Fig. 2.

Constitutive cis interaction of Ly49A-ST2 and H2D^d. (A) Expression level of Ly49A and Ly49A-ST2 on C1498-D^d target cells. FACS analyses using an anti-Ly49A mAb (A1) and anti-H2D^d (34-2-12) are shown. (B) Cis interaction assays with C1498-D^d/Ly49A-ST2 target cells. Indicated target and reporter cells were incubated overnight and subjected to CPRG assays. The (−) indicates no target or reporter cells. (C) Killing assays with the C1498-D^d/Ly49A-ST2 target cells. Killing assays were performed with indicated target cells and Ly49A-positive or Ly49A-negative LAK cells from B10 mice. Representative data from 4 independent experiments are shown. (D) Failure of Ly49A tetramer binding to C1498-D^d/Ly49A and C1498-D^d/Ly49A-ST2. Indicated cells were stained with streptavidin-phycoerythrin (SA-PE)-conjugated Ly49A or control (H2D^d/hβ2m) tetramers. The quantitative Ly49A tetramer binding to C1498-D^d coexpressing Ly49A or Ly49A-ST2 is assessed by mean fluorescence intensity (MFI). (E) Physical interaction of Ly49A-ST2 and H2D^d. C1498 cells were transduced with flag-tagged H2D^d (fH2D^d) and wild-type Ly49A or Ly49A-ST2. Lysates were immunoprecipitated by anti-flag mAb, and interrogated for Ly49A by Western blotting with anti-Ly49A polyclonal Abs under reducing conditions. Representative data from 3 independent experiments are shown. (F) Reduced Ly49A tetramer binding to primary LAK cells expressing H2^d and Ly49A. Indicated retrovirus and lentivirus constructs were transduced into day 3 LAK cells from B10.D2, respectively. Thy1.1-positive cells were gated and stained with SA-PE-conjugated Ly49A or control (H2D^d/hβ2m) tetramers (Left), and with biotinylated A1 mAb followed by SA-PE (Right). Gray shades represent the overlaid histogram of the tetramer binding to the pMX-IRES-Thy1.1-transduced cells. No Ly49A tetramer binding was observed in LAK cells from B10 mice.