A. The workflow for evaluating CAR-mediated early activation of CAR T cells, e.g. CD19CAR Tregs, involves co-incubating CD19CAR Tregs with irradiated CD19-expressing K562 for 48h, followed by flow cytometry to detect upregulation of the activation marker CD71 (transferrin receptor). Continued expansion of the cells for 9 days allows for assessment of CAR-mediated expansion (CAR Treg cell number), as well as assess the phenotypic stability of the CAR Tregs (e.g. FOXP3 expression). B. CD19CAR-mediated activation of human CD4+ T cells, CD8+ T cells, and Treg cells after 48h co-incubation with either irradiated K562 or CD199-K562, as assessed by flow cytometry with anti-human CD71 FITC. Histograms represent CD71 surface expression of live CD4+GFP+ cells.