Article Figures & Data
Figures
- FIGURE 1.
Structure of the A7 retrovirus. A modified SAMEN′ pg1 Moloney murine leukemia virus backbone. Expression of the TCR α-chain is driven by the promoter in 5′ long terminal repeat (LTR), which also drives expression of the neomycin phosphotransferase gene (neo) via an internal ribosomal entry site (IRES). β-chain expression is driven by the hybrid HTLV-I/SV40 SRα promoter. SD, splice donor site; SA, splice acceptor site; pA, polyadenylation signal; Ψ+, packaging signal. Arrows denote transcription start sites.
- FIGURE 2.
Cytokine release and lysis assays for representative transduced PBL clones. A, Human IFN-γ release measured in culture supernatants from clones incubated with unpulsed T2 cells or T2 cells pulsed with either decreasing concentrations of the m9-27 peptide or the irrelevant g9-209 peptide. Recognition of HLA-A2+ and HLA-A2− melanoma lines was also tested. Specific recognition is defined as >100 pg/ml of IFN-γ released and >2-fold above background release by relevant controls. ∗, Values were above the maximum standard (2000 pg/ml) used in the assay. B, Specific lysis of T2 cells pulsed with either m9-27 peptide or the irrelevant g9-209 peptide, and of melanoma lines 888 Mel (HLA-A2−) and 888-A2+ (HLA-A2+), by transduced clones in 4-h 51Cr release assays.
Tables
Stimulators HLA-A2 Responders (pg/ml IFN-γ released) Medium 65 SHAMa 65 TRANa 1235 TILb 1520 TILb Medium − 0.0 60.7 16.1 63.1 29.5 T2 alone + 0.7 62.9 65.8 51.8 140.9 T2+ g9-209 + 0.4 70.1 80.9 45.4 12875.0 c T2+ m9-27 + 0.6 54.6 980.0 8082.5 158.1 397 MEL − 0.0 67.4 71.1 42.0 21.6 397 A2 MEL + 1.6 74.8 129.8 2210.0 1717.5 624-28 MEL − 0.3 58.2 131.1 36.6 15.6 624-38 MEL + 12.6 59.0 205.0 5612.5 3515.0 1300 MEL + 0.4 50.2 243.6 11902.5 13627.5 Stimulators HLA-A2 Medium 66 SHAM 66 TRAN 67 SHAM 67 TRAN 1235 TIL 1520 TIL Medium − 1.5 8.2 13.2 8.3 14.7 39.5 72.5 T2 alone + 1.6 15.7 15.7 15.5 16.5 16.4 109.6 T2+ g9-209 + 0.4 13.3 15.2 16.7 12.2 32.6 >2000 T2+ m9-27 + 1.6 15.3 506.5 13.2 214.3 >2000 119.3 397 MEL − 1.1 15.3 56.8 19.5 46.1 34.8 19.9 397 A2 MEL + 1.5 11.7 74.6 13.8 51.0 >2000 >2000 1011 MEL − 1.1 11.7 17.7 9.9 15.0 34.9 170.8 888 A2 MEL + 1.2 39.3 603.1 31.4 473.9 >2000 >2000 1558 MEL + 0.8 91.9 292.0 57.6 174.0 226.5 252.0 1300 MEL + 0.0 56.8 249.1 51.0 163.1 >2000 >2000 SK23 MEL + 1.6 20.9 281.4 22.1 177.9 >2000 >2000 -
a Abbreviations: SHAM, untransduced; TRAN, transduced.
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b 1235 TIL is a MART-1-reactive culture that recognizes the m9-27 peptide. 1520 TIL is a gp100-reactive culture and recognizes the g9-209 peptide.
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c Bold results represent specific IFN-γ release that is >50 pg/ml and at least 2-fold over background secretion. Values marked >2000 were in excess of the maximum standard used in that assay. Results presented are representative of multiple experiments.
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Culture Phenotypea Total No. of Clones IFN-γ Release vs. T2 + m9-27b IFN-γ Release vs. Tumorc Specific Lysis of T2 + m9-27d Specific Lysis of 888-A2e PBL-65 CD4 4 4 0 0 0 CD8 8 8 1 2 2 CD4/CD8 1 1 1 1 0 PBL-66 CD4 0 – – – – CD8 9 9 9 9 8 PBL-67 CD4 0 – – – – CD8 12 12 8 12 5 -
a Cell-surface immunofluorescence analysis. CD4/CD8: cloid containing both CD4+ and CD8+ T cells.
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b Specific IFN-γ release measured in culture supernatants from clones cocultured with m9-27 peptide-pulsed T2 cells.
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c Specific IFN-γ release measured in culture supernatants from clones cocultured with HLA-A2+ melanoma cell lines.
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d,e Specific lysis measured in 4-h 51Cr release assays at an E:T ratio of 80:1. Clones that were considered to be specific exhibited at least 15% lysis over background (T2 + m9-27, range 18–100%, mean 63.5%; 888-A2+ MEL, range 15–85%, mean 42.1%). In all experiments, background lysis of T2 + g9-209 or 888 MEL was <10%.
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