Production of BMP4 By Endothelial Cells Is Crucial for Endogenous Thymic Regeneration
Authors: Wertheimer Tobias , Velardi Enrico , Brede Christian , Xiao Shiyun , Kloss Christopher C , Beilhack Andreas , Manley Nancy , Butler Jason M. , Rafii Shahin , van den Brink Marcel , Dudakov Jarrod A 
Published: October 2019

Abstract
Endogenous thymic regeneration is a crucial function that allows for renewal of immune competence following immunodepletion caused by cytoreductive chemotherapy or radiation; however, the mechanisms governing this regeneration remain poorly understood. Moreover, 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. We have recently described a central role for group 3 innate lymphoid cells (ILC3) in a complex cellular and molecular network that drives endogenous thymic regeneration (Dudakov 2012 Science 336:91). Although IL-22 contributes considerably towards thymic regeneration and mice deficient for IL-22 lag behind WT controls in recovery of thymic function, there is still some tissue regeneration in these mice, suggesting that other regeneration pathways also play a role. Unlike other lymphoid cells, ILC3 were extremely radio-resistant with little if any depletion of cells after even lethal doses of total body irradiation (TBI). However, comprehensive analysis of all thymus-resident cell subsets revealed that ILCs were not the only damage-resistant population in the thymus as endothelial cells (ECs) were also remarkably resistant to damage in multiple clinically relevant models of acute tissue injury including corticosteroids, chemotherapy and sublethal total body irradiation (SL-TBI, 550cGy) (Fig. 1a).
Thymopoiesis is dependent on the close interaction between developing thymocytes and the non-hematopoietic stromal microenvironment, which includes highly specialized thymic epithelial cells (TECs) and ECs. While the role of TECs has been well studied, the contribution of ECs to thymopoiesis and thymic regeneration remains largely unclear. Here we demonstrate that rather than just being passive conduits that deliver oxygen and nutrients, ECs are active participants in organ function producing distinct paracrine factors that orchestrate thymic repair. Using a technique whereby ECs are transduced with the adenoviral gene E4ORF1 - ECs could be expanded ex vivo (exEC) and, when administered to mice after SL-TBI, significantly boost recovery of thymic function (Fig. 1b). Intriguingly, this trophic effect was only observed when exEC were derived from the thymus but not when they were derived from heart or kidney (Fig. 1b). Mechanistically, in vivo administration of exEC(Thy) induced the expression by TECs of Foxn1 (Fig. 1c), a key transcription factor required for thymus organogenesis, maintenance and regeneration. In vitro co-culture assays revealed that conditioned media (CM) from exEC derived from the thymus, but not the heart or the kidney, could induce Foxn1 expression by TECs (Fig. 1d), in addition to the FOXN1 target genes Kitl and Dll4; a Notch ligand itself critical for T cell development. These findings suggest that thymus-derived exEC produce a soluble factor that contributes toward thymic regeneration via activation of Foxn1. Transcriptome analysis of highly purified thymic ECs after SL-TBI identified that, among other things, expression of Bmp4 was significantly increased, offering a potential mechanism by which thymic ECs mediate their regeneration. Consistent with this hypothesis, not only could recombinant BMP4 promote the expression of Foxn1 by TECs in vitro, induction of Foxn1 by CM from exEC(Thy) was abrogated by Noggin, an inhibitor of BMP4 signaling (Fig. 1e). Moreover, exEC(Thy) produced significantly more BMP4 compared to exEC derived from the heart or kidney; and silencing Bmp4 expression by shRNA within exEC(Thy) limited their capacity to mediate exogenous thymic regeneration and failed to induce expression of Foxn1 and Dll4. Finally, strengthening its role in endogenous regeneration, administration of a pharmacologic BMP inhibitor inhibited thymic regeneration after SL-TBI; and inducible deletion of Bmp4 specifically in ECs reduced thymic regeneration after SL-TBI (Fig. 1f).
These studies not only detail a novel pathway promoting endogenous thymic regeneration, but also offer an innovative clinical approach to enhance T cell immunity in recipients of allo-HSCT and for individuals with T cell deficiencies due to aging, infectious disease, and common cancer treatments such as chemo- and radiation-therapy.

Disclosures
Rafii: Angiocrine Bioscience: Other: Founder of Angiocrine Biosceince, which is developing the technology behind endothelial cell propagation. van den Brink:Boehringer Ingelheim: Consultancy, Other: Advisory board attendee; Regeneron: Honoraria; Merck: Honoraria; Tobira Therapeutics: Other: Advisory board attendee; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.