Daudi K. Langat

Synthesis of β2-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 β2‐microglobulin (β2m) light‐chain proteins. Although comparatively low levels of β2m messenger RNA (mRNA) were identified by real‐time reverse transcription–polymerase chain reaction, immunoprecipitation studies failed to detect β2m protein even when specific mRNA was doubled by transduction of a lentivirus‐β2m complementary DNA into vCTB cells. No abnormalities were identified in the translational start codon of vCTB cell β2m mRNA and differentiation into syncytium did not promote β2m synthesis. The failure of vCTB cells to exhibit β2m in vitro was paralleled by a lack of detectable β2m in vCTB cells in vivo. Lack of the β2m protein could be the result of low levels of β2m transcripts or of as yet unidentified translational defects. Experiments with recombinant ectodomains of LILRB indicate that β2m‐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 β2m‐free homodimeric form of HLA‐G5 synthesized in primary vCTB cells could comprise a particularly effective tolerogenic molecule at the maternal–fetal interface.

Novel HLA-G-Binding Leukocyte Immunoglobulin-Like Receptor (LILR) Expression Patterns in Human Placentas and Umbilical Cords

Human placentas are sources of cytokines, hormones and other substances that program receptive cells. One of these substances is HLA-G, which influences the functioning of both leukocytes and endothelial cells. In this study, we investigated the possibility that these and/or other types of cells in extraembryonic fetal tissues might respond to HLA-G by interacting with one or another of the leukocyte immunoglobulin-like receptors (LILR). LILRB1 is expressed by most leukocytes and LILRB2 is expressed primarily by monocytes, macrophages and dendritic cells. Analysis of term placentas by immunohistochemistry and Real Time PCR demonstrated that LILRB1 and LILRB2 protein and specific messages are produced in the mesenchyme of term villous placenta but are differently localized. LILRB1 was abundant in stromal cells and LILRB2 was prominent perivascularly. Neither receptor was identified in trophoblast. Further investigation using double label immunofluorescence indicated that placental vascular smooth muscle but not endothelia exhibit LILRB2. Term umbilical cord exhibited the same LILRB2 patterns as term placenta. Samples obtained by laser capture dissection of vascular smooth muscle in umbilical cords demonstrated LILRB2 mRNA, and double label immunofluorescence showed that cord vascular smooth muscle but not endothelium exhibited LILRB2 protein. The presence of LILRB1 in placental stromal cells and LILRB2 in vascular smooth muscle strongly suggest that HLA-G has novel functions in these tissues that could include regulation of placental immunity as well as development and function of the extraembryonic vasculature.

Do Nonhuman Primates Comprise Appropriate Experimental Models for Studying the Function of Human Leukocyte Antigen-G?

Abstract The expression and function of the human major histocompatibility complex (MHC) class Ia genes, human leukocyte antigen (HLA)-A, -B, and -C, is well-established; they are expressed in most nucleated cells and present endogenous peptides to CD8+ T cells. However, MHC class Ib genes are poorly characterized and have unknown functions. In humans, the best-characterized class Ib gene is HLA-G. This gene has a restricted tissue expression of the mRNA and a unique pattern of protein expression; it is expressed mainly in the extravillous cytotrophoblast cells in the placenta. The function of HLA-G is not clear, but its presence at the maternal-fetal interface suggests a role in protection of the semiallogeneic fetus. Whereas functional studies using in vitro models and transgenic mice provide useful insights regarding the potential function of this molecule, in vivo studies cannot be performed in humans. Nonhuman primates that are closely related to humans phylogenetically contain homologues of HLA-G. The MHC-G loci in nonhuman primates appear to have diverged from the human HLA-G. However, in the rhesus monkey (Macaca mulatta) and olive baboon (Papio anubis), a novel class Ia-related locus has been described. This gene encodes glycoproteins with characteristics that resemble those of HLA-G, including restricted tissue distribution, alternative splicing of mRNA, truncated cytoplasmic domain, and limited polymorphism. Thus, this molecule may be the functional homologue of HLA-G, and these two species may comprise appropriate models for elucidating the function of HLA-G.

Potential regulatory sequences in the untranslated regions of the baboon MHC class Ib gene, Paan-AG, more closely resemble those in the human MHC class Ia genes than those in the class Ib gene, HLA-G.

