Complementary roles of Fas-associated death domain (FADD) and receptor interacting protein kinase-3 (RIPK3) in T-cell homeostasis and antiviral immunity

Abstract

Caspase-8 (casp8) is required for extrinsic apoptosis, and mice deficient in casp8 fail to develop and die in utero while ultimately failing to maintain the proliferation of T cells, B cells, and a host of other cell types. Paradoxically, these failures are not caused by a defect in apoptosis, but by a presumed proliferative function of this protease. Indeed, following mitogenic stimulation, T cells lacking casp8 or its adaptor protein FADD (Fas-associated death domain protein) develop a hyperautophagic morphology, and die a programmed necrosis-like death process termed necroptosis. Recent studies have demonstrated that receptor-interacting protein kinases (RIPKs) RIPK1 and RIPK3 together facilitate TNF-induced necroptosis, but the precise role of RIPKs in the demise of T cells lacking FADD or casp8 activity is unknown. Here we demonstrate that RIPK3 and FADD have opposing and complementary roles in promoting T-cell clonal expansion and homeostasis. We show that the defective proliferation of T cells bearing an interfering form of FADD (FADDdd) is rescued by crossing with RIPK3(-/-) mice, although such rescue ultimately leads to lymphadenopathy. Enhanced recovery of these double-mutant T cells following stimulation demonstrates that FADD, casp8, and RIPK3 are all essential for clonal expansion, contraction, and antiviral responses. Finally, we demonstrate that caspase-mediated cleavage of RIPK1-containing necrosis inducing complexes (necrosomes) is sufficient to prevent necroptosis in the face of death receptor signaling. These studies highlight the "two-faced" nature of casp8 activity, promoting clonal expansion in some situations and apoptotic demise in others.

Fig. 1.

CD8⁺ T-cell homeostasis restored in FADDdd × RIPK3^−/− mice. (A) Spleen and lymph node cells were analyzed by flow cytometry for CD4⁺ vs. CD8⁺ populations. Numbers represent percentage of cells staining in each gate. (B) Graph displays number of CD4⁺ and CD8⁺ cells in the spleen, representative of three separate experiments. Error bars represent SEM (**P < 0.01 and ***P < 0.001 vs. WT CD8⁺). (C) Decreased CD8⁺ memory T cell population in FADDdd mice restored with RIPK3 deficiency. CD8⁺ gated splenocytes analyzed for CD44⁺CD62L⁻ (D). Thymocyte DN population shown by anti-CD4 and anti-CD8 staining; numbers represent percentage of cells per quadrant. (E) Graph displays DN4:DN3 population in thymocytes of indicated genotypes, representative of three experiments (*P < 0.05 and **P < 0.01). Error bars indicate SEM.

Fig. 2.

RIPK3 deficiency restores proliferation and survival of FADDdd CD8⁺ T cells. (A) CFSE analysis of CD8⁺ T cells treated with α-CD3 (145-2C11; 1 μg/mL) plus αCD28 (200 ng/mL). Splenocytes were stained and analyzed by cytometry after 3 d. Shown are plots for CFSE vs. cell numbers of CD8⁺ splenocytes. (B) Rescue of enhanced death phenotype of FADDdd CD8⁺ T cells by RIPK3 deficiency. Plots for CFSE vs. 7-AAD of CD8 cells; upper and lower left quadrants represent populations that have divided and are dead or alive, respectively. (C) Recovery of live CD8⁺ T cells in FADDdd × RIPK3^−/− vs. WT cultures following 3 d stimulation. Activated splenocytes treated with or without 10 μM necrostatin-1 (Nec-1) added at start of culture. Graph represents percent viable CD8⁺ cells recovered ± SEM. (D) Fold change in cell death of CD4⁺ or CD8⁺ cells upon restimulation (restim) with or without Nec-1 relative to death observed in activated cells (1 μg 1 × 10⁵ cells αCD3, 200 ng/mL αCD28) not subjected to restimulation (dotted line). Error bars represent SEM (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT restimulation). (E) Lymphoid tissue from aged (40 wk) mice of indicated genotypes. (F) FACS plots display percentage of double-negative population that are CD3⁺ B220⁺ in mice of indicated genotypes. (G) Graphs represent counts or percentage of total live cells (after RBC lysis) that are CD4⁻CD8⁻CD3⁺B220⁺. Error bars represent SEM; n = 3 of each genotype.

Fig. 3.

FADDdd × RIPK3^−/− mice exhibit normal immune response to murine hepatitis virus (MHV) infection. (A) T cells from livers of infected mice were stained with MHV-specific (S510) tetramer. Graph represents percentage of S510-positive cells of total CD8⁺ cells. (B) Nonspecific and specific target cells were labeled with CFSE^hi/low, respectively, and adoptively transferred into infected and sham mice for in vivo CTL analysis shown by FACS plots. Graph displays percent specific lysis (**P < 0.01). (C) Splenocytes from infected and sham mice were incubated with specific/nonspecific target cells for 4 h to measure in vitro CTL activity. Graph represents percent specific lysis. Considered a significant difference with respect to WT specific lysis (*P < 0.01, ***P < 0.001). (D) Livers from infected mice were harvested 7 d after infection for viral titer analysis. Error bars indicate SEM (***P < 0.001).

Fig. 4.

T-cell–intrinsic FADD and RIPK3 activity required for antiviral response to murine hepatitis virus (MHV) infection. (A) Initial splenocyte counts of infected and control mice in indicated genotypes (*P < 0.05 and **P < 0.01). (B) FADDdd × RIPK3^−/− adoptive transfer mice and controls were infected intraperitoneally with MHV. IFN-γ levels in splenocytes of infected and control mice were determined by intracellular IFN-γ staining 7 d after infection. Splenocytes were restimulated 6 h with S510 or OVA peptides and stained for CD8. IFN-γ⁺ cells were calculated by multiplying total splenocytes by percentage of IFN-γ cells (**P < 0.01 vs. infected FADDdd S510).

Fig. 5.

RIPK1/RIPK3 cleavage following TCR vs. DR ligation. (A) OT-I T cells (27) were stimulated with OVA peptide for 72 h and stimulated without or with α-Fas or left untreated (naive); immunoblots were hybridized with α-RIPK1 and α-RIPK3 to detect processing. Graphs represent percent RIPK1/3 cleavage (*P < 0.05 and **P < 0.01). (B) Reconstitution of FADD-deficient Jurkat cells (28) (FADD^−/−) with full-length FADD (FADD^REC), and blots of lysates probed with α-FADD and α-casp8. (C) Western blot of RIPK1 and RIPK3 cleavage in FADD^−/− and FADD^REC Jurkat cells; HSP90 used as loading control. RIPK1_ Cl., PARP_ Cl., cleaved RIPK1 and PARP1, respectively. (D) Parental, RIPK1^−/−; D325A RIPK1, M92G/D325A RIPK1 Jurkat cells treated with TNFα, Nec-1 (10 μM), or z-VAD-FMK (20 μM) and stained with 7AAD and annexin-V to detect death.