Adenosine from Niche-Associated Tregs Maintains Hematopoietic Stem Cell Quiescence
Authors: Hirata Yuichi , Furuhashi Kazuhiro , Ishi Hiroshi , Li Hao-Wei , Pinho Sandra , Ding Lei , Frenette Paul S. , Fujisaki Joji 
Published: April 2021

Abstract
A crucial player in immune regulation, FoxP3+ regulatory T cells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. For example, specific subsets of Tregs in the adipose tissue control metabolic indices; muscle Tregs potentiate muscle repair, and lung Tregs prevent tissue damage. These studies, together with a previous finding that Tregs are enriched in the primary site for hematopoiesis, the bone marrow (BM), prompted us to examine whether there is a special Treg population which controls hematopoietic stem cells (HSCs).
We showed that HSCs within the BM were frequently adjacent to distinctly activated FoxP3+ Tregs which highly expressed an HSC marker, CD150. Moreover, specific reduction of BM Tregs achieved by conditional deletion of CXCR4in Tregs, increased reactive oxygen species (ROSs) in HSCs. The reduction of BM Tregs further induced loss of HSC quiescence and increased HSC numbers in a manner inhibited by anti-oxidant treatment. Additionally, this increase in HSC numbers in mice lacking BM Tregs was reversed by transfer of CD150high BM Tregs but not of CD150low BM Tregs. These results indicate that CD150high niche-associated Tregs maintain HSC quiescence and pool size by preventing oxidative stress.
We next sought to identify an effector molecule of niche Tregs which regulates HSCs. Among molecules highly expressed by niche Tregs, we focused on CD39 and CD73, cell surface ecto-enzymes which are required for generation of extracellular adenosine, because 1) CD39highCD73high cells within the BM were prevalent among CD150high Tregs and 2) HSCs highly expressed adenosine 2a receptors (A2AR). We showed that both conditional deletion of CD39 in Tregs and in vivo A2AR antagonist treatment induced loss of HSC quiescence and increased HSC pool size in a ROS-dependent manner, which is consistent with the findings in mice lacking BM Tregs. In addition, transfer of CD150high BM Tregs but not of CD150low BM Tregs reversed the increase in HSC numbers in FoxP3cre CD39flox mice. The data indicate that niche Treg-derived adenosine regulates HSCs.
We further investigated the protective role of niche Tregs and adenosine in radiation injury against HSCs. Conditional deletion of CD39 in Tregs increased radiation-induced HSC apoptosis. Conversely, transfer of as few as 15,000 CD150high BM Tregs per B6 mouse (iv; day-1) rescued lethally-irradiated (9.5Gy) mice by preventing hematopoiesis failure. These observations indicate that niche Tregs protect HSCs from radiation stress.
Finally, we investigated the role of niche Tregs in allogeneic (allo-) HSC transplantation. Our previous study showed that allo-hematopoietic stem and progenitor cells but not allo-Lin+ cells persisted in the BM of non-conditioned immune-competent recipients without immune suppression in a manner reversed by systemic Treg depletion1. This observation suggests that HSCs have a limited susceptibility to immune attack, as germline and embryonic stem cells are located within immune privileged sites. Because the study employed systemic Treg depletion and non-conditioned recipients, it remains unknown whether niche Tregs play a critical role in immune privilege of HSCs and in allo-HSC engraftment following conditioning. We showed here that the reduction of BM Tregs and conditional deletion of CD39 in Tregs abrogated allo-HSC persistence in non-conditioned immune-competent mice as well as allo-HSC engraftment following nonmyeloablative conditioning. Furthermore, transfer of CD150high BM Tregs but not of other Tregs (15,000 cells/recipient; day -2) significantly improved allo-HSC engraftment. This effect of niche Treg transfer is noteworthy given that 1-5 million Tregs per mouse were required in case of transfer of spleen or lymph node Tregs. These observations suggest that niche Tregs maintain immune privilege of HSCs and promote allo-HSC engraftment.
In summary, our studies identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, numbers, stress response, engraftment, and immune privilege, further highlighting potential clinical utility of niche Treg transfer in radiation-induced hematopoiesis failure and in allo-HSC engraftment (under revision in Cell Stem Cell).
1 Fujisaki, J. et al. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature474, 216-219, doi:10.1038/nature10160 (2011).

Disclosures
No relevant conflicts of interest to declare.