Rapid cloning of gp100
specific CD8+ T cells from peripheral blood. (A.) On Day 0, PBMC from four HLA-A2+ melanoma patients underwent staining with gp100154–162 peptide/HLA-A*0201 tetramers and anti-CD8 APC to determine natural precursor frequency. None of the patients demonstrated a significant population of tetramer positive CD8+ T cells by FACS. PBMC from each patient were plated in replicate microwells (n = 96) containing ~300,000 cells and sensitized for 10 days with 1 μM of gp100154–162 peptide in the presence of IL-2 (90 IU/ml). (B.) On day 10, a sample from every microculture was screened using the qPCR assay for specific recognition of the gp100154–162 peptide versus the HIVpol peptide. The wells with the highest SI reactivity (denoted by **) were selected for limiting dilution cloning. After approximately 2 weeks, growth positive wells were functional screened for their ability to lyse peptide pulsed T2 cells. Selected T cell clones were expanded and underwent FACS analysis (C.) between days 25 and 34 to reveal highly enriched (99%) populations of gp100154–162 tetramer positive CD8+ T cells. Values in FACS dot plots correspond to the percentage of total CD8 + T cells that are tetramer-positive. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0).