The Role of c-FLIP in the Regulation of Apoptosis, Necroptosis and Autophagy in T Lymphocytes

To maintain homeostasis, T lymphocytes die through caspase–dependent apoptosis. However, blockage of caspase activity in T lymphocytes does not increase cell survival. The loss of caspase 8 activity leads to programmed necrosis (necroptosis) upon T cell receptor (TCR) stimulation in T lymphocytes. Necroptosis is correlated with excessive macroautophagy, an intracellular catabolic process characterized by the sequestration of cytoplasmic compartments through double–membrane vacuoles. Meanwhile, the proper induction of macroautophagy is required for T lymphocyte survival and function. Cellular caspase 8 (FLICE)–like inhibitory protein (c–FLIP) promotes survival in T lymphocytes. c–FLIP suppresses death receptor–induced apoptosis by modulating caspase 8 activation. Whether this modulation plays a role in the regulation of necroptosis has yet to be studied. Additionally, overexpression of c–FLIP reduces autophagy induction and promotes cell survival in cell lines. It remains unclear whether c–FLIP protects primary T lymphocytes by regulating the threshold at which autophagy occurs. In this study, c–FLIP isoform–specific conditional deletion models were used to study the role of c–FLIP in necroptosis and autophagy in primary T lymphocytes.

Our results showed that the long isoform of c–FLIP (c– FLIP_L) regulates necroptosis by inhibiting receptor interacting protein 1 (RIP–1). Upon TCR stimulation, c–FLIP_L–deficient T cells underwent RIP–1–dependent necroptosis. Interestingly, though previous studies have generally described necroptosis in the absence of caspase 8 activity and apoptosis, pro–apoptotic caspase 8 activity and the rate of apoptosis were also increased in c–FLIPL–deficient T lymphocytes. Moreover, c– FLIP_L–deficient T cells exhibited enhanced autophagy, which served a cytoprotective function.

Apoptosis can be induced by either death receptors on the plasma membrane (extrinsic pathway), or the damage of the genome and/or cellular organelles (intrinsic pathway). Previous studies in c–FLIP–deficient T lymphocytes suggested that c–FLIP promotes cell survival in the absence of death receptor signals. Independent of death receptor signaling, mitochondria sense apoptotic stimuli and mediate the activation of caspases. Whether c–FLIP regulates mitochondrion–dependent apoptotic signaling remains unknown. Here, by deleting the c–Flip gene in mature T lymphocytes, we showed a role for c–FLIP in the intrinsic apoptosis pathway. In naïve T cells stimulated with the apoptosis inducer, c–FLIP suppressed cytochrome c release from mitochondria. Bim–deletion rescued the enhanced apoptosis in c–FLIP–deficient T cells, while inhibition of caspase 8 did not. Different from activated T cells, there were no signs of necroptosis in c–FLIP–deficient naïve T cells. Together, our findings indicate that c–FLIP is a key regulator of apoptosis, necroptosis and autophagy in T lymphocytes.