not against blastoid T cells. More importantly, these peptide-stimulated PBMCs exhibited cytotoxicity against freshly prepared autologous malignant cells in an HLA-A24-restricted manner. These results may provide a scientific basis for the use of these peptides from epithelial cancer-related antigens in specific immunotherapy for patients with hematologic malignancies.

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

SAMP1/Yit mice spontaneously develop ileitis resembling Crohn's 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 remarkably increased in Peyer's 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.

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.

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 beta-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.

Detection of a set of peptide vaccine candidates for use in HLA-A31+ epithelial cancer patients.

The molecular basis of host-tumor interaction in HLA-A31+ cancer patients has not been well understood. This lack of clarification is hampering the development of specific immunotherapies for these patients. This study aimed to identify a set of CTL-epitope peptides applicable for the specific immunotherapy of cancer patients with HLA-A31 allele. HLA-A31 allele is expressed in 5-10% of the world population, with the highest expression among Brazilian Amerinds (65%), and the lowest in the Eskimo population (0%). We report herein four cDNAs encoding CTL-epitopes and 7 epitope peptides with the ability to induce HLA-A31-restricted CTLs cytotoxic to tumor cell lines in the peripheral blood mononuclear cells of HLA-A31+ cancer patients. These peptides might be useful for the development of a peptide-based immunotherapy for HLA-A31+ cancer patients.

Identification of two novel tumor-associated antigens recognized by HLA-B46-restricted cytotoxic T lymphocytes.

The objective of this study was to identify novel genes and peptides capable of inducing tumor-reactive cytotoxic T lymphocytes (CTLs) in cancer patients with an HLA-B46 allele, which is preferentially expressed in Asians. We show that two genes encoding splicing factor (SF) 2 and inosine triphosphate pyrophosphatase (ITPA) have epitopes recognized by HLA-B46-restricted and tumor cell-reactive CTL lines established from tumor-infiltrating lymphocytes of colon cancer. The SF2 is essential for constitutive pre-mRNA splicing, while the enzyme ITPA controls nucleotide levels. The mRNA expression levels of these genes were higher in tumor cells than those in normal tissues. Five peptides, three from SF2 and two from ITPA, had the ability to induce HLA-B46-restricted and peptide-specific CTLs reactive to tumor cells in peripheral blood mononuclear cells of cancer patients. These results may provide new information for better understanding of host-tumor interaction at the molecular level and the development of peptide-based immunotherapy for HLA-B46+ cancer patients.

Role of the CD5 molecule on TCR gammadelta T cell-mediated immune functions: development of germinal centers and chronic intestinal inflammation.

Although CD4(+) T cells form a major subset of TCRalphabeta T cells, only a small number of TCRgammadelta T cells express CD4. Factors contributing to the down-regulation of CD4(+) TCRgammadelta T cells have not been identified. The CD5 molecule is expressed on most TCRgammadelta T cells in the spleen, whereas only a few intestinal intraepithelial TCRgammadelta T cells express this molecule in wild-type mice and TCRbeta mutant (beta(-/-)) mice. Unexpectedly, in the present studies, the lack of CD5 led to a remarkable increase of a CD4(+) TCRgammadelta T cell subset in CD5(-/-)beta(-/-) mice. The CD4(+) TCRgammadelta T cells were also detectable in MHC II(-/-)CD5(-/-)beta(-/-) triple-mutant mice. This CD4(+) TCRgammadelta T cell subset provided help in Mycobacterium-induced germinal center (GC) formation and showed a T(h)-like cytokine profile. In contrast, CD5(+) TCRgammadelta T cells suppressed the CD4(+) TCRgammadelta T cell-mediated GC formation, presumably by eliminating this CD4(+) subset. Unlike intraepithelial gammadelta T cells, >30% of TCRgammadelta T cells in the colonic lamina propria (LP) expressed CD5. The lack of CD5 also led to increased numbers of CD4(+) TCRgammadelta T cells in the colonic LP and increased susceptibility to development of chronic colitis in beta(-/-) mice. Cell transfer studies suggest that CD5(+) TCRgammadelta T cells are capable of selectively eliminating CD4(+) TCRgammadelta T cells in the intestine. The CD4(+) TCRgammadelta T cells possess immune functions similar to CD4(+) TCRalphabeta T cells.