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Biomedical subjects

K Kuzushima

Publications and source records attributed to K Kuzushima.

At least 19 recordsLinked to original sources
PubMed2006

The human cathepsin H gene encodes two novel minor histocompatibility antigen epitopes restricted by HLA-A*3101 and -A*3303.

Minor histocompatibility antigens (mHags) play crucial roles in the induction of graft versus host disease (GVHD) and/or graft versus leukaemia (GVL) effects following human leucocyte antigen (HLA)-identical haematopoietic stem cell transplantation (HSCT). Using HLA-A*3101- and -A*3303-restricted cytotoxic T lymphocyte (CTL) clones generated from different post-HSCT recipients, we identified two novel mHag epitopes encoded by the leader sequence of cathepsin H (CTSH) isoform a. The nonameric sequence ATLPLLCAR was defined as an HLA-A*3101-restricted epitope (CTSH(R)/A31), while a decameric peptide featuring a one N-terminal amino acid extension, WATLPLLCAR, was presented by HLA-A*3303 (CTSH(R)/A33). The immunogenicity of both epitopes was totally dependent on the polymorphic C-terminal arginine residue and substitution with glycine completely abolished binding to the corresponding HLA molecules. Thus, the immunogenicity of this mHag is exerted by differential HLA binding capacity. CTSH is relatively ubiquitously expressed at protein levels, thus it may be involved in GVHD and anti-leukaemic/tumour responses. Interestingly, however, CTL clones predominantly lysed targets of haematopoietic cell origin, which could not be explained in terms of the immunoproteasome system. Although the mechanisms involved in the differential susceptibility remain to be determined, these data suggest that CTSH-encoded mHags could be targets for GVL effects.

Acute Disease↗
PubMed2005

Retroviral vector backbone immunogenicity: identification of cytotoxic T-cell epitopes in retroviral vector-packaging sequences.

Retroviral vectors are the frequently applied gene delivery vehicles for clinical gene therapy, but specificity of the immunogenicity to the protein encoded by the inserted gene of interest is a problem which needs to be overcome. Here, we describe human cytotoxic T-lymphocyte (CTL) clones recognizing epitopes derived from the protein encoded by the retroviral vector backbone, which were established during the course of our attempts to generate CTLs against cytomegalovirus (CMV) or human papilloma virus (HPV) in vitro. In the case of healthy CMV-seronegative donors, CTL lines specific for retrovirally transduced cells were generated in four out of eight donors by stimulating CD8 T cells with CD40-activated B (CD40-B) cells retrovirally transduced with CMV-pp65. Two CTL clones derived from one of the CTL lines were found to recognize epitopes from gag in the context of HLA-B(*)4403 and -B(*)4601, respectively. Similarly, an HLA-B(*)3501-restricted CTL clone from a cervical cancer patient recognized an epitope located in the junctional regions of the gag and pol sequences. These results show that polypeptides encoded by components of the retroviral vector backbone are in fact immunogenic, generating CTLs in vitro in human cells. Thus, potential CTL responses to retroviral products should also be considered in clinical settings.

Animals↗
PubMed2004

Expansion and activation of minor histocompatibility antigen HY-specific T cells associated with graft-versus-leukemia response.

The immune system of females is capable of recognizing and reacting against the male-specific minor histocompatibility antigen (mHA), HY. Thus, cytotoxic T-lymphocytes (CTLs) recognizing this antigen may be useful in eradicating leukemic cells of a male patient if they can be generated in vivo or in vitro from a human leukocyte antigen (HLA)-identical female donor. The HLA-A*0201-restricted HY antigen, FIDSYICQV, is a male-specific mHA. Using HLA-A2/HY peptide tetrameric complexes, we reveal a close association between the emergence of HY peptide-specific CD8(+) T cells in peripheral blood and molecular remission of relapsed BCR/ABL(+) chronic myelogenous leukemia in lymphoid blast crisis in a patient who underwent female-to-male transplantation. Assessment of intracellular cytokine levels identified T cells that produce interferon-gamma in response to the HY peptide during the presence of HY tetramer-positive T cells. These results indicate that transplant with allogeneic HY-specific CTLs has therapeutic potential for relapsed leukemia, and that expansion of such T cells may be involved in the development of a graft-versus-leukemia response against lymphoblastic leukemia cells.

