Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the ß2-microglobulin (ß2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized ß2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with ß2m, thus accounting for ß2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.

Signaling pathways for B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in human placenta.

The tumor necrosis superfamily (TNFSF) contains two soluble ligands that are involved in B lymphocyte development, BAFF (B cell activating factor, BlyS, TALL-1, CD257, TNFSF13B) and APRIL (a proliferation inducing ligand, CD256, TNFSF13). These two ligands signal through three receptors: the exclusive BAFF receptor (BAFF-R, CD268, TNFRSF17) and two receptors that recognize both BAFF and APRIL, TACI (transmembrane-activator-1 and calcium-modulator- and cyclophilin ligand-interactor CD267, TNFRSF13B) and BCMA (B cell maturation antigen, CD269, TNFRSF13C). All but BAFF-R are known to be synthesized in term placentas. In this study, expression of the ligands and receptors were distinguished in two embryologically discrete subpopulations of placental cells, villous cytotrophoblast (vCTB) cells and mesenchymal cells (MCs). Real-Time PCR showed that vCTB cells contain low levels of BAFF and APRIL transcripts whereas MCs contain high levels. Both Real-Time PCR and immunohistochemistry identified BAFF-R and BCMA mRNA and proteins in vCTB cells but essentially no TACI. By contrast, MCs contained readily detectable levels of all three receptors. These results illustrating potential autocrine and paracrine pathways for BAFF and APRIL signaling in human placentas suggest that lineage-specific regulation of placental cell viability, differentiation and/or other activities may be novel functions of these proteins.

Differential cellular expression of LIGHT and its receptors in early gestation human placentas.

LIGHT (homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes) is an apoptosis-inducing member of the tumor necrosis factor family of ligands. Messenger RNAs encoding LIGHT and its receptors, lymphotoxin-beta receptor (LTbetaR), decoy receptor-3 (DcR3) and herpes virus entry mediator (HVEM), are present in first trimester and term placentas. Proteins have been localized to specific cells in term but not earlier gestation placentas. Here, we have studied LIGHT and its receptors in early (6-7 weeks) and early-to-middle (8-13 weeks) gestation using immunohistology. Notable cell-specific, gestation-related features were identified. LIGHT and two of its receptors, a membrane-bound receptor that mediates apoptosis (LTbetaR) and a soluble receptor that interferes with LIGHT signaling (DcR3), were present in syncytiotrophoblast and cytotrophoblast cells in all samples but were detected in placental stromal cells only at week 8 and thereafter. HVEM, a membrane-bound receptor that protects against apoptosis, was expressed only on syncytiotrophoblast. These observations suggest that the LIGHT system may regulate early to middle stages of placental development via cell-specific, temporally programmed expression of the ligand and its receptors, and may also assist in preserving placental immune privilege.

Differential expression of human leukocyte antigen-G (HLA-G) messenger RNAs and proteins in normal human prostate and prostatic adenocarcinoma.

Human leukocyte antigen-G (HLA-G) is a major histocompatibility complex class Ib gene expressed in normal organs and in some tumors. The glycoproteins encoded by this gene are best known for their immunosuppressive properties. Because isoform-specific expression of HLA-G in male reproductive organs has not been reported, we investigated HLA-G1, -G2, -G5, -G6 mRNAs and proteins in four-to-five samples of normal prostate glands, prostates with benign prostatic hyperplasia and prostate adenocarcinomas using RT-PCR and immunohistochemistry. All tissues contained HLA-G1, -G2, -G5 and -G6 specific mRNAs, but only HLA-G5 protein was detectable. In normal prostate glands, HLA-G5 protein was prominent in the cytoplasm of tubuloglandular epithelia and in glandular secretions. Staining was reduced in samples of benign prostatic hyperplasia but remained localized to the cytoplasm of glandular epithelia and secretions. In prostatic adenocarcinomas, HLA-G5 protein was detectable mainly in the secretions. Thus, HLA-G5 but not HLA-G1, -G2 or -G6 is produced in the normal prostate and is present in prostatic secretions. In addition, normal cellular localization is disturbed in benign and malignant prostatic adenocarcinomas. The results are consistent with this molecule may influencing female immune receptivity to sperm and suggest that such immunosuppression could be disturbed in men with prostatic adenocarcinomas.

