Cellular-FLIP, Raji isoform (c-FLIP R) modulates cell death induction upon T-cell activation and infection.

Dysregulation of apoptosis caused by an imbalance of pro- and anti-apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular-FLIP (c-FLIP) proteins inhibit apoptosis directly at the death-inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c-FLIPL and c-FLIPS are well characterized, the function of c-FLIPR remains poorly understood. Here, we demonstrate the induction of endogenous murine c-FLIPR in activated lymphocytes for the first time. To analyze c-FLIPR function in vivo, we generated transgenic mice expressing murine c-FLIPR specifically in hematopoietic cells. As expected, lymphocytes from c-FLIPR transgenic mice were protected against CD95-induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T-cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c-FLIPR transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild-type mice. We conclude that c-FLIPR expression in hematopoietic cells supports an efficient immune response against bacterial infections.

A single nucleotide polymorphism determines protein isoform production of the human c-FLIP protein.

The cellular FLICE-inhibitory protein (c-FLIP) is a modulator of death receptor-mediated apoptosis and plays a major role in T- and B-cell homeostasis. Three different isoforms have been described on the protein level, including the long form c-FLIP(L) as well as 2 short forms, c-FLIP(S) and the recently identified c-FLIP(R). The mechanisms controlling c-FLIP isoform production are largely unknown. Here, we identified by sequence comparison in several mammals that c-FLIP(R) and not the widely studied c-FLIP(S) is the evolutionary ancestral short c-FLIP protein. Unexpectedly, the decision for production of either c-FLIP(S) or c-FLIP(R) in humans is defined by a single nucleotide polymorphism in a 3' splice site of the c-FLIP gene (rs10190751A/G). Whereas an intact splice site directs production of c-FLIP(S), the splice-dead variant causes production of c-FLIP(R). Interestingly, due to differences in protein translation rates, higher amounts of c-FLIP(S) protein compared with c-FLIP(R) are produced. Investigation of diverse human cell lines points to an increased frequency of c-FLIP(R) in transformed B-cell lines. A comparison of 183 patients with follicular lymphoma and 233 population controls revealed an increased lymphoma risk associated with the rs10190751 A genotype causing c-FLIP(R) expression.

Up-regulation of c-FLIP short by NFAT contributes to apoptosis resistance of short-term activated T cells.

Upon encounter with pathogens, T cells activate several defense mechanisms, one of which is the up-regulation of CD95 ligand (CD95L/FasL) which induces apoptosis in sensitive target cells. Despite expression of the CD95 receptor, however, recently activated T cells are resistant to CD95L, presumably due to an increased expression of antiapoptotic molecules. We show here that, in contrast to naive or long-term activated T cells, short-term activated T cells strongly up-regulate the caspase-8 inhibitor, cellular FLICE-inhibitory protein (c-FLIP). Intriguingly, upon activation, T cells highly induced the short splice variant c-FLIP(short), whereas expression of c-FLIP(long) was only marginally modulated. In contrast to the general view that c-FLIP transcription is controlled predominantly by nuclear factor-kappaB (NF-kappaB), induction of c-FLIP(short) in T cells was primarily mediated by the calcineurin-nuclear factor of activated T cells (NFAT) pathway. Importantly, blockage of NFAT-mediated c-FLIP expression by RNA interference or inhibition of calcineurin rendered T cells sensitive toward CD95L, as well as activation-induced apoptosis. Thus, the resistance of recently activated T cells depends crucially on induction of c-FLIP expression by the calcineurin/NFAT pathway. Our findings imply that preventing autocrine CD95L signaling by c-FLIP facilitates T-cell effector function and an efficient immune response.

Loss of caspase-9 provides genetic evidence for the type I/II concept of CD95-mediated apoptosis.

The death receptor CD95 triggers apoptosis upon formation of a death-inducing signaling complex and the activation of caspase-8. Two types of CD95-mediated apoptosis have been distinguished that differ in their efficiency of death-inducing signaling complex formation and the requirement of mitochondria for caspase activation. The validity of the type I/II model, however, has been challenged, as Bcl-2 expression or the use of various CD95 agonists resulted in different apoptosis effects. By identifying a caspase-9-deficient T cell line, we now provide genetic evidence for the two-pathway model of CD95-mediated apoptosis and demonstrate that type II cells strongly depend on caspase-9. Caspase-9-deficient cells revealed strongly impaired apoptosis, caspase activation, and mitochondrial membrane depolarization upon CD95 triggering, whereas, surprisingly, activation of Bak and cytochrome c release were not inhibited. Furthermore, caspase-9-deficient cells did not switch to necrosis, and reconstitution of caspase-9 expression restored CD95 sensitivity. Finally, we also show that different death receptors have a distinct requirement for caspase-9.