Volume 22, Issue 3, Pages 445-453.e5 (March 2018) CD150high Bone Marrow Tregs Maintain Hematopoietic Stem Cell Quiescence and Immune Privilege via Adenosine  Yuichi Hirata, Kazuhiro Furuhashi, Hiroshi Ishii, Hao Wei Li, Sandra Pinho, Lei Ding, Simon C. Robson, Paul S. Frenette, Joji Fujisaki  Cell Stem Cell  Volume 22, Issue 3, Pages 445-453.e5 (March 2018) DOI: 10.1016/j.stem.2018.01.017 Copyright © 2018 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2018 22, 445-453.e5DOI: (10.1016/j.stem.2018.01.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 1 BM Tregs Maintain HSC Quiescence and Pool Size by Reducing Oxidative Stress (A) Treg frequencies in CD4+CD3+NK1.1− T cells (left) and BM mononuclear cells (right) of mice with conditional deletion of CXCR4 in Tregs. fl/fl, FoxP3cre CXCR4fl/fl mice; fl/wt, FoxP3cre CXCR4fl/wt mice; wt/wt, control FoxP3cre CXCR4wt/wt littermates. (B) Frequencies and numbers of HSPCs and HSCs in FoxP3cre CXCR4fl/fl mice. HSPC and HSC numbers in one tibia and one femur were analyzed. These results were reproducible in five independent experiments (total 17–20 mice/group; refer to Table S1). Data were pooled from two independent experiments. (C) Ki67− cell frequencies in HSCs in FoxP3cre CXCR4fl/fl mice. The results were reproducible in two independent experiments (total 7–8 mice/group). Data from a representative experiment are shown here. (D) Donor blood chimerism in 9.5 Gy-irradiated SJL mice receiving FoxP3-YFPcre CXCR4fl/fl or FoxP3-YFPcre CXCR4wt/wt BM cells (CD45.2) together with SJL BM cells. BM cells from each donor mouse (n = 9) were transplanted into one-two recipients (total 15–17 mice/group). (E) ROS levels in HSPCs and HSCs. Flow cytometric analysis was performed following incubation of BM cells with 2 μM CellROX Deep Red (Invitrogen) for 30 min. These results were reproducible in two independent experiments (total 7–9 mice/group). Data from a representative experiment are shown here. (F) Numbers of HSCs and HSPCs in FoxP3-YFPcre CXCR4fl/fl mice receiving NAC treatment (daily; s.c. injection; 100 mg/kg for 4 weeks). These results were reproducible in two independent experiments (total 7 mice/group). Data from a representative experiment (3 mice) are shown here. s.c., subcutaneous. (G) Donor blood chimerism in lethally irradiated SJL mice receiving SJL BM cells together with BM cells of NAC-treated FoxP3-YFPcre CXCR4fl/fl or FoxP3-YFPcre CXCR4wt/wt mice. BM cells from each donor mouse (n = 6–7) were transplanted into one-two recipients (total n = 12/group). Data are presented as mean ± SD. Cell Stem Cell 2018 22, 445-453.e5DOI: (10.1016/j.stem.2018.01.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 2 Potent CD150high Tregs in the Perivascular Niche Regulate HSCs (A) In vivo imaging of Tregs (green) adjacent to CXCL12-DsRedhigh perivascular cells (purple) in the skull BM of a FoxP3-GFP × CXCL12-DsRed mouse 1 hr after intravenous injection of Alexa647-conjugated Isolectin GS-IB4. Red, sinusoidal vessels; white, bone. The white bar indicates 20 μm. (B) Distances from Tregs to CXCL12-DsRedhigh cells. We analyzed 3D image stacks of the skull BM (2,540 μm [x] × 2,570 μm [y] × 150 μm [z]) of FoxP3-GFP × CXCL12-DsRed mice or global-GFP × CXCL12-DsRed mice (control). These results were reproducible in three mice. A representative figure is shown here. (C) Treg frequencies in BM mononuclear cells in mice with conditional deletion of CXCL12 in lepr+ cells. The results were reproducible in an additional experiment. (D) Distances from CD150+CD48−Lin− HSCs or control (CD48+ or Lin+) cells with respect to the nearest observable FoxP3-GFP Tregs in the tibia BM of B6 FoxP3-GFP mice. Data from 5 FoxP3-GFP mice were pooled. (E) Histological image of CD150high Tregs (whitish-yellow) adjacent to CD150+CD48−Lin− HSCs (red). Green, FoxP3-GFP.; red, CD150; blue, CD48 or Lin. CD150+ Tregs (whitish-yellow cells) express CD150 (red), FoxP3-GFP (green), and Lin (blue). (F) Frequencies of CD150high cells among different Treg fractions (y axis) categorized by their distance (x axis) from the nearest observable HSC in the tibia BM (x axis). CD150high Tregs were defined as Tregs that expressed CD150 at levels in the top 35% of relative fluorescence units across all observable BM Tregs. p values were determined by comparing frequencies of CD150high cells in Tregs within 25 μm of HSCs with those in Tregs within more than 25 μm of HSCs. Data from three FoxP3-GFP mice were pooled. (G) Flow cytometric analysis of BM FoxP3+ Tregs. (H) Flow cytometric analysis of CD150high BM Tregs, CD150low BM Tregs, and LN Tregs. CD150high Tregs were defined as Tregs that expressed CD150 at levels in the top 30% of all BM Tregs, and CD150low Tregs cells were those in the bottom 70%. These results were reproducible in three independent experiments (total 10 mice). Data from a representative experiment (3–4 mice) are shown here. (I) HSC numbers in FoxP3cre CXCR4fl/fl mice 5 days after intravenous injection of CD150high BM Tregs, CD150low BM Tregs, or CD4+FoxP3− BM T cells (30,000 cells/mouse). Data were pooled from three independent experiments. Data are presented as mean ± SD. Cell Stem Cell 2018 22, 445-453.e5DOI: (10.1016/j.stem.2018.01.