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Competent T cell development relies on efficient functioning of the thymus, which is extremely sensitive to acute and chronic insults, but has a remarkable ability to regenerate. We identified that cell-specific metabolic remodeling triggers key molecular mechanisms driving this endogenous regeneration process, namely the transcription of Foxn1 by thymic epithelial cells (TECs). Firstly, following acute damage (sub-lethal total body irradiation) murine CD4+CD8+ (DP) thymocytes, which undergo high levels of death, have a disrupted glycolytic flux triggering a switch to OXPHOS, with reduced mitophagy and increased mitochondrial ROS, an effect not observed in TECs, which are relatively damage-resistant, suggesting metabolic stability confers protection. Furthermore, DPs preferentially undergo pyroptotic cell death after damage facilitated by increased pyruvate-induced TCA cycling, evidenced by an acute surge in caspase 1 cleavage compared to caspase 3. Importantly, pyroptotic thymocytes induce Foxn1 transcription in cortical TECs (cTECs) and in vitro screening revealed the purinergic receptors P2Y2 as a critical molecular target. Moreover, we translated this therapeutic strategy in vivo and demonstrated enhanced thymic regeneration after damage in mice treated with a P2Y2 agonist. Together these data describe an innovative mechanism of immunometabolic regulation of T cell development and tissue regeneration and provides a novel therapeutic strategy to enhance immune function.

The thymus is extremely sensitive to damage, resulting in attenuated T cell reconstitution and failure to elicit appropriate responses to infection, cancer and immunization. Although the thymus has a remarkable regenerative capacity, the mechanisms underlying this endogenous regeneration remain poorly understood. Previously we have identified two critical secreted factors that drive thymic regeneration, including BMP4 from endothelial cells (ECs) and IL-22 from dendritic cells (ILCs), which can be used as therapeutic regenerative strategies. Additionally, we have shown that steady-state CD4+CD8+ thymocytes (DPs) suppress levels of IL-23 (Science 336:91), a key regulator of IL-22; and as developing thymocytes undergo high levels of apoptosis, we hypothesize that apoptotic DPs mediate suppression of regenerative factors under homeostatic conditions, and damage-induced DP depletion removes this suppression, triggering thymic recovery. Here, we demonstrate a DP-specific reduction in cl-caspase 3 levels after acute injury in mice (total body irradiation, 550 cGy), and reduced Bmp4 expression in ECs co-cultured with apoptotic thymocytes, an effect rescued by apoptosis inhibition, using the pan-caspase inhibitor zVAD-FMK. We identified that apoptotic thymocytes mediate their suppression via TAM receptors in ECs and DCs, which subsequently activate Rho GTPases. Importantly, we show that inhibition of Rac1 activation increases Bmp4 and Il23 expression in ECs and DCs, respectively, and in vivo inhibition of Rac1 with EHT1864 enhances thymus cellularity in models of acute injury and age. These data describe a novel regulatory pathway that can be therapeutically targeted to boost T cell reconstitution after damage.