Harumi Kakuda

Expansion of Highly Cytotoxic Human Natural Killer Cells for Cancer Cell Therapy

Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Seven-day coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56(+)CD3(-) NK cells from peripheral blood (range, 5.1- to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3(+) lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2-stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer.

Replicative potential of human natural killer cells.

The replicative potential of human CD56+ CD3− natural killer (NK) cells is unknown. We found that by exposing NK cells to the leukaemic cell line K562 genetically modified to express 4‐1BB ligand and interleukin 15 (K562‐mb15‐41BBL), they expanded for up to 30 population doublings, achieving numbers that ranged from 1·6 × 105 to 1·2 × 1011% (median, 5·9 × 106%; n = 7) of those originally seeded. However, NK cells eventually became unresponsive to stimulation and died. Their demise could be suppressed by enforcing the expression of the human telomerase reverse transcriptase gene (TERT). TERT‐overexpressing NK cells continued to proliferate in response to K562‐mb15‐41BBL stimulation for more than 1 year of culture, while maintaining a normal karyotype and genotype. Long‐lived NK cells had high cytotoxicity against myeloid and T‐lineage leukaemic cells. They remained susceptible to genetic manipulation, becoming highly cytotoxic to B‐lineage leukaemic cells after expression of anti‐CD19 signaling receptors. Thus, human NK cells have a replicative potential similar to that of T lymphocytes and their lifespan can be significantly prolonged by an increase in TERT activity. We suggest that the methods described here should have many applications in studies of NK cell biology and NK cell‐based therapies.

Chimeric MLL products with a Ras binding cytoplasmic protein AF6 involved in t(6;11) (q27;q23) leukemia localize in the nucleus

In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a result of 11q23 translocations. We previously showed that the N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore suggested that it might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11) (q27;q23) translocation were analysed since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrated that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization was examined and found to be a region containing AT-hook motifs.

Interleukin 6, a possible autocrine growth and differentiation factor for the human megakaryocytic cell line, CMK

Summary. CMK is a human cell line derived from a megakaryoblastic leukaemia. It has characteristics of the megakaryocytic lineage, such as the presence of platelet peroxidase, membrane glycoproteins (GP)Ib and GPIIb/IIIa, α‐granules, and demarcation membranes. The cell line proliferates autonomously in serum‐containing medium. Here we report that the cell line expresses the gene for IL‐6 and releases small quantities of the cytokine into the medium. Addition of exogenous IL‐6 to cultures seeded into medium was found to promote growth of the cells. Conversely, addition of a neutralizing anti‐IL‐6 antibody inhibited cell growth. These data support the notion that autocrine IL‐6 is one of the factors accounting for autonomous growth of the cell line.

HIV infection of megakaryocytic cell lines.

Thrombocytopenia is a common hematologic disorder in HIV infection and occurs in both asymptomatic and AIDS patients. An autoimmune mechanism has been postulated for the platelet destruction associated with some forms of thrombocytopenia. However, recent studies revealed that megakaryocytes are susceptible to HIV infection and suggested the possibility that HIV can directly impair the platelet production from megakaryocytes. This study was designed to characterize the HIV receptor expression in megakaryocytic cells and the responsiveness to HIV infection. Four different megakaryocytic cell lines at different stages of differentiation were established from the peripheral blood of different individuals with hematologic malignancies. CMK and CMY cells (differentiated cell lines) expressed CD4, but CMS and CTS cells (poorly differentiated cell lines) did not. The HIV coreceptor CXCR4 was also expressed in CMY and CMK cells. HIV-1 (HTLV-IIIB) replicated in CMY cells persistently but not in other three cell lines. CMY cells as well as CMK cells were also susceptible to the lytic infection of HIV-2 (LAV2). Pretreatment of the CMY cells with anti-CD4 antibody inhibited the infection by both HIV-1 and HIV-2. Our results indicate that mature megakaryocytic cells express CD4 along with HIV core-ceptors and are susceptible to HIV infection.

A novel human leukaemic cell line, CTS, has a t(6;11) chromosomal translocation and characteristics of pluripotent stem cells

A novel human leukaemic cell line, designated CTS, was established from the peripheral blood of a 13‐year‐old girl suffering from acute myeloblastic leukaemia (AML) in relapse. CTS cells expressed CD7, CD13, CD33, CD34 and HLA‐DR antigens, and showed ultrastructural myeloperoxidase activity. In addition, CTS cells showed DNA rearrangements of the immunoglobulin heavy chain gene and the light κ chain gene, and deletions of the T‐cell receptor δ1 gene. Cytogenetic analysis revealed a human female diploid karyotype with a t(6;11)(q27;q23) chromosomal translocation. Molecular studies demonstrated a DNA rearrangement of the MLL gene, the expression of a truncated 11.0 kb MLL mRNA and the detection of the MLL/AF‐6 fusion transcript in CTS cells. To our knowledge, this cell line is the first report of a human leukaemic cell line with a t(6;11) chromosomal translocation. CTS cells showed no significant proliferative response to the cytokines, IL‐2, IL‐3, IL‐6, IL‐11, GM‐CSF, G‐CSF, EPO, SCF, but were induced to differentiate to the T‐cell, B‐cell, erythroid or megakaryocytic lineage in the presence of particular cytokines. This CTS cell line may provide a useful tool in the study of the oncogenesis of mixed lineage leukaemia with 11q23 abnormalities and for the analysis of growth and differentiation of pluripotent stem cells.

