Soluble HLA-G levels in heart transplant recipients: Dynamics and correlation with clinical outcomes.

PURPOSE: To describe the evolution of the serum levels of soluble HLA-G (s-HLA-G) during the first 12 months after heart transplantation (HT) and to correlate it with clinical outcomes. METHODS: Observational study based in a single-center cohort of 59 patients who underwent HT between December-2003 and March-2010. Soluble HLA-G levels were measured from serum samples extracted before HT, and 1, 3, 6 and 12 months after HT. The cumulative burden of s-HLA-G expression during the first post-transplant year was assessed by means of the area under the curve (AUC) of s-HLA-G levels over time and correlated with the acute rejection burden -as assessed by a rejection score-, the presence of coronary allograft vasculopathy (CAV) grade ≥ 1 and infections during the first post-transplant year; as well as with long-term patient and graft survival. Mean follow-up was 12.4 years. RESULTS: Soluble HLA-G levels decreased over the first post-transplant year (p = 0.020). The AUC of s-HLA-G levels during the first post-transplant year was higher among patients with infections vs. those without infections (p = 0.006). No association was found between the AUC of s-HLA-G levels and the burden of acute rejection or the development of CAV. Overall long-term survival, long-term survival free of late graft failure and cancer-free survival were not significantly different in patients with an AUC of s-HLA-G levels higher or lower than the median of the study population. CONCLUSIONS: Soluble HLA-G levels decreased over the first year after HT. Higher HLA-G expression was associated with a higher frequency of infections, but not with the burden of acute rejection or the development of CAV, neither with long-term patient or graft survival.

The HLA-G immune checkpoint: a new immuno-stimulatory role for the α1-domain-deleted isoform.

The heterogeneity of cancer cells, in part maintained via the expression of multiple isoforms, introduces significant challenges in designing effective therapeutic approaches. In this regard, isoforms of the immune checkpoint HLA-G have been found in most of the tumors analyzed, such as ccRCC, the most common human renal malignancy. In particular, HLA-G∆α1, which is the only HLA-G isoform described that lacks the α1 extracellular domain, has been newly identified in ccRCC and now here in trophoblasts. Using a cellular model expressing HLA-G∆α1, we have uncovered its specific and overlapping functional roles, relative to the main HLA-G isoform, i.e., the full-length HLA-G1. We found that HLA-G∆α1 has several particular features: (i) although possessing the α3 domain, it does not associate with ß2-microglobulin; (ii) it may not present peptides to T cells due to absence of the peptide-binding groove; and (iii) it exerts immune-stimulatory activity towards peripheral blood NK and T cells, while all known isoforms of HLA-G are immune-inhibitory checkpoint molecules. Such immune-stimulatory properties of HLA-G∆α1 on the cytotoxic function of peripheral blood NK cells are individual dependent and are not exerted through the interaction with the known HLA-G receptor, ILT2. Importantly, we are faced here with a potential antitumor effect of an HLA-G isoform, opposed to the pro-tumor properties described for all other HLA-G isoforms, which should be taken into account in future therapeutic designs aimed at blocking this immune checkpoint.

Predicting the most deleterious missense nsSNPs of the protein isoforms of the human HLA-G gene and in silico evaluation of their structural and functional consequences.

BACKGROUND: The Human Leukocyte Antigen G (HLA-G) protein is an immune tolerogenic molecule with 7 isoforms. The change of expression level and some polymorphisms of the HLA-G gene are involved in various pathologies. Therefore, this study aimed to predict the most deleterious missense non-synonymous single nucleotide polymorphisms (nsSNPs) in HLA-G isoforms via in silico analyses and to examine structural and functional effects of the predicted nsSNPs on HLA-G isoforms. RESULTS: Out of 301 reported SNPs in dbSNP, 35 missense SNPs in isoform 1, 35 missense SNPs in isoform 5, 8 missense SNPs in all membrane-bound HLA-G isoforms and 8 missense SNPs in all soluble HLA-G isoforms were predicted as deleterious by all eight servers (SIFT, PROVEAN, PolyPhen-2, I-Mutant 3.0, SNPs&GO, PhD-SNP, SNAP2, and MUpro). The Structural and functional effects of the predicted nsSNPs on HLA-G isoforms were determined by MutPred2 and HOPE servers, respectively. Consurf analyses showed that the majority of the predicted nsSNPs occur in conserved sites. I-TASSER and Chimera were used for modeling of the predicted nsSNPs. rs182801644 and rs771111444 were related to creating functional patterns in 5'UTR. 5 SNPs in 3'UTR of the HLA-G gene were predicted to affect the miRNA target sites. Kaplan-Meier analysis showed the HLA-G deregulation can serve as a prognostic marker for some cancers. CONCLUSIONS: The implementation of in silico SNP prioritization methods provides a great framework for the recognition of functional SNPs. The results obtained from the current study would be called laboratory investigations.

