High plasma levels of HLA-G are associated with low birth weight and with an increased risk of malaria in infancy.

BACKGROUND: The immunosuppressive properties of HLA-G protein can create a tolerogenic environment that may allow Plasmodium falciparum to avoid host immune responses. There are known associations between high levels of circulating soluble HLA-G (sHLA-G) and either parasite or viral infections and it has been suggested that the induction of sHLA-G expression could be a mechanism via which infectious agents subvert host immune defence. The study presented here is the first to investigate the possible association between sHLA-G and malaria or malaria related risk factors in Benin. METHODS: A parasitological and clinical follow-up of 165 mothers and their newborns from delivery through to one year of age was conducted in the Tori Bossito area of southern Benin. Plasma levels of sHLA-G were determined by ELISA in maternal peripheral and cord blood and again in infants' peripheral blood at 3, 6, 9 and 12 months of age. The associations between the levels of sHLA-G and malaria risk factors were investigated through multivariate mixed models. RESULTS: Strong correlations were observed between the maternal and cord plasma concentrations of sHLA-G. In multivariate analyses, high cord plasma levels of sHLA-G were independently associated with (i) low birth weight and (ii) an increased risk of P. falciparum infection in infancy. CONCLUSION: These results show for the first time the possible involvement of sHLA-G in generating immune tolerance during pregnancy-associated malaria. Soluble HLA-G may represent a useful marker of susceptibility to malaria in infants and be associated with the higher susceptibility to infection observed for LBW children.

Characterization of monoclonal antibodies recognizing HLA-G or HLA-E: new tools to analyze the expression of nonclassical HLA class I molecules.

Nonclassical major histocompatibility complex (MHC) class I human leukocyte antigen E (HLA-E) and HLA-G molecules differ from classical ones by specific patterns of transcription, protein expression, and immunotolerant functions. The HLA-G molecule can be expressed as four membrane-bound (HLA-G1 to -G4) and three soluble (HLA-G5 to -G7) proteins upon alternative splicing of its primary transcript. In this study, we describe a new set of monoclonal antibodies (mAbs) called MEM-G/01, -G/04, -G/09, -G/13, MEM-E/02, and -E/06 recognizing HLA-G or HLA-E. The pattern of reactivity of these mAbs were analyzed on transfected cells by flow cytometry, Western blotting, and immunochemistry. MEM-G/09 and -G/13 mAbs react exclusively with native HLA-G1 molecules, as the 87G mAb. MEM-G/01 recognizes (similar to the 4H84 mAb) the denatured HLA-G heavy chain of all isoforms, whereas MEM-G/04 recognizes selectively denatured HLA-G1, -G2, and -G5 isoforms. MEM-E/02 and -E/06 mAbs bind the denatured and cell surface HLA-E molecules, respectively. These mAbs were then used to analyze the expression of HLA-G and HLA-E on freshly isolated cytotrophoblast cells, on the JEG-3 placental tumor cell line, and on cryopreserved and paraffin-embedded serial sections of trophoblast tissue. These new mAbs represent valuable tools to study the expression of HLA-G and HLA-E molecules in cells and tissues under normal and pathologic conditions.

Polymorphic sites at the 3' untranslated region of the HLA-G gene are associated with differential hla-g soluble levels in the Brazilian and French population.

HLA-G molecule has well-recognized tolerogenic properties, and the encoding gene shows lower frequency of polymorphism at the coding region but higher variability at regulatory 5' and 3' untranslated (3'UTR) regions. At least three 3'UTR polymorphic sites have been associated with HLA-G mRNA regulation, including the 14 base pair (14bp) Insertion/Deletion, +3142C-G and +3187A-G. We studied the association of polymorphic sites at 3'UTR (sequencing analysis, encompassing the 14bp Ins-Del/+3003T-C/+3010C-G/+3027C-A/+3035C-T/+3142C-G/+3187A-G/+3196C-G polymorphic sites) with plasma soluble HLA-G levels (sHLA-G, detected by ELISA) in 187 French and 153 Brazilian healthy individuals. Allele and genotype frequencies were closely similar in both populations; however, Brazilians showed a higher HLA-G 3'UTR haplotype diversity. Considering sHLA-G levels in both populations altogether, individuals presenting 14bp Del/Del showed higher levels compared to 14bpIns/Ins genotype (P <0.05); those presenting +3010C/G showed higher levels compared to the +3010C-C genotype (P< 0.05); those presenting +3027C-C showed higher levels than the +3027A-A genotype (P< 0.05); and those bearing +3035C-C showed higher levels compared to the +3035C-T (P < 0.01) and +3035T-T (P < 0.05) genotypes. The analyses of 3'UTR haplotypes showed that UTR-1 (DelTGCCCGC) was associated with higher expression of sHLA-G, whereas UTR-5 (InsTCCTGAC) and UTR-7 (InsTCATGAC) with lower expression and other UTRs (UTR-2/3/4/6) exhibited intermediate levels. Since the differential expression of HLA-G may be beneficial or harmful depending on the underlying condition, the identification of individuals genetically programmed to differentially express HLA-G may help on defining novel strategies to control the immune response against the underlying disorder.

