2B4 Cells Research Articles

Nitric Oxide Synthase Plays a Signaling Role in TCR-Triggered Apoptotic Death

A functional role for stimulated nitric oxide (NO) production was tested in the TCR-triggered death of mature T lymphocytes. In purified peripheral human T cell blasts or the 2B4 murine T cell hybridoma, apoptotic cell death induced by immobilized anti-CD3 was blocked by inhibitors of NO synthase (NOS) in a stereospecific and concentration-dependent manner. This effect appeared to be selective since apoptotic death induced by anti-Fas Ab or the steroid dexamethasone was not affected by NOS inhibitors. TCR-stimulated expression of functional Fas ligand was attenuated in a stereospecific manner by NOS inhibitors, but these compounds did not inhibit TCR-stimulated IL-2 secretion or CD69 surface expression. Nitrosylated tyrosines, a stable marker for NO generation, were immunochemically detected in T cells using flow cytometry. TCR signals induced NO production, as measured by an increase in nitrotyrosine-specific staining. NOS enzymatic activity was detected in lysates of 2B4 cells, and Western blot analysis suggests that the activity is due to expression of the neuronal isoform of NOS. Thus, T cells have the capacity to generate NO upon Ag signaling, which may affect signal transduction, Fas ligand surface expression, and apoptotic cell death of mature T lymphocytes.

Regulation of Fas-dependent activation-induced T cell apoptosis by cAMP signaling: a potential role for transcription factor NF-kappa B.

TCR-mediated activation of T cell hybridomas induces programmed cell death by a Fas-dependent pathway. We now show that costimulation of 2B4 cells, in the absence or presence of transgenic Bcl-2, with anti-CD3 epsilon and forskolin, an activator of cAMP signaling, resulted in antagonism of Fas-dependent activation-induced cell death that was always accompanied by selective downregulation of the nuclear levels of NF-kappa B p65-p50 (RelA-p50) transcription factor. Forskolin not only inhibited activation-induced cell death and NF-kappa B activation, but also suppressed expression of Fas and Fas ligand (Fas-L). Furthermore, NF-kappa B p65 antisense oligonucleotide down-regulated nuclear levels of NF-kappa B, inhibited cell surface expression of Fas-L and apoptosis of 2B4. Collectively, these finding demonstrate a potential role of NF-kappa B in the regulation of activation-induced apoptosis in T lymphocytes.

Role of reactive oxygen intermediates in TCR-induced death of T cell blasts and hybridomas.

The functional role of reactive oxygen intermediates (ROI) in activation-induced death of mature T lymphocytes and hybridomas was tested using antioxidants and inhibitors of enzymes that generate oxidants. These agents were shown to inhibit TCR-triggered death in a concentration-dependent manner, suggesting a possible role of ROI in this death. Since the TCR-induced death of both human T blasts and the murine T cell hybridoma 2B4 involve an initial step of TCR-induced Fas ligand (FasL) up-regulation followed by a lethal step induced by Fas cross-linking, both steps were examined separately for ROI dependence. The thiol antioxidants N-acetyl cysteine and glutathione blocked Fas-induced death triggered via cross-linking either by IgM anti-Fas or cell-bound FasL, while the other inhibitors of activation-induced death did not block this late lethal step. None of the agents used blocked early events after TCR ligation, as seen by the lack of inhibition of IL-2 secretion or CD69 up-regulation. However, the nonthiol agents that blocked activation-induced death all blocked FasL up-regulation induced by TCR signals in the hybridoma, as measured by a functional assay. Agents inhibiting FasL up-regulation also inhibited activation-induced ROI generation in the hybridoma, as detected by flow cytometry using dihydrorhodamine oxidation. Furthermore, a good correlation was found between the extent of ROI generation and functional FasL expression in 2B4 cells. Thus, while ROI do not appear to act as downstream mediators of apoptotic death induced by steroid or Fas cross-linking in T cells, they are generated by TCR signaling and appear to participate in FasL up-regulation.

Bcl-2 blocks glucocorticoid- but not Fas- or activation-induced apoptosis in a T cell hybridoma.

Overexpression of Bcl-2 can prevent or markedly delay cell death induced by a variety of apoptotic stimuli. Although Fas and Fas ligand (FasL) interactions play a major role in the elimination of self-reactive T cells in the periphery, inhibition of Fas-mediated killing by Bcl-2 has not been consistently observed. The mouse T hybridoma 2B4.11 (2B4) has been a useful model to study glucocorticoid- and activation-induced apoptosis, which is mediated through Fas and FasL. Using both stable transfectants and transient transfections, overexpression of Bcl-2 or Bcl-xL readily blocked glucocorticoid-induced but not activation-induced apoptosis of 2B4 cells. Bcl-2 expression did not inhibit Fas-mediated cytotoxicity triggered by cells expressing FasL or by the transient transfection of human Fas. Similarly, overexpression of Bcl-2 in the mouse T hybridoma A1.1 did not block activation-induced/Fas-mediated apoptosis. In Jurkat cells, however, expression of Bcl-2 partially inhibited anti-Fas-induced cell death. A Bcl-2-related protein that can interfere with anti-Fas killing, the adenoviral E1B 19K, also did not block activation-induced/Fas-mediated apoptosis in 2B4 cells. In contrast, expression of CrmA, a cowpox virus protein that inhibits ICE-like protease activity, blocked activation-induced apoptosis in 2B4 cells but had little effect on Dex-mediated cytotoxicity. These results show that: 1) Bcl-2 can have strikingly different anti-cell death activity in the same cell depending upon the apoptotic stimulus, 2) distinct apoptosis signaling pathways may exist with differential sensitivity to Bcl-2 and ICE-like protease inhibitors.

