TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy
- Division of Rheumatology, Department of Medicine, Washington University School of Medicine, United States
- Department of Obstetrics and Gynecology, Washington University School of Medicine, United States
Figures
Figure 1
Peripheral conventional NK (cNK) cells extravasate into the pregnant uterus and acquire a uterine tissue-resident NK (trNK) cell phenotype.
(A) Representative flow plots depicting the presence of non-vascular CD49b+ Eomes+ cNK cells within the gravid uterus of wildtype mice intravascularly labeled with anti-CD45.2 antibody in vivo at gestational days (gds) 6.5 and 14.5 (gd 6.5: C57BL/6 dams, n=3, implantation sites n=9; gd 14.5: C57BL/6 dams, n=3, implantation sites n=9). Gating strategy: live, single cells; CD3- CD19- CD45.1- CD45.2–PE-Cy7- CD45.2–Pacific Blue+ NK1.1+ NKp46+ cells. (B) Absolute cell counts of non-vascular CD49b+ Eomes+ cNK cells within the gravid uterus of wildtype mice at gds 6.5 and 14.5. (C) Concatenated flow plots of implantation sites showing that adoptively transferred cNK cells in the pregnant uterus of wildtype dams upregulate CD49a and downregulate CD49b by gd 10.5, acquiring a CD49a+ CD49b- Eomes+ phenotype characteristic of uterine trNK cells (C57BL/6 dams n=4). Here, 2.5×106 CD45.2+ CD3- CD19- NK1.1+ NKp46+ CD49b+ splenic cNK cells were adoptively transferred into pregnant C57BL/6-CD45.1 dams at gd 0.5, and the receptor profile of these cells was subsequently assessed at gd 10.5. Gating strategy: live, single cells; CD3- CD19- CD45.1- CD45.2–PE-Cy7- CD45.2–PE+ NK1.1+ NKp46+ cells. (D) Proportion of uterine innate lymphoid cell (ILC) subsets derived from adoptively transferred splenic cNK cells in the pregnant uterus of wildtype dams. Statistics were calculated using unpaired t tests with the Mann-Whitney correction. Error bars indicate SEM; ***p<0.001.
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Figure 1—source data 1
Raw data for Figure 1B showing absolute cell counts of non-vascular CD49b+ Eomes+ conventional NK (cNK) cells within the gravid uterus of wildtype mice at gestational days (gds) 6.5 and 14.5.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig1-data1-v1.xlsx
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Figure 1—source data 2
Raw data for Figure 1D showing absolute cell counts and proportions of uterine innate lymphoid cells (ILCs) derived from adoptively transferred splenic conventional NK (cNK) cells in the pregnant uterus of wildtype dams.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig1-data2-v1.xlsx
Figure 2 with 1 supplement
Loss of TGF-β signaling in Ncr1-expressing cells impairs uterine tissue-resident NK (trNK) cell differentiation in pregnant mice.
(A) Representative histograms depicting TGF-β receptor II expression on splenic NK cells from virgin TGF-βRIINcr1∆ and wildtype mice, as well as splenic and uterine NK cell subsets from pregnant wildtype mice at gestational day (gd) 10.5 (virgin TGF-βRIINcr1∆ mice, n=2; virgin mice: C57BL/6, n=5; gd 10.5: C57BL/6 dams, n=8, implantation sites n=8). MFI, median fluorescent intensity. Gating strategy: live, single cells; CD3- CD19- CD45.1- CD45.2+ NK1.1+ NKp46+ cells. (B) Representative flow plots showing the expression of CD49a, CD49b, and Eomes across innate lymphoid cell (ILC) subsets in the pregnant spleens of littermate control and TGF-βRIINcr1∆ dams at gd 6.5 (littermates, n=6; TGF-βRIINcr1∆, n=5). Gating strategy: live, single cells; CD3- CD19- CD45.1- CD45.2+ NK1.1+ NKp46+ cells. (C) Absolute cell counts of CD49a+ Eomes+ trNK cells, CD49a+ Eomes- type 1 ILCs (ILC1s), and CD49b+ Eomes+ conventional NK (cNK) cells in the spleens of pregnant littermate control and TGF-βRIINcr1∆ dams at gd 6.5. (D) Representative flow plots showing the expression of CD49a, CD49b, and Eomes across ILC subsets in the gravid uterus of littermate control and TGF-βRIINcr1∆ dams at gd 6.5 (littermates, n=6, implantation sites n=54; TGF-βRIINcr1∆, n=5, implantation sites n=15). (E) Absolute cell counts of CD49a+ Eomes+ trNK cells, CD49a+ Eomes- ILC1s, and CD49b+ Eomes+ cNK cells in the gravid uterus of littermate control and TGF-βRIINcr1∆ dams at gd 6.5. Statistics were calculated using unpaired t tests with the Mann-Whitney correction. Error bars indicate SEM; ***p<0.001; and ****p<0.0001.
