FIG. 1.

Gene constructs and sorting reagents to MACSort TCR-engineered human T cells. (A) Schematic representation of TCRα, TCRβ, and TCRα-2A-tCD34-2A-TCRβ transgenes used to gene-engineer primary human T cells. TCR specific for gp100/A2 comprised TRAV13-1*02/J52*01/CA and TRBV27*01/J2-7*01/D2*02/CB2 (Schaft et al., 2003). tCD34 represents a truncated and functionally inert variant of CD34. T cells were transduced either with pBullet:TCRα+pBullet:TCRβ and termed “TCR T cells” or with pBullet:TCRα-2A-tCD34-2A-TCRβ and termed “TCR-tCD34 T cells.” Abbreviations: V, TCRαβ-variable domain; C, TCRαβ-constant domain; D, TCRβ-diversity domain; J, TCRαβ-joining domain; 2A, 2A sequence encoding a self-cleaving peptide; tCD34, truncated CD34 molecule. (B) Schematic illustration of reagents used to MACSort TCR-engineered human T cells. From left to right: Tetramer (Altman et al., 1996), pentamer (Sebestyen et al., 2008), Streptamer (Knabel et al., 2002; Neudorfer et al., 2007), and CD34 mAb (Stull et al., 2000). Tetramers consist of 4 gp100 peptide (YLEPGPVTA)/HLA-A2 (gp100/A2) monomers that were biotinylated and multimerized with streptavidin–phycoerythrin; pentamers consist of 5 gp100/A2 monomers that were linked to phycoerythrin and multimerized by a self-assembling, coiled-coil structure; and Streptamers consist of 8–12 gp100/A2 monomers that were Strep-tagged and multimerized with Strep-Tactin–phycoerythrin (depicted with 5 monomers). Anti-CD34 mAbs consist of heavy and light chains providing two antigen-binding sites and are directly coupled to magnetic microbeads.