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Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression ofFoxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTPγS promotes rapid regeneration of the thymusin vivofollowing acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration.SUMMARYThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration.Damage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes.Elevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage.Extracellular ATP release promotesFoxn1expression in cTECs via activation of P2Y2Therapeutic targeting of the P2Y2 receptor promotes thymic regeneration.

Prolonged lymphopenia represents a major clinical problem after cytoreductive therapies such as chemotherapy and the conditioning required for hematopoietic stem cell transplant (HCT), contributing toward the risk of infections and malignant relapse. Restoration of T cell immunity is dependent on tissue regeneration in the thymus, the primary site of T cell development; although the capacity of the thymus to repair itself diminishes over lifespan. However, although boosting thymic function and T cell reconstitution is of considerable clinical importance, there are currently no approved therapies for treating lymphopenia. Here we found that Zinc (Zn), is critically important for both normal T cell development as well as repair after acute damage. Accumulated Zn in thymocytes during development was released into the extracellular milieu after HCT conditioning, where it triggered regeneration by stimulating endothelial cell-production of BMP4 via the cell surface receptor GPR39. Dietary supplementation of Zn was sufficient to promote thymic function in a mouse model of allogeneic HCT, including enhancing the number of recent thymic emigrants in circulation; although direct targeting of GPR39 with a small molecule agonist enhanced thymic function without the need for prior Zn accumulation in thymocytes. Together, these findings not only define an important pathway underlying tissue regeneration, but also offer an innovative preclinical approach to treat lymphopenia in HCT recipients.KEY POINTSThymocytes release zinc after HCT conditioning is sensed by GPR39 and promotes epithelial repairPharmacologic stimulation of GPR39 promotes T cell reconstitution after HCT

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 molecular triggers of organotypic tissue repair are unknown. The thymus, which is the primary site of T cell development, is a model of tissue damage and regeneration as it is particularly sensitive to insult, but also has a remarkable capacity for repair. However, acute and profound damage, such as that caused by common cytoreductive therapies or age-related decline, lead to involution of the thymus and prolonged T cell deficiency, precipitating life-threatening infections and malignant relapse. Consequently, there is an unmet need to boost thymic function and enhance T cell immunity. Here, we demonstrate an innate trigger of the reparative response in the thymus, centered on the attenuation of signaling directly downstream of apoptotic cell detection as thymocytes are depleted after acute damage. We found that the intracellular pattern recognition receptor NOD2, via induction of microRNA-29c, suppressed the induction of the regenerative factors IL-23 and BMP4, from thymic dendritic cells (DCs) and endothelial cells (ECs), respectively. During steady-state, when a high proportion of thymocytes are undergoing apoptosis (as a consequence of selection events during T cell development), this suppressive pathway is constitutively activated by the detection of exposed phosphatidylserine on apoptotic thymocytes by cell surface TAM receptors on DCs and ECs, with subsequent downstream activation of the Rho GTPase Rac1. However, after damage, when profound cell depletion occurs across the thymus, the TAM-Rac1-NOD2-miR29c pathway is abrogated, therefore triggering the increase in IL-23 and BMP4 levels. Importantly, this pathway could be modulated pharmacologically by inhibiting Rac1 GTPase activation with the small molecule inhibitor EHT1864, leading to increased thymic function and T cell recovery after acute damage. In conclusion, our work not only represents a novel regenerative strategy for restoring immune competence in patients whose thymic function has been compromised due to cytoreductive conditioning, infection, or age; but also, identifies a mechanism by which tissue regenerative responses are triggered.

SUMMARY The thymus, which is the primary site of T cell development, is particularly sensitive to insult but also has a remarkable capacity for repair. However, the mechanisms orchestrating regeneration are poorly understood, and delayed repair is common after cytoreductive therapies. Here, we demonstrate a trigger of thymic regeneration, centered on detecting the loss of dying thymocytes that are abundant during steady-state T cell development. Specifically, apoptotic thymocytes suppressed production of the regenerative factors IL-23 and BMP4 via TAM receptor signaling and activation of the Rho-GTPase Rac1, the intracellular pattern recognition receptor NOD2, and micro-RNA-29c. However, after damage, when profound thymocyte depletion occurs, this TAM-Rac1-NOD2-miR29c pathway is attenuated, increasing production of IL-23 and BMP4. Notably, pharmacological inhibition of Rac1-GTPase enhanced thymic function after acute damage. These findings identify a complex trigger of tissue regeneration and offer a regenerative strategy for restoring immune competence in patients whose thymic function has been compromised., Graphical Abstract, In brief Delayed lymphopenia is a common feature of many cancer therapies that is predicated on poor regeneration of thymic function. Kinsella et al. identify a trigger of endogenous thymic repair, centered on the detection of apoptotic thymocytes, that can be exploited to improve T cell regeneration after immune-depleting therapies.

