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Letters to the Editor
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Figure 2. Niche regulatory T-cell (Tregs) transfer and A2AR agonist treatment rescued lethally irradiated mice from critical bone marrow (BM) failure. (A-C) Analysis of 9.5 Gy-irradiated B6 mice receiving tail vein injection of CD150high BM Tregs (CD150H), CD150low BM Tregs (CD150L), LN Tregs (LN) or vehicle (Control, Ctl) (30,000 cells/mouse; day -2; i.v.). CD150high Tregs were defined as Tregs with CD150 expression levels in the top 30% of BM Tregs, and CD150low Tregs were those in the bottom 30%. (A) Post-irradiation survival. Pooled from three independent experiments. (B) Hematopoietic stem progenitor cell (HSPC) numbers on day 10 after 7 Gy total body irradiation (TBI). nonRT: non-irradiated wild-type mice. N=6/group. (C) HSPC death frequencies on day 10 after 7 Gy g TBI. (D) Survival of 9.5 Gy-irradiated B6 mice receiving tail vein injection of CD150high BM Treg from Foxp3creCD39fl/wt mice (CD39KO CD150H), wild-type CD150high BM Tregs (CD150H) or vehicle (Control, Ctl) (30,000 cells/mouse; day -2; i.v.). Pooled from three independent experiments. (E) Survival of 9.5 Gy-irradiated B6 mice treated with PSB0777 (daily from day -2 till day 7; i.p.; 25 mg/dose; Tocris). Pooled from two independent experiments. Statistical analyses were performed with GraphPad Prism and Easy-R (EZR) software. Statistical significance was determined using one-way ANOVA with Bonferroni post test. For survival analysis, generalized Wilcoxon test was used, followed by Holm post-hoc multiple comparison test when needed. All data are presented as mean±standard deviation. 7-week-old mice were used in all studies. *P<0.05; ****P<0.0001.
tected donor allo-HSC from recipient immunity, enabling allo-HSC persistence without immune suppression and promoting allo-BM engraftment following non-myeloab- lative conditioning.2,3 Under non-transplantation settings, niche Treg-derived adenosine protected endogenous HSC and progenitors from oxidative stress, maintaining stem cell quiescence via adenosine 2A receptors (A2AR).2 These observations prompted us to investigate protective roles of CD150high niche Tregs and adenosine in post-irra- diation BM injury.
BM Tregs (~0.1% of BM mononuclear cells) was depleted by using previously established mice with con- ditional deletion of CXCR4 in Tregs,2 which is a chemokine receptor required for Treg homing to the BM, but not to the spleen or lymph nodes. FoxP3cre-CXCR4fl/fl mice showed from 70% to approximately 80% reduc- tions in frequencies and numbers of BM Tregs and CD150high BM Tregs, while pool sizes of spleen and lymph node Tregs were not altered.2 We assessed how this reduction of BM Tregs influences BM failure follow-
ing total body irradiation (TBI). Mortality of 9-Gy irradi- ated FoxP3cre-CXCR4fl/fl mice was significantly higher than that of control FoxP3cre mice (Figure 1A). Reduction of BM Tregs slowed down post-irradiation recovery of numbers of BM cells, cKit+Sca1+Lin- hematopoietic stem and HSPC, and CD150+CD48– cKit+Sca1+Lin– HSC, fur- ther increasing HSPC and HSC death on days 10 and 28 (Figure 1B-E and Online Supplementary Figure 1A and B). Irradiated FoxP3cre-CXCR4fl/fl mice and FoxP3cre mice were rescued by intravenous injection of HSPC (Online Supplementary Figure 1C), suggesting that the post-irradi- ation death of these models were largely attributed to hematopoiesis failure. These observations suggest that the reduction of BM Tregs exacerbated post-irradiation hematopoiesis failure which increased mouse mortality.
Next, protective roles of Treg-derived adenosine were investigated by using conditional deletion of a cell-sur- face ectoenzyme CD39 in Tregs, which is required for the generation of extracellular adenosine. 8.5 Gy-irradiated FoxP3cre-CD39fl/wt mice recapitulated all the phenotypes
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haematologica | 2021; 106(3)