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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.

T cell reconstitution, which is dependent on thymus function, is significantly delayed after hematopoietic stem cell transplant; leading to morbidity and mortality due to infection and possibly even malignant relapse. Zinc, the second most abundant trace metal in the body, is crucial for thymic function: in fact, zinc deficiency of all causes lead to thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). However, the role of zinc in thymic reconstitution after chemotherapy and hematopoietic stem cell transplantation (HSCT) has not been investigated. Using a murine model of thymic function after acute damage, we found that mice showed reduced thymic cellularity after three weeks of a zinc deficient diet, and lower regenerative capacity after a sublethal dose of total body irradiation (SL-TBI) (Fig. a). Conversely, zinc supplementation in normal dietary condition caused increased thymic regeneration at time-points after a mouse model of allogeneic-HSCT (Fig. b). As a result, mice that received zinc supplementation demonstrated more naive T cells and more recent thymic emigrants in peripheral blood (Fig. c) after allo-HSCT. Mechanistically, we found that zinc supplementation could stimulates the proliferation of thymic epithelial cells (TECs), which are the main cell type responsible for supporting for T-cell development, likely via induction in the production of BMP4, a pro-regenerative cytokine produced by endothelial cells (EC) (Fig. d). In fact, treatment with the BMP-receptor antagonist dorsomorphin abrogated the effect of zinc supplementation (Fig. e), and ex vivo propagated ECs (exECs) were directly induced to produce BMP4 when stimulated in vitro for 24 hours with zinc sulfate (Fig. f). Following damage there is a switch in the availability of zinc from inside the cellular fraction to the extracellular fraction (Fig g), suggesting that zinc is normally used and stored in developing T-cells but after damage they release zinc in the extracellular space after cell death due to TBI. This release is enough to trigger the production of regenerating factors from radio resistant cells, such as EC. Consistent with this, we found higher levels of intracellular zinc in thymocytes from zinc-supplemented mice before TBI and higher levels of zinc in the extracellular fraction after damage (Fig h). Moreover, while there was no difference in BMP4 expression in cocultures of exEC in presence of SN from control and zinc-treated mice at day 0, BMP4 levels increased in the presence of SN harvested from mice that had previously received TBI (Fig i). In conclusion, we herein demonstrate that zinc is important for thymic regeneration, and zinc supplementation offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT.

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.

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.

The thymus is not only extremely sensitive to damage but also has a remarkable ability to repair itself. However, the mechanisms underlying this endogenous regeneration remain poorly understood, and this capacity diminishes considerably with age. We show that thymic endothelial cells (ECs) comprise a critical pathway of regeneration via their production of bone morphogenetic protein 4 (BMP4) ECs increased their production of BMP4 after thymic damage, and abrogating BMP4 signaling or production by either pharmacologic or genetic inhibition impaired thymic repair. EC-derived BMP4 acted on thymic epithelial cells (TECs) to increase their expression of Foxn1, a key transcription factor involved in TEC development, maintenance, and regeneration, and its downstream targets such as Dll4, a key mediator of thymocyte development and regeneration. These studies demonstrate the importance of the BMP4 pathway in endogenous tissue regeneration and offer a potential clinical approach to enhance T cell immunity.