T cell reconstitution after transplant is critically dependent on the thymus; an inverse relationship between a transplant recipient's age and their capacity to generate T lymphocytes (in particular CD4+T cells) has been found in several studies, and thymic function pre-transplant can have a significant impact on clinical outcomes. Although the thymus has a remarkable ability to repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Despite this regenerative capacity, delayed T cell reconstitution is associated with an increased risk of infections, relapse of malignancy and the development of secondary malignancies. Therefore, there is a clinical demand for therapeutics that restore immune function after damage. Our recent studies have identified two key pathways driving thymic regeneration; centered on the secretion of BMP4 by endothelial cells (ECs) and IL-22 by innate lymphoid cells (Dudakov 2012 Science 336:91; Dudakov 2017 Blood130:933; Wertheimer 2018 Sci Immunol3:19). However, the specific regulatory mechanisms that trigger these regeneration-associated factors after damage remain unclear. Our previous work identified that the presence of homeostatic apoptotic CD4+CD8+ (DP) thymocytes, as apoptotic thymocytes form the bulk of developing T cells, suppress the production of IL-23 in dendritic cells (DCs), a key downstream mediator for IL-22, and BMP4 in ECs (Fig. 1A), and that the depletion of apoptotic thymocytes after damage precedes the production of these regenerative factors. Therefore, together with our findings that the metabolic needs of key thymus populations alter drastically following injury due to damage-induced metabolic remodeling, we hypothesized that further to the loss of DP-specific suppression, metabolic dysfunction in DPs after damage triggers mitochondrial-induced pyroptotic cell death, which can directly promote regeneration of the thymus. Consistent with this hypothesis, our preliminary data shows increased levels of cl-caspase 1 (pyroptotic caspase) and a decrease in cl-caspase 3 (apoptotic caspase) in DPs after SL-TBI (550 cGy), demonstrating a preferential induction of pyroptotic cell death in DPs after damage (Fig. 1B). Furthermore, we demonstrated an increase in extracellular lactate dehydrogenase (LDH) levels, HMGB-1 and TNF⍺[canonical damage-associated molecular patterns (DAMPs) released during ICD] acutely after damage caused by SL-TBI (Fig. 1C).Given our previous findings that stromal cells are more radio-resistant than DP thymocytes (Wertheimer 2018 Sci Immunol3:19), and evidence for mitochondrial-induced pyroptosis, we identified hyperpolarization of the mitochondrial membrane potential accompanied by increased levels of ROS in DPs, an effect not observed in TECs, suggesting metabolic stability confers protection against acute damage (Fig. 1D). Furthermore, co-culture of pyroptotic thymocytes results in increased IL12p40+ DCs and increased Foxn1 expression in TECs (Fig. 1E), strengthening our hypothesis that cell-cell communication drives thymic regeneration after damage by inducing regenerative factors as well as directly promoting TEC function via secreted factors from pyroptotic DPs. One way in which DAMPs, such as ATP, can initiate cell signaling is by the activation of cell surface purinergic receptors, including P2Y2 which is widely expressed on TECs, and here we demonstrate that in vitro treatment with ATP or P2Y2 agonist increases Foxn1 in cTECs, and P2Y2 antagonism reverses this effect (Fig 1F). As P2Y2 activation promotes Ca2+efflux from the ER, we have further demonstrated that stimulating the intracellular release of Ca2+, using tunicamycin, induced Foxn1 expression in cTECs, which was reversed upon inhibition of Ca2+release (Fig. 1G). Importantly, we demonstrate here that this pathway can be therapeutically targeted by activating P2Y2 signaling in vivo with MRS2568 or ATP enhances thymus cellularity and expands cTECs in models of acute injury (Fig. 1H&I). These findings not only reveal a novel metabolic-mediated molecular mechanism governing tissue regeneration; but also by targeting FOXN1 directly offers a potentially superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by HCT, infection or cytoreductive therapy. Disclosures No relevant conflicts of interest to declare.

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.