Characterization of an acute T lymphoblastic leukemia expressing the gamma/delta T-cell receptor

Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the gamma/delta receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a "natural killer"-like activity can be induced in these cells by IL-2.     

Immortalization of human T cells expressing T-cell receptor gamma delta by herpesvirus saimiri.

Herpesvirus saimiri (HVS) has recently been shown to immortalize human CD4+ and CD8+ T cells expressing T-cell receptor alpha beta (TCR-alpha beta) with the maintenance of their original phenotypes and functional properties. However, the immortalization of human T cells expressing TCR-gamma delta by HVS has not been successful. Here we report that HVS can also infect and immortalize human T cells expressing TCR-gamma delta. Two human TCR-gamma delta+ T-cell clones, which continuously proliferated in interleukin-2-containing culture medium without any exogenous stimulation or addition of feeder cells for more than 8 months, were established by HVS infection. Morphologically, the HVS-transformed TCR-gamma delta+ T-cell clones were granular lymphocytes which exhibited wide-range HLA-unrestricted cytotoxicity as untransformed TCR-gamma delta+ T cells. Their phenotypes and cytotoxic activities were not altered during long-term culture. The immortalization of human TCR-gamma delta+ T cells by HVS infection would be useful for functional analysis of this lymphocyte population, which is believed to play an important role in protection against various infectious diseases.     

Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice

We recently reported that the number of gamma delta T cells was increased after infection with Escherichia coli in C3H/HeN mice. We here showed that an i.p. injection with native lipid A derived from E. coli induced an increase of gamma delta T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice. The purified gamma delta T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by V gamma 6-J gamma 1/V delta 1-D delta 2-J delta 2 gene segments and proliferated in response to the native lipid A derived from E. coli in a TCR-independent manner. The lipid A-reactive gamma delta T cells bearing canonical V gamma 6/V delta 1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable. Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the gamma delta T cells to the native lipid A. TLR2-deficient mice showed an impaired increase of the gamma delta T cells following injection of native lipid A. These results suggest that TLR2 is involved in the activation of canonical V gamma 6/V delta 1 T cells by native E. coli lipid A.     

T-cell receptor gamma/delta: comparison of gene configurations and function between humans and chimpanzees

The human and chimpanzee T-cell receptor gamma-delta (TCR ) bearing cells represent a minor subset (3–8%) of T lymphocytes. In the periphery, the TCR population has a restricted combinatorial repertoire. The TCRD-V1 and-V2 gene products are expressed in a mutually exclusive fashion, whereas, the TCRD-V2 and the TCRG-V9 encoded proteins show, in general, a coordinated expression. Restriction fragment length polymorphism analysis showed conservation of the restriction sites that identify the TCRG-V9 and TCRD-V2 rearrangements. The human TCRG-V9 locus has two alleles, TCRG-V9A1 and TCRG-V9A2 differing at codon position 31. The chimpanzee TCRG-V9 gene product differs from the products of the human TCRG-V9A1 and TCRG-V9A2 allele by two and three amino acid replacements, respectively. The human and the chimpanzee TCRG-V9-TCRD-V2 lymphocytes show a similar specific proliferative and cytolytic response to human Daudi Burkitt's lymphoma cells. Therefore, the amino acid replacements found in the chimpanzee TCRG-V9 gene product do not change the superantigen specificity across this species barrier.This nucleotide sequence data reported in this paper have been submitted to the EMBL data library and have been assigned the accession number: X61069 P. Troglodytes TCR VGG.     

Synthesis of alpha and gamma interferons by a human cutaneous lymphoma with helper T-cell phenotype

A cloned human cutaneous lymphoma Hut102-B2 with helper T-cell phenotype (Leu1⁺, Leu2a^?, Leu3a⁺) was found to produce substantial quantities of interferon (IFN) on induction with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Whereas only trace amounts of IFN were secreted by Hut102-B2 cells spontaneously, up to 8000 laboratory units/ ml of IFN were synthesized under the optimal conditions of TPA induction. Characterization studies including neutralization by specific antisera to IFNs and determination of the activities in human and bovine cells disclosed that the IFN produced by Hut102-B2 cells exposed to TPA was a mixture of immune IFN (IFN-γ) and leukocyte IFN (IFN-α) made in approximately equal amounts in terms of antiviral activity.     

