Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library.

Antitumor antibodies with the same specificity as cytotoxic T lymphocytes that recognize antigenic peptides encoded by tumor-associated genes and presented by MHC class I molecules would be valuable tools to analyze the antigenicity or target tumor cells in vivo. To obtain a human antibody directed against a peptide encoded by gene melanoma-associated antigen (MAGE)-A1 and presented by HLA-A1 molecules, we selected a large phage Fab antibody repertoire on a recombinant version of the complex HLA-A1-MAGE-A1 produced by in vitro refolding. One of the selected phage antibodies shows binding to HLA-A1 complexed with the MAGE-A1 peptide, but does not show binding to HLA-A1 complexed with a peptide encoded by gene MAGE-A3 and differing from the MAGE-A1 peptide by only three residues. Phages carrying this recombinant antibody bind to HLA-A1(+) cells only after in vitro loading with MAGE-A1 peptide. These results indicate that nonimmunized phage Fab libraries are a source of antibodies with a T cell antigen receptor-like specificity. The human anti-HLA-A1-MAGE-A1 antibody described here may prove very useful for monitoring the cell surface expression of these complexes, and eventually, as a targeting reagent for the specific immunotherapy of HLA-A1 patients bearing a MAGE-A1-positive tumor.

Surface expression of HLA-C antigen by human extravillous trophoblast.

In this paper definitive evidence that the classical class I product, HLA-C, is expressed on the surface of normal trophoblast cells is provided. HLA-C transcripts were sequenced from cDNA isolated from first trimester trophoblast cells obtained by flow cytometric sorting. Both paternal and maternal alleles were transcribed. HLA-C proteins were demonstrated by biochemical analysis and found on the cell surface in association with beta(2)-microglobulin. Upregulation of cell surface HLA-C but not HLA-G expression after interferon (IFN)-gamma treatment was demonstrated by flow cytometric analysis. Immunohistology has confirmed HLA-C is expressed by all extravillous subpopulations in vivo. The question of whether trophoblast HLA-C molecules interact with decidual NK cells expressing killer Ig-like receptors (KIR) has also been addressed. Our results demonstrate that extravillous trophoblast expresses at least two HLA class I molecules, HLA-G and HLA-C on the cell surface.

Structurally diverse forms of HLA-B27 molecules are displayed in vivo in a cell type-dependent manner.

The formation of a trimeric complex, composed of heavy chain (HC), beta(2)-microglobulin (beta(2)m) and antigenic peptide, is generally believed to be a prerequisite for the expression of HLA class I molecules at the cell surface in vivo. Therefore, a possible role in immunological processes for HC/beta(2)m complexes devoid of peptide has not been seriously considered. Using a novel HLA-B*2705-transgenic rat model and monoclonal antibodies that distinguish between structurally different forms of HLA-B27 molecules, we demonstrate here that class I molecules which appear to lack antigenic peptides are expressed in abundance on a variety of cell types in lymphoid organs. These results imply a role for structurally diverse, possibly empty, MHC molecules in physiological T cell selection which has so far not been sufficiently appreciated.

The monoclonal antibody HCA2 recognises a broadly shared epitope on selected classical as well as several non-classical HLA class I molecules.

HCA2 is a widely used monoclonal antibody, thought to be highly selective for HLA-A and -G heavy chains. We demonstrate here that it also shows affinity to HLA-B73 and HLA-E molecules on intact cells. By comparing the differences in the amino acid (AA) sequences of several HLA class I alleles that are either recognised or not recognised by HCA2, a likely epitope of HCA2 has been deduced. It extends from position 76 to position 83 of the alpha1-domain. In intact cells, the solvent-exposed AA in positions 76 (Ala, Val, or Met), 80 (Asn or Thr) and 83 (Gly) are likely to constitute the recognition region. Inhibition experiments with peptides spanning the region of the alpha1-domain from position 74 to 85 of various HLA class I heavy chains prove that HCA2 recognizes a broadly shared epitope on HLA-E, -F and -G molecules as well as selected HLA-A, -B and -C antigens.

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 amino 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.

Expression of HLA class I molecules in human first trimester and term placenta trophoblast.

