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



INTRODUCTION

The proteins of the Grb2-associated binding family (Gab) support the assembly of activated signal complexes by means of framework and docking functions. Gab family members consisting of Gab1-3 contain a highly conserved N-terminal pleckstrin homology domain (PH) as well as various phosphorylation sites specific for tyrosine kinases ( 1 3 ). The PH domain mediates the recruitment of plasma membranes through interaction with lipids such as phosphatidylinositol-3,4,5-triphosphate (PIP 3 ). 1 2 4 ). In contrast to Gab1 and Gab2, Gab3 is expressed exclusively in cells of the hematopoietic lineage, with highest expression in natural killer cells (NK), mast cells and memory CD8 cells (1

9459009 + T cells (http: // biogps. org, www.immgen.org) ( 5 ). Although the functions of Gab1 and Gab2 have been defined, the functions of Gab3 are still unknown.

NK cells play an important role in recognition and Elimination of Tumor Cells, Allogeneic Cells and Pathogenically Infected Cells 6 ) Activation of NK cells can occur via various receptor pathways integrating signals derived from inhibitory and activating surface receptors ( 7 In addition, NK cells can be activated by cytokines such as interleukin-2 (IL-2) and IL-15, cytokines that share common receptor signaling components, including the common γ-Ke and the IL-2 receptor β chain (CD122) ( 8 ). Both IL-2 and IL-15 initiate priming of NK cells, resulting in an increase in NK cell effector function, while promoting expansion of peripheral NK cells ( 9 ). NK cells are found in the bloodstream and lymphoid organs, but can also be found in different tissues. Uterine NK (uNK) cells in the maternal tissue (decidua and mesometrial lymphoid aggregate of pregnancy) of the mother-fetus interface are required for the early development and vascular remodeling of the decidua basalis (DB) during pregnancy (19459006] 10 – 12 ). This unique subgroup of NK cells is highly proliferative and represents the major leukocyte subset in the early mouse and human DB and reaches a maximum of 5.5 to 9.5 in gestational day mice (gd), while their number increases in humans in the first trimester and then decreases as pregnancy progresses ( 11 ). UNK cells in the mouse can be defined by expression of a glycan recognized by Dolichos biflorus agglutinin (DBA) lectin ( 13 14 ) or expression of CD122 and NK1.1. Moreover, due to their expression of CD49a and eomesodermin (EOMES), they can be differentiated into congenital lymphoid cells 1 (ILC1), tissue-resident NK cells (trNK cells) and conventional NK cells (cNK cells) ([19459005)] 15 [FIG – 17 ). In addition to vascular remodeling, uNK cells play an essential role in regulating the invasion of fetal trophoblasts during DB development and pregnancy ( 18 ). Although the importance of uNK cells for a successful pregnancy is much appreciated, the pathways leading to their expansion and activation are still poorly defined.

Our recent studies identify a key role for Gab3 in NK cells. In particular, we show that Gab3 is essential for mitogen-activated protein kinase (MAPK) signaling downstream of the IL-2 / IL-15 receptor in NK cells. Gab3 deficiency was associated with a marked impairment in the ability to remove tumor cells in vivo, whereas IL-2 and IL-15 induced priming and expansion of NK cells was abrogated in vitro. In addition, we show a key role for Gab3 in the expansion of uNK cells, which is associated with abnormal vascular remodeling and an increased incidence of failed pregnancies characterized by stillbirth, retained placenta, maternal bleeding and undelivered fetoplacental units at the appropriate time.

19659006] Gab3 is a critical protein needed for NK cell function.

As part of our N ethyl N nitrosourea (ENU) mutagenesis approach to the identification of genes involved in NK cell-mediated missing-self-recognition ( 19 ) we identified a G3 germline mutant (A961) that could not clear any β2-microglobulin-deficient target cells while having normal antigen-specific CD8 T-cell responses after immunization (Figure 1A). Linkage analysis ( 19 ) revealed the causative mutation on chromosome X with a critical range between 0 and 93.6 Mb (Figure 1B). Subsequent sequencing of the entire exome (WES) revealed a nucleotide change [C to T at X:722788707 base pair (bp)] in Gab3 which was predicted to be harmful by sorting intolerant (SIFT) and polymorphic phenotyping (PolyPhen). In particular, the C → T missense mutation causes a single amino acid change (Arg → Cys or R27C) in the PH domain of Gab3 (Fig. 1C and S1A). Orientational studies show that the Arg residue is highly conserved in PH domains; However, for the same Gab3 residue there is a rare human missense variant (Arg → His: http://exac.broadinstitute.org/). We hypothesized that the Gab3 R27C mutation might be responsible for the observed NK cell deficiency, and to confirm the role of Gab3 in NK cell function, we generated Gab3 knockout (KO) mice using CRISPR-Cas9. In particular, lead RNAs were targeted that targeted exon 2 and caused a single nucleotide insertion, resulting in image shift, alternative translation, and premature stop (Figures S1, B to D). Gab3 KO mice were viable and confirmed the importance of Gab3 for the detection of β2M-deficient targets in vivo (Figure S1E). Characterization of other lymphocyte subgroups of Gab3 R27C and Gab3 KO mice, including CD8 + CD4 + and B Cells show no differences in the frequency or evolution of these subgroups and the normal CD8 + T-cell effector function, as determined by immunization with a transporter treated with antigen-processing (TAP) -deficient embryonic mouse Fibroblasts that express humans express adenovirus type 5 early region 1 (5E1-TAKO) cells (Figure 1A). Further characterization of peripheral NK cell populations in homozygous Gab3 R27C or Gab3 KO mice revealed no change in the frequency of NK cells in the spleen (Fig. S2A). , In addition, NK cells from Gab3 R27C and Gab3 KO showed normal NK cell maturation to mice (Figure S2B), expression of the activating receptors Ly49D and Ly49H (Fig S2C) or expression of inhibiting Ly49 receptors, although the incidence of Ly49G2 + NK cells in Gab3 R27C and Gab3 KO [19659008verringertwar] mice (Fig. S2D). However, stimulation of NK cells ex vivo using various activating stimuli (e.g., anti-NK1.1, YAC-1 tumor target cells, or combined IL-12 / IL-18 activating cytokines) gave similar interferon activity. γ (IFN-γ) production (Figure S2E). Recently, we investigated the development of NK cells in the bone marrow by flow cytometry as previously described ( 20 ). Consistent with normal peripheral NK cell counts, no changes in the frequency of NK-bound precursors or NK1.1 + NK cells were observed in the bone marrow (Figures S3, A and B).

