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Prolonged lymphopenia represents a major clinical problem after cytoreductive therapies such as chemotherapy and the conditioning required for hematopoietic stem cell transplant (HCT), contributing to the risk of infections and malignant relapse. Restoration of T-cell immunity depends on tissue regeneration in the thymus, the primary site of T-cell development, although the capacity of the thymus to repair itself diminishes over its 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 and 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.

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 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.

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.

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.

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.

Efficient loading of major histocompatibility complex class II molecules with peptides requires the invariant chain (Ii) and the class II–like molecule H-2M. Recent in vitro biochemical studies suggest that H2-M may function as a chaperone to rescue empty class II dimers. To test this hypothesis in vivo, we generated mice lacking both Ii and H-2M (Ii−/−M−/−). Antigen presenting cells (APCs) from Ii−/−M−/− mice, as compared with APCs from Ii−/− mice, exhibit a significant reduction in their ability to present self-peptides to a panel of class II I-Ab–restricted T cells. As a consequence of this defect in the loading of self peptides, CD4+ thymocyte development is profoundly impaired in Ii−/−M−/− mice, resulting in a peripheral CD4+ T cell population with low levels of T cell receptor expression. These findings are consistent with the idea that H-2M functions as a chaperone in the peptide loading of class II molecules in vivo.

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.

Forkhead winged-helix transcription factor Foxp3 serves as the dedicated mediator of the genetic program governing CD25+CD4+regulatory T cell (Tr) development and function in mice. In humans, its role in mediating Trdevelopment has been controversial. Furthermore, the fate of Trprecursors in FOXP3 deficiency has yet to be described. Making use of flow cytometric detection of human FOXP3, we have addressed the relationship between FOXP3 expression and human Trdevelopment. Unlike murine Foxp3−T cells, a small subset of human CD4+and CD8+T cells transiently up-regulated FOXP3 uponin vitrostimulation. Induced FOXP3, however, did not alter cell-surface phenotype or suppress T helper 1 cytokine expression. Furthermore, onlyex vivoFOXP3+Trcells persisted after prolonged culture, suggesting that induced FOXP3 did not activate a Trdevelopmental program in a significant number of cells. FOXP3 flow cytometry was also used to further characterize several patients exhibiting symptoms of immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) with or withoutFOXP3mutations. Most patients lacked FOXP3-expressing cells, further solidifying the association between FOXP3 deficiency and immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome. Interestingly, one patient bearing aFOXP3mutation enabling expression of stable FOXP3mutprotein exhibited FOXP3mut-expressing cells among a subset of highly activated CD4+T cells. This observation raises the possibility that the severe autoimmunity in FOXP3 deficiency can be attributed, in part, to aggressive T helper cells that have developed from Trprecursors.