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CD73 regulates HSC quiescence
expressed by CD150high Treg.7 CD39 is known to convert extracellular adenosine triphosphate (ATP) and adeno- sine diphosphate (ADP) into adenosine monophosphate (AMP) which is further hydrolyzed by the other cell-sur- face ectoenzyme, CD73, into extracellular adenosine, a purine nucleotide with various tissue-protective effects.8 The results of our study using conditional knockout (KO) of CD39 in Treg suggested that extracellular adenosine generated via CD39 on Treg protected HSC from oxida- tive stress, maintaining HSC quiescence.7 It remains unclear how the other cell-surface ectoenzyme, CD73, contributes to HSC regulation and which BM cell popu- lations regulate HSC via CD73. In addition, while the BM serves as a reservoir of memory T cells,9,10 little is known about the role of these conventional T cells in HSC regulation.
This work identified unique CD150highCD4+FoxP3- con- ventional T cells (nonTreg) which highly expressed CD73 and CD39, like CD150high Treg. Our observations in CD73 KO mice into which these CD150high T-cell populations were transferred suggest that CD73 of CD150high CD4+ nonTreg and CD150high Treg maintain HSC quiescence and abundance.
Methods
Animals
C57BL/6J mice, SJL mice, BALB/c mice, CD73 KO mice, FoxP3- YFP mice, and Lep-cre mice (Jackson Laboratory, Bar Harbor, ME, USA) were housed in a specific pathogen-free environment. CD39-flox mice were kindly provided by Dr. Simon C. Robson (Harvard Medical School). Seven-week old CD73 KO mice were analyzed. The mice were sacrificed by CO2 inhalation and cervical dislocation. Studies were conducted with approval from Institutional Review Boards and Animal Care and Use Committees at Columbia University.
Antibodies and reagents
We used FITC-conjugated Lineage monoclonal antibodies (B220, Mac1, GR-1, CD2, CD3a, CD8a, CD4, CD19 and Ter119), APC-780-conjugated cKit monoclonal antibodies, PECy7- or APC- conjugated CD39 monoclonal antibodies, FITC-conjugated FoxP3 monoclonal antibodies, PE-conjugated Ki67 monoclonal antibod- ies (all purchased from eBioscience), APC/Cy7-conjugated NK1.1 monoclonal antibodies, BV510-conjugated CD3 monoclonal anti- bodies, PE/Cy7-, or BV605-conjugated CD4 monoclonal antibod- ies, Alexa700- or Pacific blue-conjugated CD48 monoclonal anti- bodies, PerCP/Cy5.5-, APC-, or BV605-conjugated CD73 mono- clonal antibodies, PE- or PE/Cy7-conjugated CD150 monoclonal antibodies (all from Biolegend), and BV605-conjugated Sca-1 monoclonal antibodies (from BD Pharmingen or Biolegend).
N-acetyl-L-cysteine (A9165) was purchased from Sigma- Aldrich.
Competitive reconstitution assay
SJL (CD45.1) mice were irradiated at 475 cGy twice (950 cGy in total) at least 2 h apart. Two hours after the last irradiation, donor BM cells (CD45.2), together with competitor BM cells (SJL) (3 x 105/each), were injected into the tail veins of SJL recipients. Peripheral blood samples were analyzed periodically. Red blood cells were lysed with an ammonium chloride potassium buffer. The antibodies used to analyze donor chimerism were anti- CD45.1, anti-CD45.2, anti-GR1, anti-CD11b, anti-B220, and anti- TCR-b (all from Biolegend).
Flow cytometric analysis
BM cells were isolated by crushing tibiae and femora. Following treatment with a red blood cell lysis buffer (Biolegend), the cell suspension (2x106 cells) was plated onto 96-well plates and incu- bated with culture media containing 2 μM CellROX Deep Red (Invitrogen) for 30 min. Flow cytometry was performed using an LSRII (BD Biosciences), LSRFortessa (BD Biosciences), or FACSCanto (BD Biosciences) cytometer followed by analysis using FlowJo software (Tree Star Inc.).
Colony-forming assay
BM cells (2.0x104 cells/each well) were plated in six-well plates (Corning, NY, USA) containing 1 mL MethoCultTM (M3234, Stemcell Technologies Inc.) supplemented with 1% penicillin/streptomycin (Gibco), stem cell factor (50 ng/mL), inter- leukin-3 (15 ng/mL), interleukin-6 (20 ng/mL), and granulocyte- macrophage colony-stimulating factor (15 ng/mL). Colonies were maintained at 37°C in humidified incubators. Colony formation was scored on day 10.
Flow cytometry following intracellular staining of FoxP3
Intracellular FoxP3 staining was performed according to the manufacturer’s protocol (eBioscience).
Stromal cell analysis
For analysis of stromal cells, long bones were gently crushed using Hanks balanced saline solution to harvest the BM cells. Whole bone marrow was digested with collagenase IV (200 U/mL) and DNase I (200 U/mL) at 37°C for 30 min. Following treatment with a red blood cell lysis buffer (Biolegend), the cell suspension (2x106 cells) was plated onto 96-well plates and then stained with antibodies. Anti-CD140a (APA5), anti-CD140b (APB5), anti-CD45 (30F-11), anti-CD31 (390) and anti-Ter119 anti- bodies (all from Biolegend) were used to stain perivascular stromal cells.
T-cell transfer assay
HSC numbers were determined in CD73 KO mice 7 days after intravenous injection of CD150high BM Treg, CD150low BM Treg, CD150high BM nonTreg, or CD150low BM nonTreg (30,000 cells/mouse). Data were pooled from three independent experi- ments (4-10 mice/group).
Adenosine 2A receptor agonist treatment
CD73 KO mice were given PSB0777, a potent adenosine 2A receptor (A2AR) agonist, daily for 7 consecutive days (25 μg/mouse, intraperitoneally). Total HSC numbers in one tibia and one femur were analyzed 1 day after the final injection.
Statistics
Statistical analyses were performed with GraphPad Prism soft- ware (version 6.0). Statistical significance was determined using a two-tailed t-test or one-way analysis of variance (ANOVA) with a Bonferroni post-test correction. P values less than 0.05 were con- sidered to be statistically significant. All data are presented as mean ± standard deviation (SD).
Results
CD73 deletion increased hematopoietic stem cell pool size
The effect of global CD73 deletion on hematopoiesis was first analyzed. CD73 KO mice showed increases in
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