Takaaki Hato

Aurora-A kinase: A novel target of cellular immunotherapy for leukemia

Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9-amino-acid epitope (Aur-A(207-215): YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A-specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201-restricted manner. Importantly, Aur-A-specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A(207-215) epitope-specific CTL precursors are present in peripheral blood of HLA-A*0201-positive and HLA-A*2402-positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

Polymorphisms of HPA-1 Through 6 on Platelet Membrane Glycoprotein Receptors Are Not a Genetic Risk Factor for Myocardial Infarction in the Japanese Population

We examined HPA genotypes derived from polymorphisms of platelet membrane receptors in 88 Japanese patients with early-onset myocardial infarction and in 100 control subjects. The results indicated that HPA genotypes 1 through 6 are not associated with early-onset myocardial infarction.

Successful treatment of transfusion‐associated graft‐versus‐host disease

Summary. We present an immunocompetent patient with transfusion‐associated graft‐versus‐host disease (GVHD), in which chimaerism of peripheral blood lymphocytes was demonstrated by analysis of a highly polymorphic genome. The patient was treated successfully with anti‐CD 3 monoclonal antibody, OKT3 and cyclosporin A. Although it is undoubtedly important to prevent transfusion‐associated GVHD by irradiation of cellular blood components, intensive therapy with OKT3 and cyclosporin A in the early phase of onset may be effective for treatment of this potentially fatal condition. The mechanism of the effectiveness of this treatment for transfusion‐associated GVHD is discussed.

Phenotypic and genotypic analysis of chronic myelogenous leukaemia with T lymphoblastic and megakaryoblastic mixed crisis

A case of blast crisis in chronic myelogeneous leukaemia (CML) in which two distinct cell lineages were involved is presented. The phenotype of blasts in lymph nodes was T11 (CD2)+, Ia+, TdT+, suggesting T cell lineage. On the other hand, blasts in bone marrow and peripheral blood expressed platelet glycoprotein IIb/IIIa complex on their surface, suggesting megakaryocyte lineage. Cytogenetic analysis of lymph node and bone marrow cells revealed the abnormalities. inv(7) (p15q34) and t(1;3) (q23;q21), respectively, as well as the presence of the Ph1 chromosome in both cell types. Rearrangement of the T cell receptor β‐chain gene was detected in lymph node blasts, although blast cells in peripheral blood showed a germ line configuration. The involvement of T cell and megakaryocyte lineages in the blast crisis phase of CML was confirmed in our phenotypic and genotypic analysis, and the pathogenic association between blast crisis lineages and the additional chromosome abnormalities present is discussed.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Non-Hodgkin's lymphoma developing in a pacemaker pocket

A 29-year-old man developed diffuse large B-cell lymphoma in a subpectoral pacemaker pocket that 6 years previously had been created in the chest for a titanium-covered pulse generator. The patient had an 8-cm—diameter dark red tumor with necrotic tissue on a keloidal surgical scar in the left side of the chest. Left axillary lymphadenopathy also was present. Laboratory studies showed an increased level of soluble interleukin 2 receptor and a normal level of lactose dehydrogenase. A biopsy specimen showed a diffuse large B-cell phenotype and monoclonal immunoglobulin H gene rearrangement. A gallium scintigraphy study showed abnormal accumulation in the left chest and left axilla. On the basis of these findings, we diagnosed diffuse large B-cell lymphoma, stage II. The patient received THP-COP chemotherapy (pirarubicin, cyclophosphamide, vincristine, and prednisolone) and radiotherapy, achieved complete remission, and was free of disease for 16 months after treatment. This case suggests that there was a relationship between the development of non-Hodgkin’s lymphoma and the presence of chronic inflammation in the pulse generator pocket.

Identification of an epitope derived from CML66, a novel tumor‐associated antigen expressed broadly in human leukemia, recognized by human leukocyte antigen‐A*2402‐restricted cytotoxic T lymphocytes

CML66 is a newly identified differentiation antigen that is expressed broadly in human leukemia and solid tumors, but its physiological function remains unknown. In the present study, to clarify the feasibility of CML66‐targeted cancer immunotherapy, we attempted to identify cytotoxic T lymphocyte (CTL) epitopes derived from CML66. An immunogenic CML66‐derived epitope (amino acid residues 76–84; YYIDTLGRI) capable of inducing human leukocyte antigen (HLA)‐A*2402‐restricted CTL specific for this peptide was identified. CML66‐derived peptide‐specific CTL efficiently lysed human leukemia cells, but not normal cells, in a HLA‐A*2402‐restricted fashion. Quantitative real‐time polymerase chain reaction revealed that CML66 mRNA is expressed abundantly in primary acute myeloid leukemia cells, acute lymphoid leukemia cells, and chronic myelogenous leukemia cells in advanced phase, and that the expression level of CML66 mRNA in normal cells is low compared with that in leukemia cells. CML66‐specific CTL precursors were detected in the peripheral blood of patients with acute leukemia. These data indicate that the CML66‐derived epitope identified in the present study is a new target antigen for cellular immunotherapy of human leukemia. (Cancer Sci 2008; 99: 1414–1419)

