A simple rosette assay for demonstration of complement receptor sites using complement-coated zymosan beads.

In the present article we describe a simple rosette assay for detection of C3 receptor-bearing B lymphocytes with complement (C)-coated zymosan (Zy) beads. Zy coated with murine or human C bound to a distinct population of human and mouse lymphocytes as well as to the majority of lymphoblastoid cells of several human established cell lines. Rosette formation was also observed with human red blood cells, with human monocytes and neutrophils. Experiments with anti-immunoglobulin sera, with other B and T cell markers and with mouse thymocytes proved that the capacity to bind C-coated Zy is primarily a feature of B lymphocytes. The following findings suggested that C-coated Zy is bound via receptor sites for the activated components of C3: (a) Zy coated with C3-deficient serum failed to bind, (b) comparable percentages of various lymphoid and nonlymphoid cells formed rosettes with C-coated Zy as well as with antibody and C-coated sheep red blood cells, and (c) antibodies against human or murine C3 inhibited the binding of C-coated Zy.     

Effects of preincubating human peripheral blood lymphocytes on their ability to bind sheep red blood cells

The effect of preincubating human peripheral lymphocytes at 37°C or 4°C for various lengths of time on their subsequent ability to form rosettes with sheep red blood cells (SRBC) was investigated. Lymphocytes suspended in either medium or medium containing fetal calf serum (FCS) and preincubated at 37°C for 30 min exhibited marked decrease in rosette formation with a return to normal values by 120 min. However, neither lymphocytes suspended in medium and preincubated at 4°C nor lymphocytes suspended in medium containing normal human serum (HS) and preincubated at 37°C exhibited this phenomenon.     

A simple rosette assay for demonstration of complement receptor sites using complement-coated zymosan beads

In the present article we describe a simple rosette assay for detection of C3 receptor-bearing B lymphocytes with complement (C)-coated zymosan (Zy) beads. Zy coated with murine or human C bound to a distinct population of human and mouse lymphocytes as well as to the majority of lymphoblastoid cells of several human established cell lines. Rosette formation was also observed with human red blood cells, with human monocytes and neutrophils. Experiments with anti-immunoglobulin sera, with other B and T cell markers and with mouse thymocytes proved that the capacity to bind C-coated Zy is primarily a feature of B lymphocytes. The following findings suggested that C-coated Zy is bound via receptor sites for the activated components of C3: (a) Zy coated with C3-deficient serum failed to bind, (b) comparable percentages of various lymphoid and nonlymphoid cells formed rosettes with C-coated Zy as well as with antibody and C-coated sheep red bloodcells, and (c) antibodies against human or murine C3 inhibited the binding of C-coated Zy.     

Lymphocyte membrane receptors in human lymphoid leukemias.

Membrane-bound immunoglobulins, receptors for the Fc fragment of IgG and receptors for the third component of human or murine complement were used as B cell membrane markers to study peripheral blood lymphocytes from twenty-two patients with chronic lymphatic leukemia (CLL), five patients with acute lymphoblastic leukemia (ALL) and one patient with Sézary syndrome. The capacity of human T cells of forming "spontaneous rosettes" with sheep erythrocytes was employed as T cell membrane marker. In nineteen out of twenty-seven CLL or ALL cases tested a larger percentage of cells than that found in normal individuals expressed at least one of the three B cell membrane markers studied. In the patient with Sézary syndrome the percentage of cells forming "spontaneous rosettes" with sheep erythrocytes was larger than the normal, while cells bearing B cells markers were below the normal values.     

Lymphocyte subpopulations. Human red blood cell rosettes.

