Phenotypic characterization of developing NK cells in CD45-congenic HSCT. Lethally irradiated B6 (H-2^b, CD45.2) mice were transplanted with 5 × 10⁶ Ly5.2 congenic (H-2^b, CD45.1) BMCs depleted of NK and T cells. Splenocytes of B6 (H-2^b) recipients were stained for CD45.1, NK1.1, CD3, Ly49C/I, Ly49G2, Ly49A, Ly49D, CD94, DX5, NKG2A, CD27, Thy1.2, CD11b, and CD43 at day 14 after congenic BM transplantation and compared with untreated control mice. Gated CD45.1⁺NK1.1⁺CD3⁻ cells are shown. (A) Frequencies of NK cells positive for CD94, DX5, CD11b, and CD43. (B) Frequencies of CD94^dim and CD94^bright NK cells. (C) Frequencies of NK cells positive for NKG2A. (D) Proportions of NK cells expressing CD27 and CD11b. (E) Frequency of Thy1.2⁺ NK cells. (F) Thy1.2 density shown as MFI on control (tinted gray) and transplanted (solid dark line) mice. The numbers represent the percentages of cells positive for the mentioned markers or MFI values. Data are representative of 5 experiments using 3-4 mice per group in each experiment.

Long-term course of Ly49C/I and Ly49G2 expression on developing NK cells in CD45-congenic HSCT. Lethally irradiated B6 (H-2^b, CD45.2) mice were transplanted with 5 × 10⁶ Ly5.2 congenic (H-2^b, CD45.1) BMCs depleted of NK and T cells. Splenocytes of B6 (H-2^b) recipients were stained for CD45.1, NK1.1, CD3, Ly49C/I, and Ly49G2 at different time points after congenic HSCT and compared with untreated control mice. Gated CD45.1⁺NK1.1⁺CD3⁻ cells are shown. (A) Frequencies of NK-cell subsets. The numbers represent the percentages of cells in each quadrant. (B) Numbers of donor-derived NK cells after HSCT. *P < .001; **P < .01. NS indicates not significant. Data are representative of 3 experiments using 3-4 mice per group per time point in each experiment (means ± SEM).

Increased frequency of Ly49G2^high NK cells in IL-2–treated mice. B6 (H-2^b) and B10.D2 (H-2^d) mice received recombinant human IL-2. Gated splenic CD45⁺NK1.1⁺CD3⁻ cells expressing Ly49s in IL-2–treated mice versus untreated mice are shown. (A) Frequencies of NK cells positive for Ly49G2 in B6 and B10.D2 mice. (B) Intensity levels of Ly49G2 expression as determined by MFI on control (tinted gray) and treated (solid dark lines) mice. Distribution of NK-cell subsets in B6 (C) and B10.D2 (D) mice. *P < .01; **P < .001. NS indicates not significant. The numbers represent the percentages of cells or MFI values. Data are representative of 3 experiments using 3-4 mice per group in each experiment (means ± SEM).

Increased frequency of Ly49G2^high NK cells in mice infected with pathogens. B6 (H2^b) mice were infected with L monocytogenes and splenocytes were stained for CD45, NK1.1, CD3, Ly49C/I, and Ly49G2 at day 3 after infection and compared with uninfected control mice. (A) Increase in the total numbers of NK cells and (B) frequencies of Ly49G2^high cells in mice infected with L monocytogenes. B6 (H2^b) and B10.D2 (H2^d) mice were infected with MCMV and splenocytes were stained for NK1.1, TCRβ, Ly49C/I, and Ly49G2 at day 6 after infection and compared with uninfected control mice. Shown are increased percentages (C) and increased intensity levels (D) of Ly49G2 expression as determined by MFI on NK cells in MCMV-infected mice (solid dark line) compared with uninfected controls (tinted gray). *P < .001; **P < .01. The absolute numbers represent the percentages of cells or MFI values. Data are representative of 3 experiments using 3-4 mice per group in each experiment (means ± SEM).

Increased frequency of Ly49G2^high NK cells in congenic and syngeneic HSCT. Lethally irradiated B6 (H-2^b, CD45.2) mice were transplanted with 5 × 10⁶ Ly5.2 congenic (H-2^b, CD45.1) BMCs depleted of NK and T cells. Splenocytes of recipients were stained for CD45.1, NK1.1, CD3, Ly49C/I, Ly49A, and Ly49G2 at day 14 after congenic HSCT and compared with untreated control mice. Gated CD45.1⁺NK1.1⁺CD3⁻ are shown. (A) Frequencies of NK cells positive for Ly49G2, Ly49CI, and Ly49A. (B) Intensity levels of Ly49G2 expression as determined by MFI on control (tinted gray) and transplanted (solid dark line) mice. (C) Frequencies of NK-cell subsets. *P < .001. (D) NK cells (CD45.1⁺CD122⁺CD3⁻ CD19⁻) were sorted from spleen cells of control or transplanted B6 (H-2^b) mice (day 14) and used in a 4-hour killing assay against YAC-1 to assess cytotoxicity. Lethally irradiated B10 (H-2^b) and B10.D2 (H2^d) mice were transplanted with 5 × 10⁶ syngeneic BMCs depleted of NK and T cells. Splenocytes of recipients were stained for CD45, NK1.1, CD3, Ly49C/I, and Ly49G2 at day 14 after syngeneic HSCT and compared with untreated control mice. Gated CD45⁺NK1.1⁺CD3⁻ cells are shown. (E) Frequencies of NK cells positive for Ly49G2 and Ly49C/I. (F) Intensity levels of Ly49G2 expression as determined by MFI on control (tinted gray) and transplanted (solid dark line) mice. Distribution of NK-cell subsets in B10 (G) and B10.D2 (H) are shown. *P < .001; **P < .01. The numbers represent the percentages of cells or MFI values. Data are representative of 3 experiments using 3-4 mice per group in each experiment (means ± SEM).

Ly49G2 depletion results in deficient NK-cell numbers after congenic HSCT. Lethally irradiated B6 (H-2^b, CD45.2) mice were transplanted with 5 × 10⁶ Ly5.2 congenic (H-2^b, CD45.1) BMCs depleted of NK and T cells. A group of mice received purified anti-Ly49G2 (4D11) depleting Ab. Splenocytes were stained for CD45.1, CD122, Ly49C/I, Ly49G2, and Ly49A at days 14, 21, and 28 after congenic HSCT and compared with transplanted control mice. (A) Frequencies of NK-cell subsets. The numbers represent the percentages of cells in each quadrant. (B) Numbers of developing CD45.1⁺CD122⁺CD3⁻ cells. *P < .001; **P < .0001; ***P < .01. NS indicates not significant. Data are representative of 2 experiments using 3-4 mice per group per time point in each experiment (means ± SEM).

Preferential induction of Ly49G2 transcripts by HSCT and IL-2. RT-PCR (35 cycles) of Ly49 Pro1 transcripts was performed with primers specific for Ly49G2, Ly49a, and Ly49c/i. The identity of all products was confirmed by DNA sequencing. (A) Detection of Ly49 Pro1 transcripts after HSCT. RNA was isolated from the spleen, BM, or liver of mice receiving hematopoietic stem cell transfer HSCT or control mice. The numbers under each panel (Δ+HCST) indicate the average -fold induction of the transcript by HCST as measured by SYBR-Green quantitative PCR. (B) Induction of Ly49 Pro1 transcripts by in vivo IL-2 treatment. Induction (fold) in response to IL-2 treatment is shown under each panel (Δ+IL-2). Data are representative of 3 experiments.