A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Establishment and characterization of new B-cell precursor leukemia cell line NALM-35

A novel B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) cell line, NALM-35, was established from the peripheral blood of a 40-year-old woman at diagnosis of ALL. Imunophenotyping showed BCP type III characteristics including expression of TdT, CD10, CD19, CD22, CD79a and HLA class II. T-cell and myeloid-associated antigens tested were negative except CD5 and CD28. The surrogate light chains CD179a and CD179b were positive. NALM-35 cells have the morphological appearance of lymphoblasts. Cytogenetic analysis of NALM-35 revealed an abnormal karyotype with 46, XX, add(9)(p11). Southern blot analysis of the immunoglobulin genes status of NALM-35 at 10 months after establishment showed germ line configuration of the kappa and lambda light chain genes, and rearrangement of the mu heavy chain gene. DNA fingerprinting, chromosomal analysis and immunophenotyping proved that NALM-35 was clonally derived from the primary leukemia cells. The established cell line may provide a useful model system and unprecedented opportunities for analyzing the multitude of biological aspects of normal and neoplastic B-lymphocytes and their precursors.     

Differential sensitivity to x-ray of chromosomes of blood T-lymphocytes and B-and T-cell lines.

Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a singificantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia.     

Differential sensitivity to X-ray of chromosomes of blood T-lymphocytes and B- and T-cell lines

Summary Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a significantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia. Supported in part by USPHS grants CA-14413 and CA-16935.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Quantitative evaluation of leukemic mitochondria with a computer-controlled image analyzer

Mitochondria from 25 patients with acute lymphoblastic leukemia (ALL) and 25 patients with acute myelogenous leukemia (AML) were compared in terms of their number, area, and shape index using a computer-controlled image analyzer. The number of mitochondria was greater in the AML than in the ALL patients. However, their size, as measured in electron micrographic profiles was similar in the two groups, in disagreement with conventional reports that mitochondria are small in granulocytes but large in lymphocytes. Two ALL patients had giant mitochondria. The mitochondria of the ALL cells were more irregular than those of the AML cells, and furthermore, within the ALL group, the degree of the irregularity was greater in those with a poor prognosis than in those in longstanding remission. The number of mitochondria was significantly greater in B-cell ALL than in null cell and T-cell ALL.     

bcr rearrangement and C-abl gene expression in Ph1-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts

bcr gene rearrangement and c-abl gene expression were analyzed in a patient with Philadelphia chromosome (Ph1)-positive hybrid acute leukemia with simultaneous proliferation of lymphoid and myeloid blasts. These data were compared with those from a patient with chronic myelogenous leukemia (CML) in mixed crisis. The leukemic cells of both patients showed immuno-phenotypic profiles such as non-T, non-B common ALL with some MPO-positive leukemic cells and rearranged JH genes. On analysis of molecular events associated with the Ph1 chromosome, the leukemic cells of a patient with CML in mixed crisis showed bcr rearrangement and an 8.5-kb bcr-abl chimeric mRNA, but those of a patient with Ph1-positive hybrid acute leukemia showed no 8.5-kb bcr-abl mRNA, as previously reported in a number of Ph1-positive acute lymphoblastic leukemia (ALL) cases. These results revealed that the molecular event found in Ph1-positive ALL is not only restricted to lymphoid lineage but may play an important role in the proliferation of the myeloid lineage.     