The role of HLA-G in human pregnancy.

Pregnancy in mammals featuring hemochorial placentation introduces a major conflict with the mother's immune system, which is dedicated to repelling invaders bearing foreign DNA and RNA. Numerous and highly sophisticated strategies for preventing mothers from rejecting their genetically different fetus(es) have now been identified. These involve production of novel soluble and membrane-bound molecules by uterine and placental cells. In humans, the placenta-derived molecules include glycoproteins derived from the HLA class Ib gene, HLA-G. Isoforms of HLA-G saturate the maternal-fetal interface and circulate in mothers throughout pregnancy. Uteroplacental immune privilege for the fetus and its associated tissues is believed to result when immune cells encounter HLA-G. Unequivocally demonstration of this concept requires experiments in animal models. Both the monkey and the baboon express molecules that are similar but not identical to HLA-G, and may comprise suitable animal models for establishing a central role for these proteins in pregnancy.

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.

Methods for evaluating histocompatibility antigen gene expression in the baboon.

A wide variety of techniques has been developed for qualitative and quantitative analysis of gene expression in human cells and tissues. Two commonly used methods are reverse-transcription (RT)-polymerase chain reaction (PCR) to analyze the transcribed messenger RNAs (mRNA) and immunohistochemistry to detect the translated proteins. These techniques can be modified and adapted for use in analyzing gene expression in animal models. In particular, as a result of the close phylogenetic relationship between humans and nonhuman primates, human reagents, especially antibodies, cross-react with nonhuman primate tissues. However, the results are not always satisfactory as some antibodies may cross-react with irrelevant antigens in these tissues. In this chapter, we describe the use of RT-PCR and immunohistochemical techniques to analyze expression of Paan-AG, a novel class lb major histocompatibility complex antigen in the olive baboon (Papio anubis) placenta. We used Paan-AG-specific primers to amplify Paan-AG transcripts from baboon placenta, and generated Paan-AG isoform-specific polyclonal antibodies for use in immunohistochemistry.

In vitro models for studying human uterine and placental macrophages.

Human monocytes and macrophages, which are also called mononuclear phagocytes, represent a major arm of the innate immune system. These cells not only protect against infection but are also central to tissue remodeling and production of chemokines, cytokines, and growth factors. Tissue macrophages reside in the human placenta and uterine decidua throughout pregnancy, where they comprise part of the host defense network and facilitate placental and extraembryonic development. The purpose of this chapter is to describe methods for establishing useful models of human uteroplacental macrophages: (1) differentiated U937 myelomonocytic cells, (2) peripheral blood monocytes, (3) peripheral blood monocyte-derived macrophages, (4) decidual macrophages, and (5) placental macrophages.

Analysis of the soluble isoforms of HLA-G mRNAs and proteins.

The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.

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.

Cell-specific expression of B lymphocyte (APRIL, BLyS)- and Th2 (CD30L/CD153)-promoting tumor necrosis factor superfamily ligands in human placentas.

Apoptosis-inducing tumor necrosis factor (TNF) ligands and receptors have been reported in human placentas, but the expression patterns of family members lacking this function [a proliferation-inducing ligand (APRIL), B lymphocyte stimulator (BLyS), CD30L/CD153, CD40L/CD154, TNF-related activation-induced cytokine, CD27L/CD70, OX40L, activation-inducible TNF receptor ligand (AITRL)] are incompletely documented or unknown. We therefore investigated expression of these eight ligands and nine of their receptors (B cell maturation antigen, transmembrane activator and calcium-modulator and cyclophilin ligand-interactor, CD30, CD40, receptor activator of nuclear factor-kappaB, osteoprotegerin, CD27, OX40/CD134, AITR). Analysis by reverse transcriptase-polymerase chain reaction revealed mRNAs encoding only three of the ligands (APRIL, BLyS, CD30L/CD153). Immunoblots demonstrated all three proteins in first-trimester and term placentas, and immunohistochemical experiments showed that expression was cell-specific and gestation-related. Although mRNAs encoding receptors for the three expressed ligands were absent, those encoding receptors for all of the unexpressed ligands were detectable. Collectively, the results are consistent with the postulate that nonapoptosis-inducing, placenta-derived TNF superfamily cytokines contribute to the T helper cell type 2 bias required for successful pregnancy. Patterns of placental expression of receptors suggest bidirectional maternal-fetal cytokine communication.

