TCR gene transfer of allogeneic T cells reduces the expression of the endogenous TCR and diminishes alloresponsiveness in vitro. (A) Alloreactivity after TCR gene transfer was assessed in an MLR adjusted to the specific biologic needs of preactivated CD8⁺ T cells (“In vitro cytotoxicity and MLR”). TCR-transduced (■) or nontransduced (♦) B10.A T cells were stimulated with either irradiated allogeneic B6 splenocytes or syngeneic B10.A splenocytes as controls (□/◇). Values are mean ± SE. To assess the viability of transduced vs nontransduced responders at the time of MLR readout, samples were drawn from the wells and stained for annexin V/propidium iodide. (B) Either CD8⁺GFP^lo- or CD8⁺GFP^hi-expressing T cells (B10.A) were gated on the Vα2/Vβ5 double-positive population after transduction. Transduced T cells were costained for 4 randomly chosen endogenous TCR Vβ chains. Bar graphs represent the reduction of the respective endogenous Vβ chains in percentage as calculated by the ratio of MFI (endogenous Vβ) on GFP^hi/MFI (endogenous Vβ) on GFP^lo. (C) To directly visualize a high-affinity alloreactive endogenous TCR, L^d alloantigen-specific TCR-transgeneic 2C T cells were used for transduction and consecutively gated on either GFP^lo or GFP^hi. Fluorescence-activated cell sorter plots represent the respective population expressing both of the introduced TCR chains (Vα2 and Vβ5, middle plots). Cells were analyzed for endogenous 2C TCR expression (right plots) using the 2C-specific clonotypic marker 1B2.