The NK cell granule protein NKG7 regulates cytotoxic granule exocytosis and inflammation

Abstract

Immune-modulating therapies have revolutionized the treatment of chronic diseases, particularly cancer. However, their success is restricted and there is a need to identify new therapeutic targets. Here, we show that natural killer cell granule protein 7 (NKG7) is a regulator of lymphocyte granule exocytosis and downstream inflammation in a broad range of diseases. NKG7 expressed by CD4⁺ and CD8⁺ T cells played key roles in promoting inflammation during visceral leishmaniasis and malaria-two important parasitic diseases. Additionally, NKG7 expressed by natural killer cells was critical for controlling cancer initiation, growth and metastasis. NKG7 function in natural killer and CD8⁺ T cells was linked with their ability to regulate the translocation of CD107a to the cell surface and kill cellular targets, while NKG7 also had a major impact on CD4⁺ T cell activation following infection. Thus, we report a novel therapeutic target expressed on a range of immune cells with functions in different immune responses.

Extended Data Fig. 1 |. Nkg7 is only expressed by NK cells and a subset of CD8⁺ TCRβ⁺ cells in the spleen in naive mice. t-SNE plot of splenocytes from a naive mouse, pre-gated to exclude doublets and dead cells.

The remaining cells were clustered using TCRβ-BUV737, CD4-BUV395, CD8α-PE/Cy7, CD11b-PerCP/Cy5.5, CD11c-BV785, MHC-II-Pacific Blue, B220-BV650, NK1.1-APC/Cy7 and Ly-6C-BV605. Equal numbers of cells (15,000 cells) are shown for Cre^– and Cre⁺ plots. The black oval indicates the GFP⁺ population. n = 1 per genotype, performed once. BV, brilliant violet.

Extended Data Fig. 2 |. Changes in the frequencies of Nkg7-expressing NK cells and CD8⁺ T cells during Leishmania donovani infection.

a, The gating strategy used to assess changes in the key immune cell subsets including NK cells, CD4⁺ T cells, CD8⁺ T cells, B cells, cDCs, pDCs, CD11b^hi Ly6C^int monocytes, inflammatory monocytes, macrophages, and NKT cells. b, The graphs show changes in GFP within each of the key immune cell subsets in the spleen and liver during L. donovani infection. Statistical significance was determined using the Kruskal–Wallis one-way analysis of variance (ANOVA) with Dunn’s multiple comparisons test. c, The frequencies of GFP⁺ T_H1 (gated on NK1.1⁻ TCRβ⁺ CD8⁻ CD4⁺ IFN-γ⁺ IL-10⁻) and T_R1 cells (gated on NK1.1⁻ TCRβ⁺ CD8⁻ CD4⁺ IFN-γ⁺ IL-10⁺) in the spleen and liver during the course of infection are shown. A two-way ANOVA with Sidak’s multiple comparisons test was performed to test for statistical significance. d, Changes in the frequency and total number of GFP⁺ cells in the spleen and liver over the course of infection are shown. p value is indicated where * p < 0.05. Error bars represent mean ± SEM. The data shown is representative of two independent experiments, each with n = 3 mice per genotype, per timepoint.

Extended Data Fig. 3 |. Nkg7 deficiency results in reduced CD4⁺ T cell responses during Leishmania donovani infection.

