Evidence for the separate molecular expression of four distinct polymorphic Ia epitopes on cells of DR4 homozygous individuals

The monoclonal antibodies 109d6 and IVD12 reacted with separate polymorphic Ia epitopes in immunofluorescent studies. Using a panel of lymphoblastoid B cell lines, antibody 109d6 reacted with all HLA-DR4 or DR7 positive lines in a pattern resembling the MT3 specificity recognized by human alloantisera. The antibody IVD12 reacted with all HLA-DR4 and two of three DR5 positive B cell lines suggesting that it recognized a specificity similar to MB3. The intensity of fluorescence was greater on DR5(+) cell lines than on DR4(+) cell lines relative to the amount of a nonpolymorphic Ia determinant. Among 45 unrelated control individuals reactivity with antibody 109d6 was correlated most closely with (r = 0.724) but not identical in occurrence to the MT3 specificity. Cocapping experiments demonstrated that the 109d6 epitope and the IVD12 epitope were present on independently redistributed cell surface molecules of DR4 homozygous lymphoblastoid cell lines. Furthermore, the DR4 alloantigens detected by an absorbed polyclonal human alloserum were similarly identified on molecules that were independent from those bearing either the 109d6 epitope or the IVD12 epitope. Taken together, these data indicate the existence of at least four distinct, serologically defined Ia molecular species.     

Removal of rat dendritic cells from single cell suspensions by passage through columns of Sephadex G-10

Rat T lymphocytes require dendritic cells as accessory cells in order to respond to the mitogen sodium periodate. Passage of a lymph node cell suspension through a column of Sephadex G-10 reduced the mitogenic response by greater than 90%, despite a cell recovery of 75%. An even greater reduction in the response occurred after a second passage over Sephadex G-10; addition of purified dendritic cells restored the response. Lymph node cell suspensions and preparations enriched in dendritic cells were nearly depleted of these cells by passage over Sephadex G-10. After passage of lymph node cells over Sephadex G-10, a limited number of retained cells could be recovered; these included both dendritic cells and macrophages. Enumeration of macrophages in the various passed and retained fractions by non-specific esterase staining of smears confirmed that macrophages were effectively removed from lymph node cell suspensions by repeated passage over Sephadex G-10. Cell preparations that pass through Sephadex G-10 are therefore depleted of both dendritic cells and macrophages.     

Isolation and subfractionation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation on Percoll

The use of Percoll for isolation and subfractionation of PBMC and T-lymphocytes by discontinuous and continuous density gradient centrifugation is described: PBMC were isolated from human peripheral blood by discontinuous density gradient centrifugation on Percoll. The use of Percoll instead of Ficoll-Isopaque has the advantage that Percoll, in contrast to Ficoll-Isopaque, does not alter the density of monocytes. Therefore, a better separation of lymphocytes and monocytes was achieved after subsequent continuous density gradient centrifugation on Percoll. E-RFC were isolated by discontinuous density gradient centrifugation after a first low speed centrifugation step banding lymphocytes and SRBC on a Percoll-Ficoll cushion, and a subsequent high speed centrifugation step separating high density rosettes and SRBC from low density non-E-RFC. The advantage of this procedure is the short time of performance and that there is no need to resuspend the lymphocyte/SRBC pellet. PBMC, nph.PBMC T-lymphocytes were further subfractionated by continuous density gradient centrifugation on Percoll. The method described here resulted in a good separation of lymphocytes and monocytes. However, to obtain lymphocyte fractions with minute numbers of contaminating monocytes, a depletion of monocytes prior to further subfractionation of the lymphocytes by continuous density gradient centrifugation is recommended. A marker analysis of T-lymphocytes subfractionated by continuous density gradient centrifugation on Percoll shows that high density T-lymphocytes are enriched in ANAE positive lymphocytes of type 1 and depleted of ANAE positive lymphocytes of type 2. Low density T-lymphocytes are enriched in ANAE type 2 cells and depleted of ANAE type 1 cells. On the other hand, no considerable differences were found when analyzing the T-cells from different fractions for differentiation antigens by means of monoclonal antibodies (anti Lyt 3, OKT4, and OKT8). The results may indicate that subfractionation of T-lymphocytes by continuous density gradient centrifugation on Percoll provided T-cells in different functional states rather than T-cells of distinct subclasses.     

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.     