Protease inhibitors selectively block T cell receptor-triggered programmed cell death in a murine T cell hybridoma and activated peripheral T cells.

The hypothesis that cytoplasmic proteases play a functional role in programmed cell death was tested by examining the effect of protease inhibitors on the T cell receptor-mediated death of the 2B4 murine T cell hybridoma and activated T cells. The cysteine protease inhibitors trans-epoxysuccininyl-L-leucylamido-(4-guanidino) butane (E-64) and leupeptin, the calpain selective inhibitor acetyl-leucyl-leucyl-normethional, and the serine protease inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, all showed dose-dependent blocking of the 2B4 death response triggered by the T cell receptor complex and by anti-Thy-1. These protease inhibitors enhanced rather than inhibited IL-2 secretion triggered by T cell receptor cross-linking, showing that they did not act by preventing signal transduction. Growth inhibition induced by cross-linking the 2B4 T cell receptor, measured by inhibition of thymidine incorporation, was not generally blocked by these protease inhibitors. All five of these protease inhibitors enhanced rather than blocked 2B4 cell death triggered by dexamethasone, an agent previously shown to have a death pathway antagonistic with that of the TCR. 2B4 cytolysis by the cytotoxic agents staphylococcal alpha-toxin and dodecyl imidazole, and that caused by hypotonic conditions, was not significantly affected by the five protease inhibitors tested. The selected protease inhibitors blocked both the apoptotic nuclear morphology changes and DNA fragmentation induced by T cell receptor cross-linking, and enhanced both these properties induced by dexamethasone in 2B4 cells. The T cell receptor-induced death of activated murine lymph node T cells and human peripheral blood CD4+ T cells was blocked by both cysteine and serine protease inhibitors, showing that the protease-dependent death pathway also operates in these systems.

T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway.

The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.

Characterization of the Antigen‐Specific T Cell Receptor from a Pigeon Cytochrome c‐Specific T Cell Hybrid

The monoclonal antibody, A2B4-2, specifically bound to and inhibited antigen-induced IL-2 release from the cloned pigeon cytochrome c-specific T cell hybrid, 2B4. The initial immunoprecipitation with these reagents demonstrated a protein of 85-90kd on non-reducing conditions, and two bands of 45-50 and 40-44 on reducing conditions. These data enabled us to conclude that this monoclonal antibody bound to the antigen receptor on the 2B4 cell. In this paper we have presented results that further define the receptor structure. The migration pattern of the protein on SDS-PAGE in the presence and absence of reducing agents was consistent with the interpretation that the receptor has intrachain disulfide bonds that create globular domains. Sequence data from the recently described cDNA clones that encode receptor genes confirm that there are cysteine residues in positions that could be involved in such disulfide bonding. Since both alpha and beta chains behave identically on the SDS-PAGE we predict that both chains have the same double domain structure. The antigen receptor has a heterodimeric structure. A relatively simple acidic alpha chain is bound to one of two forms of the beta chain. Some of the receptors have a beta chain equal in molecular weight to the alpha chain, while some of the beta chains (beta') are heavier and more acidic. The difference in beta chain forms appears to be due to different levels of glycosylation of this chain. The cDNA sequence data demonstrate that there are several possible carbohydrate addition sites on the protein encoded by this gene so it may be that 2B4 beta chains are present that are either completely or partially glycosylated at these sites. Finally, we have presented a preliminary experiment that indicates that a smaller 20-25kd molecule is associated with the antigen receptor. The well characterized T3 molecule appears to be in a complex with the clonotypic structure on human T cells. The crosslinking data that we present suggests that a similar molecule may be present in the murine system.

T cell clone-specific alloantisera that inhibit or stimulate antigen-induced T cell activation.

Pigeon cytochrome c-specific, IL 2-secreting T cell hybrids have been used for immunizations to generate alloantibodies against the T cell antigen receptor on these cells. The B10.A-derived cloned T cell hybrid 2B4 was emulsified in complete Freund's adjuvant and injected i.p. into several F1 strains of mice. After boosting the recipient animals with cells in incomplete Freund's adjuvant, malignant ascites developed. In the (BALB/c X AKR)F1 or (AKR X BALB/c)F1 strains, these ascites consistently contained material that specifically inhibited the antigen-induced IL 2 secretion of only the immunizing 2B4 cell. The inhibitory material was antibody that specifically bound and could be absorbed only by 2B4. A similar immunization was performed with the cell 2C2. Although this cell apparently has similar antigenic fine specificity as 2B4, high concentrations of ascites generated by 2C2 immune animals inhibited antigen-induced IL 2 release only from 2C2. At lower concentrations of ascites, this preparation synergized with antigen to increase the IL 2 release, again only from 2C2. This antibody preparation did not affect concanavalin A-induced IL 2 release. The most likely explanation for these data is that the ascites contain antibodies that react with the antigen-specific receptor on the T cell hybrids.