Human lymphocyte subpopulations: giant SRBC rosettes.

Human thymus-derived lymphocytes have the ability to form rosettes with sheep red blood cells (SRBC) in vitro. In the investigation of rosettes of peripheral blood lymphocytes of 10 normal subjects, the number of SRBC adhering to the lymphocyte in each of 100 rosettes was assessed. The percentage of rosettes with SRBC greater than or equal to 36 per rosette was only 1.2 +/- 0.5. These were defined as giant SRBC rosettes. Peripheral blood lymphocytes were stimulated in vitro by four mitogens: sodium periodate, neuraminidase plus galactose oxidase, pokeweed mitogen, and concanavalin A. The lymphocytes were then cultured at 37 degrees C. The giant rosette-forming lymphocytes became significantly increased 4 to 24 hr after stimulation, prior to the appearance of lymphoblasts or increased incorporation of tritiated thymidine. The giant rosettes were not caused by the hemagglutinating properties of pokeweed mitogen and concanavalin A that were adsorbed on the lymphocyte surfaces. This was shown by the fact that, on removal of the receptors by trypsinization, they were regenerated on culture in vitro in the absence of the mitogens. It was concluded that giant SRBC rosettes constituted a marker for some of the activated lymphocytes. Their appearance was independent of the increase in size of the cells or of DNA synthesis. These receptors were intrinsic to lymphocytes and not caused by mitogens adsorbed on their surfaces.     

E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C.Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture.This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.     

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.     

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.     

Human lymphocyte receptor movement induced by sheep erythrocyte binding: effect of temperature and neuraminidase treatment

The formation of sheep red blood cells (SRBC) rosettes by human lymphocytes was promoted by incubation at 4 °C and by treatment of the lymphocytes or SRBC by neuraminidase. On incubating the untreated SRBC rosettes at 37 °C, the rosettes dissociated by capping in which rings were converted into horseshoes and then caps. This capping was inhibited by incubation of the rosettes at 4 °C and partially by treatment of the cells with neuraminidase. During rosette formation, the proportion of caps decreased progressively during 4 °C incubation. This decrease of capping was also promoted by neuraminidase treatment. We concluded that the main reason why 4 °C and neuraminidase treatment facilitated rosette formation was by inhibition of capping.     

EA rosette forming lymphoid cells: distribution as to cell types and organs from chickens of different developmental stages.

The interaction of chicken spleen cells with sheep erythrocytes coated with chicken antibody (EA complexes) was studied using a rosette assay. The results reported in this paper indicate that subpopulations of chicken lymphocytes, monocytes, and heterophils have a receptor for EA. The formation of rosettes between chicken lymphoid cells and sheep erythrocytes (SRBC) was dependent upon the concentration of antibody used to sensitize the SRBC. In a developmental study of rosette-forming lymphocytes (RFL), the bursa was the first site of appearance of large numbers of RFL. The percentage of RFL in the bursa reached a peak at 17 days of embryonic life, and declined to a low by hatching. The percentage of RFL in the spleen, however, began to increase at the time of hatching and by 6 weeks of age the spleen far surpassed the bursa in percentage RFL. At no age were significant numbers of RFL detected in the thymus.     

Studies of the sheep red blood cell receptor using anti-lymphocyte antibodies

Human thymus derived lymphocytes (T cells) interact with sheep red blood cells (SRBC) to form rosettes. We wanted to determine whether the lymphocyte's receptor for SRBC is associated with serologically detectable cell surface antigens. Antisera were prepared by immunizing horses with either fresh human thymus (ATG) or with B lymphocytes from an established lymphoid cell line in culture (ALG). ATG, ALG or Concanavalin A (Con A) were added to lymphocyte preparations to determine their effect on rosetting. The results showed that ATG inhibited rosettes in a dose dependent manner. In contrast, both the Con A and ALG had no effect. By immunofluorescence, Con A and ALG staining cells were able to form rosettes. ATG staining cells were unable to form rosettes. Removal of the ATG receptor by capping could not restore the rosette forming capacity suggesting that inhibition was not due to steric hindrance. We conclude that antibody directed against T cells but not B cells binds to surface antigens which appear to be identical with or in close proximity to the specific SRBC receptor.     

