The death of a T-cell: expression of the CD95 ligand

CD95 (APO-1, Fas) is a member of the tumor necrosis factor (TNF) receptor superfamily expressed in various tissues; however, expression of its ligand CD95L, a type II transmembrane protein of the TNF family, is restricted to a few cell types, such as T cells, macrophages, and cells of immune-privileged tissues like testis and eye. In lymphoid tissues CD95 is constitutively expressed; by contrast, CD95L is not present in resting T cells, but is highly expressed upon T-cell activation. The binding of CD95L to CD95 results in rapid apoptosis. Many features of apoptosis in T cells induced by the CD95/CD95L system have been worked out: CD95 mediated apoptosis is now known to be a mechanism for maintenance of peripheral tolerance and for termination of an ongoing immune response.^1,2 CD95/CD95L interaction also initiates cell death in many other systems, and its deregulation leads to severe diseases.^1,2 Thus, to explain the pathophysiology of these diseases and to identify potential therapeutic targets it is important to understand the molecular mechanisms of CD95L activation.

Activation of CD95L via the Ca²⁺/calcineurin signaling pathway

The earliest signaling events upon T-cell receptor (TCR) engagement are the sequential activation of tyrosine kinases (TPKs) including Lck and ZAP-70. ZAP-70 and Lck are shown to be involved in up-regulation of CD95L expression in activation-induced apoptosis.^3,4 Both Lck and ZAP-70 are required for calcium mobilization in T cells. Increase in [Ca²⁺]_i leads to activation of the Ca²⁺- and calmodulin-dependent phosphatase calcineurin and, consequently, activates calcineurin-regulated transcription factors (Figure 1). CD95L mRNA expression and activation-induced-cell-death (AICD) in T cells can be blocked by the immunosuppressive drug Cyclosporin A (CsA), an inhibitor of calcineurin. This finding has directed the search for CsA-sensitive transcription factors involved in CD95L activation. NF-AT proteins are major targets of CsA. In resting T cells, NF-AT proteins are phosphorylated, have low affinity for DNA and reside in the cytoplasm. Activation of T cells through the TCR leads to calcineurin activation and results in rapid dephosphorylation of NF-AT proteins and their translocation to the nucleus. The dephosphorylated NF-AT proteins exert increased affinity for DNA. CsA blocks calcineurin-dependent dephosphorylation and prevents nuclear translocation of NF-AT. The first functional NF-AT binding site was identified at the −180 region of the human CD95L promoter and was shown to confer CsA-sensitive transcription to CD95L^5,6 (Figure 1). Following the identification of the NF-AT site, a cis-acting motif in the −120 region was found to bind members of the Early Growth Response Gene (Egr1, 2, 3 and 4) family of transcription factors (Figure 1). Among these factors, Egr2 and Egr3 were proposed to confer most of the activation-induced expression to CD95L.^7,8 Although Egr factors activate transcription in a CsA-insensitive manner, Egr2 and Egr3 themselves are NF-AT target genes and their expression is sensitive to CsA^7,8 (Figure 1). A recent study proposed that activation of CD95L is sequentially regulated by NF-AT and Egr3 and the effect of NF-AT on CD95L expression is largely indirect acting through its induction of Egr2 and Egr3 transcription.⁹ However, the bulk of studies favors a direct role of NF-AT in CD95L activation.^5,6,10,11,12 In addition, it was found that both the −180 NF-AT and the −120 Egr regions are Egr/NF-AT composite sites for NF-AT and Egr transcription factors.¹¹ Three such Egr/NF-AT composite sites have been identified in the CD95L promoter (−680, −180 and −120) (Figure 1). NF-AT and Egr were shown to display a cooperative and synergistic activation of the CD95L promoter at these three sites.¹¹

Figure 1

A schematic view of signaling pathways and transcription factors that influence CD95L expression. A representation of identified regulatory regions of the human CD95L promoter is also shown

Activation of CD95L via the PKCθ signaling pathway

The protein kinase C (PKC) iso-form PKCθ was found to play an important role in induction of CD95L expression following TCR/CD3 stimulation.¹³ Of the various PKC isoforms present in T cells, PKCθ is essential for TCR-mediated T-cell activation and is required for TCR-induced NF-κB activation in mature T lymphocytes.¹⁴ Normally, NF-κB remains sequestered in an inactive state by the cytoplasmic inhibitor-of-κB (IκB) proteins. T-cell activation leads to phosphorylation, ubiquitinylation, and the subsequent degradation of IκB proteins. The DNA-binding subunits of NF-κB (p65, p50) immigrate into the nucleus and activate expression of target genes (Figure 1). Several studies have indicated that high expression of CD95L in activated T cells requires NF-κB. Two NF-κB binding sites are localized at the −530 and −50 region of the human CD95L promoter¹⁵ (Figure 1). Mutation at each of these sites resulted in 30–70% reduction of promoter activity indicating that NF-κB is required for optimal activation of the CD95L promoter.¹⁵ Another NF-κB binding site has been reported at the −980 region of the CD95L promoter.¹⁶ The −980 NF-κB binding sequence described by this study, however, differs from the DNA sequence described by other groups. Conflicting results have been obtained by mutation or deletion studies of the −980 site that argue against the role of this site in expression of CD95L.¹⁵

