Antigen-mediated T cell expansion regulated by parallel pathways of death

Abstract

T cells enigmatically require caspase-8, an inducer of apoptosis, for antigen-driven expansion and effective antiviral responses, and yet the pathways responsible for this effect have been elusive. A defect in caspase-8 expression does not affect progression through the cell cycle but causes an abnormally high rate of cell death that is distinct from apoptosis and does not involve a loss of NFkappaB activation. Instead, antigen or mitogen activated Casp8-deficient T cells exhibit an alternative type of cell death similar to programmed necrosis that depends on receptor interacting protein (Ripk1). The selective genetic ablation of caspase-8, NFkappaB, and Ripk1, reveals two forms of cell death that can regulate virus-specific T cell expansion.

Fig. 1.

Reduced accumulation of caspase-8-deficient T cells. (A) Purified T cells were labeled with CFSE and cultured for 3 days (representative of 10 experiments). (B) Casp8^f/f and Casp8^f/f;Cd4Cre mice were infected with LCMV and splenic T cells were stained for intracellular IFNγ after restimulation in vitro (representative of 4 experiments). (C) OT-I;Casp8^f/f and OT-I;Casp8^f/f;Cd4Cre T cells were adoptively transferred into congenic mice, immunized, and analyzed for live CD8⁺Vα2⁺CD45.2⁺ T cells. Standard error was calculated for each condition (representative of 3 experiments).

Fig. 2.

Caspase-8 deficient T cell death is distinct from apoptosis. (A) CD8 T cells were stimulation with anti-CD3 and anti-CD28 and stained with Annexin V and 7AAD (representative of 4 experiments with each experiment normalized to the percentage of WT death at day 1). (B) Purified CD8 T cells were cultured and measured for TUNEL (representative of 9 experiments).

Fig. 3.

Loss of caspase-8 in T cells does not affect NFκB signaling. (A) Cytoplasmic and nuclear lysates from anti-CD3 and anti-CD28 stimulated cells were resolved and immunoblotted with the antibodies listed above (representative of 5 experiments). (B) Nuclear lystates were assessed for NFκB nuclear DNA-binding activity by EMSA. Supershifts were performed on lysates from 24-h stimulated cells (above) (representative of 5 experiments). (C) Gene expression levels of NFκB targets were plotted between unstimulated and stimulated Casp8^f/f and Casp8^f/f;Cd4Cre T cells. (D) CD8 T cells from Casp8^f/f, Casp8^f/f;Cd4Cre, and NFκB-deficient mice were stimulated for 3 days and analyzed for CFSE and TUNEL (representative of 3 experiments).

Fig. 4.

Caspase-8-deficient death can be rescued by necrostatin-1. Purified T cells were cultured in media alone or stimulated for 3 days in the presence of vehicle (DMSO), necrostatin-1, or 7-Cl-O-Nec-1 (representative of 10 experiments).

Fig. 5.

Ripk1 activity is inducible and inhibited by necrostatin. (A) Purified T cells were stimulated 48 h in the presence of vehicle or 7-Cl-O-Nec-1. Kinase reactions were performed with Histone H1 as the substrate (representative of 4 experiments). (B) Specific activity was determined by plotting the ratio of Histone H1 phosphorylation to the amount of immunoprecipitated Ripk1. These data are compiled from 4 experiments. (C) Purified T cells were stimulated, resolved, and immunoblotted with the antibodies listed above (representative of 12 experiments).

Fig. 6.

Ripk1 knockdown rescues a caspase-8 deficiency. (A) Ripk1 deletion of day 7 sorted OT-I T cells (purity 98%). Average for mi30 transduction was 11,640 arbitrary density units; average for Ripk1-mi30 was 359. (B) Splenocytes were gated on live GFP⁺CD8⁺Vα2⁺CD45.2⁺ T cells and plotted with the standard error for 4 mice each (representative of 3 experiments).