Figure 2

Engineered Tregs express CAR and FoxP3. (A) Antibodies from the 8.18 C5 hybridoma cross-reacted with murine myelin as demonstrated by immunohistochemistry (red colour expression). (B) CD4⁺T cells transduced with CARαMOG-FoxP3 lentivectors (CAR Tregs) were stained with fluorescein isothiocyanate (FITC)-conjugated antibodies and analyzed for surface expression of the scFv by flow cytometry. (C) FoxP3 copy number in transduced cells analyzed by quantitative PCR analysis. Expression of FoxP3 is significantly higher than both CD4-mock and naïve CD4⁺T cells (P < 0,05) as analyzed by Mann-Whitney using GraphPad prism software. The expression experiments were repeated for every cell generation with similar result. (D) CAR Tregs were mixed with αCD3/IL-2-stimulated T cells at a1:2 ratio and analyzed for suppressive ability in a thymidine-based assay. CAR Tregs were able to suppress activated T-cells (P < 0.05). CAR Tregs suppressed αCD3/IL-2-stimulated T cells in the presence of activated macrophages (P < 0.05) or MOG-expressing cells (P < 0.05). The experiments were repeated and statistical differences analyzed by Mann-Whitney test using GraphPad prism software.