Is antigen presentation the primary function of HLA-G?

Human histocompatibility leukocyte antigen (HLA)-G is an antigen-presenting molecule. This review discusses the possibility that this might not be its primary function. HLA-G indeed modulates innate immunity by interacting with immunoglobulin-like receptors and by regulating HLA-E expression and its subsequent interaction with CD94/NKG2 receptors. HLA-G also down-modulates both CD8(+) and CD4(+) T-cell responsiveness.

The full length HLA-G1 and no other alternative form of HLA-G is expressed at the cell surface of transfected cells.

In contrast to HLA class Ia, the HLA-G class Ib transcripts can be alternativeley spliced to yield several isoforms including four potentially membrane-bound variants, namely HLA-G1, -G2, -G3 and G4. It is so far unclear whether each of these splice variants lacking one or two external domains is properly translated and expressed at the cell surface. We used targeted Enhanced Green Fluorescence Protein (EGFP)-HLA-G fusion cDNA to track HLA-G isoform expression in living murine (L-human beta2m) and human (JAR) transiently transfected cells. It was demonstrated that the four HLA-G1, -G2, -G3, and -G4 isoforms were translated in these transfectants by the means of (i) Western blotting analysis, using an anti-EGFP mAb; (ii) intracellular double labeling flow cytometry analysis, using the EGFP natural fluorescence and phycoerythrin-labeled HCA2 anti-HLA-G mAb; and (iii) immunocytochemistry on isolated acetone fixed transfectants with the use of different anti-HLA-G mAbs. Cell surface flow cytometry analysis using the HCA2 mAb revealed that only the HLA-G1 isoform was expressed as a membrane-bound protein. Two color confocal microscopy performed on fixed, permeabilized cells further showed that the EGFP green fluorescence co-localized with anti-calnexin rhodamine fluorescence in the four HLA-G isoform transfectants but only in HLA-G1 transfectant was the green EGFP fluorescence also detectable at the outer part of the cells, suggesting that the HLA-G2, -G3, and G4 were retained in the endoplasmic reticulum. Such intracellular retention of the three shorter forms of HLA-G suggest that they may play a role in regulating cell surface expression either of the full length HLA-G1 form or of HLA-E.

Soluble HLA-G1 at the materno-foetal interface--a review.

HLA-G differs from the other MHC class I genes. This includes a unique promoter region, a restricted constitutive tissular distribution, the translation of different membrane-bound and soluble isoforms, a shortened cytoplasmic tail and a minimal polymorphim. Soluble HLA-G1 is an immunosuppressive molecule inducing apoptosis of activated CD8(+) T cells and down-modulating CD4(+) T cell proliferation. Soluble HLA-G1 may also contribute to the control of implantation.

Macro and microheterogeneity in normal endothelial cells: differential composition of luminal glycocalyx and functional implications.

Endothelial cells (EC) are involved in various physiological and pathological processes through the expression of their surface glycoproteins. They are covered by the glycocalyx, composed of glucidic residues from cell surface membrane glycoproteins, glycoplipids and proteoglycans. Glucidic sequences can be specifically characterized by their binding to lectins. Eight lectins were used to investigate the distribution and regulation of EC surface glucidic residues in various blood vessels of adult and newborn pigs. EC lectin binding was compared to von Willebrand factor (vWF) expression as EC reference marker. Six out of eight lectins (BSI-B4, DBA, EEA, HP, MAL I and PNA) were helpful for this determination. Considering only the intensity of labelings, vWF and DBA gave the best stainings of adult pig ECs. In newborn pigs, the best labelings were obtained with EEA and MAL I. Furthermore, the distribution of lectin binding to ECs and EC vWF expression was heterogeneous depending on the EC location along vascular tree and age. Beside this macroheterogeneity this study highlights a microheterogeneity of EC lectin binding and vWF expression in situ, defined as a staining of equal intensity by individual ECs, scattered among negative ones, in a given vascular segment. EC surface sugar residues were differently modulated in newborn and adult pig ECs and differently according to EC vWF expression. The functional involvement of EC glycocalyx was reflected by EC lectin binding in the spleen and liver. This study emphasizes the high level of EC heterogeneity for various markers. The EC macro- and microheterogeneity reflect the "plasticity" or "unstability" of EC phenotypes and suggests that ECs are subject to several levels of regulation and are probably grouped in functional clusters to best adjust their functions to microenvironmental requirements. This concept must be considered in further investigations notably in in vitro studies where EC phenotype can be altered.