The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells

Background aimsCytokine-induced killer (CIK) cells may serve as an alternative approach to adoptive donor lymphocyte infusions (DLI) for patients with acute leukemia relapsing after haplo-identical hematopoietic stem cell transplantation (HSCT). We investigated the feasibility of enhancing CIK cell-mediated cytotoxicity by interleukin (IL)-15 against acute myeloid and lymphoblastic leukemia/lymphoma cells.MethodsCIK cells were activated using IL-2 (CIK_IL-2) or IL-15 (CIK_IL-15) and phenotypically analyzed by fluorescence-activated cell sorting (FACS). Cytotoxic potential was measured by europium release assay.ResultsCIK_IL-2 cells showed potent cytotoxicity against the T-lymphoma cell line H9, T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT-4 and subtype M4 acute myeloid leukemia (AML) cell line THP-1, but low cytotoxicity against the precursor B (pB)-cell ALL cell line Tanoue. IL-15 stimulation resulted in a significant enhancement of CIK cell-mediated cytotoxicity against acute lymphoblastic leukemia/lymphoma cell lines as well as against primary acute myeloid and defined lymphoblastic leukemia cells. However, the alloreactive potential of CIK_IL-15 cells remained low. Further analysis of CIK_IL-15 cells demonstrated that the NKG2D receptor is apparently involved in the recognition of target cells whereas killer-cell immunoglobulin-like receptor (KIR)-HLA mismatches contributed to a lesser extent to the CIK_IL-15 cell-mediated cytotoxicity. In this context, CD3 ⁺ CD8 ⁺ CD25 ⁺ CD56^? CIK_IL-15 cell subpopulations were more effective in the lysis of AML cells, in contrast with CD56 ⁺ CIK_IL-15 cells, which showed the highest cytotoxic potential against ALL cells.ConclusionsThis study provides the first evidence that CIK_IL-15 cells may offer a therapeutic option for patients with refractory or relapsed leukemia following haplo-identical HSCT.     

Partial versus productive immunoglobulin heavy locus rearrangements in chronic lymphocytic leukemia: implications for B-cell receptor stereotypy

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level.     

New chromosomal translocations correlate with specific immunophenotypes of childhood acute lymphoblastic leukemia

Cytogenetic analysis of leukemic cells obtained at diagnosis from 122 patients with childhood acute lymphoblastic leukemia (ALL) disclosed chromosomal translocations in 36 cases. Two new nonrandom translocations were identified and found to be associated with specific immunophenotypes of the disease. The first, identified in 4 of 16 cases of T-cell ALL positive for sheep erythrocyte receptors (E+), involved the short arm (p) of chromosome 11 and the long arm (q) of chromosome 14 and was designated t(11;14) (p13;q13). The second, found in 7 of 23 cases with a pre-B-cell phenotype, involved the long arm of chromosome 1 and the short arm of chromosome 19; it was designated t(1;19) (q23;p13.3). A third abnormality involving a common breakpoint on chromosome 12 (band p 12) was also identified. These two new differentiation-specific translocations suggest a mechanism for aberrant expression of genes that influence lymphoid cell growth and development, as well as leukemogenesis.     

Telomeric fusion in pre-T-cell acute lymphoblastic leukemia

Summary Telomeric fusion, a rare phenomenon, was observed in malignant cells from the peripheral blood of an 18-year-old male with rapidly progressive pre-T-cell acute lymphoblastic leukemia (ALL). Only two comparable cases, both with B-cell ALL, have been reported with telomeric fusion in neoplasia. All of the leukemic cells examined from our patient had two chromosome abnormalities consisting of partial triplication (trp) of chromosome 2 and a derivative chromosome 3. Approximately a third of the leukemic cells showed in addition telomere-telomere fusions. These involved the telomeric regions of 1p, 2p, 4q, 5q, 7q, 10q, 11q, 12p, 15p, 21p, and Xq and 3p of the derivative (3). The findings in this case suggest that telomeric fusion may function as a mechanism for the development of chromosome rearrangements that may play a role, albeit rarely, in human neoplasia.     