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Figure 2—source data 1
Raw data for Figure 2D measuring TGF-β receptor II expression (MFI) on splenic NK cells from virgin TGF-βRIINcr1∆ and wildtype mice, as well as splenic and uterine NK cell subsets from pregnant wildtype mice at gestational day (gd) 10.5.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig2-data1-v1.xlsx
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Figure 2—source data 2
Raw data for Figure 2C and E showing absolute cell counts of CD49a+ Eomes+ tissue-resident NK (trNK) cells, CD49a+ Eomes- type 1 innate lymphoid cells (ILC1s), and CD49b+ Eomes+ conventional NK (cNK) cells in the gravid uterus and spleens of pregnant littermate control and TGF-βRIINcr1∆ dams at gestational day (gd) 6.5.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Ncr1iCre expression does not alter uterine innate lymphoid cell (ILC) composition or early pregnancy outcomes.
(A) Representative flow plots showing the expression of CD49a, CD49b, and Eomes across ILC subsets in the spleens of wildtype C57BL/6 CD45.1 and Ncr1iCre dams at gestational day (gd) 6.5 (C57BL/6 CD45.1, n=2; Ncr1iCre, n=3). Gating strategy: live, single cells; CD3- CD19- CD45.1- CD45.2+ NK1.1+ NKp46+ cells. (B) Absolute cell counts of CD49a+ Eomes+ tissue-resident NK (trNK) cells, CD49a+ Eomes- ILC1s, and CD49b+ Eomes+ conventional NK (cNK) cells in the spleens of pregnant wildtype C57BL/6 CD45.1 and Ncr1iCre dams at gd 6.5. (C) Representative flow plots showing the expression of CD49a, CD49b, and Eomes across ILC subsets in the gravid uterus of wildtype C57BL/6 CD45.1 and Ncr1iCre dams at gd 6.5 (C57BL/6 CD45.1, n=3, implantation sites n=21; Ncr1iCre, n=3, implantation sites n=21). (D) Absolute cell counts of CD49a+ Eomes+ trNK cells, CD49a+ Eomes- ILC1s, and CD49b+ Eomes+ conventional NK (cNK) cells in the gravid uterus of wildtype C57BL/6 CD45.1 and Ncr1iCre dams at gd 6.5. (E) Number of implantation sites and (F) fetal resorption rates in Ncr1iCre dams at gd 6.5 were comparable to those measured in wildtype C57BL/6 CD45.1 dams (C57BL/6 CD45.1, n=3; Ncr1iCre dams, n=3). Resorption rates (RR) were calculated as: RR(%) = (number of resorbed implantation sites/number of total implantation sites) × 100. Statistics were calculated using unpaired t tests with the Mann-Whitney correction.
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Figure 2—figure supplement 1—source data 1
Raw data for Figure 2—figure supplement 1B and D showing absolute cell counts of CD49a+ Eomes+ tissue-resident NK (trNK) cells, CD49a+ Eomes- type 1 innate lymphoid cells (ILC1s), and CD49b+ Eomes+ conventional NK (cNK) cells in the spleens and gravid uterus of pregnant wildtype C57BL/6 CD45.1 and Ncr1iCre dams at gestational day (gd) 6.5.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
Raw data for Figure 2—figure supplement 1E and F showing the number of implantation sites and fetal resorption rates at gestational day (gd) 6.5 in wildtype C57BL/6 CD45.1 dams and Ncr1iCre dams.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig2-figsupp1-data2-v1.xlsx
Figure 3
Impaired TGF-β-dependent uterine tissue-resident NK (trNK) cell differentiation leads to adverse pregnancy outcomes characterized by reduced litter sizes.
