The detection of HLA-DR, MB and MT determinants on purified class II molecules by inhibition of microcytotoxicity

An assay has been developed which makes it possible to determine the HLA allospecificities carried by molecules in purified fractions of detergent lysates from EBV-transformed human lymphocytes. It is based on inhibition of the standard microlymphocytotoxic test used for identifying HLA class I and II antigens with alloantisera. Soluble cell membrane products from EBV-transformed cell lines homozygous for the HLA region gave specific inhibition of standard typing antisera. The test requires preincubation of microliter volumes of soluble antigen preparations maintained in 0.05% NP-40 with selected antisera prior to adding EBV-transformed cells as target cells. It was possible using this assay to follow isolation of the structurally related human class II molecules bearing the MB and DR specificities. Detergent lysates of cells were fractionated on affinity columns prepared from monoclonal antibodies directed against distinct class II antigens. Eluates from these columns contained the expected DR and MB specificities. The assay is easy to perform, highly reproducible and allows multiple determinations.     

In vitro response of peripheral blood mononuclear cells to phytohemagglutinin and interleukin-2

The serological determination of class II antigens is still a mandatory test prior to allotransplantation. It is known that these antigens are normally expressed on B lymphocytes and monocytes. The B lymphocytes that constitute 10% to 15% of total blood lymphocytes are the cells currently used for HLA-DR typing. To avoid HLA-DR typing difficulties, or even impossibilities that are frequently encountered among some patient groups, we studied the response of peripheral blood mononuclear cells--as an alternative source of cells for class II antigen typing--to in vitro mitogen and interleukin-2 activation and propagation. Although the patients included in this study were selected having previously known HLA-DR typing difficulties, all could be adequately typed by this method.     

Immunological studies of trophoblast antigens: no evidence for human leucocyte antigen (HLA) linkage

Parental disparity for trophoblast-lymphocyte crossreactive (TLX) antigens may promote successful pregnancy. A TLX antigen system has been defined on peripheral blood lymphocytes by heteroantisera. More recently, we have reported additional activity against antigens on B lymphocytes alone termed trophoblast-B lymphocyte crossreactive (TBX) antigens. In the present study we have investigated ten TLX sera in order to determine if their target antigens are linked to the human leucocyte antigen (HLA) gene complex. The sera showed no selective activity when tested against target B lymphocytes from ten normal donors. Cytotoxic activity of TLX antisera against peripheral blood lymphocytes from six normal donors was not reduced when the class I HLA antigens of the target cells were blocked with a monoclonal antibody (PA 2.6). Similarly the cytotoxic activity of both TBX antisera against B lymphocytes from six normal donors was not decreased when class II HLA antigens were blocked by a monoclonal antibody (FMC 4). Within a family the cytotoxic activity of the TLX antisera was absorbed equally by lymphocytes from siblings who shared neither HLA haplotype. Antibody content in TLX and TBX antisera is not directed toward the classically defined HLA class I or class II antigens and is not linked to the HLA gene complex.     

Expression of class I and class II markers on populations of leukemic cells

The study of class I and class II antigen expression on leukemic cells brought the following conclusions: most of the leukemic cells show a slower number of class I antigenic sites than normal peripheral blood lymphocytes (PBL) but, in most cases, this does not hinder HLA typing; contrarily to normal PBL, leukemic cells seem to carry "non HLA" antigens (and/or non classical HLA antigens) which are probably responsible of the false positive reactions frequently observed at the time of HLA typing; most of the leukemic cell types express DR antigens (except those belonging to the T lineage) but DQ antigen expression (and in some cases MT antigen expression) varies depending on the cell type studied: well defined on mature B hemopathies, DQ expression is often lower than DR expression on acute leukemic cell types.     

A one-step monoclonal antibody typing procedure that simplifies HLA class I and class II typing

Abstract: We have developed monoclonal antibodies to most HLA specificities, making it possible for us to devise a simple, rapid, one-step micro-cytotoxicity test. The test is performed by adding 1 μl of cells to 1 μl of antibody-complement mixture predotted on the microtest tray. The reactions are read following a 1-hour incubation period (30 minutes in some instances). The analysis of reactions seen on testing 105 class I antibodies and 50 class II antibodies is shown. A comparison of typing by the standard NIH method and the new one-step procedure showed a > 96% concordance in the 500 T cells and 200 B cells we examined. Class I and class II typing could be performed using B cells, thus obviating the need to isolate both T and B cells for HLA typing.     

HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation - a fast and reliable technique

This paper describes a new cell isolation and HLA typing technique, which permits cell separation and HLA class I or class II typing to be performed in 70 min. Magnetic monodisperse microspheres (Dynabeads TM) were coated with monoclonal antibodies (MAbs) specific for the CD8 T cell antigen or for HLA class II monomorphic epitopes. They could then be used to obtain HLA class I or class II positive cells directly from ACD blood in approximately 15 min by the use of magnetic separation. The cells (attached to the microspheres) were subsequently used in microcytotoxic HLA typing (total incubation time of 55 min) using acridin orange/ethidiumbromide to stain viable (yellow) and dead (red) cells. It was found that this immunomagnetic (IM) HLA typing technique was specific, has a sensitivity superior to that observed for conventional microcytotoxicity assays and gave low background staining. IM HLA-ABC typing of 50 healthy donors and 10 patients and IM HLA-DR typing of 25 healthy donors and 30 patients gave results corresponding well with that obtained independently by conventional HLA typing (concordancy rates 92-100%). Furthermore, the IM HLA typing technique permitted reliable HLA class II typing of blood cells from six patients where conventional HLA class II typing was impossible. The IM HLA typing technique also enables HLA class I and II typing to be quickly and reliably performed on cells from ACD blood of cadaveric donors.     

A simple and rapid method for the detection of lymphocytotoxic antibodies using cell panels frozen on Terasaki plates

A 40 cell panel of lymphocytes selected for HLA-A,B and DR antigens, frozen in Terasaki microtest trays can be used routinely to identify the presence of specific HLA antibodies within two hours. Blind testing using well defined HLA typing sera showed that specificities could be identified to a high degree of significance using this method. The method has proved successful for screening for T cell, including HLA-A,B and B cell, including HLA-DR antibodies. The method is particularly useful for the routine tissue typing laboratory as the frozen panel can be used without the need for complicated and time-consuming cell washing procedures which have been the downfall of previously published methods.     

Divergent effects of thyroid HLA-antigens on T and natural killer (NK) cell cytotoxicity.

We have used non-autoimmune non-neoplastic human thyroid cells to explore the role of surface class I and DR antigens on these cells' sensitivity towards T and Natural Killer (NK) cell cytotoxicity. Non-treated thyrocytes expressed class I but no DR antigens. Following incubation with gamma-interferon (gamma-IFN) class I antigens were markedly elevated and DR expression was induced. Whereas non-treated thyrocytes were minimally lysed by sensitized T cells, they served as appropriate targets for NK cells. Following incubation with gamma-IFN, the thyroid cells became highly sensitive to T cell lysis, with no significant reduction in their vulnerability to NK cell killing. The addition of monoclonal anti class I or DR antigens, or brief acid treatment which specifically eliminates class I molecules, inhibited T cell cytotoxicity but enhanced the sensitivity to lysis by NK cells. Thus, the presence of HLA antigens on the same thyroid cells have an opposite effect on two major cytotoxi mechanisms. Our findings are relevant within the context of recent suggestions of intervening with target HLA antigens for the management of autoimmune and malignant diseases.     

Genetically Appropriate Expression of HLA and DR (IA) Alloantigens on Human Melanoma Cell Lines

Studies of the expression of HLA and DR alloantigens on cultured human melanoma cells in comparison with those expressed on peripheral lymphocytes and B-cells derived from the same patients have shown that the HLA-A,B, and C locus antigens expressed on the cultured tumor cells were consistent with those expected from the typing of peripheral blood lymphocytes. One melanoma cell line failed to express all the HLA antigens expected from donor typing. All five of the lines tested also expressed DR antigens and in three instances these could be demonstrated to have genetically consistent allotypes. However, in preliminary studies stimulation of allogeneic lymphocytes by the DR positive melanoma cells could not be demonstrated.     

Proliferative T cell clones directed at a human minor antigen

Clones directed against a human minor antigen were isolated from an interleukin-2-dependent cytotoxic T cell line. As expected, some clones exhibiting cytotoxicity for HLA-identical minor antigen-positive cells were detected. In addition, some clones were detected which, though not cytotoxic, were able to proliferate in the primed lymphocyte typing (PLT) test in response to stimulation by cells from some HLA-identical sibs and certain DR 2+/ve unrelated cells. These clones expressed the Leu 3a cell surface antigen. DR 2 was one of the class II determinants expressed by the responding cell used to generate the cytotoxic T lymphocyte line. These results are consistent with the interpretation that helper T cells are able to recognize minor antigens in the context of self-class II HLA determinants and respond in PLT.     