Human acute lymphoblastic leukemia (ALL) blasts as accessory cells during T-cell activation: differences between patients in costimulatory capacity affect proliferative responsiveness and cytokine release by activated T cells

The ability of acute lymphoblastic leukemia (ALL) blasts to mediate costimulatory signals during T-lymphocyte activation was investigated in an experimental model in which monoclonal T-cell populations were stimulated with standardized activation signals (anti-CD3 and anti-CD28 monoclonal antibodies; phytohemagglutinin, PHA). Leukemia cells from 12 consecutive ALL patients with high peripheral blood blast counts were studied. Proliferative T-cell responses were detected for a majority of these patients when irradiated leukemia blasts were used as accessory cells during activation. T-cell cytokine release was also observed for most patients when using nonirradiated ALL accessory cells. Low or undetectable cytokine levels were usually observed for CD8+ clones, whereas the CD4+ clones often showed a broad cytokine response with release of interleukin-2 (IL-2), IL-4, IL-10, IL-13 and interferon gamma(IFN-gamma) in the presence of the ALL accessory cells. ALL blasts were also able to function as allostimulatory cells for normal peripheral blood mononuclear responder cells. However, both T-cell proliferation and cytokine release showed a wide variation between ALL patients. The accessory cell function of ALL blasts showed no correlation with the release of immunomodulatory mediators (IL-2, IL-10, IL-15) or the expression of any single adhesion/costimulatory membrane molecule (CD54, CD58, CD80, CD86) by the blasts. We conclude that for a majority of patients, native ALL blasts can mediate costimulatory signals needed for accessory cell-dependent T-cell activation, but differences in costimulatory capacity between ALL patients affects both the proliferative responsiveness and cytokine release by activated T cells.     

JAK2 Translocations in hematological malignancies: Review of the literature

JAK2 is a cytoplasmic tyrosine kinase whose gene is located on chromosome 9p24. It is involved in the regulation of different cytokines and growth factors and plays an important role in the diagnosis and treatment of myeloproliferative neoplasms (Smith et al., 2008). Translocations involving the JAK2 locus are uncommon with just a few cases described in the literature, and they usually lead to a fusion protein with JAK2 (Patnaik et al., 2010). Chromosome 9p24 abnormalities have been described in myeloid and lymphoid neoplasms including chronic myelogenous leukemia (CML), acute megakaryoblastic leukemia, CD10+ B-cell acute lymphoblastic leukemia, T-cell ALL and chronic myeloproliferative disorders (CMD) (Smith et al., 2008; Lacronique et al., 1997). Although the breakpoints of each translocation are known, characterization of the partner gene has not been done in many of the cases reported due to insufficient sample or other factors. In the present study we review all translocations involving JAK2 that have been reported in the literature.     

Antibody-induced antigenic modulation is antigen dependent: characterization of 22 proteins on a malignant human B cell line

Expression of several of the surface antigens on normal and malignant hematopoietic cells is reduced or is modulated by incubation with specific antibodies. Although antigenic modulation provides a means by which cells can escape antibody-mediated immune destruction, the physiologic significance and frequency of this phenomenon are both poorly understood. To begin to address these issues, we identified and characterized surface antigens on the malignant B cell line Laz 221 established from a patient with acute lymphoblastic leukemia (ALL). Indirect immunofluorescence analysis with the use of 26 hematopoietic cell populations and immune precipitation studies with the use of iodinated ALL cells indicate that 163 monoclonal antibodies (MoAb) identify 22 different proteins on this cell line, including at least six previously described surface molecules. Seven of these antigens are expressed by all nucleated cells examined, whereas only the mu chain of immunoglobulin is B cell specific. Incubation of specific MoAb with cultures of Laz 221 cells at 37 degrees C reduces or modulates surface expression of five of these 22 antigens (p45, immunoglobulin mu chain, transferrin receptor, common ALL antigen (CD10), and p105). Studies that made use of multiple MoAb specific for the same antigen suggest that the capacity for antigenic modulation is an intrinsic property of individual antigens. These studies also suggest that the murine immune response to shared human antigens varies from one immunizing cell population to another. For example, three of the antigens present on Laz 221 cells were only identified by MoAb raised to the Burkitt's cell line Ramos and vice-versa. Only one of these six shared antigens is present in greater amounts on the immunogenic cell population. Immunogenicity of individual human antigens in the mouse may be a function of their cell surface environment.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.