Cellular FLIP (Long Form) Regulates CD8+ T Cell Activation through Caspase-8-Dependent NF-B Activation
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免疫学杂志 2005年第9期
Abstract
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-B, 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-B 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-B. 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-B activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells.
Introduction
The potential role of death receptors or downstream caspases in cell processes such as growth, differentiation, or effector function, has received growing recognition in a variety of cell types. Fas has been reported to be involved with hepatocyte regeneration (1), neurite dendrite outgrowth (2), fibroblast proliferation (3), cardiac hypertrophy (4), and T cell costimulation (5). The concept that signaling molecules in the Fas pathway are important to development is underscored by the finding that mice genetically deficient for either Fas-associated death domain protein (FADD),3 caspase-8, or cellular FLIP (c-FLIP), are all embryonically lethal due to a cardiac malformation (6). Furthermore, T cells from humans or mice lacking functional caspase-8 manifest a profound proliferation defect (7), consistent with earlier findings that caspase inhibitors block proliferation of human T cells (8, 9). Exactly how death receptors or associating caspases might promote cell growth and differentiation is unknown, as are the potential caspase target(s) for these processes.
We considered c-FLIP long form (c-FLIPL) as a caspase substrate that is potentially involved with cell growth. c-FLIPL is a homolog of caspase-8 in possessing two death effector domains but lacks a functional caspase domain due to a mutation of a critical cysteine to tyrosine in the enzymatic domain (10). As such, c-FLIPL acts as a competitive inhibitor for recruitment of caspase-8 to the death effector domain of FADD following Fas ligation (11). However, subsequent studies demonstrated the ability of c-FLIPL to bind adaptor proteins that can link to the NF-B and ERK pathways. These adaptors include receptor-interacting protein (RIP)1, TNFR-associated factor (TRAF)2, and Raf-1 (12). Increased expression of c-FLIPL in T cell lines augmented IL-2 production, and in transgenic mice, c-FLIPL enhanced T cell proliferation (13). In addition, c-FLIPL has a known caspase cleavage site at Asp376 resulting in caspase-8-dependent cleavage of full-length 55-kDa c-FLIPL to p43FLIP (14, 15, 16). The functional significance of c-FLIPL cleavage is unknown.
The current studies sought to define how increased c-FLIPL expression results in enhanced T cell growth and whether cleavage of c-FLIPL is required for this function. We observe that mice transgenic for c-FLIPL in the T cell compartment have a decreased activation threshold to Ags bearing decreased TCR affinity and are less dependent on CD28 costimulation. The increased proliferation is due to augmented expression of CD25, consistent with known increased activation of ERK and NF-B by c-FLIPL. These effects of c-FLIPL are independent of Fas expression. Finally, the ability of c-FLIP to recruit RIP1 is greatly increased after c-FLIPL is cleaved to p43FLIP. These findings suggest that, during T cell activation, c-FLIPL may be a critical caspase substrate that helps promote the activation of NF-B.
Materials and Methods
Mice
c-FLIPL was expressed transgenically in T cell compartment as previously reported (13). Briefly, FLAG-tagged mouse FLIPL cDNA was inserted into a target vector containing the -globin promoter and a downstream human CD2 locus enhancer element. Transgenic mice were screened by PCR of ear DNA using the following primers: 5' primer, 5'-GGAGCCAGGGCTGGGCATAAAA-3'; and 3' primer, 5'-GACTCACCCTGAAGTTCTCAGGATCC-3'.
Immunoblot using anti-FLIP mAb (Dave-2; Apoxis) further confirmed expression of the transgene at levels 8- to 10-fold greater than wild-type c-FLIPL. The c-FLIPL-transgenic (Tg) mouse strain has been backcrossed to C57BL/6 mice (The Jackson Laboratory) for nine generations. Mice were maintained at the University of Vermont Animal Facility (American Association for the Accreditation of Laboratory Animal Care approved), and experiments were conducted in accordance with Institutional Animal Care and Use Committee-approved protocols.
OT-1 mice bear a transgenic TCR that recognizes chicken OVAp restricted to class I MHC, Kb, and were kindly provided by Drs. F. Carbone and M. Bevan (University of Washington, Seattle, WA) (17). OT-1 mice were maintained by breeding TCR transgenic male mice to normal C57BL/6 females. Offspring were screened for the clonotype TCR using anti-V2 mAb.
CD8+ T cell purification and culture
Spleen cells after hemolysis by Gey’s solution were combined with lymph node cells followed by negative selection to enrich for CD8+ cells. Cells were incubated with Abs to CD4 (GK1.5), MHC class II (3F12), NK1.1 (PK136), and CD11b (all from BD Biosciences) for 30 min to remove, respectively, CD4+ cells, B cells, NK cells, and macrophages. Samples were washed and then incubated with goat anti-rat/mouse IgG-labeled magnetic beads (Qiagen) for 45 min followed by magnetic field separation. CD8+ T cell purity was confirmed by the flow cytometry and was routinely >90%.
T cells were activated in culture medium (RPMI 1640 supplemented with penicillin (200 μg/ml; Sigma-Aldrich), streptomycin (200 μg/ml; Sigma-Aldrich), glutamine (4 mM; Sigma-Aldrich), 2-ME (50 μM; Sigma-Aldrich), HEPES (10 mM; Sigma-Aldrich), and 8% FBS (Intergen)) using plate-bound anti-CD3 (5 μg/ml; clone 145-2C11), anti-CD28 ascites (1:500), and recombinant human IL-2 (50 U/ml; Cetus), or OVAp or low-affinity variant G4 (SIIGFEKL) in the case of OT-I mice, for 2 days. For proliferation studies, cell were pulsed at this time with [3H]thymidine for an additional 18 h before harvest. Cells propagated for longer periods were removed from anti-CD3-coated wells on day 2 and fed with fresh culture medium containing IL-2.
Cytokine ELISA
Purified CD8+ T cells were cultured at 106/ml with plastic-bound anti-CD3 (5 μg/ml; 145-2C11) and soluble anti-CD28 (1/500 dilution of ascites; 37.51) for 48 h, and 72 h. Quantification of cytokines (IL-2, IL-4, and IFN-) in cell culture supernatants was performed using a sandwich ELISA as described (18).
RNA preparation and RNase protection assay
Total RNA was prepared from cultured CD8+ T cells using Ultraspec (Biotecx) according to the manufacturer’s recommendation. Cytokine RNA levels were determined by RNase protection assay using the RiboQuant multiprobe kit (BD Biosciences). Five micrograms of total RNA was hybridized overnight with a 32P-labeled RNA probe, which had been synthesized from the multicytokine template set, after which free probe and other ssRNA were digested with RNase.
Abs and flow cytometry
Monoclonal anti-murine CD8 conjugated to Red613 was purchased from Invitrogen Life Technologies. Monoclonal anti-murine CD4 conjugated to TriColor or PE was purchased from Caltag Laboratories. Monoclonal anti-murine V2 conjugated to PE, monoclonal anti-murine CD69 conjugated to PE, monoclonal anti-murine CD80 (B7.1) conjugated to FITC, monoclonal anti-murine CD86 (B7.2) conjugated to PE, and monoclonal anti-murine H-2 Kb conjugated to biotin were purchased from BD Biosciences.
For flow cytometry, 750,000 cells were incubated in 0.1 ml of PBS containing 0.5% BSA fraction V, 0.001% (w/v) sodium azide (PBS-azide) (Sigma-Aldrich), and the Abs listed above at 4°C for 30 min. After washing with PBS-azide, cells were fixed in 1% methanol-free formaldehyde (Ted Pella) in PBS-azide. Samples were stored at 4°C until they were analyzed with a Coulter Elite flow cytometer calibrated using DNA check beads (Coulter).
AP-1-, NFAT-, and NF-B-luciferase reporter mice and luciferase activity
AP-1-luciferase transgenic mice carry the luciferase gene driven by four human collagenase 12-O-tetradecanoylphorbol-13-acetate-responsive elements, which have high affinity for the AP-1 complex, in the context of the rat minimal prolactin promoter, linked to the luciferase gene (19). The NFAT-luciferase reporter mice bear three copies of the NFAT binding sequence from the IL-2 gene linked to the luciferase gene (20). The NF-B-luciferase reporter mice have the luciferase gene controlled by two copies of B sequences from the Ig enhancer (21). Purified CD8+ T cells were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1/500 dilution of ascites). At 48 h, and 72 h following activation, cells were harvested, washed with PBS, and lysed. The lysates were then analyzed using luciferin (Promega) and measured in a luminometer for 10 s. Four measurements were made for each sample. Results are presented as the mean (±SEM) with background subtracted.
Caspase activity assay
Total cellular caspase activity was quantitated using DEVD-rhodamine (Promega) according to the manufacturer’s protocol. In brief, viable cells were isolated using centrifugation over Lympholyte M (Cedarlane) and were titrated as indicated in 100 μl of culture medium, and an equal volume of DEVD-rhodamine was added to the cells according to the manufacturer’s protocol. As the DEVD substrate is cleaved by caspases, rhodamine is released and measured by a fluorescent spectrophotometer at 2 h.
Western blot analysis
Cells were washed once in ice-cold PBS and solubilized in lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% glycerol, 2 mM DTT, protease inhibitor mixture (Complete; Boehringer Mannheim). Postnuclear lysate proteins (40 μg per lane) were separated in 12.5% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Hybond-ECL; Amersham), and blots were blocked and probed with the indicated Abs in 4% nonfat milk in TBS/0.1% Tween 20. Immunoreactive proteins were visualized using HRP-labeled conjugates (Jackson ImmunoResearch Laboratories) and ECL blotting substrate (Amersham Biosciences). Abs used were specific for caspase-8 and c-FLIP (Apoxis), and RIP1 (BD Biosciences).
