T-lymphocyte subpopulations identified by monoclonal antibodies after human bone-marrow transplantation

Peripheral T-lymphocyte reconstitution after bone-marrow transplantation, 12 allogeneic and 13 autologous, was studied by indirect immunofluorescence assay using mouse monoclonal antibodies. Abnormal counts were detected in the two major sub-populations of T-cells i.e. helper and T cytotoxic-suppressor lymphocytes, defined by monoclonal antibodies (alpha Leu 3a and B 9.2), and in DR antigen-positive T-cells. The pattern of T-lymphocytes replenishment was identical for both types of transplant, and was not affected by Graft Versus Host disease (GVHD).     

Lymphocyte subsets in human adenoids and tonsils. Rosette formation, alpha-naphthyl acetate esterase cytochemistry, monoclonal antibodies and peanut lectin reactivity

The distribution of mononuclear cell subsets has been studied in human adenoids, tonsils and peripheral blood (PB) by evaluating the presence of surface immunoglobulins, E-rosette formation, receptors for IgG Fc and for complement, alpha-naphthyl acetate esterase (ANAE) cytochemistry, reactivity with peanut lectin (PNA) and with monoclonal antibodies (McAb) (OK panel). Adenoids and tonsils, compared to PB, contain (1) fewer macrophages and T cells but more B cells; (2) higher proportions of ANAE negative, complement receptors and Ia-like antigens bearing T lymphocytes; (3) higher percentages of cells reacting with the McAbs OKT9 and OKT10 ("immature" lymphoid cells). In both adenoids and tonsils, clusters, formed by a central heavily ANAE stained interdigitating cell surrounded by lymphocytes with a sickle-shaped ANAE reaction, were found. Analogous clusters have been previously described in mice and human thymus. Two major hypotheses could be put forward: (1) adenoids and tonsils contain "immature" lymphoid cells undergoing education process, or (2) the above organs contain lymphocytes activated by a constant exposure to bacterial antigens or mitogens.     

An ultrastructural study with enzyme-labeled antibody technique on immunoglobulin-containing cells in human tonsils, especially in germinal centers.

Localization of IgG, IgA and IgM in human palatine tonsils, especially in germinal centers, was studied with the electron microscopical enzyme-labeled antibody method. The large germinal center cells differentiate into two kinds of cells within the germinal center; one was the medium-sized germinal center cells not engaging in intracytoplasmic production of immunoglobulins and another was the immature cells producing at least one of the three classes of immunoglobulins, especially IgM. The latter continued to maturate and developed into the intermediate-matured cells and probably into the plasmocytes. The three classes of immunoglobulins were also deposited in the form of admixtures in the intercellular spaces among the constituent cells of the germinal centers, mainly attaching on the cell membrane of desmodendric cells. In addition, some of the deposits were found freely in the intercellular spaces. Some differences between the immunoglobulin-containing cells outside and within the germinal centers were pointed out.     

Three monoclonal antibodies identifying antigens on all equine T lymphocytes, and two mutually exclusive T-lymphocyte subsets.

The aim of this study was to produce monoclonal antibodies (mAb) recognizing equine lymphocyte surface antigens. Fusions were conducted using BALB/c mice hyperimmunized with equine thymocytes. Hybridoma supernatants were screened by flow cytometry and positive hybridomas were cloned twice by limiting dilution. These mAb were then characterized for tissue distribution by immunohistology and flow cytometry, and by precipitation and analysis of the lymphocyte antigens which they recognized. Three mAb (CVS5, CVS4 and CVS8) are described which recognize only T lymphocytes in peripheral blood. Two-colour immunofluorescent studies showed that CVS5 recognized all T lymphocytes and that CVS4 and CVS8 recognized two mutually exclusive subsets of CVS5-positive cells. In the thymus there was a large population of CVS4/CVS8 double-positive cells. Immunohistochemical staining with these mAb was restricted to T-lymphocyte areas. CVS4 and CVS5 precipitated molecules of 58,000 and 69,000 MW, respectively, in both reducing and non-reducing conditions. CVS8 precipitated two molecules of 32,000 and 39,000 MW in reducing conditions, and one molecule of 69,000 MW in non-reducing conditions. This evidence suggests that CVS5, CVS4 and CVS8 recognize the equine homologues of CD5, CD4 and CD8, and that the characteristics of these antigens are similar to those of other species.     

