AAV transduction of HEK-293T and T cells

Three different doses of AAVs were administered: 1 × 10⁴ AAVs/cell was considered AAV low (L), 1.5 × 10⁴ AAVs/cell was considered AAV middle (M), and 1 × 10⁵ AAVs/cell was considered AAV high (H). The day before transduction, HEK-293T cells were trypsinized and counted, and 1–4 × 10⁵ cells were plated in 2.0–3.0 ml complete culture medium and incubated at 37 °C overnight. Sixteen hours later, purified AAV (AAV-CD4CAR or AAV-CD20CAR) was added to each well at different doses by gentle swirling/mixing. Twenty-four hours post-transduction, fresh media containing 20% FBS was added, and the cells were cultured for 2–3 days and then analyzed by FACS, or fluorescence microscopy.

The T cells were activated and infected with AAV-CAR as described [51]. Briefly, 10⁶ cells were plated on day 0 and activated for 2 days in the presence of 300 IU/ml IL-2 and 1 μg/ml anti-human CD3 antibody. On day 2, purified AAV (AAV-CD4CAR or AAV-CD20CAR) was added to each well at different doses by gentle swirling/mixing and incubated at 37 °C in the presence of 5% CO₂. The next morning following transduction, fresh media containing 20% FBS was added, and the cells were cultured for 2–3 days and then analyzed by FACS or fluorescence microscopy.