c-FLIP(S) reduces activation of caspase and NF-kappaB pathways and decreases T cell survival.

Effective stimulation of NF-kappaB in T cells following TCR ligation requires the activity of caspase-8. The active caspase-8 complex includes the paracaspase, MALT1, and Bcl-10, which connect to the NF-kappaB pathway. It has been less clear what regulates the level of caspase-8 activity during T cell activation. A likely candidate is cellular FLIP (c-FLIP), an enzymatically inert caspase-8 homologue. Two alternatively spliced forms of c-FLIP exist, a long form (c-FLIP(L)) and a short-form (c-FLIP(S)). The latter lacks the C-terminal caspase-like domain. c-FLIP(L) can heterodimerize with and activate caspase-8 through an activation loop in the C terminus of c-FLIP(L). Here we show that, in contrast to c-FLIP(L), c-FLIP(S) inhibits activation of caspase-8 in T cells, and consequently reduces recruitment of MALT1 and Bcl-10 to the active caspase complex. This results in reduced activity of NF-kappaB. Consequently, T cells from c-FLIP(S)-transgenic mice undergo more rapid cell death both spontaneously and after activation. The findings suggest that c-FLIP(S) functions to reduce the expansion of T cells during an immune response.

Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation.

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.