High levels of tumor antigen-reactive clones persist in the blood of ablated patients after adoptive transfer. (A) Elevated numbers of lymphocytes in the peripheral blood of select patients following cell transfer contract to normal levels. Approximately 1 week after transfer, absolute lymphocyte counts (ALCs) were measured from peripheral blood from patients 9 (▵), 10 (▧), 20 (♦), 21 (•), 23 (x), and 28 (▪). The x-axis represents days relative to cell transfer, with day 0 representing the day of cell infusion. Latest time points analyzed for patients 9, 10, and 23 were days 124, 524, and 161 after infusion, respectively. (B) CD8⁺ T-cell frequencies are generally elevated after cell transfer and diminish temporally. The frequency of CD8⁺ cells was measured on gated viable lymphocytes. (C) High frequencies of tumor antigen-specific CD8⁺ T cells persist in the peripheral blood of select patients. Tumor antigen-specific CD8⁺ T cells were identified using MART-1: 27-35(27L) peptide containing HLA-A^*0201 tetramer complexes or Vβ-specific antibodies. (D) High numbers of melanoma-specific CD8⁺ T cells persist in the blood of metastatic melanoma patients receiving ACT. Absolute numbers of tumor antigen-specific CD8⁺ T cells were calculated as number of CD8⁺ lymphocytes per mm³ times percent tetramer or Vβ-specific antibody positive. (E-F) PBLs from treated patients maintain tumor antigen-specific reactivity. Posttreatment PBL from patients 9 (d 56), 10 (d 524) 20 (d 56), 21 (d 63), 23 (d 28), and 28 (d 97) was tested for IFN-γ secretion in response to T2 targets cells pulsed with 1 μM gp100:209-217 or MART-1:27-35 (M1) peptide, or 10 μM MART-1:27-35 (M10) peptide (E) or melanoma cell lines 888 (HLA-A2^- MART-1⁺), 2098 (HLA-A2⁺ MART-1^-), 526 (HLA-A2⁺ MART-1⁺), or 624 (HLA-A2⁺ MART-1⁺) by standard ELISA assay (F).