Transfection studies
Human embryonic kidney 293 and 293T cells were seeded on 60-mm culture plates (3 ml/plate) the day before transfection, and transfected by the calcium phosphate method with various expression vectors. The cells were harvested 16 h after transfection and washed with PBS, and lysed with lysis buffer. After repeated centrifugation, postnuclear lysates were precleared with Sepharose 6B for 1 h and then incubated with anti-FLAG M2 agarose (Sigma-Aldrich) for 3 h. Agarose beads were washed four times with the lysis buffer. Immunoprecipitates and cell lysates were analyzed by Western blotting. 293T cells were seeded on a 24-well culture plate (0.6 ml/plate) the day before transfection, and transfected by the calcium phosphate method with various expression vectors, together with the NF-B-driven luciferase reporter plasmid and the -galactosidase plasmid. Postnuclear lysates were subjected to the luciferase assay and -galactosidase assay, which was used to normalize transfection efficiency.
Biotin-VAD-fmk caspase precipitation assay
Viable T cells (freshly isolated or day 4 T cell blasts) were incubated with 10 μM biotin-VAD-fmk (Enzyme System Products). Cell membranes were disrupted using lysis buffer containing 20 μM biotin-VAD. Lysate (600 μg) was then precleared by rocking with 40 μl of agarose beads (Santa Cruz) at 4°C for 2 h. Supernatant was then incubated with 30 μl of streptavidin-Sepharose beads (Zymed) on a rocker at 4°C overnight. Beads were washed five times in lysis buffer without Complete protease inhibitor. Beads were then boiled in loading buffer. Beads were removed by centrifugation, and immunoblot analysis was then performed on the supernatants.
Results
CD8+ T cells from c-FLIPL-Tg mice manifest increased CD28-independent proliferation to low-dose Ag peptide or low-affinity peptide variant
Although caspase-8 has been demonstrated to be required for activation of both human and mouse T cells (7, 22), the process that initiates the caspase activity is not known, nor is the caspase-8 substrate(s) that is responsible for promoting activation. We considered the possibility that c-FLIPL might provide both of these functions because it can both heterodimerize with and activate caspase-8, and is also cleaved by caspase-8 (23). We have previously observed that CD8+ T cells from mice transgenic for c-FLIPL (c-FLIPL-Tg) in the T cell compartment manifest hyperproliferation upon activation (13). We sought to extend these studies to determine the mechanism of increased proliferation, and whether caspase activity and c-FLIPL cleavage are in any way linked to these functions. As previously reported, CD8+ T cells from c-FLIPL-Tg mice had increased proliferation in response to low-dose CD3 stimulation (Ref. 13 ; Fig. 1A). At doses of anti-CD3 of 5 μg/ml or higher, control and c-FLIPL-Tg CD8+ T cells manifested similar rates of incorporation of [3H]thymidine. This reflects the fact that, although cell cycling of c-FLIPL-Tg CD8+ T cells was still faster than wild-type CD8+ cells even at higher concentrations of anti-CD3 (see Fig. 4A below), increased cell death of c-FLIPL-Tg CD8+ T cells balanced this property (A. Dohrman, unpublished observations). Hyperproliferation was also apparent even in the absence of CD28 costimulation (Fig. 1B).
FIGURE 1. Increased proliferation of c-FLIPL-Tg CD8+ T cells to low-dose anti-CD3, Ag peptide, and low-affinity peptide variant. A and B, Purified CD8+ T cells (5 x 104/well) from c-FLIPL-Tg mice or normal littermate controls (NLC) were activated with various concentrations of plate-bound anti-CD3 Ab in the presence of constant anti-CD28 (1/500 dilution of ascites) (A) or in the absence of anti-CD28 (B). Proliferation was measured by [3H]thymidine incorporation during the final 18 h of a 72-h culture period. C and D, T cells from OT-1 or OT-1 x c-FLIPL-Tg mice (5 x 104/well) were activated with either high-affinity OVAp (C) or low-affinity variant SIIGFEKL (G4) (D) in the presence of irradiated B6 spleen cells (3 x 105/well), and proliferation was measured by [3H]thymidine incorporation during the final 18 h of a 72-h culture period. These findings were consistent in five experiments.
FIGURE 4. Increased cell cycling of c-FLIPL-Tg T cells is CD25-dependent. A—C, CD8+ T cells were activated via anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) and analyzed on day 3 for DNA content by propidium iodide (A), for surface expression of CD25 (B), and DNA content gated on the CD25+ subset (C). The gate for 2N DNA was established using naive noncycling T cells. D and E, Purified CD8+ T cells from normal littermate control (NLC) mice (D) or c-FLIPL-Tg mice (E) were activated via CD3/CD28 for 3 days in the presence of the indicated concentrations of control IgG or blocking anti-CD25 antibody. The results were consistent in three experiments.
The augmented proliferation was also observed with Ag stimulation by crossing the c-FLIPL-Tg mouse to the OT-I mouse that bears a transgenic TCR reactive to OVAp restricted to H-2Kb (17). This system allowed a better definition of the effects of decreasing the MHC/peptide affinity for TCR. The OT-I TCR binds H-2Kb/SIINFEKL with an affinity of 6.5 μM, whereas the peptide variant SIIGFEKL (G4) binds with a lesser affinity of 10 μM (24). OT-1 x c-FLIPL-Tg T cells manifested increased proliferation to OVAp over a broad dose range (Fig. 1C). This was particularly pronounced with low-affinity G4, to which OT-I T cells proliferated only minimally, whereas OT-1 x FLIP-Tg T cells had a considerably stronger response (Fig. 1D). The dramatic difference in the proliferation intensity between OVAp and G4 reflects the nonlinear relation between affinity of the TCR interaction and effector function, as observed previously with OT-1 T cells (17). As noted in Fig. 1, A and C, at high doses of anti-CD3 (5 μg/ml) or OVAp (10–8 M), there was no difference in [3H]thymidine incorporation. This most likely resulted from the increased cell cycling of c-FLIPL-Tg T cells being balanced by increased death, due to increased caspase-8 activation by c-FLIPL (see Fig. 6).
FIGURE 6. Caspase activity of CD8+ T cells from c-FLIPL-Tg mice is increased and independent of Fas expression. A, Caspase activity was measured by rhodamine release from DEVD in freshly isolated CD8+ cells, and day 3 blasts were activated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) from normal littermate controls (NLC) and c-FLIPL-Tg mice. B, Caspase activation as measured by Western blot showing cleavage of caspase-8 and c-FLIPL. The levels of c-FLIPL in T cells from transgenic mice was 8- to 10-fold increased over normal littermate controls by densitometry. C and D, Absence of Fas in lpr mice does not reverse the hyperproliferation (C) or increased caspase activity (D) of c-FLIPL-Tg CD8+ T cells. Purified CD8+ cells were stimulated with the indicated concentrations of anti-CD3 (C) or 5 μg/ml anti-CD3 (D) plus anti-CD28 (1/500 dilution of ascites) as in Fig. 1A. These results were similar in four additional experiments.
Hyperproliferation of c-FLIPL-Tg CD8+ T cells is due to increased expression of CD25
IL-2 signaling represents one of the principal growth pathways for T cells. Production of IL-2 and expression of the high-affinity IL-2R-chain (CD25) were thus examined on c-FLIPL-Tg CD8+ T cells. In parallel with the differences in proliferation, IL-2 production by c-FLIPL-Tg CD8+ T cells was higher than from the equivalent population from normal littermate controls at low-dose anti-CD3, but became equivalent at higher anti-CD3 concentrations (Fig. 2A). To determine whether this completely explained the difference in proliferation, exogenous IL-2 was added to T cells activated by either low-dose anti-CD3 or with OT-I cells stimulated with G4 peptide. Although exogenous IL-2 increased the proliferation of both populations, it remained higher in the c-FLIPL-Tg CD8+ T cells (Fig. 2, B and C).
FIGURE 2. Increased production and responsiveness to IL-2 by c-FLIPL-Tg CD8+ T cells. A, Purified CD8+ T cells (106/ml) were stimulated with the indicated concentrations of plate-bound anti-CD3 and a constant amount of anti-CD28. After 48 h, supernatants were removed and analyzed for IL-2 protein by ELISA. The findings were consistent in two experiments. B, CD8+ T cells were activated with low-dose anti-CD3 (0.5 μg/ml) in the presence of the indicated concentrations of rIL-2, and proliferation was measured after 3 days. C, T cells from OT-1 or OT-1 x c-FLIPL-Tg mice were activated with G4 peptide (10–7 M) in the presence of the indicated concentrations of IL-2, and proliferation was measured after 3 days. Similar results were found in four additional experiments.
The persistence of increased proliferation of c-FLIPL-Tg CD8+ T cells despite addition of exogenous IL-2 suggested that the augmented proliferation of c-FLIPL-Tg T cells was not due solely to a difference in IL-2 production, but resulted in part also from increased sensitivity to IL-2. This possibility was investigated further through an analysis of expression of CD25 and the activation marker CD69 on c-FLIPL-Tg CD8+ T cells following TCR stimulation. As shown in Fig. 3, following activation of purified T cells via CD3/CD28, these activation markers were more prominently expressed over several days by T cells from c-FLIPL-Tg mice than from control mice. By days 3 and 4 after activation, the percentage of CD25+ cells became similar in the two groups, but this was anticipated because during this period the CD25+ cells would have overgrown any cells lacking CD25. After CD25 levels began to decline on day 6 in control CD8+ T cells, the enhanced expression of CD25 became once again apparent in the c-FLIPL-Tg CD8+ T cells. However, during the entire period of analysis, the mean fluorescence intensity of CD25 expression was higher on the c-FLIPL-Tg CD8+ T cells (Fig. 3A, right panel).
FIGURE 3. Enhanced expression of activation markers by stimulated CD8+ T cells from c-FLIPL-Tg mice. CD8+ T cells from c-FLIPL-Tg mice were stimulated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites), and surface expression of activation molecules CD25 and CD69 was measured by mean fluorescent intensity (MFI) by flow cytometry on the days indicated. Similar results were obtained when results were expressed as percent positive cells. The studies were performed three times with similar results.