[Phadiatop in the diagnosis of skin allergy to pneumoallergens]. / Le phadiatop dans le diagnostic de l'allergie cutanée aux pneumallergènes.

Usually, Phadiotop is presented as an in vitro multitest to pneumoallergens that are indicated in respiratory allergy. Now, in skin allergy atopic dermatitis and reaginic urticaria-Quincke's oedema, the responsibility of pneumoallergens, particularly mites, is clear. 47 Subjects were studied. Clinical history, skin test nd specific IgE gave confirmation of allergy to pneumoallergens. This was proved by the subsequent clinical development, with, in most cases, spectacular improvement after desensitization.8 section. We have compared Phadiotop with these different clinical criteria, skin tests and Cap Rast IgE. The results favour Phadiotop and confirm its value and indication in skin allergy to pneumoallergens.

[Multitest Fx5 in food allergy]. / Le Multitest Fx5 dans l'allergie alimentaire.

A comparative study was made of three in vivo and in vitro diagnostic methods for food allergy: Fx5 multitest (Pharmacia); Measurement of specific IgE CAP (Pharmacia); Skin tests (Prick Tests). 20 patients, from 3 to 71 years (mean 24.4 years), were selected by clinical suggestion (asthma, rhinitis, atopic dermatitis, urticaria and/or Quincke's oedema). The Fx5 test used six food allergens: wheat, egg, cow's milk, soya, peanut and fish. The Cap Rast for each substance was evaluated, as was Fx5, by a radio-immunological method. The Prick Tests made with the six allergens used were considered to be positive when the diameter of the weal was greater than that produced by a reference test with histamine. The results were considered as a comparison between Fx5 and Cap Rast to each of the foods, between Fx5 and prick Test with five foods and finally between CAP RAST and Prick Test. Correlation Fx5--Cap rast was better and more useful in the diagnosis of food allergy than skin tests.

HLA-G unique promoter region: functional implications.

In contrast to the highly polymorphic HLA class Ia genes that exhibit a broad somatic tissue distribution, the restricted constitutive expression of HLA-G to trophoblast and a subset of thymic epithelial cells suggests tight transcriptional control of this MHC class Ib gene. Transactivation of MHC class I genes is mediated by three major regulatory modules present in their promoter region namely enhancer A, ISRE, and SXY. The 220-bp promoter sequence of HLA-G comprises modified enhancer A and SXY modules and lacks the ISRE which renders this gene unresponsive to NK-kappaB, IRF1, and class II transactivator DNA-binding factors. A number of other HLA-G upstream regulatory elements have recently been described. Using different transgenic HLA-G mouse models under the control of the HLA-G promoter, several groups have shown by in situ hybridization and/or qualitative or quantitative RT-PCR that constitutive HLA-G transcriptional expression in placental tissue decreased with gestational time. This suggests that once the placenta is fully formed, the functions of HLA-G might not be so crucial.

Placental HLA-G protein expression in vivo: where and what for?

In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.

Comparative reactivity of different HLA-G monoclonal antibodies to soluble HLA-G molecules.

Different HLA-G monoclonal antibodies (mAbs) were first evaluated for their capability to identify soluble HLA-G (sHLA-G) in ELISA. Three of them, namely 87G, BFL.1 and MEM-G/9, when used as coating mAbs together with W6/32 capture mAb, identified beta2-microglobulin (beta2m)-associated-sHLA-G but not soluble HLA-B7 (sHLA-B7) in cell culture supernatants from transfected cells. By comparison, the anti-HLA class I mAb 90 did recognize both sHLA-G and sHLA-B7. By using these HLA-G mAbs, sHLA-G was identified in amniotic fluids as well as in culture supernatants of first trimester and term placental explants but not in cord blood. Intron 4-retaining sHLA-G isoforms were identified in some amniotic fluids by the use of an intron 4-specific mAb (16G1). Reactivity of these different HLA-G mAbs was then compared to determine their respective binding sites on soluble and membrane-bound HLA-G. Using both ELISA and flow cytometry analysis, we showed that they did not compete with each other, which suggested that they did not recognize the same determinants. Finally, we report that two mAbs directed against the alpha1 domain of HLA class I heavy chain (mAb 90 and YTH 862) did compete with 87G, therefore demonstrating that this latter mAb recognized an epitope localized on this external domain of HLA-G.