Adolescent↗
PubMed2004

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA-peptide complexes.

BACKGROUND: Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS: CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT: Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION: This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.

Antigens, Viral↗
PubMed2004

Identification of an epitope from the epithelial cell adhesion molecule eliciting HLA-A*2402-restricted cytotoxic T-lymphocyte responses.

Because the epithelial cell adhesion molecule (Ep-CAM) is expressed in almost all carcinomas and human leucocyte antigen (HLA)-A*2402 is the most common allele in many ethnic groups, including Japanese, the identification of peptide sequences, which elicit HLA-A*2402-restricted Ep-CAM-specific cytotoxic T-lymphocyte (CTL) responses, would facilitate specific immunotherapy for various histological types of carcinomas. An epitope was identified through the following steps: (i) computer-based epitope prediction from the amino acid sequence of Ep-CAM, (ii) major histocompatibility complex (MHC) stabilization assay to determine the affinity of the predicted peptide with HLA-A*2402 molecules, (iii) stimulation of CD8+ T cells with peptide-pulsed dendritic cells and (iv) testing the CTL specificity by means of enzyme-linked immunospot (ELISPOT) assays, CTL assays and MHC/peptide-tetramer staining. Peripheral CD8+ T cells of four of five healthy donors after three rounds of stimulation with the peptide Ep-CAM173-181 (RYQLDPKFI) secreted interferon-gamma in ELISPOT assays when exposed to the peptide. A CTL clone specific to the peptide efficiently lysed Ep-CAM-expressing cancer cell lines in an HLA-A*2402-restricted fashion. Endogenous processing and presentation of the peptide in a lung cancer cell line were confirmed by means of cold target inhibition assays. The CTL clone was also lytic to normal bronchial epithelial cells but to a lesser extent at low effector: target ratios. All these data suggest that the peptide-specific CTL responses may play some roles both in anti-cancer and autoimmune reactions. The peptide should prove useful to study anti-Ep-CAM CTL responses among population possessing HLA-A*2402.

Biomarkers, Tumor↗
PubMed2001

Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8(+) T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay.

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the application, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL epitopes in the context of given MHC molecules of interest through multiple screenings consisting of a computer-assisted algorithm and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)-specific CTL epitope, QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was identified from 83 candidate peptides. The results indicate that the CMV-specific CTL response is highly focused to pp65 in the context of HLA-A*2402. Endogenous processing and presentation was confirmed using a peptide-specific CD8(+) T-cell clone as the effectors and autologous fibroblast cells infected with recombinant vaccinia virus expressing pp65 gene or CMV as antigen-presenting cells. Flow cytometric analysis of intracellular interferon-gamma production revealed 0.04% to 0.27% of CD8(+) T cells in peripheral blood of HLA-A24(+) and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T-cell clone and 0.79% of CD8(+) T cells in peripheral blood from a seropositive donor. The peptide could be a useful reagent to study CTL responses to CMV among populations positive for HLA-A*2402.

Algorithms↗
PubMed2001

Impaired cytotoxic T lymphocyte response to Epstein-Barr virus-infected NK cells in patients with severe chronic active EBV infection.

Clinical evidence of a relationship between severe chronic active Epstein-Barr virus (EBV) infection and clonal expansion of EBV-infected T or NK cells has been accumulated. In order to clarify pathogenesis of EBV-infected cell proliferation in patients with severe chronic active EBV infection, cytotoxic T lymphocyte (CTL) responses of two patients against B-lymphoblastoid cell lines (B-LCL) and EBV-infected NK cells were examined in comparison with those of HLA-identical healthy siblings. Unexpectedly, patients' CTL activities induced by mixed culture with autologous B-LCLs were markedly reduced, although uncontrolled EBV-related B-cell proliferations have never been experienced. In contrast, limiting dilution analysis demonstrated that B-LCL-specific CTL precursor (CTLp) frequencies of patients were comparable to those of their healthy sisters. The existence of normal levels of B-LCL-specific T cell responses was confirmed by flow-cytometric analysis of IFN-gamma-producing T cells after stimulation with B-LCLs. Infected NK-cell-specific CTLp frequencies of the patients were at undetectable levels despite their expression of latent membrane protein (LMP) 1, suggesting mechanisms to escape immunologic surveillance. In the patients' HLA-identical healthy sisters, infected NK-cell-specific CTLps were detected, and infected NK-cell-specific CTL clones could be established. From these findings, two treatment options for severe chronic active EBV infection are offered for consideration: adoptive transfer of in vitro-cultured CTL, and bone marrow transplantation from HLA-identical donors.

