Characterization of Ag specificity using TCR150-transgenic lymphocytes. (A) Specific lysis of parental THP-1 and mel624.38 (black symbols) or HMMR-specific shRNA-treated THP-1 or mel624.38 (gray symbols) by TCR150-transgenic PBLs, measured in a standard chromium-release assay. The HMMR^low cell line MCF-7 (black triangles) and mock PBLs with THP-1 or mel624.38 (open symbols) served as controls. (B) IFN-γ secretion (ng/mL) of HMMR-specific TCR150-transgenic, GFP-transduced, or mock-treated PBLs (2 × 10⁵) after stimulation with autologous, HLA-A2⁺ mature DCs (5 × 10⁴). DCs were pulsed with ivt-RNA encoding full-length HMMR_1-725 or deletion mutants encoding aa 1-170, aa 1-108, aa 1-66, or aa 1-17, respectively. (C) IFN-γ release (ng/mL) by 2 × 10⁵ lymphocytes (mock treated, GFP transduced, or TCR150 transgenic) after stimulation with 5 × 10⁴ HLA-A2⁺ DCs, electroporated with HMMR-derived peptides. Left block depicts DCs pulsed with a long 17-mer (HMMR_1-17) and corresponding nonamers; the right block shows DCs loaded with 20 μg of the peptides HMMR_1-8, HMMR_1-9, HMMR_1-10, and irrelevant, tyrosinase-derived YMD peptide. (D) IFN-γ secretion (pg/mL) of TCR150-transgenic PBLs after stimulation with primary ALL cells. Left block shows HMMR⁻ samples; right block depicts HMMR⁺ samples. All samples except ALL14 were electroporated with ivt-RNA encoding HLA-A*02:01:01:01 and expression was assessed by flow cytometry (approximately 50% positive cells; data not shown). Both HLA-A2⁻ (parental; open bars) and transient HLA-A2⁺ cells (filled bars) were analyzed for stimulatory capacity in 24-hour cocultures with TCR150-transgenic PBLs at an effector-to-target ratio of 40:1 using 2 × 10³ target cells.