Characterization of 17 Human Immunodeficiency Virus-1 Carrier Cell Lines with T cell, Myelomonocyte, or Megakaryocyte Lineages
Yoshinobu MatsuoTakashi KamiyaShuji NakamuraNaoki YamamotoFumio UnoMasayoshi YoshidaS. NiiM KurimotoJ Minowada
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Abstract:
Of 29 hematopoietic cell lines tested for susceptibility to human immunodeficiency virus (HIV)-1HTLV-IIIB infection, all CD4+ cell lines became infected. Continuous culturing of infected cell lines resulted in nine HIV-1 carrier cell lines, including, for the first time, an HIV-1 carrier megakaryoblastic cell line, MEG-01/HIV. The immunophenotypic profiles of a total of 17 HIV-1 carrier cell lines (nine newly and eight previously established cell lines) were compared with their respective parental noninfected cell lines. Except for total absence of CD4 expression, the expression of other antigens was variable among the 17 HIV-1 carrier cell lines. Persistent and consistent replication of infectious HIV-1 was detected in all of them in varying quantities. The great variability observed in both the altered marker expression, with respect to that of the noninfected parental cell lines, and in the quantities of persistently produced infectious HIV-1 was, nevertheless, specific to the individual cell lines. Furthermore, the present study demonstrates that there is no apparent correlation in the quantity of HIV-1 produced to either T cell, myelomonocytic cell, or megakaryocytic cell types. Instead, the results suggest that a particular interaction between HIV-1 and individual clonal cell lines may provide insight into the extremely complex immune dysregulation associated with the pathogenesis of acquired immune deficiency syndrome. Thus, the 17 HIV-1 carrier cell lines of diverse origin presented here provide valuable and unique models for further understanding acquired immune deficiency syndrome pathogenesis at the cellular and molecular levels. [P.S.E.B.M. 1993, Vol 202]Keywords:
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A novel surface molecule, Tp90, is described which appears to be involved in an antigen-independent pathway of human T lymphocyte activation. The Tp90 molecule was identified by a monoclonal antibody (mAb), MX20, obtained from a fusion using spleen cells of a mouse immunized with cells from two T cell leukemia lines, Jurkat and HPB-ALL. Biochemical data show that Tp90 is distinct and physically independent from the structures already known to be involved in T cell activation, namely T11, T44 or T3/TCR. These results were confirmed by antibody-induced antigen modulation experiments. Modulation of Tp90 had no effect on the expression of T3 and of the T cell receptor. Conversely, the expression of Tp90 was not affected by modulation of the T3/TCR molecular complex by either anti-T3 or anti-TCR antibody. Functional studies showed that anti-Tp90 mAb MX20 induced high levels of interleukin 2 production in Jurkat cells. Modulation of the T3/TCR complex significantly decreased the response of Jurkat cells to stimulation by antibody MX20, suggesting that the T3/TCR complex regulates the ability of the Tp90 molecule to induce IL 2 synthesis. In addition to its effect on Jurkat cells, anti-Tp90 mAb was found to be mitogenic for peripheral blood T cells. As the magnitude of the proliferative response elicited by anti-Tp90 mAb was lower than that induced by anti-T3 mAb, the possibility was considered that only a subpopulation of T cells is reactive with anti-Tp90. Indeed as determined by FACS analyses, only 3-14% of E-rosette-positive cells were stained with mAb MX20. In addition, multicolor flow cytometry analysis showed that the Tp90+ cells belong preferentially to the CD8 subset.
