In this issue of Blood, Iovino et al describe a new function for zinc as a damage-associated ion released by dying thymocytes, triggering a signaling cascade that promotes T-cell reconstitution.1 

The production of new naïve T cells relies on their development and carefully regulated selection in the thymus. However, thymopoiesis is a fragile process sensitive to multiple insults and even normal aging as part of age-related thymic involution. Clinically, thymic fragility is most apparent after bone marrow or hematopoietic stem cell transplantation (HSCT), when impaired thymopoiesis can lead to delayed and defective immune reconstitution as well as to immune dysregulation.2 Beyond their basic immunological significance, better understanding thymic damage and repair mechanisms could identify new therapeutic opportunities to promote de novo T-cell reconstitution.

In this issue of Blood, Iovino et al uncover a dual role for zinc as an essential trace element in developing T cells as well as a signaling molecule released into the extracellular space during thymic damage to activate regeneration mechanisms (see figure).1 Zinc ions (Zn2+) are normally concentrated actively into intracellular compartments.3 Bioinformatic predictions indicate that up to 5% to 10% of the human proteome may have zinc-binding properties, and indeed zinc is critical for the function of numerous enzymes, transcription factors, and signaling molecules, among others.3 Clinically, zinc deficiency induces multiple defects, including prominent immunological abnormalities and thymic atrophy. Consistent with these observations, Iovino et al carefully documented the impact of experimental zinc deficiency on thymopoiesis in mice, showing early and high sensitivity of the thymus to zinc depletion, out of proportion to other organs.1 Perhaps not surprisingly, zinc-deficient mice had delayed T-cell recovery after thymic injury in the setting of total body irradiation with or without HSCT. However, this observation came with an interesting twist: after thymic damage, zinc ions were released from dying thymocytes into the extracellular space and triggered production of BMP4, a member of the bone morphogenetic protein family previously reported by the same group to promote regeneration of thymic epithelial cells (TECs) and T-cell reconstitution.4 Produced by radiation-resistant endothelial cells, BMP4 stimulates TECs to express the master transcription factor Foxn1, which induces expression of Notch ligands, chemokines, cytokines, and other factors critical for TEC interactions with developing T cells. BMP4 levels depended on zinc, because thymi from zinc-deficient mice had less BMP4 protein, which could, in turn, be rescued by zinc sulfate supplementation. Mechanistically, Zn2+ induced BMP4 expression in endothelial cells by triggering signaling through a G protein-coupled receptor called GPR39 previously identified as a zinc-sensing receptor.5 Thus, zinc ions functioned essentially as a damage-associated signal, adding to a list of damage-associated molecular patterns (DAMP) that can transmit information about multiple aspects of disrupted tissue integrity.

Zinc functions as a damage-associated signal after thymic damage. Zinc ions (Zn2+) are normally highly concentrated intracellularly, and zinc plays a critical role in thymopoiesis (baseline). After damage, dying thymocytes release Zn2+ into the extracellular space, activating endothelial cells via the G protein-coupled receptor GPR39. GPR39 signaling triggers the release of BMP4, which sustains regeneration of cortical and medullary thymic epithelial cells (cTEC, mTEC) to promote de novo T-cell production. Professional illustration by Patrick Lane, ScEYEnce Studios.

Zinc functions as a damage-associated signal after thymic damage. Zinc ions (Zn2+) are normally highly concentrated intracellularly, and zinc plays a critical role in thymopoiesis (baseline). After damage, dying thymocytes release Zn2+ into the extracellular space, activating endothelial cells via the G protein-coupled receptor GPR39. GPR39 signaling triggers the release of BMP4, which sustains regeneration of cortical and medullary thymic epithelial cells (cTEC, mTEC) to promote de novo T-cell production. Professional illustration by Patrick Lane, ScEYEnce Studios.

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From a therapeutic perspective, the critical requirement for zinc in T-cell development has already inspired clinical trials of zinc supplementation to promote T-cell reconstitution after autologous HSCT, with early results from small numbers of patients suggesting a potential benefit.6 Past observations documenting a high prevalence of clinical zinc deficiency in HSCT recipients provides further support to this intervention.7 However, the newly discovered function of zinc as a damage-associated signal generates new predictions that could inspire changes in this strategy. As opposed to zinc supplementation starting only after HSCT, it might be essential to “preload” recipients with zinc so that potent zinc-mediated damage signals can be released at the time of thymic injury. Interestingly, Iovino et al also explored a creative alternative strategy relying on a small molecule agonist of GPR39.1 Their preclinical data in mice suggest that this approach stimulates the critical GPR39/BMP4 regenerative cascade efficiently while bypassing zinc release as a signaling event and thus the need for zinc “preloading.”

Beyond the discovery of the zinc/GPR39 signaling axis in the thymus, several open research questions warrant further investigation. Although substantial circumstantial evidence supports a critical role for zinc-responsive GPR39 in endothelial cells that is consistent with past findings, the data do not include direct in vivo genetic evidence so far; in particular, TECs also express high levels of GPR39, and a role for GPR39 signaling in these cells cannot be ruled out. From a translational perspective, one important limitation of the preclinical data available in mice about zinc supplementation and GPR39 agonism is that they were restricted to a sublethal irradiation model of thymic injury and to models of allogeneic HSCT in which T cells were depleted from the inoculum. This strategy avoided induction of T-cell–mediated graft-versus-host disease, which can prominently affect the thymus. Thus, even if the authors showed that zinc deficiency could worsen thymic recovery in the setting of thymic graft-versus-host disease, it is unclear so far if zinc supplementation or GPR39 agonism will significantly improve thymic function in the presence of immune-mediated thymic injury. Past work from the authors showed that other important regeneration pathways are defective in the presence of thymic graft-versus-host disease, including release of interleukin-22 by innate lymphoid cells.8 Thus, zinc supplementation or GPR39 agonism will have to override the impact of other defects that curtail thymic regeneration in T-cell–rich allogeneic transplants, and more studies along these lines will be interesting. Including graft-versus-host disease as a parameter is relevant translationally because delayed T-cell reconstitution after autologous HSCT only causes relatively moderate clinical problems and because T-cell–depleted allogeneic HSCT does not represent the most commonly performed type of hematopoietic cell transplantation in patients. In terms of the overall clinical relevance of defective thymopoiesis after HSCT, another important consideration is a potential upper age limit in thymic recovery; indeed, past clinical observations in recipients of autologous HSCT have suggested markedly decreased thymic rebound in patients older than 50 years, although new effective therapeutic interventions could, in principle, change this ceiling.9 Conversely, immune-mediated damage to secondary lymphoid organs has also been reported in preclinical models and may contribute significantly to immune dysfunction after allo-HSCT in patients.10 Thus, interventions to boost T-cell production must be integrated with other approaches to remediate the complex immune dysregulation experienced by allo-HSCT recipients.

Altogether, Iovino et al provide new findings with basic immunological significance that nominate zinc as a new DAMP in the thymus.1 Furthermore, their detailed identification of signaling cascades supporting thymic regeneration informs molecular models and could inspire new therapeutic concepts after allo-HSCT and beyond.

Conflict-of-interest disclosure: Research in the Maillard laboratory is supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant R01-AI091627) and the Leukemia and Lymphoma Society (grant TRP-6583-20). E.P. is supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant F30-AI136325). I.M. has received research funding from Regeneron and Genentech and is a member of the scientific advisory board for Garuda Therapeutics. This funding is unrelated to the contents of this commentary.

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