(A) Chemical structure of 4-hydroxy-3-nitrophenyl acetyl (NP). Flow cytometry analysis of (B) NP67-PE staining of γδ T cells from C57BL/6 or G8/Rag2−/− mouse splenocytes and PE staining of γδ T cells from B6 splenocytes; (C) staining of 58α-β- cells expressing an NP-specific γδ TCR, 1G9, with NP43-CGG-Cy5 or CGG-Cy5, showing staining in relation to γδ TCR expression (left) or as a histogram (right); (D) staining of 58α-β- cells expressing an NP-specific γδ TCR, 1E3, with NP43-CGG-Cy5, NP26-BSA-Cy5, or BSA-Cy5 (left) and NP67-PE alone, NP67-PE with a 20-fold molar excess of anti-NP Fab, or PE (right). (E) IL-2 production by 1E3/58α-β- cells activated by the indicated amount of plate-bound NP25-KLH, KLH (light gray bars), or 0.1 μg/ml anti-CD3. (F) Sensorgram and steady state analysis of NP43-CGG (0–7 μM) binding to soluble 1G9 TCR measured by surface plasmon resonance. Apparent KD was determined by steady state analysis of SPR measurements (circles). Equal concentrations of un-modified CGG were tested (squares), as well as NP43-CGG with a PE-specific γδ TCR, MA2 (triangles).