Seed 1 x 10^5 HEK293A cells in 2 ml cell culture media (DMEM, 10% FBS, 5% PenStrep) in one well of a 6-well plate and incubate the cells for 6 - 14 hr at 37 °C in a 10% CO2 incubator to allow them to adhere. Cells should then be at approximately 50% confluency. Lipofection: Transfer 6 μl of jetPEI reagent into 94 μl of 50 μM NaCl, vortex briefly. Add the mixed solution to 100 μl linearized adenovirus vector while vortexing and incubate this transfection mix for 15 - 30 min at room temperature.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGFbeta and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown. Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44(low), CD62L(high)) and resting (CD25(-), CD69(-)) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.