Inhibition of human T lymphocyte E rosette formation by neutrophils and hydrogen peroxide. Differential sensitivity between helper and suppressor T lymphocytes

Co-incubation of human mononuclear cells with small numbers of autologous polymorphonuclear leukocytes resulted in a ratio-dependent inhibition of T lymphocyte rosette formation with sheep erythrocytes (SRBC). Inhibition by polymorphonuclear leukocytes was reversed with catalase, implicating H2O2 or associated products as the suppressive agent(s). Exposing T lymphocytes directly to micromolar concentrations of H2O2 also inhibited subsequent rosette formation. Inhibition was dose-dependent between 40 and 320 microM H2O2 and was not due to cytotoxicity at those H2O2 concentrations. Kinetic studies demonstrated that after exposing T lymphocytes to H2O2 a proportion of cells appeared to be relatively resistant in that they retained their ability to form E rosettes. T cell phenotype analysis of these cells showed that, in contrast to untreated T cells, H2O2-treated T rosette-forming cells were almost exclusively of the helper/inducer phenotype. Analysis with the monoclonal antibody 4B4 revealed that H2O2-resistant T rosette-forming cells contained significantly fewer 4B4-positive cells than predicted for the T helper population, indicating that H2O2-resistant T cells might be a subset of helper/inducer T lymphocytes. The interaction between H2O2 and T cells was rapid but did not lead to loss of Leu-5b binding sites. Preliminary functional analysis indicates that interleukin 2 production and phytohemagglutinin-induced proliferation by the relatively H2O2-resistant T cells is similar to untreated T cells. In contrast, H2O2-treated T cells which lost their ability to form E rosettes produced undetectable levels of interleukin 2 and proliferated poorly in response to phytohemagglutinin. Finally, mononuclear cells pretreated with H2O2 and subsequently stimulated with mitogens demonstrated a preferential expansion of the T helper population with concomitant loss of T suppressors. Our results show that, although neutrophil-released H2O2 inhibits effective interactions between SRBC and T lymphocyte sheep erythrocyte receptors, there appears to be a population of T helper cells which is relatively resistant to H2O2 both in terms of SRBC rosette formation and response to mitogens. These data suggest that at sites of inflammation phagocyte-released H2O2 could exert immunoregulatory effects on T cells by altering T cell subset survival and allowing the function of a particular lymphocyte population to predominate.     

Natural cytotoxic reactivity of human lymphocytes against a myeloid cell line: characterization of effector cells.

Using a series of techniques to identify and deplete various peripheral blood lymphocyte subpopulations, we studied the cytotoxic reactivity of normal individuals against the myeloid cell line K-562 in a 4-hr 51chromium-release assay. Depletion of lymphocytes bearing complement receptors had a variable, usually negligible effect on cytotoxicity. In contrast, depletion of lymphocytes bearing Fc receptors abrogated target cell lysis. Separation of lymphocytes with high-affinity binding of sheep red blood cells (SRBC) evidenced by rosette formation at 29 degrees C yielded a population of rosette-forming cells containing few cytotoxic cells, whereas separation of total E-RFC under optimal rosetting conditions produced a rosette fraction containing a major proportion of the effector cells. These data indicate that the cytotoxic lymphocyte in this system is Fc receptor positive, largely complement receptor negative, and may possess low density or low affinity receptors for SRBC.     

Coelomocytes of earthworms: the T-cell-like rosette.

The coelomocytes forming spontaneous rosettes with sheep red blood cells (SRBC) were studied under various experimental conditions in lumbricus terrestris. The percentage of rosettes did vary with the pH, increased with the incubation period and remained constant at 4°C and at room temperature. The viability of the coelomocytes was a prerequisite for the formation of rosettes.The phenomenon was found to be stable, repeatable, and specific, thus suggesting rosettes are probably not an artefact.The coelomocytes forming rosettes were basophilic, nongranulated, and nonadhering cells with a large nucleus and little cytoplasm closely resembling vertebrate T-lymphocytes. The adhering cells did not form rosette.These results suggest that the coelomocytes forming rosettes could well be the evolutionary precursors or analogs of immunocyte rosettes forming cells of the vertebrates. Whether or not the coelomocyte receptors for the SRBC are similar to those of the T-cells of the vertebrate remains to be established.     

树鼩(Tupaia belangeri)淋巴细胞的自体花结

参与人自体花结(A花结)形成的分子(如CD2/LFA-3),与免疫细胞的粘附和激活有关。我们曾发现,人和猴淋巴细胞表面的树鼩红细胞(TRBC)受体不同于绵羊红细胞(SRBC)受体(CD2),可能是一种新的白细胞分化抗原。花结试验表明,树鼩的外周血淋巴细胞(TPBL)和胸腺细胞都能形成A花结,结花率分别为20.9％和11.1％;而绵羊红细胞花结(E花结)形成率分别是20.9％和1.1％。以四种单克隆抗体(McAb)(Leu 5,0-275,AICD2.1和E2 McAb)进行树鼩A花结和E花结的抑制与抗原调变试验,结果表明,这些抗体对树鼩的A花结都没有明显的抑制或调变作用,但对E花结的抑制及调变作用明显。说明TPBL表面的TRBC受体不同于SRBC受体,与CD2/LFA-3及E2分子无关。因此,TPBL的A花结与E花结形成机制不同。     

Carbohydrate specificity of T-cell cytophilic chicken anti-SRBC IgM antibodies.