Expression of HLA class I molecules in trophoblast cells from various locations in normal human first trimester and term placenta was investigated by immunohistochemistry with a panel of monoclonal antibodies against the heavy chains or complete HLA class I molecules complexed with beta2-microglobulin. These reagents were also employed to distinguish between the products of different HLA class I loci. In addition to previously characterized reagents, a novel monoclonal antibody against HLA-A molecules (TU155) was used. Various choriocarcinoma and transfected cell lines served as controls for the specificities of the monoclonal antibodies. Cells in close contact with maternal cells, such as invading trophoblast cells and cells of the basal plate, expressed beta2-m micro globulin in association with HLA-G and HLA-C heavy chains. These class I heavy chains may also have been present as isolated molecules, although not in each of the cells. In contrast, cells of the chorion laeve exclusively expressed HLA-G, and not HLA-A, -B, or -C antigens. Our data support the often discussed immune protective function and the regulatory function of the HLA-G molecule, during invasion. In addition, by using monoclonal antibodies HCA2 (anti-HLA-A and -G), HC10 (anti-HLA-B and -C), TU149 (anti-HLA-B, -C, and some -A alleles), SFR8-B6 (anti-HLA-Bw6 and some -C), LA45 (some HLA-A and -B), TU48 (anti-HLA-Bw4 and some -A), and TU155 (anti-HLA-A), we show the presence of HLA-C molecules in all extravillous trophoblast cells of the cell columns and in the basal plate; the trophoblast cells of the chorion laeve lack this antigen. The function of this molecule is not clear, although a protective function against natural killer cell activity in the endometrium is postulated.

MHC class II antigen expression is increased in different forms of urticaria.

Urticarial reactions encompass a variety of inflammatory and immunological reactions. In order to clarify specific aspects of these processes, we analyzed the distribution and sequential expression of major histocompatibility complex II (MHC class II) molecules in tissue sections from different types of whealing reactions. Using immunohistochemical techniques and monoclonal antibodies, expression of HLA-DR, HLA-DP, and HLA-DQ was examined on resident and infiltrating cells in different skin cell compartments, comparing early with longer-lasting wheals and lesional with uninvolved skin. Sequential biopsies were studied in cold urticaria (CU). No increase of MHC class II molecule expression was found in early prick test wheals to common inhalant allergens. In CU, however, sequential biopsies demonstrated an up-regulation of MHC class II molecules within 30 min after elicitation. This was more pronounced in longer-lasting urticaria lesions of acute, chronic recurrent and delayed pressure urticaria, with HLA-DR and, to a lesser degree, HLA-DP and HLA-DQ being noted on cell infiltrates, on vascular endothelia and around nerves and sweat glands. Nonelesional skin in these types of urticaria also showed increased MHC class II expression. Longer-lasting urticarial wheals are thus associated with up-regulation of MHC class II molecules on resident and infiltrating cells, suggesting an involvement of these molecules in the pathomechanisms of these types of urticarial lesions.

Genetic markers on chromosome 19p and prenatal diagnosis of HLA class II-deficient combined immunodeficiency.

HLA class II-deficient combined immunodeficiency (CID) is an inherited disease characterized by a total lack of HLA class II gene expression, due to a regulatory defect affecting these genes. In the family investigated the disease phenotype occurs parallel to an abnormal structural feature of the CD23 antigen. We sequenced parts of the FCER2 gene coding for CD23 and found a restriction fragment length polymorphism (RFLP) that cosegregates with the disease. Analysis of recombinant haplotypes by microsatellites mapping to the chromosomal region 19p13.3 suggests that the disease locus maps between FCER2 and the microsatellite marker D19S424, probably close to D19S216 and D19S177. These data may offer the possibility of a rapid and early prenatal diagnosis of a subgroup of patients with HLA class II-deficient CID.

Use of human leukocyte-specific monoclonal antibodies for clinically immunophenotyping lymphocytes of rhesus monkeys.

