NY-ESO-1–redirected T cells efficiently kill MM cells. Shown are results from TCR TR, SE, CE, and untransduced lymphocytes (mock-trd) cells tested by IFN-γ Elispot assay against T2 cells pulsed with decreasing concentrations of the NY-ESO-1_157-165 HLA-A2 restricted peptide or the irrelevant WT1_126-134 HLA-A2 restricted peptide as negative control (A) or against U266 (HLA-A2^pos and NY-ESO-1^pos) and MM.1S (HLA-A2^neg and NY-ESO-1^neg; negative control) MM cell lines (B). Specific spots are shown on the y-axis as spots produced in the presence of stimulators minus the spots produced by the effectors alone. Mean and SEM are shown, at the E/T ratio of 1. (C-D) Cytotoxic assay with TCR edited, TR, and untransduced T cells. (C) Shown is the functional activity measured by a ⁵¹Cr release assay for the lysis of labeled T2 cells pulsed with a concentration of the NY-ESO-1_157-165 HLA-A2 restricted peptide of 1 µM, at decreasing E/T ratios or against U266 and MM.1S (negative control) MM cell lines (D) at the E/T ratio of 50:1. Mean and SEM of the percentages of lysis are shown. (E-G) Four-day coculture assay. TCR TR, SE, CE, and untransduced lymphocytes were cultured with U266 or MM.1S (E/T ratio = 1:5). After 4 days, residual U266 cells (CD138^pos/CD3^neg), MM.1S cells (CD38^pos/CD3^neg), and T lymphocytes (CD138^neg/CD38^neg/CD3^pos) were counted and analyzed by FACS. (E) Expansion of NY-ESO-1–redirected T cells and control untransduced T cells (mock-trd) in response to NY-ESO-1–expressing (U266) or nonexpressing (MM.1S) cells measured as fold increase at the end of culture. (F) Antimyeloma effect by T cells measured as elimination index (see “Methods”). Mean and SEM are shown. (G) Representative plots of T-cell coculture with MM.1S (upper panel) and U266 (lower panel) at the E/T ratio of 1:5. All functional studies have been performed with 3 distinct donors. TR and SE cells were sorted for high expression of the NY-ESO-1_157-165 dextramer before functional assays. All effectors were tested after 1 cycle of cell stimulation through a rapid expansion protocol.²⁴