] fig. 1 Identification of Gab3 as critical determinant of NK cell function, antitumor reactions and pregnancy.

( A ) Identification of an ENU germline mutant (A961) with impaired NK cell function compared to a littermate control (A962). Control and ENU mice were immunized with 5E.1-TAKO cells. Seven days after immunization, mice were given intravenous control splenocytes [carboxyfluorescein diacetate succinimidyl ester (CFSE)–low] NK targets (β2M – / – splenocytes; CFSE medium) and CD8 + targets (EBI [19459007)injiziert] 192-200 loaded splenocytes (CFSE-high) After 2 days, the frequency of the target populations in the blood was determined by flow cytometry ( B ) Gross mapping on 34 mice (15 control mice and 19 mutated mice) under Use of 150 genome-wide SNPs identified the causal mutation that resides on chromosome X. ( C ) WES identifies a C → T nucleotide change at position X: 722788707 bp representing a single residue change (Arg 27 → Cys 27 ) in the PH domain of Gab3. ( D and E ) C57BL / 6 (  Embedded Image ), Gab3 R27C ([19659021)] Emb Embedded Image ” src=”https://immunology.sciencemag.org/sites/default/files/highwire/immunology/4/38/eaav3866/F1/embed/inline-graphic-2.gif”/> Embedded Image   Embedded Image

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1 Identification of Gab3 as a critical determinant of NK cell function, antitumor responses, and pregnancy.

( A ]) Identification of an ENU germline mutant (A961) with impaired NK cell function in comparison to a littermate control (A962) Control and ENU mice were challenged with 5E.1-TAKO Cells immunized Seven days after immunization, control splenocytes were injected intravenously into the mice [carboxyfluorescein diacetate succinimidyl ester (CFSE)–low] NK targets (β2M – / – splenocytes; CFSE medium) and CD8 + targets (EBI 192-200 -loaded splenocytes, CFSE-high). After 2 days, the frequency of target populations in the blood was determined by flow cytometry. ( B ) gross mapping to 34 mice (15 control mice and 19 mutant mice) using 150 genome-wide SNPs, identif ( C ) WES identified a C → T nucleotide change at position X: 722788707 bp, which caused a single residue alteration (Arg 27 → Cys 27 ) in the PH domain of Gab3. D and E ) C57BL / 6 (  Embedded Image ), Gab3 R27C ([19659021)] Embedded Image ” src=”https://immunology.sciencemag.org/sites/default/files/highwire/immunology/4/38/eaav3866/F1/embed/inline-graphic-2.gif”/> Gab3 KO Embedded Image ” src=”https://immunology.sciencemag.org/sites/default/files/highwire/immunology/4/38/eaav3866/F1/embed/inline-graphic-3.gif.backup.1564768680.7377″/> and NK Cell – Depleted Embedded Image   Embedded Image [Micewereinjectedintravenouslywith1×10<sup> 5 </sup> B16-F10 melanoma cells, and the mice were euthanized after 3 weeks and the tumor burden was determined (bars represent the mean ± SEM) NS, not significant. <strong> F </strong>) incidence of dystocia in WT, <em> Gab3- <sup> R27C- </sup></em>  and <em> Gab3- <sup> KO mice </sup></em> . <strong> G </strong> and <strong> H </strong>) Representative images of stillborn puppies, retained placenta (G) and maternal hemorrhage (H) of a <em> Gab3 <sup> KO </sup></em>   Woman with dystocia. The statistical analysis was performed using a one-way Tuovo Posttest ANOVA. * <em> P </em> <0.05, *** <em> P </em> <0.001 and **** <em> P </em> <0.0001. </p><div><script async src=

In vivo Functional Gab3 is Essential for Tumor Cell Clearance and Successful Pregnancy

Lack of self-recognition is an important mechanism by which NK cells recognize tumor cells. We therefore investigated whether Gab3 R27C and Gab3 KO [19659008] mice are susceptible to melanoma tumor exposure in vivo. Specifically, we injected wild-type (WT), NK cell-poor, Gab3 R27C and Gab3 KO mice with 1 × 10 5 B16 -F10 melanoma tumor cells intravenously. After 3 weeks, we quantified pulmonary tumor nodules, and it was found that both Gab3 R27C and Gab3 KO mice had a significantly higher tumor burden in the lung than WT control mice (Fig. D and E). The highest tumor burden was in the Gab3 KO mice, which achieved tumor numbers similar to those of NK cell-poor mice, while Gab3 R27C exhibited an intermediate phenotype (Fig. 1), D and E).