The enhanced proliferative capacity of c-FLIPL-Tg CD8+ T cells was further shown to reside within the CD25+ fraction. T cells from c-FLIPL-Tg mice or normal littermate controls were activated for 3 days and then stained for expression of surface CD25, and cell cycling was defined by uptake of propidium iodide. As shown in Fig. 4, A and B, c-FLIPL-Tg T cells demonstrated an increased proportion of CD25+ cells after activation, and cell cycling was confined to the CD25+ subset. Furthermore, within the CD25+ fraction of activated T cells, there was little difference in cell cycle rates between c-FLIPL-Tg vs littermate control mice (Fig. 4C). Thus, the increased proliferation of c-FLIPL-Tg T cells was also due in part to increased expression of CD25, and rather than IL-2-independent proliferation. This was also consistent with the inhibition of proliferation of c-FLIPL-Tg T cells by blocking CD25 engagement (Fig. 4, D and E). These findings support the view that the increased cell cycling of T cells from c-FLIPL-Tg mice is due to increased signal pathways that lead to increased CD25 expression, rather than effects directly on downstream cell cycle regulators that would be independent of CD25 expression.
In contrast to the increased production of IL-2 by c-FLIPL-Tg CD8+ T cells, levels of IFN- were somewhat decreased, whereas IL-4 production was slightly increased (Fig. 5A). This was consistent at all doses of anti-CD3 tested (data not shown). This T cytotoxic 2 pattern was confirmed by RNase protection analysis (Fig. 5B). A similar pattern of cytokine production has been observed in the CD4+ subset of c-FLIPL-Tg mice (18).
FIGURE 5. T cytotoxic 2 pattern of cytokine production by c-FLIPL-Tg CD8+ T cells. Purified CD8+ T cells (106/ml) were stimulated with anti-CD3 (5 μg/ml) plus anti-CD28 (1/500 dilution of ascites)/CD28, and supernatants were examined on days 2 and 3 for production of IL-4, IL-2, and IFN-, by ELISA of culture supernatants (A), and RNase protection assay of RNA from the same cells (B). Similar results were observed in two additional experiments.
c-FLIPL-Tg CD8+ T cells exhibit enhanced caspase and NF-B activities that are independent of Fas
In considering the potential signaling pathways influenced by c-FLIPL in primary CD8+ T cells, two aspects of c-FLIPL function were examined. First, we previously observed that transfection of cell lines with c-FLIPL manifested increased ERK and NF-B activities due to the association of c-FLIPL with, respectively, Raf-1 and RIP1 (12). The second aspect was that c-FLIPL is known to associate with and actually activate caspase-8 (23). c-FLIPL is also a potential substrate of caspase-8 (14, 15, 16). We therefore investigated to what extent these pathways might be related and contribute to the phenotype of c-FLIPL-Tg T cells, and whether this required the presence of Fas.
Consistent with the ability of c-FLIPL to activate caspase-8, c-FLIPL-Tg T cells manifested more caspase activity than littermate control mice (Fig. 6A). This was apparent even in fresh resting T cells, as well as in activated day 3 blasts. These findings were supported by the observation that caspase-8 was more extensively cleaved in c-FLIPL-Tg T cells, especially in the resting state (Fig. 6B). Resting wild-type T cells contained largely full-length p55caspase-8, but following their activation, caspase-8 cleavage became progressively apparent over the subsequent 3 days. By contrast, c-FLIPL-Tg T cells demonstrated extensive cleavage of caspase-8 when freshly isolated, which did not increase as dramatically with activation as with wild-type T cells. This was likely due to increased death of c-FLIPL-Tg T cells (A. Dohrman, unpublished observations), which would tend to constantly eliminate those T cells with the highest levels of caspase activity. Because dead cells were always intentionally eliminated from these analyses, it would remove this subset and further caspase-8 cleavage would not be so apparent. Another issue is that caspase-8 can be active in its full-length form when complexed to c-FLIPL (23), so analysis of only the degree of caspase-8 cleavage may underestimate the amount of active caspase-8. The increased caspase activity in c-FLIPL-Tg T cells was corroborated by increased and early cleavage of c-FLIPL, a known caspase-8 substrate (Fig. 6B).
Neither the hyperproliferation nor the enhanced caspase activation conferred by c-FLIPL required Fas. B6 c-FLIPL-Tg mice were crossed with B6 lpr mice, and T cell proliferation was examined in response to varying doses of anti-CD3. As shown in Fig. 6C, in the absence of Fas, purified CD8+ T cells from c-FLIPL-Tg/lpr mice proliferated more extensively than those from age- and sex-matched littermate control lpr mice. This was similar to that observed earlier with c-FLIPL-Tg and wild-type mice. In parallel with these findings, c-FLIPL-Tg/lpr CD8+ T cells contained more caspase activity than lpr T cells (Fig. 6D).
The presence of caspase activity was actually required for the proliferative response of c-FLIPL-Tg CD8+ T cells, because the caspase blockers z-Val-Ala-Asp(OCH3)-fluoromethylketone (z-VAD-fmk) and QVD-OPh largely inhibited both proliferation (Fig. 7A) and CD25 expression (B) in a dose-dependent manner, both in wild-type and c-FLIPL-Tg CD8+ T cells. Similar results were observed for the CD4+ subset (data not shown). These agents were not merely toxic to lymphocytes, because there was no increase in dead cells in cultures containing caspase blockers compared with unstimulated T cells, and the delayed addition of z-VAD by even 24 h after activation resulted in substantially less inhibition of T cell growth (data not shown). Caspase activity is therefore required particularly during the initial 24 h of T cell activation. Furthermore, the augmented caspase activity of c-FLIPL-Tg T cells is at least partly responsible for their increased proliferative capacity.
FIGURE 7. Caspase-dependent proliferation and CD25 expression by T cells from c-FLIPL-Tg mice. Purified CD8+ T cells from normal littermate controls (NLC) or c-FLIPL-Tg mice were stimulated with 5 μg/ml anti-CD3 plus anti-CD28 (1/500 dilution of ascites) in the presence of the pancaspase blockers z-VAD-fmk or QVD-OPh, or controls DMSO or zFA. A, Proliferation measured by [3H]thymidine incorporation during the final 18 h of a 72 h culture period. B, Surface CD25 expression on day 3. These findings were consistent in two experiments.
Because the signal pathways that involve regulation of CD25 and cytokines include the transcription factors NF-B, AP-1, and NFAT, we examined these pathways by crossing the c-FLIPL-Tg mice with luciferase reporter mice transgenic for the DNA binding site for each of these transcription factors (19, 20, 21). Maximal luciferase activity in T cells from these mice is detectable 2–3 days after activation, the time required for resting T cells to become metabolically active, and closely parallels DNA binding by electromobility shift assay (19, 20, 21, 25) (M. Rincon, unpublished observations). Luciferase activity was measured following activation of purified CD8+ T cells with anti-CD3/CD28. An increase in NF-B activity was observed on days 2 and 3 in CD8+ T cells from the NF-B x c-FLIPL-Tg mice, and was statistically significant (Fig. 8A). This was not merely due to the greater ability of c-FLIPL-Tg T cells to produce IL-2, because the amount of anti-CD3 (5 μg/ml) was a dose at which no difference in IL-2 production was observed (see Fig. 5). Furthermore, addition of saturating levels of exogenous IL-2 yielded persistent increases in NF-B activity in c-FLIPL-Tg CD8+ T cells. By contrast, there were no significant differences in AP-1 or NFAT activity (Fig. 8A). These findings were supported by observations of greater phosphorylation and decreased levels of the NF-B inhibitor, IB, in c-FLIPL-Tg T cells compared with wild-type T cells at all time points examined (Fig. 8B). Examination of more proximal signals in the TCR pathway revealed no difference in either CD3 phosphorylation or Ca2+ flux following TCR stimulation (data not shown). These studies thus focused attention on the possible link between c-FLIPL and the NF-B pathway.
FIGURE 8. Increased NF-B activity by c-FLIPL-Tg CD8+ T cells. A, c-FLIPL-Tg mice were crossed by mice transgenic for the DNA binding sites of AP-1, NF-B, and NFAT linked to a luciferase reporter. Purified CD8+ T cells were activated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) and measured on days 1, 2, and 3 for luciferase activity. Results represent mean ± SD of three separate experiments. *, Statistically significant differences (p < 0.05). B, CD8+ T cells were used either freshly isolated or following activation with the same concentrations of anti-CD3/CD28 as in A for 2 or 3 days, and cell lysates were analyzed by Western blot for expression of phospho-IB (p-IB), total IB, and actin. The findings were consistent in three studies.
The requirement of caspase activity for T cell proliferation raised the possibility of a caspase substrate whose cleavage would promote NF-B activation. We have previously observed that, although transient transfection with both c-FLIPL and p43FLIP N-terminal fragment induces NF-B activity, caspase inhibition by z-VAD blocked NF-B activity by c-FLIPL but not by p43FLIP (26). We have also observed in transient transfection studies that c-FLIP associates with RIP1, a known activator of NF-B (12). We therefore compared the ability of c-FLIPL vs p43FLIP to recruit RIP1. 293 T cells were transfected with c-FLIPL or p43FLIP. Coimmunoprecipitation studies demonstrated that endogenous RIP1 associated with p43FLIP but not with full-length c-FLIPL (Fig. 9A). Furthermore, the association of p43FLIP with RIP1 required the presence of caspase-8 because this association did not occur in a caspase-8-low variant of 293 cells, but was restored with cotransfection of caspase-8 (Fig. 9B). Moreover, a dominant-negative form of RIP1 (RIP1559–671) was able to inhibit p43FLIP-induced NF-B activity, attesting to the view that this function of c-FLIP was indeed working via RIP1 (Fig. 9C).
FIGURE 9. Requirement of caspase-8 to promote association of RIP1 with p43FLIP and activation of NF-B. A and B, 293T cells (A) or caspase-8-low variant 293 cells (B, left panel) were transfected with FLAG-tagged caspase-8, p43caspase-8, c-FLIPL, or p43FLIP for 16 h, and cell lysates were precipitated using anti-FLAG followed by Western blot detection of RIP1. B, Right panel, Transfection of caspase-8 into caspase-8-low variant 293 cells restores association of RIP1 with p43FLIP. Coimmunoprecipitation of p43FLIP was performed as in A and B in the absence (–) or presence (+) of transfected caspase-8. C, Dominant-negative RIP1559–671 was transfected using the amounts indicated into 293T cells along with p43FLIP followed by measurement of NF-B-luciferase activity. D, Purified c-FLIPL-Tg T cells, either freshly isolated (D0) or activated with anti-CD3/CD28 plus IL-2 for 4 days (D4), were incubated for 15 min with biotin-VAD-fmk (10 μM) and then lysed. Lysates were subjected to a preclear with Sepharose beads and then incubated with avidin-Sepharose. Precipitates were analyzed by immunoblot for caspase-8, c-FLIP, and RIP1. Shown are avidin-Sepharose precipitates compared with whole-cell lysates (WCL) as a reference. E, CD8+ T cells from NF-B-luciferase mice were activated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) for 48 h in the absence or presence of caspase blockers z-VAD-fmk or QVD-OPh (each at 100 μM) and then assayed for luciferase activity. The results were similar in two additional studies.
