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


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.


NK subsets CD27 Reproduction Alymphoid mice Cytotoxicity 



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 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. (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 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)
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 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)


  1. 1.
    Moffett A, Loke C (2006) Immunology of placentation in eutherian mammals. Nat Rev Immunol 6:584–594PubMedCrossRefGoogle Scholar
  2. 2.
    Ashkar AA, Di Santo LP, Croy BA (2000) Interferon γ contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation during normal murine pregnancy. J Exp Med 192:259–270PubMedCrossRefGoogle Scholar
  3. 3.
    Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I et al (2006) Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med 12:1065–1074PubMedCrossRefGoogle Scholar
  4. 4.
    Romero R, Kusanovic JP, Chaiworapongsa T, Hassan SS (2011) Placental bed disorders in preterm labor, preterm PROM, spontaneous abortion and abruption placentae. Best Pract Res Clin Obstet Gynaecol 25:313–327PubMedCrossRefGoogle Scholar
  5. 5.
    Hiby SE, Walker JJ, O’shaughnessy KM, Redman CW, Carrington M, Trowsdale J, Moffett A (2004) Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med 200:957–965PubMedCrossRefGoogle Scholar
  6. 6.
    Quenby S, Nik H, Innes B, Lash G, Turner M, Drury J, Bulmer J (2009) Uterine natural killer cells and angiogenesis in recurrent reproductive failure. Hum Reprod 24:45–54PubMedCrossRefGoogle Scholar
  7. 7.
    Rieger L, Segerer S, Bernar T, Kapp M, Majic M, Morr AK, Dietl J, Kämmerer U (2009) Specific subsets of immune cells in human decidua differ between normal pregnancy and preeclampsia—a prospective observational study. Reprod Biol Endocrinol 7:132PubMedCrossRefGoogle Scholar
  8. 8.
    Ljunggren HG, Malmberg KJ (2007) Prospects for the use of NK cells in immunotherapy of human cancer. Nat Rev Immunol 7:329–339PubMedCrossRefGoogle Scholar
  9. 9.
    Vivier E, Tomasellom E, Baratin M, Walzer T, Ugolini S (2008) Functions of natural killer cells. Nat Immunol 9:503–510PubMedCrossRefGoogle Scholar
  10. 10.
    Lanier LL, Le AM, Phillips JH, Warner NL, Babcock GF (1983) Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol 131:1789–1796PubMedGoogle Scholar
  11. 11.
    Ritson A, Bulmer JN (1987) Endometrial granulocytes in human decidua react with a natural-killer (NK) cell marker, NKH1. Immunology 62:329–331PubMedGoogle Scholar
  12. 12.
    Sedlmayr P, Schallhammer L, Hammer A, Wilders-Truschnig M, Wintersteiger R, Dohr G (1996) Differential phenotypic properties of human peripheral blood CD56dim and CD56bright natural killer cell subpopulations. Int Arch Allergy Immunol 110:308–313PubMedCrossRefGoogle Scholar
  13. 13.
    Walzer T, Blery M, Chaix J, Fuseri N, Chasson L, Robbins SH, Jaeger S, André P, Gauthier L, Daniel L et al (2007) Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46. Proc Natl Acad Sci USA 104:3384–3389PubMedCrossRefGoogle Scholar
  14. 14.
    Yadi H, Burke S, Madeja Z, Hemberger M, Moffett A, Colucci F (2008) Unique receptor repertoire in mouse uterine NK cells. J Immunol 181:6140–6147PubMedGoogle Scholar
  15. 15.
    Blois SM, Barrientos G, Garcia MG, Orsal AS, Tometten M, Cordo-Russo RI, Klapp BF, Santoni A, Fernández N, Terness P et al (2008) Interaction between dendritic cells and natural killer cells during pregnancy in mice. J Mol Med 86:837–852PubMedCrossRefGoogle Scholar