Prolonged T cell reconstitution after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Therefore, strategies to enhance thymic reconstitution in allo-HSCT recipients are clinically desirable, although currently limited. Zinc, the second most abundant trace metal in the body, plays an important role in T cell homeostasis and thymic function. In a mouse model of allo-HSCT (Fig. a), we demonstrated that zinc supplementation can significantly improve thymic regeneration (Fig. b). Importantly, these findings in thymus were translated to the periphery as mice that received zinc supplementation showed increased numbers of naïve T cells as well as increased recent thymic emigrants (demonstrated using RAG2-GFP BM donors) model (Fig. c-d) 5-8 weeks after allo-HSCT. We have previously demonstrated that endothelial cells (EC), which are extremely resistant to damage, can promote endogenous thymic regeneration after acute injury via their production of BMP4, a growth factor that targets thymic epithelial cells (TECs), a key population crucial for T cell development. Interestingly, when stimulated in vitro for 24 hours with zinc sulphate, ECs could be directly induced to produce BMP4, but not when exposed to increased zinc import with the cell-permeable zinc pyrithione (Fig. e). This latter finding suggests a role for extracellular zinc in stimulating the endogenous response to damage. To explore this, we first measured the content of zinc in whole mouse thymus by mass spectrometry. Interestingly, when we examined a lysate of the entire thymus, total zinc concentration sharply declined early after total body radiation (TBI), followed by a steady increase that mirrored the reconstitution of the thymic cellularity (Fig. f). However, if we looked at zinc in the extracellular fraction of thymic dissociation (referred to as supernatants, SN), we saw that zinc increased significantly after TBI, revealing an inverse correlation with thymic cellularity (Fig. g), and providing a rationale for how zinc might contribute to the endogenous regenerative response. Given these findings, we hypothesized that zinc is normally used and stored in the T-cell precursors, a population of highly-replicating cells that account for approximately 98% of thymic cellularity in young mice and require the import of intracellular zinc for their proliferation. T-cell precursors are radio-sensitive and might release zinc in the extracellular space after cell death due to TBI, thereby triggering the production of regenerating factors from radio resistant cells, such as EC. Zinc supplementation could help this loop by increasing endogenous zinc levels. This hypothesis was confirmed when we co-cultured EC in presence of thymic SN there was no difference in BMP4 expression in cocultures with SN from control and zinc-treated mice at day 0, whereas BMP4 increased in presence of SN harvested from mice that had previously received TBI and even more when mice also received zinc supplement (Fig h). In conclusion, our findings demonstrate that zinc supplementation can improve T-cell regeneration in mice receiving allo-HSCT by reinforcing endogenous mechanisms of thymic regeneration. These results could be readily clinically translated into better outcomes for recipients of allo-HCT. Figure Disclosures No relevant conflicts of interest to declare.

Although the thymus has a remarkable capacity for repair following acute injury, such as that caused by the conditioning required for successful hematopoietic cell transplant (HCT), the mechanisms underlying this endogenous regeneration remain poorly understood. Delayed T cell reconstitution occurs following thymus insult and can exceed more than a year post-transplant due to a delay in full recovery of thymic output, function and T cell repertoire. Therefore, strategies to enhance T cell reconstitution post-transplant represents a rational approach to significantly improve the overall outcome of allo-HCT. We propose that enhancing thymic function will boost T cell reconstitution and substantially increase immune responses following allo-HCT. Our recent studies have identified two critical pathways that govern thymic regeneration; centered on secretion of BMP4 by endothelial cells (ECs) and IL-22 by innate lymphoid cells (Dudakov 2012 Science 336:91; Dudakov 2017 Blood 130:933; Wertheimer 2018 Sci Immunol 3:19). However, the specific regulatory mechanisms that trigger these regeneration-associated factors (RAFs) after damage remain unclear. Given that our prior work revealed that the presence of DP thymocytes suppresses the production of RAFs like IL-23, a key downstream mediator of IL-22; and the high basal rate of thymocyte apoptosis, as apoptotic thymocytes form the bulk of developing T cells, we hypothesized that apoptotic DP thymocytes were mediating this suppression of RAFs under homeostatic conditions. Upon injury, loss of DP thymocytes leads to reduced apoptotic signaling and reduced suppression of RAFs, triggering thymic recovery (Fig 1A). Consistent with this hypothesis, our preliminary data shows a significantly reduced number of apoptotic thymocytes after total body irradiation (TBI, 550 cGy), as measured by cleaved caspase 3 levels (Fig 1B). Additionally, co-culture of apoptotic thymocytes results in reduced Bmp4 expression in ECs, which is rescued by inhibition of thymocyte apoptosis using the pan-caspase inhibitor zVAD-FMK (Fig 1C). One way in which apoptotic thymocytes could induce this suppression of RAFs is via TAM receptor activation, which is supported by our data demonstrating increased Bmp4 expression in ECs treated with a pan-TAM receptor antagonist and subsequently co-cultured with apoptotic thymocytes (Fig 1D). Interestingly, TAM receptors can activate Rac1, a Rho GTPases involved in actin cytoskeletal rearrangement; converging neatly on our previous data showing that inhibition of Rac1 with small molecule inhibitors led to robust induction of Bmp4 and Il23 expression. Therefore, we propose that in steady-state, apoptotic thymocytes activate TAM receptors on ECs and DCs and induce intracellular activation of Rac1, which ultimately suppresses the production of BMP4 and IL-23; but after damage, when the number of apoptotic thymocytes drops precipitously, this suppression is abrogated, allowing for thymic regeneration (Fig 1E). Importantly, we demonstrate here that this pathway can be therapeutically targeted, as inhibition of Rac1 in vivo with EHT1864 enhances thymus cellularity in models of acute injury (Fig. 1F), and age (Fig. 1G). As post-transplant T cell deficiency is associated with an increased risk of infections, relapse of malignancy, and the development of secondary malignancies, identifying molecular targets to enhance thymic recovery will aid in the development of therapeutics with imminent clinical need. These findings not only reveal a novel molecular mechanism governing tissue regeneration, but also offer a potentially superior therapeutic strategy for boosting thymic regeneration and T cell reconstitution after damage such as that caused by allo-HCT, infection or cytoreductive therapy. Disclosures No relevant conflicts of interest to declare.