Human Leukocyte Antigens (HLA) Class I and Class II on Sperm Cells Studied at the Serological,Cellular, and Genomic Levels

ABSTRACT: The expression of human leukocyte antigens (HLA) on highly purified human ejaculated sperm cells was studied using the sensitive enzyme-linked immunosorbent assay (ELISA) technique and a wide panel of monoclonal antibodies to class I and class II HLA. In addition, the stimulatory capacity of these cells was tested in mixed cultures of lymphocytes and spermatozoa, and the levels of RNA homologous to the HLA class I and class II genes were determined. The results obtained using the ELISA indicate that the class I and class II HLA serologically defined antigens are weakly expressed on the cell surface of the mature spermatozoa. Highly purified sperm cells consistently stimulated heterologous lymphocytes but not when HLA-DR compatibility was observed between stimulator and responder. The proliferative response of lymphocytes induced by sperm cells was lower than the response obtained in a lymphocyte-lymphocyte combination, though the kinetics of the response were similar in both cases. In addition, it was found that spermatozoa contained RNA species homologous to HLA class II DRβ and DQβ genes sequences but not to HLA class I sequences. The levels of these RNA species were significantly reduced after interferon stimulation. Lymphocytes that served as positive control were found to contain RNA complementary to both HLA class I and class II genes.     

Flow cytometric detection of HLA antibodies using a spectrum of microbeads

We describe here the use of HLA antigen coated beads for specificity and class determination of HLA antibodies by flow cytometry. The HLA specificity of antibodies was determined by use of beads containing eight levels of fluorescence. HLA antigens isolated from eight cultured cells were coated onto these beads so that each bead was the equivalent of one cell. By using four sets of eight beads, an equivalent of 32 cells could be examined in four test tubes. A total of 76 class I and 25 class II specificities could be determined by the 32 class I bead-panel and 32 class II bead-panel used, respectively. We noted no cross-reactivity of reactions between class I and II. The sensitivity of the test was shown to be higher than that of the standard cytotoxicity by dilution experiments and detection of additional cross-reacting antigens. By use of these coated beads, we achieved improved standardized detection of HLA antibodies. Antigen-coated beads have several advantages over the use of spleens or lymphocytes. (a) A highly selected panel of antigens can be routinely used. (b) Class I and class II antibodies can be readily distinguished from each other, even when they are present as mixtures in one serum. (c) Non-HLA antibodies are not detected because the beads do not have any other antigens than HLA on them. (d) The quantity of antigens coated on beads is more uniform than that found in cells from different individuals. (e) Beads are more convenient for storage and daily use.     

A rapid and efficient method for purification of rat B cells for complement-dependent cytotoxicity testing

The development of an effective technique for the enrichment of rat B cells is described. This protocol is an adaptation of the ‘panning’ technique employing anti-Ig-coated plastic dishes. Rat B cells are bound directly to anti-rat Ig-coated wells in Terasaki test plates where they are typed by complement-dependent cytotoxicity. This technique enriches class II (Ia-like) antigen-positive B cells, facilitating detection with antisera specific for rat class II alloantigens. The utility of this technique in the cell surface phenotyping of minor lymphocyte subpopulations is discussed.     

In situ characterization of the cell-surface antigens of the mononuclear cell infiltrate and bile duct epithelium in primary biliary cirrhosis

To analyze the tissue distribution of mononuclear cells and HLA antigens in primary biliary cirrhosis, we studied liver biopsies of 12 patients at different stages of the disease, using the avidin-biotin-peroxidase technique and monoclonal antibodies directed against T and B lymphocytes, T-cell subsets, macrophages, NK/K cells, dendritic cells, and HLA class I and II antigens. To evaluate the proportion of activated T cells we used anti-interleukin-2-receptor antibodies and a double-staining technique for T cells and class II HLA antigens. In all biopsies activated T cells predominated in the portal areas and around the damaged bile ducts. T4 cells almost always outnumbered T8 cells. While B cells, NK/K cells, and dendritic cells were always scarce, macrophages constituted about 30% of the cellular infiltrate. Biliary epithelium, which normally expresses HLA class I antigens, displayed mainly HLA class II antigens. The predominance of T4 cells around the bile ducts, which express class II antigens, suggests that class II-restricted T4 lymphocytes may mediate liver damage in primary biliary cirrhosis.     

A microELISA assay for detection of anti-HLA activity of mouse monoclonal antibodies using an Astroscan 2100 automated plate reader.