Human red blood cells (HRBC) even without prior neuraminidase treatment, could form rosettes with human peripheral blood lymphocytes in vitro. The optimum conditions for forming these rosettes were a pH of 7-0 and a medium with 5% bovine serum albumin (BSA). Rosette proportions became much less at a different pH or using lower concentrations of BSA, or replacing BSA with foetal calf sera (FCS) or human sera. Rosette formation was also promoted by prior treatment of HRBC or lymphocytes with neuraminidase. Mixed rosettes of HRBC and sheep red blood cells (SRBC) showed that HRBC receptors were detectable only on lymphocytes that possessed SRBC receptors, suggesting that HRBC rosette-forming cells were probably thymus-derived (T) cells. Next, the properties of human red blood cell (HRBC) and sheep red blood cell (SRBC) rosette-forming cells were investigated by comparing the ability of human peripheral blood lymphocytes to form these two types of rosettes after treatment with various inhibitory reagents. HRBC rosettes were relatively more resistant to inhibition with: (1) proteolytic agents, such as trypsin, alpha-chymotrypsin and pronase; (2) anti-thymocyte serum (ATS); (3) metabolic inhibitors, such as sodium azide and 2,4-dinitrophenol (DNP); (4) cytochalasin B. On further incubation after trypsinization, the lymphocytes recovered some ability to form SRBC rosettes, but continued to lose more of their capability to form HRBC rosettes. All these results were regarded as circumstantial evidence that the HRBC rosettes might represent a subpopulation of human T lymphocytes.     

Spontaneous and PHA-induced rosetting of human blood, tonsil lymphocytes and MLC blasts with sheep, human and horse erythrocytes.

Compared to peripheral blood lymphocytes the ability of human tonsil T cells and MLC blasts to bind sheep, human and horse erythrocytes was found to be increased. Tonsil and MLC T cells were able to bind sheep red blood cells without any cold incubation, i.e. they were 'early' rosettes, and higher percentage of human and horse erythrocyte rosettes were formed by these cells. Low doses of phytohaemagglutinin increased the proportion of rosettes between peripheral blood, tonsil, MLC cells and human and horse erythrocytes. PHA acted only on T cells, and not on B cells, lymphoblastoid B and other cell lines. On the ground of the stronger rosetting property of MLC blasts and tonsil cells, it is likely that the T cells responsible for binding of horse and human erythrocytes after PHA treatment are 'early' or 'active' rosetting cells.     

Rosette formation by human T and B lymphocytes

Human peripheral lymphocytes, identified as T or B cells with a fluorescent anti-globulin serum, were studied in two varieties of rosette formation: (1) with sheep red corpuscles, a phenomenon shown by a large proportion of human lymphocytes and totally different from that observed in the mouse; and (2) with human red cells sensitized with incomplete anti-Rh. Lymphocytes forming sheep cell rosettes were never fluorescent; i.e. they were T cells. 73 % of lymphocytes forming sensitized-cell rosettes were fluorescent B cells. Horse anti-human lymphocyte serum inhibits the first but not the second variety of rosette formation.     

Receptors for IgG molecules on human lymphocytes forming spontaneous rosettes with sheep red cells

A mixed rosette techinque with sheep red blood cells (SRBC) and ox erythrocytes heavily coated with rabbit antibody (EA) was employed to simultaneously idnetify human peripheral blood T lymphocytes and IgG receptor-bearing cells. The findings of a noticeable proportion of ?mixed resettes”? in peripheral blood lymphocytes freshly drawn from normal individuals and of an even higher number of these mixed rosettes in cell suspension kept in culture media supplemented with fetal calf serum provide evidence for the capacity iof human T cells to express membrane receptors for antigen-antibody complexes.     

Observations on rabbit thymocytes and peripheral T cells. I. Anomalous results in the mixed antiglobulin reaction caused by non-specific adsorption of sheep immunoglobulin.

The "single-stage" mixed antiglobulin reaction (MAR) was carried out with rabbit thymocytes. This test involved treating the cells with either sheep or goat anti-rabbit globulin sera, and subsequently reacting them with indicator erythrocytes coated with rabbit immunoglobulin (Ig) so as to form rosettes. An unexpectedly high number (up to 38%) of thymocytes reacted, although the rosettes were weaker than those given by peripheral B lymphocytes. When blood and lymph node lymphocytes or thymus cells which had already been treated with sheep anti-rabbit globulin serum were subsequently exposed to rabbit anti-sheep Ig serum and then rosetted with indicator cells coated with ox Ig (cross-reacts with sheep Ig) almost 100% reaction was obtained in each of the cell suspensions. This was designated the "two-stage" MAR. The anomalous results, both in the one-stage and two-stage MAR, were abolished by pepsin-treating the sheep anti-rabbit globulin serum; thus indicating that sheep Ig is adsorbed non-specifically via the Fc part of the molecules to the surface of rabbit thymocytes and peripheral T lymphocytes.     

