Figure 3
CTL clones from multimer-sorted cells of HLA-A2+ and HLA-A2− donors primed with RNA-pulsed DCs. (A) Cytotoxic activity of 5 CTL clones from HLA-A2+ and 7 CTL clones from HLA-A2− donors is shown against Mel-A375 (HLA-A2+tyrosinase−) and Mel-93.04A12 (HLA-A2+tyrosinase+) melanoma lines at E:T of 5:1. CTL clones showed significant differences in lysis of HLA-A2+tyrosinase− vs HLA-A2+tyrosinase+ melanoma lines (nonparametric Wilcoxon signed rank test, *P < .05). Allo-restricted clones displayed greater mean percentage-specific lysis than self-restricted clones (52.8% vs 28.5%; nonparametric Mann-Whitney U test; *P < .05). The patient-derived IVS-B clone showed 22.5% specific lysis (♦), and the peptide-primed CTL clone Tyr-F8 showed 14.4% specific lysis (▲). (B) IFN-γ secretion (picograms per milliliter) by the same clones after coculture with Mel-A375 (HLA-A2+tyrosinase−) and Mel-93.04A12 (HLA-A2+tyrosinase+) lines was measured by ELISA. Allo-restricted clones showed higher mean IFN-γ secretion than self-restricted clones (1639 pg/mL vs 561 pg/mL; nonparametric Mann-Whitney U test; **P < .005). The IVS-B clone (♦) released 106 pg/mL IFN-γ and Tyr-F8 (▲) released 129 pg/mL. (C) IFN-γ secretion (picograms per milliliter) by 7 allo-restricted (●) and 3 self-restricted (○) T-cell clones in coculture with a panel of tumor cell lines shown from left to right: a breast carcinoma line MaCa1 (HLA-A2−tyrosinase−); a melanoma line SK-Mel-28 (HLA-A2−tyrosinase+); Mel-A375 (HLA-A2+tyrosinase−), a renal cell carcinoma line RCC-26 (HLA-A2+tyrosinase−), a pancreas carcinoma line PancTu 1 (HLA-A2+tyrosinase−), a stable HLA-A*0201 transfectant of MaCa1/A2 (HLA-A2+tyrosinase−), and a tongue carcinoma line UTS CC 1588 (HLA-A2+tyrosinase−); and the melanoma cell lines Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29, and WM-266-4 (all HLA-A2+tyrosinase+). The leftmost values designated with T show the background levels of cytokine secreted by the CTL in the absence of stimulating cells. (D) 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.

CTL clones from multimer-sorted cells of HLA-A2+ and HLA-A2− donors primed with RNA-pulsed DCs. (A) Cytotoxic activity of 5 CTL clones from HLA-A2+ and 7 CTL clones from HLA-A2− donors is shown against Mel-A375 (HLA-A2+tyrosinase−) and Mel-93.04A12 (HLA-A2+tyrosinase+) melanoma lines at E:T of 5:1. CTL clones showed significant differences in lysis of HLA-A2+tyrosinase− vs HLA-A2+tyrosinase+ melanoma lines (nonparametric Wilcoxon signed rank test, *P < .05). Allo-restricted clones displayed greater mean percentage-specific lysis than self-restricted clones (52.8% vs 28.5%; nonparametric Mann-Whitney U test; *P < .05). The patient-derived IVS-B clone showed 22.5% specific lysis (♦), and the peptide-primed CTL clone Tyr-F8 showed 14.4% specific lysis (▲). (B) IFN-γ secretion (picograms per milliliter) by the same clones after coculture with Mel-A375 (HLA-A2+tyrosinase−) and Mel-93.04A12 (HLA-A2+tyrosinase+) lines was measured by ELISA. Allo-restricted clones showed higher mean IFN-γ secretion than self-restricted clones (1639 pg/mL vs 561 pg/mL; nonparametric Mann-Whitney U test; **P < .005). The IVS-B clone (♦) released 106 pg/mL IFN-γ and Tyr-F8 (▲) released 129 pg/mL. (C) IFN-γ secretion (picograms per milliliter) by 7 allo-restricted (●) and 3 self-restricted (○) T-cell clones in coculture with a panel of tumor cell lines shown from left to right: a breast carcinoma line MaCa1 (HLA-A2−tyrosinase−); a melanoma line SK-Mel-28 (HLA-A2−tyrosinase+); Mel-A375 (HLA-A2+tyrosinase−), a renal cell carcinoma line RCC-26 (HLA-A2+tyrosinase−), a pancreas carcinoma line PancTu 1 (HLA-A2+tyrosinase−), a stable HLA-A*0201 transfectant of MaCa1/A2 (HLA-A2+tyrosinase−), and a tongue carcinoma line UTS CC 1588 (HLA-A2+tyrosinase−); and the melanoma cell lines Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29, and WM-266-4 (all HLA-A2+tyrosinase+). The leftmost values designated with T show the background levels of cytokine secreted by the CTL in the absence of stimulating cells. (D) 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.

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