Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.     

Cell subpopulations in the paracortical area of the normal human lymphnode defined by monoclonal antibodies

The cell subpopulations of normal human lymphnode were studied by immunohistochemical and immunoelectronmicroscopy techniques carried out by using monoclonal antibodies. OKT3 positive cells were demonstrated overwhelming in comparison to both OKT4 positive cells and OKT8 positive cells; furthermore la positive cells, dendritic in shape, were observed. Our results, compared to literature data, confirm that paracortex of limphnode is a T-dependent area. Moreover a possible role of the la positive dendritic cells in the immunological education of T helper-inducer lymphocytes was hypotised. Finally, our immunoelectronmicroscopic findings prove that OKT4 positive cells and OKT8 positive cells show different ultrastructural patterns.     

A method for the identification of human peripheral blood T lymphocytes by sequential immunogold and esterase double staining

A double staining method involving the sequential use of monoclonal OKT hybridoma antibodies applied in the colloidal immunogold method and followed by a simultaneously capturing azo dye method for the detection of acid alpha-naphthyl acetate esterase (ANAE) is described. Mononuclear leukocytes isolated from human peripheral blood using a Ficoll-Hypaque density gradient were stained. M-pattern ANAE-positive monocytes (diffuse staining) were excluded from the lymphocyte counts. 80 +/- 5% of all lymphocytes were T-pattern ANAE positive (dot-like staining) and 77 +/- 3% were OKT3 positive. 86 +/- 6% of all ANAE-positive T-pattern lymphocytes were also OKT3 positive, and 89 +/- 6% of all OKT3-positive lymphocytes were also ANAE positive. This indicates that ANAE is a good marker for total human T lymphocytes. 53 +/- 10% of human peripheral blood lymphocytes were OKT4 positive and 87 +/- 8% of all OKT4-positive lymphocytes were also ANAE positive. 30 +/- 6% of all lymphocytes were OKT8 positive, and only 26 +/- 18% of all OKT8-positive lymphocytes were ANAE negative. This indicates that ANAE cannot be used to distinguish T-helper and T-suppressor lymphocytes as identified by monoclonal antibodies.     

Glucocorticoid receptors in subpopulations of human lymphocytes defined by monoclonal antibodies

Glucocorticoid receptors (GR) were investigated in subpopulations of lymphocytes identified by monoclonal antibodies. Purified T (OKT3+) and non-T lymphocyte subpopulations were isolated from human peripheral blood using Degalan bead columns coated with rabbit anti-human IgG. Purified subpopulations of OKT4+ and OKT8+ lymphocytes were obtained by coating the nonadherent population (T cells) from the first column with OKT4+ or OKT8+ and pouring it into a second Degalan column, coated with goat anti-mouse IgG. GR content and affinity were analyzed by a whole cell assay with [3H]dexamethasone as tracer. The numbers of GR in lymphocyte subpopulations (OKT3+ cells, non-T cells, OKT4+, and OKT8+ cells) were nearly equal. It is concluded that the differential effects of glucocorticoids on the circulatory kinetics of OKT4+ and OKT8+ cells probably are not related to differences in glucocorticoid receptors of these T-cell subpopulations.     

Three distinct antigen systems on human B cell subpopulations as defined by monoclonal antibodies

Three novel antigen systems (L22, L23, and L24) expressed on human B cell subpopulations were identified by using TB1-2C3, TB1-2B3, and TB1-3C1 monoclonal antibodies, respectively. L22 was expressed on a minor subpopulation of B cells in human lymphoid tissues and in the peripheral blood. These B cells associated with L22 were resting small B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. This antigen was absent from all cultured hemopoietic cell lines including B cell-derived cell lines as well as from all human B cell malignancies, except for B cell-type chronic lymphocytic leukemia and hairy cell leukemia. L23 and L24, on the other hand, existed on approximately two-third of B cells in blood and lymphoid tissues. These L23 and L24 antigens were expressed largely on small lymphocytes located in the mantle zone of lymphoid follicles and to a lesser extent on large blastic cells within lymphoid germinal centers. L23 and L24, like L22, seem to disappear from B cells during their differentiation into antibody-secreting cells, because they were not expressed on normal and neo-plastic plasma cells. This is additionally confirmed by the observation that L23 and L24 were expressed little or not at all on pokeweed mitogen-activated and Epstein-Barr virus-transformed B cells, and were absent from some of B cell malignancies that have been thought to correspond to the later stages of B cell development. Although L23, but not L22 and L24, was faintly expressed on mature granulocytes and monocytes, none of L22, L23, or L24 existed on human thymus and T cells. Immunoprecipitation studies showed that L23 and L24 were different molecular species consisting of a single glycoprotein with m.w. of 205,000 and 145,000, respectively. L22 antigen is presently under study.     

