Identification and retroviral expression of an FVIII-2191-2220–specific TCR in primary CD4 T cells. (A) Scheme of FVIII-2191-2220–specific TCR cloning from a hemophilia A subject’s T-cell clone. The cloning procedure is described in “Methods.” Briefly, to amplify TCR cDNA from the clone, poly C oligonucleotide was linked onto the 3′ terminus of total cDNAs by terminal deoxynucleotidyl transferase. The variable region was amplified using seminested PCR with a poly GI primer and 2 reverse primers (pC1 and pC2) corresponding to 2 different 5′ upstream coding regions of the α and β chain constant regions. (B) Amplification of V regions of the 17195TCR using seminested PCR. Primary amplification and nested amplification were carried out with pC1 and pC2 (A). PGI was commonly used as the forward primer for the primary and nested PCR step. Bold arrows indicate the predicted sizes of the amplified Vα and Vβ PCR products. (C) Retroviral expression construct of the FVIII-2191-2220–specific TCR (17195TCR). To build the FVIII-2191-2220–specific TCR, the variable regions (Vα and Vβ) from the FVIII-2191-2220–specific T-effector clone were combined with human constant regions (Cα and Cβ) extracted from the NCBI database. To produce the individual α and β TCR chains from a single transcript, a P2A cleavage peptide was inserted between the α and β chain sequences. Expression of the GFP reporter is controlled by IRES, which is located downstream of the TCR construct.