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 3 Adenosine Generated via CD39 of Niche Tregs Maintains HSC Quiescence and Pool Size by Reducing Oxidative Stress (A) Flow cytometric analysis of CD39 and CD73 expression levels by CD150high BM Tregs, CD150low BM Tregs, and whole BM cells. (B) Frequencies of CD39highCD73high cells in different BM cell populations in B6 mice. Treg: CD4+CD3+NK1.1-FoxP3GFP+ cells. Non-Treg: CD4+CD3+NK1.1-FoxP3GFP− cells. CD8 T: CD8+CD3+NK1.1-T cells. B cell: B220+ cells. HSPC: KSLs. CD39highCD73high cells correspond to the cell population of the upper right quadrant in (A). These results were reproducible in two independent experiments (total 8 mice). A representative figure from one experiment (4 mice) is shown here. (C) HSC numbers in NAC-treated or non-treated FoxP3cre CD39fl/wt mice. NAC treatment was given orally via drinking water (1 mg/mL; 4 weeks). These results were reproducible in two independent experiments (total 8–10 mice/group; refer to Table S2). Data analyzed by one-way ANOVA with Bonferroni posttest. (D) Donor blood chimerism in lethally irradiated mice receiving wild-type SJL BM cells (CD45.1) together with BM cells of NAC-treated or non-treated FoxP3cre CD39wt/wt mice (CD45.2). BM cells from each donor mouse (total 7–9 mice/group) were transplanted into one-two recipient (total 12–18 mice/group). Analysis was performed 5 months after transplantation. (E) ROS levels in HSCs in FoxP3cre CD39fl/wt mice. These results were reproducible in two independent experiments (9–10 mice/group total). A representative figure from one independent experiment is shown here. (F) Expression levels of adenosine 2A receptors. MFI, mean fluorescence intensity. Experiment includes 4 mice. (G) HSC numbers in mice that received ZM241385 (daily; 200 μg/mouse; intraperitoneally [i.p.] injection; 21 days) and/or NAC treatments (1 mg/mL in drinking water). These results were pooled from two independent experiments (total 8 mice/group). (H) Donor blood chimerism in lethally irradiated mice transplanted with SJL BM cells (CD45.1) together with BM cells isolated from B6 mice (CD45.2) that received ZM241385 and/or NAC treatments. Analysis was performed 4 months after transplantation. These results were pooled from three independent experiments (12–20 mice/group total). (I) ROS levels in HSCs in ZM241385-treated mice. These results were reproducible in two independent experiments (8 mice/group total). A representative figure from one independent experiment is shown here. Data are presented as mean ± SD. Cell Stem Cell 2018 22, 445-453.e5DOI: (10.1016/j.stem.2018.01.017) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 4 The Role of Niche Tregs and Adenosine in Allo-HSC Engraftment (A) Allo- or syn-donor chimerism in the peripheral blood of FoxP3cre CXCR4fl/wt or FoxP3cre CXCR4wt/wt mice 9 and 24 weeks after transplantation. Recipient mice (B6) received 3 Gy irradiation followed by intravenous injection of MHC-mismatched B10.A BM cells or syngeneic B6 SJL BM cells (2 × 107/each). Anti-CD8 monoclonal antibody (mAb) was i.p. injected on day –1 and anti-mouse CD40L mAb on day 0. Experiment included four recipients/group. Data were reproduced by an additional experiment (5 mice/group). (B) Allo-donor blood chimerism in FoxP3cre CD39wt/wt recipient mice. Experiment included five recipients/group. (C) Allo-donor chimerism in recipients receiving adoptive transfer of CD150high or CD150low BM Tregs or LN Tregs (30,000 cells/mouse). B6 wild-type recipients received 2.5 Gy irradiation followed by intravenous injection of MHC-mismatched BALB/c BM cells (2 × 107 each). Anti-CD8 mAb was i.p. injected on day −1 and anti-mouse CD40L mAb on day 0. Tregs were injected into the tail veins of the recipients on day −2. Analysis was performed 7 weeks after transplantation. Data were pooled from two independent experiments (six recipients/group total). Data are presented as mean ± SD. Cell Stem Cell 2018 22, 445-453.e5DOI: (10.1016/j.stem.2018.01.017) Copyright © 2018 Elsevier Inc. Terms and Conditions