Chimeric receptor design. (A) Schematic diagram of the chimeric receptors. The TNP-specific CR encompasses an scFv derived from the anti-TNP mAb, Sp6. In the tripartite configuration, the scFv is joined to a short portion (lacking the ligand-binding site) of the extracellular, transmembrane, and cytoplasmic domains of CD28 fused to the FcRγ chain. In the control CR configuration, the cytoplasmic domain of the CD28 molecule was deleted. (B) Chimeric receptor transgene constructs. Construct sequences that served to generate the transgenic mice were placed under the control of the human CD2 promoter/enhancer that directs expression only in T and NK cells. CYT indicates cytoplasmic domain; H, hinge domain; L, immunoglobulin leader; LCR, locus control region; P, promoter; pL, plasmid sequence; TM, transmembrane domain; V_H and V_L, immunoglobulin heavy- and light-chain variable domains, respectively; ΔCD28, deleted CD28 domain containing part of the extracellular and the transmembrane domain, and lacking the cytoplasmic signaling moiety. (C). Surface expression of anti-TNP CR in T (top row) and NK (bottom row) cells in the spleen of transgenic mice. Bulk splenocytes from WT or transgenic mice were double-stained with PE-conjugated rat anti–mouse Thy1.2 or PE-conjugated anti-NK1.1 and either biotinylated anti–Sp6-idiotype mAb GK-20.5 (bold lines) or matching biotinylated isotype control anti-DNP mAb (dashed lines), followed by secondary staining with fluorescein isothiocyanate (FITC)–conjugated streptavidin. Histograms were generated by gating on Thy1.2⁺ (top row) or NK1.1⁺ (bottom row) lymphocytes.