CD8+ T cells are critical in the balance between fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that Tim-3+PD-1+CD8+ T cells from decidua greatly outnumbered those from peripheral blood during human early pregnancy. Co-culture of trophoblasts with CD8+ T cells upregulated PD-1+ and/or Tim-3+ immune cells. Furthermore, the population of CD8+ T cells co-expressing PD-1 and Tim-3 was enriched within the intermediate memory subset in decidua. This population exhibited high proliferative activity and Th2-type cytokine producing capacity. Blockade of Tim-3 and PD-1 resulted in decreased in vitro proliferation and Th2-type cytokine production while increased trophoblast killing and IFN-γ producing capacities of CD8+ T cells. Pregnant CBA/J females challenged with Tim-3 and/or PD-1 blocking antibodies were more susceptible to fetal loss, which was associated with CD8+ T-cell dysfunction. Importantly, the number and function of Tim-3+PD-1+CD8+ T cells in decidua were significantly impaired in miscarriage. These findings underline the important roles of Tim-3 and PD-1 pathways in regulating decidual CD8+ T-cell function and maintaining normal pregnancy.

Rheb is a Ras family GTPase, which binds to and activates mammalian target of rapamycin complex 1 (mTORC1) when GTP loaded. Recently, cancer genome sequencing efforts have identified recurrent Rheb Tyr35Asn mutations in kidney and endometrial carcinoma. Here we show that Rheb-Y35N causes not only constitutive mTORC1 activation, but sustained activation of the MEK-ERK pathway in a TSC1/TSC2/TBC1D7 protein complex and mTORC1-independent manner, contributing to intrinsic resistance to rapamycin. Rheb-Y35N transforms NIH3T3 cells, resulting in aggressive tumor formation in xenograft nude mice, which could be suppressed by combined treatment with rapamycin and an extracellular signal-regulated kinase (ERK) inhibitor. Furthermore, Rheb-Y35N inhibits AMPKα activation in response to nutrient depletion or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), leading to attenuated phosphorylation of BRAF-S729 and retained mitogen-activated protein kinase (MAPK) activation. Finally, we demonstrate that Rheb-WT can bind AMPK to facilitate AMPK activation, whereas Rheb-Y35N competitively binds AMPK, impairing AMPK phosphorylation. In summary, our findings indicate that Rheb-Y35N is a dominantly active tumor driver that activates both mTORC1 and MAPK to promote tumor growth, suggesting a combination of mTORC1 and MAPK inhibitors may be of therapeutic value in patients whose cancers sustain this mutation.

BackgroundLAIR-1 can be triggered by collagen and its intracellular ITIMs are crucial for the inhibitory signal to NK cells. In order to investigate the regulation mechanisms of LAIR-1/collagen on NK cells, we aimed to construct LAIR-1 over-expressed NK-92 cells with which STATs related analysis was performed.MethodsLentivirus-mediated transfection was used to construct LAIR-1 over-expressed NK-92 cells. The expression of LAIR-1 in the constructed NK-92 cells was identified by QPCR and Western blot analysis. Immunofluorescence test was also used to observe the efficiency of lentivirus-mediated expression of LAIR-1 in NK-92 cells. The LAIR-1 over-expressed NK-92 cells were cultured in the IL-2 free medium for 48 hours and then different concentrations of IL-2 were added into the culture system and the cells were collected after 30 mins. The expression of STAT1, p-STAT1, STAT4, and p-STAT4 were analysed by Western blot analysis.ResultsQPCR and Western blot analysis results showed the over-expressed LAIR-1 in NK-92 cells. Immunofluorescence result showed high efficiency of LAIR-1 lentiviral infection. Western blot results showed IL-2 could increase the phosphorylation of STAT1 in a dose-dependent manner, but not STAT4.ConclusionLAIR-1 over-expressed NK-92 cells were constructed successfully and the IL-2 treatment experiment showed different roles of STAT1 and STAT4 in the regulation of NK cells.AcknowledgementsSupported by a project grant from the Natural Science Foundation of Shandong Province (ZR2015JL027) and National Natural Science Foundation of China (81370730, 81571512, 81273200, and 31300751).