CRTAM binds Necl-2. (A) Conjugate analysis. Daudi, Daudi-CRTAM, or Daudi–Necl-5 and P815–Necl-2 were tested. Daudi cells were stained with anti–human CD45, while P815 cells were CFSE labeled. A high amount of conjugation (10%-25% of total cells) was reproducibly detected only when CRTAM-Daudi cells were coincubated with Necl-2–P815. The experiment represents 1 of 3 separate assays with comparable results. (B) Incubation of CRTAM-Daudi with mAb Cr24.1 prior to conjugation reduces conjugates between CRTAM-Daudi and Necl-2–P815. An isotype-matched control antibody had no effect (data not shown). One of 2 representative experiments is shown. (C) Soluble Necl-2–hFc binds to CRTAM-Daudi and soluble CRTAM-hFc binds to Necl-2–Daudi. Binding of soluble molecules to transfected cell lines was dramatically reduced by the addition of CRTAM mAb but not by an isotype-matched control antibody (data not shown). Soluble molecules did not bind to Daudi cells, and a control soluble molecule (NKp44-hFc) did not bind CRTAM- or Necl-2–transfected cells. One of 3 experiments with comparable results is shown. MFI indicates mean fluorescence intensity. (D) Detachment profile of CRTAM-Daudi cells interacting with surface-immobilized Necl-2–hFc. The percentage of CRTAM-Daudi or mock-Daudi cells capable of binding to Necl-2–hFc as a function of increasing wall shear stress. Cells were allowed to settle onto the indicated surface-immobilized protein substrates for 10 minutes prior to the application of flow (0.5-16 dyn cm^–2). ▪ indicates CRTAM-Daudi/Nec1-2–hFc; □, CRTAM-Daudi/Nec1-2–hFc + Cr24.1; ○, Daudi/Nec1-2–hFc; and ▵, CRTAM-Daudi/hIgG1. Values represent the mean ± SD for 2 experiments performed in duplicate.