Figure 6. NKG2D-deficiency does not impair NK cell functions in vitro or in vivo.

(A-C) IL-2 activated splenic NK cells (sorted NK1.1⁺CD3⁻ cells in panel A, unsorted cells in panels B-C from Klrk1^−/− mice fail to lyse RMA-Rae1ε target cells, and show reduced lysis of YAC-1 and C1498 target cells. Lysis in the presence of NKG2D mAb is shown for Klrk1^+/+ (open squares) and Klrk1^−/− (open circles) effector cells in panels A and B. (D) Normal lysis of class I-low RMAS cells by Klrk1^−/− IL-2 activated NK cells. Results represent means ±SD obtained with Klrk1^−/− mice (neo cassette-retained) and littermate controls. (E) Splenocytes from Klrk1^−/− mice (neo cassette deleted) and littermate controls (n=5 for each genotype) were stimulated in vitro for 5 hours on plates coated with NK1.1 mAb (PK136, 40 μg/ml), Ly49D mAb (SED85, 10 μg/ml) or control mouse IgG (40 μg/ml) in the presence of Golgi-plug before staining and analysis. In parallel, NK cells were stimulated with or without a mixture of PMA and ionomycin. Intracellular IFN-γ was detected by flow cytometry on gated NK1.1⁺CD3⁻ cells, except for the anti-NK1.1-stimulated cells, which were gated on DX5⁺CD3⁻ cells. Results represent means +/− SD (n=5). (F) In vivo rejection of B2m^−/− bone marrow cells by Klrk1^−/− mice. A mixture of CFSE (5 μM) labeled BM cells from C57Bl/6-Ly5.2 and B2m^−/− C57Bl/6-Ly5.1 mice was injected i.v. in irradiated Klrk1^+/+ (n=4), Klrk1^−/− (n=6), negative control C57Bl/6 B2m^−/− (n=2), or NK-depleted Klrk1^−/− (n=3) recipients. Similar results were obtained in one additional independent experiment.