Figure 3. CAR expression and function in peripheral T lymphocytes is fully restored by 2-MRE copy regulation.

(A) Lentiviral vectors used in these experiments express 19z1 CAR driven by hPGK promoter under regulation by either no (19z1₀), 2 (19z1₂), or 4 (19z1₄) copies of miR-181a–specific MRE. (B) Mean fluorescence intensity (MFI) expression of 19z1 in DN, DP, CD4SP, and CD8SP thymocytes as well as CD4⁺ and CD8⁺ splenocytes normalized to expression in CD4^–CD8^– splenocytes for each mouse. Data are averaged from 4 mice harboring each of vectors 19z1₀, 19z1₂, and 19z1₄ analyzed 3–4 months after BM transplantation. Error bars denote SEM. (C) Expression of 19z1 in peripheral blood 19z1⁺CD3⁺ T cells in a total of 23 mouse chimeras harboring vectors 19z1₀, 19z1₂, and 19z1₄ analyzed 4 weeks after BM transplantation. Expression levels in CD3^– cells indicate comparable levels of gene transfer. Error bars denote SD. Repeated analysis at week 10 after BM transplantation (not shown) corroborated the data presented here. (D) T cells expressing a miR-181–regulated 19z1 receptor specifically lysed a hCD19⁺ tumor cell line. ⁵¹Cr-release assays of T cells isolated from spleens of mouse chimeras harboring vectors 19z1₀, 19z1₂, and 19z1₄ targeting EL4-hCD19⁺ tumor cells or unmodified EL4 (hCD19^–) tumor cells, as well as T cells from a mouse reconstituted with untransduced BM cells. E/T ratio, effector-to-target ratio of CD8⁺19z1⁺ T cells to tumor cells.