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.

Delayed T cell recovery after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Optimal immune reconstitution requires a functional thymus, however, at the present time strategies to improve thymic reconstitution are limited. Zinc (Zn) is the second most abundant trace metal in the body, binding to more than 300 proteins. Zn deficiency is a clinical condition causing immunosuppression and thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). Furthermore, the role of Zn in tissue regeneration after damage has been clearly demonstrated in liver, skin, and intestinal diseases. In a pilot clinical trial, we previously demonstrated that patients receiving oral Zn supplementation after autologous HSCT showed increased thymic-dependent T cell reconstitution in the absence of adverse clinical events. Although a clear clinical benefit was observed, the mechanisms underlying this process are poorly understood. Thus, we used a murine model to evaluate the effect of Zn supplementation in thymic reconstitution after acute damage. Using a model of thymic damage caused by sub-lethal total body irradiation (SL-TBI, 550 cGy), we found that mice that received Zn supplementation demonstrated increased thymic cellularity when compared to untreated age-matched mice (Fig. 1a). Importantly, this finding was also confirmed in a clinically-applicable model of MHC-matched allogeneic HSCT (Fig. 1b). We have previously demonstrated endothelial cells (EC), which are extremely resistant to damaged, are able to trigger thymic endogenous reconstitution after damage by producing regenerative factors such as BMP4, which targets thymic epithelial cells (TECs), a key population crucial for T cell development. Interestingly, in our model of Zn administration, we found an increase in the number of regeneration-initiating ECs (Fig. 1c), and increased proliferation of TECs (Fig. 1d), which can occur in response to BMP4. Consistent with the hypothesis that Zn supplementation is activating the BMP4 pathway, thymic levels of BMP4 were increased at day +10 in zinc-treated mice (Fig. 2a, 2b), and, when stimulated in vitro for 24 hours with Zn sulfate, ex vivo propagated ECs (exECs) were directly induced to produce BMP4 (Fig. 2c), suggesting a likely mechanism by which Zn supplementation promotes thymic reconstitution. EC also express on their surface the Zn receptor GPR39 (Fig 3a), which is able to translate extracellular Zn signals in intracellular changes, and upregulate GPR39 expression after damage (Fig 3b). In conclusion, we demonstrate a mechanism by which zinc supplementation can improve thymic function and offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT.

Intestinal microbes provide multicellular hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community. Regulatory T cells (Treg cells) expressing transcription factor Foxp3 have a key role in limiting inflammatory responses in the intestine. Although specific members of the commensal microbial community have been found to potentiate the generation of anti-inflammatory Treg or pro-inflammatory T helper 17 (TH17) cells, the molecular cues driving this process remain elusive. Considering the vital metabolic function afforded by commensal microorganisms, we reasoned that their metabolic by-products are sensed by cells of the immune system and affect the balance between pro- and anti-inflammatory cells. We tested this hypothesis by exploring the effect of microbial metabolites on the generation of anti-inflammatory Treg cells. We found that in mice a short-chain fatty acid (SCFA), butyrate, produced by commensal microorganisms during starch fermentation, facilitated extrathymic generation of Treg cells. A boost in Treg-cell numbers after provision of butyrate was due to potentiation of extrathymic differentiation of Treg cells, as the observed phenomenon was dependent on intronic enhancer CNS1 (conserved non-coding sequence 1), essential for extrathymic but dispensable for thymic Treg-cell differentiation. In addition to butyrate, de novo Treg-cell generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of histone deacetylase (HDAC) inhibition, but not acetate, which lacks this HDAC-inhibitory activity. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory mechanisms.

The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T(reg) cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400-800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T(reg) cell biology.

Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by common cancer therapies such as cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Despite this capacity, prolonged T cell deficiency is a major clinical hurdle in recipients of hematopoietic stem cell transplantation (HSCT) and can precipitate high morbidity and mortality from opportunistic infections, and may even facilitate malignant relapse. Our recent studies have revealed that innate lymphoid cells (ILCs) and endothelial cells (ECs), through their production of the regeneration-associated factors (RAFs) IL-22 and BMP4, respectively, have profound reparative effects in the thymus after acute injury; and can be utilized individually as therapeutic strategies of immune regeneration (Dudakov 2012 Science 336:91; Dudakov 2017 Blood 130:933; Wertheimer 2018 Sci Immunol 3:19). These two pathways act by stimulating thymic epithelial cells (TECs), a heterogeneous population of stromal cell in the thymus critical for thymopoiesis. However, the regulation of these endogenous regenerative responses is still 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. Analysis of thymic recovery following acute injury in mice deficient for NOD2 revealed increased intrathymic BMP4 and IL-23 (Fig. 1a), a key regulator of IL-22 production, and commensurate improved ability to regenerate (Fig. 1b). Although NOD2 is expressed ubiquitously across all populations within the thymus, in regeneration-initiating DCs and ECs, but not thymocytes (damage-targets), we identified a specific reduction in the expression of miR29c after damage, which previous reports suggest could mediate NOD2-induced suppression of IL-23 (Brain 2013 Immunity 39:521). Consistent with these findings, miR29c expression was decreased in the thymus of NOD2-deficient mice (Fig. 1d), and overexpression of miR29c in either ECs or DCs reduced their expression of Bmp4 or Il23, respectively (Fig. 1e). Canonical ligands for NOD2 are peptidoglycans found in the cell wall of bacteria; however, these are unlikely to serve as a NOD2 activator in the thymus since it is typically thought of as a sterile organ. One recently described alternate function of NOD2 is as a cytosolic sensor of activated Rho GTPases. The Rho GTPase family is responsible for a wide range of physiological processes, including the intrathymic regulation of b-selection and positive selection, and inhibition of Rho GTPase signaling is of considerable clinical interest. Consistent with a role in suppressing regeneration, unbiased transcriptome analysis revealed significant downregulation of many members of the RhoGTPase family after damage, corresponding to the increase in production of RAFs. Importantly, suggestive of a potential clinical application, pharmacological suppression of RhoGTPase in vitro significantly induced the expression of Bmp4 in ECs, and Il23 in DCs (Fig. 1f), as well as suppressing the expression of miR29c (Fig. 1g). Although several pathways have been described as contributing to endogenous thymic regeneration, the specific mechanisms regulating their induction has been poorly understood. Here we reveal a common mechanism triggering production of multiple distinct regeneration pathways such as those centered on production of BMP4 and IL-22. Therefore, the mechanistic and pre-clinical studies described not only define an important regulatory mechanism governing endogenous tissue regeneration, but could also offer an innovative therapeutic strategy to boost thymic function and T cell reconstitution in recipients of allo-HSCT, as well as for individuals with T cell deficiencies due to aging, autoimmune diseases, genetic causes, infectious disease, shock, radiation injury (nuclear accident, terrorism) and common cancer treatments such as chemo- and radiation-therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Delayed T cell reconstitution after allogeneic hematopoietic stem cell transplant (allo-HSCT) is an important contributor to transplant-related morbidity and mortality due to infection and malignant relapse. Optimal T cell recovery requires a functional thymus, and strategies to enhance T cell reconstitution have the potential to improve overall outcome in allo-HSCT recipients, however, at the present time such strategies are limited. Hence one of the most significant clinical challenges is the need for rapid regeneration of thymopoiesis following induced immunodepletion and transplantation. Zinc is the second most abundant trace metal in the body, binding to more than 300 proteins involved in DNA synthesis and repair, gene transcription, cell proliferation as well as differentiation and apoptosis. Zinc deficiency (ZD) is a clinical condition causing immunosuppression and thymic atrophy with a consequent reduction in the number of circulating recent thymic emigrants (RTEs). Furthermore, mild ZD is one of the causes of the reduction in thymic function in the elderly and the role of zinc in tissue regeneration after damage has been clearly demonstrated in liver, skin, and intestinal diseases. In a pilot clinical trial, we demonstrated that patients receiving oral zinc supplementation after autologous HSCT showed increased thymic-dependent T cell reconstitution in the absence of adverse clinical events (Iovino 2018, Leuk Res). Although a clear clinical benefit was observed, the mechanisms underlying this process are poorly understood. Thus, we used a murine model to evaluate the effect of zinc supplementation in thymic reconstitution after acute damage. Using a model of thymic damage caused by sub-lethal total body irradiation (SL-TBI, 550 cGy), we found that mice that received zinc supplementation demonstrated increased thymic cellularity when compared to untreated age-matched mice (Fig. 1a). Importantly, this finding was also confirmed in a clinically-applicable model of MHC-matched allogeneic HSCT (Fig. 1b). We have previously demonstrated endothelial cells (EC), which are extremely resistant to damaged, are able to trigger thymic endogenous reconstitution after damage by producing regenerative factors such as BMP4, which targets thymic epithelial cells (TECs), a key population crucial for T cell development (Wertheimer 2018, Science Immunol). Interestingly, in our model of zinc administration, we found an increase in the number of regeneration-initiating ECs (Fig. 1c), and increased proliferation of TECs (Fig. 1d), which can occur in response to BMP4. Consistent with the hypothesis that zinc supplementation is activating the BMP4 pathway, when stimulated in vitro for 24 hours with supraphysiological doses of zinc sulfate, ex vivo propagated ECs (exECs) were directly induced to produce BMP4 (Fig. 1e), suggesting a likely mechanism by which zinc supplementation promotes thymic reconstitution. In conclusion, we demonstrate a mechanism by which zinc supplementation can improve thymic function and offers an innovative therapeutic strategy to improve T cell reconstitution in patients receiving allo-HSCT. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