In Vitro studies on human B and T cell purified populations. Stimulation by mitogens and allogeneic cells, and quantitative binding of phytomitogens.

The property of T cells to form rosettes with sheep red blood cells has been used to separate peripheral blood lymphocytes into purified T- and B-cell suspensions after density gradient centrifugation. A study of lymphocyte markers has shown that 2-6 per cent of E rosettes only were recovered in the B cell-enriched population. Lymphocyte stimulation in vitro was obtained with PHA, con A and PWM in unseparated and T-cell populations, but never in B-cell suspensions. Experiments of recombination between the two purified fractions have demonstrated that 10% of T cells added to B cells were able to induce a response to PHA and PWM. Otherwise, only T cells responded to allogenic stimulation. Lastly, B and T cells seemed to bind qualitatively and quantitatively the same mitogens on their membranes.     

Analysis with monoclonal antibodies of human lymphoid cells forming rosettes with rabbit red blood cells.

Rabbit red blood cells have previously been shown to rosette with a subpopulation of thymocytes and with mitogen activated peripheral lymphocytes but not with unstimulated lymphocytes. Using monoclonal antibodies and double marker assays we studied the phenotype of these cells. In thymus, over 90% of rosetting cells express antigens of immature thymocytes (HTA1, OKT6). A proportion of the rosetting cells shows in addition antigens of mature thymocytes (OKT3, UCHT1). These cells probably correspond to a stage of intrathymic maturation between common and mature thymocytes. Virtually all rosetting cells are T cells and express an antigen related to T cell activation (TAC) when lymphocytes are activated by mitogens like PHA or Con A. Few rosetting cells are Ia positive. Two other antigens (OKT9, OKT10) known to be associated with proliferating and immature cells, are found in variable proportions on rosetting cells. After stimulation with allogeneic lymphocytes, fewer rosettes are detected than after stimulation by mitogens. Cells activated by a soluble antigen (PPD) and forming rosettes with rabbit red blood cells have a helper phenotype (Leu3a positive). Screening of leukaemia cell samples revealed that only cells from patients with T-ALL form rosettes with rabbit red blood cells. Rosette formation is almost totally inhibited by a polyclonal anti-thymocyte serum and two monoclonal antibodies (OKT11A,Lyt3) which have been shown to block rosettes with sheep erythrocytes.     

Lymphocyte binding of aggregated IgG and surface Ig staining in chronic lymphocytic leukaemia

Eleven selected patients with chronic lymphocytic leukaemia were evaluated for lymphocyte binding of aggregated IgG and surface Ig staining in order to classify them into B and T cell types. Ten of the eleven patients bound aggregates and stained for surface Ig. In the individual ten patients the number of cells binding aggregates was high (88–100%, mean 96%) whereas the number staining for surface Ig was more variable (8–100%, mean 62%). Parallel and double labelling experiments with aggregates and sheep red blood cell rosettes, a human T cell marker, provided evidence that aggregates were binding to B cells only, even when surface Ig was not detectable. Aggregates did not bind to human thymocytes. Evidence was presented that lymphocytes from some cases of CLL have low but not absent amounts of surface Ig that may be only partially detected by fluorescence techniques. Aggregate binding appears to be a more sensitive method for the detection of B lymphocytes than surface Ig staining.In one of the eleven patients the leukaemic cells were negative in the aggregate binding test. Separate studies on this case also indicated an absence of surface Ig staining and a high percentage of cells forming sheep red blood cell rosettes. It would appear that this case represented a T cell leukaemia.     

Subpopulations of human lymphocytes differing in receptors to mouse and sheep red blood cells

A segregation procedure of human lymphocytes by successive rosetting with mouse and sheep red blood cells, and separation of rosettes by Ficoll Hypaque gradient centrifugation, is described. This method yields four lymphocyte subpopulations: lymphocytes with mouse and sheep receptors (M+E+), with mouse receptor only (M+E-), with sheep receptor only (M-E+), and without any receptors (M-E-). Presence of surface membrane receptors: E, C3, Fc, and DR on the cells in the subpopulations have indicated that M-E+ and M+E+ cells are T cells, M+E- cells are B cells and M-E- cells are nonhomogeneous, consisting of B cells, T cells, monocytes, and granulocytes.     