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Objective:To establish a multi-drug resistance(MDR) cell line Jurkat/Doxorubicin(DOX) derived from human T lymphocyte leukemia cell line Jurkat,and to compare the biological characteristics between Jurkat/DOX and Jurkat cell line.Methods:Jurkat cells were induced with long-term,singular,and dose-increasing DOX addition to MDR cell line Jurkat/DOX.The biological characteristics and the spectrum of MDR of Jurkat/DOX were evaluated by MTT assay.The intracellular accumulation of DOX was detected by flow cytometer and laser scanning confocal microscope.The expression of mdr1 was analyzed by RT-PCR.Western blot was also performed to detect the expression of P-gp.Results:MDR cell line Jurkat/DOX was established by long-term,intermittent,singular and dose-increasing DOX induction.The growth of Jurkat/DOX was slower than that in Jurkat cell line.Jurkat/DOX cells were resistant not only to the original DOX,but also to many other anticancer drugs such as CTX,VCR,and DNR.Intracellular accumulation of DOX was significantly less than that in parent cells.The expression of mdr1 gene and P-gp protein were up-regulated in Jurkat/DOX cell line.Conclusion:The MDR cell line Jurkat/DOX was successfully established,it has the property of resistant to many anticancer drugs,and the resistant maybe closely related to the over-expression of P-gp.
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Human T cells play an important role in pathogen clearance, but their aberrant activation is also linked to numerous diseases. T cells are activated by the concurrent induction of the T cell receptor (TCR) and one or more costimulatory receptors. The characterization of signaling pathways induced by TCR and/or costimulatory receptor activation is critical, since these pathways are excellent targets for novel therapies for human disease. Although studies using human T cell lines have provided substantial insight into these signaling pathways, no comprehensive, direct comparison of these cell lines to activated peripheral blood T cells (APBTs) has been performed to validate their usefulness as a model of primary T cells.We used quantitative biochemical techniques to compare the activation of two widely used human T cell lines, Jurkat E6.1 and HuT78 T cells, to APBTs. We found that HuT78 cells were similar to APBTs in proximal TCR-mediated signaling events. In contrast, Jurkat E6.1 cells had significantly increased site-specific phosphorylation of Pyk2, PLCgamma1, Vav1, and Erk1/Erk2 and substantially more Ca2+ flux compared to HuT78 cells and APBTs. In part, these effects appear to be due to an overexpression of Itk in Jurkat E6.1 cells compared to HuT78 cells and APBTs. Both cell lines differ from APBTs in the expression and function of costimulatory receptors and in the range of cytokines and chemokines released upon TCR and costimulatory receptor activation.Both Jurkat E6.1 and HuT78 T cells had distinct similarities and differences compared to APBTs. Both cell lines have advantages and disadvantages, which must be taken into account when choosing them as a model T cell line.
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Ligands, including antigen, which interact with the T3/T cell antigen receptor (Ti) induce an increase in cytoplasmic free calcium ((Ca/sup + +/)/sub i/). However, such an increase in (Ca/sup + +/)/sub i/ is not sufficient to result in T cell activation. Activation can occur if such ligands, which increase (Ca/sup + +/)/sub i/, are added in the presence of phorbol myristate acetate (PMA). This suggests that interactions involving other T cell surface receptors might function in a manner analogous to PMA and play an important role in T cell activation. Using the human leukemic T cell line Jurkat, they demonstrated that 9.3, a monoclonal antibody specific for a 90 kD homodimer expressed on human T cells (Tp44), synergized with some ligands interacting with T3/Ti on Jurkat in the induction of IL-2 production from Jurkat. Thus, 9.3 synergized with PHA, ConA, or sepharose beads coupled with anti-T3 or anti-Ti but not with calcium ionophores or soluble anti-T3 or anti-Ti. Furthermore, at high concentrations only, 9.3 could also synergize with PMA in the activation of Jurkat and T3/Ti negative mutants of Jurkat. No detectable activation of protein kinase C was induced by 9.3 alone. These studies suggest that T cell activation maymore » result from the simultaneous triggering of several distinct T cell surface molecules.« less
