Margaret G. Petroff

HLA-G and immune tolerance in pregnancy

Multiple mechanisms underlie the surprising willingness of mothers to tolerate genetically different fetal tissues during pregnancy. Chief among these is the choice of HLA‐G, a gene with few alleles, rather than the highly polymorphic HLA‐A and ‐B genes, for expression by the placental cells that interface directly with maternal blood and tissues. Novel aspects of this major histocompatibility complex class Ib gene include alternative splicing to permit production of membrane and soluble isoforms, deletions that dampen responses to interferons, and a shortened cytoplasmic tail that affects expression at the cell surface. Placental cells migrating into the maternal uterus synthesize both membrane and soluble isoforms, which interact with inhibitory receptors on leukocytes such as ILT2 and ILT4. Cytotoxic T lymphocytes either die or reduce production of one of their major coreceptor/activator cell surface molecules, CD8; natural killer cells are immobilized and mononuclear phagocytes are programmed into suppressive modes characterized by high production of anti‐inflammatory cytokines. The idea that placental HLA‐G proteins facilitate semiallogeneic pregnancy by inhibiting maternal immune responses to foreign (paternal) antigens via these actions on immune cells is now well established, and the postulate that the recombinant counterparts of these proteins may be used as powerful tools for preventing immune rejection of transplanted organs is gaining in popularity.—Hunt, J. S., Petroff, M. G., McIntire, R. H., Ober, C. HLA‐G and immune tolerance in pregnancy. FASEB J. 19, 681–693 (2005)

B7 Family Molecules Are Favorably Positioned at the Human Maternal-Fetal Interface

Abstract The human placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. We investigated the pattern of expression of the B7 family of immunomodulatory molecules B7-H1 (PD-L1), B7-2 (CD86), and B7-1 (CD80) at the term maternal-fetal interface. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that B7-H1 mRNA is abundant in term placenta and that cytotrophoblasts are sources of this message. Immunohistochemistry demonstrated that B7-H1 is constitutively expressed by the syncytiotrophoblast and by extravillous cytotrophoblasts, both of which are juxtaposed to maternal blood and tissue. By contrast, placental stromal cells, including macrophages, lacked the protein. Expression of B7-H1 protein was low in first-trimester placenta compared to second- and third-trimester tissue (P < 0.05) and was enhanced in cultured cytotrophoblasts by treatment with either interferon-γ or epidermal growth factor (P < 0.05), suggesting that one or both of these mediators regulates B7-H1 expression in the placenta. RT-PCR and immunofluorescence analysis of term placental tissue revealed different patterns of expression of the immunostimulatory protein, B7-2. In contrast to B7-H1, B7-2 mRNA and protein were absent in cytotrophoblast cells but present in maternal macrophages and some fetal macrophages. The B7-1 mRNA and protein were absent at the maternal-fetal interface. These studies document expression of the B7 family proteins at the maternal-fetal interface and demonstrate that B7-H1 is positioned such that it could facilitate protection of fetal cells against activated maternal leukocytes. Conversely, B7-2 was absent on trophoblasts and was appropriately localized to fetal and maternal macrophages, which may participate in antigen presentation.

Altered subcellular localization of transcription factor TEAD4 regulates first mammalian cell lineage commitment

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.