Identification and function of T cells in the peripheral blood of patients with hypogammaglobulinaemia

Patients with common variable immune deficiency may have depressed blast transformation to PHA in the peripheral blood and depressed delayed hypersensitivity skin reactions at a time when their percentage of sheep red cell rosettes (presumptive measure of T cells) is normal. This may indicate the presence of two types of T cells in human peripheral blood.     

Human peripheral lymphocytes bearing both B-cell complement receptors and T-cell characteristics for sheep erythrocytes detected by a mixed rosette method

Human peripheral lymphocyte preparations were tested with a mixed rosette method for the presence of lymphocytes bearing both the complement receptors characteristic of B lymphocytes, and the capacity of T lymphocytes to spontaneously bind with sheep red blood cells (SRBC). The large and oval shaped pigeon red blood cells (PRBC) which do not form T-cell rosettes were utilized as indicators for rosette formation with the complement receptor-bearing B cell. In every individual (an average of 2·06%) lymphocytes were observed which formed rosettes with both SRBC and PRBC indicators. These findings show that independent markers for both T and B cells may be present on the surface of the same lymphocytes.     

Spontaneous rosette formation by mouse L cells.

Mouse fibroblasts form spontaneous rosettes with sheep erythrocytes and with red cells from several other species. They do not form C3 or Fc rosettes, and they do not bind small amounts of aggregated gammaglobulin. Spontaneous rosette formation is maximum at 37 degress and minimum at 4 degrees. The reaction is complete in 30 min. Antisera to fibroblast membrane components do not inhibit rosette formation. Rosetting is inhibited by fixation of the cells with formaldehyde or glutaraldehyde as well as by killing with phenol or by prolonged incubation with sodium azide. The rosette reaction is inhibited by vinblastine but not by cytochalasin B. From these data on the kinetics, temperature and drug sensitivities it is suggested that T cells and fibroblasts form spontaneous rosettes by different mechanisms.     

Rosette formation with goat erythrocytes. A marker for human T lymphocytes.

We demonstrate the use of goat erythrocytes in a rosette procedure for the classification of human lymphocytes. The population is almost perfectly overlapping with the lymphocytes which form rosettes with sheep red blood cells. 70·2 ± 7·5% of peripheral lymphocytes form rosettes with goat erythrocytes and less than 1% of these cells have surface immunoglobulins. Enrichment of goat rosette-forming cells results in a population with an increased percentage of both goat and sheep rosettes. This population retains activity to the T-cell mitogens Con A and PHA, while the cells depleted of goat rosettes have greatly diminished responses to these same mitogens. Tonsil and spleen lymphocytes form 50·2 ± 6·8% and 24% of goat rosettes respectively, while peripheral blood lymphocytes from patients with CLL rarely form goat rosettes. Cell lines maintained in vitro rosetted with goat cells in a parallel fashion to sheep cells. Thus T-cell lines, such as Molt-3, which form rosettes with SRBC also rosette with GRBC, while sheep rosette-negative lines, i.e. Molt-4, are negative for both erythrocytes. B-lymphoid cell lines were negative, as were several lymphoma cell lines. There was a slight variation in the binding of goat cells, depending on the source of the goat. Thus, as in sheep rosettes, some animals were better sources than others, although all the animals tested formed rosettes.Human lymphocytes are capable of binding goat red cells. The cells which bind to the erythrocytes seem identical to those binding sheep red blood cells, and should be considered as a T-cell population. Preliminary inhibition data suggests that the receptor on T cells is the very same structure for both erythrocytes.     

