Histone deacetylase inhibitors promote mice corneal allograft survival through alteration of CD4+ effector T cells and induction of Foxp3+ regulatory T cells

Trichostatin A (TSA) is classical Histone deacetylase inhibitors (HDACIs) II which is used in treatment of advanced cutaneous T-cells lymphoma. Our works focused on the roles of TSA on immuno-modulatory. We found that the TSA could induce resting Teff cells into apoptotic cell death and inhibit Teff cells proliferation in a dose-dependent manner. We also observed down-regulation effects of various costimulatory/adhesion molecules on Teff cells and up-regulation of Foxp3 expression on CD4+ CD25+ T cells. Treatment with TSA could improve mice corneal allograft survival by promoting the proportions and allosuppressive function of CD4+ CD25+ regulatory T cells. Our findings suggest that the use of TSA allows the beneficial pharmacological effect on CD4+ CD25- T activation in vitro and enhancement of Foxp3+ Treg cells in vivo.     

CD3 receptor modulation in Jurkat leukemic cell line

CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low) (217.6), CD3+(217.9) or CD3(low) (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.     

Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.     

Early stimulation by EGF plus insulin of rRNA, c-fos, and actin mRNA expression: Inhibition by cytochalasin D

Stimulation of membrane ruffling is one of the first events induced by addition of growth factors to quiescent cultures. In order to assess the importance of intact cytoskeleton in induction, by EGF + insulin, of early events such as stimulation of rRNA, c-fos, and actin mRNA expression, we studied the effect of cytochalasin D (CD) on these metabolisms. We observed that CD slightly increased rRNA synthesis in nonstimulated cells; conversely, it decreased rRNA synthesis in cells stimulated by EGF + insulin. The maximal inhibition observed was 60%. The c-fos mRNA was not expressed in control cells and was accumulated in cells stimulated by the mixture of EGF + insulin; this accumulation was inhibited by CD. Actin mRNA was expressed in control cells and its expression was stimulated by EGF + insulin. Addition of CD decreased actin mRNA accumulation in stimulated cells but increased this accumulation in unstimulated cells. Our results, taken together, show that CD specifically affected the stimulation of rRNA and mRNA expression induced by growth factors and suggest that intact cytoskeleton and possibly membrane ruffling favored this stimulation.     

Early activation events render T cells susceptible to HIV-1-induced syncytia formation. Role of protein kinase C.

In human immunodeficiency virus-1 (HIV-1)-infected cell cultures, cell-to-cell fusion and the formation of multinucleated giant cells (syncytia) are induced as a consequence of interactions between the viral envelope glycoprotein on infected cells and cell surface CD4 molecules on uninfected cells. Although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein expressing (env+) cells, freshly isolated, unstimulated CD4+ T cells do so more slowly. In these studies, we sought to explore the role of T cell activation in rendering CD4+ T cells susceptible to HIV-1-mediated syncytia formation. Our results indicate that within 2 h of exposure to immunologic stimuli, CD4+ T cells acquire the ability to form syncytia with HIV-1 env+ cells. Both cholera toxin, an inhibitor of protein kinase C (PKC) through its effects on inositol triphosphate and diacylglycerol production, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a noncompetitive inhibitor (with respect to ATP) of PKC, prevented unstimulated but not previously stimulated CD4+ T cells from forming syncytia with HIV-1 env+ cells. 1-Oleoyl-2-acetyl glycerol, an analog of the PKC activator, diacylglycerol, enhanced syncytia formation whereas ionomycin, a calcium ionophore, had no effect. These results suggest that activation of PKC is essential for previously unstimulated CD4+ T cells to become fusogenic.     

