A physical linkage map of HLA-A, -G, -7.5p, and -F

The class I region of the human leukocyte antigen (HLA) complex includes genes encoding the classical transplantation antigens (HLA-A, -B, -C), at least three nonclassical class I genes (HLA-E, -F, and -G), and many class I pseudogenes (including HLA-7.5p). We have used probes from DNA within or flanking the HLA -A, -F, -G, and -7.5p genes to construct a physical linkage map that places the HLA-F, -G, and -7.5p loci in order with respect to HLA-A. The map was constructed using clamped homogeneous electric field pulsed-field gel electrophoresis. DNA was isolated from LCL 721 (A1:B8, A2:B5), a human Epstein-Barr virus-transformed lymphoblastoid cell line (LCL), and from two gamma-irradiation-induced mutants of LCL 721 lacking complementary class I haplotypes. The physical linkage data place HLA-G closest to HLA-A and place HLA-7.5p between HLA-G and HLA-F. The map constructed supports a maximum distance of 490 kilobases between HLA-A and HLA-F.     

The short cytoplasmic tail of HLA-G determines its resistance to HIV-1 Nef-mediated cell surface downregulation

During infection with the human immunodeficiency virus type 1 (HIV-1), selective downregulation of major histocompatibility complex (MHC) class I molecules by Nef protein allows infected cells to be protected from natural killer (NK) cell lysis and to escape the HIV-specific cytotoxic T-lymphocyte response. The nonclassical MHC class I molecule human leukocyte antigen (HLA)-G is mainly expressed in placental tissues and in thymic epithelial cells. Using chimeric molecules and flow cytometry, we show that in contrast with HLA-A2, the non classical MHC class I molecule HLA-G is resistant to Nef-induced cell surface downregulation solely because of the length of its intracytoplasmic domain. Moreover, confocal microscopy analysis indicates that Nef does not delocalize HLA-G molecules from the cell surface, whereas HLA-G molecules extended with the cytoplasmic tail of HLA-A2 accumulate intracellularly with Nef. Together, these data demonstrate that the short cytoplasmic tail of HLA-G confers resistance to Nef-induced downregulation and intracellular accumulation. This resistance may have functional consequences during the course of HIV infection.     

HLA non-A,B,C class I genes: Their structure and expression

Clearly, the human genome includes a group of genes closely related to but distinct from the HLA class I genes encoding the HLA-A, -B, and -C major transplantation antigens. These non-A,B,C class I genes, designated as HLA-E, HLA-F, and HLA-G, are on the short arm of chromosome 6 and part of the HLA class I gene family. Although the human HLA-E, -F, and -G genes have features in common with the murine Qa- and Tla-genes, e.g. little allelic polymorphism, their relationship to the murine Qa- and Tla-region genes remains unclear. It has been suggested that the nonclassical MHC class I molecules function as ligands for gamma-delta T lymphocytes. The speculation is supported by the recent reports of a murine Qa-1 restricted gamma-delta T cell hybridoma and recognition of a TL antigen by gamma delta T cell receptors. The amino acid sequences of the HLA-E, -F, and -G encoded proteins suggest that each protein is likely to fold three-dimensionally into a structure very similar to HLA-A2 and has a capability of presenting a bound peptide at the cell surface. In light of the possible role of bound peptide in the expression of a class I molecule at the cell surface, it is interesting to note that the HLA-E and HLA-F molecules, even in association with beta 2-microglobulin, could not be detected at the cell surface of a transfected B-LCL. In contrast, the HLA-G molecule was found at the surface of transfected B-LCLs. Both HLA-E and HLA-F are less similar in sequence to HLA-A,B,C than is HLA-G. One explanation would be that the HLA-E and -F molecules have a mutation such that they are no longer able to bind peptide. If the HLA-G molecule does function to present peptide to T lymphocytes, there are features unique to HLA-G that should impact on its ability to perform this function. Both the analysis of HLA-G RNA and protein in trophoblasts indicate that HLA-G, unlike HLA-A, -B, -C, is relatively nonpolymorphic. Since HLA-A,B,C polymorphism is thought to increase the number of different peptides that these molecules can bind, HLA-G is likely to be able to bind a relatively limited variety of peptides. HLA-G also differs from HLA-A, -B, and -C in that it seems to only be expressed by placental amniochorionic trophoblasts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Is olfactory detection of human cancer by dogs based on major histocompatibility complex-dependent odour components? – A possible cure and a precocious diagnosis of cancer