The rhesus monkey (Macaca mulatta) is an important experimental animal frequently utilized for studies of infectious diseases, immunity, hematopoiesis, and transplantation. Since the structure of cell surface molecules is phylogenetically conserved, monoclonal antibodies raised against human leukocyte antigens can sometimes recognize the homologous determinant on monkey leukocytes. To facilitate better utilization of this animal model, we tested 89 commercially available monoclonal antibodies which define 27 human cell surface antigens for reactivity with rhesus monkey PBL. Certain antigens which delineate clinical useful lymphocyte subsets such as CD2, CD4, CD8, CD14, CD16, CD20, and MHC class II are apparently well conserved since most human cell-specific antibodies identified the homologous cell subset in monkeys. However, other antigens such as CD3, CD19, CD45, and CD56 were identified infrequently by human cell-specific antibodies. FITC-modification of antibodies which had no effect on their binding to human cells occasionally inhibited antibody binding to monkey cells. Nevertheless, an adequate number of cross-reactive monoclonal antibodies was identified to allow gating of lymphocytes for accurate flow cytometric analysis and quantitation of the major lymphocyte subsets of the rhesus monkey. The T lymphocyte subset distribution in blood and lymphoid tissue of rhesus monkeys was similar to man. However, the B subset was significantly larger in monkeys. The daily variation in absolute PBL subset size was marked and found to be due mainly to daily fluctuations in total lymphocyte number.

A subset of HLA-B27 molecules contains peptides much longer than nonamers.

An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only approximately 5-20% of the cell surface HLA-B27 was used for large-scale purification of these molecules. Subsequent mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive population contained peptides primarily from 900 to 4000 Da in size (approximately 8-33 amino acid residues), whereas the major HLA-B27 population contained peptides in the mass range of 900-1400 Da (approximately 8-12 amino acid residues). Thus, a subset of HLA-B27 molecules binds to peptides much longer than nonamers. Typical HLA-B27-binding peptides contain arginine in position 2. Further analysis by Edman sequencing of the pooled bound peptides revealed that the major population contained substantial amounts of arginine at positions 1 and 9 (40-50%) and exclusively arginine at position 2, as expected. The minor population of peptides also contained detectable amounts of arginine at these positions, but at the level of only approximately 10%; no marked enrichment at any position was observed. These long HLA-B27-bound peptides could represent either intermediates in the formation of nonamers or adventitiously bound peptides. Lastly, in the TAP2 mutant cell line BM36.1 transfected with HLA-B*2705, MARB4-reactive HLA-B27 molecules were absent from the cell surface, indicating that the peptide transporter was required for delivery of the long peptides. Thus, during the folding of class I heavy chains, peptides of diverse lengths are available and participating.

Microheterogeneity in HLA-B35 alleles influences peptide-dependent allorecognition by cytotoxic T cells but not binding of a peptide-restricted monoclonal antibody.

Strong peptide dependency of HLA-B*3501-specific alloreactive T-cell clones was observed in the recognition of cells bearing closely related B35 variants. The single amino acid exchange in the beta-pleated sheet of B*3503 completely abolished the responses of all clones, whereas an amino acid exchange in the alpha 2 helix of the newest B35 member (B*3508) only altered allorecognition of one T-cell clone, demonstrating the differential impact of these positions on peptide binding to B35 molecules. In contrast to T cells, a mAb (TU165) recognizing the B35 specificity in a peptide-dependent manner bound to the B35 variants irrespective of their sequence heterogeneity. However, quantitative binding differences were detected with cells bearing the same B35 alleles. This is most likely due to variations in the amount of peptide(s) that associates with B35 and forms the ligand seen by this mAb. These results reveal how naturally occurring single amino acid substitutions have led to generation of functionally distinct molecules of another multimember HLA class I cluster.

Soluble T cell receptor-like properties of an HLA-B35-specific monoclonal antibody (TU165).