(A) Number of live pups at first parturition from littermate control and TGF-βRIINcr1∆ dams (littermates, n=7; TGF-βRIINcr1∆, n=7). (B) Pup birth weight in grams (g) from pups birthed by littermate control and TGF-βRIINcr1∆ dams (littermates, n=68; TGF-βRIINcr1∆, n=31). (C) Gestational period in days for littermate control and TGF-βRIINcr1∆ dams (littermates, n=7; TGF-βRIINcr1∆, n=7). Statistics were calculated using unpaired t tests with the Mann-Whitney correction. Error bars indicate SEM; ***p<0.001.
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Figure 3—source data 1
Raw data for Figure 3A–C showing litter size, pup birth weight (g), and gestation period in littermate control and TGF-βRIINcr1∆ dams.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig3-data1-v1.xlsx
Figure 4
TGF-β-dependent uterine tissue-resident NK (trNK) cell differentiation required for proper spiral artery remodeling and fetal survival.
(A) At gestational day (gd) 10.5, TGF-βRIINcr1∆ dams had fewer implantation sites than littermate control dams (littermates, n=6; TGF-βRIINcr1∆, n=7). (B) Fetal resorption rates in littermate control and TGF-βRIINcr1∆ dams at gd 10.5, showing increased resorptions in conditional knockout dams (littermates, n=6; TGF-βRIINcr1∆, n=7). Resorption rates (RR) were calculated as: RR(%) = (number of resorbed implantation sites/number of total implantation sites) × 100. (C) Representative images of gd 10.5 decidual spiral arteries from three littermate control and three TGF-βRIINcr1∆ dams stained with Masson’s Trichrome (littermates, n=6; TGF-βRIINcr1∆, n=7; scale bar, 100 μm). (D) Spiral artery wall-to-lumen ratio at gd 10.5 implantation sites from littermate control and TGF-βRIINcr1∆ dams. Increased wall-to-lumen ratio in TGF-βRIINcr1∆ dams indicative of impaired spiral artery remodeling (littermates, n=6, decidual spiral arteries n=257; TGF-βRIINcr1∆, n=7 decidual spiral arteries n=305). Statistics were calculated unpaired t tests with the Mann-Whitney correction. Error bars indicate SEM; **p<0.01; and ****p<0.0001.
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Figure 4—source data 1
Raw data for Figure 4A and B showing implantation site numbers and fetal resorption rates at gestational day (gd) 10.5 in littermate control and TGF-βRIINcr1∆ dams.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig4-data1-v1.xlsx
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Figure 4—source data 2
Raw data for Figure 4D showing spiral artery wall-to-lumen ratio at gestational day (gd) 10.5 implantation sites from littermate control and TGF-βRIINcr1∆ dams.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig4-data2-v1.xlsx
Figure 5
Adoptive transfer of splenic conventional NK (cNK) cells partially reconstitutes uterine tissue-resident NK (trNK) cells and rescues mid-gestational pregnancy defects in TGFBRIINcr1∆ dams.
(A) Representative flow plots showing the expression of CD49a, CD49b, and Eomes across CD45.1+ innate lymphoid cell (ILC) subsets in gestational day (gd) 10.5 implantation sites of TGF-βRIINcr1∆ dams reconstituted with splenic CD45.1+ conventional NK (cNK) cells. Briefly, 3.0×106 splenic CD45.1+ cNK cells were adoptively transferred into TGF-βRIINcr1∆ dams at gd 0.5. By gd 10.5, a portion of adoptively transferred cNK cells in the pregnant uterus of TGF-βRIINcr1∆ dams upregulated CD49a and downregulated CD49b, acquiring a CD49a+ CD49b- Eomes+ phenotype characteristic of uterine trNK cells (reconstituted (R)–TGF-βRIINcr1∆, n=3, implantation sites, n=25). Gating strategy: live, single cells; CD3- CD19- CD45.1+ CD45.2- NK1.1+ NKp46+ cells. (B) Absolute numbers of CD45.1+ ILC subsets in gd 10.5 implantation sites from reconstituted TGF-βRIINcr1∆ dams (R–TGF-βRIINcr1∆, n=3, implantation sites, n=25). (C) Proportion of uterine CD45.1+ ILC subsets derived from adoptively transferred splenic cNK cells in gd 10.5 implantation sites from TGF-βRIINcr1∆ dams (R–TGF-βRIINcr1∆, n=3, implantation sites n=25). (D) Number of implantation sites and (E) fetal resorption rates in reconstituted TGF-βRIINcr1∆ dams at gd 10.5 were comparable to those measured in littermate control dams injected intravascularly with HBSS (HBSS–littermates, n=4; R–TGF-βRIINcr1∆, n=3). Resorption rates (RR) were calculated as: RR(%) = (number of resorbed implantation sites/number of total implantation sites) × 100. Statistics were calculated using unpaired t tests with the Mann-Whitney correction.
