HLA-DR antigens in Chinese-American families

This paper contains results of a study on HLA-DR antigens in Chinese-American families. DR2, DR4, DRw9, and DRw6Y were the most common DR specificities encountered, and DR1 occurred with the lowest frequency. All recognized DR antigens were observed. The frequency of a blank allele was 6.4–12.8%. Weak serologic reactions with sera primarily of Caucasian origin were not infrequently observed. These findings suggested that ethnic-related antigens were present in this population. Two families showed segregation of a new serologic pattern based on polyspecific sera. The gene frequencies of the Bf^F allele and the GLO¹ allele were low as compared to Caucasians. A method is described for improving the yield of viable B cells from frozen B-lymphocyte preparations.     

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.     

Monoclonal antibodies to rabbit lymphoid cells: Preparation and characterization of a T-cell-specific antibody

Spleen cells of mice immunized with rabbit spleen and mesenteric lymph node (MLN) cells were fused with mutant mouse myeloma cells. Twenty-six clones which react with rabbit lymphoid cells were obtained. By membrane immunofluorescence, as analysed visually and by the fluorescence-activated cell sorter, one of these clones, 9AE10, produced an antibody that reacted with nearly all thymocytes (> 90%) and with from 46 to 78% of spleen, MLN and peripheral blood lymphocytes. Double-membrane immunofluorescence with the 9AE10 monoclonal antibody (MAb) and anti-Ig showed that 9AE10⁺ and Ig⁺ cells of spleen, MLN and peripheral blood were distinct and non-overlapping populations. Thus, the 9AE10 MAb is a T-cell-specific antibody.The 9AE10 MAb also reacted with most brain cells and with approximately 30% of bone marrow cells. Biochemical analysis of the antigen recognized by the 9AE10 MAb indicated that the antigen is a glycoprotein with an apparent molecular weight of 25,000. These data indicate that the 9AE10 MAb may be directed against a Thy-1-like antigen.     

125I anti-immunoglobulin binding assay for the detection and characterization of anti-platelet antibodies

The binding assay as described in this paper is a very versatile system, and in this study it has been evaluated specifically for the detection of allo- or autoantibodies to platelets in man. The basic assay involves the incubation of a standard number of platelets with dilutions of test sera and the detection of platelet bound immunoglobulin by a second incubation with ¹²⁵I labeled. immunoadsorbent purified rabbit F(ab') anti-human F(ab')₂ (RAH). Of most importance, by varying the number of target platelets in the titrations and looking for binding plateaus, one can readily define conditions of optimum sensitivity for particular serum/platelet combinations. In addition, the assay can be used in conjunction with quantitative absorptions to subdivide complex sera into subspecificities and to give an estimate of the relative amounts of particular antigens on different platelets or other tissue or cell suspensions. One can also use saturating concentrations of RAH in the second incubation, in which case the amount of platelet bound radioactivity is directly related to the amount of first antibody bound to the platelets, and this can be manipulated to give information about serum antibody concentrations and amounts of antigen on the target tissue. The problem of ABO antibodies in this system, optimal conditions for platelet storage for the assay, and techniques for reducing assay backgrounds resulting from immunoglobulin adsorbed to the platelet surface are all evaluated.     

Effect of cyclosporin A on the early activation of human T helper lymphocytes: inhibition of RNA-synthesis and modification of the expression of activation antigens

The ability of cyclosporin A (CS-A) to inhibit induced lymphocyte activation and to modify expression of membrane receptors was assessed on human T helper cells. Flow cytometric cell cycle analyses of acridine orange-stained cells showed that CS-A (0.5 micrograms/ml) inhibits the G0-G1 activation process of a substantial proportion of PHA- and Con A-stimulated lymphocytes. The expression of Tac, OKT9 and 4F2 antigens (previously shown to be expressed or increased on activated cells) was investigated by immunofluorescence. Fewer cells expressed the Tac and OKT9 antigens after activation in presence of CS-A, but the percentage of 4F2-positive cells remained unchanged. Analyses of receptor densities measured by fluorescence intensity revealed for all three investigated antigens a decreased receptor density on positive cells in presence of CS-A. Thus, CS-A not only inhibited cell activation (G0-G1 transition) and the expression of Tac, OKT9 and 4F2 antigens, but it also diminished the number of Tac, OKT9 and 4F2 antigens per cell. Assessing specifically the activation of OKT4 (helper) and OKT8 (cytotoxic) cells after 24 h, either by double-fluorescence or by cell fractionation with anti-OKT4 or anti-OKT8 antibodies plus complement, showed that preferentially OKT4 cell activation as well as expression of Tac and OKT9 antigens on those cells was inhibited in the presence of CS-A.     

