Homo sapiens
Protocols: Decidual samples were obtained from healthy women undergoing elective terminations in the first trimester of pregnancy. Written and informed consent was obtained in accordance with the guidelines in The Declaration of Helsinki 2000. Ethical approval for the use of these tissues was obtained from the Cambridge Local Research Ethics Committee (REC 04/Q0108/23). This now forms part of the Centre for Trophoblast Research biobank for the 'Biology of the Human Uterus in Pregnancy and Disease Tissue Bank' at the University of Cambridge. Overall biobank ethical approval is from the East of England–Cambridge Central Research Ethics Committee (17/EE/0151). Decidual mononuclear cells were routinely isolated by enzymatic digestion of decidual tissue, followed by density gradient centrifugation through Pancoll (PAN-Biotech). Decidual cell suspensions (10 million per flask) were cultured overnight to recover in 20 ml of RPMI 1640 with 10% heat-inactivated FCS and antibiotics, supplemented with 2.5 ng/ml IL-15 (Peprotech). This dose of IL-15 maintains uNK viability without activating the cells. After 12 hours, non-adherent cells (largely DL) were recovered by vigorous washing in complete medium. Uterine natural killer (uNK) cells were stimulated by incubation with 721.221 cells expressing either HLA-C*05:01 which is a C2 allotype (C2+HLA-C) or HLA-C*08:02 which is a C1 allotype (C1+HLA-C). HLA-C*08:02 differs from HLA-C*05:01 by only the C1 and C2 epitopes (amino acids at p77 and p80). All 221 cells were cultured in RPMI 1640 medium (Gibco) with antibiotics, and 10% fetal calf serum (FCS). A total RNA isolation kit (Norgen, 51800) was used to extract the nucleic acids. cDNA was generated using SMART-Seq v4 (Takara) and then adapter and index ligation was performed using the Nextera XT workflow (Illumina).