Evaluation of effect of PTEN gene deletion in mouse CD4+ Th1 clones after stimulation

PTEN is thought to play a critical role in T cell activation by negatively regulating the PI3K signaling pathway important for cellular activation, growth, and proliferation. T cells from mice in which PTEN was conditionally deleted in the thymus were reported to display CD28-independent IL-2 production and relative resistance to anergy induction. However, such observations could have stemmed from alterations in T cell development due to early deletion in thymocytes. To directly eliminate PTEN in post-thymic T cells, we utilized CAR Tg x PTENflox/flox mice which enabled gene deletion using a Cre adenovirus in vitro. Gene expression profiling revealed a small subset of induced genes that were augmented upon PTEN deletion and T cell stimulation. Our results indicate that deletion of PTEN can augment the activation of post-thymic T cells. Nonetheless, PTEN inhibition may be a viable target for immune potentiation due to increased cytokine production by activated CD4+ cells. It was of interest to determine the spectrum of transcripts regulated by PTEN using gene expression profiling. To this end, CAR Tg x PTENflox/flox Th1 clones were either treated with adeno-Cre or adeno-EV, then confirmed to have PTEN protein abscence by western blot, then stimulated for 6 hours with anti-CD3/anti-CD28 mAb- coated beads and analyzed (2 total conditions). The experiment was replicated so there are 2 samples for each condition.