Stability of receptor surface expression upon target-cell encounter.

<p>The histograms show the flow cytometric analysis of chNKG2D expression (detected in CD4^pos T cells by an anti-CD314 antibody or in CD8^pos T cells by an anti-human-IgG1-Fc antibody; log scale in all histograms; note: amplification voltage in flow cytometric analysis was lowered for lentivirally transduced T cells due to the strong expression of the receptor). <i>A,</i> comparison of lentivirally transduced and mRNA transfected T cells. 75,000 chNKG2D^pos CD4^pos T cells were cultured 24 hours after electroporation for further 24 hours in presence or absence of 200,000 STA-ET-3 cells (filled grey histograms = presence of STA-ET-3, open histograms bold line = absence of STA-ET-3, open histograms dotted line = T cells not transfected). Shown is one representative experiment out of three independent repetitions. <i>B,</i> chNKG2D down-regulation in response to different ESFT cell lines. 100,000 chNKG2D^pos CD8^pos T cells were co-cultured 50 hours after mRNA electroporation for further 24 hours with 100,000 cells of different ESFT cell lines as indicated (unmodified T cells = “without chNKG2D”; without co-culture = “without target cells”). <i>C,</i> kinetics of chNKG2D down-regulation upon target-cell encounter at different times after transfection. 20,000 chNKG2D^pos CD4^pos T cells were co-cultured with 60,000 STA-ET-3 target cells for different times (1, 4, or 48 hours) before chNKG2D expression was determined (6, 30, or 74 hours after transfection) (one experiment). <i>D,</i> comparison of differentially stabilized mRNA species. ChNKG2D^pos CD4^pos T cells were co-cultured with STA-ET-3 target cells at different effector∶target ratios and at different times after transfection. Target cells were added at the indicated ratios either two or 26 hours after transfection and chNKG2D expression was determined by flow cytometry 6, 30, 50, or 74 hours after transfection. Shown is one out of three experiments. <i>E,</i> effect of patient serum containing soluble NKG2D-Ls. 15,000 chNKG2D^pos CD4^pos T cells were cultured 24 hours after electroporation for further 24 hours with or without serum of two different neuroblastoma patients (final serum concentration 33%).