Altered NK cell development and enhanced NK cell-mediated resistance to mouse cytomegalovirus in NKG2D-deficient mice

Abstract

NKG2D is a potent activating receptor on natural killer (NK) cells and acts as a molecular sensor for stressed cells expressing NKG2D ligands such as infected or tumor-transformed cells. Although NKG2D is expressed on NK cell precursors, its role in NK cell development is not known. We have generated NKG2D-deficient mice by targeting the Klrk1 locus. Here we provide evidence for an important regulatory role of NKG2D in the development of NK cells. The absence of NKG2D caused faster division of NK cells, perturbation in size of some NK cell subpopulations, and their augmented sensitivity to apoptosis. As expected, Klrk1(-/-) NK cells are less responsive to tumor targets expressing NKG2D ligands. Klrk1(-/-) mice, however, showed an enhanced NK cell-mediated resistance to mouse cytomegalovirus infection as a consequence of NK cell dysregulation. Altogether, these findings provide evidence for regulatory function of NKG2D in NK cell physiology.

Figure 1. Generation of NKG2D-Deficient Mice

(A) Targeting of the NKG2D (Klrk1) locus. Partial restriction map of the Klrk1 locus with restriction sites for EcoR I (E) and Stu I (S) is shown. Selection markers (HSV-tk and neo), loxP sites (▶), external hybridization probes a and b, and, internal probe c are depicted. The expected sizes of the DNA fragments are indicated above the lines. (B and C) Southern blot analyses of the targeted ES clones. (B) The homologous recombination as well as the introduced mutation in the second exon was tested by EcoR I digestion and external probe a. The DNA fragments of 3.7 and 6 kb represent targeted and wt NKG2D allele, respectively. (C) The Cre-mediated deletion of the neo cassette (Δneo) in the targeted ES clones (neo) was tested by the Stu I digestion and the external probe b. The DNA fragments of 12.8, 6.9 and 14 kb representing Δneo, neo and wt alleles, respectively, are shown. (D) Flow cytometric analysis of NKG2D expression on the spleen CD3ε⁻CD19⁻NK1.1⁺ NK cells of C57BL/6 (dotted line), NKG2D^+/− (dashed line) and NKG2D^−/− (line) is shown.

Figure 2. Reduced cell numbers in the spleen of NKG2D^−/− Mice

(A) Cell numbers in the spleen and inguinal lymph nodes from 8 –10 wk old C57BL/6 (○), NKG2D^+/− () or NKG2D^−/− (●) mice are shown. (B) Percentages and absolute numbers of NK and B cells isolated from the spleen and inguinal lymph nodes of C57BL/6 (○) and NKG2D^−/− (●) mice are shown. Each symbol represents an individual mouse. Horizontal lines represent mean values.

Figure 3. Altered NK Cell Development in NKG2D^−/− Mice

Analysis of NK cell subsets in the bone marrow and spleen of 9 wk old NKG2D^−/− mice and their B6 litermates. (A) Comparison of c-Kit⁺, CD127⁺ and CD51⁺ subpopulations of CD3ε⁻CD19⁻ NK1.1⁺ NK cells. (B) The representative dot plot analyses of c-Kit, CD127 and CD51expression on CD3ε⁻ CD19⁻ CD122⁺ NK1.1⁺ NK cells are shown. Comparisons of c-Kit⁺ subpopulation of CD3ε⁻ CD19⁻ NK1.1⁻ NK cells in the bone marrow and spleen are shown (bar plots). (C) Analyses of CD27, CD11b, CD43 and KLRG1 expression on CD3ε⁻ CD19⁻ NK1.1⁺ NK cells are shown. Indicated numbers as well as bar plots (A, B, C) represent mean percentages ± SD of individual data from four independent experiments (3–5 mice per group in each).