A mouse monoclonal antibody of IgM class (TU165) was produced using Epstein-Barr virus (EBV)-infected mutant cells derived from the human BJAB-B95.8.6 cell line as immunogen. Binding studies with several HLA deletion mutant cell lines indicated that TU165 recognized the HLA-B35 molecule. In a panel of 89 EBV-transformed lymphoblastoid cell lines, all HLA-B35+ cells (n = 24) reacted with TU165 while all but two HLA-B35- lines (n = 65) were unreactive (r = 0.95). Surprisingly, peripheral blood lymphocytes of HLA-B35+ donors were unreactive; however, strong enhancement of TU165 recognition was observed with B cells of one of these individuals after transformation with EBV (B95.8 strain). Transfection of both HLA-B35 and human beta 2-microglobulin genomic DNA into mouse P815 cells led to high expression of HLA-B molecules; yet, expression of the TU165 epitope was not observed. Furthermore, the EBV-negative cell line BJAB as well as the EBV-infected (P3HR1 strain) line BJAB-HR1K were only weakly reactive, whereas the BJAB-B95.8 cell line was strongly positive. These results indicate that EBV-encoded or -controlled peptide(s) must be bound by HLA-B35 antigens to create the epitope which allows efficient binding of TU165.

Assembly and function of the two ABC transporter proteins encoded in the human major histocompatibility complex.

Presentation of cytoplasmic antigens to class I-restricted cytotoxic T cells implied the existence of a specialized peptide transporter. For most class I heavy chains, association with peptides of the appropriate length is required for stable assembly with beta 2-microglobulin. Mutant cells RMA-S and .174/T2 neither assemble stable class I molecules nor present intracellular antigens, and we have suggested that they have lost a function required for the transport of short peptides from the cytosol to the endoplasmic reticulum. The genetic defect in .174 has been localized to a large deletion in the class II region of the major histocompatibility complex, within which two genes (RING4 and RING11) have been identified that code for 'ABC' (ATP-binding cassette) transporters. We report here that the protein products of these two genes assemble to form a complex. Defects in either protein result in the formation of unstable class I molecules and loss of presentation of intracellular antigens. The molecular defect in a new mutant, BM36.1, is shown to be in the ATP-binding domain of the RING11/PSF2 protein. This is in contrast to the mutant .134, which lacks the RING4/PSF1 protein.

Monoclonal antibodies against antigens expressed by human sperm.

The production and characterization of 21 mouse monoclonal antibodies (TüS1-TüS21) with specificity predominantly for human spermatozoa antigens is described. Reactivity of cells from human ejaculates, peripheral blood and several organs was determined using the alkaline phosphatase-anti-alkaline phosphatase (APAAP)-technique as well as the indirect immunofluorescence test. 15 of the monoclonal antibodies reacted with various regions of human sperm and often also with their precursor cells in the testis. Cross-reactivity with animal spermatozoa was frequently observed.

Immunoelectron microscopy of human spermatozoa.

A number of immunocytochemical methods using various markers for electron microscopy have been developed in recent years. The immunogold technique has been especially effective in histotopochemical studies. The value of this technique for demonstrating sperm antigens results from the high electron density of gold, which makes it easily detectable under the electron microscope. The high resolution of the electron microscope permits precise localization of immunologic reactions in the sperm cell. Light microscopy findings can thus be elucidated. We tested a number of monoclonal antibodies that react with sperm antigens. Of three techniques for preparing the spermatozoa, the pre-embedding method and marking of cryoultra-microtome sections proved best.

Immunocytochemical characterization of round cells in human semen using monoclonal antibodies and the APAAP-technique.

Round cells of 42 ejaculates were characterized by their surface antigens using monoclonal antibodies and the immunocytochemical APAAP-technique. In this way immature germ cells, polymorphonuclear leukocytes, lymphocytes and their subpopulations could be distinguished. It was also attempted to correlate the incidence of these cells, sperm parameters and other clinical data.

Identification of human spermatozoa antigens using monoclonal antibodies and the alkaline phosphatase anti-alkaline phosphatase-technique.

Both cytoplasmic and surface-membrane antigens of human spermatozoa were detected by means of monoclonal antibodies (MoAbs) and of the alkaline phosphatase anti-alkaline phosphatase- (APAAP-) technique. Several advantages of this technique for the identification of sperm could be demonstrated. The labeling of cytocentrifuge preparations from 16 ejaculates proved the presence of glycosphingolipids, nuclear and mitochondrial antigens of spermatozoa. However, there were no HLA-molecules and other leukocyte antigens on sperm cells.