Extended HLA-G haplotypes in patients with age-related macular degeneration.

The study aims to determine if genetic polymorphisms in the human leukocyte antigen (HLA)-G gene are associated with age-related macular degeneration (AMD). HLA-G is important for immunological tolerance, and it is also known to have angiogenic effects. Polymorphisms in the 5'-upstream regulatory region (URR) and 3'-untranslated region (UTR) of HLA-G have been associated with a number of diseases, especially with respect to a 14 bp insertion/deletion (ins/del) polymorphism in the 3'UTR. Full gene sequencing was performed on a cohort of 146 AMD patients and 63 healthy controls aged 60 years or older and HLA-G haplotypes were determined. Analyses were performed on a publicly available gene expression dataset from the NCBI GEO database (accession number GSE29801) from which expression data for HLA-G, -C and -A were extracted. Analysis of the GEO dataset showed that both HLA-G and -C was expressed in the back of the eye and that expression was upregulated in the macular area of AMD. No differences were observed between patients and controls when analysing the distribution of haplotypes in the HLA-G promoter, coding region, 3'UTR or the 14 bp ins/del polymorphism of the 3'UTR. The increased expression of HLA-G in the macula of AMD patients indicates a role of HLA-G in the micro environment as part of the AMD pathogenesis. This is supported by the expression of HLA-C, which has previously been shown to play a role in AMD. The HLA-G haplotype distribution did not display any differences between AMD patients and controls. This article is protected by copyright. All rights reserved.

Expression and differential regulation of HLA-G isoforms in the retinal pigment epithelial cell line, ARPE-19.

The purpose of this study was to examine if HLA-G is expressed in the retinal pigment epithelium (RPE) cells of the eye. The RPE comprises the outer most layer of the retina and as such defines the interface to the blood and contributes to the immune privilege in the posterior part of the eye. One way the RPE might be regulating the immune system could be by expressing the non-classical human leukocyte antigen (HLA) molecule, HLA-G. We therefore sought to define if the RPE cell line, ARPE-19, expressed HLA-G and analyse the regulation as a response to pro-inflammatory cytokines. This was done by digital droplet PCR, measuring the gene expression of HLA-G in total RNA. The protein expression was analysed by immunohistochemistry and by immunofluorescence followed by confocal microscopy and the expression of the HLA-G isoforms was explored by fragment analysis. In the current study, we show that HLA-G is expressed by ARPE-19 cells and is upregulated as a response to pro-inflammatory cytokines. Moreover, we are the first to describe a differential regulation of the HLA-G isoforms as a direct response to stimulation. These results might indicate that HLA-G can be part of the immune privilege of the posterior part of the eye, but further experiments on primary RPE cells are needed.

Multiplex bead-based immunoassay for the free soluble forms of the HLA-G receptors, ILT2 and ILT4.

Human leukocyte antigen (HLA)-G is an immune-inhibitory molecule that exerts its function via interaction with two main inhibitory receptors: ILT2 and ILT4. This interaction is considered to be an immune checkpoint. HLA-G can be found as a soluble molecule, but it is not known if its receptors can also be found as soluble molecules. In this work, we present a multiplex luminex-based assay to measure soluble ILT2 (sILT2) and soluble ILT4 (sILT4) molecules together. It is based on two antibody pairs, GHI/75 and HP-F1-PE for ILT2 and 27D6 and 42D1-PE for ILT4. The characterization of our method reveals that it specifically detects the free soluble forms of sILT2 and sILT4, and not those complexed to HLA Class I molecules such as their ligand of highest affinity HLA-G. A study on two small cohorts of cancer patients demonstrated that soluble ILT2 and ILT4 molecules were of low abundance in the plasma of healthy controls, but that elevated levels of plasmatic sILT2 were present in non-muscle-infiltrating bladder cancer patients. This demonstrated that the titration test is indeed working, and that soluble ILT2 molecules do exist in pathological contexts, which relevance may now be sought on larger cohorts and other pathologies.

The expression and functional activity of membrane-bound human leukocyte antigen-G1 are influenced by the 3'-untranslated region.

Human Leukocyte Antigen (HLA)-G is an immunosuppressive molecule acting on both the innate and adaptive immune system. A 14 bp insertion/deletion polymorphism (rs66554220) in the 3'-untranslated region (3'UTR) of the HLA-G gene has been associated with a number of diseases, pregnancy complications, and graft rejection after organ transplantation. We have investigated the effect of HLA-G polymorphism in the 3'UTR on the processing and stability of the membrane-bound HLA-G1 (mHLA-G1) isoform, as well as its functional significance. Different HLA-G1 cDNA sequences were transduced into the human K562 cell line. Flow cytometry, immunohistochemistry, and ELISA were used to examine HLA-G1 protein expression. A quantitative RT-PCR assay was used to quantify transduced HLA-G1 DNA and mRNA transcript levels. Stability of mRNA and functional significance of HLA-G were investigated via Actinomycin D and NK cytotoxicity assays, respectively. Human leukocyte antigen-G mRNA from the 14 bp insertion K562-G1 cells showed a higher degree of stability than the other constructs, and increased mHLA-G1 expression relative to transductants lacking the 14 bp sequence. In line with this, transductants carrying the 14 bp insertion were the most efficient in inhibiting NK cytotoxicity but showed a lower soluble HLA-G1 per mHLA-G1 ratio than the HLA-G1 K562 cells lacking the 14 bp insertion. Our data suggest 3'UTR polymorphism may play an important role in HLA-G regulation with implications on a range of diseases.

Human leukocyte antigen-G in the male reproductive system and in seminal plasma.

One of the non-classical human leukocyte antigen (HLA) class Ib proteins, HLA-G, is believed to exert important immunoregulatory functions, especially during pregnancy. The presence of HLA protein in paternal seminal fluid has been suggested to have an influence on the risk of developing pre-eclampsia. We have investigated whether HLA-G protein is present in human seminal plasma and in different tissue samples of the male reproductive system. Western blot technique and a soluble HLA-G (sHLA-G) assay were used to detect sHLA-G in human seminal plasma samples. Immunohistochemical staining was performed on paraffin-embedded tissue samples. We detected sHLA-G protein in seminal plasma, and HLA-G expression in normal testis and in epididymal tissue of the male reproductive system but not in the seminal vesicle. Furthermore, the results indicated a weak expression of HLA-G in hyperplastic prostatic tissue. In summary, several of the findings reported in this study suggest an immunoregulatory role of HLA-G in the male reproductive system and in seminal plasma.