Antigen-Specific Electrophoretic Cell Separation (ASECS): Isolation of human T and B lymphocyte subpopulations by free-flow electrophoresis after reaction with antibodies

The electrophoretic mobility of human lymphocytes can be reduced by incubation with surface antigen specific antibodies under non-capping conditions. This renders subpopulations of human peripheral blood lymphocytes accessible to separation by free-flow electrophoresis.After reaction of lymphocyte preparations with anti-IgM antibody and a fluorescent second antibody, B lymphocytes showed a considerable shift in position in preparative cell electrophoresis and could be separated with high yield, purity and vitality. Similarly, a T cell subpopulation reactive with the monoclonal antibody T811 could be isolated, even though only small amounts of this antibody were bound, by using a double-sandwich method.Non-specific antibody uptake via Fc-receptors did not contribute to the observed shift of antibody-labelled cells to lower electrophoretic mobility. Flow cytometric analysis showed that cells were separated according to their antigen density.Thus cell electrophoresis can be used to separate antibody-labelled cells. With a flow rate of 100,00 cells/sec this method has a much higher separation capacity than fluorescence-activated cell sorting. The described method should be applicable to the separation of a wide range of cell populations for which specific antibodies are available.     

A method of purifying sheep sIg+ lymphocytes as a tool for class II MHC antigen analysis

A method is described for the purification of sheep lymphocytes carrying class II MHC antigens. After incubation of purified blood lymphocytes on anti-IgM-coated petri dishes, the adherent fraction contained 95% sIg-positive cells determined by immunofluorescence. When tested with cross-reacting anti-class II (bovine and human) monoclonal antibodies, more than 95% of these cells were positive either by immunofluorescence or cytotoxicity. This technique will permit studies of the polymorphism of sheep class II antigens.     

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (± weak DC) (22 MoAbs); Dr + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.     

The mode of action of the calcium ionophore A23187 on T cell proliferation. I. The ionophore does not replace lymphokines but acts via induction of IL-2 production on IL-2 responsive cells

The two compounds, the calcium ionophore A23187 and phorbol myristate acetate (PMA), which are not mitogenic for mouse thymocytes, induce proliferation when added in combination to thymocyte cultures. Short exposure to PMA renders the cells responsive to IL-2, added exogeneously. The cells rendered IL-2-responsive by PMA are enriched in the more mature peanut agglutinin (PNA)-negative population. The ionophore does not render PNA-negative thymocytes IL-2-responsive, but induces proliferation of PMA-pulsed PNA-negative thymocytes. PMA-pulsed PNA-negative thymocytes proliferating in response to either exogeneous IL-2 or ionophore express IL-2 receptors. However, the ionophore does not mimic IL-2 action but acts indirectly by induction of both IL-2 production and of IL-2 receptor expression via IL-2. This view is based on the following findings: 1) IL-2 could be detected in supernatants derived from PMA-preactivated thymocytes incubated with the ionophore; 2) The IL-2-induced proliferation, as well as the ionophore-induced proliferation, was specifically blocked by a monoclonal anti-IL-2-receptor antibody; 3) The proliferation induced by exogeneous IL-2, as well as that induced by the ionophore, could be specifically inhibited by metabolically blocked T lymphoblasts carrying IL-2 receptors competing with the responder cells for the available IL-2 added or produced in the system.     

A new HLA Bw16 subtype defined in both Negroid and Saudi Arabian populations

Serological identification of a new HLA-Bw16 subtype, B39B, was made by the analysis of reaction patterns of many alloantisera and one monoclonal antibody. The B39B pattern of reactivity was shown to be distinct from HLA-Bw38, Bw39, and 8w57. Cytotoxicity testing before and after absorption suggests that the B39B specificity belongs to the HLA-B7 cross-reactive group. The B39B was clearly demonstrated in two families. This antigen was detected in Negroids and Saudi Arabian Caucasoids but not in a large panel of British Caucasoids.     