To further determine whether c-FLIPL and RIP1 were indeed associated with active caspases in CD8+ in T cells, fresh day 0 and day 4 c-FLIPL-Tg T cell blasts were incubated with biotin-VAD followed by lysis and precipitation of active caspases with avidin-Sepharose. Similar to the findings using DEVD-rhodamine, there was little active caspase-8 precipitated from day 0 T cells compared with day 4 blasts even though the amount of total caspase-8 was identical in the two populations (Fig. 9D). The active form of caspase-8 in day 4 T cell blasts was full-length caspase-8, which is consistent with the modeling of caspase-8 with c-FLIPL (23). In addition, very little c-FLIP was associated with active caspase-8 in day 0 lysates, but increased considerably in day 4 lysates (Fig. 9D). Of interest was that the c-FLIP associated with caspase-8 was more the cleaved p43FLIP form than full-length c-FLIPL, whereas these two forms were about equally present in the whole-cell lysates. This suggests that c-FLIPL that is in close proximity with active caspase-8 is more likely to be cleaved. In addition, RIP1 was also found associated with active caspase-8 in day 4 lysates but not in day 0 lysates. Finally, to examine the effect of caspase blockage on activation of NF-B in primary T cells, we used mice transgenic for the DNA binding site for NF-B linked to the luciferase gene (21). Purified CD8+ T cells from these mice were activated with anti-CD3/CD28 for 48 h in the absence or presence of the caspase blockers, z-VAD-fmk and QVD-OPh. As shown in Fig. 9E, caspase inhibition greatly diminished the amount of NF-B activity observed in activated primary T cells. This is consistent with the requirement of caspase-dependent cleavage of c-FLIPL to recruit RIP1, and begins to provide an explanation for the caspase requirement for T cell activation.
Discussion
c-FLIPL was originally identified as an inhibitor of cell death induced by Fas through its ability to compete with caspase-8 for recruitment to FADD during Fas ligation (10). More recently, two additional functions of c-FLIPL have been discerned. The first is the association of c-FLIPL with Raf-1, thereby promoting activation of ERK, as well as its association with TRAF2 and RIP1, promoting activation of NF-B (12). The second is the ability of c-FLIPL to heterodimerize with and activate caspase-8 (23). Although the capacity of c-FLIPL to both compete with caspase-8 for recruitment to FADD, as well as activate caspase-8 by direct heterodimerization, may seem contradictory, the current findings nonetheless suggest that at least one caspase-dependent activation pathway involves c-FLIPL as both an activator and substrate of caspase-8. Following caspase-dependent cleavage of c-FLIPL, RIP1 is recruited more efficiently to p43FLIP, thereby allowing more effective activation of NF-B.
The current findings extend in several ways our previous observations that c-FLIPL can promote T cell growth. First, it demonstrates that caspase activity is increased in c-FLIPL-Tg T cells and this is required to promote proliferation of c-FLIPL-Tg T cells, functioning largely through increased expression of CD25. Second, this is the first demonstration that activated T cells contain active caspase-8 in a full-length form and that c-FLIP and RIP1 are associated with active caspases in cycling T cells, but not in naive T cells, despite similar levels of these proteins.
Fas has been demonstrated to stimulate a variety of effector functions in several cell types, including primary T cell proliferation (5), fibroblast growth (3), hepatocyte regeneration (1), neurite outgrowth (2), and up-regulation of costimulatory molecules and cytokines by dendritic cells (27). The physiological significance of these phenomena is uncertain, because Fas-deficient lpr mice do not manifest defects in T cell proliferation, nor do they have overt developmental abnormalities in other organs systems been reported in lpr mice. The signal pathway(s) responsible for this unanticipated stimulatory property of Fas is not well described. However, in the case of T cells and neurons, a link of Fas stimulation to ERK and NF-B activation has been shown (2, 12). A more compelling case has been made for the involvement of caspase activity as a requirement for T cell activation. Not only do caspase blockers inhibit proliferation of primary human T cells (8, 9), but both murine and human T cells deficient in functional caspase-8 manifest a pronounced defect in proliferation (7, 22). Although numerous caspase-8 substrates may be important for full T cell activation, the current findings indicate that c-FLIPL is likely one of these substrates.
The finding that activated c-FLIPL-Tg CD8+ T cells express higher levels of IL-2 and surface CD25, and that the increased cycling of activated c-FLIPL-Tg T cells occurs within the CD25+ subset, supports the view that c-FLIPL-induced hyperproliferation of T cells occurs primarily via augmented IL-2 signaling and less likely through alterations of other cell cycle regulatory proteins. This is also consistent with the ability of c-FLIPL to augment activation of the ERK and NF-B pathways, which are involved with induction of IL-2 and CD25 expression (28, 29, 30).
At present, it is unclear how signals initiated by TCR ligation link to those induced by c-FLIPL. A logical consideration might be through TCR up-regulation of surface FasL, which would then bind Fas and recruit c-FLIPL. However, it is clear from the current studies that c-FLIPL also augments proliferation in Fas-deficient lpr mice. Preliminary studies identify a complex of active caspase-8, c-FLIPL, and RIP1 in lpr T cell blasts (J. Russell, unpublished observations). This does not exclude the potential involvement of other death receptors in the activation of caspases in effector T cells. Future studies are underway to examine the links between TCR signals and caspase activation.
RIP1 associates directly with a heterodimer of caspase-8/c-FLIP, although it is not certain to which component of the heterodimer it binds. Given that the form of c-FLIP associated with active caspase-8 is preferentially cleaved p43FLIP (Fig. 9D), combined with the greater association of RIP1 with transfected p43FLIP rather than full-length c-FLIPL, this suggests that p43FLIP may stabilize binding of RIP1 to the caspase-8/c-FLIP heterodimer. This immediately suggests that c-FLIPL may be an important substrate for caspase-8 during T cell activation. An additional possibility is that the form of c-FLIP in the heterodimer may influence the ability of HSP90 to stabilize RIP1 (31, 32). Thus, the amount of RIP1 complexed to caspase-8/c-FLIP may reflect differences in degradation more than association.
Other studies by us have shown a similar association of TRAF2 preferentially to p43FLIP (26). c-FLIPL has a known caspase cleavage site at Asp376, which yields p43FLIP (23). This exposes a binding site for RIP1 and TRAF2 that is presumably less accessible in full-length c-FLIPL. These findings would also serve to explain the ability of caspase inhibition to block NF-B activation by c-FLIPL but not by p43FLIP (26). Differences in caspase activity between CD8+ and CD4+ T cells might also explain why we observed increased NF-B activity in CD8+ cells from c-FLIPL-Tg mice, but did not in previous studies of the CD4+ subset from c-FLIPL-Tg mice (18). In separate studies, we observe that activated CD8+ cells from c-FLIPL-Tg mice manifest greater caspase activity than activated CD4+ cells from the same mice (A. Dohrman, unpublished observations). In addition, p43FLIP lacks the domain by which c-FLIPL activates caspase-8 (23). Thus, cleavage of c-FLIPL to p43FLIP may serve not only to recruit RIP1 and TRAF2 to promote NF-B activation, it may also impede further activation of caspase-8, to decrease the risk of promoting T cell death after activation. In this regard, it is of some interest that the viral form of FLIP (v-FLIP) is truncated, containing only the two death domains (33). v-FLIP still allows recruitment of RIP1 and TRAF2, similar to p43FLIP, but does not activate caspase-8. This could provide a survival capacity of v-FLIP.
Regulation of c-FLIPL not only in T cells but also in other cell types may strongly influence the ability to either proliferate or differentiate. Blood monocytes have very low levels of c-FLIPL and are highly sensitive to Fas-induced death, whereas dendritic cells that are derived from blood monocytes using GM-CSF and IL-4 express high levels of c-FLIPL and are highly resistant to Fas-mediated apoptosis (Ref. 34 ; R. Budd, unpublished observations). Furthermore, ligation of Fas on dendritic cells promotes up-regulation of surface CD80/CD86, class II MHC, and IL-12 production (27). A murine model of cardiac hypertrophy has also implicated a role for Fas in cardiac hypertrophy in response to hypertension (4). Interestingly, cardiac myocytes are among the highest expressors of c-FLIPL, and FLIP-null mice are embryonic lethal due to a cardiac developmental abnormality (35). Thus, c-FLIP is emerging as not only an inhibitor of Fas-induced death, but also as a promoter of cell proliferation and/or differentiation based on its ability to recruit adaptor proteins linking to the ERK and NF-B pathways. That this occurs better with cleaved p43FLIP than with full-length c-FLIPL suggests that c-FLIPL is at least one caspase-8 substrate needed for optimal T cell proliferation.
Acknowledgments
We thank Colette Charland for technical assistance with flow cytometry.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health (NIH) Grants AI36333 and AI45666 (to R.C.B.). A.D. was supported by NIH Grant T32 CA09286.
2 Address correspondence and reprint requests to Dr. Ralph C. Budd, Immunobiology Program, University of Vermont College of Medicine, Given Medical Building, Burlington, VT 05405-0068. E-mail address: ralph.budd@uvm.edu
3 Abbreviations used in this paper: FADD, Fas-associated death domain protein; c-FLIP, cellular FLIP; c-FLIPL, c-FLIP long form; v-FLIP, viral FLIP; RIP, receptor-interacting protein; TRAF, TNFR-associated factor; Tg, transgenic; OVAp, OVA peptide SIINFEKL; z-VAD-fmk, z-Val-Ala-Asp(OCH3)-fluoromethylketone.
Received for publication June 23, 2004. Accepted for publication February 15, 2005.
