Tyrosinase peptide-specific CTL recognition of tumor cell lines and primary melanoma tumor cells. (A) Columns represent the amount of IFN-γ (picograms per milliliter) secreted by patient-derived IVS-B CTL, self-restricted D115 CTL, and allo-restricted T58 CTL in coculture with a panel of tumor cell lines from left to right: MaCa1 (HLA-A2−tyrosinase−); SK-Mel-28 (HLA-A2−tyrosinase+); Mel-A375, RCC-26, PancTu 1, MaCa1/A2, and UTS CC 1588 (all HLA-A2+tyrosinase−); Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29, and WM-266-4 (all HLA-A2+tyrosinase+). T indicates CTL without stimulating cells. (B) The HLA-A2+tyrosinase− tumor cell lines Mel-A375, RCC-26, and MaCa1/A2 were exogenously loaded with either 10−5 M irrelevant flu peptide (f) or 10−5 M tyrosinase peptide YMD (t), and IFN-γ secretion was measured by ELISA and given as picograms per milliliter. (C) HLA-A2 expression on primary tumor cells (passage 12) of an HLA-A2− melanoma patient after transfection with 50 μg HLA-A2 ivt-RNA and on established melanoma cell lines Mel-93.04A12 (HLA-A2+tyrosinase+), and Mel-A375 (HLA-A2+tyrosinase−) was measured by flow cytometry after staining with HLA-A2–specific monoclonal antibody. Histograms represent stained samples (filled curves) and control samples (empty curves): control curves show untransfected primary tumor cells stained with HLA-A2–specific monoclonal (left histogram) or isotype control antibodies used with the melanoma cell lines (middle and right histograms). HLA-A2 expression on primary tumor cells was detected 10 hours after electroporation. (D) The capacity of the patient-derived CTLs (IVS-B), the representative self-restricted CTLs (D115), and the representative allo-restricted CTLs (T58) to secrete IFN-γ or (E) release perforin in coculture with the melanoma cells shown above was measured in ELISPOT assays. n.d. indicates not detectable.
