FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells

Results

Since previous work established that FADD and casp8 both impact autophagic signaling (25–27), we hypothesized that FADDdd T cells might display autophagic defects. Consistent with this, we recently reported defects in regulation of S6 kinase in FADDdd transgenic T cells (16), a key intermediate in autophagy regulation (30). Further, bromodeoxyuridine (BrdU) incorporation assays revealed diminished entry of FADDdd transgenic and casp8^−/− T cells into S-phase and enhanced proportions of cells bearing subdiploid DNA content, a defect particularly acute in FADDdd-expressing CD8⁺ T cells bearing an ovalbumin (OVA)-specific OT-I TCR transgene (14, 15). To investigate the morphology of activated FADDdd T cells, naïve and 3-day activated OT1 and OT1-FADDdd (OT1-FD) T cells were subjected to transmission electron microscopy (TEM) analysis (Fig. 1A, I–V). Activated OT1-FD T cells contained numerous large, double-membrane cytoplasmic vacuoles characteristic of autophagosomes (arrowheads) and a high level of vacuolation (asterisks; Fig. 1A, IV and V). These autophagosomes/autolysosomes enclosed other intracellular organelles (Fig. 1A, V), and were mostly absent from naïve subsets and only modestly present in activated OT1 T cells [Fig. 1B and supporting information (SI) Fig. S1a]. A hallmark of autophagy, the cleavage and lipidation of Atg8/LC3 allows for its deposition into autophagosomal membranes. We observed a large increase in LC3-II, the autophagosome associated isoform, in OT1-FD T cells activated for 3 days (Fig. 1C). To further expand these results, the level of autophagy was assessed in CD4⁺ T cells. FADDdd mice were crossed onto the OTII background (31), and LC3 lipidation was assessed (Fig. 1D). While OTII-FD T cells possessed increased levels of LC3-II compared with wild-type (WT) OTII cells, the highest level of LC3 lipidation occurred in OT1-FD cells, consistent with the more profound cell cycle and survival defects in FADDdd CD8⁺ T cells compared with FADDdd CD4⁺ T cells (14). Similarly, when WT and FADDdd T cells were activated in the presence or absence of Th1 and Th2 skewing media, the presence of the transgene increased the level of LC3-II and decreased the survival (Fig. S1 b and c). No decrease in survival was apparent in resting FADDdd T cells, nor did we observe differences in survival of FADDdd T cells following culture under low-serum conditions (Fig. S2 a and b). Since 48-h culture in low serum induced only modest levels of LC3-II, our data suggests that these T cells succumb to a form of death promoted by dysregulated autophagy only after induction of proliferation (Fig. S3 a and b). Treatment with the lysosomal protease inhibitors E64d and pepstatin A (pepA) for the last four hours of culture in WT and FADDdd T cells enhanced the overall detection of LC3, showing that this increase was not a consequence of defective fusion and subsequent degradation of autophagosomes within lysosomes (32) (Fig. S4). These data demonstrate that FADD represses autophagic induction in activated T cells.