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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-B, 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-B 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-B. 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-B activation. This provides a partial explanation of why caspase activity is required to initiate proliferation of resting T cells.
Introduction
The potential role of death receptors or downstream caspases in cell processes such as growth, differentiation, or effector function, has received growing recognition in a variety of cell types. Fas has been reported to be involved with hepatocyte regeneration (1), neurite dendrite outgrowth (2), fibroblast proliferation (3), cardiac hypertrophy (4), and T cell costimulation (5). The concept that signaling molecules in the Fas pathway are important to development is underscored by the finding that mice genetically deficient for either Fas-associated death domain protein (FADD),3 caspase-8, or cellular FLIP (c-FLIP), are all embryonically lethal due to a cardiac malformation (6). Furthermore, T cells from humans or mice lacking functional caspase-8 manifest a profound proliferation defect (7), consistent with earlier findings that caspase inhibitors block proliferation of human T cells (8, 9). Exactly how death receptors or associating caspases might promote cell growth and differentiation is unknown, as are the potential caspase target(s) for these processes.
We considered c-FLIP long form (c-FLIPL) as a caspase substrate that is potentially involved with cell growth. c-FLIPL is a homolog of caspase-8 in possessing two death effector domains but lacks a functional caspase domain due to a mutation of a critical cysteine to tyrosine in the enzymatic domain (10). As such, c-FLIPL acts as a competitive inhibitor for recruitment of caspase-8 to the death effector domain of FADD following Fas ligation (11). However, subsequent studies demonstrated the ability of c-FLIPL to bind adaptor proteins that can link to the NF-B and ERK pathways. These adaptors include receptor-interacting protein (RIP)1, TNFR-associated factor (TRAF)2, and Raf-1 (12). Increased expression of c-FLIPL in T cell lines augmented IL-2 production, and in transgenic mice, c-FLIPL enhanced T cell proliferation (13). In addition, c-FLIPL has a known caspase cleavage site at Asp376 resulting in caspase-8-dependent cleavage of full-length 55-kDa c-FLIPL to p43FLIP (14, 15, 16). The functional significance of c-FLIPL cleavage is unknown.
The current studies sought to define how increased c-FLIPL expression results in enhanced T cell growth and whether cleavage of c-FLIPL is required for this function. We observe that mice transgenic for c-FLIPL in the T cell compartment have a decreased activation threshold to Ags bearing decreased TCR affinity and are less dependent on CD28 costimulation. The increased proliferation is due to augmented expression of CD25, consistent with known increased activation of ERK and NF-B by c-FLIPL. These effects of c-FLIPL are independent of Fas expression. Finally, the ability of c-FLIP to recruit RIP1 is greatly increased after c-FLIPL is cleaved to p43FLIP. These findings suggest that, during T cell activation, c-FLIPL may be a critical caspase substrate that helps promote the activation of NF-B.
Materials and Methods
Mice
c-FLIPL was expressed transgenically in T cell compartment as previously reported (13). Briefly, FLAG-tagged mouse FLIPL cDNA was inserted into a target vector containing the -globin promoter and a downstream human CD2 locus enhancer element. Transgenic mice were screened by PCR of ear DNA using the following primers: 5' primer, 5'-GGAGCCAGGGCTGGGCATAAAA-3'; and 3' primer, 5'-GACTCACCCTGAAGTTCTCAGGATCC-3'.
Immunoblot using anti-FLIP mAb (Dave-2; Apoxis) further confirmed expression of the transgene at levels 8- to 10-fold greater than wild-type c-FLIPL. The c-FLIPL-transgenic (Tg) mouse strain has been backcrossed to C57BL/6 mice (The Jackson Laboratory) for nine generations. Mice were maintained at the University of Vermont Animal Facility (American Association for the Accreditation of Laboratory Animal Care approved), and experiments were conducted in accordance with Institutional Animal Care and Use Committee-approved protocols.
OT-1 mice bear a transgenic TCR that recognizes chicken OVAp restricted to class I MHC, Kb, and were kindly provided by Drs. F. Carbone and M. Bevan (University of Washington, Seattle, WA) (17). OT-1 mice were maintained by breeding TCR transgenic male mice to normal C57BL/6 females. Offspring were screened for the clonotype TCR using anti-V2 mAb.
CD8+ T cell purification and culture
Spleen cells after hemolysis by Gey’s solution were combined with lymph node cells followed by negative selection to enrich for CD8+ cells. Cells were incubated with Abs to CD4 (GK1.5), MHC class II (3F12), NK1.1 (PK136), and CD11b (all from BD Biosciences) for 30 min to remove, respectively, CD4+ cells, B cells, NK cells, and macrophages. Samples were washed and then incubated with goat anti-rat/mouse IgG-labeled magnetic beads (Qiagen) for 45 min followed by magnetic field separation. CD8+ T cell purity was confirmed by the flow cytometry and was routinely >90%.
T cells were activated in culture medium (RPMI 1640 supplemented with penicillin (200 μg/ml; Sigma-Aldrich), streptomycin (200 μg/ml; Sigma-Aldrich), glutamine (4 mM; Sigma-Aldrich), 2-ME (50 μM; Sigma-Aldrich), HEPES (10 mM; Sigma-Aldrich), and 8% FBS (Intergen)) using plate-bound anti-CD3 (5 μg/ml; clone 145-2C11), anti-CD28 ascites (1:500), and recombinant human IL-2 (50 U/ml; Cetus), or OVAp or low-affinity variant G4 (SIIGFEKL) in the case of OT-I mice, for 2 days. For proliferation studies, cell were pulsed at this time with [3H]thymidine for an additional 18 h before harvest. Cells propagated for longer periods were removed from anti-CD3-coated wells on day 2 and fed with fresh culture medium containing IL-2.
Cytokine ELISA
Purified CD8+ T cells were cultured at 106/ml with plastic-bound anti-CD3 (5 μg/ml; 145-2C11) and soluble anti-CD28 (1/500 dilution of ascites; 37.51) for 48 h, and 72 h. Quantification of cytokines (IL-2, IL-4, and IFN-) in cell culture supernatants was performed using a sandwich ELISA as described (18).
RNA preparation and RNase protection assay
Total RNA was prepared from cultured CD8+ T cells using Ultraspec (Biotecx) according to the manufacturer’s recommendation. Cytokine RNA levels were determined by RNase protection assay using the RiboQuant multiprobe kit (BD Biosciences). Five micrograms of total RNA was hybridized overnight with a 32P-labeled RNA probe, which had been synthesized from the multicytokine template set, after which free probe and other ssRNA were digested with RNase.
Abs and flow cytometry
Monoclonal anti-murine CD8 conjugated to Red613 was purchased from Invitrogen Life Technologies. Monoclonal anti-murine CD4 conjugated to TriColor or PE was purchased from Caltag Laboratories. Monoclonal anti-murine V2 conjugated to PE, monoclonal anti-murine CD69 conjugated to PE, monoclonal anti-murine CD80 (B7.1) conjugated to FITC, monoclonal anti-murine CD86 (B7.2) conjugated to PE, and monoclonal anti-murine H-2 Kb conjugated to biotin were purchased from BD Biosciences.
For flow cytometry, 750,000 cells were incubated in 0.1 ml of PBS containing 0.5% BSA fraction V, 0.001% (w/v) sodium azide (PBS-azide) (Sigma-Aldrich), and the Abs listed above at 4°C for 30 min. After washing with PBS-azide, cells were fixed in 1% methanol-free formaldehyde (Ted Pella) in PBS-azide. Samples were stored at 4°C until they were analyzed with a Coulter Elite flow cytometer calibrated using DNA check beads (Coulter).
AP-1-, NFAT-, and NF-B-luciferase reporter mice and luciferase activity
AP-1-luciferase transgenic mice carry the luciferase gene driven by four human collagenase 12-O-tetradecanoylphorbol-13-acetate-responsive elements, which have high affinity for the AP-1 complex, in the context of the rat minimal prolactin promoter, linked to the luciferase gene (19). The NFAT-luciferase reporter mice bear three copies of the NFAT binding sequence from the IL-2 gene linked to the luciferase gene (20). The NF-B-luciferase reporter mice have the luciferase gene controlled by two copies of B sequences from the Ig enhancer (21). Purified CD8+ T cells were activated with anti-CD3 (5 μg/ml) and anti-CD28 (1/500 dilution of ascites). At 48 h, and 72 h following activation, cells were harvested, washed with PBS, and lysed. The lysates were then analyzed using luciferin (Promega) and measured in a luminometer for 10 s. Four measurements were made for each sample. Results are presented as the mean (±SEM) with background subtracted.
Caspase activity assay
Total cellular caspase activity was quantitated using DEVD-rhodamine (Promega) according to the manufacturer’s protocol. In brief, viable cells were isolated using centrifugation over Lympholyte M (Cedarlane) and were titrated as indicated in 100 μl of culture medium, and an equal volume of DEVD-rhodamine was added to the cells according to the manufacturer’s protocol. As the DEVD substrate is cleaved by caspases, rhodamine is released and measured by a fluorescent spectrophotometer at 2 h.
Western blot analysis
Cells were washed once in ice-cold PBS and solubilized in lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10% glycerol, 2 mM DTT, protease inhibitor mixture (Complete; Boehringer Mannheim). Postnuclear lysate proteins (40 μg per lane) were separated in 12.5% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Hybond-ECL; Amersham), and blots were blocked and probed with the indicated Abs in 4% nonfat milk in TBS/0.1% Tween 20. Immunoreactive proteins were visualized using HRP-labeled conjugates (Jackson ImmunoResearch Laboratories) and ECL blotting substrate (Amersham Biosciences). Abs used were specific for caspase-8 and c-FLIP (Apoxis), and RIP1 (BD Biosciences).
Transfection studies
Human embryonic kidney 293 and 293T cells were seeded on 60-mm culture plates (3 ml/plate) the day before transfection, and transfected by the calcium phosphate method with various expression vectors. The cells were harvested 16 h after transfection and washed with PBS, and lysed with lysis buffer. After repeated centrifugation, postnuclear lysates were precleared with Sepharose 6B for 1 h and then incubated with anti-FLAG M2 agarose (Sigma-Aldrich) for 3 h. Agarose beads were washed four times with the lysis buffer. Immunoprecipitates and cell lysates were analyzed by Western blotting. 293T cells were seeded on a 24-well culture plate (0.6 ml/plate) the day before transfection, and transfected by the calcium phosphate method with various expression vectors, together with the NF-B-driven luciferase reporter plasmid and the -galactosidase plasmid. Postnuclear lysates were subjected to the luciferase assay and -galactosidase assay, which was used to normalize transfection efficiency.