Given the reports made about dogs detecting cancer on the basis of odour, our hypothesis is that the volatile organic compounds produced by tumours, and detected by dogs, are products of MHC genes. Two lines of evidences support this hypothesis: (1) human body odour is genetically determined by MHC. These antigen molecules (HLA in humans) have soluble and detectable isoforms that are present in body fluids such as blood, urine and sweat; (2) there is a strong association between changes in HLA expression and cancer. Tumour transformation is frequently associated with low classical HLA class I molecules expression, namely HLA-A, HLA-B, and HLA-C. In addition, cancer is associated with high nonclassical HLA class I molecules expression, such as HLA-G and HLA-E. These evidences suggest that these HLA-associated olfactory cues of human cancer could be easily analysed, for example, by the "electronic nose", making possible a very precocious and reliable diagnostic of cancer. Because cancer immunescape mechanism is similar to that observed in the materno-fetal immune tolerance, we propose also that immunomodulatory abortifacients could be a good strategy in cancer treatment.     

HLA-G Polymorphisms: Ethnic Differences and Implications for Potential Molecule Function

PROBLEM: Human leukocyte antigen (HLA)-G is uniquely expressed on extravillous cytotrophoblasts of the placenta and is postulated to be a mediator of maternal immune tolerance. Although it was originally considered to be nonpolymorphic, variations of the HLA-G DNA sequence have been reported, and a limited number of HLA-G alleles been defined. METHOD OF STUDY: The HLA-G wild-type sequence was compared with HLA-A2 with regard to the conservation of functionally essential parts of classical HLA-I molecules. HLA-G polymorphisms were analyzed under the aspect of ethnic differences, site, and consequences for postulated molecule functions. RESULTS: HLA-G exhibits a high degree of conservation relative to HLA-A2 in functionally relevant sites of HLA-class I molecules. However, polymorpahic sites in HLA-G and classical HLA loci are not congruent. CONCLUSION: The type and localization of HLA-G polymorphisms suggest that different parts of the HLA-G molecule underlie different selective constraints.     

Enhancement of immunogenicity of Jeg3 cells by ectopic expression of HLA-A*0201 and CD80

PROBLEM: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. METHOD OF STUDY: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. RESULTS: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. CONCLUSION: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells.     

Amnion Epithelial Cells,in Contrast to Trophoblast Cells,Express All Classical HLA Class I Molecules Together With HLA-G

PROBLEM: The expression of the non-classical HLA-G gene has been shown at the protein level on trophoblast-derived embryonic tissue, like the extravillous cytotrophoblast. However, the presence of HLA-G on embryoblast-derived cells is currently controversial. The amnion epithelium is an embryoblast-derived cell layer covering the amnion cavity and is the main source for the amnion fluid. METHOD: The expression of HLA class I molecules was investigated by immunohistochemical, biochemical, and molecular biological methods in amnion membranes and amnion fluid. RESULTS: Immunohistochemically, HLA-C and occasionally also -B molecules as well as HLA-A and/or -G molecules have been identified on amnion epithelial cells. These results were extended by Western blotting with purified amnion epithelial cells where HLA-B and/ or -C, HLA-A and HLA-G antigens have been detected. As expected HLA-G mRNA was detected in amnion epithelial cells. Furthermore, classical HLA molecules as well as HLA-G were found in amnion fluid. CONCLUSION: These results show that the amnion epithelium frequently expresses classical HLA class I molecules as well as HLA-G. The expression of HLA-G antigens on amnion epithelial cells and their presence in the amnion fluid, which is continually ingested by the fetus, may be particularly relevant for the induction of peripheral tolerance.     

Characterization of an expressible nonclassical class I HLA gene.

Screening of a human cosmid library representing genomic DNA from an individual homozygous for the HLA-DR2 B7 A2 haplotype yielded 109 class I HLA-specific clones. One cosmid clone, Ice 6.23, had a full-length nonclassical class I gene within a 5.4-kb HindIII fragment. The Ice 6.23-5.4H gene was cloned into the unique NotI site of an expression vector pSV2.Not, a derivative of pSV2neo, which was constructed to contain a second SV40 early region promoter adjacent to an introduced NotI site. The resulting construct was transfected into the P815-B2M cell line, a derivative of the mouse mastocytoma P815 (HTR) line which expressed human beta2-microglobulin following stable transfection with a cloned human beta2-microglobulin gene. Following transfection the Ice 6.23-5.4 H gene was found to be expressed at both the mRNA and cell surface product levels. DNA sequencing of this gene suggests that it is allelic to the HLA-6.0 gene clone (HLA-G) of Geraghty et al. (Proceedings of the National Academy of Sciences USA, 84:9145, 1987); thereby revealing a HindIII restriction fragment length polymorphism at the HLA-G locus. An extraordinarily high degree of sequence similarity (99.92%) between these two genes, which derive from unrelated HLA haplotypes, suggests strong conservative selection pressure at the HLA-G locus. A flanking single copy sequence probe 4 kb distant from the Ice 6.23-5.4H gene was used to generate long-range restriction mapping at the HLA-G locus.     

