Generation of FoxP3-GFP-hCre BAC Tg mice. (A) The final BAC Tg construct used for generating mice. (B) Flow cytometric analysis of LN cells from 8-wk-old Tg mice indicated specific transgene expression in CD4⁺CD25^highCD127^low T cells. The percentage of cells in each quadrant is indicated. (C) Intracellular FoxP3 staining revealed that GFP expression in the thymus, LN, and spleen was restricted to FoxP3⁺ cells. (D) To monitor hCre activity in vivo, FoxP3-GFP-hCre Tg mice were crossed to YFP reporter mice. Flow cytometric analysis of GFP or YFP expression in CD4 T cells from LN or spleen shows specific and efficient reporter activation. The percentage of cells in each gated population is indicated.

Normal thymic T reg cell development and seeding of the peripheral compartments in the FoxP3-hCre Dicer^lox/lox model. (A) Flow cytometric analysis was performed on 2-wk-old FoxP3-GFP-hCre Dicer^wt/lox (Het) and FoxP3-GFP-hCre Dicer^lox/lox (KO) mice for CD4, CD8, and FoxP3 expression. CD4⁺ SP thymocytes were analyzed for concomitant GFP and FoxP3 expression, illustrating equal relative expression of FoxP3⁺ cells in the thymus in Dicer wild-type and Dicer-deficient T reg cells. The percentage of cells in each gate is indicated. (B) Similar results to A were obtained in the LN and spleen.

Phenotype of miRNA-deficient T reg cells. (A) Splenomegaly and lymphadenopathy in >5-wk-old FoxP3-GFP-hCre Dicer^lox/lox (KO) mice. Data are representative of >10 mice. (B) LN and spleen T cell subsets in 4–6-wk-old mice. Absolute numbers of CD4⁺FoxP3⁻ and CD4⁺FoxP3⁺ cells were calculated by multiplying the percentage of cells determined by FACS by the absolute cell number per tissue. Data represent means ± SD (n = 5). (C) LNs were harvested at 5 wk from FoxP3-GFP-hCre Dicer^wt/lox (Het) and FoxP3-GFP-hCre Dicer^lox/lox (KO) mice and analyzed for the indicated cell-surface markers gated on CD4⁺FoxP3⁺ lymphocytes.

miRNA-dependent T reg lineage stability. (A) Overlap of the gene expression profile of Dicer KO T reg cells and FoxP3-deficient T reg cells. LN CD4⁺YFP⁺ T cells (>95% pure) from FoxP3-GFP-hCre × ROSA26R-YFP Dicer^wt/lox (Het) and FoxP3-GFP-hCre × ROSA26R-YFP Dicer^lox/lox (KO) mice (∼30 d old) were isolated by flow cytometry. The genes expressed differentially in Dicer KO versus Het T reg cells were compared with the published data of FoxP3 KO versus FoxP3⁺ T reg cells (reference 23). Two-dimensional scatter plot of those common gene expression values is shown. Gzma, Lilrb4, Gzmk, Gzmb, Tbx21, INF-γ, Cxcl10, Ccr5, Cd55, IL4, Sesn1, and Prg4 are highlighted. (B) FoxP3 expression in sorted CD4⁺YFP⁺ cells was examined by flow cytometry analysis. Numbers on bracketed lines indicate the percentage of cells in each gated population. Each symbol represents one mouse (from 4 to 7 wk). Small horizontal lines indicate the mean. (C) Levels of mRNA for the indicated genes in CD4⁺GFP⁺ (FoxP3-expressing cells) from 5-wk-old FoxP3-GFP-hCre Dicer^lox/lox (KO) mice were determined by real-time PCR analysis. Data represent the mean ± SD. (D) IFN-γ production was examined by intracellular cytokine staining. FoxP3 expression in CD4⁺GFP⁺ was confirmed by intracellular FoxP3 staining (left, continuous lines); dotted lines indicate CD4⁺GFP⁻ cells. (right) The percentage of IFN-γ cells from several Het and KO mice. Each symbol represents one 4–6-wk-old mouse. Small horizontal lines indicate the mean. (E) CD127 expression of the same cell subset is shown. Mean, mean fluorescence intensity.

T reg cell–specific Dicer ablation results in the loss of suppressor function in vivo. (A) Flow cytometric analysis of activation markers on Teff cells reveals strong activation of nonregulatory (GFP⁻YFP⁻) cells. LN and spleen cells from 5-wk-old FoxP3-GFP-hCre × ROSA26R-YFP Dicer^wt/lox (Het) and FoxP3-GFP-hCre × ROSA26R-YFP Dicer^lox/lox (KO) mice were stained for CD4 and the indicated proteins. Teff cells were gated on CD4⁺GFP⁻/YFP⁻ lymphocytes and analyzed for CD44, CD62L, and CD69 expression. For analysis of intracellular CTLA4, cells were stained with FoxP3 antibody and gated on FoxP3⁻ cells to distinguish T reg cells from Teff cells. (B) Percentage of CD4⁺CTLA4⁺FoxP3⁻ Teff cells from wild-type and KO mice (4–6 wk old) from LNs and spleen. Each symbol represents one mouse. Small horizontal lines indicate the mean. (C) Flow cytometric analysis illustrates massive cytokine production by CD4⁺GFP⁻/YFP⁻ effector cells from 5-wk-old FoxP3-GFP-hCre × ROSA26R-YFP Dicer KO but not FoxP3-GFP-hCre × ROSA26R-YFP Dicer wild-type mice. The percentage of cells in each quadrant is indicated. (D) Paraffin-embedded histological sections of lung and liver from 5-wk-old mice were fixed and stained with hematoxylin and eosin (n > 5). (E) Frozen sections of the FoxP3-GFP-hCre Dicer^lox/lox (KO) mice in D were stained for the presence of CD4⁺ and CD8⁺ T cells. Visualization was done with DAB substrate (Vector Laboratories) and was followed by hematoxylin counterstain. Bar, 50 μm.