Damage-induced pyroptosis drives endog thymic regeneration via induction of Foxn1 by purinergic receptor activation.

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 of Foxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTPγS promotes rapid regeneration of the thymus in vivo following 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.

Activation of the zinc-sensing receptor GPR39 promotes T-cell reconstitution after hematopoietic cell transplant in mice.

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.

Attenuation of apoptotic cell detection triggers thymic regeneration after damage.

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.

Dynamics of thymus function and T cell receptor repertoire breadth in health and disease.

T cell recognition of unknown antigens relies on the tremendous diversity of the T cell receptor (TCR) repertoire; generation of which can only occur in the thymus. TCR repertoire breadth is thus critical for not only coordinating the adaptive response against pathogens but also for mounting a response against malignancies. However, thymic function is exquisitely sensitive to negative stimuli, which can come in the form of acute insult, such as that caused by stress, infection, or common cancer therapies; or chronic damage such as the progressive decline in thymic function with age. Whether it be prolonged T cell deficiency after hematopoietic cell transplantation (HCT) or constriction in the breadth of the peripheral TCR repertoire with age; these insults result in poor adaptive immune responses. In this review, we will discuss the importance of thymic function for generation of the TCR repertoire and how acute and chronic thymic damage influences immune health. We will also discuss methods that are used to measure thymic function in patients and strategies that have been developed to boost thymic function.

GCNT1-Mediated O-Glycosylation of the Sialomucin CD43 Is a Sensitive Indicator of Notch Signaling in Activated T Cells.

Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.

Fibroblastic niches prime T cell alloimmunity through Delta-like Notch ligands.

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.