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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.

Hematopoietic stem cell transplant (HSCT) is a well-established therapy with curative potential for a variety of malignant and non-malignant diseases. However, delayed T cell reconstitution is an important contributor to transplant-related morbidity and mortality due to infections and malignant relapse. Optimal T cell development requires a functional thymus and many patients who could benefit from enhancing immune regeneration after HSCT have poor thymic function due to their age or their exposure to chemo- and radiation therapy. Here, we show that zinc, which is the second most abundant trace metal in the body, is crucial for thymic function and can be used to promote T cell reconstitution after HSCT. Mice that had been fed a zinc deficient diet for 3 weeks showed reduced thymic cellularity as zinc was required for the expansion and transition from DN to DP thymocytes. Conversely, supplementation of zinc in drinking water could significantly enhance T cell reconstitution after HSCT. Mechanistically, we found a switch in the bioavailability of zinc following damage, likely due to the death of thymocytes; and its release from thymocytes was able to directly stimulate the zinc-gated receptor GPR39 on endothelial cells to directly induce the production of BMP4; which we have previously identified as a key mediator of endogenous thymic regeneration. Consistent with this hypothesis, mice that had been given a pharmacologic inhibitor of BMP4 signaling did not demonstrate any improved thymic regeneration after zinc supplementation. In conclusion, we have demonstrated that zinc is important for steady-state T cell development and zinc supplementation offers a simple but innovative therapeutic strategy to improve T cell reconstitution.

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.

Although the thymus has a remarkable capacity for repair following damage, the mechanisms underlying this endogenous regeneration remain poorly understood. Our recent studies have identified two key pathways for thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 from innate lymphoid cells (ILCs), which can be utilized individually as therapeutic strategies of immune regeneration. The specific regulatory mechanisms that trigger these regeneration-associated factors after damage remain unclear. Here we detail an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in orchestrating these distinct pathways of thymic regeneration. After acute injury, Nod2−/− mice exhibited superior overall thymic regeneration, consistent with increased levels of BMP4, IL-22, and Il-23, a key regulator of IL-22 secreted by dendritic cells (DCs). Furthermore, expression of miR29, a downstream effector of NOD2, was significantly reduced in ECs and DCs after injury, and miR29 mimics suppressed Bmp4 and Il23 expression in ECs or DCs, respectively. Consistent with a non-canonical role for NOD2 in sensing RhoGTPases, we found several RhoGTPase family members were downregulated after TBI; and modulation with small molecule inhibitors targeting Rho GTPases led to robust production of BMP4 and IL-23, with enhanced thymic regeneration after acute injury. These findings provide evidence not only of a novel master regulator of endogenous thymic regeneration, but also offer a superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by infection or cytoreductive therapy.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following stress, infection and cytotoxic cancer treatments. Although there is continuous thymic involution and regeneration in response to stress and infection in the healthy state, prolonged T cell deficiency is common after profound thymic damage, such as that caused by the conditioning required for hematopoietic stem cell transplant. Our previous studies have revealed two pathways important for thymic repair, centered around the production of IL-22 and BMP4 by innate lymphoid cells and endothelial cells (ECs), respectively. However, the specific mechanisms that trigger these pathways are poorly understood. Here we reveal an unexpected role for the pattern recognition receptor Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in governing multiple pathways of thymic regeneration. Mice deficient in NOD2 showed increased intrathymic levels of L-22, IL-23 and BMP4 and increased thymus cellularity after damage caused by total body irradiation (TBI). Although NOD2 is classically defined as a bacterial sensor, members of the Rho GTPase family have been implicated to promote NOD signaling, even in the absence of pattern recognition. Consistent with this, inhibition of RhoA or Rac1 can induce the production of BMP4 by ECs in vitro, and after TBI there is a significant reduction in these two Rho GTPases. These studies not only enhance our understanding of endogenous tissue regeneration, but in identifying a master regulator of multiple pathways, offers a superior therapeutic strategy to boost thymic function in patients whose immune system has been decimated due to age, infection or cancer therapies.

We have recently described two independent mouse models in which the administration of diphtheria toxin (DT) leads to specific depletion of regulatory T cells (Tregs) due to expression of DT receptor-enhanced GFP under the control of the Foxp3 promoter. Both mouse models develop severe autoimmune disorders when Foxp3+ Tregs are depleted. Those findings were challenged in a recent study published in this journal suggesting the expression of Foxp3 in epithelial cells as the cause for disease development. By using genetic, cellular, and immunohistochemical approaches, we do not find evidence for Foxp3-expression in nonhematopoietic cells. DT injection does not lead to a loss of epithelial integrity in our Foxp3-DTR models. Instead, Foxp3 expression is Treg-specific and ablation of Foxp3+ Tregs leads to the induction of fatal autoimmune disorders. Autoimmunity can be reversed by the adoptive transfer of Tregs into depleted hosts, and the transfer of Foxp3-deficient bone marrow into T cell-deficient irradiated recipients leads to full-blown disease development.