Maintaining a Homozygous Colony of Mice Gab3 R27C and Gab3 KO we found significant impairments to their completeness of successful pregnancies (Figs. 1, F to H ). In particular, we observed a dramatic increase in dystoccate rates in both Gab3 R27C and Gab3 KO pregnant women (ie, delivery was initiated, but delivery was not completed ) over the next 24 hours, coincident with undelivered fetoplacental units, stillbirths, bleeding, and retained placentas (Figures 1, G, and H). The relative incidence of failed pregnancies was highest among women compared to WT women in Gab3 KO with Gab3 R27C women having a median rate of failed pregnancies. Taken together, these results indicate a key role of Gab3 in the recognition of metastatic tumor cells by mature NK cells and the successful completion of childbirth at the end of pregnancy.

Gab3 is required for IL-2 and IL-15-induced patients Priming and expansion of NK cells

In connection with antitumor reactions and pregnancy, IL-2 and IL-15 are key cytokines that cause priming and expansion of NK cells ( 21 26 ). To investigate whether NK cells from Gab3 R27C and Gab3 KO mice have a modified IL-2 or IL-15 response, we have the NK cell priming and proliferation after studied IL-2 or IL-15 stimulation. Compared to WT-NK cells, Gab3 R27C and Gab3 KO NK cells showed a significant reduction in IFN-γ production when compared with anti- NK1.1 or YAC were stimulated -1 in the presence of IL-2 (Figure 2A), suggesting an impairment of the IL-2 primer.

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

(A) WT, Gab3 R27C and Gab3 KO NK cells treated with anti-NK1.1 antibody, YAC-1 cells or IL-12 / IL-18 in the presence of IL-2. Priming of the NK cells was monitored by intracellular IFN-.gamma. Staining measured ( n = 4 mice, mean ± SEM). B and C ) IL-2 or IL-15-induced NK cell expansion in vitro using WT Gab3 R27C or [19459006Gab3 KO splenocytes ( n = 6, mean ± SEM). The cells were gated on live NKp46 + / NK1.1 + cells. ( D ) Representative diagrams showing living WT, Gab3 R27C and Gab3 KO CellTrace Violet (CTV) -labeled splenocytes with exogenously expanded IL-2 or IL-15 in vitro for 5 days and stained for Ki67 ( n = 4, mean ± SEM). E ) Cell cycle analysis with EdU incorporation and 7-AAD (DNA content) on IL-2 or IL-15-induced WT, Gab3 R27C and Gab3 KO NK cells ( n = 4, mean ± SEM). Statistical analysis was performed using a two-way ANOVA with Tukey Posttest. ** P <0.01, *** P <0.001, **** P <0.0001.

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2 Gab3 is responsible for IL-2 and IL-15-induced priming and expansion of NK cells

(A) WT, Gab3 R27C and Gab3 KO NK cells treated with anti-NK1.1 antibody, YAC-1 Cells or IL-12 / IL-18 were stimulated in. The presence of IL-2. NK cell priming was assessed 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, Gab3 R27C [19659036] or Gab3 KO Splenocytes ( n = 6, mean ± SEM.) Cells were transformed into living NKp46 + / NK1.1 + cells gated WT, Gab3 R27C [1 9659035] and Gab3 KO CellTrace Violet (CTV) -labeled splenocytes expanded with exogenous IL-2 or IL-15 for 5 days and stained for Ki67 ( n = 4, mean ± SEM). E ) Cell cycle analysis with EdU incorporation and 7-AAD (DNA content) on IL-2 or IL-15-induced WT, Gab3 R27C and Gab3 KO NK cells ( n = 4, mean ± SEM). Statistical analysis was performed using a two-way ANOVA with Tukey Posttest. ** P <0.01, *** P <0.001, **** P <0.0001.

Next we have IL-2 or measured IL-15-induced expansion of NK cells in vitro. Both Gab3 R27C and Gab3 KO The NK cell expansion was significant after 1 week with the numbers of Gab3 KO decreased Gab3 R27C NK cells reduced to ~ 20 and ~ 50% of WT-NK cell counts (2, B, and C, respectively). Since decreased expansion could be due to either decreased survival or impaired proliferation, we next assessed the proliferation / viability and progression of the cell cycle. NK cells from both Gab3 R27C and Gab3 KO mice showed markedly reduced proliferation as determined by CellTrace Violet dilution and Ki67 expression ( Fig. 2D), whereas this is not apparent. Loss of viability of the NK cells was observed. Consistent with survival, we observed a normal induction of Mcl-1, a critical survival factor induced by IL-2 or IL-15 ( 27 ) in Gab R27C . and Gab3 KO NK cells in comparison to WT (Fig. S3C). Recently, we investigated the progression of the cell cycle using EdU in conjunction with 7-aminoactinomycin D (7-AAD) staining during IL-2 and IL-15 expansion. We observed significantly less Gab3 KO NK cells in the S phase, whereas more NK cells were in G 1 than WT NK cells. Similar trends were observed in the Gab3 R27C NK cells, although differences did not reach statistical significance (Figure 2E). These findings again suggest that the Gab3 R27C mutation behaves like a hypomorphic allele. These data identify a critical role for Gab3 in IL-2 and IL-15 driven priming and expansion of NK cells.