The baboon major histocompatibility complex (MHC) class Ib gene, Paan-AG, is structurally similar to the human MHC class Ia gene, HLA-A, but exhibits characteristics similar to those of the class Ib gene HLA-G. These include limited polymorphism, alternative splicing of a single message, and restricted tissue distribution, with high expression in the placenta. In order to determine whether regulatory elements controlling expression of Paan-AG resemble those of HLA-A or HLA-G, we cloned the 5′ and 3′ untranslated regions of Paan-AG. Unexpectedly, sequence comparisons showed that potential regulatory elements in Paan-AG strikingly resembled those in HLA-A and differed in major respects from those in HLA-G. Unlike HLA-G, Paan-AG contained an intact interferon-γ stimulated response element (ISRE) in the promoter. Studies using luciferase reporter assays showed that the Paan-AG ISRE was functional. The basal activity of the Paan-AG ISRE and its response to interferon-γ was similar to that of class Ia MHC genes. Further, we identified an ISRE in the 3′ untranslated region of Paan-AG that is known to be functional in HLA-A2 but is deleted in HLA-G. These experiments predict that functional studies may demonstrate differences in regulation of expression of Paan-AG and HLA-G genes, which could restrict the use of the baboon as a primate model for studying HLA-G expression and function.

Potential risks of viral infections in xenotransplantation

The shortage of cadaveric human organs for transplantation may, be alleviated by the use of xenografts as a therapeutic option for end-stage organ failure. Successful attempts have been made to prevent rejection of xenograft tissues in humans. The potential spread of animal-derived pathogens to the xenograft recipient is a complication of xenotransplantation, which must be addressed. This can be complicated further by, the presence of new pathogens, new clinical syndromes, and altered behaviour of these organisms in the immunocompromised recipient. There is concern over the possible activation of latent viruses, including retroviruses, from xenograft tissues. This paper discusses the possible dangers of transmission of animal viruses to humans via xenotransplantation.

Characterization of antigens expressed in normal baboon trophoblast and cross-reactive with HIV/SIV antibodies.

Electron microscopic studies have revealed the presence of endogenous retroviral (ERV) particles in normal primate placental tissues. These particles have ultrastructural similarities to type C retroviral particles and are mainly associated with the trophoblast. In normal human placental tissues, they have antigenic similarity with exogenous retroviruses, such as the human immunodeficiency virus (HIV), and may have a role to play in the regulation of cellular gene expression, syncytiotrophoblast formation or pregnancy-related immunosuppression. In this study, a panel of antibodies (polyclonal and monoclonal antibodies) against viral proteins (anti-HIV and anti-SIV) and endogenous retroviral (ERV) proteins were assessed by immunohistochemistry and immunoblotting, for their cross-reactivity with ERV particles isolated from normal baboon placental tissues. The antibodies (anti-HERV-K RT, anti-ERV3 env, anti-HIV-1 p17, anti-HIV-2 gp120) reacted positively with the syncytiotrophoblast and each antibody recognized one or two proteins of molecular weights (MW) 38, 58 or 64 kDa present in the baboon placental villous tissues and SIV-infected molt-4 Cl8 cells, but not in uninfected cells. The results of this study confirm the specific expression of retroviral cross-reactive antigens in normal baboon placental tissues and suggest placental cellular proteins may have antigenic similarity with those recognized by anti-HIV/SIV antibodies. The role of these retroviral-related proteins expressed at the maternal-fetal interface remain unclear.

Phylogenetic analysis of simian T lymphotropic virus type I from Kenyan olive baboons (Papio anubis), lowland Sykes monkeys (Cercopithecus mitis), and vervet monkeys (Cercopithecus aethiops pygerythrus).

781 H UM AN T CELL LEU KEM IA/lymphotropic virus type I (HTLVI) strains have a worldwide distribution and are characterized by the genetic stability of their genome and their limited horizontal transmission. Simian T cell leukemia/lymphotropic virus type I (STLV-I) strains have been found in Old World nonhuman primates in Africa and Asia. Together, these two virus groups are referred to as primate T lymphotropic viruses (PTLVs) and have been found in all three Old World African primate families: the Cercopithecidae, 2±4 the Pongidae, and the Hominidae. These viruses cannot be separate d into distinct phyloge netic lineages according to the fam ilies or species of origin, but rather accordi ng to geographical regions , which suggest s multiple interspe cies transm issions in the past. Although uncertain, it is thought that the origina l virus infected nonhu man simian ancesto rs that eventua lly infected humans. 2 Understan ding the sequence diversit y among STLV-I strains may therefor e provide valuable insight into the evolutio n of the PTLV-I group. In this study, we analyze d the HTLV-I sequenc e of 13 nonhum an primates, using HTLV-I cross-r eacting antibod ies provide d with the HTLV-I Serodia particle aggluti nation assay (Fujirebio , Tokyo, Japan). The animals were either colony born (Institu te of Primate Research [IPR], Nairobi, Kenya) or trapped in Kibwezi (eastern Kenya) and in Gilgil and Namanga (Rift Valley Province) . The animals include d four vervet monkeys (Cercopit hecus aethiop s pygerythrus ), two lowland Sykes monkeys (Cercopit hecus mitis), and seven olive baboons (Papio anubis ) (Table 1). We have focused on the diverge nt long terminal repeat (LTR) region because it is more inform ative for sequence analysi s and subtyping, and because of the availab ility of inform ation on other PTLV-I LTR sequenc es. DNA was isolated from peripheral blood mononuclear cells (PBMCs) using a QiaAmp blood kit (Qiagen GmbH, Hilden, Germany). The LTR region was amplified by the polymerase chain reaction (PCR) with primer pairs HFL1/HFL6 and HFL5/HFL6, as described. 11 PCR products were cloned using the PCR-Script SK( 1 ) cloning kit (Stratagene, La Jolla, CA) according to the manufacturer instructions. Sequencing was performed using the Applied Biosystem s model 373 automatic DNA sequencer (Applied Biosystems/Perkin-Elmer , Foster City, CA). Nucleotide sequences were aligned with CLUSTAL V together with reference sequences from the GenBank database. Kimura distance calculation, 13 bootstrap analysis, 14 and tree construction were done with the TREECON software package. Nucleotide sequences were submitted to GenBank under accession numbers AF117282±AF117294. Comparison of the LTR region of the strains revealed that the elements that are critical for viral gene expression were well conserved. No nucleotide changes were observed in the polyadenylation signal (AATAAA), the TATA box (TATAAA), and the splice donor site (TAGGTAA). In the tree containing the LTR sequence (Fig. 1), STLV and HTLV-I clusters were identified as containing sequences representative of many different subtypes.

