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. 2019 Aug 2;4(38):eaav3866.
doi: 10.1126/sciimmunol.aav3866.

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

Anna Sliz  1   2   3 , Kathryn C S Locker  1   2   3 , Kristin Lampe  1   2 , Alzbeta Godarova  4 , David R Plas  5 , Edith M Janssen  6 , Helen Jones  7   8 , Andrew B Herr  1   2   3 , Kasper Hoebe  9
Affiliations

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

Anna Sliz et al. Sci Immunol. .

Abstract

The scaffolding protein Grb2-associated binding protein 3 (Gab3) is a member of the Gab family, whose functions have remained elusive. Here, we identify Gab3 as a key determinant of peripheral NK cell expansion. Loss of Gab3 resulted in impaired IL-2 and IL-15-induced NK cell priming and expansion due to a selective impairment in MAPK signaling but not STAT5 signaling. In vivo, we found that Gab3 is required for recognition and elimination of "missing-self" and tumor targets. Unexpectedly, our studies also revealed that Gab3 plays an important role during pregnancy. Gab3-deficient mice exhibited impaired uterine NK cell expansion associated with abnormal spiral artery remodeling and increased trophoblast invasion in the decidua basalis. This coincided with stillbirth, retained placenta, maternal hemorrhage, and undelivered fetoplacental units at term. Thus, Gab3 is a key component required for cytokine-mediated NK cell priming and expansion that is essential for antitumor responses and limits trophoblast cell invasion during pregnancy.

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Figures

Figure 1.
Figure 1.. Identification of Gab3 as a critical determinant of NK cell function, anti-tumor responses and pregnancy.
a) Identification of an ENU germline mutant (A961) exhibiting impaired NK cell function compared to a litter mate control (A962). Control and ENU mice were immunized with 5E.1 TAKO cells. Seven days p.i., mice were injected i.v. with control splenocytes (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) Whole exome sequencing identified a C→T nucleotide change at position X:722788707bp causing a single residue change (Arg27→Cys27) in the pleckstrin-homology (PH) domain of Gab3. d,e) C57BL/6 (●),Gab3R27C (formula image), Gab3KO (formula image) and NK cell-depleted (formula image) mice were injected with 1×105 B16-F10 melanoma cells i.v.. After three weeks, mice were sacrificed, and tumor burden was determined (bars represent mean ± s.e.m). f) Frequency of dystocia observed in WT, Gab3R27C and Gab3KO mice. g,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 post-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
Figure 2.
Figure 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 IFNγ staining (n=4 mice, ± s.e.m.). b-c) IL-2- or IL-15-induced NK cell expansion in vitro using WT, Gab3R27C or Gab3KO splenocytes (n=6, mean ± s.e.m.). Cells were gated on live NKp46+/NK1.1+ cells. d) Representative plots showing live WT, Gab3R27C and Gab3KO cell-trace violet-labeled splenocytes expanded with exogenous IL-2 or IL-15 in vitro for 5 days and stained for Ki67 (n=4, mean ± s.e.m.). e) Cell cycle analysis using EDU incorporation and 7AAD (DNA content) on IL-2- or IL-15-induced WT, Gab3R27C and Gab3KO NK cells (n=4, mean ± s.e.m.). Statistical analysis was done using a two-way ANOVA with Tukey post-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Figure 3.
Figure 3.. Gab3 is selectively required for MAPK activation but not AKT or STAT5 signaling downstream of IL-2 or IL-15 activation.
a-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,c) Phosphorylation of downstream targets of PI3K including b) p70 kinase and c) AKT, and d) RpS6. e-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 post-test (experiments were performed in duplicate with at least n≥2 mice). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
Figure 4.
Figure 4.. The R27C missense mutation disrupts Gab3 PH-domain binding to PIPs and impairs recruitment to the IL2-receptor complex.
a) 3D model of the PH-domain of WT Gab3 complexed with phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) (orange & magenta). The Arg27 position (green) is predicted to interact with the phosphoinositide headgroup. b) PIP strips incubated with Gab3WT or Gab3R27C PH-domain. 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. d) ImageStream colocalization studies between CD122 and Gab3WT-GFP or Gab3R27C-GFP following 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. ImageStream data represent mean values ± s.e.m of 3 experiments with >500 NK cells per experiment. A two-way ANOVA with Tukey post-test was performed to assess significance. *P<0.05, **P<0.01, ***P<0.001.
Figure 5.
Figure 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,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,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 ± s.e.m.). f) NK cell receptor expression on gd8.5 uNK cells (CD45+/CD3−/CD122+) from WT and Gab3KO pregnant females as assessed by RNAseq analysis. g) Relative mRNA expression of surface and cytotoxicity molecules in Gab3KO uNK cells compared to WT. Heatmap graphs depict the Z-score, representing the number of standard deviations away from the mean expression. Statistical analysis was done using a one-way ANOVA (a) or two-way ANOVA (b,c) with Tukey post-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Figure 6.
Figure 6.. Functional Gab3 is required for controlling trophoblast giant cell invasion.
a) Representative H&E- or cytokeratin-7-stained images of gd12.5 placentas showing decidua basalis (DB) with spiral arteries (SpA), junctional zone (JZ) and labyrinth from WT, Gab3R27C, Gab3KO, NK cell-depleted (PK-136 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 mid sagittal sections of gd12.5 placentas stained with H&E (n=4 mice, each symbol represents an individual placenta, lines represent mean ± s.e.m.). 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 mid sagittal 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 ± s.e.m.). Statistical analysis was done using a one-way ANOVA with Tukey post-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
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