To characterize the generality of this FADD signaling axis, we analyzed the extent of LC3 processing in WT and FADD^−/− murine embryonic fibroblasts (MEFs) cultured for 24 h under normal or low-serum conditions, the latter of which induces autophagy (24). While WT MEFs responded to low serum by increasing the expression of LC3-II, FADD^−/− MEFs had elevated levels under both conditions, and this was diminished by retroviral introduction of shRNA to Atg7 (Fig. 1E). Reconstitution of FADD^−/− MEFs with full-length FADD using retroviral transduction decreased basal autophagy as measured by LC3-II processing after culture in media with 10% serum (Fig. 1F). Consistent with the T cell results, treatment with E64d and pepA led to enhanced LC3-II detection in both subsets of MEFs (Fig. 1G). Using GFP-tagged LC3, we observed a much greater proportion of punctate GFP-LC3 staining under both normal and autophagic (rapamycin-treated) conditions in FADD^−/− compared with WT MEFs (Fig. S5 a and b), suggesting that the lack of FADD in MEFs led to a similar dysregulation in autophagic signaling as observed in OT1-FD, OTII-FD, and FADDdd T cells. Although not as profound as for FADDdd T cells, FADD^−/− MEFs also displayed a reduced proliferative capacity and enhanced cell death, especially following low-serum culture (Fig. S6 A and B). Similarly, FADD- and casp8-, but not RIPK1-deficient Jurkat clones (33–35), displayed enhanced levels of LC3 processing compared to parental clones (Fig. S7). We noted only weak processing of LC3 in WT Jurkat clones in several experiments, even under low serum conditions, perhaps due to hyperactive mTOR signaling that results in these PTEN-deficient cells. Even so, since we only observed the presence of LC3-II in Jurkat T cells lacking casp8 or FADD, our results are consistent with the hypothesis that both FADD and casp8 act in concert to limit general macroautophagy in a number of cell types.

At the apex of the autophagic signaling cascade, a complex of proteins, including Atg6/Beclin-1, p150, and Vps34, is essential for autophagosome formation. To assess the role of hyperautophagy in the phenotype of FADDdd T cells, CFSE-labeled OT1 and OT1-FD T cells were activated in the presence and absence of 0.8 mM 3-MA, an inhibitor of Vps34 and autophagy (36). Low doses of 3-MA dramatically rescued cycling OT1-FD T cell accumulation, as measured by CFSE dilution and live cell recovery, while higher doses completely blocked both OT1 and OT1-FD T cell proliferation (Fig. 2A, Fig. S8 (Upper), and data not shown), consistent with the reported findings that T cells require limited autophagy for productive expansion and survival (29). Increasing doses of 3-MA had a modest effect on WT OT1 proliferation but provided a near complete rescue of cell cycle progression and survival of OT1-FD T cells (Fig. 2B). 3-MA also blocked the hyperexpression of LC3-II seen in OT1-FD T cells activated for 3d (Fig. 2C). To ensure that the rescue of OT1-FD T cells by 3-MA was not due to a potential off-target effect, we used retroviral transduction of a dominant-negative form of Vps34 (Vps34-KD) to block autophagic signaling (37). At 2-, 4-, and 6-d postinfection, cultures were harvested and live T cells were assayed for Thy1.1 expression, a marker for the proportion of cells expressing the MiT retrovirus (38). Whereas infection with Vps34-KD or empty retroviruses led to only modest changes in the proportion of Thy1.1⁺ OTI T cells over time, the proportion of Thy1.1⁺ OTI-FD T cells expressing Vps34-KD increased dramatically over the course of the assay (Fig. 2D). The mean fluorescence of Thy1.1 on recovered cells was selectively enriched and the level of LC3-II was diminished in OT1-FD T cells by Vps34-KD (Fig. 2 E and F). These results demonstrate that Vps34-KD provides a selective advantage to proliferating OTI-FD T cells. As an additional approach, we used RNA interference to knock down expression of Atg7, an essential autophagy-related gene that was hyperexpressed in OT1-FD T cells, effectively reducing LC3-II to near WT levels (Fig. 2G). Using retrovirally encoded shRNA to knock down Atg7 expression, we observed an increase in the recovery of proliferating FADDdd T cells bearing shRNA-Atg7 relative to a scrambled retroviral control after culture in puromycin to enrich for retroviral transductants (Fig. 2H). Taken together, these data show for the first time that blockade of autophagic signaling rescues the survival and proliferative defects observed in FADDdd T cells.

