A distant trophoblast-specific enhancer controls HLA-G expression at the maternal–fetal interface

HLA-G, a nonclassical HLA molecule uniquely expressed in the placenta, is a central component of fetus-induced immune tolerance during pregnancy. The tissue-specific expression of HLA-G, however, remains poorly understood. Here, systematic interrogation of the HLA-G locus using massively parallel reporter assay (MPRA) uncovered a previously unidentified cis-regulatory element 12 kb upstream of HLA-G with enhancer activity, Enhancer L. Strikingly, clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9-mediated deletion of this enhancer resulted in ablation of HLA-G expression in JEG3 cells and in primary human trophoblasts isolated from placenta. RNA-seq analysis demonstrated that Enhancer L specifically controls HLA-G expression. Moreover, DNase-seq and chromatin conformation capture (3C) defined Enhancer L as a cell type-specific enhancer that loops into the HLA-G promoter. Interestingly, MPRA-based saturation mutagenesis of Enhancer L identified motifs for transcription factors of the CEBP and GATA families essential for placentation. These factors associate with Enhancer L and regulate HLA-G expression. Our findings identify long-range chromatin looping mediated by core trophoblast transcription factors as the mechanism controlling tissue-specific HLA-G expression at the maternal–fetal interface. More broadly, these results establish the combination of MPRA and CRISPR/Cas9 deletion as a powerful strategy to investigate human immune gene regulation

Human Cytomegalovirus Inhibits Cytokine-Induced Macrophage Differentiation

Human cytomegalovirus (HCMV) infection in immunocompromised patients is associated with impaired immunological function. Blood monocytes, which differentiate into macrophage effector cells, are of central importance for immune reactivity. Here, we demonstrate that HCMV transiently blocks cytokine-induced differentiation of monocytes into functionally active phagocytic macrophages. In HCMV-treated cultures, the cells had classical macrophage markers but lacked the classical morphological appearance of macrophages and had impairments in migration and phagocytosis. Even at very low multiplicities of infection, macrophage differentiation was almost completely inhibited. The inhibition appeared to be mediated by a soluble factor released upon viral treatment of monocytes. Human immunodeficiency virus or measles virus had no such effects. These findings suggest that HCMV impairs immune function by blocking certain aspects of cytokine-induced differentiation of monocytes and demonstrate an efficient pathway for this virus to evade immune recognition that may have clinical implications for the generalized immunosuppression often observed in HCMV-infected patients

Purification of primary human placental leukocytes to study maternal-fetal interactions

Summary: Decidual leukocytes play key roles in maternal-fetal tolerance and immunity. Here, we present detailed methods to purify, culture, and functionally analyze human placental dNK, dTreg, dTem, and dMɸ from decidua parietalis, the maternal part of the placental membranes; decidua basalis, the maternal part of the placenta; and placental villi. These sites have high clinical relevance in the development of villitis and chorioamnionitis. This allows in-depth phenotypic and functional investigation of placental immune populations and their interactions with extravillous trophoblasts.For complete details on the use and execution of this protocol, please refer to Ikumi et al.,1 Tilburgs et al.,2 Salvany-Celades et al.,3 Crespo et al.,4 van der Zwan et al.5 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Utilizing primary HLA-G+ extravillous trophoblasts and HLA-G+ EVT-like cell lines to study maternal-fetal interactions

Summary: Fetal extravillous trophoblasts (EVTs) are the most invasive cells of the placenta and play a key role in modulating maternal immune responses. Here, we present a protocol to purify and culture human leukocyte antigen-G (HLA-G)+ EVTs. We describe steps for tissue dissection, tissue digestion, density gradient centrifugation, and cell sorting, and we provide detailed methods to determine EVT function. HLA-G+ EVTs are isolated from two maternal-fetal interfaces: the chorionic membrane and the basalis/villous tissue. This protocol allows in-depth functional investigation of maternal immune interactions with HLA-G+ EVTs.For complete details on the use and execution of this protocol, please refer to Papuchova et al. (2020),1 Salvany-Celades et al. (2019),2 Tilburgs et al. (2015),3 Tilburgs et al. (2015),4 van der Zwan et al. (2018).5 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Three Types of Functional Regulatory T Cells Control T Cell Responses at the Human Maternal-Fetal Interface

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