Adolescent↗
PubMed2001

Severe hepatitis caused by Epstein-Barr virus without infection of hepatocytes.

Although hepatitis is a common feature of primary Epstein-Barr virus (EBV) infection, severe liver injury is rare and its pathogenesis is unclear. A previously healthy girl developed severe hepatitis with prolonged jaundice. Serologic examination showed that she had primary infection with EBV. An extremely high Epstein-Barr viral load was observed in her peripheral blood. The viral load decreased in parallel with symptomatic improvement. Histologic examinations showed spotty necrosis of the liver parenchyma and infiltration by CD8(+) T cells. The CD8(+) T cells, not hepatocytes, were positive for EBV. Possible mechanisms of viral hepatitis without infection of hepatocytes are discussed.

Antigens, Viral↗
PubMed2001

Diagnostic testing in Epstein-Barr virus infection.

Laboratory diagnosis of Epstein-Barr virus (EBV) infection is improving with the development of new technologies. Quantification of the virus by real-time polymerase chain reaction (PCR) and evaluation of EBV-specific T cells, especially by tetrameric human leukocyte antigens, are noteworthy candidates for monitoring procedures in clinical laboratories involved in the management of transplant recipients. Standardization of PCR is essential for improving the quality of these monitoring procedures.

DNA, Viral↗
PubMed2000

Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins in insect cells.

The insect-baculovirus expression system has proved particularly useful for producing recombinant proteins that are biologically active. Overexpression of foreign proteins using the recombinant baculovirus system is often accompanied by aggregation of the overexpressed protein, which is thought to be due to a limitation of the translated protein folding in the infected cells. Co-infection of a recombinant baculovirus capable of expressing the human chaperone Hsp70 slightly increased the solubility of the overexpressed Epstein-Barr virus replication protein, BZLF1. Co-expression of Hsp70 and its co-factor, Hsdj or Hsp40, was here found to improve the solubility of the target protein several fold. Thus, a baculovirus expression system producing these molecular chaperones may find application for improved production of target foreign gene products in insect cells.

Animals↗
PubMed2000

Longitudinal dynamics of Epstein-Barr virus-specific cytotoxic T lymphocytes during posttransplant lymphoproliferative disorder.

Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD) is a serious complication after allogeneic bone marrow transplantation (BMT). Dynamics of EBV-specific cytotoxic T lymphocytes (CTL), which are important in controlling EBV during the LPD, have not been fully elucidated. A patient with Wiskot-Aldrich's syndrome was diagnosed as suffering from LPD on day 47 after BMT. Fluorescence-activated cell sorter (FACS) analysis for interferon-gamma production revealed that >70% of the patient's CD8(+) T cells were EBV specific. The patient's lymphocytes were directly cytotoxic to donor-derived EBV-positive lymphoblastoid cells, which was blocked by an anti-class I antibody. EBV-specific CD8(+) T cell counts declined in parallel with EBV genome load, and full recovery of LPD was obtained with relaxation of immunosuppressive drugs. The results illustrate longitudinal dynamics of EBV-specific CTL during the posttransplant LPD; they also illustrate the advantages of using FACS analysis for EBV-specific CTL to make decisions about treatment.

Antigens, Surface↗
PubMed2000

Early intervention in post-transplant lymphoproliferative disorders based on Epstein-Barr viral load.

Using a real-time quantitative PCR assay, we identified two patients with EBV-related lymphoproliferative disorders at a very early stage. Both had received an unmanipulated bone marrow transplant with anti-thymocyte globulin for conditioning. To estimate virus-specific immunity, the frequencies of EBV-specific CD8+ T cells were measured using an enzyme-linked immunospot assay. The frequencies of EBV-specific CD8+ T cells of the two were 3.2 and 7.7%, respectively, which had possibly expanded in vivo. After withdrawing the immunosuppressive agents or administering donor lymphocytes transfusion, their symptoms regressed in parallel with the viral load.

