Rosetting of human red blood cells to thymocytes and thymus-derived cells

Human thymocytes bind with human red blood cells (HRBC) and neuraminidase-treated HRBC (nHRBC) forming rosettes. Peripheral blood lymphocytes and tonsillar lymphocytes bind to nHRBC but not HRBC. The conditions which lead to nHRBC rosette formation are similar to those required for rosette formation with sheep red blood cells (SRBC). The rosetting of human lymphocytes to nHRBC does not require serum but is prevented by pretreatment of the lymphocytes with sodium azide, proteolytic enzymes, 50°C for 7 min, or anti-T cell (but not anti-B cell) serum. Peripheral lymphocytes from patients with chronic lymphatic leukaemia do not rosette with either HRBC or nHRBC. Therefore, the nHRBC rosetting lymphocyte is a living T cell. It is proposed that this in vitro test may simulate an in vivo mechanism for immune surveillance.     

The lack of specificity of the sheep erythrocyte-T lymphocyte rosetting phenomenon.

One subpopulation of lymphocytes, thymus-derived (T) cells, is identified by its characteristic ability to form spontaneous rosettes with sheep erythrocytes (SRBC). However, the mechanism by which the rosettes form remains unknown. To gain more insight into the specificity of this phenomenon, the ability of SRBC to rosette with cells other thah lymphocytes was studied. All of the cell types utilized in this study (L cells, monkey liver cells, HeLa cells, and human liver, bladder, lung, parathyroid, and dermal fibroblasts), except neoplastic B lymphocytes, rosetted with SRBC. Viability was not a factor in the rosette formation. These findings suggest that the process of T lymphocyte-SRBC rosette formation is not due to a T cell-specific membrane receptor or antigen but may be due to a widely distributed basic substructure of the cell membrane.     

Studies of the presence of membrane receptors for complement,IgG and the sheep erythrocyte rosetting capacity on the same human lymphocytes

Normal peripheral blood lymphocytes were tested by a mixed rosette method, employing different sized erythrocytes as indicators to identify lymphocytes simultaneously possessing membrane markers found commonly on B and T cells. Only small populations of these lymphocytes were detected regulary in normal lymphocyte preparations. One type of lymphocyte (ranged from 0.5%-8%) was shown to possess the following markers: receptors for human and rabbit IgG, receptors for the third complement component C3b and C3b inactivator-cleaved C3b (C3d), and the capacity to rosette spontaneously with uncoated sheep erythrocytes (SRBC). Another lymphocyte cell type was shown to possess both SRBC and IgG receptors but lack membrane immunoglobulins and complement receptors. This population was detected in lymphocyte preparations depleted of complement receptor cells, and an increased number of these cells was found in rosette preparations incubated with human serum. The possible presence of some lymphocytes possessing both complement and SRBC receptors and lacking other markers was considered. The possibility that these small populations of human lymphocytes are sub-populations of T cells with certain cytotoxic function is postulated.     

In vitro monocyte-lymphocyte interaction influenced by d-penicillamine.

When put into 37 degrees C incubation, the E rosettes dissociate spontaneously and the sheep erythrocytes (SRBC) form caps at one pole of the lymphocytes. This process is associated with changes in cell morphology such as uropod formation or membrane budding. The disintegration of E rosettes and the capping of SRBC can be retarded by addition of cytochalasin B plus colchicine, chlorpromazine or sodium azide. These findings suggest a pivotal role of the cytoskeleton in the dissociation process of E rosettes. However, other mechanisms of disintegration are to be considered since none of the drugs can prevent the dissociation of E rosette entirely.     

Arming of Lymphoid Cells by IgG Antibodies Treated with Protein A from Staphylococcus aureus

Mouse spleen lymphocytes treated with rabbit IgG anti-sheep erythrocytes (SRBC) complexed with protein A of Stapbylococcus aureus (SpA) form rosettes with SRBC. The attachment of SRBC to lymphocytes was due to the binding of the SpA-IgG antibody complex to the surface of the lymphocytes and was thus considered 'arming' of the cells. Normal mouse spleen cells 'armed' with SpA-rabbit IgG anti-chicken erythrocytes (CRBC) kill specifically ⁵¹Cr-labeled CRBC 'in vitro' in the absence of free antibodies. The killing by these 'armed' cells is an effect of the cell-bound SpA-IgG antibody complex. Both the SRBC rosette formation and the cell-mediated CRBC killing was dependent on the concentration of the SpA-IgG antibody complexes used for 'arming' the cell. A 100-fold increase in rosette formation or in killing of target cells was recorded for lymphocytes treated with SpA-IgG antibody complexes in comparison with cell treated with noncomplexed IgG antibodies. The specific binding of SpA-IgG antibody complexes to the Fc receptors of mouse spleen cells was demonstrated by inhibition studies. More than 60% inhibition of the rosette formation and in the killing of target cells was shown for cell treated With normal rabbit IgG or its Fc fragment before addition of the SpA-IgG antibody complex.     

