Fig. 2. Both HLA-A2⁺ and A2⁻ peripheral T cells can recognize A2/MART1 when transduced with chain-centric SIG35α.

(A), Both HLA-A2⁺ and A2⁻ peripheral T cells become A2/MART1-reactive upon transduction of chain-centric SIG35α. Peripheral CD8⁺ T cells freshly isolated from two HLA-A2⁺ donors (#1 and #2) and two A2⁻ donors (#3 and #4) were retrovirally transduced with ΔNGFR or SIG35α/ΔNGFR and stained with A2/MART1 multimer or A2/HIV multimer in conjunction with anti-CD8 mAb and anti-NGFR mAb. Data shown are gated on ΔNGFR⁺ cells. Data of donors #1 and #3 are representative of three independent experiments and data of donors #2 and #4 are representative of two independent experiments. (B), SIG35α-transduced A2/MART1 CD8⁺ T cells expand in an A2/MART1-specific manner. A2⁺ and A2⁻ CD8⁺ T cells transduced with SIG35α/ΔNGFR were stimulated with wt-aAPC or mut-aAPC pulsed with wild-type A2/MART1 peptide once a week. Between stimulations, the T cells were supplemented with IL-2 (10 IU/ml) and IL-15 (10 ng/ml) every 3 days. Data depict A2/MART1 multimer staining performed following the first and second stimulations using wt-aAPC and the second and third stimulations utilizing mut-aAPC. Data shown are gated on ΔNGFR⁺ cells. Representative multimer-staining data from one of two HLA-A2⁺ donors and one of two A2⁻ donors are shown. (C), Peripheral T cells transduced with SIG35α are highly avid for A2/MART1 recognition. CD8⁺ T cells following stimulation with wt-aAPC pulsed with wild-type A2/MART1 peptide were used as responder cells in IFN-γ ELISPOT analysis. T2 cells pulsed with 10 μg/ml A2/HIV control peptide or wild-type A2/MART1 peptide were used as stimulator cells (top). The A2⁺ MART1⁻ melanoma line, A375, and the A2⁺ MART1⁺ melanoma line, Malme-3M, were used as stimulator cells (bottom). Data shown are representative of two independent experiments. All experiments were carried out in triplicate and error bars depict SD. *P < 0.05, **P < 0.01, ***P < 0.001. (D), Peripheral CD8⁺ T cells isolated from an A2⁻ donor #3 were transduced with ΔNGFR, SIG35α/ΔNGFR, SIG35αN/ΔNGFR, DMF5α/ΔNGFR, or DMF5αS/ΔNGFR and stained by A2/MART1 multimer or A2/HIV multimer in conjunction with anti-CD8 mAb and anti-NGFR mAb as described in Fig. 1. Data shown are gated on ΔNGFR⁺ cells.