EAC Rosettes Research Articles

Rosette formation by buffalo (Bos bubalis) T and B lymphocytes

Buffalo (Bos bubalis) lymphocytes were purified and tested for their E and EAC rosette forming capacity as a marker for the detection of T and B cells, respectively. Sheep erythrocytes were found to form 17.7 per cent of E rosette with buffalo lymphocytes. This population of lymphocytes is believed to be T cell. Erythrocytes of guinea-pig, rabbit, hamster, rat, chicken, dog and donkey formed a lower percentage of rosettes. Five to 18.5 per cent of SRBC-EAC rosettes were detected with buffalo lymphocytes which are believed to be B cells.

Detection of Human B Cells and Chronic Myelocytic Leukemic Cells by Rosette Formation with Sheep Erythrocytes and Fresh Human Sera

A new method of detecting the C3b receptor is reported. A particular merit of this method is that anti-RBC rabbit antiserum is not required. Rosettes were formed with human B lymphocytes, B lymphoblasts and granulocytes, using sheep erythrocytes (SRBC) sensitized with fresh human serum (FHS). T lymphocytes and T lymphoblasts did not form rosettes. The percentage of cells forming rosettes with this method approximated the percentage of rosettes formed with EACm. However, FHS coated SRBC did not react with most cells of B cell type chronic lymphocytic leukemia (CLL), whereas EACm rosette formations showed a definite reaction. On the other hand, 34--58% of cells of chronic myelocytic leukemia (CML) bound with the indicator red cells. SRBC sensitized with fresh rabbit or guinea pig serum formed rosettes with PBL, tonsil cells, B lymphoblasts and granulocytes. Complement and IgM antibody were required for this reaction, as in EAC rosette formation.

γ3-Heavy-chain disease ( γ3-HCD) in a young patient with Down syndrome. Study of peripheral blood lymphocytes and of susceptibility to infection

A 19-year-old male patient with Down syndrome was found to have γ 3-heavy-chain disease (γ-HCD) which probably started at the age of 12 years and presented with recurrent infections, anemia, and peripheral blood lymphocytosis. The patient later developed splenomegaly and leukocytosis, with up to 95% atypical large lymphocytes in the peripheral blood. Sixty-nine to seventy-five percent of purified peripheral blood lymphocytes formed E rosettes and 52–66% of the cells formed EAC rosettes. Sixty-two percent of lymphocytes had surface immunoglobulins, predominantly of the IgG class. The results did not change after overnight shedding in a serum-free medium. Thus, a significant subpopulation of lymphocytes were of the so-called D-cell type. Peripheral blood lymphocytes, regardless of shedding, did not respond to mitogenic stimulation and did not react in MLC. Without shedding the cells died in 24–72 hr. A potent lymphocytotoxin reacting with autologous and with allogeneic lymphocytes was detected in the blood. A cold-reacting granulocytotoxin and a warm-reacting monocytotoxin were also found. Since eluate from the lymphocytes killed granulocytes and vice versa, it may be that the same cytotoxin was directed against antigenic determinant(s) shared by both types of cells. The Sia (euglobulin) precipitate obtained from the serum consisted of IgG and IgM but was devoid of γ-HCD protein and had cytotoxic activity as well. This activity was abolished by 2-mercaptoethanol. Purified γ-HCD protein did not have cytotoxic activity. The exact nature of the cytotoxin remains to be elucidated. Impaired bactericidal activity of the serum and impairment of phagocytosis were observed in our patient and may explain his susceptibility to infections. In 10 of 12 cultured peripheral blood lymphocytes, trisomy 21 was found. Since 95% of peripheral blood lymphocytes were morphologically abnormal, it is conceivable that the genetic marker of Down syndrome was retained by γ-HCD-related lymphocytes.

