Mette Dahl

Human leucocyte antigen class Ib molecules in pregnancy success and early pregnancy loss

BACKGROUND The human leucocyte antigen (HLA) class Ib molecules, HLA-E, -F and -G, are expressed at the materno-fetal interface. Because of the apparent immunoregulatory functions of these proteins, they may be involved in successful acceptance of the semi-allogenic fetus during pregnancy. METHODS The literature on polymorphisms of the three genes, expression patterns of the proteins, and interactions with immune cell receptors have been evaluated to elucidate whether HLA-E, -F and -G are involved in the pathogenesis of some cases of recurrent miscarriages and unexplained infertility. RESULTS AND CONCLUSIONS The HLA class Ib molecules seem to induce suppression of the maternal immune system, but are not necessarily fundamental factors for pregnancy success. However, evidence points towards low expression of these proteins, especially HLA-G, being associated with reduced fertility. To clarify the functions of HLA-E, -F and -G future studies need to link investigations of the polymorphisms in these genes to measurements of protein levels, and examine the role of these proteins in the complex interplay of immune cells and cytokines at the materno-fetal interface.

Soluble Human Leukocyte Antigen-G in Seminal Plasma is Associated with HLA-G Genotype: Possible Implications for Fertility Success

We have previously shown that human seminal plasma contains immunomodulatory soluble HLA‐G (sHLA‐G). We investigated whether sHLA‐G levels in seminal plasma are associated with a specific 14 base pair (bp) insertion/deletion (ins/del) polymorphism in the 3′‐untranslated region of the HLA‐G gene and/or with the outcome of assisted reproduction treatments (ART) in couples attending a fertility clinic.

The many faces of human leukocyte antigen-G: relevance to the fate of pregnancy.

Pregnancy is an immunological paradox, where fetal antigens encoded by polymorphic genes inherited from the father do not provoke a maternal immune response. The fetus is not rejected as it would be theorized according to principles of tissue transplantation. A major contribution to fetal tolerance is the human leukocyte antigen (HLA)-G, a nonclassical HLA protein displaying limited polymorphism, restricted tissue distribution, and a unique alternative splice pattern. HLA-G is primarily expressed in placenta and plays multifaceted roles during pregnancy, both as a soluble and a membrane-bound molecule. Its immunomodulatory functions involve interactions with different immune cells and possibly regulation of cell migration during placental development. Recent findings include HLA-G contributions from the father and the fetus itself. Much effort has been put into clarifying the role of HLA-G during pregnancy and pregnancy complications, such as preeclampsia, recurrent spontaneous abortions, and subfertility or infertility. This review aims to clarify the multifunctional role of HLA-G in pregnancy-related disorders by focusing on genetic variation, differences in mRNA stability between HLA-G alleles, differences in HLA-G isoform expression, and possible differences in functional activity. Furthermore, we highlight important observations regarding HLA-G genetics and expression in preeclampsia that future research should address.

Associations between fetal HLA-G genotype and birth weight and placental weight in a large cohort of pregnant women – Possible implications for HLA diversity

Birth weight and placental weight are crucial parameters for the survival of fetuses and newborns in mammals. High variation in the MHC is important for an effective adaptive immune response. The maternal immune system must be controlled in relation to the semi-allogenic fetus. The immunoregulatory HLA/MHC class Ib gene, HLA-G, is strongly expressed on extravillous trophoblast cells. We investigated birth weight and placental weight of the newborns in mothers heterozygous for an HLA-G 14bp insertion (Ins)/deletion (Del) gene polymorphism. Separate analyses for pregnancies without preeclampsia (n=185), pregnancies complicated by preeclampsia (n=101), and both groups combined, were performed. Interestingly, we observed the highest mean birth weight and placental weight in homozygous 14bp Del/Del newborns, and the lowest in 14bp Ins/Ins newborns (P=0.008 and P=0.009). The 14bp Del/Del genotype is also associated with high expression of HLA-G on the trophoblast membrane. In theory, fetuses and newborns with intermediate weights and sizes would be an optimal compromise for both the fetus/father and the mother compared with very high and low weights. If such fetuses/newborns more often are heterozygous at the HLA-G gene locus, then newborns with two distinct HLA haplotypes are favored, leading to a higher degree of HLA diversity. The results of the study may indicate that a compromise between an intermediate birth weight and placental weight, induction of maternal tolerance by a fetal-derived non-polymorphic HLA class Ib molecule, and favoring of HLA heterozygous offspring, have evolved in humans.

Evaluation of a competitive enzyme-linked immunosorbent assay for measurements of soluble HLA-G protein