A rapid rosette technique for quantitation and separation of mononuclear cell subsets using monoclonal antibodies

We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG. Rosette-forming cells (RFC) may then be counted and separated from non-rosetting MNC by Isopaque-Ficoll gradient centrifugation. The yield and viability are close to 100% after ORBC lysis. Adherent cells do not interfere. Isolated T8⁺ and Leu3a⁺ cells were further tested: the purity was 97–99%, and the cells were functionally intact with respect to their modulating activity on the generation of immunoglobulin-secreting cells by MNC after stimulation with pokeweed mitogen.     

Distribution of T-lymphocyte subsets in different portions of sarcoid granulomas: immunohistologic analysis with monoclonal antibodies

The numbers and the distribution of T-lymphocyte subpopulations in lymph node granulomas from 11 patients with sarcoidosis were studied in cryostat sections by an immunoperoxidase technique. Greater numbers of helper T lymphocytes (Leu-3+) were found at the periphery than in the central portion of the same granuloma. Most of the suppressor T lymphocytes (OKT8+) were present at the periphery of the granulomas. In addition, the Leu-3a/OKT8 ratio varied from 0.7 to 1.8 in the outer compartment, while in the central portion of the granuloma much higher values (3 to 20) were found. These different distribution patterns of T-lymphocyte subsets provide evidence for two different compartments with different immune reactions in sarcoid granulomas.     

Definition of glomerular antigens by monoclonal antibodies produced against a human glomerular membrane fraction.

Experimental animal models of glomerulonephritis (GN) produced by direct antibody binding to non-basement membrane glomerular capillary wall antigens do not to date have human parallels. To examine the potential for this form of humoral glomerular injury in man, we sought to define discrete human non-GBM glomerular antigenic targets using hybridoma technology. Mice were immunised intraperitoneally with 20-100 micrograms of a human glomerular membrane fraction (HGMF). Six fusions have yielded 12 stable reagents defined by positive glomerular indirect immunofluorescence (IF) and microELISA using HGMF as the screening antigen. Subclass analysis of ascitic McAbs indicated several IgG1, one IgG2b, and three IgM reagents. Distinctive IF patterns of reactivity with epithelial, endothelial or mesangial structures have been observed, with or without peritubular capillary, tubular basement membrane and vessel wall reactivity. Seven normal non-renal human organs and the kidneys of rat, rabbit and sheep have shown patterns characteristic of each individual McAb, restricted to human or with species cross reactivity. To partially characterise McAb-reactive antigens, detergent-solubilised renal cortex and collagenase-solubilised GBM (CS-GBM) extracts have been probed by immunoblot. A unique McAb 7-5Q, reactive with glomerular and tubular epithelial structures, binds major bands of approximately 107 KD and 93 KD in detergent solubilised cortex and a single band of similar size by immunoprecipitation (110 KD). 5-3A (a human-restricted linear-reacting McAb) binds bands of 20-200 KD (major band 58 KD) in CS-GBM. In conclusion, distinct species-restricted and more broadly disposed glomerular epitopes are definable in man by McAbs and are potential targets for humoral injury. Purification of these antigens will allow assay for circulating putative nephritogenic auto-antibody and potentially, McAbs may be useful in screening urine for evidence of occult structural renal disease.     

Monoclonal anti-human T-lymphocyte antibodies: enumeration and characterization of T-cell subsets.

The complement-mediated lysis of human lymphocytes by three monoclonal anti-human T-cell antibodies OKT3.PAN, OKT4.INd and OKT8.SUP was studied. The percentages of Ficoll-Hypaque-isolated mononuclear cells lysed by these antibodies were respectively: 65% for OKT3.PAN, 39% for OKT4.IND and 20% for OKT8.SUP. Optimal lymphocytotoxic reactions were noticed when unabsorbed rabbit serum was used as the source of complement (C). Addition of heat-inactivated human, mouse and newborn calf sera but not of foetal calf serum inhibited the lytic activity of the antibodies. Treatment of peripheral mononuclear blood cells with OKT3.PAN and C abrogated their mitotic response to PHA and Con-A. Sheep erythrocyte rosetting lymphocytes (E+ cells) treated with OKT4.IND or OKT8.SUP and C exhibited no marked changes in responsiveness to PHA, Con-A or allogeneic non-T cells. However, only E+ cells enriched with OKT4.IND-reactive cells responded to purified protein derivative, proliferated in the autologous mixed lymphocyte reaction and were highly sensitive to hydrocortisone suppression when stimulated by PHA. Our data indicate that these monoclonal antibodies can be regarded as invaluable tools for enumeration, characterization and functional assessment of human T cells and their subclasses.     