Blood lymphocytes in myeloma patients; high percentage of complement receptor bearing cells is accompanied by decreased anti-immunoglobulin (Ig) binding capacity

Receptor functions of peripheral blood lymphocytes of 60 myeloma patients have been studied. Cell surface immunoglobulin, spontaneous rosette formation with SRBC, complement receptors using EAC rosette formation and uptake of radioiodinated antibodies against human Ig have been examined and compared to the results obtained with cells from normal individuals. Also the response to polyclonal B cell activators has been studied. Significant differences between normal individuals and myeloma patients have been found for the numbers of EAC rosette forming cells. The binding capacity for radiolabelled anti-Ig antibodies showed a strong tendency to low values in the patient group. Our results are compatible with the idea that cells with an exclusive capacity for complement binding are increased in myeloma patients. A high number of EAC binding cells has been found also in bone marrow aspirates of myeloma patients showing infiltration by tumor cells. Therefore, it seems possible that the site primarily affected from disease is the main source of complement receptor bearing cells. No correlation has been seen between the response to polyclonal B cell activators or the concentration of normal serum Ig to the frequency of EAC binding cells in individual patients (r s 0.07 and 0.11, respectively).

Changes in peripheral blood leukocyte counts, lymphocyte subpopulations, and in vitro transformation after heart valve replacement

We studied the effects of elective open-heart surgery with cardiopulmonary bypass on peripheral blood leukocyte counts, lymphocyte subpopulations, and in vitro transformation in 21 patients with heart valve disease. Peripheral blood showed significant neutrophilia with increases in the number of stab cells for up to 14 days postoperatively. In contrast, total lymphocyte count and the number of lymphocyte subclasses, as characterized by E, EA, and EAC rosette formation and surface membrane immunoglobulin markers, were significantly diminished on the second but not on the seventh postoperative day. Significant increases in the relative amount of atypical lymphocytes, characterized by both morphology and autoradiography, occurred for up to 14 days postoperatively. The transformation response of lymphocytes to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) was significantly decreased 2 days postoperatively, but values greater than the preoperative level were seen again 5 days later. The mixed lymphocyte culture (MLC) response was significantly decreased on the second day postoperatively, but returned to the preoperative level 5 days later. The stimulatory ability of lymphocytes in MLC and their response to purified tuberculoprotein were significantly lower than normal for up to 14 days after the operation. There were no differences in the changes of these parameters between patients perfused with bubble and membrane oxygenators, or between patients given conventional fluid therapy (approximately 6 kcal, per kilogram of body weight per day) and parenteral nutrition (approximately 40 kcal. per kilogram of body weight per day) for 4 days postoperatively. The results show a significant decline of lymphocyte count and impairment of their function immediately after open-heart surgery. The oxygenator type and the postoperative parenteral nutrition have no effect on these changes.

Einfluß der Therapie auf die Verteilung lymphoider Zellen im Venenblut von Patientinnen mit Mammakarzinomen

Rosetting properties (E, EAh, EAox, EAC rosettes) and presence of surface immunoglobulins (SIg) were examined on peripheral blood lymphocytes from 30 breast cancer patients immediately prior to therapy and 4 weeks thereafter. Therapy consisted of limited radical surgery followed by combined X-ray and telecobalt radiotherapy. The results were compared to patients who had received the same treatment 1 year ago (n = 13), 2 years ago (n = 13) and 3 to 10 years ago (n = 20). All irradiated patients exhibited a considerable leuko- and lymphopenia with a particular decrease of E and EAh rosettes, and a concommittant relative increase of EAox and EAC rosettes. SIg positive cells showed no significantly different percentages before and after therapy although in absolute counts they were similarly reduced as the other subpopulations after radiotherapy. The possible prognostic influence of radiation induced lymphopenia is discussed without coming to clear conclusions.

