TCR^neg T cells can be enriched by depletion of CD3ϵ⁺ T cells. (A) CD3 expression before and after depletion using CD3-specific paramagnetic beads. Flow cytometry reveals expression of CD3ϵ in CD4⁺ and CD8⁺ T cells 15 days after stimulation by OKT3-loaded aAPC (9 days after ZFN transfection). Numbers in the bottom-right quadrant represent the percentage of CD3ϵ^neg T cells. Representative results using in vitro numerically expanded T cells. (B) CD3ϵ^neg T cells can be further enriched by additional round of depletion with CD3-specific paramagnetic beads. Flow cytometry revealing expression of CD3ϵ in CD4⁺ and CD8⁺ T cells after 2 rounds of depletion of CD3ϵ^pos T cells. Numbers in the lower right quadrant represents the percentage of CD3ϵ negative cells in CD4⁺ and CD8⁺ T-cell populations. (C) Vβ repertoire analysis in T cells modified with ZFNs. The Vβ usage clonogram was analyzed by a panel of TCR-specific mAbs, costained with CD4 and CD8. Percentage of specific Vβ⁺ T-cell fractions within CD4 and CD8 gating is shown. The nomenclatures of Vβ repertoire shown are based on Arden et al.⁵⁰  Representative data from 3 independent experiments are shown.