Production and use of human monoclonal anti-D antibodies

Rhesus haemolytic disease of the newborn is a condition which can result in intrauterine or perinatal death. Although the passive administration of therapeutic anti-D post-partum is a most effective method for the prevention of this condition, there is currently a shortage of immune plasma for the preparation of the therapeutic anti-D immunoglobulin product. In addition the availability of anti-D for use in blood grouping has also been reduced. The advances made in recent years in the techniques for the production of human monoclonal antibodies raise the possibility that human monoclonal anti-D-based products may provide solutions to both of these problems. There are now a number of reports of the production of stable cell lines secreting high titre human anti-D. In this review we consider the various strategies used in the production of human monoclonal anti-D-secreting cell lines, the basic properties of these reagents and their potential usefulness in blood grouping, in therapy and as research tools.     

Characteristics and functional aspects of nonlymphoid cells in rat germinal centers, recognized by two monoclonal antibodies ED5 and ED6

In this study two newly developed monoclonal antibodies, ED5 and ED6, are described, which specifically recognize nonlymphoid cells in B cell follicles of spleen and lymph nodes. Enzyme and immunocytochemical techniques demonstrated that ED5 and ED6 stain two different types of reticulum cells. The staining patterns were compared with those of the monoclonal antibody Ox2. ED5+ cells are able to retain immune complexes and are considered to be the follicular dendritic cell; in contrast ED6+ lack this capacity. Peyer's patches contain many ED6+ and Ox2+ cells but are completely devoid of ED5+ cells. After the application of immunomodulatory agents, like X-irradiation, cyclophosphamide and lipopolysaccharide, ED5+ and ED6+ cells remain detectable. The nature of the new type of dendritic reticulum cell in lymphoid follicles, recognized by ED6, is discussed.     

Distribution pattern of lymphocyte subpopulations in human tonsils as analysed by monoclonal antibodies with double immunoenzymatic labeling

Summary The distribution of B- and T-lymphocytes, T-cell subpopulations, including natural killer cells and monocytes/macrophages, was studied in cryostat sections of human tonsils by the avidin-biotin-peroxidase technique using monoclonal antibodies. A double immunostaining procedure was also developed to detect two types of lymphocytes on one single section simultaneously using horseradish peroxidase and alkaline phophatase as labeling enzymes.The primary follicles and the germinal centers of the secondary follicles were mainly found to be positive for B-cells. T-cells were predominantly localized in the follicular caps and in the interfollicular areas. The ratio of helper/inducer cells to suppressor/cytotoxic cells was in favour of helper T-cells. Both subpopulations were also predominant in follicular caps and interfollicular areas.The quantity of natural killer cells was very variable and nearly all were localized exclusively in the germinal centers.Monocytes/macrophages were only seen occasionally in the interfollicular areas. The double-immunoenzymatic labeling was useful for the visualization of combinations of antigens, however, without demonstrating the presence of two different surface antigens on one single cell.     

T-cell subsets in melanoma patients evaluated by anti-T-cell monoclonal antibodies

Functional T-cell subpopulations have been evaluated in the peripheral blood of 124 melanoma patients (71 non-metastatic and 53 metastatic) using monoclonal antibodies: OKT3, OKT4, OKT8, every 2 months for 1 year. The levels of OKT3+ cells were significantly lower in metastatic patients than in normal controls and they decreased in the advanced phases of the disease. Percentages and absolute OKT4+ cell values were reduced in metastatic patients only, while a significant reduction in OKT8+ cells was noted in both long-surviving, non-metastatic and metastatic patients. The ratio of OKT4+/OKT8+ cells was increased in non-metastatic patients and in patients remaining metastasis-free for 4 years after resection of their metastases. Patients with prolonged survival show normal T-helper cells and low T-suppressor cells with a significant increased ratio OKT4/OKT8. In the patients who developed visceral metastases and in those who died the progressive reduction in total T cells is mainly due to the decrease in OKT4+ cells.     