a, The liver and spleen weights of WT and Nkg7^−/− mice during L. donovani infection. A two-way ANOVA with Sidak’s multiple comparisons test was used to determine statistical significance. Data is representative of two experiments, where n = 3 naive WT and Nkg7^−/− mice, and n = 5 WT and 4 Nkg7^−/− mice at days 14, 28 and 58 p.i. groups. b and c, The frequency and total number of conventional (Foxp3⁻) CD4⁺ T cells in the liver (b) and spleen (c) at day 14 p.i. are shown. Statistical significance was determined using a two-way ANOVA with Tukey’s multiple comparisons test. The data shown is representative of two independent experiments, each with n = 3 naive WT and Nkg7^−/− mice, and n = 5 WT and 4 Nkg7^−/− mice at day 14 p.i.. d, The expression of Ifng and Tnf mRNA by spleen or liver CD4⁺ T cells in naive or infected (day 14 p.i.) mice was determined by RT-qPCR. A two-way ANOVA with Sidak’s multiple comparisons test was used to determine statistical significance. n = 4 naive and 5 infected mice in each group. e, The representative histograms show PD-1, CTLA-4, and ICOS staining on CD4⁺ T cells in the liver at day 14 p.i. The graphs indicate the frequencies of PD-1⁺, CTLA-4⁺, and ICOS⁺ CD4⁺ T cells. Statistical significance was determined using the Mann–Whitney test. Data is derived from one experiment, where n = 3 naive WT and Nkg7^−/− mice, and n = 5 infected WT and 4 infected Nkg7^−/− mice. f, The frequency and total number of I-A^bPEPCK_335–351 (tetramer)-PE⁺ cells in the liver of naive and infected (day 14 p.i.) mice are shown. A two-way ANOVA with multiple comparisons test was used to test for statistical significance. n = 4 WT or Nkg7^−/− naïve, and 5 WT or 6 Nkg7^−/− infected mice. Data is representative of two independent experiments. g, Representative plots depict the differences in T_H1 (IFN-γ⁺ T-bet⁺ cells) frequencies in WT or Nkg7^−/− I-A^bPEPCK_335–351 (tetramer)-PE⁺ cells. The frequencies and numbers are shown in the accompanying graphs below. Statistical significance was determined using the Mann–Whitney test. n = 5 mice per group. Data is representative of two independent experiments. h, The frequency of WT or Nkg7^−/− CD4⁺ TCRβ⁺ cells and I-A^bPEPCK_335–351 (tetramer)-PE⁺ cells expressing IL-6-stimulated phosphorylated (p)-STAT3 are shown. Statistical significance was determined using the Mann–Whitney test. n = 5 mice per group. Data is representative of two independent experiments. p value is indicated where * p < 0.05. Error bars represent mean ± SEM.

Extended Data Fig. 4 |. The absence of NKG7 results in decreased CD8⁺ T cell cytotoxicity during PbA infection.

a, The graphs show the frequency and total number of NK cells, CD4⁺ T cells and CD8⁺ T cells in the brain of naive and infected (peak of ECM) WT (n = 3 naive and 5 infected) and Nkg7^–/– (n = 3 naive and 5 infected) mice. Cell subsets frequencies are expressed as a percentage of CD45⁺ cells. Data is representative of two independent experiments. b, Representative flow cytometry plots were gated on lymphocytes, singlets, live cells, NK1.1-APC/Cy7⁻ TCRβ-BUV737⁺, CD8α-PerCP/Cy5.5⁺ cells and the frequencies of CD11a⁺ CD49d⁺ cells and Granzyme B⁺ cells are shown. n = 3 naïve and 4 infected mice per strain. Data is representative of two independent experiments. c, The graph shows the proportions of PbT-I^WT and PbT-IΔ^Nkg7 cells in the spleen of naive (n = 4) or infected mice at peak of ECM (n = 5). d, The frequencies of splenic PbT-I^WT and PbT-IΔ^Nkg7 transgenic CD8⁺ T cells expressing Granzyme B or Perforin, in the presence of monensin, are shown. n = 4 naïve and 5 infected mice at peak of ECM. e, The representative histograms show differences in the expression of CD107a by splenic PbT-I^WT and PbT-IΔ^Nkg7 cells incubated with monensin or stimulated with PMA and ionomycin in the presence of monensin. The frequency and MFI of CD107a expression is shown in the accompanying graphs. n = 4 naïve and 5 infected mice at peak of ECM. Statistical significance in all graphs was determined using a two-way ANOVA with Tukey’s (a) or Sidak’s (b–e) multiple comparisons test.

Extended Data Fig. 5 |. Nkg7-deficiency results in increased cancer metastasis in experimental models.

a, A correlation (r) between the moving average of a 19-gene natural killer (NK) cell signature genes and NKG7 expression in n = 472 samples from TCGA:SKCM dataset. b, The graphs indicate differences in the number of lung metastases between WT and Nkg7^−/− mice following injection of RM-1 prostate carcinoma cells (n = 6 WT and 7 Nkg7^−/−) and spontaneous metastasis of E0771 mammary carcinoma cells (n = 19 WT and 21 Nkg7^−/−, from two pooled experiments). The survival of WT and Nkg7^−/− mice treated with either cIg (n = 7 WT and 13 Nkg7^−/−) or α-asGM1 (n = 6 WT and 12 Nkg7^−/−) in an intraperitoneal RMA-s lymphoma model was also assessed. Statistical significance between groups was tested using the Log-rank (Mantel–Cox) test. Additionally, the difference in percentage of tumour-free mice between WT (n = 17) and Nkg7^−/− (n = 14) mice following MCA-induced fibrosarcoma generation is shown. The log-rank (Mantel–Cox) test was used to determine statistical significance. ** and *** represents p < 0.01 and 0.001 respectively. ns, not significant. c, The lungs of WT and Nkg7^−/− mice, injected with LWT1 cells, were assessed for differences in the frequency and total cell number of hematopoietic cells, at 14 days post-injection. d, The frequency and total cell number of NK cells and T cells were quantified in the lungs of mice injected with LWT1 cells, at 14 days post-injection. e, The differences in the frequency of NK cells at different stages of maturation, based on CD27 and CD11b expression at 14 days post-injection of LWT1 cells is shown. The data shown in C-E is representative of two independent experiments where n = 5 mice per group. The Mann–Whitney test was used to determine statistical significance. p values are shown as follows: * p < 0.05 and ** p < 0.01.

