Mice generated to lack all NK-related Ly49 molecules using CRISPR have NK cells that display a less mature phenotype and impaired KLRG1 expression.

(A) Genetic map of the Ly49 locus of wildtype C57BL/6 and Ly49KO mice and Sanger sequencing of the fusion sequences in the Ly49KO mice. (B) Ly49 receptor expression on splenic NK cells of the indicated mice. (C) Surface receptor expression on splenic NK cells from indicated mice. (D) Expression of the maturation markers CD27 and CD11b on splenic NK cells from indicated mice. MFI, median fluorescent intensity. Error bars indicate SEM; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

NK cell licensing and rejection of MHC-I deficient target cells is defective in Ly49KO mice.

Splenocytes from the indicated mice were stimulated with plate bound anti-NK1.1 (A) or PMA/ionomycin (B) and IFNγ production by NKG2A-NK cells and analyzed by flow cytometry. (C) In vivo cytotoxicity assay against H-2Kb, H-2Db and full MHC-I deficient targets. Splenocytes from WT, H-2Kb, H-2Db and MHC-I deficient mice were differentially labeled with CFSE, CTV, and CTFR as indicated. Mixture of labeled target cells were injected i.v. into wildtype, Ly49KO, and anti-NK1.1-depleted mice. Target cells were analyzed in spleens by flow cytometry 2 days after challenge. KODO, H-2Kb x H-2Db knock out; MHC-I KO, KODO x B2m knockout; MFI, median fluorescent intensity. Error bars indicate SEM; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Ly49A is efficiently expressed in Ncr1-Ly49A knockin mice and rescues KLRG1 expression in NK cells.

Flow cytometric analysis of NK cells in D8-KODO, Ly49KO D8-KODO, and Ly49KO/Ly49A KI D8-KODO mice (A) Ly49A expression in NKp46+ and NKp46- NK1.1+ NK cells in bone marrow and spleen of the indicated mice. (B) Expression of the maturation markers CD27 and CD11b on NK cells in spleen, bone marrow, and liver of indicated mice. (C) KLRG1 expression by NK cells in spleen, bone marrow, and liver of indicated mice. MFI, median fluorescent intensity. Error bars indicate SEM; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Expression of Ly49A in isolation is sufficient for NK cell licensing and missing-self rejection.

Splenocytes from the indicated mice were stimulated with plate bound anti-NK1.1 (A) or PMA/ionomycin (B). IFNγ production and degranulation (CD107a) by NKG2A-NK cells were analyzed by flow cytometry. (C) Splenocytes from D8-KODO and KODO mice were differentially labeled with CFSE and CTV as indicated. A mixture of labeled target cells was injected i.v. into D8-KODO, Ly49KO D8-KODO, and Ly49KO/Ly49A KI D8-KODO mice. Specific rejection of target cells was analyzed in spleens by flow cytometry 2 days after challenge. Error bars indicate SEM; ns, not significant; ***p < 0.001, and ****p < 0.0001.