(A) qPCR analysis of miRNA expression in flow cytometer-sorted YFP⁺CD4⁺ T cells cultured in vitro for 5 days. Bars represent miRNA expression relative to sno202; error bars represent range for replicate qPCR reactions.
(B) Proliferation of RFP⁺CD4⁺ T cells labeled with CFSE and analyzed by flow cytometry at the indicated times in culture. Cells are from Dgcr8^fl/fl Cd4-cre R26-tdRFP mice (gray lines) or Dgcr8^+/fl Cd4-cre R26-tdRFP mice (black lines).
(C–E) Cytokine production by restimulated YFP⁺ CD4⁺ T cells cultured in nonpolarizing (ThN) or Th2 cell-type conditions (C); CD4⁺ T cells cultured in Th1 cell-type conditions (D); and Dgcr8^fl/fl Cd4-cre YFP⁺CD4⁺ T cells cultured ±5 μg/mL anti-IFN-γ (E). Data are representative of at least three independent experiments.

(A) Cytokine production of YFP⁺ or RFP⁺CD4⁺ T cells from Dgcr8^fl/fl Cd4-cre R26-tdRFP and Dgcr8^+/fl Cd4-cre R26-YFP mice. YFP and RFP (left) were used to distinguish Dgcr8^+/fl and Dgcr8^fl/fl cells, respectively (right). Cells were mixed in a 4:1 ratio (Dgcr8^fl/fl:Dgcr8^+/fl) and co-cultured in 10 units/mL IL-4. Data are representative of three independent experiments.
(B) Intracellular protein expression of cocultured Dgcr8^fl/fl Cd4-cre R26-tdRFP (Dgcr8^fl/fl) and Dgcr8^+/fl Cd4-cre R26-YFP (Dgcr8^+/fl) cells after 68 hr of stimulation (anti-CD3 and anti-CD28).
(C) IFNGR1 and T-bet expression in freshly isolated CD4⁺ T cells.
(D) Phosphorylated STAT1 expression in freshly isolated CD4⁺ T cells ± 10 ng/mL IFN-γ for 15 min in coculture. Shaded histograms represent cells not treated with IFN-γ; Dgcr8^+/fl Cd4-cre YFP⁺ (black) or Dgcr8^fl/fl Cd4-cre RFP⁺ (gray).

(A) miRNA expression from deep sequencing analysis of small RNAs in wild-type CD4⁺ T cells stimulated in vitro for 44 hr. Shaded region marks miRNAs selected for screening. Unshaded region represents all miRNAs sequenced at least once. Bars are average frequency of each miRNA among all genome-matching small RNA sequences from two independent small RNA libraries.
(B) Schematic of workflow for miRNA functional screen in DGCR8-deficient CD4⁺ T cells (from Dgcr8^fl/fl Cd4-cre R26-tdRFP mice).
(C) Proliferation index for each miRNA was determined (see Experimental Procedures) and used to calculate proliferation Z scores (Z = x-mean/SD where x represents the normalized proliferation index for each individual miRNA). All miRNAs in the miR-17 and miR-92 seed families that were screened are highlighted.
(D) miR-17 and miR-92 seed family miRNAs (ordered from highest to lowest Z score).
(E) Proliferation of CFSE-labeled, RFP⁺ cells 65 hr posttransfection with individual miR-17~92 cluster miRNAs or control miRNA (representative histograms in left panel). Proliferation index data were normalized to cells transfected with control miRNA. Values are means ± SD from five independent transfections in three independent experiments; **p < 0.01; ANOVA Tukey’s post-hoc test.

(A) Z scores for frequency of IFN-γ production among restimulated DGCR8-deficient cells transfected with individual miRNAs as described in Figure 3B. miRNAs with a proliferation score of Z < −1.5 or Z > 1.5 were not included in the IFN-γ analysis because of possible indirect effects of survival or proliferation on cytokine production.
(B) Intracellular cytokine stains for IFN-γ and IL-4 in transfected and restimulated DGCR8-deficient CD4⁺ T cells. Data are representative of three independent experiments.
(C) Frequency of IFN-γ production among Dgcr8^fl/fl Cd4-cre CD4⁺ T cells transfected with increasing concentration of miR-29b. Data are representative of two independent experiments; error bars represent range from two independent transfections.
(D) Summary of cytokine production by miR-29b-transfected and restimulated Dgcr8^fl/fl Cd4-cre CD4⁺ T cells. Values are means ± SD from four biological replicates in two independent experiments; ***p < 0.001; Student’s two-tailed t test.
(E) Proliferation analysis by CFSE dilution of transfected Dgcr8^fl/fl Cd4-cre cells on day 5.
(F) Cell viability analysis of transfected Dgcr8^fl/fl Cd4-cre cells on day 5. Numbers indicate percentage of total events that are viable.
(G and H) IFN-γ production by restimulated, C57BL/6 CD4⁺ cells cultured in 100 pg/mL IL-12 or Th1 cell-type conditions and transfected with miR-29b or control miRNA. Values in (H) represent mean ± SD from three independent transfections; data are representative of two independent experiments.