Extended Data Fig. 6 |. Nkg7^–/– NK cells do not have reduced abilities to conjugate with target cells or to form synapses.

a and b, The representative histograms show expression of DNAM-1 (CD226), NKG2D (CD314), CD11a, Granzyme B, and Perforin by WT (n = 6) or Nkg7^−/− (n = 5) NK cells in the naive state (a) or when activated with rIL-2 (b). The MFI for the expression of each marker in naive NK cells is also shown. Data is pooled from 2 independent experiments. Sv, Streptavidin. c, Representative plots depict the frequency of cell conjugates formed when CellTrace Violet (CTV)-labelled WT or Nkg7^−/− NK cells were co-cultured with carboxyfluorescein succinimidyl ester (CFSE)-labelled YAC-1 target cells for 30 minutes at an E:T ratio of 1:2. The frequency of conjugated NK cells at 5, 15, 30, and 60 minutes is shown in the accompanying graph. n = 3 mice per group. Data is pooled from 2 independent experiments. d, Representative images of effector NK cell–YAC-1 target cell conjugates visualised using an Amnis® ImageStream®XMark II after in vitro co-culture of WT or Nkg7^–/– cells with target cells for 15 minutes. The graphs show the MFI of Phalloidin or LFA-1 at the interface between effector and target cells. Data obtained from one experiment.

Fig. 1 |. NKG7 is highly upregulated in splenic CD4⁺ T cells during L. donovani infection.

a, Upregulated genes found in CD4⁺ T cells isolated from mouse spleen at day 56 p.i. and in human PBMCs from patients with visceral leishmaniasis at the time of admission to clinic for treatment. b, Cellular locations of proteins encoded by the upregulated genes in a are indicated on the cellular map using information obtained from the Gene Ontology Cellular Component knowledge base. c, The protein structure of NKG7 generated using I-TASSER. Predicted extracellular loops (indicated by black arrows) of human and mouse NKG7 are highlighted in green and purple, respectively. d, Top: validation of NKG7 upregulation in patients with visceral leishmaniasis before treatment (day 0; n = 14) compared with the same patients after treatment (day 30) and endemic controls (EC; n = 14) by RT-qPCR. Bottom: RT-qPCR validation was also performed in conventional T cells (T_conv) and T_reg cells from the spleen and liver of naive and infected (day 56 p.i.) mice. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons test (top) or two-way ANOVA with Šídák’s multiple comparisons test (bottom). Center lines indicate median values, box limits indicate upper and lower quartiles, and whiskers indicate maximum and minimum measures. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2 |. Nkg7 expression is enriched in NK cells at steady state and inducible in CD4⁺ T cells.

a, A mouse expressing the Cre gene behind the promoter of Nkg7 was generated (B6.Nkg7-cre) and crossed to a membrane reporter (B6.mT/mG) to generate Nkg7 reporter mice (Nkg7-cre × mT/mG). Validation of GFP expression was performed on splenic NK cells in naive Cre⁺ mice. b, t-SNE plot of splenocytes from a naive mouse, pre-gated to exclude doublets, dead cells and NK1.1-APC/Cy7⁻TCR-β-BUV737⁻ cells. The remaining cells were clustered using NK1.1-APC/Cy7, TCR-β-BUV737, CD4-BUV395 and CD8α-PE/Cy7. Equal numbers of cells (50,000 cells) are shown for Cre⁻ and Cre⁺ plots. n = 1 per genotype, performed once. c, The expression of Nkg7 (GFP⁺) under T_H0 (anti-CD3 + anti-CD28 + recombinant IL-2), T_H1 (recombinant IL-12 + anti-IL-4 + T_H0 conditions), T_R1 (recombinant IL-27 + T_H0 conditions), T_reg (recombinant IL-27 + recombinant TGF-β + T_H0 conditions), T_H2 (recombinant IL-4 + anti-IFN-γ + T_H0 conditions) and T_H17 (recombinant IL-6 + recombinant IL-1β + recombinant IL-23 + anti-IFN-γ + anti-IL-4 + T_H0 conditions) cell polarizing conditions. n = 1 per genotype. Plots are representative of two independent experiments. d, Expression of Nkg7 (GFP⁺) when recombinant IL-27 was titrated, or when recombinant TGF-β was titrated in the presence of recombinant IL-27. n = 1 per genotype, performed once. T_H, T helper cells; T_R1, type 1 regulatory cells; T_reg cells, inducible regulatory T cells. See also Extended Data Fig. 1.