Fixation of passively sensitized lymphocytes by formaldehyde resulted in amplified erythrocyte rosette formation. The cytophilic anti-SRBC antibodies were of the IgM class and they attached selectively to thymocytes and T lymphocytes. Anti-SRBC sera from approximately 30% of immunized chickens gave rosette counts between 500–1500 per 10⁴ cells whereas the remainder gave values considerably lower. The onset and peak of the cytophilic IgM response were reached later than that of IgM haemagglutinating antibodies and there was little correlation between the count of cytophilic rosettes and haemagglutinjn titres in individual chickens. On the basis of competitive inhibition of rosette formation by a hog blood group substance it is suggested that the cytophilic antibodies have binding site specificity for a saccharide on the surface of SRBC.     

Cultivation with K562 cells leads to blastogenesis and increased cytotoxicity with changed properties of the active cells when compared to fresh lymphocytes.

Lymphocytes from healthy donors were cultivated with K562, a cell line sensitive to natural cytotoxicity and lacking HLA antigens. Blastogenesis and cytotoxicity were generated in the presence of the serum of the lymphocyte donor. The anti K562 killing effect of the cocultivated populations exceeded considerably that of the fresh lymphocytes. In the majority of cases lymphocytes cultured alone had no cytotoxic activity.We have attempted to characterize the active lymphocyte subset, using the fractionation procedure designed for fresh lymphocytes. Separation of T cells on the basis of SRBC rosetting was not efficient because a high proportion of E rosettes remained in the interface when centrifuged on the Ficoll. Furthermore a high number of E rosetting cells adhered to nylon wool.The cytotoxic potential of various fractions correlated to the presence of blasts and EA positive cells. The least active population was the non-adherent, E rosette sedimented one. The cytotoxicity was not restricted to the sensitizing K562 cell line.     

Human monocyte-lymphocyte interaction: a new technique.

A rosette-type assay of the physical interaction between lymphocytes and monocytes after treatment with neuraminidase-galactose oxidase (NGAO) is reported. Monocyte-lymphocyte (ML) rosette formation and subsequent lymphocyte proliferation occurred when either lymphocytes or homologous monocytes were treated with NGAO and cultured together. Maximal ML rosette formation took place at 37 degrees C 4 hr after culture in media containing 10% serum at lymphocyte to monocyte ratios of 10:1 to 20:1. The percentage of rosette formation correlated with the extent of thymidine incorporation when increasing concentrations of NGAO were used. When NGAO-treated monocytes were added to untreated T and non-T lymphocytes, they bound preferentially to T lymphocytes and induced proliferation only in the T subpopulation. These results indicate that the ML rosette assay measures a highly specific monocyte-lymphocyte physical interaction after a mitogenic stimulus which is an early event in lymphocyte activation since it reflects the degree of subsequent lymphocyte proliferation.     

Spontaneous rosette and rosette-inhibition tests on fresh and cryopreserved lymphocytes.

Peripheral blood lymphocytes from 13 healthy adults were compared in the fresh state and after controlled freezing in liquid nitrogen. The mean percentage of cells forming spontaneous rosettes with sheep red blood cells was 52% in the fresh samples and 57% in frozen-thawed material. These figures are not significantly different. Fresh and frozen cells proved equally sensitive to the action of an antilymphocyte globulin preparation in inhibiting rosette formation. It is concluded that valid results may be obtained through the use of frozen preserved lymphocytes in rosette and rosette-inhibition tests.     

Antigen-reactive cell opsonization: a mechanism of antibody-mediated immune suppression.

Antisera against sheep red blood cells (SRBC) specifically suppressed the direct anti-SRBC plaque-forming cell (PFC) response in mice when passively administered with the antigen. The suppressive activity of mouse and rabbit anti-SRBC sera was found to correlate with anti-SRBC opsonic activity but not with hemagglutination or hemolysin titers. Macrophage depletion of mice, using carrageenan treatment, inhibited antibody-mediated immune suppression. When mice immunized with SRBC were given ¹²⁵I-labeled Udr, radiolabeled spleen lymphocytes were obtained which specifically formed rosettes with SRBC. These radiolabeled antigen-reactive cells (1ARC) were specifically opsonized in mice treated with antigen-antibody complexes but not in mice treated with antigen or antibody alone. These results suggest that antibody-mediated immune suppression may be due to specific opsonization (and subsequent destruction) of ARC in the presence of antigen-antibody complexes.     