Adult T cell leukemia associated with eosinophilia: analysis of eosinophil-stimulating factors produced by leukemic cells.

The mechanism of eosinophilia in a patient with adult T cell leukemia (ATL) was investigated. A 61-year-old woman with ATL presented marked eosinophilia. No parasite infections or allergic diseases were found in this patient. The number of eosinophils fluctuated in parallel with that of ATL cells during her clinical course. The patients serum and the culture supernatant of ATL cells showed eosinophil colony-stimulating activity. Northern blot analysis of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5), which are known eosinophil CSFs, showed that only GM-CSF but not IL-3 or IL-5 was expressed in freshly separated and cultured ATL cells. Since neutrophil and monocyte numbers did not increase, it is suggested that GM-CSF and unknown cytokines other than IL-3 and IL-5 produced by ATL cells synergistically stimulated eosinophil precursors in the present case.

Suppression of integrin activation by the membrane-distal sequence of the integrin alphaIIb cytoplasmic tail.

Integrin cytoplasmic tails regulate integrin activation including an increase in integrin affinity for ligands. Although there is ample evidence that the membrane-proximal regions of the alpha and beta tails interact with each other to maintain integrins in a low-affinity state, little is known about the role of the membrane-distal region of the alpha tail in regulation of integrin activation. We report a critical sequence for regulation of integrin activation in the membrane-distal region of the alphaIIb tail. Alanine substitution of the RPP residues in the alphaIIb tail rendered alphaIIbbeta3 constitutively active in a metabolic energy-dependent manner. Although an alphaIIb/alpha6Abeta3 chimaeric integrin, in which the alphaIIb tail was replaced by the alpha6A tail, was in an energy-dependent active state to bind soluble ligands, introduction of the RPP sequence into the alpha6A tail inhibited binding of an activation-dependent antibody PAC1. In alphaIIb/alpha6Abeta3, deleting the TSDA sequence from the alpha6A tail or single amino acid substitutions of the TSDA residues inhibited alphaIIb/alpha6Abeta3 activation and replacing the membrane-distal region of the alphaIIb tail with TSDA rendered alphaIIbbeta3 active, suggesting a stimulatory role of TSDA in energy-dependent integrin activation. However, adding TSDA to the alphaIIb tail containing the RPP sequence of the membrane-distal region failed to activate alphaIIbbeta3. These results suggest that the RPP sequence after the GFFKR motif of the alphaIIb tail suppresses energy-dependent alphaIIbbeta3 activation. These findings provide a molecular basis for the regulation of energy-dependent integrin activation by alpha subunit tails.

Cooperative Role of the Membrane-proximal and -distal Residues of the Integrin β3 Cytoplasmic Domain in Regulation of Talin-mediated αIIbβ3 Activation

Integrin cytoplasmic tails regulate integrin activation that is required for high affinity binding with ligands. The interaction of the integrin β subunit tail with a cytoplasmic protein, talin, largely contributes to integrin activation. Here we report the cooperative interaction of the β3 membrane-proximal and -distal residues in regulation of talin-mediated αIIbβ3 activation. Because a chimeric integrin, αIIbβ3/β1, in which the β3 tail was replaced with the β1 tail was constitutively active, we searched for the residues responsible for integrin activation among the residues that differed between the β3 and β1 tails. Single amino acid substitutions of Ile-719 and Glu-749 in the β3 membrane-proximal and -distal regions, respectively, with the corresponding β1 residues or alanine rendered αIIbβ3 constitutively active. The I719M/E749S double mutant had the same ligand binding activity as αIIbβ3/β1. These β3 mutations also induced αVβ3 activation. Conversely, substitution of Met-719 or Ser-749 in the β1 tail with the corresponding β3 tail residue (M719I or S749E) inhibited αIIbβ3/β1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type αIIbβ3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced αIIbβ3 activation. These results suggest that the β3 membrane-proximal and -distal residues cooperatively regulate talin-mediated αIIbβ3 activation.