Biotin-VAD-fmk caspase precipitation assay
Viable T cells (freshly isolated or day 4 T cell blasts) were incubated with 10 μM biotin-VAD-fmk (Enzyme System Products). Cell membranes were disrupted using lysis buffer containing 20 μM biotin-VAD. Lysate (600 μg) was then precleared by rocking with 40 μl of agarose beads (Santa Cruz) at 4°C for 2 h. Supernatant was then incubated with 30 μl of streptavidin-Sepharose beads (Zymed) on a rocker at 4°C overnight. Beads were washed five times in lysis buffer without Complete protease inhibitor. Beads were then boiled in loading buffer. Beads were removed by centrifugation, and immunoblot analysis was then performed on the supernatants.
Results
CD8+ T cells from c-FLIPL-Tg mice manifest increased CD28-independent proliferation to low-dose Ag peptide or low-affinity peptide variant
Although caspase-8 has been demonstrated to be required for activation of both human and mouse T cells (7, 22), the process that initiates the caspase activity is not known, nor is the caspase-8 substrate(s) that is responsible for promoting activation. We considered the possibility that c-FLIPL might provide both of these functions because it can both heterodimerize with and activate caspase-8, and is also cleaved by caspase-8 (23). We have previously observed that CD8+ T cells from mice transgenic for c-FLIPL (c-FLIPL-Tg) in the T cell compartment manifest hyperproliferation upon activation (13). We sought to extend these studies to determine the mechanism of increased proliferation, and whether caspase activity and c-FLIPL cleavage are in any way linked to these functions. As previously reported, CD8+ T cells from c-FLIPL-Tg mice had increased proliferation in response to low-dose CD3 stimulation (Ref. 13 ; Fig. 1A). At doses of anti-CD3 of 5 μg/ml or higher, control and c-FLIPL-Tg CD8+ T cells manifested similar rates of incorporation of [3H]thymidine. This reflects the fact that, although cell cycling of c-FLIPL-Tg CD8+ T cells was still faster than wild-type CD8+ cells even at higher concentrations of anti-CD3 (see Fig. 4A below), increased cell death of c-FLIPL-Tg CD8+ T cells balanced this property (A. Dohrman, unpublished observations). Hyperproliferation was also apparent even in the absence of CD28 costimulation (Fig. 1B).
FIGURE 1. Increased proliferation of c-FLIPL-Tg CD8+ T cells to low-dose anti-CD3, Ag peptide, and low-affinity peptide variant. A and B, Purified CD8+ T cells (5 x 104/well) from c-FLIPL-Tg mice or normal littermate controls (NLC) were activated with various concentrations of plate-bound anti-CD3 Ab in the presence of constant anti-CD28 (1/500 dilution of ascites) (A) or in the absence of anti-CD28 (B). Proliferation was measured by [3H]thymidine incorporation during the final 18 h of a 72-h culture period. C and D, T cells from OT-1 or OT-1 x c-FLIPL-Tg mice (5 x 104/well) were activated with either high-affinity OVAp (C) or low-affinity variant SIIGFEKL (G4) (D) in the presence of irradiated B6 spleen cells (3 x 105/well), and proliferation was measured by [3H]thymidine incorporation during the final 18 h of a 72-h culture period. These findings were consistent in five experiments.
FIGURE 4. Increased cell cycling of c-FLIPL-Tg T cells is CD25-dependent. A—C, CD8+ T cells were activated via anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) and analyzed on day 3 for DNA content by propidium iodide (A), for surface expression of CD25 (B), and DNA content gated on the CD25+ subset (C). The gate for 2N DNA was established using naive noncycling T cells. D and E, Purified CD8+ T cells from normal littermate control (NLC) mice (D) or c-FLIPL-Tg mice (E) were activated via CD3/CD28 for 3 days in the presence of the indicated concentrations of control IgG or blocking anti-CD25 antibody. The results were consistent in three experiments.
The augmented proliferation was also observed with Ag stimulation by crossing the c-FLIPL-Tg mouse to the OT-I mouse that bears a transgenic TCR reactive to OVAp restricted to H-2Kb (17). This system allowed a better definition of the effects of decreasing the MHC/peptide affinity for TCR. The OT-I TCR binds H-2Kb/SIINFEKL with an affinity of 6.5 μM, whereas the peptide variant SIIGFEKL (G4) binds with a lesser affinity of 10 μM (24). OT-1 x c-FLIPL-Tg T cells manifested increased proliferation to OVAp over a broad dose range (Fig. 1C). This was particularly pronounced with low-affinity G4, to which OT-I T cells proliferated only minimally, whereas OT-1 x FLIP-Tg T cells had a considerably stronger response (Fig. 1D). The dramatic difference in the proliferation intensity between OVAp and G4 reflects the nonlinear relation between affinity of the TCR interaction and effector function, as observed previously with OT-1 T cells (17). As noted in Fig. 1, A and C, at high doses of anti-CD3 (5 μg/ml) or OVAp (10–8 M), there was no difference in [3H]thymidine incorporation. This most likely resulted from the increased cell cycling of c-FLIPL-Tg T cells being balanced by increased death, due to increased caspase-8 activation by c-FLIPL (see Fig. 6).
FIGURE 6. Caspase activity of CD8+ T cells from c-FLIPL-Tg mice is increased and independent of Fas expression. A, Caspase activity was measured by rhodamine release from DEVD in freshly isolated CD8+ cells, and day 3 blasts were activated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) from normal littermate controls (NLC) and c-FLIPL-Tg mice. B, Caspase activation as measured by Western blot showing cleavage of caspase-8 and c-FLIPL. The levels of c-FLIPL in T cells from transgenic mice was 8- to 10-fold increased over normal littermate controls by densitometry. C and D, Absence of Fas in lpr mice does not reverse the hyperproliferation (C) or increased caspase activity (D) of c-FLIPL-Tg CD8+ T cells. Purified CD8+ cells were stimulated with the indicated concentrations of anti-CD3 (C) or 5 μg/ml anti-CD3 (D) plus anti-CD28 (1/500 dilution of ascites) as in Fig. 1A. These results were similar in four additional experiments.
Hyperproliferation of c-FLIPL-Tg CD8+ T cells is due to increased expression of CD25
IL-2 signaling represents one of the principal growth pathways for T cells. Production of IL-2 and expression of the high-affinity IL-2R-chain (CD25) were thus examined on c-FLIPL-Tg CD8+ T cells. In parallel with the differences in proliferation, IL-2 production by c-FLIPL-Tg CD8+ T cells was higher than from the equivalent population from normal littermate controls at low-dose anti-CD3, but became equivalent at higher anti-CD3 concentrations (Fig. 2A). To determine whether this completely explained the difference in proliferation, exogenous IL-2 was added to T cells activated by either low-dose anti-CD3 or with OT-I cells stimulated with G4 peptide. Although exogenous IL-2 increased the proliferation of both populations, it remained higher in the c-FLIPL-Tg CD8+ T cells (Fig. 2, B and C).
FIGURE 2. Increased production and responsiveness to IL-2 by c-FLIPL-Tg CD8+ T cells. A, Purified CD8+ T cells (106/ml) were stimulated with the indicated concentrations of plate-bound anti-CD3 and a constant amount of anti-CD28. After 48 h, supernatants were removed and analyzed for IL-2 protein by ELISA. The findings were consistent in two experiments. B, CD8+ T cells were activated with low-dose anti-CD3 (0.5 μg/ml) in the presence of the indicated concentrations of rIL-2, and proliferation was measured after 3 days. C, T cells from OT-1 or OT-1 x c-FLIPL-Tg mice were activated with G4 peptide (10–7 M) in the presence of the indicated concentrations of IL-2, and proliferation was measured after 3 days. Similar results were found in four additional experiments.
The persistence of increased proliferation of c-FLIPL-Tg CD8+ T cells despite addition of exogenous IL-2 suggested that the augmented proliferation of c-FLIPL-Tg T cells was not due solely to a difference in IL-2 production, but resulted in part also from increased sensitivity to IL-2. This possibility was investigated further through an analysis of expression of CD25 and the activation marker CD69 on c-FLIPL-Tg CD8+ T cells following TCR stimulation. As shown in Fig. 3, following activation of purified T cells via CD3/CD28, these activation markers were more prominently expressed over several days by T cells from c-FLIPL-Tg mice than from control mice. By days 3 and 4 after activation, the percentage of CD25+ cells became similar in the two groups, but this was anticipated because during this period the CD25+ cells would have overgrown any cells lacking CD25. After CD25 levels began to decline on day 6 in control CD8+ T cells, the enhanced expression of CD25 became once again apparent in the c-FLIPL-Tg CD8+ T cells. However, during the entire period of analysis, the mean fluorescence intensity of CD25 expression was higher on the c-FLIPL-Tg CD8+ T cells (Fig. 3A, right panel).
FIGURE 3. Enhanced expression of activation markers by stimulated CD8+ T cells from c-FLIPL-Tg mice. CD8+ T cells from c-FLIPL-Tg mice were stimulated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites), and surface expression of activation molecules CD25 and CD69 was measured by mean fluorescent intensity (MFI) by flow cytometry on the days indicated. Similar results were obtained when results were expressed as percent positive cells. The studies were performed three times with similar results.