Figure 4. Faster division of NK cells in NKG2D^−/− mice

(A and B) C57BL/6 (B6, ◇) and NKG2D^−/− (◆) mice were pulsed with BrdU in the drinking water for 11 days. (A) A decay of BrdU⁺ NK cells in the bone marrow and spleen at day 0, 15 and 21 is shown. Half times (t_1/2) of the decay as well as goodness-of-fit (r²) of the linear regressions are indicated. Representative of three independent experiments (3–4 mice per group in each) is shown. (B) Analyses of c-Kit and CD11b markers on BrdU⁺ NK subsets after 11 days of the BrdU treatment (day 0) are shown. (C) Higher susceptibility of NKG2D^−/− NK cells to apoptosis. Freshly isolated cells from the spleen of C57BL/6 or NKG2D^−/− mice were cultured either without or with rIL-15 (0,5 or 5 ng/ml). Apoptotic cells were determined upon 14h of culture by Annexin V staining. Representative data of three independent experiments (3–5 mice per group in each) is shown.

Figure 5. Functional properties of NKG2D^−/− NK cells

(A) NK cytotoxic assay. Equilibrated NK cells from the spleen of NKG2D^−/− and C57BL/6 mice were incubated with 10⁴ CFSE labeled target cells for 4 h at 37°C and in the presence of rIL-2 (1000 U/ml). Triplicates of samples for each E:T ratio were analyzed by flow cytometry. Representative of three independent experiments are shown. (B) NK cells from the spleen of NKG2D^−/− and C57BL/6 mice were incubated with the tumor cell targets in 1:1 ration for 7 h. The IFNγ production was determined by the intracellular staining. Dot plot analysis shown here is representative of three independent experiments (3 mice per group in each). (C) NK cells were in vitro stimulated either by immobilized mAbs (αNK1.1, αLy49H or αLy49D), incubation with CHO cell line, rIL-12 or PMA/Ionomycin. After an incubation period of 9 h, intracellular IFNγ was determined. When NK cells were stimulated with αNK1.1, they were stained with αDX5 mAb. Representative of four independent experiments (3 mice per group in each) is shown.

Figure 6. Enhanced resistance of NKG2D^−/− mice to MCMV infection

(A) Groups of NKG2D^−/− (●), NKG2D^+/− (), and C57BL/6 (○) mice were treated either with PBS, αNK1.1 (PK136) or αNKG2D (C7) prior to i.v. infection with 5 × 10⁵ PFU Δm157 MCMV. Virus titers determined in the spleen and liver 4 days after the infection are shown. Each symbol represents an individual mouse. Median values are indicated as horizontal lines. Representative of six independent experiments is shown. (B) Survival of NKG2D^−/− (●), NKG2D^+/− (), and C57BL/6 (○) mice upon the infection with either 2 or 3 × 10⁵ PFU of SGV is shown. Control mice treated with mock infected salivary gland homogenate (SGH, dashed line) as well as infected and NK cell depleted mice are shown (dotted line). Numbers of mice per group are indicated. Data is representative of three independent experiments. (C) Kinetics of NK cells in the spleen of NKG2D^−/− and C57BL/6 mice is shown. Results are expressed as mean ± SD of at least five mice per group. (D) Percentages of Ly49H⁺ or Ly49A⁺ NK cell populations in the spleen of the Δm157 MCMV infected mice are depicted for each indicated time point. Representative of two independent experiments is shown (C, D).

Figure 7. The maturation kinetics and functional properties of NKG2D^−/− NK cells during the MCMV infection

Analyses of (A) CD11b/CD43 and (B) CD27/CD11b expression on the spleen NK cells at indicated time points after infection with Δm157 MCMV. Representative dot plots of two independent experiments (3–4 mice per group in each) are depicted. Results are expressed as mean ± SD. (C) Analysis of IFNγ-producing NK cells isolated from NKG2D^−/− and C57BL6 mice at indicated time points after the infection is shown. Freshly isolated spleen cells were incubated for 7 h in the presence of rIL-2 (500 U/ml) and tested for IFNγ production by intracellular staining. (D) Results of NK cell killing of either uninfected or Δm157 MCMV infected IC-21 cell targets are shown. The target cells were infected with 1 PFU/cell and incubated 14 h before the staining. Triplicates of samples for each E:T ratio were analyzed by flow cytometry. A representative of three independent experiments is shown.