  16. 16.
    Lin Y, Zhong Y, Saito S, Chen Y, Shen W, Di J, Zeng S (2009) Characterization of natural killer cells in nonobese diabetic/severely compromised immunodeficient mice during pregnancy. Fertil Steril 91:2676–2686PubMedCrossRefGoogle Scholar
  17. 17.
    Lin Y, Li C, Shan B, Wang W, Saito S, Xu J, Di J, Zhong Y, Li DJ (2011) Reduced stathmin-1 expression in natural killer cells associated with spontaneous abortion. Am J Pathol 178:506–514PubMedCrossRefGoogle Scholar
  18. 18.
    Hayakawa Y, Smyth MJ (2006) CD27 dissects mature NK cells into two subsets with distinct responsiveness and migratory capacity. J Immunol 176:1517–1524PubMedGoogle Scholar
  19. 19.
    Vossen MT, Matmati M, Hertoghs KM, Baars PA, Gent MR, Leclercq G, Hamann J, Kuijpers TW, van Lier RA (2008) CD27 defines phenotypically and functionally different human NK cell subsets. J Immunol 180:3739–3745PubMedGoogle Scholar
  20. 20.
    Chiossone L, Chaix J, Fuseri N, Roth C, Vivier E, Walzer T (2009) Maturation of mouse NK cells is a 4-stage developmental program. Blood 113:5488–5496PubMedCrossRefGoogle Scholar
  21. 21.
    De Colvenaer V, Taveirne S, Delforche M, De Smedt M, Vandekerckhove B, Taghon T, Boon L, Plum J, Leclercq G (2011) CD27-deficient mice show normal NK-cell differentiation but impaired function upon stimulation. Immunol Cell Biol 89:803–811PubMedCrossRefGoogle Scholar
  22. 22.
    Colucci F, Caligiuri MA, Di Santo JP (2003) What does it take to make a natural killer? Nat Rev Immunol 3:413–425PubMedCrossRefGoogle Scholar
  23. 23.
    Hanna J, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, Katz G, Haimov-Kochman R, Fujii N, Yagel S et al (2003) CXCL12 expression by invasive trophoblasts induces the specific migration of CD16 human natural killer cells. Blood 102:1569–1577PubMedCrossRefGoogle Scholar
  24. 24.
    Carlino C, Stabile H, Morrone S, Bulla R, Soriani A, Agostinis C, Bossi F, Mocci C, Sarazani F, Tedesco F et al (2008) Recruitment of circulating NK cells through decidual tissues: a possible mechanism controlling NK cell accumulation in the uterus during early pregnancy. Blood 111:3108–3115PubMedCrossRefGoogle Scholar
  25. 25.
    Mazurier F, Fontanellas A, Salesse S, Taine L, Landriau S, Moreau-Gaudry F, Reiffers J, Peault B, Di Santo JP, de Verneuil H (1999) A novel immunodeficient mouse model—RAG2 × common cytokine receptor gamma chain double mutants—requiring exogenous cytokine administration for human hematopoietic stem cell engraftment. J Interferon Cytokine Res 19:533–541PubMedCrossRefGoogle Scholar
  26. 26.
    Girard MP, Tam JS, Assossou OM, Kieny MP (2010) The 2009 A (H1N1) influenza virus pandemic: a review. Vaccine 28:4895–4902PubMedCrossRefGoogle Scholar
  27. 27.
    de Fougerolles AR, Baines MG (1987) Modulation of the natural killer cell activity in regnant mice alters the spontaneous abortion rate. J Reprod Immunol 11:147–153PubMedCrossRefGoogle Scholar
  28. 28.
    Takeda M, Yamada H, Iwabuchi K, Shimada S, Naito M, Sakuragi N, Minakami H, Onoé K (2007) Administration of high-dose intact immunoglobulin has an anti-resorption effect in a mouse model of reproductive failure. Mol Hum Reprod 13:807–814PubMedCrossRefGoogle Scholar
  29. 29.
    Zhang J, Chen Z, Smith GN, Croy BA (2010) Natural killer cell triggered vascular transformation: maternal care before birth? Cell Mol Immunol 8:1–11PubMedCrossRefGoogle Scholar
