Cellular FLIP long form (c-FLIPL) was originally identified as an inhibitor of Fas (CD95/Apo-1). Subsequently, additional functions of c-FLIPL were identified through its association with receptor-interacting protein (RIP)1 and TNFR-associated factor 2 to activate NF-κB, as well as by its association with and activation of caspase-8. T cells from c-FLIPL-transgenic (Tg) mice manifest hyperproliferation upon activation, although it was not clear which of the various functions of c-FLIPL was involved. We have further explored the effect of c-FLIPL on CD8+ effector T cell function and its mechanism of action. c-FLIPL-Tg CD8+ T cells have increased proliferation and IL-2 responsiveness to cognate Ags as well as to low-affinity Ag variants, due to increased CD25 expression. They also have a T cytotoxic 2 cytokine phenotype. c-FLIPL-Tg CD8+ T cells manifest greater caspase activity and NF-κB activity upon activation. Both augmented proliferation and CD25 expression are blocked by caspase inhibition. c-FLIPL itself is a substrate of the caspase activity in effector T cells, being cleaved to a p43FLIP form. p43FLIP more efficiently recruits RIP1 than full-length c-FLIPL to activate NF-κB. c-FLIPL and RIP1 also coimmunoprecipitate with active caspase-8 in effector CD8+ T cells. Thus, one mechanism by which c-FLIPL influences effector T cell function is through its activation of caspase-8, which in turn cleaves c-FLIPL to allow RIP1 recruitment and NF-κB activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells.
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-B signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that isActivation of caspases has traditionally been associated with oligomerization of death receptors and induction of cell death (1). It has been determined more recently that caspase activity is also required for the induction of proliferation by primary naïve human and murine T cells (2-4). Inhibition of caspase activity by the pan-caspase blocker, Z-VAD-fmk, 4 or the caspase-8-specific blocker, IETD-fmk, greatly reduced production of IL-2 and proliferation following TCR ligation (2, 3). It was subsequently shown that a non-functional mutation in the caspase-8 gene in humans (5, 6) or the deletion of caspase-8 in murine T cells (7) resulted in an immunodeficiency syndrome characterized by markedly decreased production of IL-2 and proliferative capacity of T cells. It remained uncertain from these studies how caspase activation was linked to T cell antigen receptor (TCR) ligation, what were the regulatory protein(s) and substrate(s) of caspase activity, and whether this required the presence of the death receptor Fas (CD95/APO1). Equally puzzling was how the activation of caspase-8 might be limited to avoid induction of apoptosis. We recently observed that following T cell activation, cleavage occurs of certain known caspase substrates, such as c-FLIP-Long form (c-FLIP L ) and RIP1, although another caspase-8 substrate associated with cell death, Bid, was not cleaved (4). This suggested that active caspase-8 may become sequestered in a specific site within T cells after activation.We considered the possibility of c-FLIP L as both an activator of caspase-8 and an early caspase-8 substrate after T cell activation. c-FLIP L is homologous to caspase-8 in containing two death effector domains, but bears a mutation in the caspase domain, which renders it enzymatically inactive (8). Following ligation of Fas, c-FLIP L is co-recruited with caspase-8 to the death-inducing signal complex (1,8). In addition, c-FLIP L is able to directly heterodimerize with and activate caspase-8 in its full-length form, independently of Fas (9 -11). c-FLIP L , furthermore, contains a caspase cleavage site at Asp 376 that yields processed p43 FLIP (12, 13). As such, c-FLIP L may be an early caspase-8 substrate during T cell activation. c-FLIP L also associates with RIP1 and TRAF2 to promote activation of NF-B (14). Mice overexpressing c-FLIP L in the T cell compartment manifest augmented IL-2, and enhanced proliferation (15, 16). Collectively, c-FLIP L may provide an important link in our understanding of how TCR stimulation activates caspase-8, what is the caspase-8 substrate(s), and how caspase activity may link to NF-B activation.
Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)–restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4−CD8−B220+ T cells occurs more frequently from a CD8+ precursor than from CD4+ T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8+ cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC–restricted TCR. The findings show that CD4−CD8−B220+ T cells preferentially derive from a CD8+ precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.
Caspase activity is required not only for the death of T cells, but also for their activation. A delicate balance of caspase activity is thus required during T cell activation at a level that will not drive cell death. How caspase activity is initiated and regulated during T cell activation is not known. One logical candidate for this process is cellular FLIP long form (c-FLIPL), because it can block caspase-8 recruitment after Fas (CD95) ligation as well as directly heterodimerize with and activate caspase-8. The current findings demonstrate that after T cell activation, caspase-8 and c-FLIPL associate in a complex enriched for active caspases. This occurs coincidently with the cleavage of two known caspase-8 substrates, c-FLIPL and receptor interacting protein 1. Caspase activity is higher in wild-type CD8+ than CD4+ effector T cells. Increased expression of c-FLIPL results in augmented caspase activity in resting and effector T cells to levels that provoke cell death, especially of the CD8 subset. c-FLIPL is thus not only an inhibitor of cell death by Fas, it can also act as a principal activator of caspases independently of Fas.
Cellular FLIP long form (c-FLIPL) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIPL in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIPL with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIPL-transgenic mice. In this study we show that activated CD4+ T cells from c-FLIPL-transgenic mice produce increased amounts of Th2 cytokines and decreased amounts of Th1 cytokines. This correlates with increased serum concentrations of the Th2-dependent IgG1 and IgE. The Th2 bias of c-FLIPL-transgenic CD4+ T cells parallels impaired NF-κB activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN-γ and increased Th2 cytokines. The Th2 bias of c-FLIPL-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIPL can influence cytokine gene expression to promote Th2-driven allergic reaction, in addition to its traditional role of blocking caspase activation induced by death receptors.
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