Human T-cell Blasts Research Articles

Liver X receptors interfere with cytokine-induced proliferation and cell survival in normal and leukemic lymphocytes

Liver X receptors (LXRs) are nuclear receptors regulating lipid and cholesterol metabolism. Recent data indicate an additional role of LXR in immunity by controlling dendritic cell and T-cell function and in breast and prostate cancer cells. Here, we show that LXR activation interferes with IL-2 and IL-7-induced proliferation and cell cycle progression of human T-cell blasts mainly through inhibited phosphorylation of the retinoblastoma protein and decreased expression of the cell cycle protein cyclin B. Comparable results were obtained with IL-2-dependent chronic lymphoblastic leukemia (CLL) T cells. Furthermore, we show for B-CLL cells that LXR are functionally active and inhibit expression of survival genes bcl-2 and MMP-9, and significantly reduce cell viability, suggesting an interference of LXR with cytokine-dependent CLL cell survival. In conclusion, our data reveal LXR as a potent modulator of cytokine-dependent proliferation and survival of normal and malignant T and B lymphocytes. This novel LXR action could find clinical application in immunosuppressive and antileukemic therapies.

WR1065 mitigates AZT‐ddI‐induced mutagenesis and inhibits viral replication

The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)-didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 muM AZT-ddI (equimolar) for 3 days with or without 150 muM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 muM AZT in the absence or presence of 5-1,000 muM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum antiviral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation.

Antiretroviral activity of the aminothiol WR1065 against Human Immunodeficiency virus (HIV-1) in vitro and Simian Immunodeficiency virus (SIV) ex vivo

BackgroundWR1065 is the free-thiol metabolite of the cytoprotective aminothiol amifostine, which is used clinically at very high doses to protect patients against toxicity induced by radiation and chemotherapy. In an earlier study we briefly reported that the aminothiol WR1065 also inhibits HIV-1 replication in phytohemagglutinin (PHA)-stimulated human T-cell blasts (TCBs) infected in culture for 2 hr before WR1065 exposure. In this study we expanded the original observations to define the dose-response curve for that inhibition, and address the question of additive effects for the combination of WR1065 plus Zidovudine (AZT). Here we also explored the effect of WR1065 on SIV by examining TCBs taken from macaques with well-established infections several months with SIV.ResultsTCBs from healthy human donors were infected for 2 hr with HIV-1, and viral replication (p24) was measured after 72 hr of incubation with or without WR1065, AZT, or both drugs. HIV-1 replication, in HIV-1-infected human TCBs, was inhibited by 50% at 13 μM WR1065, a dose at which 80% of the cells were viable. Cell cycle parameters were the same or equivalent at 0, 9.5 and 18.7 μM WR1065, showing no drug-related toxicity. Combination of AZT with WR1065 showed that AZT retained antiretroviral potency in the presence of WR1065. Cultured CD8+ T cell-depleted PHA-stimulated TCBs from Macaca mulatta monkeys chronically infected with SIV were incubated 17 days with WR1065, and viral replication (p27) and cell viability were determined. Complete inhibition (100%) of SIV replication (p27) was observed when TCBs from 3 monkeys were incubated for 17 days with 18.7 μM WR1065. A lower dose, 9.5 μM WR1065, completely inhibited SIV replication in 2 of the 3 monkeys, but cells from the third macaque, with the highest viral titer, only responded at the high WR1065 dose.ConclusionThe study demonstrates that WR1065 and the parent drug amifostine, the FDA-approved drug Ethyol, have antiretroviral activity. WR1065 was active against both an acute infection of HIV-1 and a chronic infection of SIV. The data suggest that the non-toxic drug amifostine may be a useful antiretroviral agent given either alone or in combination with other drugs as adjuvant therapy.