The human leukocyte antigen (HLA) class Ib molecule, HLA-G, has gained increased attention because of its assumed important role in immune regulation. The HLA-G protein exists in several soluble isoforms. Most important are the actively secreted HLA-G5 full-length isoform generated by alternative splicing retaining intron 4 with a premature stop codon, and the cleavage of full-length membrane-bound HLA-G1 from the cell surface, so-called soluble HLA-G1 (sHLA-G1). A specific and sensitive immunoassay for measurements of soluble HLA-G is mandatory for conceivable routine testing and research projects. We report a novel method, a competitive immunoassay, for measuring HLA-G5/sHLA-G1 in biological fluids. The sHLA-G immunoassay is based upon a competitive enzyme-linked immunosorbent assay (ELISA) principle. It includes a recombinant sHLA-G1 protein in complex with β2-microglobulin and a peptide as a standard, biotinylated recombinant sHLA-G1 as an indicator, and the MEM-G/9 anti-HLA-G monoclonal antibody (mAb) as the capture antibody. The specificity and sensitivity of the assay were evaluated. Testing with different recombinant HLA class I proteins and different anti-HLA class I mAbs showed that the sHLA-G immunoassay was highly specific. Optimal combinations of competitor sHLA-G1 and capture mAb concentrations were determined. Two versions of the assay were tested. One with a relatively wide dynamic range from 3.1 to 100.0 ng/ml, and another more sensitive version ranging from 1.6 to 12.5 ng/ml. An intra-assay coefficient of variation (CV) of 15.5% at 88 ng/ml and an inter-assay CV of 23.1% at 39 ng/ml were determined. An assay based on the competitive sHLA-G ELISA may be important for measurements of sHLA-G proteins in several conditions: assisted reproduction, organ transplantation, cancer, and certain pregnancy complications, both in research studies and possibly in the future also for clinical routine use.

Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies

Circular RNAs (circRNAs) are covalently closed endogenous molecules with tissue- and disease-specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The molecules are formed by a backsplicing event linking the 3′-end of an exon to the 5′-end of the same or an upstream exon, and they exert diverse regulatory functions important in carcinogenesis. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Here, we demonstrate that circRNAs can be accurately quantified without enzymatic reactions or bias using color-coded probes (NanoString technology). First, we performed high-throughput RNA sequencing (RNA-seq) of several mantle cell lymphoma and multiple myeloma cell lines to profile the genome-wide landscape of circRNA expression. We detected several circRNAs known to be deregulated in other cancers and identified a novel circRNA from the IKZF3 gene. Based on these data, we selected 52 unique circRNAs for which we designed color-coded probes spanning their specific backsplicing junctions. These circRNAs were quantified in cell lines and patient samples from several different B-cell malignancies (mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia) simultaneously using the NanoString technology. The circRNA expression profiles obtained could distinguish different B-cell malignancies, and confirmed the presence of the novel circRNA derived from IKZF3. The NanoString assays were specific for circRNA detection and data were more reproducible and quantitatively more accurate than RNA-seq data. In addition, we obtained high-quality data on severely degraded RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. Together, we provide a map of circRNA expression in B-cell malignancies and present an enzyme-free digital counting methodology, which has the potential to become a new gold standard for circRNA quantification.Endogenous circular RNA (circRNA) expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations. Here, the authors provide a map of circRNA expression in B-cell malignancies based on high-throughput RNA sequencing and present an enzyme-free digital counting methodology, which has the potential to become the gold standard for circRNA quantification.

Long Non-Coding RNAs Guide the Fine-Tuning of Gene Regulation in B-Cell Development and Malignancy

With the introduction of next generation sequencing methods, such as RNA sequencing, it has become apparent that alterations in the non-coding regions of our genome are important in the development of cancer. Particularly interesting is the class of long non-coding RNAs (lncRNAs), including the recently described subclass of circular RNAs (circRNAs), which display tissue- and cell-type specific expression patterns and exert diverse regulatory functions in the cells. B-cells undergo complex and tightly regulated processes in order to develop from antigen naïve cells residing in the bone marrow to the highly diverse and competent effector cells circulating in peripheral blood. These processes include V(D)J recombination, rapid proliferation, somatic hypermutation and clonal selection, posing a risk of malignant transformation at each step. The aim of this review is to provide insight into how lncRNAs including circRNAs, participate in normal B-cell differentiation, and how deregulation of these molecules is involved in the development of B-cell malignancies. We describe the prognostic value and functional significance of specific deregulated lncRNAs in diseases such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma and multiple myeloma, and we provide an overview of the current knowledge on the role of circRNAs in these diseases.

Abstract LB-394: Profiling of endogenous circular RNA molecules in formalin-fixed paraffin-embedded tissues from patients with B-cell malignancies using an enzyme-free digital counting method

Circular RNAs (circRNAs) are covalently closed endogenous RNA molecules with tissue- and disease specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Based on high-throughput RNA sequencing (RNA-seq) data, we designed assays for analyzing 52 unique circRNAs simultaneously, using a digital, enzyme-free technology termed NanoString, in cell lines and paired fresh frozen and formalin-fixed, paraffin-embedded (FFPE) patient samples (including mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia). The data obtained using NanoString were compared to RNA-seq and reverse transcription-qPCR (RT-qPCR) data obtained on the same samples. RNA-seq profiling revealed a high expression of circRNAs in B-cell malignancies and the NanoString circRNA expression profiles were able to distinguish different B-cell malignancies. We detected circRNAs known to be deregulated in other cancers, including ciRS-7, circHIPK3 circCCDC66, circCDYL, circZKSCAN1 and circFBXW7, and identified a novel circRNA from the IKZF3 oncogene. NanoString data were more reproducible and quantitatively accurate than RNA-seq data and the technology works in particular well for low quality RNA samples. Together, we demonstrate that the NanoString technology enables specific, sensitive and accurate quantification of circRNAs in FFPE samples and provide a map of circRNA expression in B-cell malignancies. Citation Format: Lasse Sommer Kristensen, Mette Dahl, Maria S. Andersen, Iben Daugaard, Thomas B. Hansen, Kirsten Gronbaek, Jorgen Kjems. Profiling of endogenous circular RNA molecules in formalin-fixed paraffin-embedded tissues from patients with B-cell malignancies using an enzyme-free digital counting method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-394.