NKG2A/CD94 Is a New Immune Receptor for HLA-G and Distinguishes Amino Acid Differences in the HLA-G Heavy Chain.

Natural killer (NK) cell therapies are a tool to antagonize a dysfunctional immune system. NK cells recognize malignant cells, traffic to a tumor location, and infiltrate the solid tumor. The immune checkpoint molecule human leukocyte antigen (HLA)-G is upregulated on malignant cells but not on healthy surrounding cells, the requirement of understanding the basis of receptor mediated events at the HLA-G/NK cell interface becomes obvious. The NK cell receptors ILT2 and KIR2DL4 have been described to bind to HLA-G; however, their differential function and expression levels on NK cell subsets suggest the existence of an unreported receptor. Here, we performed a ligand-based receptor capture on living cells utilizing sHLA-G*01:01 molecules coupled to TriCEPS and bound to NK cells followed by mass spectrometric analyses. We could define NKG2A/CD94 as a cognate receptor of HLA-G. To verify the results, we used the reciprocal method by expressing recombinant soluble heterodimeric NKG2A/CD94 molecules and used them to target HLA-G*01:01 expressing cells. NKG2A/CD94 could be confirmed as an immune receptor of HLA-G*01:01. Despite HLA-G is marginal polymorphic, we could previously demonstrate that the most common allelic subtypes HLA-G*01:01/01:03 and 01:04 differ in peptide repertoire, their engagement to NK cells, their catalyzation of dNK cell proliferation and their impact on NK cell development. Continuing these studies with regard to NKG2A/CD94 engagement we engineered recombinant single antigen presenting K562 cells and targeted the surface expressed HLA-G*01:01, 01:03 or 01:04 molecules with NKG2A/CD94. Specificity and sensitivity of HLA-G*01:04/NKG2A/CD94 engagement could be significantly verified. The binding affinity decreases when using K562-G*01:03 or K562-G*01:01 cells as targets. These results demonstrate that the ligand-receptor assignment between HLA-G and NKG2A/CD94 is dependent of the amino acid composition in the HLA-G heavy chain. Understanding the biophysical basis of receptor-mediated events that lead to NK cell inhibition would help to remove non-tumor reactive cells and support personalized mild autologous NK cell therapies.

Soluble HLA-G expression levels and HLA-G/irinotecan association in metastatic colorectal cancer treated with irinotecan-based strategy.

We here explore the soluble Human Leukocyte Antigen-G (sHLA-G) expression level as clinical biomarker in metastatic colorectal cancer (mCRC). To this aim the sHLA-G protein was measured in plasma samples of 40 patients with mCRC treated with the FOLFIRI (irinotecan (CPT-11) plus 5-fluorouracil (5-FU) and leucovorin (LV)) regimen. The results suggest a link between HLA-G levels and irinotecan (CPT-11) pharmacokinetic, leading to hypothesize a molecular interaction between sHLA-G and CPT-11. This interaction was confirmed experimentally by fluorescence spectroscopy. HLA-G is known to exist in a number of polymorphs that affect both the protein expression levels and its peptide-binding cleft. The interaction between HLA-G polymorphs and CPT-11 was explored by means of computational modelling, confirming the hypothesis that CPT-11 could actually target the peptide binding cleft of the most common HLA-G polymorphs.

Structures of the four Ig-like domain LILRB2 and the four-domain LILRB1 and HLA-G1 complex.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs), also known as CD85 and immunoglobulin-like transcripts (ILTs), play pivotal roles in regulating immune responses. These receptors define an immune checkpoint that immune therapy can target. Through cis or trans interactions with human leukocyte antigen (HLA)-G, the two most abundantly expressed inhibitory LILRs, LILRB1, and LILRB2 (LILRB1/2, also known as CD85j/d and ILT2/4), are involved in immunotolerance in pregnancy and transplantation, autoimmune diseases, and immune evasion by tumors. Although the discrete domains of LILRB1/2 are clear, the assembly mode of the four extracellular Ig-like domains (D1, D2, D3, and D4) remains unknown. Previous data indicate that D1D2 is responsible for binding to HLA class I (HLA-I), but the roles of D3D4 are still unclear. Here, we determined the crystal structure of the four Ig-like domain LILRB2 and four-domain LILRB1 in complex with HLA-G1. The angles between adjacent domains and the staggered assembly of the four domains suggest limited flexibility and limited plasticity of the receptors during ligand binding. The complex structure of four-domain LILRB1 and HLA-G1 supports the model that D1D2 is responsible for HLA-I binding, while D3D4 acts as a scaffold. Accordingly, cis and trans binding models for HLA-I binding to LILRB1/2 are proposed. The geometries of LILRB1/2 in complex with dimeric and monomeric HLA-G1 suggest the accessibility of the dimeric receptor, which in turn, transduces more inhibitory signals. The assembly of LILRB1/2 and its binding to HLA-G1 could aid in the design of immune regulators and benefit immune interference.