Cell cycle regulation by FasL and Apo2L/TRAIL in human T-cell blasts. Implications for autoimmune lymphoproliferative syndromes

The Fas-FasL pathway plays an important role in the homeostasis of mature lymphocytes, with defects causing autoimmune lymphoproliferative syndromes (ALPS). Human T-cell blasts are not sensitive to FasL or Apo2L/TRAIL-induced apoptosis unless they get reactivated, but either of those ligands inhibits their growth in the absence of cell death induction due to a cell cycle arrest in S-G2/M. In the present work, we have studied the mechanism(s) by which FasL or Apo2L/TRAIL regulate T-cell blast cell cycle in healthy donors and in two types of ALPS patients. Our data indicate that in human CD8+ T-cell blasts, Fas ligation, and especially Apo2L/TRAIL induce the p53-dependent decrease in cyclin-B1 levels. However, the induction of the negative cell cycle regulator p21WAF1 by FasL or Apo2L/TRAIL in either CD4+ or CD8+ T-cell blasts seems to be the main regulatory mechanism. This mechanism is dependent on caspase activation and on H2O2 generation. The increase in p21 levels by FasL or Apo2L/TRAIL is concomitant with p53 increases only in CD8+ T-cell blasts, with p21 levels maintained high for longer times than p53 levels. In CD4+ T-cell blasts p21 levels are controlled through a transient and p53-independent mechanism. The present results suggest that the etiology of ALP syndromes could be related not only to defects in apoptosis induction, but also in cell cycle regulation.

The induction of Bim expression in human T-cell blasts is dependent on nonapoptotic Fas/CD95 signaling

AbstractThe BH3-only protein Bim is required for maintaining the homeostasis of the immune system, since Bim regulates the down-modulation of T-cell responses, mainly through cytokine deprivation. Using T-cell blasts from healthy donors and also from patients with autoimmune lymphoproliferative syndromes (ALPSs) due to homozygous loss-of-function mutation of FasL (ALPS-Ic) or heterozygous mutation in the Fas/CD95 death domain (ALPS-Ia), it is shown that the induction of Bim expression during the process of human T-cell blast generation is strictly dependent on FasL/Fas-mediated signaling. The main pathway by which Fas signaling regulates the levels of Bim expression in human T-cell blasts is the death-domain– and caspase-independent generation of discrete levels of H2O2, which results in the net increase of Foxo3a levels. The present results connect the 2 main pathways described until the moment for the control of T-cell responses: death receptor–mediated activation-induced cell death and apoptosis by cytokine deprivation.

Killing of human immunodeficiency virus-infected primary T-cell blasts by autologous natural killer cells is dependent on the ability of the virus to alter the expression of major histocompatibility complex class I molecules

AbstractIn the current study, we evaluated whether the capacity of HIV to modulate major histocompatibility complex (MHC) class I molecules has an impact on the ability of autologous natural killer (NK) cells to kill the HIV-infected cells. Analysis of HIV-infected T-cell blasts revealed that the decrease in MHC class I molecules on the infected cell surface was selective. HLA-A and -B were decreased on cells infected with HIV strains that could decrease MHC class I molecules, whereas HLA-C and -E remained on the surface. Blocking the interaction between HLA-C and -E and their corresponding inhibitory receptors increased NK cell killing of T-cell blasts infected with HIV strains that reduced MHC class I molecules. Moreover, we demonstrate that NK cells lacking HLA-C and -E inhibitory receptors kill T-cell blasts infected with HIV strains that decrease MHC class I molecules. In contrast, NK cells are incapable of destroying T-cell blasts infected with HIV strains that were unable to reduce MHC class I molecules. These findings suggest that NK cells lacking inhibitory receptors to HLA-C and -E kill HIV-infected CD4+ T cells, and they indicate that the capacity of NK cells to destroy HIV-infected cells depends on the ability of the virus to modulate MHC class I molecules. (Blood. 2004;104:2087-2094)

Human T cells resistant to complement lysis by bivalent antibody can be efficiently lysed by dimers of monovalent antibody

