The c-FLIPL cleavage product p43FLIP promotes activation of extracellular signal-regulated kinase (ERK), nuclear factor κB (NF-κB), and caspase-8 and T cell survival.

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.

Reduced immune response to Borrelia burgdorferi in the absence of γδ T cells.

Little is known regarding the function of γδ T cells, although they accumulate at sites of inflammation in infections and autoimmune disorders. We previously observed that γδ T cells in vitro are activated by Borrelia burgdorferi in a TLR2-dependent manner. We now observe that the activated γδ T cells can in turn stimulate dendritic cells in vitro to produce cytokines and chemokines that are important for the adaptive immune response. This suggested that in vivo γδ T cells may assist in activating the adaptive immune response. We examined this possibility in vivo and observed that γδ T cells are activated and expand in number during Borrelia infection, and this was reduced in the absence of TLR2. Furthermore, in the absence of γδ T cells, there was a significantly blunted response of adaptive immunity, as reflected in reduced expansion of T and B cells and reduced serum levels of anti-Borrelia antibodies, cytokines, and chemokines. This paralleled a greater Borrelia burden in γδ-deficient mice as well as more cardiac inflammation. These findings are consistent with a model of γδ T cells functioning to promote the adaptive immune response during infection.

Activation of gamma delta T cells by Borrelia burgdorferi is indirect via a TLR- and caspase-dependent pathway.

Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are gammadelta T cells. gammadelta T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human gammadelta T cells of the Vdelta1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine gammadelta T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of gammadelta T cells, are both caspase-dependent.

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.

Proteolytic regulation of nuclear factor of activated T (NFAT) c2 cells and NFAT activity by caspase-3.

The nuclear factor of activated T (NFAT) cell family of transcription factors is important in regulating the expression of a broad array of genes, including cytokines, T cell surface receptors, and other transcription factors. NFATc1 and NFATc2 are two principal NFAT members that are expressed in peripheral T cells. Levels of NFAT expression in T cells are partly transcriptionally regulated, but less is understood regarding their post-transcriptional control. We show here that NFATc1 and NFATc2 are rapidly degraded in apoptotic T cells. NFATc2 is highly sensitive to cleavage by caspase-3, whereas NFATc1 is only weakly sensitive to caspase-3 or caspase-8. Two potential caspase-3 cleavage sites were identified in the N-terminal transactivation domain. These sites were confirmed by in vitro caspase cleavage assays. Abolition of NFATc2 cleavage by mutation of these two cleavage sites resulted in augmented NFAT transcriptional activity. Furthermore, NFAT activity could be augmented in wild-type effector T cells by inhibition of caspase activity. Of particular interest was that non-apoptotic T cells from cellular FLIP long transgenic (c-FLIP(L)-Tg) mice that manifest elevated caspase activity have greatly reduced levels of NFATc2 protein and NFAT transcriptional activity. Our findings reveal a new post-transcriptional regulation of NFATc2 that operates, not only during apoptosis, but also in non-apoptotic effector T cells.

Fas ligand deficiency impairs host inflammatory response against infection with the spirochete Borrelia burgdorferi.

Lyme disease represents a complex response to Borrelia burgdorferi that involves both bacterial factors as well as host responses. This results in an inflammatory reaction at several sites, including the synovial lining of joints. Synovial tissues of inflamed joints contain cells expressing high levels of Fas and Fas ligand (FasL). Although Fas stimulation is typically associated with cell death, it can also transmit stimulatory signals to certain cell types. Among these are dendritic cells and macrophages, which are abundant in inflamed synovium. To better assess the role of FasL in the pathogenesis of Lyme arthritis, we evaluated the response to B. burgdorferi infection in C3H/HeJgld mice that bear a nonfunctional mutation in FasL. Compared to wild-type C3H+/+ mice, C3Hgld mice had a similar bacterial burden and antibody response 2 weeks and 4 weeks following infection, but they manifested a significantly reduced Borrelia-specific cytokine response. In addition, C3Hgld mice developed a greatly reduced incidence and severity of arthritis. The findings document a contribution of FasL to the host inflammatory response to B. burgdorferi.

Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase-8.

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-FLIP(L)), 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-FLIP(L) associate in a complex enriched for active caspases. This occurs coincidently with the cleavage of two known caspase-8 substrates, c-FLIP(L) and receptor interacting protein 1. Caspase activity is higher in wild-type CD8(+) than CD4(+) effector T cells. Increased expression of c-FLIP(L) results in augmented caspase activity in resting and effector T cells to levels that provoke cell death, especially of the CD8 subset. c-FLIP(L) 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) regulates CD8+ T cell activation through caspase-8-dependent NF-kappa B activation.

Cellular FLIP long form (c-FLIP(L)) was originally identified as an inhibitor of Fas (CD95/Apo-1). Subsequently, additional functions of c-FLIP(L) were identified through its association with receptor-interacting protein (RIP)1 and TNFR-associated factor 2 to activate NF-kappaB, as well as by its association with and activation of caspase-8. T cells from c-FLIP(L)-transgenic (Tg) mice manifest hyperproliferation upon activation, although it was not clear which of the various functions of c-FLIP(L) was involved. We have further explored the effect of c-FLIP(L) on CD8(+) effector T cell function and its mechanism of action. c-FLIP(L)-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-FLIP(L)-Tg CD8(+) T cells manifest greater caspase activity and NF-kappaB activity upon activation. Both augmented proliferation and CD25 expression are blocked by caspase inhibition. c-FLIP(L) itself is a substrate of the caspase activity in effector T cells, being cleaved to a p43(FLIP) form. p43(FLIP) more efficiently recruits RIP1 than full-length c-FLIP(L) to activate NF-kappaB. c-FLIP(L) and RIP1 also coimmunoprecipitate with active caspase-8 in effector CD8(+) T cells. Thus, one mechanism by which c-FLIP(L) influences effector T cell function is through its activation of caspase-8, which in turn cleaves c-FLIP(L) to allow RIP1 recruitment and NF-kappaB activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells.

Effector CD4+ T cells generate intermediate caspase activity and cleavage of caspase-8 substrates.

Caspase-8 activation promotes cell apoptosis but is also essential for T cell activation. The extent of caspase activation and substrate cleavage in these divergent processes remains unclear. We show that murine effector CD4(+) T cells generated levels of caspase activity intermediate between unstimulated T cells and apoptotic populations. Both caspase-8 and caspase-3 were partially activated in effector T cells, which was reflected in cleavage of the caspase-8 substrates, c-FLIP(L), receptor interacting protein 1, and to a lesser extent Bid, but not the caspase-3 substrate inhibitor of caspase-activated DNase. Th2 effector CD4(+) T cells manifested more caspase activity than did Th1 effectors, and caspase blockade greatly decreased initiation of cell cycling. The current findings define the level of caspase activity and substrates during initiation of T cell cycling.

Cellular FLIP long form-transgenic mice manifest a Th2 cytokine bias and enhanced allergic airway inflammation.

Cellular FLIP long form (c-FLIP(L)) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIP(L) in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIP(L) with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIP(L)-transgenic mice. In this study we show that activated CD4(+) T cells from c-FLIP(L)-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-FLIP(L)-transgenic CD4(+) T cells parallels impaired NF-kappa B activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN-gamma and increased Th2 cytokines. The Th2 bias of c-FLIP(L)-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIP(L) 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.