Fig 2: Effect of global CD73 deficiency on stroke outcome after tMCAO(A) Generation of CD73−/− mice by crossing CD73fl/fl(Nt5etm2Jsc) mice with a “deleter” cre-transgenic mouse strain (Tg(CMV-cre)1Cgn) mediating the deletion of loxP-flanked genes in all tissues including germ cells (Schwenk et al., 1995).(B) Verification of Nt5e deletion in CD73−/− mice by comparing CD73 cell surface staining on CD73−/− and CD73fl/fl peritoneal macrophages (n = 3/group) and on CD73−/− and CD73fl/fl oligodendrocytes (n = 2/group).(C) Physiological parameters of CD73−/− and CD73fl/fl mice: regional cerebral blood flow (rCBF) in the ipsilateral and contralateral MCA territory measured by laser Doppler (n = 8–12/group), rectal body temperature at filament insertion and removal (n = 12–13/group), peripheral oxygen saturation at the hind paw at filament insertion and removal (n = 12–13/group). Data are presented as mean ± SD, unpaired two-sided Student’s t test, ∗ p ≤ 0.05, ns = not significant.(D) Representative TTC images and edema-corrected infarct volume measured by TTC staining of brain sections three days after tMCAO of CD73−/− and CD73fl/fl mice (n = 12–13/group). Data are presented as mean ± SD, unpaired two-sided Student’s t test, ns = not significant.(E) Representative TTC images and edema-corrected infarct volume measured by TTC staining of brain sections three days after tMCAO of anti-CD73 and isotype control treated C57BL/6J mice (n = 8–9/group). Data are presented as mean ± SD, unpaired two-sided Student’s t test, ns = not significant.(F) Physiological parameters of anti-CD73 and isotype control treated C57BL/6J mice: regional cerebral blood flow (rCBF) in the ipsilateral and contralateral MCA territory measured by laser Doppler (n = 5–8/group) and peripheral oxygen saturation at the hind paw at filament insertion and removal (n = 5–8/group). Data are presented as mean ± SD, unpaired two-sided Student’s t test, ns = not significant.

Fig 3: Schematic of cellular and molecular mechanisms through which Nox2 in CD4+ T cells regulates Ang II–induced hypertension and heart remodeling.Deficiency of Nox2 in Tregs results in increased numbers of Tregs in the heart and vessels and protects against the development of hypertension, interstitial cardiac fibrosis, and cardiomyocyte hypertrophy. Mechanistically, the deficiency of Nox2 in Tregs enhances their suppressive function through an increase in nuclear levels of FoxP3 and NF-κB activation, and higher levels of GITR, CTLA-4, CD39, CD73, and CD25. The higher CD25 levels may drive an increased STAT5 phosphorylation under IL-2 stimulation, and lead to a positive feedback promoting suppression. The deficiency of Nox2 also drives a change in balance between Tregs and Th17 cells to a more antiinflammatory profile, with higher IL-10 and lower IL-17 levels.

Fig 4: Tregs deficient in Nox2 are more suppressive than WT Tregs.(A and B) In vitro suppression assay using Tregs purified from spleen and lymph nodes of Nox2-deficient mice (Nox2–/y) and WT littermate controls. Cells were stimulated with APCs and anti-CD3ε Ab. Representative plots in B show proliferation of Teffs after 3 days of stimulation; numbers at the top are the ratio of Tregs to Teffs. Mean data are shown in A. The “IC50” is the ratio of Tregs/Teffs at which there was 50% suppression of Teff proliferation. (C) Levels of IL-10 in culture supernatants determined by cytometric bead array. The cell combinations that were cocultured are shown at the bottom. (D–G) Baseline levels of mRNA for CTLA-4, CD39, CD73, and GITR in Tregs purified from spleen and lymph nodes. *P < 0.05 for highlighted comparisons, by 2-way ANOVA (A), 1-way ANOVA followed by Tukey’s post-test (C), or unpaired t test (D–G); n = 3–6 per group.

Fig 5: TGFβ-blockade ablates myofibroblasts and induces expansion of CD73+ CAFs.a Superimposed t-SNE projections of CAF-curated single cell RNAseq data from isotype- and anti-TGFβ-treated mice. n = 4 mice per group from 2 independent experiments. b Representative t-SNE plots highlighting CAF clusters in isotype- and anti-TGFβ-treated mice. c Frequency of CAF subsets within the overall CAF cluster. Each column represents a mouse. n = 4 mice per group from 2 independent experiments. d Quantification of CAFs expressing αSMA from dissociated 4T1 tumors of isotype- and anti-TGFβ-treated mice by flow cytometric analysis. Each dot represents a mouse. Mean is depicted. n = 10 mice per group. Data are representative of four independent experiments. ****p < 0.0001 (unpaired, two-tailed t-test with Welch’s correction). e Violin plot depicting expression of Nt5e in ilCAFs. n = 4 mice per group from 2 independent experiments. f Representative flow cytometric analysis of CD73 expression on CAFs from dissociated 4T1 tumors from isotype- and anti-TGFβ-treated mice. g Quantification of CAFs expressing CD73 as a frequency of single cells from dissociated 4T1 tumors of isotype- and anti-TGFβ-treated mice by flow cytometric analysis. Each dot represents a mouse. Mean is depicted. n = 10 mice per group. Data are representative of four independent experiments. ****p < 0.0001 (unpaired, two-tailed t-test with Welch’s correction). h Pseudotime analysis overlaid on UMAP projection depicting divergent trajectories among the five CAF subsets. Each dot represents a cell, and each curve represents a lineage.