We have previously shown that bivalent human gamma1 CD3 monoclonal antibody (mAb) is ineffective at mediating lysis of human T cells with human complement. In this paper we have used genetic engineering and sulfur chemistry to prepare 2 types of human gamma1 CD3 mAb dimer, with the aim of improving complement lysis activity. The IgG molecules forming the dimers were linked together at their C-termini by stable bismaleimide thioether bridges. The first dimer was composed of 2 bivalent mAb molecules. This dimer proved incapable of lysing human T-cell blasts with human, rabbit, or guinea pig complement. The second dimer consisted of 2 molecules of a monovalent derivative (possessing a single Fab domain) of the bivalent mAb. This dimer was highly lytic with human complement, with a lytic titer 64-fold greater than that of the nondimerized monovalent mAb. The maximum level of lysis of human T-cell blasts achieved with this monovalent mAb dimer was equal to that obtained with the therapeutic antilymphocyte mAb alemtuzumab, but its lytic titer was 4-fold greater. The monovalent mAb dimer was also found to be lytic in the presence of rabbit and guinea pig complement. Dimerization of monovalent antibodies may provide a general strategy for improving the cytolytic activity of other mAbs that are normally unable to induce lysis with complement. The monovalent CD3 mAb dimer may have potential for development as an agent for immunotherapy of T-cell leukemia.

Luteolin, an Abundant Dietary Component is a Potent Anti-leishmanial Agent that Acts by Inducing Topoisomerase II-mediated Kinetoplast DNA Cleavage Leading to Apoptosis

Plant-derived flavonoids, which occur abundantly in our daily dietary intake, possess antitumor, antibacterial, and free radical scavenging properties. They form active constituents of a number of herbal and traditional medicines. Several flavonoids have been shown to exert their action by interacting with DNA topoisomerases and promoting site-specific DNA cleavage. Therefore, flavonoids are potential candidates in drug design. We report here that, although the flavonoids luteolin and quercetin are potent antileishmanial agents, luteolin has great promise for acting as a lead compound in the chemotherapy of leishmaniasis, a major concern in developing countries. Kinetoplast DNA (kDNA) minicircle cleavage in drug-treated parasites was measured by electrophoresis of the total cellular DNA, followed by Southern hybridization using 32P labeled kDNA as a probe. Cell cycle progression and apoptosis were measured by flow cytometry using propidium iodide and fluorescein isothiocyanate (FITC)-labeled Annexin V. Luteolin and quercetin inhibited the growth of Leishmania donovani promastigotes and amastigotes in vitro, inhibited DNA synthesis in promastigotes, and promoted topoisomerase-II-mediated linearization of kDNA minicircles. The IC50 values of luteolin and quercetin were 12.5 microM and 45.5 microM, respectively. These compounds arrest cell cycle progression in L. donovani promastigotes, leading to apoptosis. Luteolin has no effect on normal human T-cell blasts. Both luteolin and quercetin reduced splenic parasite burden in animal models. Luteolin and quercetin are effective antileishmanial agents. Quercetin has nonspecific effects on normal human T cells, but luteolin appears nontoxic. So, luteolin can be a strong candidate for antileishmanial drug design.

Leishmania donovani parasites interact with gamma/delta+ human peripheral blood T cells and induce susceptibility to NK cell-mediated lysis.

We recently reported that Leishmania donovani infect the human T-cell line in vitro. To examine whether primary human T cells could be infected by this parasite, a direct interaction of the peripheral blood T cells with L. donovani was examined. The percentage of gamma/delta+ T cells was markedly increased when in vitro generated normal human T-cell blasts were cultured with L. donovani amastigotes. About 30% of the gamma/delta+ T cells in the parasite exposed T-cell blasts expressed parasite antigens intracellularly without detectable intracellular parasites. Parasite exposed T-cell blasts had a reduced surface expression of HLA-DR and were lysed by the sorted CD56+ cells. In contrast, neither L. donovani amastigotes nor T-cell blasts exposed to heat killed amastigotes and/or were sensitive to the NK cell-mediated lysis. Of interest is that about 10% CD3+ peripheral blood T cells in two out of three Indian Kala-azar patients tested expressed intracellular L. donovani antigens.

