Figure 5. Suppressive function of TCR-engineered Tregs.

(A) Validation of the expression of gp100-TCR-engineered Tregs. gp100-TCR overexpressing Tregs were stained with mTCR constant β chain antibody or gp100-dextramer and analyzed by FACS. (B) Tregs_TCR-gp100 upregulate GARP upon peptide activation. Tregs expressing gp100-TCR were stained with a GARP antibody 2 days after gp100 presentation by T2 cells or DCs. A dose-dependent upregulation of GARP on Tregs_TCR-gp100 is shown. The data are representative from three different experiments from multiple donors. (C–F) Tregs_TCR-gp100 suppress the proliferation of T_{N TCR-gp100} and T_{N TCR-SL9} cells in vitro. At 2 weeks post activation, expanded gp100-TCR-transduced Tregs were sorted by FACS, using RFP expression as a marker. IL-7 cultured, gp100- or SL9-TCR-engineered, and CellTrace violet dye-labeled T_N cells were used as target cells. Suppression assay was set up at Tregs:target 1∶1 ratio, either using same peptide (C, D) or different peptides (E, F). The proliferation of target cells was determined at day 5 post activation. (C, D) Tregs_TCR-gp100 were mixed with labeled T_{N TCR-gp100} in the presence of 10 nM gp100 presented by either T2 cells (Exp.1) or DCs (Exp.2). (E,F) Tregs_TCR-gp100 were mixed with labeled T_{N TCR-SL9} when gp100 and SL9 were presented by either T2 cells (Exp. 3) or HLA-A*0201⁺ DCs (Exp. 4). In Exp.4, DCs were preloaded with gp100 (10,000 nM) 1 day in advance and were washed three times before co-culturing with Tregs and T_N cells. In Exp.5, Tregs_TCR-gp100 were activated with gp100 preloaded DCs and T_{N TCR-SL9} were activated with SL9 presented by T2 cells. In Exp.6, suppression assay was set up as in Exp.3 except that autologous DCs of Tregs (HLA-A*0201⁻) were added to the co-culture. The data are representative from three different experiments from multiple donors.