Research ArticleNK CELLS

Gab3 is required for IL-2– and IL-15–induced NK cell expansion and limits trophoblast invasion during pregnancy

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Science Immunology  02 Aug 2019:
Vol. 4, Issue 38, eaav3866
DOI: 10.1126/sciimmunol.aav3866
  • Fig. 1 Identification of Gab3 as a critical determinant of NK cell function, antitumor responses, and pregnancy.

    (A) Identification of an ENU germline mutant (A961) exhibiting impaired NK cell function compared with a littermate control (A962). Control and ENU mice were immunized with 5E.1 TAKO cells. Seven days post immunization, mice were injected intravenously with control splenocytes [carboxyfluorescein diacetate succinimidyl ester (CFSE)–low], NK targets (β2M−/− splenocytes; CFSE-medium), and CD8+ targets (EBI192–200–loaded splenocytes; CFSE-high). After 2 days, the frequency of target populations in blood was determined by flow cytometry. (B) Coarse mapping on 34 mice (15 control and 19 mutant mice) using 150 genome-wide SNPs, identified the causal mutation to reside on chromosome X. (C) WES identified a C→T nucleotide change at position X:722788707 bp causing a single-residue change (Arg27→Cys27) in the PH domain of Gab3. (D and E) C57BL/6 (Embedded Image), Gab3R27C (Embedded Image), Gab3KO (Embedded Image), and NK cell–depleted (Embedded Image) mice were injected with 1 × 105 B16-F10 melanoma cells intravenously. After 3 weeks, mice were euthanized, and tumor burden was determined (bars represent mean ± SEM). N.S., not significant. (F) Frequency of dystocia observed in WT, Gab3R27C, and Gab3KO mice. (G and H) Representative images of stillborn pups, retained placentas (G), and maternal hemorrhaging (H) of a Gab3KO female with dystocia. Statistical analysis was performed using one-way ANOVA with Tukey posttest. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

  • Fig. 2 Gab3 is required for IL-2– and IL-15–induced priming and expansion of NK cells.

    (A) WT, Gab3R27C, and Gab3KO NK cells stimulated with anti-NK1.1 antibody, YAC-1 cells, or IL-12/IL-18 in the presence of IL-2. NK cell priming was measured by intracellular IFN-γ staining (n = 4 mice, mean ± SEM). (B and C) IL-2– or IL-15–induced NK cell expansion in vitro using WT, Gab3R27C, or Gab3KO splenocytes (n = 6, mean ± SEM). Cells were gated on live NKp46+/NK1.1+ cells. (D) Representative plots showing live WT, Gab3R27C, and Gab3KO CellTrace Violet (CTV)–labeled splenocytes expanded with exogenous IL-2 or IL-15 in vitro for 5 days and stained for Ki67 (n = 4, mean ± SEM). (E) Cell cycle analysis using EdU incorporation and 7-AAD (DNA content) on IL-2– or IL-15–induced WT, Gab3R27C, and Gab3KO NK cells (n = 4, mean ± SEM). Statistical analysis was done using a two-way ANOVA with Tukey posttest. **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 Gab3 is selectively required for MAPK activation but not Akt or STAT5 signaling downstream of IL-2 or IL-15 activation.

    (A to G) WT, Gab3R27C, and Gab3KO NK cells stimulated directly ex vivo with IL-2 or IL-15 at various time points. (A) Phosphorylation of STAT5. (B to D) Phosphorylation of downstream targets of PI3K including (B) p70 kinase, (C) Akt and (D) RpS6. (E to G) Phosphorylation of MAPK signaling including (E) ERK, (F) p38, and (G) JNK. (H) Proposed working model of where Gab3 functions downstream of the IL-2/IL-15 receptor signaling complex. Statistical analysis was done using a two-way ANOVA with Tukey posttest (experiments were performed in duplicate with at least n ≥ 2 mice). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 4 The R27C missense mutation disrupts Gab3 PH domain binding to PIPs and impairs recruitment to the IL2-receptor complex.

    (A) Three-dimensional model of the PH domain of WT Gab3 complexed with PIP3 (orange and magenta). The Arg27 position (green) is predicted to interact with the phosphoinositide headgroup. (B) PIP strips incubated with Gab3WT or Gab3R27C PH domain. LPA, lysophosphatidic acid; LPC, lysophosphatidylcholines; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine. (C) Representative images of Gab3KO NK cells transfected with Gab3WT-GFP or Gab3R27C-GFP fusion proteins to assess Gab3 colocalization with CD122 upon stimulation with IL-2. BF, brightfield. (D) ImageStream colocalization studies between CD122 and Gab3WT-GFP or Gab3R27C-GFP after various incubation times with IL-2. Colocalization was defined by the intensity of Gab3 in CD122+ vesicles, the diameter and area of Gab3/CD122+ vesicles, and colocalization assessed by bright detail similarity (BDS). ImageStream data represent mean values ± SEM of three experiments with >500 NK cells per experiment. A two-way ANOVA with Tukey posttest was performed to assess significance. *P < 0.05, **P < 0.01, and ***P < 0.001. MFI, mean fluorescence intensity.