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Clinical reports indicate that the development of drug resistance to AZT after chronic administration is common. In order to study this phenomenon, the T-cell line Jurkat E6-1 was treated continuously in vitro with low, gradually increased, concentrations of azidothymidine (AZT). Initially, 1 microM AZT significantly retarded the cell line from reaching confluence. However, after 10 weeks the T-cell line was able to grow in 10 microM AZT without any evidence of growth inhibition. Subsequently, cell isolates could grow continuously in the presence of 20, 50, and 100 microM AZT without growth inhibition. These T-cell lines (Jurkat E6-1/AZT-10, Jurkat E6-1/AZT-20, Jurkat E6-1/AZT-50, and Jurkat E6-1/AZT-100) were tested for AZT anabolism using purified [3H]AZT, and the results were compared to the wild-type untreated Jurkat E6-1 cell line. Similar intracellular AZT anabolites concentrations were determined in all cell lines. However, a four- to sixfold lower cellular concentration of mono-, di-, and triphosphate anabolites of AZT was determined in the Jurkat E6-1/AZT-10 cell line after 1 microM AZT incubation and 6.5-fold lower after 10 microM AZT treatment. In general, a five- to sixfold reduction in the phosphorylation rates were estimated in the AZT resistant T-cell line. Pharmacology studies of [3H]AZT in the Jurkat E6-1/AZT-100 cell line showed a much lower level of activation of the pro-drug (28-fold), due to lack of thymidine kinase (TK) activity when compared to the Jurkat E6-1/AZT-10 T-cell line. A similar level of resistance was obtained at the thymidylate (dTMP) kinase level. Concurrently an additional mode of resistance (407-fold) was seen on the incorporation of the AZT triphosphate anabolite (AZTTP) into cellular DNA. The formation of this cell line in a period of < or = 4 months coincides with the evidence of the clinical development of "resistance" to AZT in patients who receive the drug continuously. In addition, these T-cell lines have been infected with HIV, and studies on the development of collaterally sensitive regimens are under way.
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Experiments from flight- and ground-based model systems suggest that unexpected alterations of the human lymphoblastoid cell line Jurkat, as well as effects on cell growth, metabolism, and apoptosis, can occur in altered gravity conditions. Using a desktop random positioning machine (RPM), we investigated the effects of simulated microgravity on Jurkat cells and their multidrug-resistant subline, Jurkat/A4 cells. The viability of Jurkat/A4 cells decreased after simulated microgravity in contrast with the Jurkat cells. At the same time, the viability between the experimental Jurkat cells and control Jurkat cells was not significantly different. Of note, Jurkat cells appeared as less susceptible to apoptosis than their multidrug-resistant clone Jurkat/A4 cells, whereas cell-cycle analysis showed that the percentage of Jurkat/A4 cells in the S-phase was increased after 72 and 96 h of RPM-simulated microgravity relative to their static counterparts. The differences in Jurkat cells at all phases between static and simulated microgravity were not significant. The surface expression of the intercellular adhesion molecule 3 (ICAM-3)—also known as cluster of differentiation (CD)50—protein was changed for Jurkat/A4 cells following exposure to the RPM. Changes in cell morphology were observed in the Jurkat/A4 cells after 96 h of RPM-simulated microgravity. Thus, we concluded that Jurkat/A4 cells are more sensitive to RPM-simulated microgravity as compared with the parental Jurkat cell line. We also suggest that intercellular adhesion molecule 3 may be an important adhesion molecule involved in the induction of leukocyte apoptosis. The Jurkat/A4 cells with an acquired multidrug resistance phenotype could be a useful model for studying the effects of simulated microgravity and testing anticancer drugs.
