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

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An Erratum to this article was published on 17 May 2013

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.

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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.

Author information

Authors and Affiliations

  1. Brain Body Institute, Department of Medicine, McMaster University, Hamilton, Canada

    Khalil Karimi & Huang Ho

  2. Laboratory for Experimental Feto-Maternal Medicine, Department of Obstetrics and Fetal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    María Emilia Solano, Kurt Hecher & Petra Clara Arck

  3. Department of Pathology and Molecular Medicine, Institute of Molecular Medicine and Health, McMaster University, Hamilton, ON, Canada

    Ali A. Ashkar

  4. Institute of Sports Medicine, Prevention and Rehabilitation, Paracelsus Medical University Salzburg, Salzburg, Austria

    Eva-Maria Steidle

  5. Brain Body Institute, Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, Canada

    Karen-Anne McVey Neufeld

  6. Brain Body Institute, Department of Pathology, McMaster University, Hamilton, Canada

    John Bienenstock

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  1. Khalil Karimi
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Correspondence to Khalil Karimi or Petra Clara Arck.

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Figure 1

Frequencies 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. (A–D) 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)

Figure 2

Absolute 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)

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)

Figure 4

Immunohistochemistry 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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Karimi, K., Solano, M.E., Ashkar, A.A. et al. Regulation of pregnancy maintenance and fetal survival in mice by CD27low mature NK cells. J Mol Med 90, 1047–1057 (2012). https://doi.org/10.1007/s00109-012-0872-5

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