CD28 and RHOA mutants synergize in promoting IL-2 transcription and IL-2 secretion. (A) In vitro secretion of IL-2. A total of 200 000 Jurkat cells per well, transduced with the lentiviruses indicated by the color code, were seeded and stimulated with either immunoglobulin G (IgG) control (5 μg/mL); anti-CD3 (5 μg/mL) with crosslinker (5 μg/mL); anti-CD3 and anti-CD28 and crosslinker (1 μg/mL or 5 μg/mL); or with PMA (20 ng/mL) and IONO (1 μM). Twenty-four hours later, supernatants were collected, and IL-2 was measured by enzyme-linked immunosorbent assay. Data are represented as mean ± SEM from 5 independent experiments conducted in quadruplicates. Significant differences in IL-2 secretion were determined using 2-way ANOVA with Tukey multiple comparison test (∗P ≤ .05; ∗∗∗P ≤ .01; ∗∗∗P ≤ .001). (B) Time course of IL-2 expression. A total of 2 × 106 Jurkat cells per well bearing the indicated lentiviruses were seeded and stimulated with anti-CD3, anti-CD28, and crosslinker at 5 μg/mL during indicated periods of time. RNAs were extracted, and reverse transcription was performed. qPCR was performed using TaqMan reagents and a QuantStudio 5 machine. Data are presented as mean ± SEM from 3 independent experiments. AUCs and 95% CIs are indicated. AUC, area under the curve; CI, confidence interval; IONO, ionomycin.