The enhanced proliferative capacity of c-FLIPL-Tg CD8+ T cells was further shown to reside within the CD25+ fraction. T cells from c-FLIPL-Tg mice or normal littermate controls were activated for 3 days and then stained for expression of surface CD25, and cell cycling was defined by uptake of propidium iodide. As shown in Fig. 4, A and B, c-FLIPL-Tg T cells demonstrated an increased proportion of CD25+ cells after activation, and cell cycling was confined to the CD25+ subset. Furthermore, within the CD25+ fraction of activated T cells, there was little difference in cell cycle rates between c-FLIPL-Tg vs littermate control mice (Fig. 4C). Thus, the increased proliferation of c-FLIPL-Tg T cells was also due in part to increased expression of CD25, and rather than IL-2-independent proliferation. This was also consistent with the inhibition of proliferation of c-FLIPL-Tg T cells by blocking CD25 engagement (Fig. 4, D and E). These findings support the view that the increased cell cycling of T cells from c-FLIPL-Tg mice is due to increased signal pathways that lead to increased CD25 expression, rather than effects directly on downstream cell cycle regulators that would be independent of CD25 expression.
In contrast to the increased production of IL-2 by c-FLIPL-Tg CD8+ T cells, levels of IFN- were somewhat decreased, whereas IL-4 production was slightly increased (Fig. 5A). This was consistent at all doses of anti-CD3 tested (data not shown). This T cytotoxic 2 pattern was confirmed by RNase protection analysis (Fig. 5B). A similar pattern of cytokine production has been observed in the CD4+ subset of c-FLIPL-Tg mice (18).
FIGURE 5. T cytotoxic 2 pattern of cytokine production by c-FLIPL-Tg CD8+ T cells. Purified CD8+ T cells (106/ml) were stimulated with anti-CD3 (5 μg/ml) plus anti-CD28 (1/500 dilution of ascites)/CD28, and supernatants were examined on days 2 and 3 for production of IL-4, IL-2, and IFN-, by ELISA of culture supernatants (A), and RNase protection assay of RNA from the same cells (B). Similar results were observed in two additional experiments.
c-FLIPL-Tg CD8+ T cells exhibit enhanced caspase and NF-B activities that are independent of Fas
In considering the potential signaling pathways influenced by c-FLIPL in primary CD8+ T cells, two aspects of c-FLIPL function were examined. First, we previously observed that transfection of cell lines with c-FLIPL manifested increased ERK and NF-B activities due to the association of c-FLIPL with, respectively, Raf-1 and RIP1 (12). The second aspect was that c-FLIPL is known to associate with and actually activate caspase-8 (23). c-FLIPL is also a potential substrate of caspase-8 (14, 15, 16). We therefore investigated to what extent these pathways might be related and contribute to the phenotype of c-FLIPL-Tg T cells, and whether this required the presence of Fas.
Consistent with the ability of c-FLIPL to activate caspase-8, c-FLIPL-Tg T cells manifested more caspase activity than littermate control mice (Fig. 6A). This was apparent even in fresh resting T cells, as well as in activated day 3 blasts. These findings were supported by the observation that caspase-8 was more extensively cleaved in c-FLIPL-Tg T cells, especially in the resting state (Fig. 6B). Resting wild-type T cells contained largely full-length p55caspase-8, but following their activation, caspase-8 cleavage became progressively apparent over the subsequent 3 days. By contrast, c-FLIPL-Tg T cells demonstrated extensive cleavage of caspase-8 when freshly isolated, which did not increase as dramatically with activation as with wild-type T cells. This was likely due to increased death of c-FLIPL-Tg T cells (A. Dohrman, unpublished observations), which would tend to constantly eliminate those T cells with the highest levels of caspase activity. Because dead cells were always intentionally eliminated from these analyses, it would remove this subset and further caspase-8 cleavage would not be so apparent. Another issue is that caspase-8 can be active in its full-length form when complexed to c-FLIPL (23), so analysis of only the degree of caspase-8 cleavage may underestimate the amount of active caspase-8. The increased caspase activity in c-FLIPL-Tg T cells was corroborated by increased and early cleavage of c-FLIPL, a known caspase-8 substrate (Fig. 6B).
Neither the hyperproliferation nor the enhanced caspase activation conferred by c-FLIPL required Fas. B6 c-FLIPL-Tg mice were crossed with B6 lpr mice, and T cell proliferation was examined in response to varying doses of anti-CD3. As shown in Fig. 6C, in the absence of Fas, purified CD8+ T cells from c-FLIPL-Tg/lpr mice proliferated more extensively than those from age- and sex-matched littermate control lpr mice. This was similar to that observed earlier with c-FLIPL-Tg and wild-type mice. In parallel with these findings, c-FLIPL-Tg/lpr CD8+ T cells contained more caspase activity than lpr T cells (Fig. 6D).
The presence of caspase activity was actually required for the proliferative response of c-FLIPL-Tg CD8+ T cells, because the caspase blockers z-Val-Ala-Asp(OCH3)-fluoromethylketone (z-VAD-fmk) and QVD-OPh largely inhibited both proliferation (Fig. 7A) and CD25 expression (B) in a dose-dependent manner, both in wild-type and c-FLIPL-Tg CD8+ T cells. Similar results were observed for the CD4+ subset (data not shown). These agents were not merely toxic to lymphocytes, because there was no increase in dead cells in cultures containing caspase blockers compared with unstimulated T cells, and the delayed addition of z-VAD by even 24 h after activation resulted in substantially less inhibition of T cell growth (data not shown). Caspase activity is therefore required particularly during the initial 24 h of T cell activation. Furthermore, the augmented caspase activity of c-FLIPL-Tg T cells is at least partly responsible for their increased proliferative capacity.
FIGURE 7. Caspase-dependent proliferation and CD25 expression by T cells from c-FLIPL-Tg mice. Purified CD8+ T cells from normal littermate controls (NLC) or c-FLIPL-Tg mice were stimulated with 5 μg/ml anti-CD3 plus anti-CD28 (1/500 dilution of ascites) in the presence of the pancaspase blockers z-VAD-fmk or QVD-OPh, or controls DMSO or zFA. A, Proliferation measured by [3H]thymidine incorporation during the final 18 h of a 72 h culture period. B, Surface CD25 expression on day 3. These findings were consistent in two experiments.
Because the signal pathways that involve regulation of CD25 and cytokines include the transcription factors NF-B, AP-1, and NFAT, we examined these pathways by crossing the c-FLIPL-Tg mice with luciferase reporter mice transgenic for the DNA binding site for each of these transcription factors (19, 20, 21). Maximal luciferase activity in T cells from these mice is detectable 2–3 days after activation, the time required for resting T cells to become metabolically active, and closely parallels DNA binding by electromobility shift assay (19, 20, 21, 25) (M. Rincon, unpublished observations). Luciferase activity was measured following activation of purified CD8+ T cells with anti-CD3/CD28. An increase in NF-B activity was observed on days 2 and 3 in CD8+ T cells from the NF-B x c-FLIPL-Tg mice, and was statistically significant (Fig. 8A). This was not merely due to the greater ability of c-FLIPL-Tg T cells to produce IL-2, because the amount of anti-CD3 (5 μg/ml) was a dose at which no difference in IL-2 production was observed (see Fig. 5). Furthermore, addition of saturating levels of exogenous IL-2 yielded persistent increases in NF-B activity in c-FLIPL-Tg CD8+ T cells. By contrast, there were no significant differences in AP-1 or NFAT activity (Fig. 8A). These findings were supported by observations of greater phosphorylation and decreased levels of the NF-B inhibitor, IB, in c-FLIPL-Tg T cells compared with wild-type T cells at all time points examined (Fig. 8B). Examination of more proximal signals in the TCR pathway revealed no difference in either CD3 phosphorylation or Ca2+ flux following TCR stimulation (data not shown). These studies thus focused attention on the possible link between c-FLIPL and the NF-B pathway.
FIGURE 8. Increased NF-B activity by c-FLIPL-Tg CD8+ T cells. A, c-FLIPL-Tg mice were crossed by mice transgenic for the DNA binding sites of AP-1, NF-B, and NFAT linked to a luciferase reporter. Purified CD8+ T cells were activated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) and measured on days 1, 2, and 3 for luciferase activity. Results represent mean ± SD of three separate experiments. *, Statistically significant differences (p < 0.05). B, CD8+ T cells were used either freshly isolated or following activation with the same concentrations of anti-CD3/CD28 as in A for 2 or 3 days, and cell lysates were analyzed by Western blot for expression of phospho-IB (p-IB), total IB, and actin. The findings were consistent in three studies.
The requirement of caspase activity for T cell proliferation raised the possibility of a caspase substrate whose cleavage would promote NF-B activation. We have previously observed that, although transient transfection with both c-FLIPL and p43FLIP N-terminal fragment induces NF-B activity, caspase inhibition by z-VAD blocked NF-B activity by c-FLIPL but not by p43FLIP (26). We have also observed in transient transfection studies that c-FLIP associates with RIP1, a known activator of NF-B (12). We therefore compared the ability of c-FLIPL vs p43FLIP to recruit RIP1. 293 T cells were transfected with c-FLIPL or p43FLIP. Coimmunoprecipitation studies demonstrated that endogenous RIP1 associated with p43FLIP but not with full-length c-FLIPL (Fig. 9A). Furthermore, the association of p43FLIP with RIP1 required the presence of caspase-8 because this association did not occur in a caspase-8-low variant of 293 cells, but was restored with cotransfection of caspase-8 (Fig. 9B). Moreover, a dominant-negative form of RIP1 (RIP1559–671) was able to inhibit p43FLIP-induced NF-B activity, attesting to the view that this function of c-FLIP was indeed working via RIP1 (Fig. 9C).
FIGURE 9. Requirement of caspase-8 to promote association of RIP1 with p43FLIP and activation of NF-B. A and B, 293T cells (A) or caspase-8-low variant 293 cells (B, left panel) were transfected with FLAG-tagged caspase-8, p43caspase-8, c-FLIPL, or p43FLIP for 16 h, and cell lysates were precipitated using anti-FLAG followed by Western blot detection of RIP1. B, Right panel, Transfection of caspase-8 into caspase-8-low variant 293 cells restores association of RIP1 with p43FLIP. Coimmunoprecipitation of p43FLIP was performed as in A and B in the absence (–) or presence (+) of transfected caspase-8. C, Dominant-negative RIP1559–671 was transfected using the amounts indicated into 293T cells along with p43FLIP followed by measurement of NF-B-luciferase activity. D, Purified c-FLIPL-Tg T cells, either freshly isolated (D0) or activated with anti-CD3/CD28 plus IL-2 for 4 days (D4), were incubated for 15 min with biotin-VAD-fmk (10 μM) and then lysed. Lysates were subjected to a preclear with Sepharose beads and then incubated with avidin-Sepharose. Precipitates were analyzed by immunoblot for caspase-8, c-FLIP, and RIP1. Shown are avidin-Sepharose precipitates compared with whole-cell lysates (WCL) as a reference. E, CD8+ T cells from NF-B-luciferase mice were activated with anti-CD3 (5 μg/ml) and CD28 (1/500 dilution of ascites) for 48 h in the absence or presence of caspase blockers z-VAD-fmk or QVD-OPh (each at 100 μM) and then assayed for luciferase activity. The results were similar in two additional studies.