Functional properties of human B cell subpopulations defined by monoclonal antibodies HB4 and FMC7

Human B cell subpopulations can be distinguished by the expression of B cell-associated antigens. mAb directed against these structures allow the isolation and subsequent functional analysis of such subsets. Blood B cells from healthy individuals were separated on basis of the expression of the antigens recognized by the mAb HB4 and FMC7. The B cells present in the thus obtained populations were analyzed for their ability to secrete IgM and IgG after stimulation in vitro with polyclonal B cell activators (PWM, Staphylococcus aureus Cowan I), antigens (tetanus toxoid, type 4 pneumococcal polysaccharides), and soluble B cell differentiation factors. The results suggest that B cells capable of Ig and anti-tetanus toxoid or anti-type 4 pneumococcal polysaccharide antibody production after in vitro culture are localized in a relative small B cell subpopulation carrying the FMC7 determinant but lacking HB4. This holds true for both the IgM- and IgG-secreting B cells. These data further suggest that the majority of B cells found in the blood and which can be characterized as being surface IgM+/IgD+ HB4+ are immature cells unable to respond to differentiation-inducing signals.     

T cell maturation: thymocyte and thymus migrant subpopulations defined with monoclonal antibodies to MHC region antigens

The maturation sequences of thymocytes is known to some extent: A generative layer of subcapsular large lymphoblasts gives rise to a major population of small cortical thymocytes and a minor population of midsize medullary thymocytes. The relative contribution of these three populations to the peripheral T cell populations is not yet known. In this study, subcapsular lymphoblasts, cortical small cells, medullary cells, and thymic emigrant cells have all been analyzed by immunofluorescence for expression of the antigens H-2D, I-A, H-2K, and TL. H-2D is expressed brightly on all subcapsular large cells, dimly on cortical small cells, and brightly on all migrants, cortisone-resistant thymocytes (CRT), and peripheral T cells. I-A can be detected at low levels on 30 to 50% of cells in all the thymic subpopulations, and on 30 to 50% of migrants and peripheral T cells. Fifty to 80% of small cortical cells do not express detectable H-2K, but all the other subpopulations, both inside and outside the thymus, stain uniformly quite brightly. TL3 is expressed on 70 to 80% of subcapsular and cortical thymocytes, 30 to 40% of CRT, is undetectable on migrants but can be seen at low levels on 10 to 20% of spleen and lymph node T cells. The possibility that some or all of these antigens represent stable markers of separate lineages rather than unstable, stage-specific markers is discussed.     

Heterogeneity of human T4+ inducer T cells defined by a monoclonal antibody that delineates two functional subpopulations

A monoclonal antibody termed anti-T4 that detected approximately 60% of peripheral blood T lymphocytes was shown to define the human inducer population. In the present study, we characterized three additional monoclonal antibodies, anti-T4A, anti-T4B, and anti-TQ1, that were reactive with a similar percentage of T lymphocytes. Anti-T4A, anti-T4B, and anti-T4 delineated identical cell populations, while those defined by anti-TQ1 differed in several respects: 1) Anti-TQ1 stained a minority (less than 7%) of thymocytes, whereas the other antibodies stained a majority (80%); 2) Anti-TQ1 reacted with 70 to 85% of T4+ lymphocytes, but also stained 50% of T cells within the T4- (T8+) cytotoxic/suppressor subset; 3) The antigen defined by anti-TQ1 was not restricted in its expression to T cells; it defined a fraction of normal B and null lymphocytes as well as non-T cell lines. In vitro studies indicated that the subpopulations of T4+ T lymphocytes delineated by anti-TQ1 were functionally distinct. Although T4+TQ1+ and T4+TQ1- T cells proliferated in an equal fashion to soluble antigen and alloantigen, only the T4+TQ1+ subset was responsible for maximal proliferation in autologous MLR. This T4+TQ1+ subset contained a population of lymphocytes reactive with the previously defined JRA autoantibody. In contrast, the T4+TQ1-, but not the T4+TQ1+, subset provided the majority of T cell help for B cell immunoglobulin production in a pokeweed-driven system. We conclude that the subpopulation of T4+ inducer cells responsible for maximal helper activity in T-B interactions is restricted to a minor subpopulation of T4+ lymphocytes.     

Survival and PHA-stimulation of gamma-irradiated human peripheral blood T lymphocyte subpopulations

Human peripheral blood T lymphocyte subpopulations were identified and isolated on the basis of their ability to bind IgG (T-G), IgM (T-M), or neither immunoglobulin class (T-null). Lymphocytes were exposed to 0, 0.5, 1.0, 2.5 or 5.0 Gy of 60Co gamma-rays either as a T-cell suspension or as separated T cell subsets. Survival curves, determined 5 days after irradiation, revealed that each subset has radiosensitive and radioresistant portions, and that the T-G cell is the most sensitive subset. Mitotic indices of 48-h cultures showed that the response of unirradiated T lymphocytes to PHA varied greatly among the subsets, the highest indices being obtained for the T-M and the lowest for the T-G cells. With the possible exception of the T-G cells, the subsets are relatively resistant to mitotic effects of gamma-rays. T-G cells suppress the PHA-induced mitotic response of the other T lymphocyte subsets, and this suppressor effect is radiosensitive, being abolished by 1.0 Gy. It is concluded that lymphocytes exposed to greater than or equal to 1 Gy of gamma-rays will have very few dividing B lymphocytes or T-G cells. This together with radiation-induced loss of T-G suppressor action means that the predominant lymphocyte types in mitosis after greater than or equal to 1 Gy are the radioresistant T-M and T-null cells.     