Fig. 3 |. Nkg7 is expressed by mouse spleen and liver CD4⁺ T cells during L. donovani infection.

a, Nkg7 reporter mice were infected with L. donovani and the overall expression of GFP was assessed in the liver and spleen before infection (naive) and at days 14, 28 and 58 p.i. The sizes of the pie charts and the associated frequency values represent the relative percentage of GFP⁺ cells in the liver (above timeline) and spleen (below timeline). Within these pie charts, each slice represents the proportion of the indicated immune cell subset that made up the GFP⁺ population. Measurements were taken from distinct samples at each time point. The data shown are representative of two independent experiments, each consisting of n = 3 mice per genotype, per time point. cDCs, conventional dendritic cells; NKT cells, natural killer T cells; pDCs, plasmacytoid dendritic cells. b, Confocal immunofluorescence microscopy was used to determine the tissue localization of Nkg7-expressing (GFP⁺) cells in the liver of mice at day 28 p.i. The co-localization of Nkg7 and CD4 expression is shown in the merged image. Scale bar: 50 μm. The numbers of CD4⁺NKG7⁺ cells within inflammatory foci (granulomas), relative to surrounding tissue, are shown in the accompanying graph (right). Statistical significance was determined using the Mann–Whitney U-test. ****P < 0.0001. See also Extended Data Fig. 2.

Fig. 4 |. Nkg7 deficiency promotes elevated parasite burdens during L. donovani infection.

a, WT and Nkg7^−/− mice were infected with L. donovani and parasite burdens were measured in the liver and spleen at days 14, 28 and 56 p.i. Statistical testing was performed using a two-way ANOVA with Šidák’s multiple comparisons test. LDU, Leishman–Donovan units. b, Quantification of serum pro-inflammatory cytokines in infected mice. Statistical testing was performed using a two-way ANOVA with Šidák’s multiple comparisons test. MCP-1, monocyte chemoattractant protein 1. c, Total number of leukocytes in the liver and spleen at day 14 p.i. Statistical testing was performed using the Mann–Whitney U-test. d, Day 14 p.i. liver parasite burdens in Rag1^−/− mice that received CD4⁺ T cells isolated from either WT (n = 5) or Nkg7^−/− (n = 5) mice. Statistical testing was performed using the Mann–Whitney U-test. e, Frequency and total number of T_H1 cells (gated on lymphocytes, singlets, live cells, NK1.1-APC/Cy7⁻TCR-β-BUV737⁺, CD4-BUV395⁺CD8α-Alexa Fluor 700⁻, Foxp3-Alexa Fluor 488⁻ and IFN-γ-APC⁺IL-10-PE⁻ cells) in the livers of naive mice and infected mice at day 14 p.i. Statistical testing was performed using the two-way ANOVA with Šidák’s multiple comparisons test. f, Top: representative flow cytometry plots were gated on lymphocytes, singlets, live cells, NK1.1-APC/Cy7⁻TCR-β-BUV737⁺ and CD4-BUV395⁺CD8α-Alexa Fluor 700⁻ cells. Bottom: graphs showing the frequency and total number of CD11a⁺CD49d⁺CD4⁺ T cells in the liver at day 14 p.i. Statistical testing was performed using the Mann–Whitney U-test. Cy, Cyanine; FITC, fluorescein isothiocyanate; PE, phycoerythrin. g, Frequency (top) and median fluorescence index (MFI; bottom) of phosphorylated STAT4 (p-STAT4) within CD4⁺TCR-β⁺ (polyclonal) and I-A^bPEPCK_335–351 (tetramer) PE⁺ cells in the liver at day 14 p.i. Statistical significance was determined using the Mann–Whitney U-test. Representative plots (middle) show the expression of p-STAT4 upon treatment with recombinant IL-12. n = 5 WT and n = 6 Nkg7^−/− mice. Data are representative of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Error bars represent means ± s.e.m. The data shown in a–c, e and f are representative of two independent experiments, each with n = 3 naive WT and Nkg7^−/− mice and n = 5 WT and n = 4 Nkg7^−/− mice at days 14, 28 and 58 p.i. Measurements in a and b were obtained from distinct samples at each time point. See also Extended Data Fig. 3.