Polymorphisms in the Paan-AG promoter influence NF-κB binding and transcriptional activity

The human leukocyte antigen-G (HLA-G) gene encodes a protein that is highly expressed at the human maternal–fetal interface during pregnancy and may be critical to the survival of the semiallogenic fetus. A unique feature of this gene is a 13-bp deletion in the proximal promoter that renders it unresponsive to transactivation by the nuclear factor-κB (NF-κB). We previously showed that the proximal promoter of Paan-AG, the functional homologue of HLA-G in the olive baboon (Papio anubis), is intact. We cloned the promoters of two putative Paan-AG alleles (AG1 and AG2) and identified a number of regulatory elements including two κB sites. In the current study, binding and activity of the two κB elements in each putative allele were assessed by electrophoretic mobility shift and supershift assays. Functional activity was determined using luciferase reporter assays. The κB1 and κB2 elements in AG1 bound NF-κB with similar affinity. In contrast, the κB1 element of AG2 bound NF-κB with a much higher affinity than AG-1 κB1 (a 30-fold increase), whereas κB2 did not bind. Mutagenesis analysis showed that the difference in binding intensities was due to two nucleotides in the 3′ end of κB1. Similarly, failure of AG2 κB2 binding was a result of the last nucleotide in the 3′ end that differed from the consensus; mutating this nucleotide to match the consensus reestablished binding. Functional activity of the two putative alleles also differed; AG1 luciferase activity was consistently lower than that of AG2. Mutating the last two nucleotides in the 3′ end of AG1 κB1 resulted in increased luciferase activity to levels comparable to that of AG2. Overall, these results show that in vitro variations in the promoter region may influence transcription of Paan-AG.

IMMUNOHISTOCHEMICAL LOCALIZATION OF RETROVIRAL-RELATED ANTIGENS EXPRESSED IN NORMAL BABOON PLACENTAL VILLOUS TISSUE

Abstract: Endogenous retroviral particles (ERVs) have been detected in the genome of all eukaryotes. They are generally non‐pathogenic except in mice where they have been found to induce tumors and immunological disorders. The ERVs have morphological features consistent with type‐C retroviral particles and are commonly expressed in normal placental villous tissues. ERVs may have a role in the regulation of placental gene expression, syncytiotrophoblast formation, or pregnancy‐related immunosuppression. In this study, well‐characterized antibodies (monoclonal and polyclonal antibodies) raised against retroviral proteins (anti‐HIV and anti‐SIV) and endogenous retroviral (ERV) particles were assessed for their cross‐reactivity (by using immunohistochemistry) with normal baboon placental and other adult tissues. The monoclonal antibodies to exogenous retroviral proteins (anti‐HIV‐2 gp120, anti‐HIV‐1 gp41, anti‐SIVmac p27, anti‐HIV‐1 RT, and anti‐HIV‐2 core protein) showed specific immunohistochemical reactivity with the syncytiotrophoblast. Antibodies to endogenous retroviral gene products (anti‐ERV3 env, anti‐HERV‐K RT, and anti‐HERV‐K env) also reacted in a similar manner and did not cross‐react with other adult tissues. These studies have shown that retroviral‐cross‐reactive proteins are expressed in baboon placental syncytiotrophoblast and may have a role to play at the feto‐maternal interface.

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.