COX2 expression and cell proliferation are NFATc3 dependent in primary T blasts. Primary T cells were stimulated with PHA (5 μg/mL) and maintained in medium containing IL2 (50 U/mL) for 10 days. The cells were then infected with KDSc, KD-c1, or KD-c3 LV vectors as indicated. (A) shRNA-infected T cells were stimulated by plating onto anti-CD3–coated wells (1μg/mL) for 2 hours, and the expression of COX2 (A) (n = 7) and IL2 (B) (n = 8) mRNA was determined by qPCR. ***P < .001. CD4⁺ (C) and CD8⁺ (D) blasts were isolated from the infected T-cell blast population and stimulated with plate-bound anti-CD3 for 2 hours. COX2 mRNA expression was analyzed by qPCR. Data are from 1 representative experiment of 3 performed. (E) Infected T-cell blasts were labeled with CellTrace Violet, plated onto wells coated with anti-CD3 plus anti-CD28 (1 μg/mL of each), and proliferation was analyzed by flow cytometry 1 and 4 days after treatment. The proliferating cells lose fluorescent compound in each division. Numbers indicate the per-centage of proliferating cells (generation > 1) after 1 or 4 days. A representative experiment is shown.