Journal: EMBO Molecular Medicine
Article Title: The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation
Figure Lengend Snippet: Down-regulation of NKG2D by the MICA-129Met and the MICA-129Val isoforms on CD8 + T cells and impairment of subsequent co-stimulation via NKG2D NKG2D expression on purified CD8 + T cells exposed to L-MICA-129Met ( n = 19) or L-MICA-129Val clones ( n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8 + T cells (2.5 × 10 5 ) were co-cultured with 5 × 10 4 target cells and analyzed for NKG2D expression after gating on CD3 + CD8 + T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D + CD8 + T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P -values are indicated. Purified CD8 + T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG 1 ) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D + cells (lower panel) are shown ( n = 6). Differences between the groups were analyzed by t -tests, and the P -values are indicated. These CD8 + T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8 + T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated. The MFI of CFSE in unstimulated CD8 + T cells (control) was set to 100% in individual experiments ( n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8 + T cells pre-exposed to anti-NKG2D and isotype control (mIgG 1 ) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.
Article Snippet: In other experiments, purified CD8+ T cells were labeled by the dye CFSE (C-1157, Invitrogen, Karlsruhe, Germany) before culture on the coated plates.
Techniques: Expressing, Purification, Clone Assay, Flow Cytometry, Cytometry, Cell Culture, Staining, Positive Control