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The immunosuppressive molecule HLA-G and its clinical implications

Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex (MHC) class I molecule that, through interaction with its receptors, exerts important tolerogenic functions. Its main physiological expression occurs in placenta where it seems to participate in the maternal tolerance toward the fetus. HLA-G has been studied as a marker of pregnancy complications such as abortion or pre-eclapmsia. Although HLA-G is not expressed in most adult tissues, its ectopic expression has been observed in some diseases such as viral infections, autoimmune disorders, and especially cancer. HLA-G neo-expression in cancer is associated with the capability of tumor cells to evade the immune control. In this review, we will summarize HLA-G biology and how it participates in these physiopathological processes. Special attention will be paid to its role as a diagnostic tool and also as a therapeutic target.

Study of Circulating MicroRNA-125b Levels in Serum Exosomes in Advanced Melanoma

CONTEXT Malignant melanoma is an aggressive tumor that produces exosomes, which contain microRNAs (miRNAs) that could be of utility in following tumoral cell dysregulation. MicroR-125b is a miRNA whose down-regulation seems to be implicated in melanoma progression. OBJECTIVE To analyze miR-125b levels in serum, and in exosomes obtained from serum, from patients with advanced melanoma. DESIGN Serum samples were obtained from 21 patients with advanced melanoma, from 16 disease-free patients with melanoma, and from 19 healthy volunteers. Exosomes were isolated from serum by precipitation, and miR-16 and miR-125b levels were quantified by real-time polymerase chain reaction. RESULTS MicroR-16, but not miR-125b, was detected in all samples, and miR-16 levels were significantly higher in serum than they were in exosomes. MicroR-16 expression levels did not differ significantly between the 2 groups (patients with melanoma and healthy donors). There was a significant relationship between miR-125b and miR-16 levels in exosomes. Additionally, miR-125b levels in exosomes were significantly lower in patients with melanoma compared with disease-free patients with melanoma and healthy controls. CONCLUSIONS Exosomes can provide a suitable material to measure circulating miRNA in melanoma, and miR-16 can be used as an endogenous normalizer. Lower levels of miR-125b in exosomes obtained from serum are associated with advanced melanoma disease, probably reflecting the tumoral cell dysregulation.

Evaluation of multiple serum markers in advanced melanoma.

The aim of this retrospective study was to analyse in advanced melanoma the potential tumor markers S-100B, melanoma inhibiting activity protein (MIA) and YKL-40 compared to LDH. Serum levels of S-100B, MIA, LDH and YKL-40 were measured in 110 patients with advanced melanoma (36 in stage IIIB/C and 74 in stage IV), in 66 disease-free patients and in 65 healthy controls. Results show that S-100B, MIA and LDH levels were significantly higher in patients with advanced melanoma than in disease-free patients or healthy controls. The combination of S-100B plus MIA had the best diagnostic sensitivity, and the addition of LDH did not further increase this sensitivity. MIA was an independent prognostic factor of overall survival. Patients with both S-100B and MIA elevated had a significant shorter survival than those with both S-100B and MIA under the cut-off. YKL-40 levels did not differentiate patients with advanced melanoma from controls. We concluded that the combination of MIA plus S-100B showed a better prognostic value in advanced melanoma compared to LDH.

Circulating melanoma exosomes as diagnostic and prognosis biomarkers

BACKGROUND Malignant melanoma is an aggressive cancer with an increasing incidence. Exosomes are actively secreted microvesicles, whose characteristics reflect those of the cell they are originated in. The aim of this study was to identify and evaluate the presence of the melanoma biomarkers MIA, S100B and tyrosinase-related protein 2 (TYRP2) in exosomes and their potential clinical utility. METHODS Serum samples were obtained from stage IV melanoma patients, melanoma-free patients and healthy controls. Exosomes were precipitated and TYRP2, MIA and S100B concentrations were quantified in serum, exosomes, and exosome-free serum. RESULTS Both MIA and S100B were detected in exosomes and correlated significantly with serum concentrations (S100B: r=0.968; MIA: r=0.799; p<0.001). MIA and S100B concentrations in exosomes were significantly higher in melanoma patients than in healthy controls and disease-free patients. However, TYRP2 concentrations in exosomes did not differ between these three groups. ROC curves analysis rendered AUCs for MIA of 0.883 (p<0.01) and of 0.840 for S100B (p<0.01). Patients with exosome MIA concentration higher than 2.5 μg/L showed shorter median survival related to those with lower level (4 versus 11 months; p<0.05). CONCLUSIONS MIA and S100B can be detected in exosomes from melanoma patients and their quantification presents diagnostic and prognostic utility.