Gab3 is selectively required for MAPK signaling downstream of IL-2 / IL-15 receptors.

IL-15 – / – and IL-15Rα – / – mice show a marked reduction in peripheral NK cell numbers ( 28 31 ). While our studies demonstrate a role for Gab3 in the IL-15-induced expansion of NK cells, peripheral NK cell numbers remain unchanged in Gab3-deficient mice. We therefore hypothesized that Gab3 is required for a selective pathway downstream of the IL-2 / IL-15 receptor. Activation of the IL-2 / IL-15 receptor triggers three major signaling pathways, including the JAK (Janus kinase) / STAT5 (transcriptional 5 transducers and activators), the phosphoinositide 3-kinase (PI3K), and the MAPK route ( 32 33 ). We examined the downstream signaling in WT, Gab3- R27C- and Gab3- KO- NK cells with IL-2 or IL-15 by evaluation the phosphorylation of STAT5 (JAK / STAT) pathway), Akt, p70 S6 kinase, mTORc1, ribosomal protein S6 (RpS6) and ERK (extracellular signal regulated kinase), JNK (c-Jun N-terminal kinase) and p38 (MAPK) Way) at different times. Both IL-2 and IL-15 induced robust and normal activation of STAT5, Akt and p70 S6 kinase in WT, Gab3 R27C and Gab3 KO NK cells (Fig. 3, A to C). In contrast, both Gab3 R27C and Gab3 KO NK cells demonstrated a serious defect in the activation of MAPK pathways (ie, phospho-ERK, phospho- JNK, and phospho-p38) (Figure 3, E to G). The Gab3 R27C showed a partial reduction, while the Gab3 KO showed an almost complete lack of MAPK activation, confirming that the Gab3 R27C allele behaves like a hypomorphic allele. These studies also showed a partial but significant reduction in the phosphorylation of RpS6 (Figure 3D) in Gab3 R27C and Gab3 KO NK cells. RpS6 is a key component of the 40 S ribosome subunit required for RNA translation and cell growth, and previous studies have shown that phosphorylation of RpS6 occurs via both PI3K and ERK proteins. Way can be conveyed ( 34 35 ). The partial reduction in RpS6 phosphorylation is likely due to the selective loss of ERK, but not of PI3K, which is signaled in Gab3-deficient NK cells. These findings demonstrate a critical and selective role of Gab3 in activating the MAPK signaling pathway downstream of IL-2 / IL-15R, while Gab3 is superfluous for the JAK / STAT and PI3K signaling pathways (Figure 3H).

. 3 Gab3 is required selectively for MAPK activation, but not for Akt or STAT5 signaling after IL-2 or IL-15 activation.

( A to G ) WT, Gab3 R27C and Gab3 KO NK cells, which stimulated directly at different times ex vivo with IL-2 or IL-15. (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 for the function of Gab3 downstream of the IL-2 / IL-15 receptor signaling complex. Statistical analysis was performed using a two-way ANOVA with Tukey Posttest (experiments were performed on at least two n ≥ 2 mice). * P <0.05, ** P <0.01, P <0.001 and **** P <0 , 0001.

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Fig. 3 Gab3 is selectively required for MAPK activation, but not for the nude or STAT5 signaling after IL-2 or IL-15 activation.

( A to G ) WT, Gab3 R27C and Gab3 KO NK cells stimulated directly at different times ex vivo with IL-2 or IL-15. (A) phosphorylation of STAT5. (B to D) phosphorylation of downstream targets of PI3K, including (B) p70 kinase) Akt and (D) RpS6. (E to G) phosphorylation of MAPK signals, including (E) ERK, (F) p38 and (G) JNK. ( H [ProposedworkingmodelforthefunctionofGab3downstreamoftheIL-2/IL-15receptorsignalingcomplexwasastatisticalanalysisusingatwo-wayTukeyPosttestANOVA(experimentswereperformedinduplicatedetwithatleast n ≥ 2 mice). * P <0.05, ** P <0.01, P <0.001 and **** P <0 , 0001.

