Fig. 5.

Regulation of IS formation by the stalk region of the NK receptor. (A) Confocal images of IS formation between H2D^d-expressing target cells and J7 cells expressing mutant Ly49A receptors. C1498 cells expressing RFP-tagged H2D^d (C1498-D^d/RFP-high) were cocultured for 2 h with J7 cells expressing the GFP-tagged Ly49A, Ly49A-ST2, ANKD-CST, or ANKD-HST receptors. Merged images are shown, and yellow indicates IS formation between reporter and target cells. (B) Decreased IS formation in cells with stalk-mutant Ly49A receptors expressed on J7 cells. The average number of synapses for each incubation period was obtained as described for synapse/J7 in Table S1. Data were combined from 9 independent experiments. Total number of synapses was not statistically significant between Ly49A and ANKD-CST at any of the time points. All other comparisons were statistically significant at all of the time points. (C) LAK cells were infected with lentivirus encoding the indicated GFP-fusion receptor. FACS profiles of LAK cells after FACS-sorting by GFP are shown. (D) IS formation (GFP clustering) was counted 30 min after incubation of C1498-D^d/RFP-high cells and LAK cells expressing GFP-fusion receptors. The P of all comparisons except for the combination of Ly49A and ANKD-CST was <0.001 (P for Ly49A and ANKD-CST was 0.087); therefore, the differences were statistically significant. Representative data from 3 independent experiments is shown.