Adult↗
PubMed2000

The Epstein-Barr virus pol catalytic subunit physically interacts with the BBLF4-BSLF1-BBLF2/3 complex.

The Epstein-Barr virus (EBV)-encoded replication proteins that account for the basic reactions at the replication fork are thought to be the EBV Pol holoenzyme, consisting of the BALF5 Pol catalytic and the BMRF1 Pol accessory subunits, the putative helicase-primase complex, comprising the BBLF4, BSLF1, and BBLF2/3 proteins, and the BALF2 single-stranded DNA-binding protein. Immunoprecipitation analyses using anti-BSLF1 or anti-BBLF2/3 protein-specific antibody with clarified lysates of B95-8 cells in a viral productive cycle suggested that the EBV Pol holoenzyme physically interacts with the BBLF4-BSLF1-BBLF2/3 complex to form a large complex. Although the complex was stable in 500 mM NaCl and 1% NP-40, the BALF5 protein became dissociated in the presence of 0.1% sodium dodecyl sulfate. Experiments using lysates from insect cells superinfected with combinations of recombinant baculoviruses capable of expressing each of viral replication proteins showed that not the BMRF1 Pol accessory subunit but rather the BALF5 Pol catalytic subunit directly interacts with the BBLF4-BSLF1-BBLF2/3 complex. Furthermore, double infection with pairs of recombinant viruses revealed that each component of the BBLF4-BSLF1-BBLF2/3 complex makes contact with the BALF5 Pol catalytic subunit. The interactions of the EBV DNA polymerase with the EBV putative helicase-primase complex warrant particular attention because they are thought to coordinate leading- and lagging-strand DNA synthesis at the replication fork.

Animals↗
PubMed1999

Antigen-driven expansion and contraction of CD8+-activated T cells in primary EBV infection.

The origin of the increased numbers of CD8+ atypical lymphocytes, expressing activated markers such as HLA-DR or CD45RO, in the peripheral blood of patients with infectious mononucleosis (IM) has been debated. Using a recently developed assay to detect intracellular accumulation of IFN-gamma in EBV-reactive T cells by FACS, we have demonstrated that 34-54% of HLA-DR+/CD8+ and 34-60% of CD45RO+/CD8+ T cells in the PBMCs of febrile patients suffering from IM are EBV-specific. The EBV-specific CD8+ T cell counts in the PBMCs of four febrile patients suffering from IM ranged between 2,260 and 8,200/microl, decreasing to 5.1% and 7.9% of the counts in the first samples over 10 days in two donors. The decline of CD8+ T cell subpopulations, namely HLA-DR+, CD45RO+, and EBV-specific T cells, was in parallel with the drop in the EBV genome load. These data indicate that the Ag-driven expansion of CD8+ T cells and subsequent contraction with the Ag decline in vivo in humans is effective for clearing virus-infected cells with minimal disturbance of the homeostasis of the immune system.

Antigens, Viral↗
PubMed1999

Rapid determination of Epstein-Barr virus-specific CD8(+) T-cell frequencies by flow cytometry.

We have developed an efficient and rapid method for detection of Epstein-Barr virus (EBV)-specific CD8(+) T-cell frequencies both in freshly isolated peripheral blood mononuclear cells (PBMCs) and in vitro established cytotoxic T lymphocyte (CTL) lines. Responder cells are thereby stimulated with an autologous lymphoblastoid cell line for 5 hours and intracellular accumulation of interferon gamma (IFNgamma) is detected by multiparameter flow cytometric analysis. EBV-specific CD8(+) T-cell frequencies ranged between 0.63% and 1. 29% in PBMCs of 5 healthy long-term EBV carriers. Using EBV-specific T-cell lines, it was shown that flow cytometric analysis is more sensitive than limiting dilution analysis for CTL precursors and enzyme-linked immunospot assay detecting IFNgamma-producing T cells. The class I restriction of IFNgamma production was confirmed using an anti-class I monoclonal antibody (MoAb). Information on other cytokine production of EBV-specific CTLs could be obtained using combinations of anti-cytokine MoAbs. The sensitive and rapid nature of the flow cytometric assay for EBV-specific CD8(+) T-cell frequency has significant advantages for evaluation of EBV-specific CD8(+) T-cell responses in PBMCs of patients with EBV-related diseases.

Antigens, Viral↗