To further determine whether c-FLIPL and RIP1 were indeed associated with active caspases in CD8+ in T cells, fresh day 0 and day 4 c-FLIPL-Tg T cell blasts were incubated with biotin-VAD followed by lysis and precipitation of active caspases with avidin-Sepharose. Similar to the findings using DEVD-rhodamine, there was little active caspase-8 precipitated from day 0 T cells compared with day 4 blasts even though the amount of total caspase-8 was identical in the two populations (Fig. 9D). The active form of caspase-8 in day 4 T cell blasts was full-length caspase-8, which is consistent with the modeling of caspase-8 with c-FLIPL (23). In addition, very little c-FLIP was associated with active caspase-8 in day 0 lysates, but increased considerably in day 4 lysates (Fig. 9D). Of interest was that the c-FLIP associated with caspase-8 was more the cleaved p43FLIP form than full-length c-FLIPL, whereas these two forms were about equally present in the whole-cell lysates. This suggests that c-FLIPL that is in close proximity with active caspase-8 is more likely to be cleaved. In addition, RIP1 was also found associated with active caspase-8 in day 4 lysates but not in day 0 lysates. Finally, to examine the effect of caspase blockage on activation of NF-B in primary T cells, we used mice transgenic for the DNA binding site for NF-B linked to the luciferase gene (21). Purified CD8+ T cells from these mice were activated with anti-CD3/CD28 for 48 h in the absence or presence of the caspase blockers, z-VAD-fmk and QVD-OPh. As shown in Fig. 9E, caspase inhibition greatly diminished the amount of NF-B activity observed in activated primary T cells. This is consistent with the requirement of caspase-dependent cleavage of c-FLIPL to recruit RIP1, and begins to provide an explanation for the caspase requirement for T cell activation.
Discussion
c-FLIPL was originally identified as an inhibitor of cell death induced by Fas through its ability to compete with caspase-8 for recruitment to FADD during Fas ligation (10). More recently, two additional functions of c-FLIPL have been discerned. The first is the association of c-FLIPL with Raf-1, thereby promoting activation of ERK, as well as its association with TRAF2 and RIP1, promoting activation of NF-B (12). The second is the ability of c-FLIPL to heterodimerize with and activate caspase-8 (23). Although the capacity of c-FLIPL to both compete with caspase-8 for recruitment to FADD, as well as activate caspase-8 by direct heterodimerization, may seem contradictory, the current findings nonetheless suggest that at least one caspase-dependent activation pathway involves c-FLIPL as both an activator and substrate of caspase-8. Following caspase-dependent cleavage of c-FLIPL, RIP1 is recruited more efficiently to p43FLIP, thereby allowing more effective activation of NF-B.
The current findings extend in several ways our previous observations that c-FLIPL can promote T cell growth. First, it demonstrates that caspase activity is increased in c-FLIPL-Tg T cells and this is required to promote proliferation of c-FLIPL-Tg T cells, functioning largely through increased expression of CD25. Second, this is the first demonstration that activated T cells contain active caspase-8 in a full-length form and that c-FLIP and RIP1 are associated with active caspases in cycling T cells, but not in naive T cells, despite similar levels of these proteins.
Fas has been demonstrated to stimulate a variety of effector functions in several cell types, including primary T cell proliferation (5), fibroblast growth (3), hepatocyte regeneration (1), neurite outgrowth (2), and up-regulation of costimulatory molecules and cytokines by dendritic cells (27). The physiological significance of these phenomena is uncertain, because Fas-deficient lpr mice do not manifest defects in T cell proliferation, nor do they have overt developmental abnormalities in other organs systems been reported in lpr mice. The signal pathway(s) responsible for this unanticipated stimulatory property of Fas is not well described. However, in the case of T cells and neurons, a link of Fas stimulation to ERK and NF-B activation has been shown (2, 12). A more compelling case has been made for the involvement of caspase activity as a requirement for T cell activation. Not only do caspase blockers inhibit proliferation of primary human T cells (8, 9), but both murine and human T cells deficient in functional caspase-8 manifest a pronounced defect in proliferation (7, 22). Although numerous caspase-8 substrates may be important for full T cell activation, the current findings indicate that c-FLIPL is likely one of these substrates.
The finding that activated c-FLIPL-Tg CD8+ T cells express higher levels of IL-2 and surface CD25, and that the increased cycling of activated c-FLIPL-Tg T cells occurs within the CD25+ subset, supports the view that c-FLIPL-induced hyperproliferation of T cells occurs primarily via augmented IL-2 signaling and less likely through alterations of other cell cycle regulatory proteins. This is also consistent with the ability of c-FLIPL to augment activation of the ERK and NF-B pathways, which are involved with induction of IL-2 and CD25 expression (28, 29, 30).
At present, it is unclear how signals initiated by TCR ligation link to those induced by c-FLIPL. A logical consideration might be through TCR up-regulation of surface FasL, which would then bind Fas and recruit c-FLIPL. However, it is clear from the current studies that c-FLIPL also augments proliferation in Fas-deficient lpr mice. Preliminary studies identify a complex of active caspase-8, c-FLIPL, and RIP1 in lpr T cell blasts (J. Russell, unpublished observations). This does not exclude the potential involvement of other death receptors in the activation of caspases in effector T cells. Future studies are underway to examine the links between TCR signals and caspase activation.
RIP1 associates directly with a heterodimer of caspase-8/c-FLIP, although it is not certain to which component of the heterodimer it binds. Given that the form of c-FLIP associated with active caspase-8 is preferentially cleaved p43FLIP (Fig. 9D), combined with the greater association of RIP1 with transfected p43FLIP rather than full-length c-FLIPL, this suggests that p43FLIP may stabilize binding of RIP1 to the caspase-8/c-FLIP heterodimer. This immediately suggests that c-FLIPL may be an important substrate for caspase-8 during T cell activation. An additional possibility is that the form of c-FLIP in the heterodimer may influence the ability of HSP90 to stabilize RIP1 (31, 32). Thus, the amount of RIP1 complexed to caspase-8/c-FLIP may reflect differences in degradation more than association.
Other studies by us have shown a similar association of TRAF2 preferentially to p43FLIP (26). c-FLIPL has a known caspase cleavage site at Asp376, which yields p43FLIP (23). This exposes a binding site for RIP1 and TRAF2 that is presumably less accessible in full-length c-FLIPL. These findings would also serve to explain the ability of caspase inhibition to block NF-B activation by c-FLIPL but not by p43FLIP (26). Differences in caspase activity between CD8+ and CD4+ T cells might also explain why we observed increased NF-B activity in CD8+ cells from c-FLIPL-Tg mice, but did not in previous studies of the CD4+ subset from c-FLIPL-Tg mice (18). In separate studies, we observe that activated CD8+ cells from c-FLIPL-Tg mice manifest greater caspase activity than activated CD4+ cells from the same mice (A. Dohrman, unpublished observations). In addition, p43FLIP lacks the domain by which c-FLIPL activates caspase-8 (23). Thus, cleavage of c-FLIPL to p43FLIP may serve not only to recruit RIP1 and TRAF2 to promote NF-B activation, it may also impede further activation of caspase-8, to decrease the risk of promoting T cell death after activation. In this regard, it is of some interest that the viral form of FLIP (v-FLIP) is truncated, containing only the two death domains (33). v-FLIP still allows recruitment of RIP1 and TRAF2, similar to p43FLIP, but does not activate caspase-8. This could provide a survival capacity of v-FLIP.
Regulation of c-FLIPL not only in T cells but also in other cell types may strongly influence the ability to either proliferate or differentiate. Blood monocytes have very low levels of c-FLIPL and are highly sensitive to Fas-induced death, whereas dendritic cells that are derived from blood monocytes using GM-CSF and IL-4 express high levels of c-FLIPL and are highly resistant to Fas-mediated apoptosis (Ref. 34 ; R. Budd, unpublished observations). Furthermore, ligation of Fas on dendritic cells promotes up-regulation of surface CD80/CD86, class II MHC, and IL-12 production (27). A murine model of cardiac hypertrophy has also implicated a role for Fas in cardiac hypertrophy in response to hypertension (4). Interestingly, cardiac myocytes are among the highest expressors of c-FLIPL, and FLIP-null mice are embryonic lethal due to a cardiac developmental abnormality (35). Thus, c-FLIP is emerging as not only an inhibitor of Fas-induced death, but also as a promoter of cell proliferation and/or differentiation based on its ability to recruit adaptor proteins linking to the ERK and NF-B pathways. That this occurs better with cleaved p43FLIP than with full-length c-FLIPL suggests that c-FLIPL is at least one caspase-8 substrate needed for optimal T cell proliferation.
Acknowledgments
We thank Colette Charland for technical assistance with flow cytometry.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health (NIH) Grants AI36333 and AI45666 (to R.C.B.). A.D. was supported by NIH Grant T32 CA09286.
2 Address correspondence and reprint requests to Dr. Ralph C. Budd, Immunobiology Program, University of Vermont College of Medicine, Given Medical Building, Burlington, VT 05405-0068. E-mail address: ralph.budd@uvm.edu
3 Abbreviations used in this paper: FADD, Fas-associated death domain protein; c-FLIP, cellular FLIP; c-FLIPL, c-FLIP long form; v-FLIP, viral FLIP; RIP, receptor-interacting protein; TRAF, TNFR-associated factor; Tg, transgenic; OVAp, OVA peptide SIINFEKL; z-VAD-fmk, z-Val-Ala-Asp(OCH3)-fluoromethylketone.
Received for publication June 23, 2004. Accepted for publication February 15, 2005.
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