The genotype of the NK cell receptor, KIR2DL4, influences INFγ secretion by decidual natural killer cells

Molecular Human Reproduction, Vol.15, No.8 pp. 489– 497, 2009 Advanced Access publication on June 9, 2009 doi:10.1093/molehr/gap039 ORIGINAL RESEARCH The genotype of the NK cell receptor, KIR2DL4, influences INFg secretion by decidual natural killer cells 1 Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North Seattle, Seattle, WA 98109, Australia 2School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, WA 6009, Australia 3Department of Clinical Immunology and Immunogenetics, PathWest, Royal Perth Hospital, Wellington St, Perth, WA 6847, Australia 4Department of Pathology, PathWest, King Edward Memorial/Princess Margaret Hospitals, Subiaco, WA 6008, Australia 5 Correspondence address. Tel: þ61-8-9224-2899; Fax: þ61-8-9224-2920; E-mail: abstract: Natural killer (NK) cells are the predominant leukocyte in first trimester decidua and play a role in vascular remodelling through interferon gamma (IFNg) secretion. Membrane expression of the killer immunoglobulin-like receptor (KIR) KIR2DL4 on peripheral blood NK (pNK) cells is controlled by the 9A/10A transmembrane genetic polymorphism. On peripheral NK cells (pNK), KIR2DL4 can only be detected on the membrane of cells from individuals with at least one copy of the 10A allele and ligation of KIR2DL4 results in IFNg secretion. In this study, we assessed KIR2DL4 expression and IFNg secretion as a result of KIR2DL4 ligation, by decidual NK (dNK) cells. The 9A/10A transmembrane polymorphism was shown to control KIR2DL4 expression by dNK, as previously shown for pNK cells. Freshly isolated dNK cells from subjects with at least one 10A allele expressed KIR2DL4 whereas those from 9A homozygous subjects did not. Although freshly isolated dNK did not secrete IFNg in response to KIR2DL4 ligation regardless of KIR2DL4 genotype, activation by in vitro culture with IL-2 enabled dNK cells from individuals with at least one 10A allele, but not those without a 10A allele, to secrete IFNg in response to KIR2DL4 ligation. This study confirms that expression of KIR2DL4 by dNK is dependent on the 9A/10A polymorphism and that this polymorphism influences IFNg secretion by dNK cells. Key words: CD158d / cell surface molecules / cytokines / KIR2DL4 / natural killer cells Introduction NK cells are the predominant lymphoid cell population at the site of implantation, and constitute 70% of the infiltrating CD45þ leukocytes in first trimester decidua (Moffett-King, 2002). Receptors that enable decidual NK (dNK) cells to recognize HLA class I molecules include the lectin-like family (CD94/NKG2), leukocyte Ig-like receptor-1, and the immunoglobulin-like receptor (KIR) family (Bashirova et al., 2006). It has been postulated that activation of dNK cells via these receptors may be an important event in human placentation. Indeed, particular combinations of maternal KIR and fetal HLA-C alleles have been shown to predispose to pre-eclampsia and recurrent miscarriage (Hiby et al., 2004, 2008), syndromes characterized by shallow placentation. The immunological events leading to pathological syndromes of pregnancy such as pre-eclampsia are not well-defined. In the mouse, it has been demonstrated that interferon gamma (IFNg) secretion by dNK cells is an important event in placentation (Croy † et al., 2002) although there is as yet no evidence of a role for IFNg in humans. As ligation of KIR2DL4 on peripheral blood NK (pNK) cells by anti-KIR2DL4 monoclonal antibody (mAb) results in IFNg secretion (Rajagopalan et al., 2001; Goodridge et al., 2007), KIR2DL4 expression on dNK cells may play a role in placentation. Little is known about the expression of KIR2DL4 on dNK cells, although it has been detected on such cells using a polyclonal antibody in a single report (Ponte et al., 1999). In contrast, much is now known about the expression of KIR2DL4 on pNK cells. KIR2DL4 is only expressed on the surface of freshly isolated CD56bright pNK cells, although it can be induced on CD56dim pNK cells by culture (Goodridge et al., 2003; Kikuchi-Maki et al., 2003). Based on the presence of a sequence of 9 or 10 adenine residues at the end of exon 6, two common allele groups, 9A and 10A, each having a gene frequency of approximately 0.5, have been described (Witt et al. 2000, 2002). The deletion of one adenine in 9A alleles results in a frame-shift and the production of either a protein with a truncated cytoplasmic tail These authors contributed equally to the work described in this manuscript. & The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: J.P. Goodridge 1,†, L.J. Lathbury 2,†, E. John 3, A.K. Charles 2,4, F.T. Christiansen 2,3, and C.S. Witt3,5 490 Materials and Methods Cells Peripheral blood and placental tissue samples were obtained with consent from 56 donors undergoing elective first trimester termination of normal pregnancy at the Marie Stopes International Clinic (Perth, Australia). Ethics approval was obtained from the Human Ethics Committee, University of Western Australia. pNK cells were isolated from blood samples by Ficoll density gradient centrifugation with RosetteSep human NK cell enrichment cocktail (Stem Cell Technologies, Vancouver, Canada). Decidual tissue was macroscopically dissected from non-decidual tissue, and mononuclear cells were isolated by mechanical disruption, followed by filtration through a 70 mm sieve and centrifugation over Ficoll. dNK cells were then obtained by incubating the decidual mononuclear cells with the RosetteSep human NK cell enrichment antibody cocktail and 200 ml peripheral blood (g-irradiated to 30 Gy to prevent proliferation of contaminating peripheral NK cells) from the tissue donor as a source of red blood cells (a specific ratio between mononuclear cells and red blood cells is required for the purification of NK cells via the RosetteSep procedure), followed by a second centrifugation over Ficoll. Due to limited cell numbers, not all of the assays described below were performed on all samples. Antibodies Expression of KIR2DL4 on the cell surface and stimulation of NK cells through KIR2DL4 was examined using several different anti-KIR2DL4 mAbs. MAb #33 (IgG1) and #64 (IgM), were both kindly provided by Dr E. Long and S. Rajagopalan (Laboratory of Immunogenetics, NIH, MD, USA). Mab mAb #2238 was purchased from R&D Systems (IgG2a, clone 181703, R&D Systems, USA). Isotype controls included IgG1 and IgM (Beckman Coulter, Australia) and IgG2a (R&D Systems). Leaf-purified anti-human CD16 (IgG1, clone 3G8, Biolegend, USA) and an equivalent IgG1 isotype control (clone MG1-45, Biolegend) were used for the in vitro stimulation of NK cells. The secondary antibody used with mAb #33 to detect KIR2DL4 was FITC-conjugated Affinipure F(Ab’)2 fragment goat anti-mouse IgG (Fcg specific, Beckman Coulter, Australia). The secondary antibody used with mAb #64 was FITC- (...truncated)