Induction of LAT phosphorylation in human NK cells by CD16, target cells, or anti-KIR/KAR antibody. (A) Human NK cells (5 × 106) were incubated with Class I–deficient 721.221 (.221) targets or targets transfected with HLA-Cw4 (.221-Cw4) cells for 10 minutes at 37°C. Phosphorylation of immunoprecipitated LAT was determined by Western blot (pTyr, top panel) and the amount of LAT in each lane verified with anti-LAT (bottom panel). (B) Human NK cells (5 × 106) were stimulated for 10 minutes at 37°C with a control IgM (MOPC104E) or anti KIR/KAR (HP-3E4). The samples were analyzed as in A except the first sample was immunoprecipitated with preimmune serum = C. (C) IL-2 expanded primary human NK cells were sorted for expression of KIR2DS4. Immediately after sorting the cells were stimulated by warming to 37°C in the presence of anti–mouse crosslinking antibody for the indicated times. Control cells had crosslinking antibody added after lysis. Phospho-LAT was assayed as in panel A.