Accessory cell function of airway epithelial cells

Accessory cell function of airway epithelial cells. We previously demonstrated that airway epithelial cells (AECs) have many features of accessory cells, including expression of class II molecules CD80 and CD86 and functional Fcgamma receptors. We have extended these studies to show that freshly isolated AECs have mRNA for cathepsins S, V, and H [proteases important in antigen (Ag) presentation], invariant chain, human leukocyte antigen (HLA)-DM-alpha and HLA-DM-beta, and CLIP, an invariant chain breakdown product. A physiologically relevant Ag, ragweed, was colocalized with HLA-DR in AECs, and its uptake was increased by granulocyte-macrophage colony-stimulating factor and IFN-gamma treatments, which had no effect on CD80 and CD86 expression. We demonstrate the presence of other costimulatory molecules, including B7h and B7-H1, on AECs and the increased expression of B7-H1 on AECs after treatment with granulocyte-macrophage colony-stimulating factor and IFN-gamma. Finally, we compared T cell proliferation after allostimulation with AECs and dendritic cells (DCs). The precursor frequency of peripheral blood T cells responding to AECs was 0.264% compared with 0.55% for DCs. DCs stimulated CD45RO(+), CD45RA(+), CCR7(+) and CCR7(-)CD4(+), and CD8(+) T cells, whereas AECs stimulated only CD45RO(+), CD45RA(-), CCR7(-), CD4(+), and CD8(+) T cells. There was no difference in cytokine production, type of memory T cells stimulated (effector vs. long-term memory), or apoptosis by T cells cocultured with AECs and DCs. The localization of AECs exposed to the external environment may make them important in the regulation of local immune responses.     

Trichostatin A improved epigenetic modifications of transfected cells but did not improve subsequent cloned embryo development

Reprogramming impairment of DNA methylation may be partly responsible for the low efficiency in somatic cell nuclear transfer. In this study, bovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was dose dependent. When the TSA concentration was over 5 ng/ml, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100 ng/ml (P < 0.01). The number of cells in the S phase was significantly decreased in the 5- to 50-ng/ml TSA-treated groups, while the majority of the cells were at the G0/G1 phases. The number of eGFP-expressed cells were approximately twofold higher in 25-ng/ml (30.5%) and 50-ng/ml (29.5%) TSA groups than the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50 ng/ml of TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50 ng/ml of TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the putative blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation, and eGFP gene expression was activated. Donor cells with reduced DNA methylation did not improve subsequent cloned embryo development; however, transgene expression was improved in cloned embryos.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

Trichostatin A-histone deacetylase inhibitor with clinical therapeutic potential-is also a selective and potent inhibitor of gelatinase A expression

Modulation of histone acetylation is currently being explored as a therapeutic strategy in treatment of cancer. Specifically, inhibition of histone deacetylase by trichostatin A (TSA) has been shown to prevent tumorigenesis and metastasis. In the present paper we demonstrate that increased histone acetylation by TSA-treated 3T3 cells decreases mRNA as well as zymographic activity of gelatinase A, a matrix metalloproteinase, which is itself, implicated in tumorigenesis and metastasis. Furthermore, TSA inhibits cytochalasin D-induced activation of gelatinase A, but TSA does not affect other members of the gelatinase A activation complex, MT1-MMP and TIMP-2. Thus, TSA is a selective and potent inhibitor of expression and activation of gelatinase A. This finding not only strengthens the rationale for continuing to investigate the therapeutic utility of TSA in cancer, but also, provides evidence that TSA inhibition of gelatinase A expression and activation can be used as a biological marker to monitor and determine end-points of clinical trials involving TSA.     

