Journal of Molecular Medicine

, Volume 90, Issue 9, pp 1047–1057 | Cite as

Regulation of pregnancy maintenance and fetal survival in mice by CD27low mature NK cells

  • Khalil Karimi
  • María Emilia Solano
  • Ali A. Ashkar
  • Huang Ho
  • Eva-Maria Steidle
  • Karen-Anne McVey Neufeld
  • Kurt Hecher
  • John Bienenstock
  • Petra Clara Arck
Original Article

Abstract

Uterine natural killer (NK) cells are pivotal for successful mammalian reproduction. However, insights on functionally distinct subpopulations of uterine NK cells are largely elusive. Furthermore, translation of findings from murine into human pregnancy has been overshadowed by the limited number of mutual phenotypic NK cell markers. We here provide evidence that a subset of murine mature NK (mNK) cells present at the feto-maternal interface, identified as CD27lowDX5+CD3neg, is pivotal in maintaining pregnancy. This mNK subset has low cytotoxic capacity, produces higher amounts of interferon (IFN)-γ, and expresses functional homologs of human NK cell immunoglobulin-like receptors. We further show that bone marrow-derived CD27low mNK cells are selectively recruited to the uterus and ameliorate the rate of fetal loss when adoptively transferred into alymphoid RAG2−/−/γc−/− mice. Additionally, expression of CD27 is down-modulated on mNK cells upon migration to the uterus. Hence, we propose the existence of a regulatory mNK cell subset, which is licensed toward successful pregnancy maintenance at the fetomaternal interface in mice. As CD27low NK cells are also present in human decidua, the CD27low NK subset may provide a tool to foster translational research in reproduction, aiming to improve pregnancy outcome in humans.

Keywords

NK subsets CD27 Reproduction Alymphoid mice Cytotoxicity 

Notes

Acknowledgments

We thank Evelin Hagen for her assistance in generating some of the data. We also thank M. Ito (Central Institute for Experimental Animals, Kawasaki, Japan) for providing us with initial breeding pairs of Rag2−/−/γc−/− mice. This work was supported by research grants provided to P.C.A. by the German Research Foundation and the Excellence Initiative of the Hamburg Foundation for Research.

Conflict of interest disclosure

The authors declare no competing financial interests.

Supplementary material

109_2012_872_MOESM1_ESM.pdf (38 kb)
Figure 1Frequencies of DX5+CD3neg NK cells (A) and (early activated) CD69+DX5+CD3neg NK cells (B) in paraaortic lymph node (LN) and spleen in virgin mice and on gestation day (gd) 0.5, 3.5, 5.5, and 7.5, as analyzed by flow cytometry. (C) Frequencies of CD27lowDX5+CD3neg NK cells in bone marrow (BM) (C) and uterus (D), obtained from virgin mice at various stages of the estrous cycle. (AD) Data are shown as mean ± SEM. (E) Representative photomircographs of vaginal smears stained for hematoxylin and eosin (H&E). “Proestrous” was identified if small nucleated epithelial cells and few leucocytes could be detected; “Estrous” das defined by cornified anucleated epithelial cells; “Metaestrous” was assigned when many leukocy tes and few cornified anucleated epithelial cells were visible; “Diestrous” was classified as many leucocytes and few nucleated epithelial cells. (PDF 38 kb)
109_2012_872_MOESM2_ESM.pdf (86 kb)
Figure 2Absolute cells numbers of CD27lowNK cells and CD27+NK cells in uterus and LN upon adoptive transfer of CD27+-enriched NK cell. (A) Absolute numbers of CD27low or CD27+NK cells in respective tissues 1 h upon transfer of CD27+NK cells. (B) Absolute numbers of CD27low or CD27+NK cells in respective tissues 40 h upon transfer of CD27+ NK cells. Data are shown as mean ± SEM. (C) Dot plots of CD11c and CD27 expression on cells derived from uterus of LN on gd 7.5 of DBA/2J-mated CBA/J females. (D) Summary of antibody clones used f or f low cytometric analyses. (PDF 85 kb)
109_2012_872_MOESM3_ESM.pdf (243 kb)
Figure 3(A) Frequencies of IFN-γ+cells among different NK cell parent populations (DX5+ vs. DX5neg in CD27low NKp46+ cells) in BM- and uterus-derived cells. Cell were harvested from DBA/2J-mated CBA/J females and analyzed by flow cytometry on gd 7.5. Data are shown as mean ± SEM. (B) Representative dot plots depicting the results shown in (A). Number of mice from which cells were obtained was n = 3. (C) Flow cytometric analysis was performed to reveal the frequency of CD27low cells among DX5+CD3neg in BM and uterus on gd 9.5 (n = 5). (D) Purity of the adoptively transferred either CD27lowDX5+CD3neg (bottom left plot) or CD27+DX5+CD3neg (bottom right plot) NK cell populations used f or adoptive transfer in Rag2−/−γc−/− mice. Dot plots in top row show the pre-sorting and post-first step of sorting cell distribution. € Frequency of DX5+CD3neg NK cells (F) and CD27lowDX5+CD3negNK cells in DBA/2Jmated CBA/J females, compared to Balb/c-mated CBA/J f emales. The flow cytometric analyses were performed on gd 9.5 and the number of females per mating combination was n = 3 in DBA/J matings and n = 3 in BALB/c matings. Data are shown as mean ± SEM. (PDF 242 kb)
109_2012_872_MOESM4_ESM.pdf (430 kb)
Figure 4Immunohistochemistry and histology analy ses: decidual-placental specimen were collected from syngeneically mated Rag2−/−/γc−/− females. The females had received an adoptive transfer of either CD27+DX5+CD3neg or CD27lowDX5+CD3neg cells on gd 7.5. Tissue specimen were harvested on gd 15.5, snap frozen in OCT and stored at −80°C until further use. Then, cryostat sections (8 μm) were prepared and slides treated with a blocking solution, followed by the incubation with the primary antibody (anti-PECAM-1, clone MEC13.3 or anti-CD34, clone RAM34, both purchased from BD Biosciences) at 4°C overnight. Sections were washed then incubated with a peroxidase complex (Vector). The signal was detected by incubating sections with diaminobenzidine (DAB; Sigma) and 0.05% hy drogen peroxide, f ollowed by light counterstaining with 0.1% Mey er’s hematoxylin. A routine H&E was performed on an additional slide for morphological orientation. (PDF 429 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Khalil Karimi
    • 1
  • María Emilia Solano
    • 2
  • Ali A. Ashkar
    • 3
  • Huang Ho
    • 1
  • Eva-Maria Steidle
    • 4
  • Karen-Anne McVey Neufeld
    • 5
  • Kurt Hecher
    • 2
  • John Bienenstock
    • 6
  • Petra Clara Arck
    • 2
  1. 1.Brain Body Institute, Department of MedicineMcMaster UniversityHamiltonCanada
  2. 2.Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Fetal MedicineUniversity Medical Center Hamburg-EppendorfHamburgGermany
  3. 3.Department of Pathology and Molecular Medicine, Institute of Molecular Medicine and HealthMcMaster UniversityHamiltonCanada
  4. 4.Institute of Sports Medicine, Prevention and RehabilitationParacelsus Medical University SalzburgSalzburgAustria
  5. 5.Brain Body Institute, Department of Psychiatry and Behavioural NeurosciencesMcMaster UniversityHamiltonCanada
  6. 6.Brain Body Institute, Department of PathologyMcMaster UniversityHamiltonCanada

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