HLA-G in reproduction: studies on the maternal–fetal interface

For more than a decade, investigators have known that membrane-bound and soluble isoforms of the HLA class Ib molecule, HLA-G, are present at the maternal-fetal interface. Although it is clear that extravillous cytotrophoblast cells are major producers, other cells may also contribute. Recent studies in our laboratory raised the question of whether soluble isoforms might reach the maternal and/or fetal blood circulation. A capture enzyme-linked immunoabsorbent assay (ELISA) identified soluble HLA-G (sHLA-G) in maternal blood throughout pregnancy but failed to detect sHLA-G in cord sera. Further studies suggested that the circulating proteins may be either free heavy chain (sHLA-G1 and/or sHLA-G2) or exclusively sHLA-G2. To study the potential function(s) of the soluble isoforms to modulate local or systemic immunity in mothers, we generated recombinant sHLA-G1 and -G2 in both prokaryotic and eukaryotic systems. Preliminary experiments conducted using DNA microarray analysis suggest that sHLA-G is capable of modulating gene expression in blood mononuclear leukocytes. Potential local targets were also identified; decidual and placental macrophages but not trophoblast cells contained mRNA encoding two of the known receptors for HLA-G, ILT2 and ILT4. Collectively, the studies are consistent with the hypothesis that sHLA-G produced at the maternal-fetal interface targets to the cells of the monocyte/macrophage lineage and modulates their functions for the benefit of pregnancy.

Decidual macrophages are potentially susceptible to inhibition by class Ia and class Ib HLA molecules

Several members of the immunoglobulin-like transcript (ILT), also called leukocyte immunoglobulin-like receptor (LIR), family of transmembrane proteins have been identified as receptors for class I HLA molecules and transduce inhibitory signals to leukocytes upon binding of these ligands. The ligands for ILT2 (LIR1/CD85j) and ILT4 (LIR2/CD85d) include HLA-A, -B, and -G, the last of which is highly expressed in fetal trophoblast cells in both membrane-bound and soluble isoforms. To investigate the potential of fetally-derived HLA class I molecules to interact with maternal macrophages through these receptors, we examined the expression patterns of ILT2 and ILT4 in decidual macrophages. Highly purified populations of decidual macrophages were obtained by fluorescence activated cell sorting and were examined by RT-PCR for these messages. Analysis of mRNA from first trimester and term macrophages, as well as the monocyte cell line U937, resulted in amplicons of similar size to those expected for ILT2 and ILT4. Sequence analysis of the amplicons revealed that the messages from decidual macrophages corresponded to ILT2 and ILT4 messages. The message amplified from the U937 cells using the ILT2 primers was also found to be identical to ILT2; however, sequence analysis revealed that the ILT4 message amplified from these cells is a truncated form of the message. Dual label flow cytometry confirmed the expression of ILT2 and ILT4 on CD14-positive first trimester decidual macrophages and U937 cells. These results reveal that inhibitory HLA receptors are expressed in decidual macrophages and suggest that HLA-G may deliver negative signals to maternal decidual macrophages through interaction with these receptors.

Recombinant HLA‐G5 and ‐G6 drive U937 myelomonocytic cell production of TGF‐β1

Throughout human pregnancy, activated maternal macrophages producing anti‐inflammatory cytokines comprise a stable cell population in the uterus. This organ is also massively infiltrated with semiallogeneic, placenta‐derived, invasive cytotrophoblast cells, which produce membrane and soluble isoforms of human leukocyte antigen (HLA)‐G. Here, we investigated the possibility that two soluble isoforms of HLA‐G, HLA‐G5 and ‐G6, program macrophage production of cytokines. The model system consisted of human U937 myelomonocytic cells treated with phorbol 12‐myristate 13‐acetate (PMA) and interferon‐γ (IFN‐γ), which induced differentiation and activation but did not affect their viability or decrease their expression of the two inhibitory immunoglobulin‐like transcript (ILT) receptors for HLA‐G, ILT2 and ILT4. Exposure of the PMA/IFN‐γ‐treated U937 cells to increasing concentrations of recombinant HLA‐G5 or ‐G6 (rG5 and rG6) stimulated effects common to the two isoforms. High doses of both significantly decreased interleukin (IL)‐10 and dramatically increased transforming growth factor‐β1. Differential effectiveness between the isoforms was demonstrated in dose‐response studies, as was differential binding to ILT2 and ILT4 in receptor‐blocking studies. No effects on production of IL‐4, IL‐1 receptor antagonist, IL‐15, tumor necrosis factor α, IL‐1β, or IL‐6 were observed. Collectively, the results are consistent with the postulate that environmental programming of decidual macrophages may be dictated in part by their proximity to soluble HLA‐G‐producing fetal cytotrophoblast cells.