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.

Evaluation of the Reactivity and Receptor Competition of HLA-G Isoforms toward Available Antibodies: Implications of Structural Characteristics of HLA-G Isoforms.

The human leucocyte antigen (HLA)-G, which consists of seven splice variants, is a tolerogenic immune checkpoint molecule. It plays an important role in the protection of the fetus from the maternal immune response by binding to inhibitory receptors, including leukocyte Ig-like receptors (LILRs). Recent studies have also revealed that HLA-G is involved in the progression of cancer cells and the protection from autoimmune diseases. In contrast to its well characterized isoform, HLA-G1, the binding activities of other major HLA-G isoforms, such as HLA-G2, toward available anti-HLA-G antibodies are only partially understood. Here, we investigate the binding specificities of anti-HLA-G antibodies by using surface plasmon resonance. MEM-G9 and G233 showed strong affinities to HLA-G1, with a nM range for their dissociation constants, but did not show affinities to HLA-G2. The disulfide-linker HLA-G1 dimer further exhibited significant avidity effects. On the other hand, 4H84 and MEM-G1, which can be used for the Western blotting of HLA-G isoforms, can bind to native HLA-G2, while MEM-G9 and G233 cannot. These results reveal that HLA-G2 has a partially intrinsically disordered structure. Furthermore, MEM-G1, but not 4H84, competes with the LILRB2 binding of HLA-G2. These results provide novel insight into the functional characterization of HLA-G isoforms and their detection systems.

A soluble recombinant form of human leucocyte antigen-G 6 (srHLA-G6).

Human Leucocyte Antigen-G (HLA-G) is a non classical major histocompatibility complex (MHC) molecule that through RNA splicing can encode seven isoforms which are membrane bound (-G1, -G2, -G3 and -G4) and soluble (-G5, -G6 and -G7). HLA-G is described as important immune suppressor endogenous molecule to favor maternal-fetal tolerance, transplant survival and tumor immune scape. HLA-G shows low protein variability and a unique structural complexity that is related with the expression of different isoforms followed by biochemical processes, such as, proteolytic cleavage, molecular interactions, and protein ubiquitination. Studies with HLA-G have shown difficult to assess the role of the individual isoforms. Thus, the aim of this work was to obtain a HLA-G6 recombinant form. The results indicated the production of high homogeneous preparations of soluble recombinant HLA-G6 (srHLA-G6) with molecular mass 23,603.76 Da, determined by MALD-TOF/TOF. In addition, native and denatured srHLA-G6 were detected by ELISA, using commercial monoclonal antibodies. Finally, we developed a suitable methodology to express srHLA-G6 that could contribute in structural and functional studies involving specific isoforms.

HLA-E regulates NKG2C+ natural killer cell function through presentation of a restricted peptide repertoire.

UNLABELLED: NK cells interact with the HLA-E molecule via the inhibitory receptor NKG2A and the activating receptor NKG2C. Hence, HLA-E can have a dual role in the immune response. In the present study, we aim to investigate the functional consequences of HLA-E for NKG2A and NKG2C expressing NK cell subsets by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. PBMC derived from healthy subjects were used as targets for isolated NK cells and NK cell activation was examined by analysis of the expression of the degranulation marker CD107a. Peptide induced HLA-E expression inhibited degranulation of NKG2A+ NK cell subsets with almost all peptides, whereas NKG2A- NKG2C+ NK cell responses were enhanced only after incubation with four peptides; 1.3-fold with CMV(I), A80 and B13 and 3.2-fold with HLA-G derived peptide. In addition, the HLA-E:G peptide complex triggered NKG2C receptor internalization, as evidenced by reduction in the percentage of NKG2C+ NK cells when incubated with the peptide, which could be restored by addition of Bafilomycin. IN CONCLUSION: in contrast to NKG2A, NKG2C is regulated by HLA-E only when HLA-E is in complex with a restricted peptide repertoire, especially in combination with the HLA-G leader peptide.

