MaGSC Stra8 SSC5 cells can be killed by CTLs despite low expression of MHC class I molecules. (A) MaGSC Stra8 SSC5 cells cultured under conditions I, II, III, and IV, in vitro differentiated maGSC Stra8 SSC5 cells (diff.), and RMA cells were analyzed for MHC class I expression (H2Kb and H2Db) by flow cytometry. The mean percentages of positive cells + SD (staining with MHC class I mAbs minus isotype control) and the mean fluorescence intensities (MFI) + SD are given as determined in 5 experiments. Significant differences between culture conditions that differed in the presence of LIF are indicated (* p < 0.05, U test). (B) Individual dot plots from these experiments are shown. The left panel shows staining with an isotype control and the right panel with an anti-H2Kb mAb. The percentages of EGFP and H2Kb (or isotype control) double positive cells are indicated in the upper right quads. (C) The evaluation of the proportion of MHC class I-positive cells in the experiments was restricted to the EGFP-positive maGSC Stra8 SSC5 cell population. Significant differences between culture conditions that differed in the presence of LIF are indicated (* p = 0.05, U test). (D) A representative experiment is shown in which the susceptibility of maGSC Stra8 SSC5 cells cultured under conditions II and III to CTLs was determined. RMA cells served as highly CTL susceptible controls. The target cells were pulsed with the SIINFEKL peptide P (0.5 μg/ml) and exposed to CTLs derived from TCR-transgenic OT-I mice. The mean of specific lysis plus SD at different effector:target ratios (10:1 to 0.16:1) in the presence or absence of the SIINFEKL peptide measured in a 51Cr release assay is shown. To confirm granule exocytosis dependency of killing EGTA was added to the test. (E) The means of relative lysis ± SD of the various target cell lines by OT-I CTLs are shown as determined in three independent experiments. The mean percentage of specific lysis of RMA cells (serving as positive control) at the highest effector to target ratio (10:1) was adjusted to 100% in each test and the relative lysis of the various target cells at different effector to target ratios was calculated.