  30. 30.
    Fu B, Wang F, Sun R, Ling B, Tian Z, Wei H (2011) CD11b and CD27 reflect distinct population and functional specialization in human natural killer cells. Immunology 33:350–359CrossRefGoogle Scholar
  31. 31.
    Boysen P, Storset AK (2009) Bovine natural killer cells. Vet Immunol Immunopathol 130:163–177PubMedCrossRefGoogle Scholar
  32. 32.
    Le Bouteiller P, Siewiera J, Casart Y, Aguerre-Girr M, El Costa H, Berrebi A, Tabiasco J, Jabrane-Ferrat N (2011) The human decidual NK-cell response to virus infection: what can we learn from circulating NK lymphocytes? J Reprod Immunol 88:170–175PubMedCrossRefGoogle Scholar
  33. 33.
    Westgaard IH, Berg SF, Vaage JT, Wang LL, Yokoyama WM, Dissen E, Fossum S (2004) Rat NKp46 activates natural killer cell cytotoxicity and is associated with FcepsilonRIgamma and CD3zeta. J Leukoc Biol 76:1200–1206PubMedCrossRefGoogle Scholar
  34. 34.
    Narni-Mancinelli E, Chaix J, Fenis A, Kerdiles YM, Yessaad N, Reynders A, Gregoire C, Luche H, Ugolini S, Tomasello E et al (2011) Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor. Proc Natl Acad Sci USA 108:18324–18329PubMedCrossRefGoogle Scholar
  35. 35.
    Carayannopoulos LN, Barks JL, Yokoyama M, Riley JK (2010) Murine trophoblast cells induce NK cell interferon-gamma production through KLRK1. Biol Reprod 83:404–414PubMedCrossRefGoogle Scholar
  36. 36.
    Oh MJ, Croy BA (2008) A map of relationships between uterine natural killer cells and progesterone receptor expressing cells during mouse pregnancy. Placenta 29:317–323PubMedCrossRefGoogle Scholar
  37. 37.
    Karimi K, Blois SM, Arck PC (2008) The upside of natural killers. Nat Med 14:1184–1185PubMedCrossRefGoogle Scholar
  38. 38.
    Salcedo M, Diehl AD, Olsson-Alheim MY, Sundbäck J, Van Kaer L, Kärre K, Ljunggren HG (1997) Altered expression of Ly49 inhibitory receptors on natural killer cells from MHC class I-deficient mice. J Immunol 158:3174–3180PubMedGoogle Scholar
  39. 39.
    Clark DA, Keil A, Chen Z, Markert U, Manuel J, Gorczynski RM (2003) Placental trophoblast from successful human pregnancies expresses the tolerance signaling molecule, CD200 (OX-2). Am J Reprod Immunol 50:187–195PubMedCrossRefGoogle Scholar
  40. 40.
    Zhang J, Wie H, Wu D, Tian Z (2007) Toll-like receptor 3 agonist induces impairment of uterine vascular remodeling and fetal losses in CBA x DBA/2 mice. J Reprod Immunol 74:61–67PubMedCrossRefGoogle Scholar
  41. 41.
    Lin Y, Zeng Y, Di J, Zeng S (2005) Murine CD200+ CK7+ trophoblasts in a poly (I:C)-induced embryo resorption model. Reproduction 130:529–537PubMedCrossRefGoogle Scholar
  42. 42.
    Hendriks J, Gravestein LA, Tesselaar K, van Lier RA, Schumacher TN, Borst J (2000) CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol 1:433–440PubMedCrossRefGoogle Scholar
  43. 43.
    Plaks V, Sapoznik S, Berkovitz E, Haffner-Krausz R, Dekel N, Harmelin A, Neeman M (2011) Functional phenotyping of the maternal albumin turnover in the mouse placenta by dynamic contrast-enhanced MRI. Mol Imaging Biol 13:481–492PubMedCrossRefGoogle Scholar
  44. 44.
    Hu B, He Y, Wu Y, Bao G, Liu H, Welniak LA, Murphy WJ (2010) Activated allogeneic NK cells as suppressors of alloreactive responses. Biol Blood Marrow Transplant 16:772–781PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Khalil Karimi
    • 1
    Email author
  • 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
    Email author
  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

Personalised recommendations