AICD is also a major cause of depletion of CD4⁺ T cells in HIV-1 infected individuals. Increased expression of both CD95 and CD95L has been observed in HIV-1 infected individuals. HIV-1 Tat protein is essential for efficient HIV-1 gene expression and has been implicated in sensitization of CD95-mediated apoptosis and in upregulation of CD95L expression in T cells.¹⁷ Ectopic expression of Tat protein in T cells results in enhanced binding of NF-κB to its target DNA.¹⁵ The NF-κB sites of the CD95L promoter may contribute the Tat-enhanced CD95L expression, at least in part.¹⁵

Activation of CD95L via the Ras signaling pathway

Activation of T cells via TCR engagement also leads to a Ras-activated cascade of kinase activity including Raf, MEK, ERK and p38 mitogen-activated protein kinase (MAPK) (Figure 1). It has been shown that the Ras signaling pathway is required for optimal TCR-mediated expression of CD95L.^18,19 The Ras pathway is involved in induction of AP-1 (Fos/Jun) transcription factors. An AP-1 binding site was recently localized in the +90 region of the CD95L promoter²⁰ (Figure 1). This AP-1 site was originally identified to be essential for anti-cancer-drug-induced CD95L expression, and we found that it is also involved in upregulation of CD95L expression in T cells (unpublished data). A distal AP-1 binding site at the −950 region which was shown to respond to DNA-damaging-agent-induced CD95L expression by another group, however, is not required for T-cell activation-induced CD95L promoter activity.¹⁶

The role of constitutive transcription factor SP-1

SP-1 is constitutively present in T cells and preferentially interacts with GC rich DNA sequences. All NF-κB and Egr/NF-AT composite sites contain GC rich DNA sequences and are found to interact with SP-1^10,12,15 (Figure 1). SP-1 has been implicated in maintaining the basal level of CD95L in T cells and in constitutive expression of CD95L in other tissues.

Differential regulation of CD95L expression in Th1 and Th2 cells

During T-cell development, CD4⁺ T-helper (Th) cell differentiate in response to antigen stimulation into different types of Th cells that can be grouped based on their cytokine expression patterns. Th1 cells produce IFN-γ, TNF and IL-2 are important for the cell-mediated immune response against intracellular pathogens. Th2 cells produce IL-4, IL-5, IL-6 and IL-10, and are involved in humoral immunity. Activated Th1 cells are shown to express CD95L and are sensitive to CD95-mediated apoptosis, whereas Th2 cells express less or no CD95L and are resistant to AICD. A recent report by Rengarajan et al proposed that enriched Egr3 expression in Th1 cells and deficient expression of Egr3 in Th2 cells may account for differential regulation of CD95L expression in Th1 and Th2 cells.⁹ However, conflicting results concerning this proposal are reported by another group showing that Egr3 is not expressed in Th1 cells and, instead, is expressed in Th2 cells.¹² Thus, the lack of CD95L expression by Th2 cells is apparently not due to a deficiency of Egr3. Absence of Egr3 in Th1 cells shown in this study further argues against the role of Egr3 in preferential expression of CD95L in Th1 vs Th2 cells. Thus, the molecular basis for the differential expression of CD95L in Th1 and Th2 cells remains unknown.

Taken together, activation-induced CD95L expression in T cells involves multiple transcription factors. NF-AT and Egr proteins seem to play a major role for CD95L expression upon TCR-stimulation. However, other factors may be also important. For example, in HIV-1 infected individuals, NF-κB activity may be enhanced through virus infection and, consequently, may lead to increased CD95L expression and accelerated apoptosis. T-cell apoptosis can also be elicited by other stimuli, which change the oxidation/reduction (redox) status of the cell such as UV irradiation, chemotherapeutic agents, and stress. Activities of NF-kB and AP-1 are known to be largely affected by the cellular redox status. In these cases, NF-κB and AP-1 may exert a major role in induction of the CD95L promoter. Thus, different sets of transcription factors may be used for CD95L expression in T cells in different situations.