Association between sHLA-G and HLA-G 14-bp deletion/insertion polymorphism in Crohn's disease.

The aim of this study was to evaluate the association between the HLA-G 14-bp deletion/insertion (Del/Ins) polymorphism and soluble (s) HLA-G production in patients with Crohn's disease (CD). We analyzed also the sHLA-G molecules by ELISA and western blot in plasma samples. Among unselected patients, the 14-bp Del/Ins polymorphism was not significantly associated with increased CD risk neither for alleles (P = 0.371) nor for genotypes (P = 0.625). However, a significant association was reported between the 14-bp Del/Ins polymorphism and CD, in particular in young-onset CD patients for alleles [P = 0.020, odds ratio (OR) = 2.438, 95% confidence interval (CI): 1.13-5.25] but not with adult-onset CD patients. A significant association was reported concerning the genotype Ins/Ins for young-onset CD patients (P = 0.029, OR = 3.257, 95% CI: 1.08-9.77). We observed also a significant increase in sHLA-G measured by ELISA in CD patients compared to controls (P = 0.002). The 14-bp Del/Del and 14-bp Del/Ins genotypes are the high HLA-G producers. Among sHLA-G(positive) patients, 43% of subjects present dimers of HLA-G. The presence of dimers seems to be related to the advanced stages of the disease. The 14-bp Del/Ins polymorphism is associated with an increased risk of CD particularly in young-onset CD patients and controls sHLA-G plasma levels. Dimers of sHLA-G are frequent in advanced disease stages. The above findings indicate that the genetic 14-bp Del/Ins polymorphism in exon 8 of the HLA-G gene is associated with the risk of CD and suggest a role for sHLA-G as a prognostic marker for progressive disease.

[HLA-G: from feto-maternal tolerance to organ acceptance]. / HLA-G : de la tolérance fœto-maternelle à l'acceptation d'organe.

HLA-G is a nonclassical class I molecule that differs from classical antigens by its restricted expression, very low polymorphism, expression of 7 different protein isoforms, and immune tolerance-inducing activity. HLA-G plays a key role in feto-maternal tolerance. Its interaction with three specific receptors expressed on immune cells (T, B, natural killer and antigen-presenting cells) allows it to act at all levels of the immune response. HLA-G can also be expressed by tumor cells and their microenvironment, endowing them with significant local tolerance. The same is true in some inflammatory and viral diseases.

Soluble human leukocyte antigen-g5 activates extracellular signal-regulated protein kinase signaling and stimulates trophoblast invasion.

Soluble human leukocyte antigen-G (HLA-G) is a non-classical class Ib HLA molecule that is secreted from blastocysts. Soluble HLA-G modulates the immune tolerance of the mother and can be used as a prognostic factor for the clinical pregnancy rate. However, the underlying mechanism of how soluble HLA-G5 affects pregnancy remains largely unknown. We hypothesized that soluble HLA-G5 promotes successful implantation and pregnancy by modulating trophoblast invasion through receptor binding and activation of extracellular signal-regulated protein kinase (ERK) signaling pathway. Recombinant HLA-G5 protein over-expressed in E. coli BL21 was purified to near homogeneity. We studied the expression of HLA-G5 and its receptors, the leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) and killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), in primary trophoblasts and trophoblastic (JAr and JEG-3) cell lines by florescence-labeled HLA-G5. HLA-G5 was detected in the primary trophoblasts and JEG-3 cells. The LILRB1 and KIR2DL4 receptors were expressed in both primary trophoblasts and trophoblastic cell lines. HLA-G5 stimulated cell invasion (p<0.05) and increased urokinase (uPA) and matrix metalloproteinases (MMPs) transcripts and their activity (p<0.05) in trophoblastic cells. HLA-G5 activated the ERK signaling pathway and induced ERK1/2 phosphorylation in the trophoblastic cell lines. Addition of ERK inhibitors (U0126 and PD98059) nullified the stimulatory effect of HLA-G5 on trophoblastic cell invasion. Taken together, HLA-G5 induced trophoblast invasion by binding to KIR2DL4 and LILRB1, by increasing uPA and MMPs expressions and by activating the ERK signaling pathway.

Quantified colocalization reveals heterotypic histocompatibility class I antigen associations on trophoblast cell membranes: relevance for human pregnancy.

Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G, -E, and -F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C, -E, and -G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G, -E, and -C colocalize with each other and have the potential to form preferential heterotypic associations.

HLA-G regulates the invasive properties of JEG-3 choriocarcinoma cells by controlling STAT3 activation.

The expression of human leucocyte antigen-G (HLA-G) in trophoblasts plays a crucial role in successful embryonic implantation, and reduced HLA-G expression might contribute to adverse obstetric outcomes. In this study, we silenced HLA-G expression using RNA interference in JEG-3 cells, resulting in a notably attenuated invasion capacity of the cells in a Transwell assay; however, no alterations in cell proliferation or apoptosis were observed. The down-regulation of HLA-G dampened the activation of signal transducer and activator of transcription 3 (STAT3), whereas the up-regulation of HLA-G promoted STAT3 activation and invasion in JEG-3 cells treated with human galectin-1. Most importantly, interleukin-6 (IL-6), but not galectin-1, was shown to rescue invasion deficiency in a dose-dependent manner. Thus, we demonstrate that HLA-G is able to regulate JEG-3 cell invasion by influencing STAT3 activation, which may underlie the implantation defects accompanying HLA-G hypo-expression in pre-eclampsia.

Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding.

Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.

Synthetic HLA-G proteins for therapeutic use in transplantation.

The human leukocyte antigen (HLA)-G is a tolerogenic molecule, whose expression by allografts is associated with better acceptance. An increasing interest in producing HLA-G as a clinical-grade molecule for therapy use is impaired by its complexity and limited stability. Our purpose was to engineer simpler and more stable HLA-G-derived molecules than the full-length HLA-G trimolecular complex that are also tolerogenic, functional as soluble molecules, and compatible with good manufacturing practice (GMP) production conditions. We present two synthetic molecules: (α3-L)x2 and (α1-α3)x2 polypeptides. We show their capability to bind the HLA-G receptor LILRB2 and their functions in vitro and in vivo. The (α1-α3)x2 polypeptide proved to be a potent tolerogenic molecule in vivo: One treatment of skin allograft recipient mice with (α1-α3)x2 was sufficient to significantly prolong graft survival, and four weekly treatments induced complete tolerance. Furthermore, (α1-α3)x2 was active as a soluble molecule and capable of inhibiting the proliferation of tumor cell lines, as does the full length HLA-G trimolecular complex. Thus, the synthetic (α1-α3)x2 polypeptide is a stable and simpler alternative to the full-length HLA-G molecule. It can be produced under GMP conditions, it functions as a soluble molecule, and it is at least as tolerogenic as HLA-G in vivo.

HLA-G 2012 conference: the 15-year milestone update.

The non-classical human leukocyte antigen (HLA) Class I molecule HLA-G is best known for its tolerogenic function at the maternal-fetal interface, where it protects the fetus from destruction by the immune system of its mother. Yet, HLA-G has been the topic of intense investigations and its functions reach much further than originally believed. International conferences on HLA-G have taken place every 3 years since 1998, and the Sixth International Conference on HLA-G, that took place in Paris in July 2012. It counted 180 attendees from 28 countries, 35 speakers in plenary sessions, and 63 presentations of research in symposia and poster sessions, bringing new insight in HLA-G research. Here we summarize the major advances on the function and nature of HLA-G molecule that were reported, with particular interest on the findings in new mechanisms of action through regulatory cells, its relevance in cancer as well as in the molecular structure and functions of HLA-G, which are key for its clinical application.

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 ß-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.

Reassessment of HLA-G isoform specificity of MEM-G/9 and 4H84 monoclonal antibodies.

Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule thought to play a key role in maternal-fetal tolerance. Although initial studies suggested that HLA-G expression is restricted to extravillous cytotrophoblasts, expression was subsequently reported in a wide variety of other human tissues and tumor cells. However, consensus as to the validity of these collective findings has proven difficult because the antibodies used to define the temporal and spatial expression patterns of HLA-G remain incompletely characterized. The aim of our study was to reassess two of the most widely used HLA-G antibodies (MEM-G/9 and 4H84) in HLA-G-positive (JEG-3 and HLA-G transduced) and -negative (dermal fibroblast, mesenchymal stem cell, K562, and Jurkat) lines using flow cytometry, immunofluorescence, and western blotting. We found that MEM-G/9 recognized HLA-G3 by flow cytometry, indicating that its epitope is present on the α1 domain of HLA-G. Although 4H84 preferably recognized unfolded HLA-G-free chains, it showed strong non-specificity under certain methodological conditions.