Fig. 5 |. Nkg7 promotes parasite accumulation in tissues and the onset of ECM following P. berghei ANKA infection.

WT and Nkg7^−/− mice were infected with P. berghei ANKA, which causes ECM. a, ECM scores obtained as repeated measures at each time point. b, Survival analysis between WT and Nkg7^−/− mice. Statistical testing was performed using the log-rank (Mantel–Cox) test. c, Levels of pRBCs in the circulation, obtained as repeated measures at each time point. Statistical significance was determined using a two-way ANOVA with Šidák’s multiple comparisons test. d,e, Parasite biomass was quantified using luciferase-expressing P. berghei ANKA parasites, in the bodies (d) and brains (e) of infected mice. n = 4 WT and n = 5 Nkg7^−/− mice. Statistical significance was determined using the Mann–Whitney U-test. f, Frequency of H2-K^b-NVF (tetramer) PE⁺ cells within the brain CD8⁺ T cell population at the peak of ECM. Statistical significance was determined using the Mann–Whitney U-test in one experiment where n = 3 mice per group. g, Proportion of co-transferred transgenic PbT-I^WT and PbT-IΔ^Nkg7 cells as a frequency of TCR-β⁺CD8⁺ cells in the brains of naive and infected mice (at peak ECM). h, Frequencies of granzyme B⁺ or perforin⁺ PbT-I^WT and PbT-IΔ^Nkg7 cells in the brain after incubation with monensin. Statistical significance was determined using a two-way ANOVA with Šidák’s multiple comparisons test. i, Top: representative histograms illustrate CD107a (LAMP-1) expression by PbT-I^WT and PbT-IΔ^Nkg7 cells in the brain after incubation with monensin or stimulation with PMA and ionomycin (iono) in the presence of monensin. Bottom: graphs of the histograms present the frequency and MFI of CD107a expression by PbT-I^WT and PbT-IΔ^Nkg7 cells. Data in a–e are representative of three independent experiments. n = 5 mice per group in a–c. The experiments in g–i were performed once where n = 4 naive and n = 5 infected mice per strain. Error bars represent means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See also Extended Data Fig. 4.

Fig. 6 |. NKG7 co-localizes with cytotoxic vesicles expressing CD107a.

a, Representative images showing the co-localization of NKG7 and LysoTracker in WT splenocytes transduced (bottom), or not (top), to express NKG7–GFP. Scale bar: 10 μm. b, Splenocytes from PbT-I^WT T cell transgenic mice were also transduced to express NKG7–GFP. The representative images depict the co-localization of NKG7 and the cytotoxic granule-associated protein CD107a. Scale bar: 10 μm. AF647, Alexa Fluor 647. c, Comparison of cytotoxic ability between PbT-I^WT and PbT-IΔ^Nkg7 transgenic CD8⁺ T cells against NVF peptide-pulsed splenocytes in vitro. Data were obtained from one experiment. Statistical significance was determined using a two-way ANOVA with effector-to-target ratio and group as variables. Data are representative of two independent experiments. *P < 0.05.

Fig. 7 |. Nkg7 deficiency increases metastatic burden.