  • Fig. 5 Gab3 is required for expansion of CD122+NK1.1+uNK cells during pregnancy.

    (A) Frequency of CD122+/NK1.1+ and DBA+ uNK cells in gd8.5 implantation sites from Gab3KO or WT control females as quantified by flow cytometry (n = 6). (B and C) Frequency of ILC1, cNK, and trNK cells in WT and Gab3KO implantation sites as defined by CD49a and EOMES expression using flow cytometry. (D and E) Phosphorylation of ERK and STAT5 in CD45+/CD122+/CD3/NK1.1+ uNK (D) and DBA+ uNK cell subsets (E) isolated from gd8.5 implantation sites from Gab3KO females stimulated ex vivo with IL-15 (n = 3, mean ± SEM). (F) NK cell receptor expression on gd8.5 uNK cells (CD45+/CD3/CD122+) from WT and Gab3KO pregnant females as assessed by RNA-seq analysis. (G) Relative mRNA expression of surface and cytotoxicity molecules in Gab3KO uNK cells compared with WT. Heat map graphs depict the z score, representing the number of SDs away from the mean expression. Statistical analysis was done using a one-way ANOVA (A) or two-way ANOVA (B and C) with Tukey posttest. *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 6 Functional Gab3 is required for controlling TGC invasion.

    (A) Representative hematoxylin and eosin (H&E)– or cytokeratin-7–stained images of gd12.5 placentas showing DB with spiral arteries (SpA), junctional zone (JZ), and labyrinth from WT, Gab3R27C, Gab3KO, NK cell–depleted (PK136 treatment) WT, or Gab3KO injected with WT NK cells at gd9.5 females. (B) Spiral artery remodeling was assessed by measuring the vessel-to-lumen ratio of spiral arteries on midsagittal sections of gd12.5 placentas stained with H&E (n = 4 mice; each symbol represents an individual placenta, lines represent mean ± SEM). ns, not significant. (C) Loss of Gab3 is associated with increased TGC invasion within the maternal spiral arteries. The average depth of cytokeratin-7+ cell layer in SpA walls were quantified on midsagittal sections of gd12.5 placentas stained with cytokeratin-7 antibody and a hematoxylin counterstain (n = 4 mice; each symbol represents an individual placenta, lines represent mean ± SEM). Statistical analysis was done using a one-way ANOVA with Tukey posttest. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/38/eaav3866/DC1

    Materials and Methods

    Fig. S1. Generation of Gab3-deficient mouse models.

    Fig. S2. Loss of Gab3 does not affect peripheral NK cell numbers or their maturation.

    Fig. S3. Loss of Gab3 does not affect NK cell development in the bone marrow.

    Fig. S4. The R27C missense mutation disrupts the PH domain binding to phosphotyidylinositols.

    Fig. S5. Altered gene expression in gd8.5 uNK cells (CD45+/CD3/CD122+) isolated from Gab3KO compared with WT uNK cells.

    Fig. S6. Loss of Gab3 has limited effect on the decidual depth, labyrinth, or junctional zone area at gd12.5.

    Fig. S7. Functional Gab3 is required for controlling trophoblast invasion.

    Fig. S8. WT mice treated with anti-NK1.1 antibodies (PK136) have efficient depletion of NK cells in the spleen and placentas at gd12.5.

    Table S1. Raw data in Excel spreadsheet.

    References (6467)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Generation of Gab3-deficient mouse models.
    • Fig. S2. Loss of Gab3 does not affect peripheral NK cell numbers or their maturation.
    • Fig. S3. Loss of Gab3 does not affect NK cell development in the bone marrow.
    • Fig. S4. The R27C missense mutation disrupts the PH domain binding to phosphotyidylinositols.
    • Fig. S5. Altered gene expression in gd8.5 uNK cells (CD45+/CD3/CD122+) isolated from Gab3KO compared with WT uNK cells.
    • Fig. S6. Loss of Gab3 has limited effect on the decidual depth, labyrinth, or junctional zone area at gd12.5.
    • Fig. S7. Functional Gab3 is required for controlling trophoblast invasion.
    • Fig. S8. WT mice treated with anti-NK1.1 antibodies (PK136) have efficient depletion of NK cells in the spleen and placentas at gd12.5.
    • References (6467)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Raw data in Excel spreadsheet.

    Files in this Data Supplement:

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