Surface Markers on Human B and T Lymphocytes

Almost 100% of peripheral T lymphocytes are shown to have the capacity to form rosettes with human lymphoblastoid B-cell lines, predominantly at 4°C and with lines having surface-bound IgG. Blast-transformed T cells retained this capacity and formed rosettes even at 37°C Unstimulated T cells bound less readily to B-cell blasts, stimulated by pokeweed mitogen for 72 hr. Even though rosettes, formed at 4°C, were stable for several hours at 72°C, no T-cell-mediated cytotoxicity could be detected during overnight incubation. Extreme pH values and trypsinization decreased rosette formation, whereas neuraminidase treatment enhanced the reaction. Rosette formation was independent of bivalent cations and unimpaired in the presence of inhibitors (NaF. NaN₃), undiluted human or fetal call sera, protein A, sonicated sheep erythrocyte membranes, and normal or heat-aggregated human IgG. Anti-Ig, anti β₂-microglobulin, or anti-T cell sera did not influence rosette formation.     

The predominant lymphocyte in most thymomas and in nonneoplastic thymus from patients with myasthenia gravis is the cortical thymocyte

Cell suspensions prepared from 12 specimens of nonneoplastic thymus (6 normal and 6 from patients with myasthenia gravis) and from 17 thymomas were investigated with a panel of monoclonal antibodies. The great preponderance of thymocytes from the 12 nonneoplastic specimens and from 13 of the 17 thymomas (2 of 3 predominantly lymphocytic tumors and 11 of 12 mixed tumors) displayed the surface phenotype of cortical or common thymocytes. These cells formed rosettes with unsensitized sheep erythrocytes (E-rosettes) at both 4 and 37 degrees C, and reacted with the following monoclonal antibodies: OKT1 (thymic and peripheral T cells), OKT6 (common thymocytes), OKT10 (replicating lymphoid cells), OKT11 (sheep cell receptor), and both OKT4 (inducer-helper T cells) and OKT8 (cytotoxic-suppressor T cells). Few B cells (lymphocytes with either immunoglobulin or Ia-like antigen on the cell surface), and few cells with receptors for transferrin and interleukin 2 were detected. Thymocytes from 3 of the 4 remaining thymomas (2 predominantly epithelial tumors and 1 mixed tumor) displayed surface marker characteristics of medullary thymocytes or peripheral T cells; i.e., they were reactive with OKT1, OKT3 (peripheral T cells), OKT11, and either OKT4 or OKT8, and were also E-rosette positive only at 4 degrees C and TdT negative. Thymocytes from the final tumor, a lymphocytic thymoma, exhibited an intermediate phenotype. Thus, almost all mixed (11 of 12) and lymphocytic (2 of 3) thymomas were composed predominantly of cortical thymocytes, while the medullary cell was the rule in the two tumors that were predominantly epithelial.     

The role of E receptors in the attachment of thymocytes and T lymphocytes to human target cells

Human thymocytes, activated T lymphocytes, and neuraminidase-treated T cells possess the distinct capacity of forming conjugates with various human cell lines. The present study investigated whether E receptors, which endow human T cells with their capacity to bind sheep red blood cells (SRBC), are involved in this phenomenon. Monoclonal antibodies to human T cells and various simple sugars were studied for their effect on the attachment of human T cells to target cells. A-22, a monoclonal antibody to the E receptor, inhibited the formation of E rosettes by T cells and SRBC, and reacted in immunofluorescent-staining assays with the majority of human thymocytes and peripheral T cells, and with T-cell lines capable of forming E rosettes. When human thymus cells were treated with A-22 antibody they showed a reduction of up to 70% in their capacity to attach to the GM-4762 lymphoblast cell line and the K-562 myeloid line. Antibody treatment of the target cells, rather than of the thymus cells, had no effect on the formation of conjugates between thymus cells and target cells. Treatment of thymus cells with various monoclonal antibodies to T cells which do not react with the E receptor had no inhibitory effect. The exposure of human thymus cells to various simple sugars (D-mannose, D-fucose, galactose, and lactose) markedly reduced their capacity of forming conjugates with target cells. Exposure of neuraminidase-treated peripheral blood lymphocytes and of activated T cells to A-22 antibody inhibited their attachment to human target cells. The present study suggests that E receptors play a role in the attachment of human thymus cells and activated T cells to other human cells, and raises the possibility that these T-cell receptors may be involved in the process of recognition of "self" structures by human T lymphocytes.