ZNF384-related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype

Fusion genes involving ZNF384 have recently been identified in B-cell precursor acute lymphoblastic leukemia, and 7 fusion partners have been reported. We further characterized this type of fusion gene by whole transcriptome sequencing and/or polymerase chain reaction. In addition to previously reported genes, we identified BMP2K as a novel fusion partner for ZNF384. Including the EP300-ZNF384 that we reported recently, the total frequency of ZNF384-related fusion genes was 4.1% in 291 B-cell precursor acute lymphoblastic leukemia patients enrolled in a single clinical trial, and TCF3-ZNF384 was the most recurrent, with a frequency of 2.4%. The characteristic immunophenotype of weak CD10 and aberrant CD13 and/or CD33 expression was revealed to be a common feature of the leukemic cells harboring ZNF384-related fusion genes. The signature gene expression profile in TCF3-ZNF384-positive patients was enriched in hematopoietic stem cell features and related to that of EP300-ZNF384-positive patients, but was significantly distinct from that of TCF3-PBX1-positive and ZNF384-fusion-negative patients. However, clinical features of TCF3-ZNF384-positive patients are markedly different from those of EP300-ZNF384-positive patients, exhibiting higher cell counts and a younger age at presentation. TCF3-ZNF384-positive patients revealed a significantly poorer steroid response and a higher frequency of relapse, and the additional activating mutations in RAS signaling pathway genes were detected by whole exome analysis in some of the cases. Our observations indicate that ZNF384-related fusion genes consist of a distinct subgroup of B-cell precursor acute lymphoblastic leukemia with a characteristic immunophenotype, while the clinical features depend on the functional properties of individual fusion partners.

Expression of multidrug resistant gene (mdr-1/P-glycoprotein) in a megakaryoblastic cell line, CMK, and its enhancement during megakaryocytic differentiation.

Multidrug resistance is a severe clinical problem in the chemotherapy of malignant disease. Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often resistant to many anti-cancer chemotherapeutic drugs. Here we report the expression of the mdr-1/P-glycoprotein in a cell line, CMK, established from a patient with AMKL. Expression of mdr-1 mRNA in CMK11-5 cells, a well differentiated subline, was higher than in CMK6 cells, a poorly differentiated subline. The level of P-glycoprotein was also higher in CMK11-5 cells. The cytokines interferon-gamma (IFN-gamma), GM-CSF and IL-3, which were shown to induce megakaryocytic differentiation of CMK cells, enhanced the expression of the mdr-1 mRNA and levels of P-glycoprotein. These results imply that differentiated megakaryocytic cells may have higher levels of the P-glycoprotein expression, suggesting a possible normal physiological function of P-glycoprotein in mature megakaryocytes.

Genetic Modification of Natural Killer Cells for Leukemia Therapies

Natural killer (NK) cells have the capacity to recognize and kill a wide range of cancer cells. However, many cancer cells are resistant to NK cell cytotoxicity, mainly because they express molecules which inhibit NK cell activation. Previous studies have shown that enforced expression of chimeric receptors composed of single-chain variable domain of murine antibodies and human signaling molecules can redirect the specificity of T lymphocytes. The success of this approach depends on the identification of a suitable target molecule on cancer cells and on the ability of the receptor to deliver appropriate activation signals. We developed a method to express chimeric receptors in NK cells. Considerable NK cell expansion was obtained by co-culturing peripheral blood cells with the leukemia cell line K562 modified to express membrane bound-interleukin 15 and the ligand for the costimulatory molecule 4-1BB. Expanded NK cells were then transduced with anti-CD19 receptors which deliver activation signals through CD3ζ and 4-1BB. NK cells expressing these receptors became highly cytotoxic against NK-resistant CD19+ leukemic cells. We here review the methodologies for expanding and redirecting the specificity of NK cells, explain the rationale for NK-cell therapies of leukemia and lymphoma, describe potential targets for genetically-modified NK cells, and discuss future clinical applications of NK cell expansion and genetic modification in cancer therapy.

Expression of MDR-1/P-Glycoprotein in Childhood Acute Megakaryoblastic Leukemia Cells

Multidrug resistance is a major clinical problem in chemotherapy of malignant disease. Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often more resistant to many anticancer chemotherapeutic drugs compared to other types of childhood leukemia. There have been reports of the increased expression in hematologic malignancy of multidrug resistant (mdr-1) gene, which encodes for a transmembrane glycoprotein P-glycoprotein that acts as an efflux pump for structurally unrelated chemotherapeutic drugs. We investigated the malignant cells of 15 newly diagnosed childhood AMKL patients by immunocytochemical analysis and found P-glycoprotein expression in all samples from these patients. RNA prepared from five patients at the time of presentation confirmed the expression of mdr-1 specific message in all cases by Northern blot analysis. These results imply that malignant cells from all childhood AMKL might express the mdr-1/P-glycoprotein.