1,25-Dihydroxyvitamin D3 induces a myelomonocytic phenotype with enhanced effector cell function in the HL-60 promyelocytic leukemia cell line

Human promyelocytic leukemia (HL-60) cells were induced by 1,25-dihydroxyvitamin D3 (calcitriol) to differentiate and examined using a panel of monoclonal antibodies (MoAbs) and functional assays. Although morphologically and histochemically these cells appeared to be of the monocyte-macrophage phenotype, there was a decline in Fc receptors for IgGl and no induction of class II HLA antigens. There was, however, dramatic induction of the antigen detected by the myeloid-specific MoAb AML-2-23. These data suggest that the phenotypic changes induced by calcitriol in HL-60 cells are consistent with myelomonocytic differentiation in that the resultant cells possess characteristics of both monocytes (morphology, non-specific esterase staining, high levels of AML-2-23 reactivity) and granulocytes (PMN 29 binding, decreased Fc receptors for IgGl, absence of class II HLA antigens). Perhaps more important, the ability of calcitriol-treated cells to perform antibody-dependent cellular cytotoxicity and phagocytosis was markedly augmented. Lysis of antibody-coated erythrocytes by HL-60 cells increased from 5% in controls to 30-35% with calcitriol treatment for 4 days. This enhanced effector cell function was seen despite a decline in Fc receptors measured by cytofluorography. These data suggest that calcitriol may be involved in both differential and functional activation of myeloid cells.     

Evaluation of flow cytometry and fluorescence microscopy for the estimation of bovine mononuclear phagocytes

Bovine blood mononuclear cells were isolated by density gradient centrifugation on Ficoll-Paque. Phagocytic mononuclear cells were characterized functionally by ingestion of fluorescent latex beads. After incubation with beads the cells were treated with Triton X-100 and propidium iodide (PI) to stain DNA. Cells were analyzed with a FACS-III instrument connected to a Nuclear Data-6660 multiparameter computer system. The computer was used to evaluate the 2 parameter histograms in order to enumerate the percentage of cells with different numbers of associated beads. With this system we also obtained information about cell concentration and number of beads per cell. Results from flow cytometry and manual counting by fluorescence microscopy were compared and good correlation (r = 0.91) was obtained. During the first hours of incubation latex beads adhered to cell surfaces as demonstrated by FCM histograms and fluorescence microscopy. Blood mononuclear phagocytes have to be incubated for several hours before significant phagocytotic activity can be detected.     

An indirect rosette technique for the identification and separation of human lymphocyte populations by monoclonal antibodies: a comparison with immunofluorescence methods

A simple indirect rosette technique is described which allows the rapid enumeration, morphological identification and separation of monoclonal-antibody (MoAb)-defined human lymphocyte populations. The method is based on the binding to MoAb-stained cells of marker ox red blood cells coated with anti-mouse immunoglobulin. When compared with indirect immunofluorescence staining assessed by microscopy, the rosette method is quicker, shows greater sensitivity and is less subjective. Purification of helper (OKT4), suppressor (OKT8) or B-cell (BA1) subpopulations by positive or negative enrichment of rosetted cells gives purity and recoveries comparable with that of the fluorescence-activated cell sorter (FACS). Such purification is readily performed without loss of viability or function and is more easily done sterilely than with the FACS.     

A new brain cell surface glycoprotein identified by monoclonal antibody

Of 207 monoclonal antibodies produced against cultured mouse cerebellar cells, 16 reacted with cerebellar cell surfaces and 4 reacted with glycoproteins. One of them, called anti-BSP-3 (Brain cellSurfaceProtein-3) defines a 48,000 molecular weight protein which can be iodinated at the surface of cultured cerebellar cells. Lectin-binding and sugar incorporation studies established the glycoprotein nature of the antigen. Astroglia (glial fibrillary acidic protein-positive cells) in primary cerebellar cultures were labelled intensely for this antigen by the indirect immunofluorescence method while neuronal cells and their processes were more weakly labelled. Fibronectin-positive cells were negative for BSP-3. In cerebellar sections using the immunoperoxidase method at both the optical and electron microscope levels, the difference in staining intensity between astrocytes and neuronal cells was not significant: in Purkinje cells and in the large neurones present in the deep cerebellar nuclei the immunoperoxidase percipitate was confined to the plasma membrane while in both astrocytes and granule cells cytoplasmic labelling was also observed. Oligodendrocytes do not appear to react with the anti-BSP-3 monoclonal antibody; neither do endothelial or leptomeningeal cells.The availability of a monoclonal antibody produced by a stable hybridoma line will be a powerful tool in attempts to purify the BSP-3 antigen and to elucidate its function.     

