Ccl3 secreted by NK cells was sensed by NK and NKT cells in vitro. Purified NK cells from CCL3-EASER mice were expanded for 6 days with mrIL-2 and cocultured 1:1 for 4 h with possible Ccl3-receiving cells in a 96-well/plate precoated with purified anti-NK1.1 antibody (clone PK136). In all cases, purity of cocultured cells was >85%. (A) Scheme of the experiment. (B–E) Representative contour plots of the coculture of EASER-NK cells with (B) NK/NKT cells; (C) OVA-activated CD4⁺ OT-II T cells; (D) OVA-activated CD8⁺ OT-I T cells; and (E) thioglycolate elicited peritoneal macrophages. EASER-NK cells are indicated with an orange-shadowed gate, Ccl3 recipients with a gray-shadowed gate (exceptionally, recipient NK cells are indicated with a blue-shadowed gate, and NKT cells with a light-blue-shadowed gate), Venus⁺ cells with a green-shadowed gate; tdTomato⁺ cell with a red-shadowed gate; and tdTomato⁺Venus⁺ cells with a yellow-shadowed gate. (F) Transmigration assay of CD45.1 WT NK cells (top chamber) toward CD45.2 EASER-NK cells activated with plate-bound anti-NK1.1 mAb (bottom chamber) (panel 1). Ccl3-Venus production control and Venus/tdTomato negative control cultures are shown in panels 2 and 3, respectively. Venus uptake control in panel 4. Representative dot plots indicate gating of: EASER-NK cells (orange-shadowed gate) and non-reporter CD45.1 NK cells (blue-shadowed gate), Venus⁺ cells with a green-shadowed gate, tdTomato⁺ cell with a red-shadowed gate, and tdTomato⁺Venus⁺ cells with a yellow-shadowed gate. Data in B–E are representative of three independent experiments (n = 3–5 mice per group). Data in F is representative of two independent experiments. BFA, brefeldin A; Mon, monensin.