HLA-G expression is induced in Epstein-Barr virus-transformed B-cell lines by culture conditions

Human leukocyte antigen-G (HLA-G) belongs to the nonclassical HLA class I family of genes presently designated as class Ib genes. It was found to be expressed mainly in placental tissue and in the thymus. Expression of HLA-G is induced by lymphokines such as interleukin-10 and has been associated with the escape of tumor cells from immune surveillance or with inhibition of graft rejection. In this report, Epstein-Barr virus-transformed B-cell lines established from peripheral blood lymphocytes from healthy volunteers were studied. Our results show that EBV-transformed B-cell lines, but not freshly separated peripheral blood lymphocytes, can be induced to express HLA-G either by subjecting the cultures to nutrient deficiency to hypoxia or to both, however, not all cell lines responded equally to stress conditions. The association of HLA-G with certain cancer transformations may suggest that the resistance to HLA-G expression could be related to susceptibility to the development of malignancy.     

Immune modulation of HLA-G dimer in maternal-fetal interface

HLA-G is a non-classical human MHC class I molecule, which has several characteristics distinct from classical MHC, such as low polymorphism and restricted tissue distribution. HLA-G is expressed on placenta, thymus and some tumors. At the maternal-fetal interface, trophoblasts do not express major classical MHC class I molecules (MHCI), HLA-A and -B, to prevent normal T cell responses. Instead, HLA-G is expressed and can suppress a wide range of immune responses by binding to inhibitory immune cell surface receptors, such as leukocyte Ig-like receptor (LILR) B1 and LILRB2. HLA-G exists in various forms, including beta2m-associated or -free disulfide-linked dimers that can be expressed either at the cell surface or in soluble form. However, until recently the physiological role of these different molecular forms has been unclear. In this issue of the European Journal of Immunology, one article demonstrates that the disulfide-linked homodimer of beta2m-associated HLA-G is the major fraction expressed by trophoblast cells. The HLA-G dimer modulates the function of LILRB1-expressing antigen-presenting cells by principally binding to LILRB1. On the other hand, another recent report showed that beta2m-free disulfide-linked HLA-G dimers are produced by villous cytotrophoblast cells. Taken together, these results provide strong evidence in support of the hypothesis that HLA-G dimers play a role in immune suppression at the maternal-fetal interface. Further in-depth investigation will help to clarify the precise mechanism of HLA-G receptor recognition and signaling in vivo and the role of these interactions in successful reproduction. See accompanying article: (http://dx.doi.org/10.1002/eji.200737089).     

Amino acid composition of alpha1/alpha2 domains and cytoplasmic tail of MHC class I molecules determine their susceptibility to human cytomegalovirus US11-mediated down-regulation

During co-evolution with its host, human cytomegalovirus has acquired multiple defense mechanisms to escape from immune recognition. In this study, we focused on US11, which binds to MHC class I heavy chains and mediates their dislocation to the cytosol and subsequent degradation by proteasomes. To examine which domains of class I heavy chains are involved in this process, we constructed chimeric HLA molecules of US11-sensitive and -insensitive class I molecules (HLA-A2 and HLA-G, respectively). Pulse-chase experiments were performed to evaluate protein stability and interactions between class I heavy chains and US11. Flow cytometry was employed to assess the effect of US11 on surface expression of the different chimeras. Our results indicate that the alpha1 and alpha2 domains of HLA molecules are important for the affinity of US11 association. However, the degradation efficiency seems to rely mostly on cytosolic tail residues. We found that the nonclassical HLA-G molecule is insensitive to US11-mediated degradation solely because it lacks essential tail residues. A deletion of the last two tail residues in full-length MHC class I molecules already caused a severe reduction in degradation efficiency. Altogether, our data provide new insights into the mechanism by which US11 down-regulates MHC class I molecules.