R27C Mutation Disrupts the Phosphoinositide Lipid Binding of the Gab3 PH Domain

Based on the three-dimensional modeling of the PH domain in complex with PIP 3 we predicted that the R27C mutation would be the ability would interfere with the PH domain to recognize and bind lipids (Figure 4A). In this way, we generated and purified PH domains from WT Gab3 or R27C Gab3 and evaluated their ability to bind to different phosphatidylinositols and membrane lipids using PIP strips incubated with the WT or R27C PH domain of Gab3 (Figure 4B). WT bound the monophosphorylated (PIP) as well as the bisphosphorylated (PIP 2 ) and triphosphorylated (PIP 3 ) phosphatidylinositols (4B). In contrast, lipid binding was significantly disrupted in the presence of the R27C mutation; For lipids strongly binding WT Gab3 only minimal binding was observed (Figure 4B). To confirm the PIP specificity and quantitate differences between the lipid binding capacities of WT or R27C-Gab3 domains, we repeated the protein-lipid overlay assay using serial dilutions of specific phosphatidylinositols. The strongest binding of the Gab3 PH domain was observed for PIP 2 species, with highest binding for PtdIns (3,4) P 2 and PtdIns (3,5) P [observed] 2 while limited binding of these lipids to the Gab3 R27C PH domain was observed (Figure S4). These studies suggest that the R27C missense mutation might hinder the recruitment of Gab3 into the membrane and / or the IL-2 / IL-15R complex following IL-2 / IL-15 stimulation. To test this hypothesis, we investigated the localization of WT or R27C-Gab3 protein in quiescent and IL-2 / IL-15-activated NK cells. Specifically, we generated lentiviral vectors expressing green fluorescent protein (GFP) -coupled Gab3 WT or Gab3 R27C mutant protein, and then primary Gab3 KO NK transfect cells to assess protein localization. Following cytokine stimulation, Gab3 colocalization with the IL2Rβ (CD122) component was timed using ImageStream. At rest, WT Gab3-GFP or Gab3 R27C -GFP fusion proteins displayed similar expression levels, while no significant differences in cell distribution and limited colocalization were observed with the IL2Rβ component (Figures 4, C and D) ). Nach der Stimulation mit IL-2 unterscheidet sich die zelluläre Verteilung des Gab3 R27C -Proteins signifikant von der von WT Gab3. Während WT Gab3 schnell an CD122 + (IL-2Rβ) -Stellen rekrutiert wird (Abb. 4, C und D), wurde diese Rekrutierung im Fall von Gab3 R27C -GFP weitgehend aufgehoben . Daher legen diese Ergebnisse nahe, dass die Gab3 R27C -Variante in der PH-Domäne die Phosphoinositid-Lipidbindung beeinträchtigt, wodurch die Rekrutierung des Gerüstproteins Gab3 an den IL-2 / IL-15-Rezeptorkomplex aufgehoben wird, was letztendlich zu einem führt MAPK-Signalisierung konnte nicht aktiviert werden.

500 NK-Zellen pro Experiment. Eine Zwei-Wege-ANOVA mit Tukey-Posttest wurde durchgeführt, um die Signifikanz zu bewerten. * P <0,05, ** P <0,01 und *** P <0,001. MFI, mittlere Fluoreszenzintensität. "Class =" Fragmentbilder colorbox-load "rel =" Galerie-Fragmentbilder-474654054 "data-figure-caption ="

4 Die R27C-Missense-Mutation unterbricht die Bindung der Gab3-PH-Domäne an PIPs und beeinträchtigt die Rekrutierung an den IL2-Rezeptorkomplex.

( A ) Dreidimensionales Modell der PH-Domäne von WT Gab3, komplexiert mit PIP 3 (Orange und Magenta). Es wird vorausgesagt, dass die Arg 27 -Position (grün) mit der Phosphoinositid-Kopfgruppe interagiert. ( B ) Mit Gab3 WT oder Gab3 R27C PH-Domäne inkubierte PIP-Streifen. LPA, Lysophosphatidsäure; LPC, Lysophosphatidylcholine; PE, Phosphatidylethanolamin; PC, Phosphatidylcholin; PA, Phosphatidsäure; PS, Phosphatidylserin. ( C ) Repräsentative Bilder von Gab3 KO NK-Zellen, die mit Gab3 WT -GFP oder Gab3 R27C -GFP-Fusionsproteinen transfiziert wurden Bewertung der Gab3-Kolokalisation mit CD122 nach Stimulation mit IL-2. BF, Hellfeld. ( D ) ImageStream-Kolokalisierungsstudien zwischen CD122 und Gab3 WT -GFP oder Gab3 R27C -GFP nach verschiedenen Inkubationszeiten mit IL-2. Die Kolokalisation wurde durch die Intensität von Gab3 in CD122-Vesikeln + den Durchmesser und die Fläche von Gab3 / CD122-Vesikeln + und die Kolokalisation durch die Ähnlichkeit von hellen Details (BDS) bestimmt. ImageStream-Daten repräsentieren Mittelwerte ± SEM von drei Experimenten mit> 500 NK-Zellen pro Experiment. Eine Zwei-Wege-ANOVA mit Tukey-Posttest wurde durchgeführt, um die Signifikanz zu bewerten. * P <0,05, ** P <0,01 und *** P <0,001. MFI, mittlere Fluoreszenzintensität.

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4 Die R27C-Missense-Mutation unterbricht die Bindung der Gab3-PH-Domäne an PIPs und beeinträchtigt die Rekrutierung in den IL2-Rezeptorkomplex.

( A ) Dreidimensionales Modell der mit PIP 3 komplexierten PH-Domäne von WT Gab3 (orange und magenta) Die Position von Arg 27 (grün) soll mit der Phosphoinositid-Kopfgruppe interagieren. ( B ) Mit Gab3 WT oder Gab3 R27C inkubierte PIP-Streifen ] PH-Domäne, LPA, Lysophosphatidsäure, LPC, Lysophosphatidylcholine, PE, Phosphatidylethanolamin, PC, Phosphatidylcholin, PA, Phosphatidsäure, PS, Phosphatidylserin. ( C ) Repräsentative Bilder von Gab3 [19459 NK cells transfected with Gab3WT-GFP or Gab3R27C-GFP fusion proteins to assess Gab3 colocaliz ation w ith 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.