Immunosuppressive HLA-G molecule is upregulated in alveolar epithelial cells after influenza A virus infection.

Influenza virus type A (IAV) infections constitute an important economic burden and raise health-care problems. Host defense mechanisms usually clear IAV infections after a few days by exploiting a variety of cellular immune responses. However, increasing the production of immunosubversive molecules is a mechanism by which viruses escape host surveillance. In this regard, the nonclassical HLA class I molecule HLA-G displays strong tolerogenic properties. We show here that several strains of IAV differently upregulate HLA-G expression, at both the mRNA and protein levels, in alveolar epithelial cells. Thus the virulence of IAV may be caused by the capability of different strains to upregulate HLA-G allowing their escape from host immune responses.

Myocardial HLA-G reliably indicates a low risk of acute cellular rejection in heart transplant recipients.

BACKGROUND: Human leukocyte antigen-G (HLA-G), a non-classical MHC I protein with restricted tissue expression, plays an essential role in immune tolerance and has been negatively associated with acute and chronic rejection after heart transplantation. We assessed myocardial HLA-G expression in adult heart transplant patients in an attempt to determine the value of this protein in identifying patients with a low risk of acute cellular rejection. METHODS: Two groups of patients were included in this study. Group A (non-rejecting) included 29 patients who had no moderate or severe rejection episodes (ISHLT Grade <2R) post-transplant. Group B (rejecting) included 38 patients with at least two moderate or severe rejection episodes (Grade >or=2R) within a 1-year period. Expression of HLA-G in three myocardial biopsies post-transplant from each patient was determined through immunohistochemical staining. RESULTS: In Group A, 86% of patients had HLA-G(+) biopsies compared with 11% of patients in Group B (p < 0.001; sensitivity 86%, specificity 87%). Whereas 60% of non-rejecting HLA-G(+) patients had at least two positive biopsies, all rejecting HLA-G(+) patients had only one positive biopsy. CONCLUSIONS: There is a significant negative association between myocardial HLA-G expression and acute cellular rejection after cardiac transplantation. Detection of HLA-G appears to reliably indicate a low risk of developing moderate or severe allograft rejection and may, subsequently, allow for a reduced immunosuppressive regimen.

Soluble human leukocyte antigen-G5 in septic shock: marked and persisting elevation as a predictor of survival.

OBJECTIVE: Although it is established that septic shock induces immunosuppression, the mechanisms for this phenomenon remain poorly understood. Human leukocyte antigen-G exerts strong inhibitory effects that are directed at different arms of the immune system. The main objective of the current study was to measure human leukocyte antigen-G (soluble and membrane proteins) in septic shock. DESIGN: Observational study. SETTING: Adult intensive care units in a university hospital. PATIENTS: Sixty-four consecutive patients with septic shock (7 days of follow-up). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We measured plasma human leukocyte antigen-G5 (with enzyme linked immunosorbent assay) and human leukocyte antigen-G1 (with flow cytometry) expression on circulating leukocytes. As early as days 1-2 after the onset of shock, we observed a marked elevation of soluble human leukocyte antigen-G5 in patients: 60 ng/mL (34-146) as median (Q1-Q3) (reference values <5 ng/mL). This increase was stable over time. Most important, we also found at days 1-2 a significant difference between survivors and nonsurvivors: 109 ng/mL (43-183) vs. 37 ng/mL (19-61), respectively (p = .003). This difference remained significant until day 7. Receiver operating characteristic curve analysis showed that human leukocyte antigen-G5 was a good predictor of outcome (areas under curves: 0.76 and 0.84 at days 1-2 and days 3-4, respectively, p < .001). Adjusted logistic regression analysis suggested that human leukocyte antigen-G5 at days 3-5 was a better prognostic marker than decreased monocyte human leukocyte antigen-DR and/or severity score. CONCLUSIONS: The present results show a marked elevation of soluble human leukocyte antigen-G5 protein during septic shock. We may hypothesize that given its potent inhibitory properties and its association with survival, human leukocyte antigen-G5 has an important role in the numerous negative feedback signals that limit the process of inflammation during septic shock.