Observation of E and EAC Rosette Formation in Human Peripheral Blood Lymphocytes with Mixed Drugs Reaction

Observation of E and EAC Rosette Formation in Human Peripheral Blood Lymphocytes with Mixed Drugs Reaction

Antibody‐independent activation of complement by human peripheral B lymphocytes

Normal human peripheral blood lymphocytes were shown to activate complement in normal human serum (NHS). This activation led to C3 fixation on the cell membrane, which in turn was visualized by fluorescence. The reaction occurred in the absence of detectable amounts of antibodies, since an agammaglobulinemic patient's serum also supported complement activation, and the results were unaltered by absorption of normal human serum. Trypsin treatment of lymphocytes to remove possible complement activators bound to the cell membrane, did not have any effect on the complement activation by lymphocytes. Complement membrane fluorescence was abolished in the presence of EDTA or ethyleneglycolbis(aminoethylether) tetraacetate (EGTA) supplemented by Mg++ions indicating that the classical pathway of complement activation was involved in the reaction. Experiments performed with T and B-purified populations showed that the phenomenon was confined to the B cell fraction. Complement deposition did not diminish the number of EAC rosettes, and viability remained unchanged after exposure of lymphocytes to normal human serum.

Stimulating properties of lynestrenol on normal human blood T-lymphocytes and other leucocytes

The influence of lynestrenol, a synthetic progesterone-like drug, has been investigated on normal human blood leucocytes in vitro. Lynestrenol significantly increased the percentage of active T rosettes (a test for T-cell competence) at concentrations of 10 −1 to 10 −4 μg/ml (controls: 16.5%; drug at 10 −1 μg/ml: 23.6%; P<0.001). It did not alter the percentage of total T rosettes (a quantitative measure of T cells) and the percentage of EAC rosettes. It increased the non-adherent cells in the leucocyte adherence inhibition test (LAI) over a dose range between 10 −1 and 10 −3 μg/ml (controls: 360,000 compared to 517,000 in the presence of 10 −2 μg/ml; P<0.001) suggesting some leucocyte activation. Finally, it also increased the phagocytic properties of monocytes. These results indicate the lynestrenol acts upon normal human blood T lymphocytes, monocytes and possibly neutrophils. Therefore, lynestrenol appears to posses immunostimulatory properties.

Prospects and Limitations of Systematic Monitoring in Patients with Laryngeal Cancer

The authors report the first results concerning the systematic use of "immunological monitoring" in patients suffering from well-developed laryngeal cancer. The authors have also studied, in 50 cases in different stages of development, the activity of T-cells using the cutaneous tests (BCG, PPD, PHA and SK-SD), the E-rosette test and the PHA-induced lymphocytic blastization test (it is well known that the T-cells are responsible for a cytotoxic effect against neoplastic cells). The authors have also studied the activity of B-lymphocytes by means of the EA and EAC rosette test and of IgG, IgA, IgM and complement serum titles. They have observed a progressive depression of the T-cell activity after surgical intervention, whereas the B-cell activity in several cases tends to increase. The authors discuss these immunological results and the possibility that these findings may represent the basis for a complementary immunotherapy following the surgical treatment and/or radiotherapy or chemotherapy. Finally, the authors discuss the possibilities and the limitations of these immunological methods from the point of view of clinical application.

Preparation, characterization, and functions of rabbit lymph node cell populations: II. Preparation of KLH primed B memory cells by EAC rosetting and Ficoll-Hypaque centrifugation

Complement receptor lymphocytes (CRL) were isolated from lymph nodes of rabbits immunized 6 days earlier with keyhole limpet hemocyanin. Isolation was accomplished by rosetting with sheep erythrocytes sensitized with antibody and horse complement (EAC) followed by Centrifugation on Ficoll-Hypaque (F-H). The CRL population comprised about 30% of the starting LNC and was highly enriched for surface immunoglobulin (SIg) bearing cells. By the criterion of [ 3H]thymidine incorporation, CRL responded poorly to Concanavalin A (ConA), but extensively to goat anti-rabbit Fab′ (AFab′) ; indeed, the CRL responded better than unfractionated LNC to AFab′. Upon addition of KLH, the CRL produced much less antibody and total Ig than unfractionated LNC. KLH-challenged T cells prepared on anti-Fab′ columns also synthesized minimal amounts of antibody and Ig. Antigenic challenge of a mixture of CRL and T cells synthesized 3 to 5 times more antibody and Ig than the sum of the individual CRL- and T cell-mediated syntheses. Moreover, the KLH concentration dependency, kinetics, and ratio of 19S 7S antibody responses were similar for the unfractionated LNC and the reconstituted mixture of CRL plus T cells. Thus in all examined parameters the reconstituted responses were indistinguishable from the responses of the unfractionated LNC. Therefore, the CRL and T cell populations can be utilized individually and in combination to study their roles in the in vitro induction and regulation of the anamnestic response to KLH.

CELL SURFACE MARKERS IN CORD BLOOD LEUCOCYTES AFTER STIMULATION WITH LIPOPOLYSACCHARIDE B

T- and B-lymphocyte markers were studied in cord blood cells cultured with lipopolysaccharide B (LPS). Cells cultured with leucoagglutinin (LA) and pokeweed mitogen (PWM) were used as controls. LPS-induced lymphoblasts were negative for surface Ig, positive for intracellular Ig and did not form rosettes with sheep red blood cells (SRBC). LA-activated cells formed rosettes with SRBC, while PWM cultures showed a varying proportion of surface Ig-positive or SRBC rosetting cells, dependent on the time of culture. About 50% of both LA- and LPS-activated lymphoblasts formed EA rosettes (specific for Fc receptors) and EAC rosettes (specific for complement receptors). The response of foetal cells to LPS was reduced when lymphocytes obtained from Isopaque-Ficoll gradients were passed through nylon wool columns, whereas this procedure led to an increased response to LA. Thus LPS-activated foetal leucocytes are B lymphocytes expressing intracellular but not surface Ig.

Leukemic lymphosarcoma (LLS) with monoclonal IgM: idiotypic specificity on the cell surface and in the cytoplasm of lymphosarcoma cells

A case of leukemic lymphosarcoma with a small amount of monoclonal IgM in the serum is described. Immunologic investigations showed that about 10% of the abnormal cells formed EAC rosettes, that most of these cells had IgMlambda on the cell surface, and that 40% of them contained IgMlambda in the cytoplasm. Monoclonal IgM was detected in the 19S fraction of the serum despite the lack of monoclonal change as determined by serum electrophoresis or immunoelectrophoresis. The common idiotypic specificity was found in the monoclonal serum IgM, surface Ig, and cytoplasmic Ig of lymphosarcoma cells using antiidiotypic antiserum prepared against purified serum IgM. After chemotherapy, cells with idiotypic determinants disappeared and the serum IgM changed from a monoclonal to a polyclonal distribution.

Characteristics of Complement Receptor-Bearing Cells in the Spleens of Tumor-Bearing Mice

In the course of mammary tumor development, a population of nylon nonadherent cells with CR appears in the spleens of tumor-bearing mice although none are ever detected in normal mice. These cells apparently arise in response to immunologic stimulation. In a series of studies we have further characterized subsets of T cells (CR+ and CR-) with regard to their responses to mitogens in the lymphocyte transformation assay. Nylon column nonadherent cells from the spleens of tumor-bearing mice were rosetted in a complement receptor assay using EAC rosetting, and CR+ cells were separated from CR- by centrifugation in a discontinuous Ficoll gradient. CR+ T cells responded strongly to PHA and Con A and in addition responded to LPS, an activity not usually associated with conventional T cells. In contrast, CR- T cells from tumor-burdened mice responded to PHA but failed to respond to Con A or LPS.

Inosiplex, A Stimulating Agent for Normal Human T Cells and Human Leukocytes

The influence of inosiplex upon various in vitro leucocyte assays was studied in normal individuals. It was found that the drug increases the response of bidirectional and unidirectional mixed lymphocyte cultures at concentrations of 200, 300, and 500 microgram/ml. It also significantly increases the percentage of active T rosettes (concentration range: 50 to 500 microgram/ml) and the percentage of autologous red cell T rosettes (concentration: 100 microgram/ml). In contrast, inosiplex did not modify the percentage of total T rosettes and EAC rosettes. Inosiplex increases the number of nonadherent leucocytes in the leucocyte adherence inhibition test at a concentration range between 100 and 300 microgram/ml. Finally, inosiplex also increases the percentage of monocytes phagocytizing yeast at a concentration between 50 and 500 microgram/ml. These data indicate that inosiplex enhances the function of normal human T cells, monocytes, and possibly neutrophils. Therefore inosiplex appears to have immunostimulant properties.

Immunopathology of Follicular Lymphomas

To the Editor: In their article in the March 2 issue of the Journal Warnke and Levy propose that reactive lymphoid follicles can be differentiated from lymphoma nodules by the characterization of immunoglobulin light chains in direct immunofluorescence. There are several arguments against this approach. First of all, some lymphomas may be biclonal rather than monoclonal.1 For example, we recently studied a lymphoid lung mass from a patient with well differentiated lymphocytic lymphoma containing two populations of cytologically identical cells: one consisted of IgMκ cells that formed EA but not EAC rosettes, and the other IgGλ cells positive for EAC . . .

Ultrastructure of immunoblastic sarcoma as observed by freeze-fracturing

Malignant tumor cells have an enhanced rate of cell division and of metabolic activities of cells. We studied IgG positive immunoblastic sarcoma cells by means of freeze-fracturing and found significant structural differences between these tumor cells and control B-cells.Lymphnodes infiltrated with sarcoma cells were dissected from the cervical areas of two patients. The sarcoma cell suspension was obtained from these lymphnodes. Sarcoma cells had fine granules stained with PAS and mothyl green pyronin in cytoplasma. Most of sarcoma cells were positive fluorescent (SIgG bearing). Alpha-naphthyl-acetate-esterase test was negative or false positive. Single cell suspension of lymphnodes was obtained from patients of non-lymphatic diseases. EAC rosette forming cells and adherent cells to nylon column fiber were used as control B-cells. The pellet of sarcoma cells and control B-cells were fixed with 2. 5% glutaraldehyde solution. Freeze-fracture was done as described previously. Under the electron microscope, EAC rosette forming cells were selected as control B-cells.

High terminal deoxynucleotidyl transferase activity in pediatric patients with acute lymphocytic and acute myelocytic leukemias

Specificity of TdT5 as a marker for ALL was evaluated by determining its activity in cells from normal control subjects and from 35 pediatric patients with ALL, AML, Hodgkin's disease and disseminated Burkitt's lymphoma. We evaluated the DNA polymerase activity, cell surface phenotypes (E rosettes, EAC rosettes, Smlg and la-like, HTLA and cALL antigens), and hematological and cytochemical characteristics in both the normal and patient groups. DNA polymerase alpha + beta and DNA polymerase gamma activity were indiscriminately high in all immature cells as found in ALL, AML, Burkitt's lymphoma and phytohemagglutinin-stimulated normal lymphocytes, when compared to mature leukocytes found in normal individuals or in patients whose cancer was in remission. High TdT activity was found in 24 of 26 T and non-T/non-B ALL patients in active phase as well as in two of three AML patients one of whom had Auer rods. Thus, TdT, although valuable for monitoring ALL patients, may have limitations in separating AML from ALL.

Lymphocyte isolation, rosette formation, and mitogen stimulation in rhesus monkeys

Over 70%, of rhesus monkey peripheral mononuclear cells were isolated on sodium metrizoate-ficoll gradients with greater than 98% purity. Rhesus blood contained 47.8% active E, 58.2% total E, and 30.2% EAC rosette forming cells. Optimal conditions for mitogen studies were determined using phytohemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide and streptolysin 0.

Binding of circulating immune complexes to human peripheral blood lymphocytes: Effect of complement

The mode of binding of immune complexes, with and without bound complement components, to normal human peripheral blood lymphocytes was examined. Heat-aggregated human IgG (agg IgG) was employed as an in vitro model of immune complexes. Immune complexes without bound complement or with bound C1, C4, or C2 inhibited antibody-dependent cellular cytotoxicity (ADCC) and EA rosette formation but not rosette formation with EAC. In contrast, immune complexes with bound C3b blocked EAC rosette formation but inhibited ADCC and EA rosette formation little if at all. These findings demonstrate that agg IgG, and thus immune complexes, without fixed complement or those with bound C1, C4, or C2, preferentially interact with the lymphocyte Fc receptor and that C3b-bound immune complexes bind to the lymphocyte surface C3b receptor.