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CD73 regulates HSC quiescence
were shown to overlap exclusively with leptin receptor- positive (lepr+) perivascular niche cells.2,3 CD39 and CD73 expression was observed on CD31+ vasculature and CD45- CD31-CD140a+CD140b+ cells, but not on CD45-CD31- CD140a-CD140b- cells (Figure 3G,H, Online Supplementary Figure S3F). The frequencies of CD39high and CD73high cells within CD31+ vasculature and CD45-CD140a+CD140b+ cells were comparable to those in CD150low Treg, and lower than those in CD150high Treg and CD150high nonTreg (Figure 3B,G-H, Online Supplementary Figure S3A). Taken together, these observations indicate that, while various BM cell populations expressed CD39 and/or CD73, CD73high cells were frequently found within CD150high Treg and CD150high nonTreg.
The increase of hematopoietic stem cells in CD73 knockout mice was reversed by transfer of CD150high regulatory T cells and CD150high non-regulatory T cells and by in vivo adenosine receptor agonist treatment
To assess the role of CD150high Treg and CD150high CD4+ nonTreg in CD73-mediated HSC regulation, we analyzed how transfer of these T-cell populations influences HSC. Transfer of CD150high Treg and of CD150high CD4+ nonTreg significantly reversed the increase of HSC in CD73 KO mice relative to control wildtype mice. In contrast, the size of the HSC pool was not significantly altered by transfer of CD150neg-low nonTreg or CD150low Treg (Figure 4A). These observations are consistent with our previous
observations that transfer of CD150high Treg reversed the increase of HSC in mice with conditional deletion of CD39 in Treg.7 These results suggest that CD150high Treg and CD150high nonTreg contribute largely to CD73-medi- ated HSC regulation.
The downstream signaling of CD73 in HSC regulation was further assessed. HSC showed higher levels of expression A2AR than various other BM cell populations (Figure 4B). In vivo A2AR agonist treatment significantly reversed the increase of HSC in CD73 KO mice (Figure 4C). These observations suggest that A2AR signaling plays an important role in CD73-mediated HSC regula- tion.
Discussion
This study identified CD73 and CD150high nonTreg as important regulators of HSC quiescence and abundance in the adult BM. Global CD73 deletion increased ROS levels in HSC, and HSC numbers. This increase of HSC was reversed by anti-oxidant treatment, suggesting that CD73 maintains HSC quiescence by preventing oxidative stress. Because HSC did not express CD73 but CD39, CD73- mediated HSC regulation is driven by the microenviron- ment. While various BM cells showed intermediate levels of CD73 expression, CD73high cells were frequently found within unique CD150high Treg and CD150high nonTreg.
AC
B
Figure 4. CD73 of CD150high regulatory T cells and CD150high non-regulatory T cells regulates hematopoietic stem cells via adenosine 2A receptors. (A) Numbers of hematopoietic stem cells (HSC) in CD73 knockout (KO) mice 7 days after intravenous injection of CD150high bone marrow (BM) regulatory T cells (Treg), CD150low BM Treg, CD150high BM nonTreg, or CD150low BM nonTreg (30,000 cells/mouse). Data were pooled from three independent experiments (total 4-10 mice/group) and ana- lyzed by one-way analysis of variance (ANOVA) with the Bonferroni multiple comparison test. (B) Levels of adenosine 2A receptor (A2AR) expression in different BM cell populations. MFI: mean fluorescence intensity. Four mice were used in the experiment. (C) HSC numbers in CD73 KO mice that received daily intraperitoneal injections of PSB0777 (25 μg/mouse) for 7 days. The total HSC numbers in one tibia and one femur were analyzed 1 day after the final injection. The results from two independent experiments were pooled (total 4-10 mice/group).
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