CD3+CD4low and CD3+CD8low are induced by HLA-G: novel human peripheral blood suppressor T-cell subsets involved in transplant acceptance.

HLA-G is a tolerogenic molecule whose detection in sera and within allografted tissues is associated with better graft acceptance. HLA-G mediates T-cell differentiation into suppressor cells, which are thought to promote tolerance. Here, we investigated such T cells phenotypically and functionally and assessed their clinical relevance in the peripheral blood of patients who have undergone transplantation. Our results demonstrate that HLA-G expressed by antigen-presenting cells or present as soluble protein down-regulates the expression of CD4 and CD8 on allostimulated T cells at both transcriptional and posttranslational levels. These CD3(+)CD4(low) and CD3(+)CD8(low) T-cell subsets are characterized by an increased proportion of cells expressing CD45RA and HLA-DR, and a decreased number of cells expressing CD62L. In addition, these HLA-G-induced CD3(+)CD4(low) and CD3(+)CD8(low) subpopulations are Foxp3-negative suppressor T cells whose function involves IL-10. Biologic relevance came from analysis of patients who underwent transplantation, with high HLA-G plasma concentrations associated with better graft survival. Peripheral blood from these patients contains increased levels of IL-10 concomitantly to an enhanced representation of CD3(+)CD4(low) and CD3(+)CD8(low) T cells compared with HLA-G-negative patients who underwent transplantation and healthy individuals. These data define novel immunosuppressive subpopulations of peripheral blood T cells induced by HLA-G with potent implications in peripheral tolerance.

Evidence to support the role of HLA-G5 in allograft acceptance through induction of immunosuppressive/ regulatory T cells.

The soluble HLA-G5 isoform encoded by intron-4 retaining spliced transcript has been previously detected in vivo in sera and grafts from transplanted patients who had significantly better graft acceptance. These findings led us to investigate the role of HLA-G5 in tolerance induction in vitro and its biological relevance in allograft acceptance in vivo. We demonstrated that engagement of Ig-like transcript-2 and Ig-like transcript-4 receptors by HLA-G5 is involved in inhibition of T cell alloproliferative responses. Naive T cells sensitized in vitro with HLA-G5, for as little as 18 h, 1) lost their ability to respond to subsequent allogeneic stimulus, and 2) acquired regulatory properties because they inhibited the reactivity of other T cells. These HLA-G5-induced T cells act in an Ag-nonspecific fashion and through soluble factors. Biological relevance was provided by ex vivo analyzes of samples from liver-kidney cotransplanted patients who had high HLA-G5 serum levels and no graft rejection. We showed that addition of HLA-G5-containing sera from these patients inhibited T cell alloresponses and that serum HLA-G5 was responsible for this inhibition. Notably, PBMC from transplanted patients exposed to high levels of circulating HLA-G5 did not respond to allostimulation and inhibited alloreactivity of other T cells. These results demonstrate that HLA-G5-mediated tolerance involves the induction of immunosuppressive T cells. These findings provide evidence supporting the tolerogenic properties of HLA-G and emphasize its potential application as a relevant therapeutic candidate capable of limiting allograft rejection.

HLA-G1-expressing antigen-presenting cells induce immunosuppressive CD4+ T cells.

We recently reported that HLA-G1-transfected antigen-presenting cells (HLA-G1+ APCs) were capable of inhibiting alloproliferative responses. The aim of the present work was to further study the function and the mechanisms of action of HLA-G1+ APCs. We show here that HLA-G1+ APCs are immunoinhibitory cells that (i) inhibit the proliferation of CD4+ T cells, (ii) shed HLA-G1 molecules that might provide extra, non-antigen-specific, inhibitory or proapoptotic signals, (iii) induce CD4+ T cell anergy, or at least long-term unresponsiveness, and (iv) cause the differentiation of CD4+ T cells into suppressive cells. Thus, HLA-G+ APCs might (i) be involved in the direct suppression of immune responses and (ii) contribute to long-term efficient immune escape or tolerance.