Inhibitory effect of sheep erythrocyte fragments on rosette formation of human T lymphocytes with sheep red blood cells.

The effect of sheep red blood cells (SRBC) fragments on rosette formation of human peripheral T lymphocytes with SRBC was evaluated on the active and total T-rosette tests. The rosetting capacity of active rosette-forming cells was selectively and nearly completely inhibited by the pretreatment of lymphocytes with SRBC fragments. The decrease in total rosettes by blocking with SRBC fragments was almost parallel to that of active rosettes. SRBC fragments had no inhibitory effect on the rosetting capacity of a lymphocyte population in which active rosette-forming cells were removed by gradient centrifugation. These results suggested that active rosette-forming cells in human T lymphocytes have the receptors of high affinity for SRBC and these receptors readily bind SRBC fragments, resulting in block of rosette formation.     

Enumeration of T cells, B cells and monocytes in the peripheral blood of normal and lymphocytotic cattle.

The rosette-forming capacity of bovine peripheral blood lymphocytes (PBL) was determined with dextran and 2-aminoethylisothiouronium bromide (AET)-treated sheep erythrocytes (SRBC). Both dextran and AET-enhanced rosette formation; however, AET-treated SRBC detected a larger percentage of rosette-forming cells and thus was used in this study. The specificity of rosette formation by bovine thymus-derived (T) lymphocytes was shown by (1) demonstration of rosettes and surface-membrane immunoglobulins sIg) on different cells in PBL and nylon-wool fractionated lymphocyte populations and (2) rosette formation by a large percentage (83--90%) of thymocytes from three bovine foetuses and two 14-month-old heifers. A procedure was also developed to identify bovine monocytes by latex phagocytosis and 10--30% latex-ingesting cells were detected in PBL preparations isolated by Ficoll-Hypaque flotation. The frequency of sIg-bearing latex-ingesting, and sIg-bearing latex non-ingesting cells in bovine peripheral blood was also determined. These procedures were utilized to determine the distribution of T and bone-marrow derived (B) lymphocytes in peripheral blood of normal and lymphocytotic cattle. PBL from twenty normal cattle contained approximately 63% T and 11% B (sIg+ latex non-ingesting) lymphocytes. In peripheral blood of three cattle with persistent lymphocytosis, a prodromal stage of bovine leukaemia, the percentage of B cells was elevated approximately to 59% whereas T lymphocytes decreased to 35%, thus providing additional evidence that persistent lymphocytosis is a B-cell disease.     

The effect of cytochalasin B and vinca alkaloids on EA- and EAC-rosette formation and on the binding of heat-aggregated human IgG by human lymphocytes

We report the effect of cytochalasin B and vinca alkaloids on EA- and EAC-rosette formation and on the binding of heat-aggregated human IgG by human lymphocytes. Cytochalasin B inhibits EA- but not EAC-rosette formation, and neither colchicine nor vinblastine have a measurable effect on either reaction. Also, cytochalasin B inhibits the binding of heat-aggregated IgG by human lymphocytes. The inhibition caused by cytochalasin B is reversible, and the cells recover to normal values within 30 min.     

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.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

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.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Characterization of immunological depression in spontaneously hypertensive rats

Immunocompetent cell functions were evaluated in spontaneously hypertensive rats (SHR). Hematological studies revealed decreased absolute numbers of lymphocytes and increased numbers of polynucleic cells in the peripheral blood of SHR. The SHR had a reduced number of immature T lymphocytes in their thymuses in comparison with an original strain of Wistar rats, as detected by the rosette formation test with guinea pig erythrocytes. The antibody response to sheep red blood cells (SRBC) of the 3-month-old SHR was profoundly depressed and was about one-tenth that of the Wistar rats. Cell cooperation experiments suggest that the T lymphocytes of the SHR were selectively impaired in antibody responses to SRBC in cooperation with B lymphocytes. B lymphocytes from the bone marrow of the SHR were not affected and produced normal numbers of plaque-forming units. Cyclophosphamide treatment, which selectively depletes suppressor T lymphocytes, did not enhance the delayedtype hypersensitivity response to SRBC in SHR. This may rule out the possibility of the involvement of the suppressor mechanism in the T cell depression of the SHR.     

Characterisation and separation of human lymphocytes forming mouse red cell rosettes.

Rosette formation between human lymphocytes and mouse red blood cells (MRBC) was examined as a marker for B lymphocytes and as a method of B lymphocyte separation. A small proportion of lymphocytes formed spontaneous rosettes with MRBC (mean value 6%) and the number was considerably increased by pretreating the lymphocytes with neuraminidase (mean value 16%). Double marker tests demonstrated that lymphocytes forming MRBC rosette were immunoglobulin (Ig) bearing cells, with a high proportion of IgM bearing cells, but not all Ig bearing cells formed MRBC rosettes. Lymphocyte populations enriched with T or B lymphocytes, by SRBC rosette sedimentation and nylon column filtration, gave values for MRBC rosettes consistent with a subpopulation of Ig bearing cells. Separation of MRBC rosette-forming cells gave a relatively poor degree of separation and rerosetting with MRBC produced variable results.     

