Article Figures & Data
Figures
- FIGURE 1.
CD94/NKG2A continuously recycles between the cell surface and cellular interior. A, NKL or peripheral blood NK cells were incubated with PE-conjugated anti-CD94 mAb (HP-3B1) or anti-NKG2A mAb (Z199) at 37°C (▪) or 4°C (♦) for different periods of time. Cells were then washed and analyzed by flow cytometry. MFIs were plotted vs time of incubation. B, NK cells (upper panel) or NKL cells (lower panel) were incubated with Alexa 488-conjugated or Alexa 594-conjugated anti-NKG2A mAb at 37°C or 4°C for 1 h, fixed, and examined by confocal microscopy. C, NKL cells were surface biotinylated with NHS-SS-biotin and incubated for various time periods at 37°C, followed by treatment with MESNA to (Figure legend continues) remove extracellular biotin groups. After quenching with iodoacetamide, the cells were lysed in 1% Triton X-100. CD94/NKG2A was immunoprecipitated with anti-CD94 mAb (HP-3B1) or anti-NKG2A mAb (Z199), electrophoresed on SDS-PAGE, immunoblotted with streptavidin-HRP, and developed by chemiluminescence. Membranes were stripped and reprobed with an anti-NKG2A mAb (8E4), followed by goat anti-mouse-HRP. Densitometric analyses represent the mean of three independent experiments. The background at time 0 was subtracted, and the amount of internalized CD94/NKG2A was normalized to the total loaded amount of CD94/NKG2A as detected by immunoblotting. The data for anti-CD94 and anti-NKG2A were combined and plotted vs time. D, NKL cells were analyzed in the recycling assay as described in Material and Methods. MFIs were plotted vs time of incubation.
- FIGURE 2.
CD94/NKG2A and CD94/NKG2C/DAP12 are both long-lived receptors with different intracellular trafficking patterns. A, Untreated NKL cells and cells pretreated with CHX (50 μg/ml) were analyzed for constitutive exocytosis in the recycling assay as described in Material and Methods. MFIs were plotted vs time of incubation. All the experiments were performed three times. B, The effect of BrefA (10 μg/ml) on the cell surface expression of CD94, NKG2A, and CD71 expressed by NKL cells was analyzed by flow cytometry. C, Intracellular distribution of (Figure legend continues) CD94/NKG2A and CD71 in NKL cells. NKL cells were allowed to internalize anti-NKG2A Alexa 488 (green) and transferrin Alexa 594 (red) and then were analyzed by confocal microscopy. Upper panel, The internalization at different periods of time; lower panel, a representative cell at a higher magnification. D, The effect of Bref A (10 μg/ml) on the cell surface expression of CD71 and CD94 expressed by Ba/F3 cells transfected with CD94/NKG2C/DAP12 was analyzed by flow cytometry. All the experiments were performed at least three times.
- FIGURE 3.
CD94/NKG2A trafficking is independent of ligation by ligand. A, RBL-2H3 cells transfected with CD94/NKG2A were incubated with PE-conjugated anti-CD94 mAb or anti-NKG2A mAb using the same conditions listed for Fig. 1A. Cells were then washed and analyzed by flow cytometry. B, RBL-2H3 cells were transfected with CD94 and NKG2A-EGFP. Frames depict identical fields of view at different times, showing the interaction of intracellular vesicles containing CD94/NKG2A-EGFP with the plasma membrane (see supplemental data). C, RBL-2H3 cells transfected with CD94/NKG2A-EGFP were incubated with Alexa 594-conjugated anti-NKG2A mAb (red) for 1 h at 37°C or 4°C, fixed, and examined (Figure legend continues) by confocal microscopy. Colocalization is shown by the yellow color. D, CD94/NKG2A internalization by transfected RBL-2H3 cells was determined as described in Fig. 1C. E, RBL-2H3 cells transfected with CD94/NKG2A were incubated with 721.221 or 721.221-Cw3 cells at E:T ratio of 1:2 for 3 min at 37°C. Cells were then lysed and CD94/NKG2A was immunoprecipated with anti-NKG2A mAb (Z199). After SDS-PAGE, blots were probed with anti-phosphotyrosine mAb (4G10) or with anti-SHP-1 mAb and developed with secondary Ab-HRP. Blots were stripped and reprobed with anti-NKG2A mAb (8E4) to determine the total loaded amount of CD94/NKG2A. F, CD94/NKG2A internalization by transfected RBL-2H3 cells was determined as described in Fig. 1C, except that RBL-2H3 cells were incubated in the presence of 721.221 cells or 721.221-Cw3 cells (E:T ratio is 1:1).
- FIGURE 4.
CD94/NKG2A is internalized and recycles in the absence of inhibitory signals. RBL-2H3 cells transfected with CD94/NKG2A containing Tyr for Phe substitutions in its ITIMs were used in this experiment. A, Uptake of PE-conjugated anti-NKG2A mAb was monitored as described in Fig. 1A at 4°C and 37°C. B, internalization was examined as described in Fig. 1C.
- FIGURE 5.
CD94/NKG2A trafficking can be inhibited by depletion of cellular ATP and by impairing cytoskeleton polymerization. A, NKL cells were pretreated with 80 mM sodium azide for 30 min. PE-conjugated anti-CD94 mAb was added and cells were incubated at 37°C and 4°C. Cells were then washed and analyzed by flow cytometry as in Fig. 1. Similar results were obtained with RBL-2H3 cells transfected with CD94/NKG2A (data not shown). B, As described for A, except cells were pretreated with 20 nM LatA.
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