Immunohistochemical characterization,distribution and ultrastructure of lymphocytes bearing the gamma/delta T-cell receptor in the human gut

The phenotypic characterization and distribution of lymphocytes bearing the gamma/delta T-cell receptor (TCR) in the human gut were investigated by an immunohistochemical technique. A mirror section technique and double staining method were used for the phenotypic analysis. Intraepithelial delta-positive cells were almost all CD8-positive and rarely negative for both CD4 and CD8. On the other hand, lymphocytes bearing TCR gamma/delta in the lamina propria were largely negative for both CD4 and CD8. The ratio of delta-positive to CD3-positive cells amongst intraepithelial lymphocytes was larger in the lower intestine. Delta-positive cells were also observed in paracortical areas of lymphoid follicles. Immunoelectron microscopic observation revealed granular structures in these delta-positive cells, which are also present in large granular lymphocytes. The role of lymphocytes bearing TCR gamma/delta in mucosal immune responses in the human gut are discussed.     

Efficient propagation of human herpesvirus 6B in a T-cell line derived from a patient with adult T-cell leukemia/lymphoma.

The efficient propagation of the OK strain of the B variant of human herpesvirus 6 (HHV-6B) was demonstrated in a line of T cells, TaY, established from the peripheral blood lymphocytes of a patient with adult T-cell leukemia/lymphoma (ATL). Growth of TaY cells depends on the presence of IL-2 and the cells harbor HTLV-I genomes. A severe cytopathic effect (CPE) was observed in many HHV-6B(OK)-infected TaY cells one week after infection. The release of virus from HHV-6B(OK)-infected TaY cells [TaY(OK)] was first detected after three days and increased rapidly for up to seven days after infection, as demonstrated by PCR. The titer of HHV-6B(OK) in the supernatant was comparable to the value of 10(3.5) TCID50/ml obtained with PHA-activated cord blood lymphocytes (CBL) that had been infected with HHV-6B(OK). The replication of the virus was shown to depend to a considerable extent on cell viability. Electron microscopy revealed many herpesvirus-type capsid- and enveloped-viruses in the nuclei and cytoplasm of degenerated cells in TaY(OK) cultures. The U1102 strain of HHV-6A and the Z29 strain of HHV-6B also infected TaY cells productively, as detected by PCR and an immunofluorescence test. These results suggest that the activation of CD4+ T lymphocytes with mitogens such as PHA or IL-2 and the expression of some cellular gene or the HTLV-I gene might be essential for efficient propagation of HHV-6B. TaY cells should play an important role in future investigations of cell-virus interactions and genetic variations or cell tropism of HHV-6 isolates since no cell line that shows propagation of both HHV-6A and HHV-6B has been reported to date.     

Selection and molecular characterization of a lipoxygenase-free soybean mutant line induced by gamma irradiation

Key message

A lipoxygenase-free soybean mutant line (H70) induced by gamma ray was selected and its detailed information about the lipoxygenase was analyzed by comparison of DNA sequence.

Abstract

Soybean seeds contain three lipoxygenase enzymes, which induce a beany or grassy flavor. The elimination of lipoxygenases can reduce the poor stability and off-flavors of soybean oil and protein products. In this study, we selected a soybean mutant (H70) in which the three lipoxygenases had been mutated using gamma rays. To obtain detailed information about the lipoxygenase, we investigated the sequences of the Lox1, Lox2 and Lox3 genes in H70 compared to the original cultivar, Hwanggum. Comparisons of the sequences of the Lox1 and Lox2 genes in H70 with those in a line with normal lipoxygenase (HG) showed that the mutations in these genes affected a highly conserved group of six histidine residues necessary for enzymatic activity. Lox1 in H70 contained a 74 bp deletion in exon 8, creating a stop codon that prematurely terminates translation. A single point mutation (T-A) in exon 8 of Lox2 changed histidine (H532, one of the iron-binding ligands essential for Lox2 activity) to glutamine. The mutation in the Lox3 gene in H70 was a single-point mutation in exon 6 (A-G), which changed the amino acid from histidine to arginine. This amino acid alteration in Lox3 was located in the N-terminal barrel, which might play a role in molecular recognition during catalysis and/or proteolysis. These results suggest that gene analysis based on DNA sequencing could be useful for elucidating the lipoxygenase content in soybean mutant lines. Additionally, the soybean mutant line selected in this study could be used to develop soybean cultivars with improved flavor.     

