Pretreatment Of Jurkat Cells Research Articles

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells.

The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.

Immunomodulatory effects of therapeutic gold compounds. Gold sodium thiomalate inhibits the activity of T cell protein kinase C.

Previous studies have shown that the gold compounds, gold sodium thiomalate (GST) and auranofin (AUR), which are effective in the treatment of rheumatoid arthritis, inhibit functional activities of a variety of cells, but the biochemical basis of their effect is unknown. In the current studies, human T cell proliferation and interleukin 2 production by Jurkat cells were inhibited by GST or AUR at pharmacologically relevant concentrations. Because it has been documented that protein kinase C (PKC) is involved in T cell activation, the capacity of gold compounds to inhibit PKC partially purified from Jurkat cells was assayed in vitro. GST was found to inhibit PKC in a dose-dependent manner, but AUR caused no significant inhibition of PKC at pharmacologically relevant concentrations. The inhibitory effect of GST on PKC was abolished by 2-mercaptoethanol. To investigate the effect of GST on the regulation of PKC in vivo, the levels of PKC activity in Jurkat cells were examined. Cytosolic PKC activity decreased slowly in a concentration- and time-dependent manner as a result of incubation of Jurkat cells with GST. To ascertain whether GST inhibited PKC translocation and down-regulation, PKC activities associated with the membrane and cystosolic fractions were evaluated after phorbol myristate acetate (PMA) stimulation of GST incubated Jurkat cells. Translocation of PKC was markedly inhibited by pretreatment of Jurkat cells with GST for 3 d, but the capacity of PMA to down-regulate PKC activity in Jurkat cells was not altered by GST preincubation. The functional impact of GST-mediated downregulation of PKC in Jurkat cells was examined by analyzing PMA-stimulated phosphorylation of CD3. Although GST preincubated Jurkat cells exhibited an increased density of CD3, PMA-stimulated phosphorylation of the gamma chain of CD3 was markedly inhibited. Specificity for the inhibitory effect of GST on PKC was suggested by the finding that GST did not alter the mitogen-induced increases in inositol trisphosphate levels in Jurkat cells. Finally, the mechanism of the GST-induced inhibition of PKC was examined in detail, using purified PKC subspecies from rat brain. GST inhibited type II PKC more effectively than type III PKC, and also inhibited the enzymatic activity of the isolated catalytic fragment of PKC. The inhibitory effect of GST on PKC activity could not be explained by competition with phospholipid or nonspecific interference with the substrate. These data suggest that the immunomodulatory effects of GST may result from its capacity to inhibit PKC activity.

Cholera toxin modulates the T cell antigen receptor/CD3 complex but not the CD2 molecule and inhibits signaling via both receptor structures in the human T cell lymphoma Jurkat.

The human T cell lymphoma Jurkat can be activated by stimuli directed either against the T cell antigen receptor-CD3 antigen complex (TcR/CD3) or the CD2 molecule. Stimulation of cells via the TcR/CD3-complex or via the CD2 molecule increases inositol phosphates and cytoplasmic free calcium. Pretreatment of Jurkat cells with cholera toxin leads to a decrease of TcR/CD3 expression on the surface of the cells, while the expression of CD2 is unaffected. In contrast to this distinct effect on the receptor expression, signaling via both pathways is inhibited by cholera toxin. The most convincing explanation for the cholera toxin-mediated inhibition of signaling is that cholera toxin interrupts the signaling pathways at a point where both, stimulation via TcR/CD3 and via CD2, use the same route. The earliest common point of the two signaling pathways, at least in the Jurkat cell line, seems to be the CD3 complex because after its down-regulation (and functional inactivation) both pathways of activation are interrupted.