Figure 2

TGF-β and retinoic acid promote CD73 expression. Cells were isolated from the spleen, MLN, LP, and IEL of (a) littermate control and CD4-cre TGF-βRII^lox/lox mice or (b) control and vitamin A-deficient mice. Cells were stained for TCRβ, CD4, CD73, and Foxp3 and analyzed by flow cytometry. Histograms show the expression of CD73 on CD4⁺ Foxp3⁻-gated cells. Graphs represent the percentage of CD73⁺ cells among CD4⁺ Foxp3⁻ T lymphocytes (left panels) or CD4⁺ Foxp3⁺ T lymphocytes (right panels). Data are pooled from three independent experiments (n=11). (c) CD4⁺ CD25⁻ T lymphocytes were isolated from the spleen of littermate control and CD4-cre TGF-βRII^lox/lox mice and stimulated with anti-CD3/CD28 in the absence or presence of TGF-β and/or RA. 24 h later, TCRβ⁺ CD4⁺ Foxp3⁻-gated cells were analyzed for CD73 by flow cytometry. Histograms show the expression of CD73. The results are representative of three independent experiments. (d, e) CD4⁺ GFP⁻ T lymphocytes were isolated from the spleen of Foxp3^eGFP mice and were activated as in c. (d) After 24 h, CD4⁺ GFP⁻ T lymphocytes were sorted by flow cytometry. Cell lysates were analyzed with western blotting for phosphorylated Smad3 (p-Smad3) and Smad3. Actin served as a loading control. The results are representative of three independent experiments. (e) Cultures were supplemented with TGF-β receptor kinase inhibitor (SB431542), p38 inhibitor (SB203580), MAPK/ERK kinase inhibitor (PD98059), JNK inhibitor (SP600125) and PI3 kinase inhibitor (LY294002). Data are expressed as MFI of CD73 expression by CD4⁺ Foxp3⁻ CD73⁺ T lymphocytes and are representative of three independent experiments. Graph bars represent the median with interquartile range. The non-parametric Mann–Whitney U-test was used (*P<0.05, ***P<0.001). GFP, green fluorescent protein; IEL, intraepithelial lymphocyte; LP, lamina propria; MFI, mean fluorescence intensity; MLN, mesenteric lymph node; RA, retinoic acid; TGF-β, transforming growth factor-β.

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