Phenotypic and functional characterization of pulmonary macrophages subpopulations after intratracheal injection of Paracoccidioides brasiliensis cell wall components

A shift in the activation of pulmonary macrophages characterized by an increase of IL-1, TNF-α and IL-6 production has been induced in mice infected with Paracoccidioides brasiliensis. It is still unclear whether a functional shift in the resident alveolar macrophage population would be responsible for these observations due to the expression of cell surface molecules. We investigated pulmonary macrophages by flow cytometry from mice treated with P. brasiliensis derivatives by intratracheal route. In vivo labeling with the dye PKH26GL was applied to characterize newly recruited pulmonary macrophages from the bloodstream. Pulmonary macrophages from mice inflamed with P. brasiliensis derivatives showed a high expression of the surface antigens CD11b/CD18 and CD23 among several cellular markers. The expression of these markers indicated a pattern of activation of a subpopulation characterized as CD11b⁺ or CD23⁺, which was modulated in vitro by IFN-γ and IL-4. Analysis of monocytes labelled with PKH26GL demonstrated that CD11b⁺ cells did infiltrate the lung exhibiting a proinflammatory pattern of activation, whereas CD23⁺ cells were considered to be resident in the lung. These findings may contribute to better understand the pathology of lung inflammation caused by P. brasiliensis infection.     

Isolation of mast cells from murine peritoneal cell populations by velocity sedimentation at unit gravity

A method for isolation of mast cells from murine peritoneal cell suspensions is described. Mast cells are isolated by velocity sedimentation at unit gravity on a linear gradient (generated in a discontinuous step fashion) of colloidal silica particles coated with polyvinyl pyrrolidone (Percoll) in Fischer's medium supplemented with 2% fetal bovine serum. The overall recovery of the peritoneal cells layered on the gradient is 90–95%, while the yield of mast cells is 77% with an average purity of 95%.     

Identification of live cells for flow cytometric analysis of lymphoid subset proliferation in low viability populations

Combined analysis of single cell DNA content and immunofluorescence by flow cytometry is complementary to tritiated thymidine analysis of cellular proliferation, allowing detailed dissection of particular cell types in a mixed population which respond proliferatively to selective stimuli. However, in vitro culture of primary immune cells (e.g., mouse spleen or lymph node) for periods of 24-72 h frequently results in a considerable fraction of non-viable cells which bind antibodies non-specifically, resulting in altered immunofluorescence distributions, inaccurate distinctions between positive and negative cells, and sometimes in misleading DNA distributions. Forward angle light scatter cannot readily be used to distinguish live from dead cells in this case because of the heterogeneous size distributions characteristic of cultured populations. We describe a method which uses treatment with DNAase prior to immunofluorescence staining to allow more accurate distinction between live and dead cells. This treatment markedly reduces the intensity of DNA staining for non-viable cells, providing complete live/dead discrimination and improved ability to analyze the proliferative status of specific cell subtypes in low viability cultures.     

A simple technique for evaluation of methods of cell separation

A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.     

Monoclonal antibody detection of carbohydrate-defined and protein-defined H-2Kk antigens

Three different monoclonal anti-H-2K^k antibodies, 27R9, 30R3 and 11-4 were examined for the biochemical nature of the antigenic determinants they recognize. When these were compared on the basis of their sensitivity to pronase and various glycosidases, 27R9 was shown to bind to protein-defined H-2K^k antigens, while 30R3 and 11-4 bound to H-2 antigens defined by carbohydrate. From sugar inhibition studies, and treatments with specific glycosidases, d-mannose appears to be the immunodominant sugar involved in the antigenic site recognized by 30R3, while several sugars, namely sialic acid, d-mannose and α-and β-linked d-galactose would appear to be components of the antigenic site bound by 11-4. The carbohydrate determinants appear to be present on glycolipid molecules, since both the 30R3 and 11-4 antibodies could be inhibited by glycolipid extracts from spleen cells of the appropriate H-2 haplotype, as well as from several other strains of mice previously shown to be cross-reactive targets for these antibodies. This finding is supported by evidence that the molecule carrying the protein-defined antigen is distinct from that carrying the carbohydrate-defined antigens. The results are discussed in the light of current information on the nature of glycolipid Ia antigens, as well as the role of H-2 antigens in T-cell interactions.     