Serological precipitation method for studying biosynthesis and secretion of immunoglobulins by human peripheral blood lymphocytes

A reproducible, serological method to quantify radiolabeled immunoglobulin (Ig) synthesized and secreted by human peripheral blood lymphocytes is described. In contrast to indirect precipitation (rabbit antihuman Ig and goat antirabbit Ig), the direct coprecipitation method using human IgG and rabbit antihuman IgG F(ab)₂ showed much smaller, non-specifically precipitable radioactivity (i.e., 2～14%) without affecting Ig radioactivity.     

Recognition of HLA-B27 and related antigen by a monoclonal antibody

A monoclonal antibody that binds specifically to HLA-B27, B7, and B22 is described. Binding to B27 appeared to be slightly stronger than to B7 and stronger than to B22 in an indirect binding assay, but no difference in B7 and B27 binding could be detected by Scatchard analysis. No distinction could be made between B27 on cells from normal and from ankylosing spondylitis patients in any assay system. The antibody, which was not cytotoxic, blocked complement-dependent cytolysis mediated by human HLA typing sera specific for B7 and B27. Competitive binding studies with other monoclonal antibodies showed that ME1 could block the binding of antibodies that recognized different antigenic sites on HLA. ME1 did not bind to Klebsiella pneumoniae. This reagent will be useful in further analysis of the relationship between B27 and ankylosing spondylitis.     

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one M_r 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent M_r 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32–34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.     

Characterization and expression of the HLA-DC antigens defined by anti-Leu 10

The expression of HLA-DC antigens on peripheral blood mononuclear cells, in tonsil and lymph node tissue sections, on tumor cell lines, and on activated T cells was studied using monoclonal antibody, anti-Leu 10. Anti-Leu 10 reacts with HLA-DC molecules on homozygous B cell lines expressing HLA-DR 1,2,4,5,6,8, and 9. It reacts with heterozygous B lymphocytes expressing DR7 and DRw10, suggesting it also recognizes HLA-DC molecules linked to DRw10. The HLA-DC molecules detected by anti-Leu 10 are expressed on all Ig-positive and DR-positive peripheral B lymphocytes and an apparent subpopulation of DR-positive periperal blood monocytes. Two-color immunofluorescence experiments using phycoerythrin-anti-HLA-DR (L243) and FITC-anti-Leu 10 demonstrated a correlation of the amounts of HLA-DR and DC antigens expressed on B lymphocytes. Cells expressing relatively low or high amounts of one Class II molecule express respectively low or high amounts of the other Class II molecule. Anti-Leu 10 reacted with all B lymphocyte derived tumor cell lines not with lines of myeloid or erythroid origin, and with only one T cell derived line, HUT-78 which has an activated T cell phenotype. Consistent with this result, anti-Leu 10 binding suggested the presence of HLA-DC on activated T cells in lymphoid tissue, in addition to staining B cells. HLA-DC was also detected on mitogen and MLC activated T cells by anti-Leu 10 binding. Anti-Leu 10 is, therefore, a useful reagent for further studies of the role of HLA-DC in T cell activation and in normal B cell and monocyte functions.     

Antibody producing human-human hybridomas. I. Technical aspects

Technical aspects of generation of antibody-secreting human-human hybridomas are evaluated as based on 100 human-human fusions with a human B-lymphoma cell line (RH-L4) or the SKO-007 myeloma cell line as malignant fusion partners, and compared with similar fusion conditions in the mouse hybridoma system. The yield of hybrids was significantly lower when normal peripheral blood lymphocytes were used as fusion partners as compared with spleen lymphocytes, but could be substantially improved by increasing the amount of mitotic active B-lymphocytes by mitogen stimulation of the lymphocytes, preferably in HAT medium, prior to fusion. Furthermore, human hybrids grew slower and had a higher degree of chromosomal instability than usually observed in the mouse hybridoma system. Thus, out of 72 fusions, only 3 stable hybrids with antibody production against a predefined antigen were established. The importance of improved sources of human B-lymphocytes for human-human hybridoma production is discussed and methods of obtaining such improvement suggested.