Immunomodulatory molecules are released from the first trimester and term placenta via exosomes

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 co-localization 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 h. 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.

The immunomodulatory proteins B7-DC, B7-H2, and B7-H3 are differentially expressed across gestation in the human placenta.

Placental trophoblast cells form a cellular barrier between the potentially immunogenic fetus and maternal leukocytes. Trophoblasts subvert maternal immunity by producing surface-bound and soluble factors that interact with maternal leukocytes. Here, we describe the distribution of three members of the expanding family of B7 immunomodulatory molecules: B7-DC, B7-H2, and B7-H3. B7-DC and B7-H3 inhibit antigen-stimulated lymphocyte activation while B7-H2 serves in a regulatory capacity, often promoting a Th2 immunophenotype. First trimester and term placentas, purified trophoblast cells, choriocarcinoma cell lines, and human umbilical vein endothelial cells were analyzed for B7 family RNA and protein expression. Transcripts and proteins for all three B7s were present throughout gestation but were differentially expressed within the trophoblast and the stroma. Whereas B7-DC was prominent on the syncytiotrophoblast of early placenta, it was absent from the trophoblast at term. In contrast, B7-H2 and B7-H3 were prominent on the extravillous trophoblast throughout gestation. Lastly, stromal cells, including macrophages and endothelial cells, differentially expressed B7-DC, B7-H2, and B7-H3, depending on gestational age. Thus, all three of these newly discovered B7 proteins are differentially positioned at the maternal-fetal interface such that they could steer maternal leukocytes away from a harmful immune response and toward a favorable one.

Isolation and culture of term human trophoblast cells.

Experimentation with most human cell types is restricted to the use of cell lines, and this limits our ability to extrapolate interpretations to the in vivo condition. However, in studying human trophoblast cells, we have a unique opportunity to obtain large quantities of readily available human tissue. In this chapter, we outline the methodology for purification of human trophoblast cells from term placentas. The procedures are based on enzymatic dissociation of villous placental tissue, followed by gradient centrifugation and immunomagnetic bead purification. Purity may be assessed by immunocytochemistry or flow cytometry using a number of markers to identify both cytotrophoblast cells and cellular contaminants. The resulting cytotrophoblast cell populations have excellent viability and purity, and may be subjected to long-term culture.

Maternal PD-1 regulates accumulation of fetal antigen-specific CD8+ T cells in pregnancy

The failure to reject the semi-allogeneic fetus suggests that maternal T lymphocytes are regulated by potent mechanisms in pregnancy. The T cell immunoinhibitory receptor, Programmed Death-1 (PD-1), and its ligand, B7-H1, maintain peripheral tolerance by inhibiting activation of self-reactive lymphocytes. Here, we investigated the role of the PD-1/B7-H1 pathway in maternal tolerance of the fetus. Antigen-specific maternal T cells both proliferate and upregulate PD-1 in vivo at mid-gestation in response to paternally inherited fetal antigen. In addition, when these cells carry a null deletion of PD-1, they accumulate excessively in the uterus-draining lymph nodes (P<0.001) without a concomitant increase in proliferation. In vitro assays showed that apoptosis of antigen-specific CD8(+) PD-1(-/-) cells was reduced following peptide stimulation, suggesting that the accumulation of these cells in maternal lymph nodes is due to decreased cell death. However, the absence of neither maternal PD-1 nor B7-H1 had detectable effects on gestation length, litter size, or pup weight at birth in either syngeneic or allogeneic pregnancies. These results suggest that PD-1 plays a previously unrecognized role in maternal-fetal tolerance by inducing apoptosis of paternal antigen-specific T cells during pregnancy, thereby controlling their abundance.