If T cells require specific interactions with MHC-bound peptides during positive selection, then the specificities of T cells selected by one peptide should be distinct from those selected by another. We have examined positive selection of CD4 T cells in four strains of mice, each overexpressing a different peptide–1-A b (A b ) complex. We show that a subset of CD4 T cells is selected by the overexpressed peptide and that the specificities of the CD4 T cells, as measured by reactivity to wild-type antigen-presenting cells, vary greatly depending on which peptide is overexpressed. These differences in specificity are mediated through positive selection not negative selection. Each of the four peptide–A b complexes appears to adopt a different conformation, and these differences correlate with the differences in reactivity. Our results suggest that individual peptide–MHC complexes positively select different subsets of self-MHC-reactive T cells and that the conformation of the peptide–MHC complex may contribute to this process.

We report a strategy for high through-put sequence analyses of large MHC class II-bound peptide repertoires which combines automated electrospray ionization tandem mass-spectrometry with computer-assisted interpretation of the tandem mass spectra using the algorithm SEQUEST. This powerful approach discerned 128 peptide sequences displayed by the murine MHC class II molecule I-Abin activated B cells and macrophages, including a surprisingly large number of peptides derived from self cytosolic proteins. Mice lacking the chaperone molecule H-2M were used to generate T cells specific for selected self peptides. Functional T cell analyses of ex vivo antigen-presenting cells indicated that peptides originating from cytosolic proteins are efficiently presented by splenic and thymic dendritic cells, but less so by resting B cells or thymic cortical epithelial cells. These results suggest that central tolerance to at least some MHC class II-bound self peptidesderived from cytosolic proteins exists in vivo.

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high-avidity interaction of T cell receptors with the endogenous peptide: MHC class II complexes expressed on bone marrow (BM)-derived antigen-presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of E alpha52-68: I-A(b) and class II-associated invariant chain peptide (CLIP): I-A(b) complexes in thymic epithelial cells and in the bone-marrow derived splenic APC, i.e. B cells, was studied using YAe and 30-2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM-derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of E alpha52-68: I-A(b) complexes in thymic epithelial cells is approximately 5-10% of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I-A(b) complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.

To define the intracellular site of assembly of endogenous peptide-MHC class II complexes, an immunochemical approach was undertaken employing a monoclonal antibody specific for an endogenous peptide-class II complex in combination with subcellular fractionation. Here, we show that newly synthesized MHC class II molecules, upon exit from the Golgi, are delivered into a dense endocytic compartment (MIIC) distinct from late endosomes and lysosomes. Endogenous peptide-class II complexes are initially formed in this compartment and subsequently traffic through late endosomal vesicles prior to cell surface expression. Exogenous antigen delivered via immunoglobulin receptors is targeted to MIIC en route to lysosomes after passing through early and late endosomes. Processing of an endocytosed antigen was observed in this compartment. Our results suggest a specific role for MIIC in the processing of endogenous and exogenous proteins as well as the assembly of peptide-MHC class II complexes.

In the course of infection or autoimmunity, particular transcription factors orchestrate the differentiation of T(H)1, T(H)2 or T(H)17 effector cells, the responses of which are limited by a distinct lineage of suppressive regulatory T cells (T(reg)). T(reg) cell differentiation and function are guided by the transcription factor Foxp3, and their deficiency due to mutations in Foxp3 results in aggressive fatal autoimmune disease associated with sharply augmented T(H)1 and T(H)2 cytokine production. Recent studies suggested that Foxp3 regulates the bulk of the Foxp3-dependent transcriptional program indirectly through a set of transcriptional regulators serving as direct Foxp3 targets. Here we show that in mouse T(reg) cells, high amounts of interferon regulatory factor-4 (IRF4), a transcription factor essential for T(H)2 effector cell differentiation, is dependent on Foxp3 expression. We proposed that IRF4 expression endows T(reg) cells with the ability to suppress T(H)2 responses. Indeed, ablation of a conditional Irf4 allele in T(reg) cells resulted in selective dysregulation of T(H)2 responses, IL4-dependent immunoglobulin isotype production, and tissue lesions with pronounced plasma cell infiltration, in contrast to the mononuclear-cell-dominated pathology typical of mice lacking T(reg) cells. Our results indicate that T(reg) cells use components of the transcriptional machinery, promoting a particular type of effector CD4(+) T cell differentiation, to efficiently restrain the corresponding type of the immune response.

The enzymes that degrade proteins to peptides for presentation on MHC class II molecules are poorly understood. The cysteinal lysosomal proteases, cathepsin L (CL) and cathepsin S (CS), have been shown to process invariant chain, thereby facilitating MHC class II maturation. However, their role in Ag processing is not established. To examine this issue, we generated embryonic fibroblast lines that express CL, CS, or neither. Expression of CL or CS mediates efficient degradation of invariant chain as expected. Ag presentation was evaluated using T cell hybridoma assays as well as mass spectroscopic analysis of peptides eluted from MHC class II molecules. Interestingly, we found that the majority of peptides are presented regardless of CL or CS expression, although these proteases often alter the relative levels of the peptides. However, for a subset of Ags, epitope generation is critically regulated by CL or CS. This result suggests that these cysteinal proteases participate in Ag processing and generate qualitative and quantitative differences in the peptide repertoires displayed by MHC class II molecules.

The role of self-peptides in positive selection of CD4 + T cells has been controversial. We show that some self-peptides are presented by the MHC class II molecule I-A b in mice lacking Ii or H-2M but not in mice expressing a transgene-encoded peptide fused to I-A b . In experiments using specific antibodies to block selection, these low-abundance self-peptides were implicated in the positive selection of some CD4 + T cells in H-2M −/− mice. However, all three mutant backgrounds failed to positively select two class II–restricted transgenic T cell receptors. Our findings suggest that minor components of the self-peptide repertoire can contribute to positive selection of a significant number of CD4 + T cells. In addition, the data suggest that T cell receptor repertoires selected in wild-type mice and in mice displaying limited spectra of self-peptides are distinct.

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP: MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP: MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Ii peptide bound to the peptide-binding groove.

We have cloned a cDNA encoding a receptor identical to the native Mr 80,000 glycoprotein that binds interleukin (IL) 1 alpha and -beta in murine T cells. Chinese hamster ovary (CHO) cells transfected with this T-cell IL-1 receptor (IL-1R) [CHO(IL-1R)] cDNA express approximately 100,000 IL-1Rs per cell, compared to the less than 100 receptors present on control CHO cells. For two functional responses to IL-1, prostaglandin synthesis and cytokine secretion, CHO(IL-1R) cells were 1000 times more sensitive to IL-1 alpha than were control CHO cells. Northern blot analysis and antibody precipitation demonstrated that one of the cytokines induced was granulocyte colony-stimulating factor and that mRNA levels for this cytokine were increased in CHO(IL-1R) cells by IL-1 alpha concentrations that had no effect on control cells. To establish the role of the recombinant receptors in signal transduction, an IL-1R cDNA modified by deletion of the predicted cytoplasmic domain was expressed in the CHO cell line termed CHO(IL-1R delta CT). CHO(IL-1R delta CT) cells expressed approximately 100,000 high-affinity IL-1 binding sites per cell, but these cells were less sensitive than control lines to IL-1, as measured by prostaglandin and cytokine release. These results show that the IL-1R cDNA encodes the entire functional receptor and that the cytoplasmic domain is required for signal transduction but not ligand binding.