A microenzyme-linked immunosorbent assay (microELISA) method has been developed using an Astroscan 2100 system automated plate reader which was initially designed for tissue typing by a two colour fluorescent microcytotoxicity assay. A 96-well plate ELISA used for screening mouse monoclonal antibodies raised against surface HLA antigens has been modified for use with the Astroscan plate reader and 72-well typing trays. The existing substrate 4-methylumbelliferyl-beta-D-galactoside (4MUG) has been replaced with fluorescein-di-beta-D-galactopyranoside (FDG), to provide a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes have also been reduced tenfold for use with Terasaki microtest plates. The assay now has the major advantage of requiring only 5 microliters of test supernatant allowing hybridomas to be screened earlier during a fusion and on a wider cell panel. The use of the large panel which includes B lymphoblastoid cell lines (B-LCL) and mouse L cell transfectants expressing HLA genes, reduces the length of time the hybridomas need to be kept in tissue culture before selection. Other advantages include the reduction in the number of target cells required, smaller volumes of reagents throughout the assay and the ability to screen cytotoxic as well as non-cytotoxic monoclonal antibodies. The sensitivity of this microELISA proved to be comparable with the original assay and so provides an efficient screening method for monoclonal antibodies.     

A sensitive and specific ELISA using monoclonal capture antibodies for the detection of HLA antigens in blood stains

A double antibody ELISA technique is described to detect HLA antigens in extracts of blood stains. The assay involves capture of free HLA determinants using an immobilized monoclonal antibody directed against monomorphic regions of HLA class I and HLA class II antigens. The captured antigens are then detected using alloantisera directed against the polymorphic regions of the captured HLA entities. The technique is able to detect specific HLA-A, B, and DR antigens in extracts prepared from blood smears as well as from dried and freshly thawed lymphocytes. The assay may be of potential use in forensic medicine, particularly in instances where extraction of nucleic acids for fingerprinting is not feasible.     

Typing for HLA class II at the product level

Class II antigens were isolated from consanguineous homozygous typing cells by sequential immunoprecipitation with the MoAbs: 7.3.19.1 (anti-DRw52-like), B.8.11.2 (anti-DR backbone) and 7.5.10.1 (anti-HLA class II backbone). Depending on the DR serotype of the cell line used, two or three families of class II antigens could be isolated [1]. For each homozygous typing cell the different families of class II antigens were analysed on 1D-IEF gels. Charge heterogeneity showed that the different haplotypes are distinct in electrophoretic beta chain patterns. For each homozygous typing cell at least one beta chain was observed that possessed a haplotype unique pI. This means that typing for HLA class II at the product level is possible.     

Improved flow cytometry based cytotoxicity and binding assay for clinical antibody HLA crossmatching

The presence of preformed donor-specific HLA antibodies leads to early antibody mediated kidney allograft rejection. Therefore, detection and avoidance of donor reactive HLA antibodies prior to transplantation is of outmost importance in order to minimize the risk of rejection. Detection of pre-formed HLA antibodies is currently performed using complement-dependent cytotoxicity (CDC) assay alone or together with a flow cytometry based crossmatch (FCXM). This study was initiated to further evaluate our recently developed flow cytometry based procedure for determination of both cytotoxicity of and IgG binding to donor-derived lymphocytes by HLA antibodies. Highly enriched immuno-magnetic bead purified T and B lymphocytes were used as target cells for patient sera using 96-well plates. Importantly, the assay shows high sensitivity and specificity as determined by HLA typed donor cells and serum with defined HLA antibody IgG and C1q. Based on this and additional data generated in this paper, such as evaluation of appropriate serum and complements incubation times and assay reproducibility and stability, will enable us to more rapidly implement this assay in our clinical laboratory routines. In addition, we demonstrate that FCtox crossmatching of deceased donor cells has superior specificity compared to conventional CDC assay especially regarding high frequencies of false-positive reactions.     

Ankylosing spondylitis,HLA-B27 and Klebsiella. II. Cross-reactivity studies with human tissue typing sera.

Human monospecific HLA B27 typing sera have been shown to have increased binding activity for klebsiella extracts by haemagglutination (P less than 0.001), radiobinding assay (P less than 0.025) and radiolabelled antigen competition assay (P less than 0.02) when compared to non-B27 tissue typing sera. These observations are in agreement with those of studies using rabbit sera, suggesting that HLA B27 lymphocytes may exhibit partial cross-reactivity with bacterial antigens found in some Gram-negative microorganisms such as klebsiella. It is suggested ankylosing spondylitis may occur as a result of immunological damage following infection by Gram-negative bacteria carrying antigens having stereochemical similarity to self antigens.