Inhibition of cyclosporin A/FK506 resistant, lymphokine-induced T-cell activation by phenothiazine derivatives.

Earlier studies from our laboratory have demonstrated that phenothiazine derivatives are capable of inhibiting mitogen-induced activation of human T-cells and thymocytes. Similar to cyclosporin A, phenothiazine derivatives exert these inhibitory effects by decreasing the accumulation of lymphokine-specific mRNA. However, proliferation of T-cell blasts and of unfractionated human thymocytes can also be induced by interleukin 2. Since activation of T-cells via the interleukin 2 receptor seems to be resistant to the action of cyclosporin A, the present study was designed to investigate whether lymphokine-induced activation could be inhibited by phenothiazine derivatives. The effects of the phenothiazine derivatives chlorpromazine and/or fluphenazine have been studied and compared to the action of cyclosporin A and FK506 in human thymocytes, human T-cell blasts and in the human T-cell line H33-HJ JA1, which is an interleukin 2 producing cell line derived from Jurkat cells. As evidenced by the incorporation of [3H]-thymidine, cyclosporin A (1 microgram/ml) and FK506 (100 ng/ml) have no or only marginal inhibitory capacity on interleukin 2-induced proliferation in all T-cell systems tested. By contrast, phenothiazine derivatives (fluphenazine > chlorpromazine) exert a dose-dependent inhibition of the activation of these cells in pharmacologically relevant micromolar concentrations. Similar results were obtained by measuring the production of interferon-gamma in the supernatants of interleukin 2-induced human thymocytes. Our results suggest that the use of phenothiazines might be helpful in immunosuppressive regimens.

Dibutyryl guanosine cyclic monophosphate causes proliferation of interleukin-2-dependent T-cells in the presence of suboptimal levels of interleukin-2

The effect of dibutyryl guanosine cyclic monophosphate (db-cGMP) on proliferation of an interleukin-2 (IL-2)-dependent T-cell line was investigated. Dibutyryl cGMP added to HT-2 cell cultures without IL-2 or in the presence of an optimal IL-2 concentration (60 U/ml) had no effect on proliferation. The same amount of db-cGMP (10 −5M) added to cultures in the presence of suboptimal amounts of IL-2 (3–6 U/ml) produced significant increases in proliferation. In contrast, db-cAMP caused substantial inhibition of HT-2 cells cultured with IL-2. Similar effects were observed using human T-cell blasts produced after 72 h stimulation with phytohemagglutin. These results suggest that cyclic GMP may play a role in the cellular response of IL-2 responsive cells to IL-2.

Interleukin-2 secretion by synovial fluid lymphocytes in rheumatoid arthritis.

Interleukin-2 (IL-2) receptor bearing cells and soluble IL-2, measured in a bioassay with IL-2 dependent human T-cell blasts, were recognized in synovial fluid, but not in the peripheral blood of patients with rheumatoid arthritis (RA). After stimulation in vitro with appropriate concentrations of the mitogen concanavalin A (Con-A), comparable proportions of IL-2 receptor (IL-2R) bearing cells were seen in cultures of synovial fluid lymphocytes (SFL) and in cultures of peripheral blood lymphocytes (PBL). On the other hand, higher levels of secreted IL-2 were found in SFL cultures compared to corresponding PBL cultures of RA patients and normal donors. Specificity of the IL-2 bioassay was confirmed by blocking the T-cell blast proliferation (induced by SFL culture supernatants), by 83 +/- 4%, after addition of a monoclonal anti-IL-2R antibody. Despite the high levels of soluble IL-2, only a weak proliferative response was observed in the corresponding SFL cultures.

Expression of murine interleukin-2 cDNA in E. coli and biological activities of recombinant murine interleukin-2.