Loss of Gab3 impairs uNK cell function and expansion

Given the increased frequencies of dystocia and pregnancy-associated abnormalities seen in Gab3R27C and Gab3KO females (Fig. 1, F to H), we sought to identify the underlying mechanisms for these observations. The uNK population includes unique subsets that can be defined by a variety of surface markers, including NK1.1, CD122, DBA lectin binding, CD49a, and EOMES (13141617). Given the observed pregnancy complications, we posited that uNK cell expansion/function may be impaired during early gestation in Gab3R27C and Gab3KO mice. Flow cytometric analysis of WT and Gab3KO gd8.5 implantation sites revealed a markedly reduced frequency of CD45+CD122+ CD3DBA NK1.1+ NK cells (down to ~30% of WT levels) in Gab3KO mice, whereas the CD45+CD122+ CD3DBA+ NK1.1 population remained relatively unperturbed (Fig. 5A), suggesting that the NK1.1+/CD122+ uNK subset specifically requires Gab3 for its expansion early during pregnancy. Further, characterization of the innate lymphoid populations to differentiate ILC1, cNK cells from trNK cells based on CD49a and EOMES expression (1516), revealed a selective reduction in the number of trNK cells in Gab3KO compared with WT implantation sites (Fig. 5, B and C). The frequency of cNK and ILC1 cells at gd8.5 remained relatively unperturbed compared with WT implantation sites (Fig. 5, B and C). Previous work suggested only trNK cells to show evidence of proliferation (17). Ex vivo activation of isolated uNK cells with IL-15 further confirmed a selective defect in the NK1.1+/CD122+ NK cells showing reduced ERK (but not STAT5) phosphorylation, whereas pERK in DBA+NK1.1/CD122+ NK cells was unaffected (Fig. 5, D and E).

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.

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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.

We then assessed whether the remaining uNK cells in Gab3KO mice are different in terms of their transcriptional program compared with WT uNK cells. Specifically, we isolated gd8.5 uNK cells from implantation sites from pregnant WT and Gab3KO females based on the expression of CD45.2+/CD3/CD122+ (fig. S5a) and performed RNA sequencing (RNA-seq) analysis. Differential expression analysis identified a total of 613 genes with a P value of <0.05 that were differentially expressed between Gab3KO and WT uNK cells (249 genes were up and 364 genes were down in Gab3KO uNK cells) (fig. S5B). Gene enrichment analyses (ToppFun) identified gene sets with reduced expression in Gab3KO uNK cells that were involved in (i) eukaryotic translation, (ii) citric acid and respiratory electron transport, and (iii) NK cell–mediated cytotoxicity (fig. S5, C to F). The reduced expression of gene sets in translation/elongation and metabolism are consistent with the signaling defects and reduced Rps6 phosphorylation observed in IL-15–stimulated NK cells (Fig. 3D) and suggest a reduced uNK cell growth/expansion in the DB. In addition, Gab3KO uNK cells exhibited significant changes in the RNA expression of surface NK cell receptors and noncytoxic granzymes such as GzmdGzmeGzmFGzmGand GzmN (Fig. 5, F and G, and fig.S5F). These granzymes are thought to be involved in tissue remodeling and are highly expressed in trNK cells (15). Thus, their reduced expression is consistent with the overall reduction in trNK cells observed in Gab3KO implantation sites. Last, a modest but overall reduced level of chemokine (C-C motif) ligand 1 (Ccl1), migration and invasion inhibitory protein (Miip), and macrophage migration inhibitory factor (Mif) were observed for uNK cells in the DB of Gab3KO females (fig. S5G). No differences were observed in Ifng expression levels and angiogenic factors (i.e., VegfaVegfband Vegfc) between WT and Gab3KO uNK cells. In contrast, uNK cells from Gab3KO mice exhibited a ~10-fold increase in the expression of gonadotropin-releasing hormone 1 (fig. S5G). The latter has previously been shown to be involved in implantation and is suggested to promote the attachment and invasion of trophoblasts into the endometrium (36).

Last, uNK cells from Gab3KO implantation sites showed an increased cytokine/IFN signature (fig. S5, H and I) that correlated with increased expression of Ifne measured in total RNA of implantation site (fig. S5J); however, Ifne was not produced by uNK cells directly, given that the RNA-seq revealed no significant IFN-ε expression in either WT or Gab3KO uNK cells. A number of these IFN signature genes are part of a highly enriched pathway in ILC1s involving antigen processing and presentation of peptide via major histocompatibility complex (MHC) class II (i.e., H2-Aa, H2-DMb1, H2-Ab1, and CD74) (15). Thus, the IFN signature observed may in part reflect the relative enrichment of ILC1s in our RNA-seq analysis. Together, these data suggest that loss of Gab3 results in a reduced expansion of trNK cells in the DB. Moreover, the reduction of trNK cells affects genes implicated with important effector functions in the development of the placenta during pregnancy.

Loss of Gab3 is associated with abnormally invasive trophoblast

The role of maternally derived uNK cells during pregnancy is evident at an early stage when they expand rapidly in the DB and initiate spiral artery remodeling as well as controlling the depth and pattern of interstitial and endovascular trophoblast invasion (263740). Nonetheless, their regulatory function in both successful and unsuccessful pregnancies remains poorly understood (4144). Given the marked impact on uNK cell expansion in Gab3-deficient mice, we investigated whether this correlated with changes in placental development, spiral artery remodeling, and trophoblast giant cell (TGC) infiltration in gd12.5 placentas of Gab3-deficient mice. Histological assessment of the placentas revealed no major differences in the size of the labyrinth and junctional zone, whereas a trend for reduced decidual depth was observed, particularly in the Gab3KO compared with WT (fig. S6, A to C). The spiral artery walls appeared to be heavily invaded by TGCs, as determined by staining with cytokeratin-7, a pan-trophoblast maker (Fig. 6A) (45), resulting in significantly smaller lumens and thicker artery walls. We therefore assessed spiral artery remodeling by measuring the vessel-to-lumen ratio (i.e., the area measurement of the whole vessel divided by the vessel lumen area) and assessed the frequency of TGCs in the spiral arteries by cytokeratin-7 staining. Gab3R27C and Gab3KO females exhibited a significantly higher vessel-to-lumen ratio (Fig. 6B) while also showing a significant increase in the number of TGCs within spiral arteries, indicating that the increased vessel-to-lumen ratio is due to the invading cytokeratin-7+ TGCs (Fig. 6C).