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Figure 5—source data 1
Raw data for Figure 5B and C showing the absolute numbers and proportion of CD45.1+ innate lymphoid cell (ILC) subsets in gestational day (gd) 10.5 implantation sites from reconstituted TGF-βRIINcr1∆ dams.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig5-data1-v1.xlsx
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Figure 5—source data 2
Raw data for Figure 5D and E showing the number of implantation sites and fetal resorption rates of reconstituted TGF-βRIINcr1∆ dams at gestational day (gd) 10.5 and littermate control dams injected intravascularly with HBSS.
- https://cdn.elifesciences.org/articles/109878/elife-109878-fig5-data2-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | CD3e American Hamster Monoclonal, APC-eFluor 780 (clone 145-2C11) | Invitrogen | Cat Number: 47-0031-82 RRID:AB_11149861 | 1:100 |
| Antibody | CD19 Rat Monoclonal, APC-eFluor 780 (1D3) | Invitrogen | Cat Number: 47-0193-82 RRID:AB_10853189 | 1:100 |
| Antibody | CD45.2 Mouse Monoclonal, Alexa Fluor 700 (104) | Invitrogen | Cat Number: 56-0454-82 RRID:AB_657752 | 1:100 |
| Antibody | CD45.2 Mouse Monoclonal, PE-Cy7 (104) | Invitrogen | Cat Number: 25-0454-82 RRID:AB_2573350 | 1:100 |
| Antibody | CD45.1 Mouse Monoclonal, APC-eFluor 780 (A20) | Invitrogen | Cat Number: 47-0453-82 RRID:AB_1582228 | 1:100 |
| Antibody | CD49b Rat Monoclonal, FITC (DX5) | Invitrogen | Cat Number: 11-5971-85 RRID:AB_465328 | 1:50 |
| Antibody | Eomes Rat Monoclonal PE-Cy7 (Dan11mag) | Invitrogen | Cat Number: 25-4875-82 RRID:AB_2573454 | 1:85 |
| Antibody | Eomes Rat Monoclonal APC (Dan11mag) | Invitrogen | Cat Number: 17-4875-82 RRID:AB_2866428 | 1:85 |
| Antibody | NKp46 Rat Monoclonal, PerCP-ef710 (29A1.4) | Invitrogen | Cat Number: 46-3351-82 RRID:AB_1834441 | 1:100 |
| Antibody | CD45.1 Mouse Monoclonal, Alexa Fluor 700 (A20) | BioLegend | Cat Number: 110724 RRID:AB_493733 | 1:100 |
| Antibody | NK1.1 Mouse Monoclonal, Brilliant Violet 650 (PK136) | BioLegend | Cat Number: 108736 RRID:AB_2563159 | 1:100 |
| Antibody | CD49a Armenian Hamster Monoclonal BV421 (Ha31/8) | BD Biosciences | Cat Number: 740046 RRID:AB_2739815 | 1:50 |
| Antibody | CD49a Hamster PE (Ha31/8) | Fisher Scientific | Cat Number: 562115 RRID:AB_11153117 | 1:50 |
| Antibody | TGF-βRII Polyclonal Goat (Biotinylated) | R&D Systems | Cat Number: BAF532 RRID:AB_2222455 | 1:100 |
| Strain, strain background (Mus musculus) | C57BL/6 | Charles River Laboratories | Stock Number 665 RRID:IMSR_CRL:027 | |
| Strain, strain background (Mus musculus) | B6.SJL-PtprcaPecpcb/BoyJ | Charles River Laboratories | Stock Number 664 RRID:IMSR_CRL:564 | |
| Strain, strain background (Mus musculus) | B6;129-Tgfbr2tm1Karl/J | The Jackson Laboratory | Strain: 012603 RRID:IMSR_JAX:012603 | |
| Software | FlowJo Software 10.8.2 | BD Biosciences | 10.8.2 RRID:SCR_008520 | |
| Software | Prism 10.4.1 | GraphPad Software | 10.4.1 RRID:SCR_002798 | |
| Software | NIS-Elements Viewer NDP.view2 4.11.0 Imaging Software | Nikon Microscope | 4.11.0 RRID:SCR_025177 |
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TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy
eLife 15:RP109878.
https://doi.org/10.7554/eLife.109878.3