Murine interleukin-2 (MIL-2) cDNA was inserted into an expression vector carrying an Escherichia coli tryptophan promoter and was expressed in E. coli. Recombinant MIL-2 produced by E. coli supported the growth of murine CTLL-2 cells, but not that of human T-cell blasts. Recombinant MIL-2 strongly inhibited the binding of recombinant human IL-2 (HIL-2) to murine responder cells, but only very weakly inhibited the binding to human responder cells. Moreover, recombinant MIL-2 induced secondary alloantigen specific cytotoxic T lymphocytes (2 degrees CTL) from memory CTL and activated natural killer (NK) cells in murine systems in the same manner as recombinant HIL-2. The results suggest that the species hierarchy (that MIL-2 derived from native cell culture does not act on human T-cells) is due to the protein moiety, not the sugar moiety, and is to be ascribed to the difference in binding affinity of MIL-2 and HIL-2 to murine and human responder cells respectively, and that recombinant MIL-2 shares identical biological and immunological activities with recombinant HIL-2. Thus, MIL-2 might be a convenient tool for extensive studies of the pharmacological and physiological activities of IL-2 in murine models.

Role of unique consecutive glutamine repeats in active murine interleukin-2 molecule.

Murine interleukin-2 (MIL-2) cDNA deleted of 11 repeats of a CAG sequence, and that encoded from 33Met to 149Gln were inserted into an expression vector carrying an Escherichia coli tryptophan promoter and were expressed in E. coli, respectively. Recombinant MIL-2 deleted of 11 glutamine repeats (MIL-2(-Gln] supported the growth of murine CTLL-2 cells but recombinant MIL-2 initiated from 34Asp (34Asp-MIL-2) did not. The growth of human T-cell blasts was not supported by MIL-2(-Gln) or 34Asp-MIL-2. MIL-2(-Gln) had identical biological and immunological activities with intact MIL-2, but 34Asp-MIL-2 did not. These results suggest that the consecutive glutamine repeats do not play a role in the biological and immunological activities of MIL-2, but that the peptide sequence around them does, and the species hierarchy that MIL-2 does not act on human lymphocyte is not due to the presence of glutamine repeats in MIL-2.

Mouse monoclonal antibodies can nonspecifically inhibit the proliferation of human T-cell growth factor-dependent T cells

A large series of mouse monoclonal antibodies was found to inhibit the proliferation of T-cell growth factor (TCGF)-dependent human T-cell blasts as measured by the incorporation of tritiated thymidine. The specificity of the antibody appeared to be irrelevant for inhibition and two T-cell-specific antibodies did not prevent the absorption of TCGF by treated T cells. It is suggested that the antibodies function by the indirect release of suppressor factors by Fc receptor-bearing TCGF-dependent cells.

Ia determinants on stimulated human T lymphocytes. Occurrence on mitogen- and antigen-activated T cells.

Human T-cell blasts were generated by stimulation with mitogens and antigens. A proportion of these blasts expressed Ia antigens detectable by immunofluorescence with both allo- and hetero-antiserums. The maximal expression of Ia antigens was delayed and usually occurred after the peak of blastogenesis. Among the three mitogens used, pokeweed mitogen (PWM) was most effective in giving a high percentage and intense Ia staining of T-cell blasts. Phytohemagglutinin and concanavalin A blasts gave weaker and lower percentages of Ia staining. Activation by alloantigens and soluble antigens such as tetanus toxoid and purified protein derivative resulted in Ia expression on T cells comparable to PWM stimulation. Depletion of Ia+ cells from freshly isolated T cells with anti-Ia and complement decreased subsequent Ia expression, suggesting that a proportion of Ia+ blasts were derived from Ia-bearing peripheral blood T cells. When the specificities of the Ia antigens on T-cell blasts were examined with alloantiserums, it was evident that the T blasts expressed similar HLA-DR determinants to those on B cells from the same donor; occasional minor differences between stimulated T cells and autologous B-cell lines or fresh B cells were encountered.