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, 108 (37), 15312-7

Complementary Roles of Fas-associated Death Domain (FADD) and Receptor Interacting Protein kinase-3 (RIPK3) in T-cell Homeostasis and Antiviral Immunity

Affiliations

Complementary Roles of Fas-associated Death Domain (FADD) and Receptor Interacting Protein kinase-3 (RIPK3) in T-cell Homeostasis and Antiviral Immunity

Jennifer V Lu et al. Proc Natl Acad Sci U S A.

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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
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 CD4CD8CD3+B220+. Error bars represent SEM; n = 3 of each genotype.
Fig. 3.
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 CFSEhi/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.
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.
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 (FADDREC), and blots of lysates probed with α-FADD and α-casp8. (C) Western blot of RIPK1 and RIPK3 cleavage in FADD−/− and FADDREC 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.

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