Figure 1.

TCR and CD28 costimulation synergistically induce miR-17∼92 expression and forced miR-17∼92 expression enables T cell activation of CD28-deficient T cells in vitro

(A and B) Naive murine CD4⁺ T cells were activated for 24 h or 48 h using the indicated combinations of plate-bound anti-CD3 and anti-CD28 stimulatory mAbs. (A) miR-17 expression assessed by qPCR. (B) Relative frequencies of cells expressing CD25 and CD69, assessed by flow cytometry.

(C–G) wt (black), CD28^−/− (purple), rescue (dark blue) CD4⁺ T cells were stimulated for 48 h with plate-bound anti-CD3 with (+) or without (−) anti-CD28. (C) Blasting of CD4⁺ T cells shown as MFI of FSC-A of the lymphocyte gate. (D) Proliferation measured by CTV dilution, gated on viable CD4⁺ T cells. Representative histograms of each genotype activated without (blank) or with (colored) anti-CD28 and quantification of proliferation index. (E and F) Expression of early activation markers CD25/CD69 as well as CD44/CD62L expression. (G) Quantification of flow cytometric intracellular IL-2 staining in CD4⁺ cells stimulated for 3 h with PMA/Iono/BFA. Data from an experiment with 3 biological replicates per condition (A and B). Data from 3 (C–G) independent experiments with 3–4 biological replicates per group. Error bars represent mean ± SD, Tukey’s or Hom-Sidak’s multiple comparison test; p values: ns = not significant, ∗<0.05 ∗∗ < 0.002 ∗∗∗<0.0002 ∗∗∗∗<0.0001.