Article Figures & Data
Figures
- Figure 1.
Structure of chNKG2D and retroviral constructs. A, schematic diagram of the wtNKG2D and chNKG2D receptors. Both receptors associate with Dap10 in the cell membrane, and the chimeric receptor has three CD3ζ immunoreceptor tyrosine-based activation motif (ITAM) regions added to the cytoplasmic (CY) domain of NKG2D. EX, extracellular domain; TM, transmembrane domain. B, amino acid sequence of the region where the three CD3ζ ITAMs is joined to the human NKG2D receptor. The three CD3ζ ITAM chains were fused to the NH2 terminal of the NKG2D in a reverse orientation (COOH terminal to NH2 terminal). C, NKG2D sequences were inserted into an empty IRES-GFP vector. The NKG2D was positioned behind the 5′-long terminal repeat (5′ LTR), and the GFP expression was controlled by the IRES.
- Figure 2.
wtNKG2D and chNKG2D receptors are efficiently expressed on the cell surface. A, Bosc23 cells were transfected with plasmids containing NKG2D-GFP and Dap10 adaptor genes. Open histograms, surface NKG2D expression was detected in the GFP+ population using a PE-conjugated NKG2D antibody; filled histograms, isotype controls. B, primary human T cells transduced with virus-containing vector, wtNKG2D, or chNKG2D were analyzed by RT-PCR for gene expression using primers specific for chNKG2D, wtNKG2D, and β-actin. C, genetically modified human T cells were stained with PE-anti-NKG2D and FITC-anti-CD4 monoclonal antibodies. Histograms, cells were gated on CD4− (CD8+) T lymphocytes.
- Figure 3.
Chimeric NKG2D expressing T-cells secrete proinflammatory cytokines after coculture with ligand-expressing tumor cells. Human T cells transduced with wtNKG2D (gray columns) or chNKG2D (black columns) were cocultured with irradiated tumor cells for 72 hours. White columns, tumor cells. Culture supernatants were analyzed for the levels of chemokines (A) CCL3 or (B) CCL5 and cytokines (C) GM-CSF and (D) TNF-α by luminex. Columns, mean of two experiments done in triplicate; bars, SD.
- Figure 4.
Specific lysis of NKG2D ligand-positive tumor cells by chNKG2D modified human CD8+ T cells. A, primary human T cells transduced with empty vector (□), wtNKG2D (⧫), or chNKG2D (▪) were used as effector cells in a 51Cr release assay. The ratios of effector to targets (E:T ratios) were 25:1, 5:1, and 1:1. chNKG2D-transduced T cells showed significantly higher (P < 0.05) cytotoxicity against NKG2D ligand-positive tumors (P815/MICA, T47D, MCF-7, Panc-1, and A375) but not ligand-negative P815 cells than vector- or wtNKG2D-transduced T cells at all ratios. B, for NKG2D blocking experiments, wtNKG2D-bearing (circle) or chNKG2D-bearing (triangle) CD8+ T cells were incubated with saturating amounts of anti-NKG2D (1D11, filled symbols) or with control Ig (open symbols) before exposure to tumor cells. Blocking antibody 1D11 significantly reduced the cytotoxicity of chNKG2D-transduced T cells against K562 and RPMI8866 cells (P < 0.05) at all ratios compared with control antibody. Points, mean of two to three experiments done in triplicate; bars, SD.
- Figure 5.
sMICA does not inhibit specific lysis of ligand expressing tumor cells at physiologic concentrations. A, lane 2, purified sMICA in the SDS-PAGE gel. Lane 1, molecular weight markers. B, B3Z, B3Z/wtNKG2D, and B3Z/chNKG2D cells were cultured in sMICA-coated plates, and the response to the ligand was measured by A570 nm (C and D). Human primary T cells transduced with empty vector (⧫), wtNKG2D (▪), or chNKG2D (▴) were preincubated with varying concentrations of sMICA and then tested in a 51Cr assay with RPMI8866 (C) or K562 (D) at an E:T ratio of 10:1. Points, mean of two experiments done in triplicate; bars, SD. *, P < 0.05, significant difference between the treated samples (in the presence of 15,000 ng/mL sMICA) and the untreated control.
Tables
- Table 1.
IFN-γ production by chNKG2D-modified human T cells
IFN-γ (pg/mL) Tumor cells Vector wtNKG2D chNKG2D No T cells — ND ND ND — P815 ND ND ND ND P815/MICA ND ND 230 ± 19 ND RPMI8866 1,062 ± 84 * 393 ± 27 13,942 ± 1,543 ND K562 111 ± 14 ND 3,964 ± 347 ND Jurkat ND ND 217 ± 23 ND T47D ND ND 252 ± 8 ND MCF-7 ND ND 987 ± 181 ND Panc-1 ND ND 1,906 ± 122 ND DU145 ND ND 1,766 ± 122 ND A375 ND ND 520 ± 39 ND PC-3 ND ND 1,504 ± 31 ND -
Abbreviation: ND, not detectable (<37 pg/mL).
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↵* A representative result (mean ± SD of triplicates) of two or three independent experiments.
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Supplementary Data, Zhang et al.
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Volume 66, Issue 11
