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During human pregnancy, proinflammatory responses in the placenta can cause severe fetal complications, including growth restriction, preterm birth, and stillbirth. Villitis of unknown etiology (VUE), an inflammatory condition characterized by the infiltration of maternal CD8+ T cells into the placenta, is hypothesized to be secondary to either a tissue rejection response to the haploidentical fetus or from an undiagnosed infection. In this study, we characterized the global TCR β-chain profile in human T cells isolated from placentae diagnosed with VUE compared with control and infectious villitis–placentae by immunoSEQ. Immunosequencing demonstrated that VUE is driven predominantly by maternal T cell infiltration, which is significantly different from controls and infectious cases; however, these T cell clones show very little overlap between subjects. Mapping TCR clones to common viral epitopes (CMV, EBV, and influenza A) demonstrated that Ag specificity in VUE was equal to controls and significantly lower than CMV-specific clones in infectious villitis. Our data indicate VUE represents an allograft response, not an undetected infection. These observations support the development of screening methods to predict those at risk for VUE and the use of specific immunomodulatory therapies during gestation to improve outcomes in affected fetuses.

The importance of fetal placental macrophages (Hofbauer cell [HCs]) is underscored by their appearance 18 d postconception and maintenance through term; however, how human HCs evolve during healthy pregnancy and how microenvironment and ontogeny impact phenotype and function remain unknown. In this study, we comprehensively classify human HCs ex vivo, interrogate phenotypic plasticity, and characterize antiviral immune responses through gestation. Activated HCs were abundant in early pregnancy and decreased by term; molecular signatures emphasize inflammatory phenotypes early in gestation. Frequency of HCs with regulatory phenotypes remained high through term. Furthermore, term HCs exhibited blunted responses to stimulation, indicating reduced plasticity. IFN-λ1 is a key placental IFN that appeared less protective than IFN-α, suggesting a potential weakness in antiviral immunity. Ligand-specific responses were temporally regulated: we noted an absence of inflammatory mediators and reduced antiviral gene transcription following RIG-I activation at term despite all HCs producing inflammatory mediators following IFN-γ plus LPS stimulation. Collectively, we demonstrate sequential, evolving immunity as part of the natural history of HCs through gestation.

Human cytomegalovirus (HCMV) is the leading cause of congenital viral infections. When a pregnant woman develops primary HCMV infection in early gestation, the risk of transplacental infection is ~40%. Studies have documented the presence of HCMV in placental macrophages (Hofbauer cells [HCs]) as a consequence of maternal viremia. However, the interplay between HCMV and placental macrophages (HCs) is poorly defined. With written informed consent, placentae were collected from 12 HIV-1/Hep B/HCMV seronegative women (>18 years). HCs were exposed to human HCMV (TB40/E). qPCR, FACS, ELISA, and Western blot analysis determined the expression of activation markers, cytokines, antiviral genes, and host proteins. Here we found that HCs isolated from early gestation (15–23 weeks) placentae are more susceptible to HCMV infection than HCs isolated from term (>39 weeks) placentae. HCMV infection of early gestation HCs led to a significant upregulation of activation markers, along with elevated secretion of proinflammatory cytokines, compared to HCMV-infected term HCs. In early gestation and term HC, HCMV rapidly induces the type I IFN pathway, leading to secretion of IFNs and strong induction of ISGs. However, HCMV diminishes type I IFN-mediated phosphorylation of STAT2, with early gestation HCs exhibiting the greatest level of inhibition. Our findings show that HCMV can subvert type I IFN signaling to support viral growth at the placenta, particularly during early gestation. Studying mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections.