Lymphocyte Rosette Research Articles

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.

Morphine and methadone impact on human phagocytic physiology.

Human subjects submitted to treatment with morphine show a severe depression of phagocytosis, killing properties and superoxide production both of their polymorphonuclear leukocytes and monocytes. Polymorphonuclear leukocyte adherence, chemotaxis, random migration, myeloperoxidase content, lysozyme content and lymphocyte Rosette E formation were poorly influenced. Methadone-treated subjects show a similar effect at phagocytic level but far less evident. These results confirm those previously found in animals and reinforce the evidence of a depressive role of morphine on phagocytic physiology.

Gp140, a C3b-binding membrane component of lymphocytes, is the B cell C3dg/C3d receptor (CR2) and is distinct from the neutrophil C3dg receptor (CR4).

gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gp140, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on EC3bi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CR1, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.

Haematology of subclinical fatty liver in dairy cows

The relationship between liver fat content and haematology was investigated in 369 cows from eight herds sampled in the second week after calving. High levels of fat in the liver were associated with a depression in total white cell count and in neutrophils, eosinophils and lymphocytes. There was no correlation between liver fat content and the percentage of E rosetting lymphocytes, packed cell volume or haemoglobin concentration. The changes in peripheral white cell counts may be related to the increased incidence of post parturient disease in cows with fatty liver.

Changes in the proportion and absolute number of T lymphocytes in piglets from birth until after weaning and in adults

The proportion of T lymphocytes was measured on cytocentrifuged leucocytes prepared directly from whole blood after rosetting with sheep red blood cells in the presence of dextran. This method gave similar results to those obtained with conventional mononuclear preparations assessed in a haemocytometer but had the advantages of simplicity, the small volume of blood required and the ease with which absolute T lymphocyte counts could be estimated. The proportion of T lymphocytes did not change between birth and 12 days after weaning and did not differ from adults. However, the number of T lymphocytes at birth was significantly lower than in adults and rose thereafter to values significantly higher than in adults by 12 days after weaning. A comparison between one-day-old piglets and adults revealed that there was no significant difference in the proportion of either T or B lymphocytes between adults and neonates. However, there was some indication that neonates were deficient in the proportion of T lymphocytes capable of binding sheep red blood cells with high affinity. After examining the alpha-naphthyl acetate esterase activity of rosetted lymphocytes it was concluded that this enzyme was not a reliable marker for pig T lymphocytes.

B-lymphocyte blastogenesis in response to periodontitis-associated bacteria. Kinetics and proportion of total response.

A role for activated B-lymphocytes in mediating the initiation and/or progression of periodontal diseases has been proposed in previous work. The present study was conducted to: (1) assess the proportion of total lymphocyte blastogenic response to selected oral bacteria which is composed of T-cell and B-cell activation, respectively, and (2) to determine whether different kinetic patterns exist for the T-cell vs. the B-cell responses to these bacteria. Using lymphocyte rosetting and culturing techniques, rosette-positive and rosette-negative lymphocyte preparations were examined for blastogenic responsiveness following stimulation with a variety of both Gram-positive and Gram-negative periodontitis-associated bacteria. Results of these studies indicated that both peripheral blood lymphocytes (PBL) from healthy adults and cord blood lymphocytes (CBL) from placental afterbirths responded to these bacteria with similar kinetic patterns. The net PBL blastogenic response appeared to consist of an early B-cell response, which peaked at Days 2 or 3 of culture, followed by a later T-cell response, which peaked at Days 5 to 6 of culture. The B-cell response appeared to be T-cell-dependent in that B-cells cultured alone showed minimal thymidine uptake over the entire 6-day period, but the addition of irradiated T-cells to these B-cell cultures resulted in a greatly enhanced B-cell response.

The effects of total-body hyperthermia combined with anticancer drugs on immunity in advanced cancer patients.

As total-body hyperthermia (TBHT) therapy may lead to a reduction in the immune response of cancer patients because of the immediate effect of heat on lymphocytes, the authors studied the immunity of advanced cancer patients receiving combined TBHT and anticancer chemotherapy. A decrease was found in their lymphocyte blastogenesis, skin reactions to PPD and PHA, lymphocyte rosette formation, IgG, and C3c component. These parameters returned to their pretreatment levels at 1 week after completion of TBHT therapy. This result indicates that there is no necessity for giving special consideration to a reduction of cell-mediated immunity in TBHT therapy.

Effect of some choline compounds on lymphocyte rosette formation in mice

Effect of some choline compounds on lymphocyte rosette formation in mice

Homogeneous populations of macrophages from histiocytic lymphoma patients as a source for macrophage subpopulations which differ in immunoregulatory properties.

The existence of subpopulations of macrophages which express a variety of regulatory activities of other branches of the immune system is suggested in a comparative study of a human macrophage long term culture ZI and a macrophage cell line DAB-1. Both cell cultures were derived from pleural effusions of patients with diffuse histiocytic lymphoma. DAB-1 cells were found to secrete factors which strongly suppress the response of normal T and B lymphocytes to the mitogens PHA, Con-A and pokeweed mitogen (PWM) (by 96-98%) and to stimulate the cytotoxic activity of NK cells. ZI cells secrete factors which have a mild inhibitory effect on the response of lymphocytes to the T-cell mitogens PHA and Con-A and a stimulatory effect on the response to PWM, whereas very little effect could be detected on the activity of NK cells. While DAB-1 cells form large clusters during growth in culture, and are capable of inducing the formation of lymphocyte rosettes around the tumor cells, ZI cells grow as a homogeneous monolayer and could not be shown to form such rosettes. The differences in the behavior of the two cell populations suggest that the malignant transformation may have affected different subsets of macrophages in each case. Cells from histiocytic lymphoma patients may therefore be a source for homogeneous subpopulations of macrophages and their isolation and propagation in culture is one approach by which such subsets can be defined characterized and classified. The biological characterization of macrophage subsets may also be of clinical importance since a transformed subset with broad suppressory activities may lead to a violet and rapidly deteriorating course of disease, as was in the case of the patient from whom the DAB-1 cell line was derived.

A case of immunodeficiency with thymoma

A well encapsulated thymoma, consisting of spindle-shaped epithelial cells and lymphoid cells, was surgically removed for a mediastinal mass. Then a 61-year-old man developed recurrent sore throats and persistent productive cough. He was diagnosed bronchiectasis by bronchogram and immunodeficiency by markedly reduced each class of Ig (120mg% IgG, 21mg% IgM, 11mg% IgA and less than 25IU/ml IgE). Numbers of peripheral blood lymph-cytes (PBL) were normal. E rosetting lymphocytes were 67% of PBL and surface Ig bearing lymphocytes were 3% of PBL. This result was suggested that Ig producing B lymphocytes decreased.Lymphocyte stimulation with mitogens revealed a normal mitotic response to PHA and PWM, but a low response to Con A. Skin tests of PPD, Candida and DNCB were negative.He developed recurrent Gram-positive coccal infection and candidiasis of respiratory tract at end stage. He died of cardiac failure.The histopathologic findings: The germinal centers of spleen and any lymph nodes were not demonstrated and cell components of these paracortical areas were markedly reduced. The funfal vegetations with weak inflammatory reaction were demonstrated in cardiac muscle, lung and kidneys.We think this syndrome is consisted of both humoral and cellular immunodeficiency.

The effect of levamisole on peripheral T-lymphocytes of patients with malignant lymphomas.

The effect of various concentrations (0.015 - 10 micrograms/ml) of Levamisole (LMS) on the peripheral lymphocytes of patients with malignant lymphomas (ML) and normal donors was investigated in vitro. The parameters studied include: E rosettes forming cells (total T lymphocytes), active E rosettes (early T lymphocytes) and DNA synthesis induced by pitogens PHA and Con A. LMS improved significantly lymphocyte response both in patients with ML and normal donors when the cells were stimulated by Con A. In both groups no significant effect was observed on the response to PHA nor on the percentage of E-rosettes, whereas the mean number of active E rosettes was significantly increased on all concentrations of the drug. While in the normal subjects a positive statistical correlation between active E-rosettes and Con A response was observed, in patients with ML an inverse correlation was found. This latter correlation was partially reversed by LMS. These results indicate that LMS, both in normals and in patients with ML, interacts with a specific cell subset (early rosetting lymphocytes), with different functions in normal and pathological situations. The drug shows an effect on the immunological handicap of lymphocytes of patients with ML.

Cyclosporin A inhibits ganglioside-stimulated lymphocyte rosette formation in multiple sclerosis.

Brain gangliosides have been found to stimulate active lymphocyte rosette formation in patients with multiple sclerosis during an active phase of the illness. On antigenic stimulation, a soluble factor is released which enhances active rosette formation. These reactions do not occur in multiple sclerosis patients during remission, in other neurological diseases or in controls that we have tested. More than one antigen may act on sensitized lymphocytes. The ganglioside-mediated response of lymphocytes from multiple sclerosis patients is blocked in vitro by Cyclosporin A.

Phenotypes of human natural killer cell populations detected with monoclonal antibodies.

In our recent studies, human natural killer (NK) cell activity was found to be decreased 2- to 4-fold after treatment of monocyte-depleted peripheral mononuclear cells with monoclonal antibody OKM1 and complement (C). The present study was undertaken to determine whether there is an additional population of NK cells that is OKM1-, since treatment with OKM1 and C decreased, but did not eradicate, NK cell activity. Treatment of lymphocytes with monoclonal antibody OKT11A, which reacts with all sheep red blood cell rosetting lymphocytes, and C also decreased NK cell activity. Although approximately 90% of OKT11A+ cells are OKT3+, NK cell activity resides within the OKT11A+ cell population, which is OKT3- since OKT3-cell depletion fails to decrease NK cell activity. Double fluorescence analysis of OKT3-depleted lymphocytes revealed that 54% of the OKM1+ cells are OKT11A- and 45% of the OKT11A+ cells are OKM1-, thus demonstrating that within the OKT3-depleted population, approximately one-half the OKM1+ cells are OKT11A- and vice versa. Treatment of lymphocytes with OKM1 together with OKT11A and C decreased NK cell activity against 3 NK-sensitive leukemia lines--K562, MOLT-4, and HSB-2--more than did treatment with either antibody alone; virtually no lytic activity was retained after elimination of OKM1+ and OKT11A+ cells. The results thus provide strong evidence that there is at least 2 populations of human NK cells; one is OKM1+ and the other is OKT11A+

Detection and characterization of lymphocytes bearing receptors for peanut agglutinin by a specific rosetting technique

Murine lymphocytes bearing receptors for peanut agglutinin (PNA) have been visualized using a specific rosetting technique. The lymphocytes were incubated with PNA and mixed with neuraminidase-treated sheep red blood cells. The percentage of the PNA rosetting lymphocytes found in the various organs studied was dependent upon the PNA concentration. In the spleen, the PNA rosetting lymphocytes were primarily T cells with low PNA concentration while both T and B lymphocytes were rosetted with high PNA concentration. In the Fetal liver and thymus PNA rosetting lymphocytes were present in small amounts early in fetal life, increasing and reaching adult levels by late fetal life. The present study shows that the rosetting technique is more sensitive than previously described techniques for the detection of various lymphocyte subpopulations having receptors for PNA.

Isolation of lymphocyte membrane complement receptor type two (the C3d receptor) and preparation of receptor-specific antibody.

A glycoprotein binding complement component C3d was isolated from media used for culture of Raji human lymphoblastoid cells. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gas/liquid chromatography indicated that the C3d-binding glycoprotein consisted of a single polypeptide chain with extensive intrachain disulfide bonds, a molecular weight of 72,000, and several different bound carbohydrates. Several lines of evidence indicated that this medium-derived C3d-binding protein originated from membrane complement receptor type two (CR2, the C3d receptor), presumably shed during membrane turnover. The C3d-binding protein bound to sheep erythrocytes coated with C3d (EC3d) but not to sheep erythrocytes coated with C3b (EC3b). Antisera, prepared by immunization with the purified C3d-binding glycoprotein, inhibited lymphocyte rosette formation with EC3d but not with EC3b. Analysis by sodium dodecyl sulfate gel electrophoresis of the radiolabeled and solubilized lymphocyte antigens reactive with the anti-C3d-binding protein sera revealed a single-chain cell-surface protein of molecular weight 72,000 that was apparently identical to the isolated C3d-binding protein. Parallel assay of lymphocytes for Cr2 by direct immunofluorescence with F(ab')2 anti-C3d-binding protein (anti-CR2) and rosette formation with EC3d indicated that both assays had the same specificity and nearly the same sensitivity. With both systems CR2 expression was limited to B cells, and was undetectable on T cells, monocytes, or neutrophils.

Specificities and some properties of human T and B lymphocyte antisera.

Antisera were prepared in rabbits by immunization with either human peripheral blood T lymphocytes (PTL) or tonsil B lymphocytes (TBL). Specificities of sequentially absorbed antisera were examined by cytotoxicity testing, immunoperoxidase staining and absorption study. Anti-PTL serum (ATS) reacted with E rosetting lymphocytes in the normal population. ATS was also reactive with some of lymphoid neoplasms, whether neoplastic cells carried E receptors or not. This serum did not lyse any of myelogenous leukemia cells. When antiserum was used in the immunoperoxidase staining, ATS strongly stained paracortical cells in the lymph node and medullary thymocytes in the thymus. On the other hand, anti-TBL serum (ABS) was reactive with lymphocytes corresponding to the percentage of EAC binding lymphocytes in the normal population. ABS reacted not only with the neoplastic cells carrying C3 receptors and/or surface immunoglobulin, but also with non T . non B-acute lymphoblastic leukemia cells and a small number of acute myelogenous leukemia cells. In the immunoperoxidase staining, lymph follicular cells showed positive immunoreactivity with ABS, but paracortical cells did not.

Lymphocyte Subpopulations in the Ferret

Several different surface markers were sought on ferret lymphocytes. Using a technique which attaches complement to zymosan particles, 16.7% of peripheral blood lymphocytes and 15% of splenic lymphocytes exhibited receptors for complement. Receptors for the Fc portion of IgG, measured by an assay using antibody coated sheep erythrocytes, were found on 11.7% of peripheral blood lymphocytes and 34.8% of splenic lymphocytes. Using an indirect fluorescent antibody technique, surface immunoglobulin was detected on 13.4% of peripheral blood lymphocytes and 10.3% of splenic lymphocytes. Spontaneous “T” lymphocyte rosettes were sought unsuccessfully using erythrocytes from 16 different animals. These studies increase our understanding of the ferret immune system and provide methods for the in vitro separation of lymphocyte subpopulations in ferrets.

Circulating immune complexes in sickle cell hepatic crises.

Circulating immune complexes in sera from sickle cell anemia (SCA) patients were investigated using inhibition of complement-dependent lymphocyte rosette formation (EAC rosette inhibition) and the ant

Antigenic Cross-Reactivity of Sperm and T Lymphocytes

Since seminal components can mediate immunosuppression in vitro, it is possible that some antigen(s) may be common to both the reproductive and immunologic systems. In a group of 70 couples with unexplained infertility, 50 "autoimmune" males and 40 "isoimmune" females had lower than normal percentages of total T cells (mean values +/- standard error of the mean 63 +/- 2% and 60 +/- 2% for immune males and females, respectively, versus 77 +/- 2% and 78 +/- 5% for 50 normal males and females, respectively; P < 0.001). Sheep red blood cell (SRBC) rosetting of lymphocytes was significantly reduced when SRBC were preincubated with sperm extracts (61 +/- 4% versus 9 +/- 2%; P < 0.001) but not when SRBC were incubated with normal serum or when lymphocytes were preincubated with sperm extracts. Antisperm antibody titers in the patients' sera (48 +/- 13) were correlated with their antithymocyte antibody titers (18 +/- 3) (P < 0.01). Moreover, antithymocyte antiserum (titer 1024) cross-reacted with sperm extract (titer 128), and vice versa. This cross-reactivity was significantly reduced by absorption of the sera with sperm cells (P < 0.001), thymocytes (P < 0.001), or white blood cells (P < 0.005). Absorption of autoimmune sperm extracts and seminal plasmas with thymocytes or sperm cells reduced the Coombs' titers, especially immunglobulin G (P < 0.01) and immunoglobulin A (P < 0.025). Similar results were obtained in passive hemagglutiation, immunofluorescence, and cytotoxicity assays. We conclude that sperm and T cells share a common antigen(s).

OKT3: A monoclonal anti-human T lymphocyte antibody with potent mitogenic properties

OKT3, a monoclonal anti-human T cell antibody (IgG2), was found to induce DNA synthesis in human peripheral lymphocyte cultures. OKT3 induced maximal mitogenesis at a concentration of 10 to 20 ng/ml and was about 20-fold more potent than PHA as a mitogen. No high-dose inhibition of thymidine incorporation was noticed at concentrations up to 2.5 microgram OKT3/ml. The monovalent Fab fragment of OKT3 was also mitogenic but about 100 times less potent than the parent IgG. OKT3 appeared to be a T lymphocyte mitogen as only sheep red blood cell rosetting lymphocytes were responsive. Quantitative studies on the binding of 125I-labeled Fab fragment of OKT3 to human lymphocytes showed an average of 5.1 x 10(4) receptor sites/cell with an association of about 10(8) M-1 at 37 degrees C, with no heterogeneity of the cell binding sites. These data suggest a strong interaction of the monoclonal OKT3 with a limited number of identical T cell membrane receptors. As this interaction can trigger mitogenesis, the cell membrane determinant recognized by OKT3 could be described as a "T cell stimulation receptor." The mitogenecity of the lymphocytes is not solely dependent on cross-linking of these receptors.