The CD7(-) subset of CD4(+) memory T cells is prone to accelerated apoptosis that is prevented by interleukin-15 (IL-15)

The CD7(-) subset of CD4(+) T cells reflects a stable differentiation state of post-thymic helper T cells with CD45R0(+)CD45RA(-) 'memory' phenotype. Here we report that CD4(+)CD7(-) T cells are prone to increased spontaneous apoptosis in vitro compared to CD4(+)CD7(+) T cells. Spontaneous apoptosis is prevented by IL-15, but not by IL-2. Moreover, IL-15 increases Bcl-2 and decreases CD95/Fas expression of CD7(-), but not of CD7(+) T cells. Because IL-15 is physiologically not secreted but expressed in a membrane-bound form, we cocultured T cells with TNF-alpha stimulated fibroblasts that expose membrane IL-15. TNF-alpha stimulated fibroblasts rescue CD4(+)CD7(-) T cells from apoptosis whereas unstimulated fibroblasts do not. Rescue from apoptosis requires cell-cell contact and is abolished by addition of neutralizing antibodies to IL-15. We conclude that membrane IL-15 prevents accelerated apoptosis of CD4(+)CD7(-) T cells. This mechanism may contribute to accumulation of CD7(-) T cells in chronic inflammatory skin lesions.     

Inhibition of telomerase activity and human telomerase reverse transcriptase gene expression by histone deacetylase inhibitor in human brain cancer cells

The aim of the present study is to investigate the effect of histone deacetylase inhibitor, trichostatin A (TSA) on the cell growth, apoptosis, genomic DNA damage and the expression of telomerase and associated factors in human normal and brain cancer cells. Here, human normal un-transformed fibroblasts (MRC-5), human normal hTERT-immortalised fibroblasts (hTERT-BJ1) and human brain cancer cell lines (glioblastoma cell line, A-172 and medulloblastoma cell line, ONS-76) were treated with 0.5–3.0 μM TSA for 24 h. Exposure to TSA resulted in apoptosis in a dose-dependent manner in the brain cancer cells. Glioblastoma cell line (A-172) displayed higher sensitivity to TSA-induced cell killing effect and apoptosis than the medulloblastoma cell line (ONS-76). The brain cancer cell lines and hTERT-BJ1 cell line displayed significant inhibition in telomerase activity and hTERT mRNA level after 2 μM TSA treatment. Elevated expressions of p53 and p21 with a decrease in cyclin-D level supported the observation on cell cycle arrest following TSA treatment. Upregulation of Bax and cytochrome c correlated with the apoptotic events in TSA-treated cells. This study suggests that telomerase and hTERT might be the primary targets of TSA which may have the potential to be used as a telomerase inhibitor in cancer therapy.     

Trichostatin A Improved Epigenetic Modifications of Transfected Cells but did not Improve Subsequent Cloned Embryo Development

Reprogramming impairment of DNA methylation may be partly responsible for the low efficiency in somatic cell nuclear transfer. In this study, bovine fibroblast cells were transfected with enhancer green fluorescence protein (eGFP), and then treated with a histone-deacetylase inhibitor, trichostatin A (TSA). The results showed that the effect of TSA on transfected cells was dose dependent. When the TSA concentration was over 5 ng/ml, cell proliferation was significantly inhibited. The majority of the cells died when TSA reached 100 ng/ml (P < 0.01). The number of cells in the S phase was significantly decreased in the 5- to 50-ng/ml TSA-treated groups, while the majority of the cells were at the G0/G1 phases. The number of eGFP-expressed cells were approximately twofold higher in 25-ng/ml (30.5%) and 50-ng/ml (29.5%) TSA groups than the control (15.0%). Reduced DNA methylation and improved histone acetylation were observed when the cells were treated with 10 to 50 ng/ml of TSA. Transfer of the TSA-treated cells to enucleated recipient oocytes resulted in similar cleavage rates among the experimental groups and the control. Cells treated with 50 ng/ml of TSA resulted in significantly lower blastocyst development (9.9%) than the other experimental and the control groups (around 20%). Analysis of the putative blastocysts showed that 86.7% of the embryos derived from TSA-treated cells were eGFP positive, which was higher than that from untreated cells (68.8%). In conclusion, treatment of transfected cells with TSA decreased the genome DNA methylation level, increased histone acetylation, and eGFP gene expression was activated. Donor cells with reduced DNA methylation did not improve subsequent cloned embryo development; however, transgene expression was improved in cloned embryos.