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OBJECTIVE: To study the role of MEKK2 on the production of IL-2 in Jurkat cells stimulated by PHA/anti-CD28 antibody. METHODS: The MEKK2 and JNK kinase activities were measured in both dominant negative MEKK2 Jurkat (dnMEKK2 Jurkat) cells and parental Jurkat cells. The AP(1) and IL-2 promotor activities were measured by luciferase activity assay. The IL-2 mRNA and protein were detected by RT-PCR and Western blot. RESULTS: After stimulation by PHA/anti-CD28, JNK was activated in parental Jurkat cells but not in dnMEKK2 Jurkat cells. The luciferase report gene activities of AP1 and IL-2 promotors were increased by 4- and 5-folds in parental cells whereas only by 1 fold in dnMEKK2 Jurkat cells. The level of IL-2 mRNA and IL-2 protein were increased in parental Jurkat cells but not in dnMEKK2 Jurkat cells. CONCLUSION: MEKK2 plays an important role on the production of IL-2 in Jurkat cell stimulated with PHA/anti-CD28 antibody. It is a potential drug target for the treatment of GVHD and autoimmune disease.
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Objective:To investigate the expression regulation of PD-L1(Programed Death Ligand-1) in Jurkat cell and correlation between the expression of PD-L1(Programed Death Ligand-1,B7-H1) and the activity and apoptosis of Jurkat cell.Methods:IFN-γ and the siRNA(small interfering RNA) targeting to PD-L1 were used to control the expression of PD-L1 in the Jurkat cell.RT-PCR,PCR were used to measure the expression of IL-2 and flow cytometry was used to detected the apoptosis of Jurkat cell.Results:Semi-quantitative β-actin,according template amount to PCR,was used to choose siRNA-A from three different siRNA(siRNA-A,siRNA-B,siRNA-C) which can efficiently and specifically inhibit the expression of PD-L1 in Jurkat Cell.80nM siRNA-A transfect Jurkat Cell,after 24h,the expression of PD-L1 decrease obviously.2000U/ml IFN-γ induced Jurkat Cell,24h later,PD-L1 up-regulated.The expression of PD-L1 in Jurkat Cell negatively correlates with the level of IL-2 secreted by Jurkat Cell by RT-PCR and PCR.And the result from flow cytometry showed increased expression of PD-L1 can reduce the apoptosis of Jurkat Cell.Conclusions:Synthesized siRNA for PD-L1,after transfecting Jurkat cell,could specifically inhibit the expression of PD-L1.IFN-γ in vitro could stimulate Jurkat cell and increase the expression of PD-L1.PD-L1 signal negatively correlates with IL-2 secreted by Jurkat cell,and PD-L1 could inhibit the apoptosis of Jurkat cell.
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To investigate the effect of oridonin on the human acute lymphocytic leukemia cell line Jurkat and its mechanism.Jurkat cells were cultured in vitro and treated with various concentrations (0, 1.25, 2.5, 5, and 10 μmol/L) of oridonin for different lengths of time (24, 48, and 72 hours). The proliferation of Jurkat cells was analyzed by MTT assay. The changes in nuclear morphology were evaluated by fluorescence microscopy at 12 hours after treatment with various concentrations of oridonin. The expression levels of Brg1, P53, and C-myc were determined by semi-quantitative Western blot in Jurkat cells treated with various concentrations of oridonin for 24 hours or 5 μmol/L oridonin for various lengths of time (0, 2, 6, 12, and 24 hours). The expression levels of P53 and C-myc and proliferation of Jurkat cells were evaluated after Brg1 expression was knocked down by Brg1-specific siRNA.Compared with the control group, the proliferation of oridonin-treated Jurkat cells was significantly inhibited in a concentration- and time-dependent manner (P<0.05). According to the florescence microscopic analysis, oridonin treatment led to nuclear pyknosis in Jurkat cells. Compared with the control group, Jurkat cells treated with 5 μmol/L oridonin had reduced expression of Brg1 and C-myc but elevated expression of P53. Brg1 knock-down led to a significant reduction in proliferation of Jurkat cells (P<0.05), up-regulated expression of P53, and down-regulated expression of C-myc.Oridonin can inhibit the proliferation of Jurkat cells, probably via the Brg1 signaling pathway.
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In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.
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