Propagation of TCR^neg CAR⁺ T cells on CD19 expressing aAPC. (A) Sustained proliferation of TCR^neg CAR⁺ T cells. CAR⁺ T cells with (TRAC and TRBC) or without (parental and no mRNA) TCR modification by ZFNs were stimulated with γ-irradiated CD19⁺ aAPCs every 2 weeks. Viable T cells were enumerated every 7 days and inferred total numbers were calculated. Representative data from 3 independent experiments are shown. (B) Analysis of TCR^neg CAR⁺ T cells after propagation. Flow cytometry analysis of CD3ϵ expression (top panel), αβTCR and γδTCR expression (middle panel), and subset analysis for memory pool (bottom panel) of TCR^negCAR⁺ T cells after 28 days of propagation on aAPCs. Numbers are percent expression for the quadrant. (C) Vβ repertoire analysis in TCR^neg CAR⁺ T cells after propagation on aAPCs. The Vβ usage clonogram was analyzed by a panel of TCR-specific mAbs, costained with CD4 and CD8. Percentage of identified Vβ⁺ T-cell fractions within CD4 and CD8 flow cytometry gates is shown. The nomenclatures of Vβ repertoire shown are based on Arden et al.⁵⁰  Representative data from 3 independent experiments are shown.