Figure 1.
Figure 1. ULBP1 and ULBP2, but not ULBP3, bind to murine NKG2D and elicit killing by murine NK cells in vitro. (A) Soluble forms of ULBP1 and ULBP2, but not ULBP3, bind to cells transfected with murine NKG2D/DAP10. CV-1 cells were cotransfected with cDNAs encoding human (gray histograms) or murine (black histograms) NKG2D and flag-tagged human DAP10. Cells were stained with anti-FLAG followed by anti–murine IgG-PE, anti–murine IgG-PE alone, or with ULBP1-Fc, ULBP2-Fc, ULBP3-Fc, or p7.5-Fc negative control protein followed by anti–human IgG-PE, and analyzed by flow cytometry. (B) Murine NKG2D-Fc binds to cells transfected with ULBP1 or ULBP2, but not ULBP3. EL4 cells were transduced with cDNAs encoding ULBP1, ULBP2, or ULBP3. Cells were incubated with murine NKG2D-Fc (thick line), human NKG2D-Fc (thin line), or negative control p7.5-Fc (filled histogram) followed by anti–human IgG-PE and analyzed by flow cytometry. (C) Soluble forms of ULBP1 and ULBP2, but not ULBP3, bind to murine NK cells. Splenocytes from C57BL/6 SCID mice were cultured in rhuIL-15 to enrich NK cells. Cells were incubated with the indicated Fc proteins, followed by anti–human IgG-PE and analyzed by flow cytometry. More than 90% of the cells in the live gate were positive for NK1.1, an NK-cell marker, but did not stain with isotype-matched negative control antibody. (D) Enhanced in vitro cytotoxicity of murine NK cells against RMA cells transduced with ULBP1 or ULBP2, but not ULBP3. RMA cells expressing vector alone (▪), ULBP1 (•), ULBP2 (▴), ULBP3 (○), or RAE-1β (□) as a positive control were tested as targets in cytotoxicity assays using murine NK cells as effectors. (E) In vitro cytotoxicity of murine NK cells against EL4 cells transduced with ULBP1 (•) is blocked by 20 μg/mL M315 rat IgG1 anti–murine NKG2D monoclonal antibody (▪) but not by 20 μg/mL M139 isotype control (▴). Each data point in panels D-E represents the average of triplicate samples, and error bars represent 1 SD from the mean for triplicate wells. Results shown in panels A-E are each representative of 2 or more independent experiments.

ULBP1 and ULBP2, but not ULBP3, bind to murine NKG2D and elicit killing by murine NK cells in vitro. (A) Soluble forms of ULBP1 and ULBP2, but not ULBP3, bind to cells transfected with murine NKG2D/DAP10. CV-1 cells were cotransfected with cDNAs encoding human (gray histograms) or murine (black histograms) NKG2D and flag-tagged human DAP10. Cells were stained with anti-FLAG followed by anti–murine IgG-PE, anti–murine IgG-PE alone, or with ULBP1-Fc, ULBP2-Fc, ULBP3-Fc, or p7.5-Fc negative control protein followed by anti–human IgG-PE, and analyzed by flow cytometry. (B) Murine NKG2D-Fc binds to cells transfected with ULBP1 or ULBP2, but not ULBP3. EL4 cells were transduced with cDNAs encoding ULBP1, ULBP2, or ULBP3. Cells were incubated with murine NKG2D-Fc (thick line), human NKG2D-Fc (thin line), or negative control p7.5-Fc (filled histogram) followed by anti–human IgG-PE and analyzed by flow cytometry. (C) Soluble forms of ULBP1 and ULBP2, but not ULBP3, bind to murine NK cells. Splenocytes from C57BL/6 SCID mice were cultured in rhuIL-15 to enrich NK cells. Cells were incubated with the indicated Fc proteins, followed by anti–human IgG-PE and analyzed by flow cytometry. More than 90% of the cells in the live gate were positive for NK1.1, an NK-cell marker, but did not stain with isotype-matched negative control antibody. (D) Enhanced in vitro cytotoxicity of murine NK cells against RMA cells transduced with ULBP1 or ULBP2, but not ULBP3. RMA cells expressing vector alone (▪), ULBP1 (•), ULBP2 (▴), ULBP3 (○), or RAE-1β (□) as a positive control were tested as targets in cytotoxicity assays using murine NK cells as effectors. (E) In vitro cytotoxicity of murine NK cells against EL4 cells transduced with ULBP1 (•) is blocked by 20 μg/mL M315 rat IgG1 anti–murine NKG2D monoclonal antibody (▪) but not by 20 μg/mL M139 isotype control (▴). Each data point in panels D-E represents the average of triplicate samples, and error bars represent 1 SD from the mean for triplicate wells. Results shown in panels A-E are each representative of 2 or more independent experiments.

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