Nitric oxide produces HLA-G nitration and induces metalloprotease-dependent shedding creating a tolerogenic milieu

Human leucocyte antigen G (HLA‐G) is a tolerogenic molecule that protects the fetus from maternal immune attack, may favour tumoral immunoescape and is up‐regulated in viral and inflammatory diseases. The aim of this work was to discover if nitric oxide (NO) could affect HLA‐G expression or function because NO is an important modulator of innate and adaptive immunity. For this purpose HLA‐G expression and function were analysed following treatment with a NO donor or a peroxynitrite donor in various cell lines expressing HLA‐G either spontaneously or upon transfection. Results showed NO‐dependent nitration of both cellular and soluble HLA‐G protein, but not all HLA‐G moieties underwent nitration. Endogenous biosynthesis of NO by both U‐937‐HLA‐G1 and M8‐HLA‐G5 stable transfectants also caused HLA‐G nitration. The NO decreased total HLA‐G cellular protein content and expression on the cell surface, while increasing HLA‐G shedding into the culture medium. This effect was post‐transcriptional and the result of metalloprotease activity. By contrast, NO pretreatment did not affect HLA‐G capability to suppress NK cytotoxicity and lymphocyte proliferation. Our studies show that NO regulates the availability of HLA‐G molecules without modifying their biological activities.

Immunosuppression Routed Via the Kynurenine Pathway: A Biochemical and Pathophysiologic Approach

In the past years, it has been shown that kynurenines pathway is a regulator of both the innate and the adaptive immune responses. Particularly, the initial enzyme of this pathway, indoleamine 2,3-dioxygenase (IDO), is implicated in maintaining tolerance during pregnancy, and also can be expressed in tumors to avoid the immune attack. In this chapter, we will describe how the kynurenine pathway affects the immune system with important implications both in physiology and in pathology. The incorrect activation or blockade suppressive properties of the kynurenine pathway are also implicated in a number of other diseases such as AIDS or autoimmune diseases.

MULTIMERIC STRUCTURES OF HLA-G ISOFORMS FUNCTION THROUGH DIFFERENTIAL BINDING TO LILRB RECEPTORS

The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α1–α2–α3 non-covalently bound to β-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α1–α3 structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α1–α3-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.

Linking Two Immuno-Suppressive Molecules: Indoleamine 2,3 Dioxygenase Can Modify HLA-G Cell-Surface Expression

Abstract Nonclassical human leukocyte antigen (HLA) class I molecule HLA-G and indoleamine 2,3 dioxygenase (INDO) in humans and mice, respectively, have been shown to play crucial immunosuppressive roles in fetal-maternal tolerance. HLA-G inhibits natural killer and T cell function by high-affinity interaction with inhibitory receptors, and INDO acts by depleting the surrounding microenvironment of the essential amino acid tryptophan, thus inhibiting T cell proliferation. We investigated whether HLA-G expression and INDO function were linked. Working with antigen-presenting cell (APC) lines and monocytes, we found that functional inhibition of INDO by 1-methyl-tryptophan induced cell surface expression of HLA-G1 by HLA-G1-negative APCs that were originally cell-surface negative, and that in reverse, the functional boost of INDO by high concentrations of tryptophan induced a complete loss of HLA-G1 cell surface expression by APCs that were originally cell-surface HLA-G1-positive. This mechanism was shown to be posttranslational because HLA-G protein cell contents remained unaffected by the treatments used. Furthermore, HLA-G cell surface expression regulation by INDO seems to relate to INDO function, but not to tryptophan catabolism itself. Potential implications in fetal-maternal tolerance are discussed.

In vivo identification of an HLA‐G complex as ubiquitinated protein circulating in exosomes

The nonclassical human leukocyte antigen‐G (HLA‐G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA‐G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA‐G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA‐G by ELISA. Complexed HLA‐G was detected in exosomes, which indicates an intracellular origin of these forms. 2D‐PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA‐G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA‐G complexes. Immunoblot analysis of exudates and exosomes with an anti‐ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA‐G. HLA‐G ubiquitination could be reproduced in vitro in HLA‐G1‐transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA‐G forms in vivo that open a new perspective in the study of HLA‐G function and analysis.

Some Basic Aspects of HLA-G Biology

Human leukocyte antigen-G (HLA-G) is a low polymorphic nonclassical HLA-I molecule restrictively expressed and with suppressive functions. HLA-G gene products are quite complex, with seven HLA-G isoforms, four membrane bound, and other three soluble isoforms that can suffer different posttranslational modifications or even complex formations. In addition, HLA-G has been described included in exosomes. In this review we will focus on HLA-G biochemistry with special emphasis to the mechanisms that regulate its expression and how the protein modifications affect the quantification in biological fluids.