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, Gab3R27CGab3KONK 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.

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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, Gab3R27CGab3KONK 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 sig nif icant. (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.

These observations extended to gd18.5, at which point we observed invasion of TGCs into the uterine wall (fig. S7A). In addition, we observed a significant expansion of trophoblasts in the labyrinth and junctional zone of both Gab3R27C and Gab3KO placentas leading to an overall increase in the depth of labyrinth, junctional zone, and overall placenta (fig. S7, B to D). In the DB, we observed discontinuous trophoblast invasion, with select areas showing deep invasion reaching the uterine wall in Gab3R27C and Gab3KO placentas. By analyzing serial sections stained for proliferin, we next determined the smallest distance between areas of trophoblast invasion and the uterine wall for WT, Gab3R27Cand Gab3KO placentas. Placentas from Gab3KO mice but also Gab3R27C mutant mice had an average minimal distance of ~130 μm between trophoblasts and the uterine wall, whereas in WT placentas, the average minimal distance was ~490 μm, more than triple the distance observed in both Gab3R27C and Gab3KO placentas (fig. S7, A and E). Together, these data suggest that loss of Gab3 results in a failure to control trophoblast invasion throughout pregnancy, ultimately leading to continuous trophoblast invasion toward the uterine wall up to term.

To investigate the causal role of uNK cells in abnormal arterial remodeling and invasion of TGCs, we depleted uNK cells at gd0.5, gd3.5, and gd8.5 in pregnant WT females using depleting anti-NK1.1 antibodies (fig. S8). Subsequently, we examined both arterial remodeling and the presence of TGCs in the spiral arteries at gd12.5 using cytokeratin-7 immunohistochemistry. We observed a significant increase in the frequency TGCs in spiral arteries, whereas the vessel-to-lumen ratio was also similar to Gab3KO placentas (Fig. 6, A to C).

We next tested whether the adoptive transfer of WT NK cells into pregnant Gab3KO females could correct the increased trophoblast invasion in the maternal spiral arteries. Specifically, we isolated splenic NK cells from WT mice that were expanded in vitro with IL-15 for 4 days. Subsequently, 4 million NK cells were injected intravenously into pregnant Gab3KO females at gd9.5. The recipient females were euthanized 3 days after injection (gd12.5), and arterial remodeling and trophoblast invasion in the spiral arteries were assessed after hematoxylin and eosin and cytokeratin-7 staining. Intriguingly, both the vessel-to-lumen ratio and the average depth of cytokeratin-7+ cell layers in the spiral artery walls were reduced to WT levels (Fig. 6, A to C).

Together, these studies confirm the critical role of uNK cells in limiting trophoblast invasion and promoting spiral artery remodeling in the DB and reveal that Gab3 loss of function abrogates uNK cell expansion in the DB resulting in abnormal spiral artery remodeling and trophoblast infiltration that may ultimately impede a successful pregnancy outcome.

DISCUSSION

Although the functions of Gab family members Gab1 and Gab2 have been defined, the biological function of Gab3 has remained entirely elusive. Here, we identify Gab3 as a critical determinant of IL-2– and IL-15–induced activation of NK cells. Strikingly, consistent with a reduced ability to eradicate missing-self targets, we observed that Gab3 loss of function in vivo leads to a markedly impaired recognition and elimination of metastatic tumor cells. Moreover, our studies identify Gab3 to be important during pregnancy, because pregnant Gab3-deficient females exhibited reduced expansion of uNK cells, abnormal arterial remodeling, and increased trophoblast invasion into the DB. At the cellular/molecular level, we identified a clear defect in IL-2/IL-15–induced NK cell priming and expansion. Both cytokines share receptor signaling components including the IL2Rβ chain (CD122) and the common γ chain (CD132). Our studies reveal that Gab3 is selectively required for the activation of MAPK signaling pathways downstream of the IL-2/IL-15 receptor, whereas the STAT5 and PI3K signaling pathways were unaffected. As such, Gab3-deficient mice deviate from complete IL-15–deficient mice, retaining a relative normal peripheral development and survival of NK cells (284647).

A previous study performed an immune analysis of independently generated Gab3 KO mice, and the authors reported no obvious immune phenotype (5). However, the study limited immune analysis to macrophages and T cells, whereas no assessment of NK cell function was reported. The latter may be challenging when working with a mixed C57BL/6J/129SvJ background causing variable background-specific NK cell receptor expression. Our current studies involve two independent mouse models on the C57BL/6J background, a complete loss-of-function knockout and an ENU germ line carrying a hypomorph R27C missense mutation, both corroborating the critical role of Gab3 in NK cells, antitumor responses, and successful pregnancy.