Lymphocyte subpopulations and T-cell subsets in human oral cancer tissues: immunohistologic analysis by monoclonal antibodies

A series of T-cell-specific monoclonal antibodies (Leu-1, Leu-2a, and Leu-3a) and B-cell-specific monoclonal antibody (HLB-1) were used to detect the localization and intensity of infiltration of lymphocyte subpopulations and T-cell subsets in frozen sections of 17 patients with the oral cancer. The vast majority of the lymphocyte infiltrates in the oral cancer tissues were reactive with Leu-1. In contrast, B cells were detectable with HLB-1 in only 2 of 17 cases. Leu-2a-positive cells were dominant in four cases, whereas Leu-3a positive cells were dominant in only three cases. In seven cases, both cells infiltrated to the same degree. Leu-2a positive cells tended to be dominant in the cases with earlier clinical stages.     

T-Lymphocyte subpopulations in Crohn's disease: Definition by monoclonal antibodies

Summary Thirty-two patients with Crohn's disease (CD) and 32 age- and sex-matched controls were studied for T lymphocytes and T-lymphocyte subpopulations in the peripheral blood, using monoclonal antibodies defining the helper/inducer and the suppressor/cytotoxic compartment. T cells were reduced in patients with CD (P<0.05), and this reduction was more pronounced in patients with active discase (P<0.01). This T-cell deficiency involves helper and suppressor cells proportionally. The proportion of helper and suppressor cells in CD was independent of disease activity and therapy. We conclude that the T-cell deficiency in CD is a secondary event, and that there is no derangement in the immunoregulatory ratio (helper to suppressor cells) in CD which might have served as an explanation for a possible autoimmune mechanism in the pathogenesis of this disease.     

Characterization by monoclonal antibodies of lymphocyte subsets present in B-enriched suspensions

B lymphocyte enriched suspensions isolated by E rosette depletion (E⁻ cells) or by nylon fiber adherence (adherent cells) were identified by their cellular composition using different T and B cell markers (SI_g, E receptor, T3, T4, T8 and Ia-like antigens). The cells were isolated from peripheral blood both of healthy donors and uremic dialysis patients. A variable proportion of non-B cells was found in some preparations. This contaminant was represented mainly by Null cells in E⁻ lymphocyte suspensions and by T cells in the adherent population. Contaminating T lymphocytes were most frequently found in adherent cell preparations from uremic individuals and appeared to be an heterogeneous population including variable proportions of T4, T8 and Ia positive T cells. A significant increase of T8⁺ cells and a decrease of the ratio T4/T8 was seen among adherent T cells as compared to the normal distribution among T peripheral lymphocytes.     

Molecular identification of human T-lymphocyte antigens defined by the OKT5 AND OKT8 monoclonal antibodies

Three human lymphocyte differentiation antigens, specific of the entire T-cell population, of the helper/inducer T-cell subset, and of the cytotoxic/suppressor T-cell subset have been identified, using mouse monoclonal antibodies obtained from Dr. P. Kung. Various T-cell populations were radio-labelled, the antigens were isolated by immunoprecipitation with the monoclonal antibodies and the resulting immune complexes subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The OKT3 antigen, present on peripheral T-lymphocytes and on functionally mature thymocytes has been identified as an oligomeric protein, composed of 23,000 mol. wt subunits. The OKT4 antigen, specific for the helper/inducer subset, is a single protein of 53,000 mol. wt. The OKT8/OKT5 antigen, defining the cytotoxic/suppressor subpopulation is composed of two subunits of 31,000 and 33,000. From co-capping experiments and biochemical data, the hypothesis is established that OKT5 recognizes a dimer of 140,000 mol. wt and OKT8 recognizes a determinant present on both the monomer 70,000 and the dimer. This hypothesis could explain the OKT5⁻ OKT8⁺ phenotype of some T-cells.