a, Survival analysis between individuals in the top and bottom 25% of NKG7 expressers in the TCGA SKCM dataset, performed using a log-rank test. The shading represents the 95% confidence interval in the top and bottom quartiles. b, WT and Nkg7^−/− mice were injected with either B16F10 (n = 8 WT and n = 6 Nkg7^−/− mice) or LWT1 cells (n = 9 WT and n = 7 Nkg7^−/− mice). Lung metastatic burdens were quantified at day 14 post-injection. The data shown are representative of two independent experiments. The Mann–Whitney U-test was used to determine statistical significance. c, Left: the lungs of WT and Nkg7^−/− mice (n = 5 mice per group), injected with LWT1 cells, were assessed for differences in the frequency of IFN-γ⁺ (top) or CD107a⁺ NK cells (bottom). Right: the MFI of IFN-γ or CD107a on these cells at 14 d post-injection is also shown. The data are representative of two independent experiments. The Mann–Whitney U-test was used to determine statistical significance. d, WT and Nkg7^−/− mice injected with B16F10 cells were treated with isotype control antibodies (n = 27 WT and n = 26 Nkg7^−/−; pooled from four experiments) or antibodies against asGM1 (n = 10 WT and n = 11 Nkg7^−/−), CD8β (n = 9 WT and n = 5 Nkg7^−/−) or IFN-γ (n = 10 WT and n = 12 Nkg7^−/−) (pooled from two experiments). Lung metastases were quantified at day 14 post-injection of B16F10 cells. A two-way ANOVA with Šidák’s multiple comparisons test was used to determine statistical significance between groups. e, Differences in lung metastatic burden in Rag2^−/−γc^−/− mice that received either WT (n = 9) or Nkg7^−/− (n = 11) NK cells before injection with B16F10 cells. Controls consisted of Rag2^−/−γc^−/− mice that did not receive NK cells but were injected with either 1 × 10⁴ (n = 9) or 1 × 10⁵ (n = 13) B16F10 cells. The data are representative of two independent experiments. The Mann–Whitney U-test was used to determine statistical significance. f, Waterfall plot showing the top 50 up- and downregulated genes between high and low NKG7 expressers from the TCGA SKCM dataset. g, Top ten upstream regulator cytokines between high and low NKG7 expressers from the TCGA SKCM dataset identified by IPA. h, Top: WT and Nkg7^−/− mice injected with B16F10 cells were treated with recombinant IL-2 (n = 10 WT and n = 5 Nkg7^−/−) or IL-15/IL-15Rα (n = 11 WT and n = 5 Nkg7^−/−) and compared with PBS-treated controls (n = 10 WT and n = 5 Nkg7^−/−). Bottom: mice injected with LWT1 cells were treated with recombinant IL-2 (n = 8 WT and n = 16 Nkg7^−/−; pooled from two experiments) and compared with PBS-treated controls (n = 7 WT and n = 14 Nkg7^−/−; pooled from two experiments). A one-way ANOVA with multiple comparisons was used to test for statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. For the graphs in b–e and h, center lines indicate median values, box limits indicate upper and lower quartiles, and whiskers indicate maximum and minimum measures. See also Extended Data Fig. 5.

Fig. 8 |. NKG7 plays a role in cytotoxicity.

a, WT and Nkg7^−/− mice were subcutaneously injected with RMA-s-Rae1β cells, followed by treatment with control antibodies or antibodies against asGM1 or CD8β, and the tumor size was measured as repeated measures at each time point. A one-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance. The data shown are from one experiment performed where n = 5 mice per group. b, WT and Nkg7^−/− mice were given a subcutaneous injection of RMA-s-Rae1β cells at day 0. Mean tumor size was derived from repeated measures at each time point. Mice were treated with either control antibodies (n = 6 WT and n = 6 Nkg7^−/−) or anti-IFN-γ (n = 6 WT and n = 8 Nkg7^−/−) at days −1, 0, 7, 14 and 21. Statistical significance was determined using a two-way ANOVA with Tukey’s multiple comparisons test. The statistical significance between control antibodies and anti-IFN-γ-treated WT and Nkg7^−/− mice at day 24 is shown. Statistical significance between WT control antibodies and anti-IFN-γ groups was detected from day 14, whereas statistical significance between Nkg7^−/− control antibodies and anti-IFN-γ groups was detected on day 18. Data were obtained from one experiment. c, WT and Nkg7^−/− NK cell–mediated cytotoxicity against YAC-1 target cells or RMA-s-Rae1β in vitro, in the absence or presence of IL-2-induced activation. Statistical significance was assessed using a Mann–Whitney U-test at each effector-to-target ratio. n = 6 per group, pooled from two independent experiments. d, The difference in WT and Nkg7^−/− NK cell–mediated cytotoxicity against RMA-s-Rae1β in vivo is depicted in the representative plots (left) and the numbers of target cells remaining in the lungs of WT and Nkg7^−/− mice were quantified (right). Target cells were gated on live CD45.2⁺ cells. A Mann–Whitney U-test was used to determine statistical significance. n = 11 mice per group. Data were pooled from two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See also Extended Data Fig. 6.