V delta 1+ subset of human gamma delta T cells responds to ligands expressed by EBV-infected Burkitt lymphoma cells and transformed B lymphocytes.

Human gamma delta T cells of peripheral blood can be divided in two groups in terms of their TCR as well as their behavior upon in vitro stimulation. The major subset expresses the TCR V-segments V gamma 9 and V delta 2 and proliferates in response to ligands revealed by various microorganisms, and the cell line Daudi in addition. The minor group is less homogenous on the gamma-chain but is almost completely identified by mAb against the V delta 1 segment; there is no ligand known to promote growth of these cells. Here we demonstrate that gamma delta T cells out of this subgroup are strongly stimulated in vitro by cells from several Burkitt's lymphoma cell lines. EBV infection of the Burkitt's lymphoma cell lines enhanced the stimulatory ability towards the T cells. Although EBV infection influenced the expression of a variety of cell surface molecules including ICAM-1 and LFA-3, no correlation to the gamma delta T cell-stimulating capacity became apparent. We conclude that Burkitt's lymphoma cells and transformed B cells express ligands of cellular origin for a hitherto poorly characterized subgroup of human gamma delta T cells.     

Reversible macrophage differentiation induced in a new human myeloid cell line by gamma interferon.

A new cell line was established from the bone marrow of a patient with chronic myeloid leukemia. The cells were attributed an intermediate myeloid phenotype on the basis of their cytochemical features and membrane antigen expression. These cells respond to both chemical and physiological activators of the signal transduction pathways with growth arrest and phenotype changes. Macrophage maturation can be induced in a fraction of the cells by gamma-interferon (γ-IFN). Cells are however recruited again into the cell cycle by recultivation in γ-IFN-free medium: variants unresponsive to γ-IFN, and others which show either reversible or irreversible differentiation were isolated from the original cell line by cloning and sib-selection. These clones can be used to investigate the relationship between γ-IFN response pathways and cell proliferation.     

Intracellular calcium oscillations induced in a T-cell line by a weak 50 Hz magnetic field

Applied weak magnetic fields have been shown to affect cellular activity on several levels, but the mechanisms involved remain elusive. We have decided to study an early signal transduction event in the human T cell line Jurkat; oscillations of free [Ca²⁺]i, of the type seen by crosslinking the CD3 complex. Cells were exposed to a 50 Hz, 0.1 mT, sinusoidal magnetic field while intracellular free calcium was measured in individual cells, using fura-2 as a probe. An acute response was observed with oscillatory increases in [Ca²⁺]i, which subsided when the field was turned off. The effect of the magnetic field on [Ca²⁺]i was comparable to that achieved by an anti-CD3 monoclonal antibody. © 1993 Wiley-Liss, Inc.     

Inverse relationship between constitutive gamma interferon production and human T-cell lymphoma/leukemia virus expression in cultured T lymphocytes.

Particular interest in human T lymphocyte lymphoma/leukemia virus (HTLV) derives from the close association of HTLV with several types of human mature T lymphocyte malignancies and the strong possibility that HTLV is the causative agent of this group of leukemias and lymphomas. This is the first report to show that HTLV expression in T lymphocytes cultured in vitro is inversely proportional to constitutive gamma interferon production. Of 16 fresh T lymphocyte cultures established from patients with mature T lymphocyte neoplasias, 3 were grown continuously for over 3 years and 13 were grown for 2 to 8 months in culture. Of the 16 cultures, 9 were HTLVp19 positive and interferon negative, whereas the remaining 7 were HTLVp19 negative or weakly positive and also interferon positive (12 to 105 U/ml). The prototype HTLV-positive T-cell line (HUT102) was examined over a long-term culture and after selective cell cloning for high virus yield. Results indicate that early-passage, low-HTLV-producing HUT102 cells constitutively produced significant levels of gamma-immune interferon. In late-passage and cloned HUT102 cells, an increase in HTLV production was concordant with a decrease in constitutive interferon production and the loss of mature T lymphocyte antigens. Transformation of human umbilical cord blood lymphocytes by HTLV was possible only after cocultivation with the non-interferon, high virus-producing, cloned HUT102 T lymphocytes. The inverse relationship between interferon and HTLV production was also observed when normal human umbilical cord blood and adult T lymphocytes were transformed by HTLV and maintained in culture.     

T-cell activation induced by anti-CD3 × anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand

 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997