The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular colocalization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 hours. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment.

Decidual NK cells (dNK) are the main lymphocyte population in early pregnancy decidual mucosa. Although dNK decrease during pregnancy, they remain present in decidual tissues at term. First trimester dNK facilitate trophoblast invasion, provide protection against infections, and were shown to have many differences in their expression of NKRs, cytokines, and cytolytic capacity compared with peripheral blood NK cells (pNK). However, only limited data are available on the phenotype and function of term pregnancy dNK. In this study, dNK from human term pregnancy decidua basalis and decidua parietalis tissues were compared with pNK and first trimester dNK. Profound differences were found, including: 1) term pregnancy dNK have an increased degranulation response to K562 and PMA/ionomycin but lower capacity to respond to human CMV-infected cells; 2) term pregnancy dNK are not skewed toward recognition of HLA-C, as was previously shown for first trimester dNK; and 3) protein and gene expression profiles identified multiple differences between pNK, first trimester, and term pregnancy dNK, suggesting term pregnancy dNK are a distinct type of NK cells. Understanding the role of dNK throughout pregnancy is of high clinical relevance for studies aiming to prevent placental inflammatory disorders as well as maternal-to-fetal transmission of pathogens.

Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR ␥ chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the ''conventional'' translocon organization for TCR genes. The TCR ␥ translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the V␥ and J␥ segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.diversity ͉ translocon M odels proposed for the molecular evolution of the immune system are based on comparative studies of living species. Sharks are critical in this regard, because they belong to an ancient clade (chondrichthyans) that can be traced in the fossil record to the time of the earliest vertebrates. Approximately 450 million years ago, the gnathostomes diverged into 2 groups, the chondrichthyans and the line leading to modern teleosts and tetrapods. It can be concluded that the molecular components of the immune system are ancient and arose before this divergence. This conclusion follows from studies showing that all of the defining elements of the immune system, antibodies, T cell receptors (TCRs), MHC products, and recombination activator genes (RAG), are present in chondrichthyes (1, 2). Thus, continued studies of sharks and rays, the most distant living relatives of mammals with a vertebrate type (VDJ-C recombination) immune system, should provide insights into the molecular origins and evolution of the immune system.A surprising feature of the organization of shark Ig genes was that the V-J-C of light chains (3-5) and V-D-D-J-C of heavy chains (6-8) are arranged in individual clusters. This feature is in contrast to the translocons of higher vertebrates that consist of large arrays of multiple V segments distantly linked to several J and D segments, as well as C domains (9). Although the genes for the TCR chains ␣/␤ and ␥/␦ have been shown to be present in the skate (10) and horned shark (11, 12), the germ-line loci for these molecules have been only partially characterized. Analyses of cDNA sequences in the skate revealed multiple V region families, ...

During pregnancy, invading HLA-G+ extravillous trophoblasts (EVT) play a key role in placental development, uterine spiral artery remodeling, and prevention of detrimental maternal immune responses to placental and fetal antigens. Failures of these processes are suggested to play a role in the development of pregnancy complications, but very little is known about the underlying mechanisms. Here we present validated methods to purify and culture primary HLA-G+ EVT from the placental disk and chorionic membrane from healthy term pregnancy. Characterization of HLA-G+ EVT from term pregnancy compared to first trimester revealed their unique phenotypes, gene expression profiles, and differing capacities to increase regulatory T cells (Treg) during coculture assays, features that cannot be captured by using surrogate cell lines or animal models. Furthermore, clinical variables including gestational age and fetal sex significantly influenced EVT biology and function. These methods and approaches form a solid basis for further investigation of the role of HLA-G+ EVT in the development of detrimental placental inflammatory responses associated with pregnancy complications, including spontaneous preterm delivery and preeclampsia.

Trophoblast expression of immunomodulatory proteins in the human placenta is among the mechanisms that are critical for ensuring lymphocyte tolerance to the semi-allogeneic fetus. High levels of B7-H1 on trophoblast cells together with the known role of this protein in establishment of peripheral tolerance suggest that B7-H1 mediates immunological protection of the placenta during gestation. In this study we investigated the molecular mechanisms of regulation of B7-H1 in trophoblast cells by epidermal growth factor (EGF), a key regulator of trophoblast cell differentiation. EGF increased B7-H1 protein levels within 24 hours and mRNA levels within 4 hours of the initiation of treatment; by 24 hours B7-H1 mRNA levels were similar between control and EGF-treated cells. Analysis of two different potential promoter regions revealed strong promoter activity in response to IFN-γ. In contrast, no promoter activity could be induced by EGF, suggesting that this cytokine regulates B7-H1 expression post-transcriptionally in trophoblast cells. EGF-induced B7-H1 protein expression was completely blocked in the presence of inhibitors of the PI3Kinase/Akt/mTOR pathway, a pathway known to regulate gene expression at the translational level. Finally, analysis of monosomal and polysomal mRNA fractions of untreated and EGF-treated term trophoblast cells revealed that EGF induces a shift towards the translatable fractions and away from the untranslated fractions. These results highlight a novel mechanism for regulation of B7 family proteins in the placenta.

Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

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