The FADD death domain forms a complex with Atg5 to promote cell death, although the means by which this may occur remains uncertain (26). To more carefully classify the members of this complex, we made use of a highly specific tandem affinity purification (TAP) approach to characterize the in vivo binding partners of FADD in FADD^−/− MEFs. For binding-partner recovery, a TAP tag (39) encompassing an HA epitope, IgG-binding domain, TEV cleavage site, and calmodulin-binding domain was fused to full-length FADD (Fig. S8 (Lower)). Infection of FADD^−/− MEFs with TAP-FADD retrovirus and growth in 10% serum, a condition in which there is detectable levels of basal autophagy in MEFs, led to expression of TAP-FADD equal to endogenous FADD in WT MEFs (Fig. 3A). Following TAP purification (39), TAP-FADD containing complexes were analyzed by Western blotting, revealing the expected binding between FADD and Atg5 (Fig. 3B). We found that TAP-FADD also interacted with an Atg5:Atg12 covalently-linked complex, Atg16L, a protein bound to Atg5:12 on preautophagosomal membranes, and with casp8 and RIPK1, but not with Atg7, LC3, or tubulin. Demonstrating that Atg5:FADD complexes specifically contained these DISC molecules (1), anti-HA immunoprecipitation of HA-Atg5 retrovirus infected WT MEFs yielded FADD, casp8, and RIPK1 (Fig. 3C). These findings suggest that FADD is involved in tethering casp8 and RIPK1 to autophagosomal membranes, where cellular Atg5:Atg12/Atg16L complexes are found, leading to the induction of casp8 activity, a hypothesis supported by observation of colocalization of FADD and GFP-LC3 punctae (40). Consistent with previous observations that caspases are enzymatically active in non-apoptotic T cells following mitogen stimulation (41, 42), we observed that activation of WT T cells with anti-CD3 plus anti-CD28 for 36 h led to a significant increase in IETDase (casp8), but not DEVDase (casp3) activity. Much of this IETDase activity was blocked by 3-MA (Fig. 3D). Activation of OT1 T cells for 3 d in the presence of the pan-caspase inhibitor zVAD-FMK also led to increased levels of LC3-II (Fig. 3E), suggesting that casp8 activity represses autophagic signaling. These results are consistent with the hypothesis that autophagic signaling induces an interaction between Atg5:Atg12 and FADD that promotes the activation of casp8 in mitogenically stimulated live T cells (41, 42).

Since these results demonstrate the assembly of a RIPK1-containing, DISC-like structure via Atg5, we hypothesized that hyperautophagic cells lacking casp8 activity may die through a RIPK1-dependent/caspase-independent “necroptotic” pathway, similar to that observed when such cells are stimulated with death ligands (2). Consistent with this, OT1-FD T cell cycling (Fig. 4A) and proliferation (Fig. 4B) were rescued by the presence of the RIPK1-specific necroptosis inhibitor Nec-1 (43, 44), while the proportion of nonviable Annexin-V⁺ cells was reduced to WT levels (Fig. 4C). Nec-1 also dose-dependently restored live cell recovery (Fig. 4D), demonstrating that blockade of necroptotic signaling was sufficient to restore both survival and proliferation to OT1-FD T cells. As with 3-MA, Nec-1 reduced LC3 processing in OT1-FD T cells to WT levels (Fig. 4E), suggesting that RIPK1-dependent necroptotic signaling, or perhaps necroptosis itself, promotes autophagy. These results support the view that casp8 activity leads to the proteolytic inactivation of RIPK1, thereby preventing the induction of necroptosis by this DISC-associated serine/threonine kinase (45).

Given the potential that the failure to appropriately induce casp8 activity in OT1-FD T cells might lead to hyperactive RIPK1 signaling, we characterized the response of T cells bearing a T cell-specific deletion of casp8. Casp8^FL/FL mice (18) were bred onto a CD4-Cre background to promote deletion of casp8 during the double positive stage of thymocyte development, and the resulting T cells lack casp8 expression in peripheral CD4⁺ and CD8⁺ T cell subsets. T cells lacking casp8 bear a similarly diminished proliferative capacity as observed in FADDdd T cells (11), with the greatest defects noted in the CD8⁺ subset (16). Supporting the hypothesis that the hyperautophagic phenotype of FADDdd T cells is indeed due to an inability to activate casp8, casp8^−/− CD8⁺ T cells activated for 3 days possessed high levels of LC3-II, and this was reduced with addition of Nec-1 (Fig. 5A). While casp8^−/− CD8⁺ T cells failed to accumulate following mitogenic stimulation as expected (11), addition of Nec-1 to cultures restored normal proliferation and accumulation (Fig. 5 B and C). As observed for FADDdd T cells, Nec-1 also restored cell cycling and survival to casp8^−/− T cells, while knockdown of Atg7 led to increased live T cell recovery in the absence of casp8 (Figs. 5 D and E). Taken together, these data demonstrate that both FADD and casp8 act, likely in concert, to prevent casp8-independent cell death via the inhibition of autophagy, and suggest that RIPK1 activity may be proteolytically targeted by casp8 in this signaling paradigm.

We have found that autophagic signaling promotes the formation of a FADD, casp8, and RIPK1 containing complex in live, clonally expanding T cells and MEFS, and that FADD and casp8 are actively involved in the prevention of cell death through the attenuation of autophagic signaling. This view differs from previous reports suggesting that the interaction between FADD and Atg5 leads solely to cell death (26, 27). Recent work demonstrates that T cells require autophagy during clonal expansion (29), presumably due to acute bioenergetic stress or damaged organelles encountered during high-rate cell division (21). Here, we demonstrate that this process must be limited to prevent cell death. Although FADDdd-expressing Th1, Th2, and CD8⁺ T cells all displayed enhanced LC3-II processing compared with WT T cells, we previously established a much greater defect in FADDdd and casp8^−/− CD8⁺ vs. CD4⁺ T cells (14, 16). We surmise that autophagic signaling impacts CD8⁺ T cells more than CD4⁺ cells due to the enhanced proliferative capacity and/or metabolic activity of the former. Consistent with this, greater defects were observed in CD8⁺ vs. CD4⁺ subsets in Atg5^−/− mice (29).

Casp8 has been found to inhibit autophagy (25), likely through direct cleavage of RIPK1 (46). In this scenario, T cells induce autophagy in response to energetic demands, resulting in formation of a DISC-like complex including Atg5–12/Atg16L, FADD, casp8, and RIPK1. Active casp8 then cleaves RIPK1, thus constituting a negative feedback loop to limit autophagic induction (Fig. 5F). Since Nec-1 was shown to block necroptosis and autophagy induced directly by RIPK1 activity (45), we hypothesize that RIPK1 may influence autophagic signaling either directly, or perhaps indirectly as a response to necroptotic stress. Indeed, Nec-1 rescued the hyperactive autophagy and restored the cell cycle profile and survival capacity of actively dividing FADDdd and casp8^−/− T cells. It is important to emphasize that autophagic signaling is required in rapidly proliferating T cells (29), although the basis for this remains to be described. Given this, we chose to interfere with autophagic signaling using RNA interference, dominant-negative Vps34 and 3-MA, approaches unlikely to interfere with ALL macroautophagy. Indeed, under culture with higher levels of 3-MA, we observed reduced survival of proliferating WT T cells. Taken together, these results suggest that limited autophagy in proliferating T cells is beneficial, whereas unrestricted autophagy that occurs in the absence of FADD or casp8 signaling promotes T cell death.

Our results also imply that the interplay between autophagy and cell death is context dependent. We have found that blockade of hyperautophagy prevents cell death in proliferating T cells lacking FADD or casp8, whereas the inverse is true for TNF-receptor signaling (43), likely due to the direct recruitment and activation of RIPK1 in the latter case by the DISC. Since c-FLIP is similarly required for efficient T cell proliferation (12, 13), this DISC protein likely modulates the activation of casp8 during this process. Differential expression of c-FLIP during the course of an immune response may control the outcome of autophagy-induced casp8 activity, likely contributing to T cell homeostasis. While this hypothesis remains to be tested, our findings support the concept that FADD and casp8 modulate autophagic signaling in a manner vital to T cell clonal expansion.

Methods

Supplementary Material