Modulation of natural killing by tumor promoters: the regulatory influence of adherent cells varies with the type of target cell

We have analyzed the regulatory effects of two classes of tumor promoters, phorbol diesters and indole alkaloids, on human natural killer (NK) cell activity in vitro. In accordance with previous reports, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited natural killing against K 562 targets by unseparated mononuclear cells. Here, suppression of NK required the presence of adherent cells (macrophages). Contrary to the results obtained with K 562, tumor promoter-induced suppression of NK activity tested against U 937, another cell line of known NK susceptibility, was independent of the presence of adherent cells. Thus, NK cytotoxicity of effector cells rigorously depleted of adherent and Ia-positive cells was still inhibited when assayed against U 937, while it was generally enhanced when tested against K 562. Identical results were obtained with teleocidin and dihydroteleocidin B, two members of the recently discovered indole alkaloid class of tumor promoters. Therefore, we demonstrate that the regulatory effect of tumor promoters on human NK activity (suppression or stimulation) is determined not only by macrophages at the effector cell level but also by the type of target cell under study.     

A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17

A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kobler and Milstein [1]. This antibody recognizes a new specifity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.     

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.     

Are Granulocytes the Main Effector Cells of Natural Cytotoxicity in Chickens?

Purified peripheral blood granulocytes from chicken were tested for cytotoxic activity against two types of virus-transformed chicken cell line, LSCC-H32 and LSCC-RP9. Strong cytotoxicity could be demonstrated, as measured in a 4-hr 51Cr-release assay, especially against the fibroblastoid LSCC-H32 cells. The degree of cytotoxicity was dependent on the E:T ratio. Normal CEF cells were completely resistant to the cytotoxicity. No cytotoxicity of human granulocytes could be observed against a variety of adherent and non-adherent target cells, as measured by the same microcytotoxicity technique. The priority of granulocytes in the natural cytotoxicity in the avian system is, therefore, suggested.     

Discrimination of live and early apoptotic mononuclear cells by the fluorescent SYTO 16 vital dye

Accurate detection of apoptotic cells is important for the determination of cell viability. The aim of this study was to compare the sensitivity of the cell permeant SYTO 16 fluorescent dye for detecting early apoptotic mononuclear cells (MNCs) in normal donor blood with other apoptosis assays [i.e. Annexin-V, light scatter/7-amino-actinomycin-D (7-AAD) and chloromethyl-X-rosamine (CMXRos)] and to identify critical parameters for optimal SYTO 16 staining. Apoptosis was induced in normal human leukocytes from adult peripheral blood or cord blood, or the Jurkat T-lymphocytic cell line and assessed by fluorescence microscopy and flow cytometry. Dual labelling showed that SYTO 16 detected more apoptotic MNCs compared to Annexin-V. SYTO 16 staining intensity was consistent with the light scatter profiles expected of live, apoptotic and necrotic MNCs and was more objective than light scatter/7-AAD. CMXRos staining required considerable care and may not be a robust marker of apoptotic primary MNCs. For SYTO 16 flow cytometric analysis, the optimal conditions for staining 1x10(6) leukocytes were 4 nM SYTO 16 in the presence of 30 muM verapamil for 25-45 min at 37 degrees C in media containing calcium/magnesium supplemented with protein. A P-glycoprotein inhibitor, such as verapamil, and calcium/magnesium are essential for optimal loading of SYTO 16 into live MNCs and discrimination of apoptotic MNCs in normal blood samples. SYTO 16 is a sensitive, simple, inexpensive 'live cell' method for the discrimination of live, apoptotic and necrotic normal blood MNCs and is more sensitive for detecting apoptosis in these cells than Annexin-V or light scatter/7-AAD.     

An evaluation of serum sources used in the separation of T- and B-lymphocytes by nylon-wool columns

Gamma-globulin-free (GG—) newborn-calf, mouse and horse, as well as newborn-calf and calf serums were evaluated as substitutes for fetal bovine serum in the classical nylon-wool procedure used for the separation of T- and B-lymphocytes. The efficacy of the cell separation was evaluated by non-specific esterase staining and cytotoxicity tests for cell surface Thy 1.2 antigen and surface immunoglobulins. The results of these studies indicate that GG—newborn-calf, mouse and horse but not newborn-calf or calf serum could be substituted for fetal bovine serum. The results of these studies also indicate that the ratio of adherent to non-adherent cells is a useful indicator of the efficacy of the cell separation. A ratio of non-adherent to adherent less than 1.0 is indicative of a successful separation.     

Aging and immunity: decrease in interleukin-2 production and interleukin-2-dependent RNA-synthesis in lectin-stimulated and murine spleen cells

The RNA-content of G1 cells in lectin-stimulated spleen cell cultures of young and aged NMRI mice was determined by flow cytometry. In spleen cells of aged mice a preferential decrease of G1 cells with a high RNA-content, so-called G1b cells, was found. Since, as shown in a previous report, only cells with a high RNA-content are able to proliferate and the passage of low (G1a) to high (G1b) RNA-content is interleukin-2(IL-2)-dependent, the ability of young and old spleen cells to produce IL-2 was tested. In old spleen cells a diminished production of IL-2 was found. Addition of external IL-2, however, did not increase the proliferative capacity of old spleen cells, nor did it induce more G1b cells. Thus spleens of aged mice contain cells, which can be activated by lectin, but then fail to respond to IL-2. Both decrease in IL-2 production and receptivity for IL-2 may contribute to the diminishing immune response in aging individuals.     