Methods for the separation of lymphocytes]

The paper reviews the currently available methods for lymphocyte separation, with particular reference to their effectiveness. Procedures based on density and size, such as density gradient centrifugation and sedimentation and size filtration on columns, allow accumulation of lymphocytes of different degree of differentiation, but do not permit any quantitative separation of distinct lymphocyte populations, because density and size of cells are properties strongly varying with the degree of development and physiological state of the cells. Differences of the cells' net potential cause differential adhesion of lymphoid cells to glass or other materials, and lead to varying migration speeds in the electric field. Adherence columns afford only partial separation of T and B cells, whereas favourable results have been obtained by preparative cell electrophoresis. Special membrane structures, such as differentiation antigens including membrane-bound immunoglobulins, cell receptors and transplantation antigens make possible a specific separation of lymphocytes. Essentially, the following 5 methods are being used: 1. Cytolytic treatment of the cells with antisera against differentiation antigens in the presence of complement. 2. Rosette separation 2.1 Rosette formation with sheep erythrocytes (SE) or with antigen-coaded SE for the isolation of antigen-binding lymphocytes. 2.2 Rosette formation by antigen-antibody-complement complexes (B rosettes) 2.3 Rosette formation with SE by human T lymphocytes (T rosettes) 2.1--2.3. Separation of the rosettes from the free lymphocytes by centrifugation or sedimentation.     

The interaction of monomeric and complexed mouse monoclonal IgG with rat basophilic leukemia cells: a subset of IgG molecules can bind to RBL cell Fc receptors for IgG

Rat basophilic leukemia (RBL) cells were shown to bind mouse monoclonal (MC) IgE and certain mouse monomeric IgG1 and IgG2b monoclonal antibodies (MAb) by using a haptenated sheep red blood cell (SRBC) rosetting assay. Rosette formation was antibody concentration dependent with all three immunoglobulin isotypes, but at least 100 times more IgG than IgE was required to form a similar number of rosettes. It was shown by FACS analysis and rosette formation that a subset (8/23) of the IgG MC was able to bind to RBL cells as monomers. However, the majority 15/23 did not bind or bound weakly (less than 25% rosettes) unless in the form of antigen-antibody complexes. As complexes, all IgG subclasses except IgG3 could produce rosettes with RBL cells. None of the IgM or IgA MC tested formed rosettes, even in complexed form. By inhibition studies it is demonstrated that mouse IgG1, IgG2a, and IgG2b MC bind to the same Fc receptor. Mouse IgE was only partially able to inhibit IgG-dependent rosettes at high concentrations, and none of the IgG MC were able to inhibit IgE-dependent rosettes. These results suggest that the interaction of mouse IgG is quite specific for the RBL cell FcG receptor. Because deaggregated polyclonal mouse IgG was a weak inhibitor of MC IgG sensitization of RBL cells, the results are discussed in terms of the heterogeneity and possible abnormality of some MAb.     

Differentiation between chronic lymphocytic leukaemia (B-CLL) and non-Hodgkin lymphomas in the leukaemic phase by the mouse red blood cell rosette assay

A distinction between B-CLL and other malignant B-cell lymphomas in the leukaemic phase may be difficult. Mouse red blood cell rosette formation of lymphocytes from 97 patients with B-CLL and 19 patients suffering from other B-cell lymphoproliferative disorders was examined together with lymphocyte rosette formation of healthy controls. The majority of circulating lymphocytes of B-CLL patients formed rosettes with mouse red cells, whereas there was no relationship between the number of peripheral neoplastic B-cells and that of rosette forming cells in other lymphoproliferative diseases. The relatively simple mouse red blood cell rosette assay proved to be of value in the differentiation of otherwise nearly related conditions.     

Effect of organic solvents in the course of professional exposure on the E rosette test and skin reactions against distreptase and tuberculin

The study comprised 106 workers having professional contact with organic solvents containing benzene, toluene, and xylene during 1 to 122 months. The reduction of the total lymphocyte and the T lymphocyte count (T cells forming both "early" and "late" rosettes with sheep erythrocytes) corresponding to the prolongation of the exposure time to the solvents (the negative correlation) was observed. These alterations are not associated with disturbances of delayed skin hypersensitivity against tuberculin and distreptase. The E rosette test may serve as a practical test for monitoring the biological effects of exposure to organic solvents.