A method for simultaneous determination of rosette formation and phagocytosis by cells

A fluorescent agent insoluble in water was readily phagocytosed by 5–20% of human blood mononuclear cells, mainly monocytes. These cells were easily detected even amidst abundant rosetting erythrocytes. When rosette assays for the detection of complement or SRBC receptors on the surface of the cells were performed, only phagocytic cells exhibited the complement receptor with the method used in the study, whereas mainly non-phagocytic cells formed SRBC rosettes. The effect on the calculation of the percentage of T lymphocytes (SRBC receptor lymphocytes) of varying amounts of monocytes present in mononuclear cell population is demonstrated. The method presented here might prove useful particularly in studies on Fc or complement receptors on white blood cells.     

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.     

Enhanced rosetting of sheep erythrocytes by human peripheral blood T cells in the presence of dextran

Dextran in optimum concentration enhances the rosetting of human peripheral blood lymphocytes with sheep red blood cells (SRBC). Dextran does not cause B cells to rosette with SRBC. Enhanced rosetting is blocked by an antiserum specific for T cells. Possible mechanisms by which dextran enhances T-cell rosetting are discussed.     

Allotypic determinants on the surface of rosette-forming cells in the rabbit

Antigen-binding cells to sheep erythrocytes from non-injected rabbits and from rabbits after a single injection of sheep red blood cells (SRBC) have been detected by the immunocyto-adherence technique (rosette formation). The expression of immunoglobulin allotypic determinants on these antigen-binding cells has been investigated by inhibition of rosette formation with anti-allotype antisera. The results indicate that a and b locus allotypes are expressed by the great majority of rosette-forming cells (RFC) in the lymph nodes of injected and non-injected rabbits. Ms3 allotype is expressed by practically all RFC from non-injected rabbits, while, after injection of SRBC, a population of RFC appears, which does not express this allotype. An antiserum to d 11 consistently failed to inhibit RFC. Pretreatment of lymphocytes from rabbits heterozygous at the b locus with antiserum to only one allelic product produced an inhibition of rosette formation which was approximately half that obtained when antiglobulin reagents to both allelic products were added. The results suggest that in heterozygous animals, the two allelic markers are expressed on two different populations of antigen-binding cells. Electron microscopy studies on the morphology of RFC showed that the majority of the RFC were formed around small lymphocytes.     

Unification and Technical Aspects of Total T, Active T and B Lymphocyte Rosette Assays

Conditions for rosette formation of human adult T and B lymphocytes with unsensitized and complement coated SRBC respectively, have been examined in this collaborative study. Modified procedures for identifying, by rosette formation, the total T, active T and B-RFC lymphocytes are described. The procedures reported here yielded results for the percent of peripheral blood lymphocytes forming total T, active T and B-RFC for control individuals that are comparable with those previously reported. The advantage of the procedures described here are that they are 1) easy to perform; 2) highly reproducible; 3) allow for the preparation of a single lymphocyte concentration and common reaction mixture volume to be used in all three rosette assays and 4) allow for the removal of non-lymphocytic cells which would interfere with the determination of B-RFC. The use of these procedures would allow other laboratories to obtain similar values so results for controls and various types of patients could be compared.     

Human thymus lymphoid (HTL) tissue antigen, complement receptor and rosette formation with sheep erythrocytes of the lymphocytes from primary immunodeficiency diseases

The presence of human thymus lymphoid tissue (HTL) antigen, which we have proposed as a marker of thymus-derived lymphocytes (T cells) in human beings, complement receptor and rosette formation with sheep erythrocytes were tested on the lymphocytes from various immunodeficiency diseases. It is postulated that both T and B cells are reduced in severe combined immunodeficiency, B but not T cells are defective in infantile X-linked agammaglobulinaemia, and both T and part of B cell populations (IgA producers) are impaired in ataxia telangiectasia. The HTL antigen-positive cells (HTLC) were reduced in two cases of ataxia telangiectasia and a case of severe combined immunodeficiency. HTLC were present in numbers comparable to normal controls in two cases of X-linked agammaglobulinaemia and two cases of isolated IgA deficiency. The number of rosette-forming cells with sheep erythrocytes (RFC) in ataxia telangiectasia were rather higher than controls, which means that rosette formation is not specific to T cells, at least in some situations, although there is some evidence suggesting that it is a property of T cells in normal conditions. The complement receptor lymphocytes (CRL) have been suggested to be B cells in the mouse. However, CRL were present in normal numbers in the cases of X-linked agammaglobulinaemia. This indicates that CRL are not necessarily antibody-forming cells in man.