Death receptor-mediated apoptosis in human malignant glioma cells: modulation by the CD40/CD40L system.

CD40, a TNF-R-related cell surface receptor, is shown here to be expressed by glioma cells in vitro and in vivo. Glioma cell lines expressing low levels of CD40 at the cell surface resist cytotoxic effects of CD40L. CD40 gene transfer sensitizes glioma cells to CD40L. Inhibition of protein synthesis potentiates cell death which involves CD40 clustering and caspases 8 and 3 processing. CD40-transfected LN-18 cells acquire resistance to CD95L. In contrast, subtoxic concentrations of CD40L strongly sensitize these cells for TNF-alpha-induced apoptosis. Bispecific CD40xCD95 antibodies specifically kill glioma cells, disclosing the property of endogenous CD40 to facilitate death signalling.

Functional T-cell anergy in a case of persistent polyclonal B-cell lymphocytosis.

The T-cell population of a patient with persistent polyclonal B-cell lymphocytosis (PPBL) presenting with an intermittent Epstein-Barr virus (EBV)-associated disease was studied. Unstimulated T-cells did not express CD40 ligand (CD40L), whereas activation with IL-2 led to expression of this costimulatory molecule. CD40L expression was inhibited upon incubation with the supernatant of an EBV-positive B-cell line (SM) which had been grown spontaneously from the patient's peripheral blood cells. The supernatant of SM cells effectively inhibited cytotoxic T-cells. Elevated levels of IL-10, TNF-alpha and soluble CD40 were found in the supernatant of SM cells. Additionally, enhanced levels of LMP-1 protein were detected.

Purification of TNF binding proteins.

The finding that the two tumor necrosis factor receptors (TNFR) exist in soluble form in various body fluids not only has substantiated the paradigm of naturally existing soluble cytokine receptors but also has represented a milestone on the road to the biochemical and biological characterization of the two TNFRs. This chapter gives a simple, basic protocol for the purification of the two soluble TNFRs. The protocols found here may be easily adapted for the purification of various other soluble cytokine receptors. The purified proteins may be used in biological experiments or for the generation of specific research tools such as polyclonal or monoclonal antibodies.

TNF receptor (TNFR)-associated factor (TRAF) 3 serves as an inhibitor of TRAF2/5-mediated activation of the noncanonical NF-kappaB pathway by TRAF-binding TNFRs.

TNF family members and their receptors contribute to increased gene expression for inflammatory processes and intracellular cascades leading to programmed cell death, both via activation of NF-kappaB. TNF receptor (TNFR)-associated factors (TRAFs) are cytoplasmic adaptor proteins binding to various receptors of the TNFR family. In an attempt to delineate the role of individual TRAFs, we compared NF-kappaB activation by CD40(wt) and CD40 mutants with different TRAF recruitment patterns. Recognized only recently, NF-kappaB signaling occurs at least via two different pathways. Each pathway results in nuclear translocation of two different Reldimers, the canonical p50/RelA and the noncanonical p52/RelB. Here, we show that via TRAF6, CD40 mediates only the activation of the canonical NF-kappaB pathway. Via TRAF2/5, CD40 activates both the canonical and the noncanonical NF-kappaB pathways. We observed that TRAF3 specifically blocked the NF-kappaB activation via TRAF2/5. This inhibitory effect of TRAF3 depends on the presence of an intact zinc finger domain. Paradoxically, suppression of TRAF2/5-mediated NF-kappaB activation by TRAF3 resulted in enhanced transcriptional activity of TRAF6-mediated canonical NF-kappaB emanating from CD40. We also observed that 12 TNFR family members (p75TNFR, LTbetaR, RANK, HVEM, CD40, CD30, CD27, 4-1BB, GITR, BCMA, OX40, and TACI) are each capable of activating the alternative NF-kappaB pathway and conclude that TRAF3 serves as a negative regulator of this pathway for all tested receptors.

Two distinct transport motifs in the adenovirus E3/10.4-14.5 proteins act in concert to down-modulate apoptosis receptors and the epidermal growth factor receptor.

The adenovirus (Ad) early transcription unit E3 encodes immunosubversive functions. The E3 transmembrane proteins 10.4 and 14.5 form a complex that down-regulates the epidermal growth factor receptor and apoptosis receptors from the cell surface by diverting them to endosomes/lysosomes for degradation. The latter process protects infected cells from ligand-induced apoptosis. The mechanism by which 10.4-14.5 mediate re-routing remains elusive. We examined the role of putative YXX Phi and dileucine (LL) transport motifs within Ad2 10.4-14.5 for target protein modulation. By generating stable E3 transfectants expressing 10.4-14.5 proteins with alanine substitutions in these motifs, we show that 3 of the 5 motifs are essential for functional activity. Whereas tyrosine 74 in 14.5 appears to be important for efficient 10.4-14.5 interaction, the 122YXX Phi motif in 14.5 and the dileucine motif Leu 87-Leu88 in 10.4 constitute genuine transport motifs: disruption of either motif abolished binding to the cellular adaptor proteins AP-1 and AP-2, as shown by surface plasmon resonance spectroscopy, and caused missorting, dramatically altering cell surface appearance and the intracellular location of viral proteins. Fluorescence-activated cell sorter analysis and immunofluorescence data provide evidence that Tyr122 in 14.5 is essential for rapid endocytosis of the 10.4-14.5 complex, whereas the 10.4LL motif acts down-stream and protects 10.4-14.5 from extensive degradation by rerouting it into a recycling pathway. Infection of primary cells with adenoviruses carrying the relevant point mutations confirmed the crucial role of these transport motifs for down-regulation of Fas, TRAIL-R1, TRAIL-R2, and epidermal growth factor receptor. Thus, two distinct transport motifs present in two proteins synergize for efficient target removal and immune evasion.