A procedure for the isolation of highly purified populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood

A sequential separation protocol is described which reproducibly yields biologically active populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood. The purification protocol employed T lymphocyte rosetting with unmodified sheep red blood cells, monocyte adherence to microexudate-coated flasks and anti-immunoglobulin panning of B cells. Population purity was determined by cytofluorographic light scatter analysis, expression of cell surface markers, esterase activity and mitogen responsiveness. The sequential protocol reproducibly yielded viable and functionally active cell populations of greater than 95% purity from a single starting population of mononuclear cells.     

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--II. Evidence that the epitope recognized is involved in a late step in the cytolytic mechanism

A monoclonal antibody (RH1-38) which blocks multiple systems of cell-mediated cytotoxicity was functionally characterized. RH1-38 specifically blocks, in the absence of complement, natural killer (NK) activity (K562 targets) without any effect on NK-K562 conjugate formation. Kinetic studies suggested that the antibody blocks a step that occurs 30-120 min after effector populations are mixed with target cells. Single-cell cytotoxicity assays in agarose, combined with standard 51Cr release assays and Michaelis-Menten analysis revealed that RH1-38 markedly decreases Vmax and the number of active NK cells, again without any effect on the number of target-binding cells. The maximum recycling capacity was usually decreased, but in some experiments unchanged, in the presence of the monoclonal antibody. RH1-38 inhibited equally well whole peripheral blood mononuclear leukocytes (PBML), Percoll-fractionated lymphocytes enriched for NK activity, and interferon (IFN)-boosted NK activity. PBML exposed to RH1-38 and then washed mediated depressed NK activity which was partially reversed by subsequent treatment with IFN. These studies are most consistent with the hypothesis that RH1-38 inhibits a step late in the NK cytolytic mechanism rather than through an effect on conjugate formation. The primary effect is probably not on the IFN-generating or boosting mechanism, but a secondary effect on IFN-related mechanisms cannot be ruled out. Inhibition through an effect on a small lymphocyte modulator of NK activity is also unlikely but not rigorously excluded. Thus, RH1-38 appears to inhibit NK activity through a direct effect on NK effector cells, probably by interfering with a cell-surface molecule which is important in the expression of NK activity. The companion paper demonstrates that this monoclonal antibody immunoprecipitates a molecule which is very similar or identical to the LFA-1 antigen. Thus, RH1-38 recognizes either a novel epitope on the LFA-1 molecule or alternatively a distinct, functional killer cell surface molecule. The epitope appears to be involved in a late step in the cytolytic mechanism, possibly part of the effector cell lytic machinery.     

An improved autoradiographic technique for the detection of antibody-forming cells

An autoradiographic technique for the detection of antibody-forming cells has been developed for the assay of anti-DNP responses. The lymphoid cells suspension to be assayed was allowed to sediment on to a glass slide coated with DNP-conjugated gelatin to which the secreted antibody bound during subsequent incubation. The bound antibody and its Ig class was revealed by a second incubation using ¹²⁵I-anti-immunoglobulin reagents followed by autoradiography. Studies on the sensitivity and specificity of the method are presented and its advantages over other techniques described. The technique should be readily applicable to other haptens.     

Flow cytometric assessment of estrogen receptor beta expression in bovine blood neutrophils

The optimisation of a flow cytometric protocol for the determination of the estrogen receptor beta (ERbeta) expression in bovine blood neutrophils is described. The following final incubation conditions were obtained: fixation with 0.25% formaldehyde and 70% methanol, both for 1 h; permeabilisation with 0.05% Triton X-100, overnight labelling at 4 degrees C with the primary antibody diluted at 10 microg/ml and subsequent labelling for 30 min on ice with the fluorescein isothiocyanate-conjugated secondary antibody at 8 microg/ml. Of the three anti-human or anti-rat ERbeta primary antibodies evaluated, only PA1-311 was found to cross-react with bovine cells. Immunoblot analysis supports the obtained results. The flow cytometric technique allows reproducible quantitative determination of the ERbeta protein in neutrophils and may be a valuable tool for future expression studies in these cells of the innate immune system.