The dysregulation of Tim-3 signaling in dNK cells may be the main cause of miscarriages during early pregnancy. miscarriages and murine abortion-prone models. Moreover, T helper (Th)2-type cytokines were decreased and Th1-type cytokines were increased in Tim-3+ but not Tim-3? dNK cells from human and mouse miscarriages. Therefore, our results suggest that the Gal-9/Tim-3 signal is important for the regulation of dNK cell function, which is beneficial for the maintenance of a normal pregnancy. interferon (IFN)- secreted by the CD56brightCD27+ NK subset.18 Consequently, dNK cells have been shown to be a key regulatory subset that facilitates maternal-fetal immune tolerance. Abnormal changes in dNK cell number and function are found to be closely related with adverse pregnancy outcomes, such as recurrent spontaneous abortion. As a major contributor to innate immunity, NK cells also provide competent responses to infections, in addition to its immune regulatory actions during pregnancy. Maternal infections with bacterial or viral agents during pregnancy are associated with an increased incidence of miscarriage. Moderate inflammation is necessary to eliminate the outside invaders, but uncontrolled or exaggerated infection-triggered inflammation may be an important cause of pregnancy loss. Lipopolysaccharide (LPS) exposure resulting from microbial invasion of the endometrium has been linked to the risk of idiopathic miscarriage in a range of human and animal studies.19 Upon binding with its ligand Toll-like receptor (TLR)4, LPS initiates a robust inflammatory response, which is characterized by the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)- and IL-1, Kinesore which disturb the Th1/Th2 balance at the fetomaternal interface.20 dNK cells have also been reported to be targets of LPS, which can induce dNK cytotoxic activation.21 Therefore, as an active defender against microbial invasion, maintenance of a proper dNK cell inflammatory response is critical for a successful pregnancy during pathogen infection. T-cell immunoglobulin domain and FSCN1 mucin domain-containing molecule-3 (Tim-3), a newly defined regulatory factor, downregulates Th1 responses through transduction of apoptosis signaling by galectin-9 (Gal-9) engagement, suggesting that Tim-3 may modulate the Th1/Th2 balance.22,23 In addition to being expressed on activated T cells, Tim-3 is also constitutively expressed on cells of the innate immune system in both mice and humans. Increasing numbers of studies have shown that abnormal expression of Tim-3 is an important cause of autoimmune diseases, infections, transplantation problems and cancers. 24 Recent data have shown that NK cells can also be regulated by Tim-3. Tim-3 was found to act as a marker of activation or maturation of NK cells Kinesore and suppress NK cell cytotoxicity.25 In contrast, other reports have provided evidence that increased Tim-3 expression on NK cells leads to NK cell dysfunction in chronic virus infections, such as hepatitis B and HIV infection.26,27 Therefore, we propose that the regulatory effects of Tim-3 on NK cells are distinct in different immune microenvironments. However, Gal-9/Tim-3 signaling has not yet been found to regulate the function Kinesore of NK cells at the maternalCfetal interface. In the present study, we first detected the expression of Tim-3 in dNK cells and analyzed the cytokine profile and cytotoxicity of Tim-3+ and Tim-3? dNK cells. Then, we investigated the role of Gal-9/Tim-3 signaling in the shift from pNK cells to a dNK cell-like phenotype, as instructed by trophoblasts. Moreover, we observed the role of Gal-9/Tim-3 signaling in the cytokine production and cytotoxicity of dNK cells after LPS stimulation. Finally, the number of Tim-3+ dNK cells and the cytokine profile of Kinesore Tim-3+ and Tim-3? dNK cells in normal pregnancies and miscarriages were compared. Our data provide evidence that Gal-9/Tim-3 signaling plays an important physiological and pathological role in the regulation of dNK cell function during early pregnancy, which is also helpful for developing novel strategies to target Gal-9/Tim-3 signaling to promote maternalCfetal tolerance and prevent pregnancy loss. Materials and methods Human sample collection This study was approved by the Human Research Ethics Committee of Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China. All subjects gave informed written consent for the collection and study of.