Previous genome-wide association studies linked single-nucleotide polymorphisms (SNPs) in the promoter region of GAB3 with risk for human type 1 diabetes (4849), whereas recent studies link GAB3 overexpression with tumor cell growth (5051). The ExAC database (a database containing >80,000 WES from human patients/controls) reveals an absence of human complete loss-of-function mutations, suggesting that the latter may be incompatible with successful human survival or reproduction. Nonetheless, missense variants in human GAB3 have been identified, including a rare missense mutation affecting the same Arg27 (Arg→His: http://exac.broadinstitute.org/) that is affected in our ENU mouse model. Alignment studies show that the Arg residue is highly conserved in PH domains, and our studies suggest this residue to be critical for the interaction of the PH domain with phosphoinositides. Thus, our ENU mouse model not only presents a unique model to molecularly assess the role of the PH domain in PIP binding, it may also be predictive of a potential NK cell deficiency in humans carrying this R27H missense mutation.

The pregnancy complications in Gab3-deficient mice associated with invasive fetal trophoblasts in the uterine wall resemble the human condition placenta accreta. The latter presents a clinical condition when placental trophoblasts invade deeper into the uterine wall and fail to separate from the uterine wall. The incidence of placenta accreta has increased significantly over the past decades and is currently estimated to be 1 in ~530 births. Although this increase may be linked to the increase in cesarean delivery rate, immunological and/or genetic factors are likely to play a role as well. For instance, recent studies link the development of placenta accreta with significantly reduced numbers of uNK, whereas no significant association with the number of uterine scars was observed (5253). Whether the rare R27H missense mutation presents a hypomorph allele in humans that results in impaired uNK cell expansion like in the mouse model and/or is associated with the development of placenta accreta in humans remains to be addressed.

It is evident that uNK cells play a critical role in the DB development and determine the successful outcome of a pregnancy (112637394054). NK cell deficiencies have previously been linked with impaired regulation of trophoblast invasion in both mice and rats. Abnormal uNK cell numbers have been associated with human gestational complications, including recurrent spontaneous abortion and placenta accreta (5559). Moreover, IL-15–deficient murine models lack peripheral and uNK cells and display a robust invasive trophoblast phenotype and spiral artery remodeling similar to what is observed in Gab3-deficient mice, although significant differences exist between spiral artery remodeling in IL-15–deficient mice and rats (242660). Nonetheless, these studies corroborate the importance of IL-15 in placental development and pregnancy. Although Gab3-deficient mice exhibit reduced uNK cell numbers, they have relatively normal peripheral NK cell numbers in the blood circulation that have the potential to interact with fetal trophoblasts aligned within the spiral arteries. Given the impaired recognition and clearance of MHC-I–deficient target cells or tumor cells by Gab3-deficient NK cells, the possibility exists that this mechanism may contribute to the invasive trophoblast phenotype. Fetal trophoblasts express a unique pattern of predominantly non-MHC antigens and lack the expression of a number of classical MHC antigens (6163), thus representing a “missing-self” target. Whether peripheral NK cells within the maternal arteries play a role in containing trophoblast invasion is currently unclear and remains to be investigated.

Gab3 is expressed in a variety of cell types including CD8+ T cells and mast cells. Although we have not observed abnormal CD8+ T cell responses or changes in their development, we cannot exclude a role for these cell types in the abnormal pregnancy phenotype observed. Nonetheless, our studies establish an important role for Gab3 in uNK cell expansion and the control of decidual trophoblast invasion. Our “add-back” experiments, in which we transplant IL-15–stimulated WT NK cells in Gab3KO recipients at gd9.5, can largely overcome decidual trophoblast invasion as analyzed by gd12.5. These experiments point to an effective therapeutic approach to limit trophoblast invasion in the maternal spiral arteries that deserves further investigation.

Gab3 expression is predominantly observed in hematopoietic cells and is particularly high in NK cells. Our current study reveals a key role for Gab3 in IL-2/IL-15–induced NK cell expansion, specifically in mediating MAPK activation downstream of the IL-2/IL-15R.This pathway provides a new opportunity to selectively induce or repress NK cell function in settings of autoimmunity, antitumor immunity, transplantation, and other therapeutically important settings. Moreover, we posit that Gab3 loss of function is associated with increased trophoblast invasion and development of placenta accreta, a significant pregnancy complication in humans.

Acknowledgments: We thank M. Kofron for excellent support in conducting confocal imaging and microscopy studies. We also thank the staff of the CCHMC Core facilities including the research flow cytometry core, the Transgenic Animal and Genome Editing Core, and the pathology research core for providing exceptional technical support. We thank the Veterinary Services Core at CCHMC for their excellent animal care, and last, we thank the Genomics, Epigenomics and Sequencing Core at the University of Cincinnati for support in the RNA-seq studies. Funding: The research was funded by NIH grant P30 DK078392 (Integrative Morphology Core of the Cincinnati Digestive Disease Research Core Center), NIH grant R21 AI135380, and the Maren Foundation. Author contributions: A.S. performed and designed experiments, analyzed the data, and contributed in writing. K.C.S.L., K.L., and A.G. performed experiments. D.R.P., E.M.J., H.J., and A.B.H. helped with the experimental design and data analyses, and K.H. was involved in experimental design, data analyses, and writing of the manuscript. Competing interests: A.B.H. serves as a Scientific Advisory Board member for Hoth Therapeutics Inc. and has equity interests in Hoth Therapeutics Inc. and Chelexa BioSciences LLC. The other authors declare that they have no competing interests. Data and materials availability: The RNA-seq data is available from the Gene Expression Omnibus under accession number GSE133313. The Gab3 mutant mouse strains and the Gab3-GFP fusion proteins are available through a material transfer agreement with the Cincinnati Children’s Hospital Medical Center. Requests for mice should be directed to H.J., and requests for the fusion proteins should be directed to A.B.H. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.


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