(A) Cumulative distribution of mRNA expression changes from microarray data subsequent to transfection with miR-29b in Dgcr8^fl/fl Cd4-cre CD4⁺ T cells (left) or miR-29 inhibitor in C57BL/6 (wild-type) CD4⁺ T cells (right). Negative values indicate downregulation of mRNA after transfection. Black lines represent the entire microarray gene set, gray lines represent the subset of genes that are Targetscan computationally predicted miR-29 targets, and red lines represent the subset of predicted targets with 8-mer binding sites perfectly complementary to the entire miR-29 seed sequence.
(B) Scatter plot of the change in mRNA expression after miR-29 transfection in Dgcr8^fl/fl Cd4-cre cells (y axis) and change in expression after miR-29 inhibitor transfection in wild-type cells (x axis). Each point represents a different gene array probe. Included are predicted miR-29 targets differentially expressed (FDR < 0.1) after miR-29 transfection.
(C) Heatmap representation of changes in gene expression after transfection for three independent biological samples of each genotype. Scale is log2 fold change in array hybridization intensity compared to control transfected cells.
(D) qPCR validation of array data for miR-29 candidate targets of interest. mRNA expression was normalized to Gapdh and is relative to expression in control transfected cells. Values are means ± SD from three biological replicates.
(E and F) Primary Dgcr8^fl/fl Cd4-cre (E) or wild-type (F) CD4⁺ T cells were cotransfected with a dual luciferase reporter and miR-29b or control miRNA (E); miR-29 inhibitors or control inhibitor (F). Luciferase reporters contained the full-length mouse 3′UTR of Tbx21 or Eomes. Renilla luciferase activity was measured 24 hr after transfection and normalized to firefly luciferase activity. Values are relative to normalized luciferase in control transfected cells. Data are representative of three independent experiments and values are means ± SD from three independent transfections.
*p < 0.05, **p < 0.01; ANOVA Tukey’s post hoc test.

(A and B) CD4⁺ cells from Dgcr8^fl/fl Cd4-cre mice or Dgcr8^+/fl Cd4-cre mice were cultured in ThN conditions and stained intracellularly for T-bet and Eomes protein 24 hr after transfection. T-bet data are on day 2 and Eomes data are on day 5. Values are means ± SD from three independent transfections; **p < 0.01; ANOVA Tukey’s post hoc test.
(C) MFI of intracellular T-bet stains for Thy1.1⁺ CD45.1⁺ cells subsequent to adoptive transfer of CD4⁺ SMARTA T cells and LCMV infection. Transferred and transduced cells were identified by congenic CD45.1 and retroviral Thy1.1 expression, respectively.
(D) MFI of intracellular IFN-γ for transduced and transferred CD4⁺ SMARTA T cells (as in C). Cells were stimulated with 1 μM gp61-80 peptide. Data represent IFN-γ MFI for TNF-α-producing cells.
(E) MFI of intracellular T-bet in CD8⁺ P14 T cells after LCMV infection, analyzed as in (C).
(F) Transduced CD8⁺ P14 T cells (as in E) were analyzed for KLRG1 and IL-7Rα expression. Values are mean frequency (%) ± SD; n = 3; p < 0.05 for both populations; Student’s two-tailed t test.
Data in (C)–(F) are representative of two independent experiments; **p < 0.01; Student’s two-tailed t test.

Dgcr8^fl/fl Cd4-cre CD4⁺ T cells were transfected with control miRNA, miR-29b, siRNA targeting T-bet (siTbet), and/or siRNA targeting Eomes (siEomes). Cells were cultured in ThN conditions and transfected at 24 hr and 96 hr. T-bet (A), Eomes (B), and IFN-γ (C) protein expression was analyzed subsequent to restimulation at 120 hr. Values are means ± SD from three independent transfections; ***p < 0.001, *p < 0.05; ANOVA Tukey’s post hoc test.