Immunogold silver staining method for light microscopic detection of peripheral blood lymphocyte surface antigens with monoclonal antibodies.

Labelling of peripheral blood Lymphocytes surface antigens was carried out using the method of colloidal gold, enhanced with silver staining. Instead of PBS the minimal essential medium (MEM) according the Eagle, pH 7.2, was used rinsing of isolated lymphocytes. Visibility of positive reactions on lymphocytes at application of both mentioned media was the same. Positive reaction at demonstration of p24 BLV on cells acquired the from of black cap while the IgG expression was observed in the from of diffuse dispersion of colloidal gold on cells. Differences between the application of individual media were observed in the shape of peripheral lymphocytes in smears. Utilization of Eagle's MEM resulted in more uniform shapes and optically smooth surfaces when viewed under a light microscope.     

Phylogenic distribution of human renal antigens defined by monoclonal antibodies

The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated.     

Morphometric ultrastructural evaluation of human peripheral blood T lymphocyte subpopulations isolated on the basis of their affinity for sheep red blood cells

The present investigation were undertaken to compare on the basis of ultrastructural morphometric analysis human T lymphocyte subfractions forming rosettes with sheep red blood cells (SRBC). The following T lymphocyte subfractions were obtained: ARFC (active rosettes), T1RFC (rosettes after 1 h at 4 degrees C) and T2RFC (rosettes after 2 h at 4 degrees C). Analysis of the mean cell and nuclei volumes of lymphocyte subfractions led to the separation of two cell groups differing in volume in each donor: a cell group consisting of large cells (T1RFC) and group of small cells (ARFC and T2RFC).     

Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.     

Epitopes of human intestinal alkaline phosphatases, defined by monoclonal antibodies

Antibody additivity assayTopology     

Synthesis of prostaglandins by subpopulations of human peripheral blood monocytes

The ability of monocytes/macrophages to regulate various aspects of immunologic responses may in part depend on their release of soluble substances such as prostaglandins. Using quantitative gas-liquid chromatography/mass spectrometry, prostaglandin E₂ was found to be the major prostaglandin synthesized in culture by human peripheral blood monocytes. Subjecting these cells to discontinuous density gradient fractionation demonstrated significant differences in the synthesis of prostaglandins E₂ and E₁ among the resulting monocyte subpopulations.     

Localization of alpha 1-microglobulin (HC protein) in normal human tissues: an immunohistochemical study using monoclonal antibodies

Summary Alpha-1-microglobulin is a low molecular weight (approximately 30 000 d) glycoprotein present in biological fluids. It is heterogeneous in charge. A monoclonal antibody was used to investigate the tissue distribution of the protein in normal human tissues and cell lines by indirect immunofluorescence and immunoperoxidase techniques. The protein was demonstrable in cells of the monocyte-macrophage lineage, in thymus and T cell dependent areas of spleen, lymph node and tonsils. It was detected in several lymphoid or nonlymphoid cell lines but not in peripheral blood lymphocytes. The microglobulin was also detectable in the cytoplasm of hepatocytes. Finally, it was observed in glandular secretions (sudoral glands and mucosal glands of the digestive tract) where it may be associated with IgA. Possible explanations for the highly divergent results previously reported with polyclonal antisera to ₁ microglobulin are discussed.     

Immunohistochemical analysis of human peripheral blood and lymphoid tissues using monoclonal antibodies immunoreactive with non-lymphoid cells

Immunoperoxidase methods were used to study human peripheral blood and lymphoid tissues using a panel of monoclonal antibodies to non-lymphoid cells. The majority of peripheral blood monocytes were immunoreactive for LeuM1, LeuM2, LeuM3 and LeuM5. Rare peripheral blood monocytes were immunoreactive for R4/23. The different antibodies showed characteristic patterns of immunoreactivity in peripheral lymphoid tissues. LeuM1 was immunoreactive with scattered cells in the paracortex of lymph node and tonsil and with many cells in the marginal zone of the spleen. LeuM2 was immunoreactive with endothelial cells in lymph node and tonsil. A few cells in the red pulp of the spleen were immunoreactive for LeuM2. LeuM3 and R4/23 showed distinctive immunoreactivity in germinal centers of secondary follicles, giving a "lacy" pattern. LeuM3 was also immunoreactive with endothelium in lymph node and tonsil and with sinus lining cells in lymph node. LeuM5 was immunoreactive with macrophages in the germinal center, fibroblastic reticulum cells in the mantle zone and interdigitating reticulum cells in the paracortex of lymph node and tonsil.