Recombinant HLA-G5 and -G6 drive U937 myelomonocytic cell production of TGF-beta1.

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-gamma (IFN-gamma), 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-gamma-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-beta1. 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 alpha, IL-1beta, 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.

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.

Immunoregulatory molecules in human placentas: potential for diverse roles in pregnancy.

Molecules with immunological functions abound in hemochorial mammalian placentas where maternal blood and tissues are in direct contact with fetal placental cells. For the most part, investigators have focused on the possibility that these molecules are primarily in place for the purpose of preventing maternal immune mechanisms from attacking the genetically different fetal cells. Yet information collected in recent years indicates that these immunological mediators may serve other, non-immunological functions in placentas. In this article we discuss two families of these molecules investigated in our and other laboratories, namely the tumor necrosis factor superfamily (TNFSF) and the human leukocyte antigen (HLA) family, and present accumulating evidence for dichotomy of function during gestation.

Transcriptional regulation of VEGF-A by the unfolded protein response pathway.

BACKGROUND: Angiogenesis is crucial to many physiological and pathological processes including development and cancer cell survival. Vascular endothelial growth factor-A (VEGFA) is the predominant mediator of angiogenesis in the VEGF family. During development, adverse environmental conditions like nutrient deprivation, hypoxia and increased protein secretion occur. IRE1alpha, PERK, and ATF6alpha, master regulators of the unfolded protein response (UPR), are activated under these conditions and are proposed to have a role in mediating angiogenesis. PRINCIPAL FINDINGS: Here we show that IRE1alpha, PERK, and ATF6alpha powerfully regulate VEGFA mRNA expression under various stress conditions. In Ire1alpha(-/-) and Perk(-/-) mouse embryonic fibroblasts and ATF6alpha-knockdown HepG2 cells, induction of VEGFA mRNA by endoplasmic reticulum stress is attenuated as compared to control cells. Embryonic lethality of Ire1alpha-/- mice is due to the lack of VEGFA induction in labyrinthine trophoblast cells of the developing placenta. Rescue of IRE1alpha and PERK in Ire1alpha(-/-) and Perk(-/-) cells respectively, prevents VEGFA mRNA attenuation. We further report that the induction of VEGFA by IRE1alpha, PERK and ATF6 involves activation of transcription factors, spliced-XBP-1, ATF4 and cleaved ATF6 respectively. CONCLUSIONS/SIGNIFICANCE: Our results reveal that the IRE1alpha-XBP-1, PERK-ATF4, and ATF6alpha pathways constitute novel upstream regulatory pathways of angiogenesis by modulating VEGF transcription. Activation of these pathways helps the rapidly growing cells to obtain sufficient nutrients and growth factors for their survival under the prevailing hostile environmental conditions. These results establish an important role of the UPR in angiogenesis.

HLA-G: a human pregnancy-related immunomodulator.

In human pregnancies mothers and their embryo/fetuses are invariably genetically different. Thus, attenuation of the adaptive maternal immune response, which is programmed to reject 'foreign' entities, is required for pregnancy to be initiated and maintained. Unexpectedly, given the propensity of the immune system to dispose of non-self entities, at least 50% of expected human pregnancies reliably go forward. This indicates that to a large extent, effective systems of tolerance have evolved. Although overlapping and redundant mechanisms of tolerance have been identified, production of HLA-G by trophoblast cells derived from the external trophectoderm layer of the blastocyst appears to be of major importance. At this point in time, no pregnancies in which all of the proteins derived from the HLA-G gene are absent have as yet been reported. Many studies have shown that both membrane-bound and soluble isoforms of the proteins derived from this HLA class Ib gene are produced by placental trophoblast cells, with consequences that include but are not restricted to immune suppression at the maternal-fetal interface. Here we report new studies that are leading to a better understanding of the HLA-G proteins, their unique structures, unusual modes of regulation, diverse functions, and potential for use in diagnostic and therapeutic procedures related to suboptimal fertility in women.

