Figure 6. Persistence of and TCR isolation from T cells specific for mutated antigens.

(A) Cells obtained from pheresis in patients 2–3 months following adoptive TIL transfer were stained with the indicated MHC tetramer and analyzed for binding. The percentage of tetramer-positive cells among the gated lymphocyte population is indicated. (B) Similarly, pheresis samples from patient 3703 obtained 2, 5, 8, and 12 months following treatment were analyzed for NSDHL tetramer binding. The percentage of positive T cells among the gated lymphocytes is indicated. (C) Sorted T cells specific for mutated SRPX (left panel) were assessed for TCR BV13 and BV28 expression (right panel). (D) The α and β chains for the BV13 (CDR3b: CASSFFGSNQPQHF) and BV28 (CDR3b: CASGSSGQGEYGELFF) TCR were cloned from expanded T cells sorted from patient 3713 infusion bag cells stained with SRPX tetramer. OKT3-stimulated PBMCs were transfected with RNAs encoding the appropriate α and β pairs or with GFP. Transfected T cells were cocultured with either mutated SRPX–pulsed T2 cells (left panel) or tumor cells from patient 3713 (right panel), along with the appropriate negative controls (right panel: T2 pulsed with MART1_26-35 -2L epitope and melanoma cells from patient 3466). Bulk-treatment TILs from patient 3713 were used as a positive control for effector cells. Sixteen hours after the start of the coculture, IFN-γ secretion was measured in the culture supernatant. Results are presented as the mean ± SEM (n = 3) for 2 individual donors. P < 0.05 by Student’s t test for the difference in IFN-γ secretion compared with the negative control.