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Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.

BAX and BAK are “multidomain” proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca 2+ ] er ) that results in decreased uptake of Ca 2+ by mitochondria after Ca 2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca 2+ adenosine triphosphatase) corrected [Ca 2+ ] er and mitochondrial Ca 2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca 2+ from intracellular stores (such as arachidonic acid, C 2 -ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to “BH3-only” signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca 2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.

The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.

Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp 34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus–induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus–induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.