Programming of human monocytes by the uteroplacental environment.

During human pregnancy, monocytes recruited to the uterus (decidua) are modified to promote immune defense and semiallogeneic pregnancy. The purpose of this study was to identify decidual factors involved in programming of monocytes into decidual macrophages by comparing the surface and secretory phenotypes of resting and interferon- gamma (IFN-gamma)-activated monocytes, unfractionated decidual cells, purified term decidual macrophages, and monocyte-derived macrophages. Surface markers for antigen presentation (HLA-DR, CD86), a membrane-bound cytokine interleukin (IL)-15, leukocyte immunoglobulin-like receptors (LILRB1, LILRB2), and secreted anti-inflammatory cytokines (transforming growth factor [TGF]-beta1 and IL-10) were assessed. The results demonstrate that differentiated, activated monocytes closely resemble but are not identical to decidual macrophages. In addition to differential IFN-gamma responsiveness, decidual macrophages were smaller than monocyte-derived macrophages and produced IL-10, which monocyte-derived macrophages did not. Only the unfractionated decidual cells secreted TGF-beta1. These results suggest that activation, differentiation, and decidual signals cooperate to program monocytes into the decidual macrophage phenotype.

Synthesis of beta(2)-microglobulin-free, disulphide-linked HLA-G5 homodimers in human placental villous cytotrophoblast cells.

Human leucocyte antigen-G (HLA-G) is a natural immunosuppressant produced in human placentas that binds differently to the inhibitory leucocyte immunoglobulin-like receptors LILRB1 (ILT2) and LILRB2 (ILT4) according to its biochemical structure. To predict the binding functions of the HLA-G5 soluble isoform synthesized in placental villous cytotrophoblast (vCTB) cells, we investigated structural features of this protein. Biochemical and immunological studies showed that vCTB cell HLA-G5 heavy (H)-chain proteins are disulphide-bonded homodimers unassociated with beta(2)-microglobulin (beta2m) light-chain proteins. Although comparatively low levels of beta2m messenger RNA (mRNA) were identified by real-time reverse transcription-polymerase chain reaction, immunoprecipitation studies failed to detect beta2m protein even when specific mRNA was doubled by transduction of a lentivirus-beta2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell beta2m mRNA and differentiation into syncytium did not promote beta2m synthesis. The failure of vCTB cells to exhibit beta2m in vitro was paralleled by a lack of detectable beta2m in vCTB cells in vivo. Lack of the beta2m protein could be the result of low levels of beta2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that beta2m-free HLA-G binds strongly to LILRB2, a receptor that is expressed by macrophages. This potentially immunosuppressive cell type is abundant in the pregnant uterus. Thus, our findings are consistent with the postulate that the natural beta2m-free homodimeric form of HLA-G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal-fetal interface.

Stranger in a strange land.

Mammalian mothers and their embryos/fetuses are almost invariably genetically different, which raises the question of how the mother's immune system is diverted so as to permit cohabitation with the 'foreign' body. Several decades of research have shown that multiple cooperative systems sanction uteroplacental immune privilege. These systems include production of several varieties of soluble immunosuppressive molecules in the uterus and the placenta and strict regulation of the molecules expressed on or by placental trophoblast cells. Trophoblast, a unique lineage without counterpart in adult tissues, is in direct contact with maternal blood and tissue. The major graft rejection-promoting molecules, human leukocyte antigens (HLAs), are tightly regulated in these cells, with none of HLA-A, HLA-B, or HLA class II antigens expressed. The HLA class Ib antigens, HLA-E, HLA-F, and HLA-G, are detectable on some subpopulations. Our studies have focused on the expression, regulation, and functions of the soluble isoforms of HLA-G, which circulate in maternal blood and are present at high levels in the pregnant uterus. These isoforms are derived from the single HLA-G gene by alternative splicing and are now known to have immunosuppressive properties. Ours and other studies indicate that soluble HLA-G proteins may comprise a unique tolerogenic system for establishing local immune privilege during pregnancy.