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.     

The formation of stable E-rosettes by human T lymphocytes activated in mixed lymphocyte reactions.

The aim of the present study was to determine whether activation of human T-lymphocytes affects their interaction with sheep red blood cells (SRBC). Less than 3% of the E-rosettes formed by freshly isolated peripheral blood lymphocytes (PBL) and SRBC are stable and do not disintegrate after incubation at 37 degrees C. In contrast, about 30% of PBL kept in culture for 5 days in the presence of mitomycin C-treated allogeneic lymphocytes were found to form stable E-rosettes. Whereas no rosettes were formed by freshly isolated PBL incubated with human red blood cells at 24 degrees C, 15% of the cells recovered from mixed lymphocyte reactions (MLR) formed such rosettes. When responder PBL were maintained in culture in the absence of allogeneic stimuli the proportion of cells forming stable E-rosettes depended on the serum present in the medium. Less than 5% of the responder cells kept in medium containing human serum or in serum-free medium formed stable E-rosettes, whereas 18% of the cells maintained in medium containing fetal calf serum formed stable E-rosettes. The proportion of cells forming stable E-rosettes increased before any increase in DNA synthesis was detectable in MLR. Indeed, a high proportion of cells forming stable E-rosettes appeared in MLR taking place in serum-free medium, without any accompanying increase of DNA synthesis. Depletion of cells forming EAC'-rosettes from responder PBL increased the proportion of cells forming stable E-rosettes in MLR. Exposure of the cells recovered in MLR to specific anti-T sera inhibited the formation of both stable and regular E-rosettes. Exposure of the cells recovered in MLR to anti-Ig serum had no effect on the formation of regular rosettes. Anti-Ig serum strongly inhibited the formation of stable E-rosettes by cells grown in medium containing human serum, but had no effect on the formation of stable E-rossettes by cells grown in either serum-free medium or in serum containing fetal calf serum. It is concluded that activated human T lymphocytes are characterized by their capacity to form stable E-rosettes, resistant to incubation at 37 degrees C, and by their capacity to acquire an immunoglobulin coat, possibly by binding immunoglobulin molecules present in their environment.     

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.     

Comparative study of the T- and B-cell immunity systems in human and animal embryogenesis

Data are presented on the spleen and thymus structure, time of appearance of the lymphocytes and their heterogeneity in the liver, thymus, spleen, lymph nodes and blood of human foetuses with hemochorial placenta (3 to 34 weeks) and of the minipigs foetuses with epitheliochorial placenta (32 to 95 days). In both foetuses the first T- and B-lymphocytes are found in liver, T-lymphocytes are then found in thymus and later in spleen and lymph nodes whereas B-lymphocytes are found, after liver, in spleen. Kinetics of T- and B-lymphocytes during embryogenesis is described. Reaction of the minipig lymphocytes to mitogens was demonstrated.     

Regulation of the cytotoxic effect of human ‘normal killer cells’ on tumor cell lines by neuraminidase-treated T-lymphocytes

Summary Subpopulations of peripheral blood lymhocytes (PBL) from healthy individuals were separated according to their capacity to form various rosettes and tested for their cytotoxic activity on cell lines of urinary bladder and breast carcinomas. The subpopulation exerting the highest natural cytotoxic activity was characterized by the presence of cell surface Fc-receptors and by the lack of receptors for sheep red blood cells and for C'3 on their surface. Treatment with vibrio cholera neuraminidase (VCN) increased the cytotoxicity of unseparated PBL to a level twice as high as that of untreated PBL. The attachment of T-lymphocytes to tumor monolayers was increased several fold after VCN-treatment, while the attachment of other lymphocyte subpopulations was not. Evidence is presented that the augmentation of the cytotoxicity of PBL following VCN-treatment results from the interaction of VCN-treated T-lymphocytes, attached to target cells, with normal killer cells. It is suggested that augmentation of the activity of killer cells by T-lymphocytes may play a role in antitumor defense mechanisms.Abbreviations CMC Cell-mediated cytolysis - E-rosettes Rosettes formed with sheep red blood cells - EA-rosettes Rosettes formed with red blood cells coated with antibody - EAC'-rosettes Rosettes formed with red blood cells coated with antibody and complement - FCS Heat inactivated fetal calf serum - PBL Peripheral blood lymphocytes - RBC Red blood cells - RF-TAL E-rosette forming, target-attached lymphocytes - SRBC Sheep red blood cells - VCN Vibrio cholera neuraminidase     

Subpopulations of human thymus cells differing in their capacity to form stable E-rosettes and in their immunologic reactivity.

The majority of human thymus cells from young donors form stable E-rosettes with sheep red blood cells (SRBC) that do not distintegrate after prolonged incubation at 37 degrees C. With advancing age the proportion of thymus cells forming such rosettes decreases gradually. The thymus of a patient receiving prednisone treatment was found to contain only a few cells that formed stable E-rosettes. The minor population of thymus cells that fails to form stable E-rosettes (non-rosetting or NR cells) was isolated and tested for its cell surface markers and immunologic reactivity in vitro. Most of the NR-cells were capable of forming regular E-rosettes with SRBC at room temperature. Like the majority of human thymus cells they were sensitive to the cytotoxic effect of normal constituted less than 0.2% of the original thymus cell suspensions, but about 1 to 3% of the NR-population. Thymus cells from donors over the age of 36 and from a prednisone-treated child responded in vitro to stimulation with either phytohemagglutinin (PHA) or concanavalin A (Con A). Unfractionated thymus cells from children up to the age of 14 failed to react to either PHA or Con A, but their NR-population responded vigorously to both lectins. In contrast to unfractionated thymus cell suspensions from children, the NR fraction showed a significant reactivity in mixed lymphocyte cultures with mitomycin-C treated allogeneic lymphocytes. It is concluded that like the thymus of other species, the human thymus contains a minor population of cortisone-resistant cells endowed with many of the immunologic properties characteristic for periperal T lymphocytes.     

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.     

Human, guinea pig and rat lympocyte rosette formation with spermatozoids]

In the thymus, spleen and bone marrow of adult guinea pigs, 14--30-week human fetuses, and the peripheral blood of sterile men there were found cells capable of forming the rosettes with homo- or heterologous spermatozoa (RFC). Development of autoimmune orchitis after the trauma of the rat testis or after the guinea pig immunization with the testicular homogenate mixed with complete Freund's adjuvant caused the appearance of RFC with spermatozoa in the thymus and the spleen of rats, and an increase of their number in the lymphoid organs of guinea pigs. Such treatment did not influence the quantity of sheep-cell rosettes in the lymphoid organs of rats and guinea pigs. A possibility of using the detected capacity of animal and human lymphocytes to form spontaneous and immune rosettes with the spermatozoa to test the degree of lympocyte differentiation and their sensitisation to the spermatozoa antigens after the spermatogenesis distrubances of the autoimmune nature is discussed.     

The significance of varying SRBC/lymphocyte ratio in T cell rosette formation.

The incubation ratio of sheep red blood cells (SRBC) to lymphocytes is a critical factor in rosette formation, whereas the length of time SRBC and lymphocytes are incubated together does not significantly affect the percentage of lymphocytes forming rosettes. The graph obtained by plotting percentage of rosette formation against the ratio of SRBC to lymphocytes is similar to that resulting from the formation of bimolecular complexes. If rosette formation is analogous to formation of bimolecular complexes, maximal rosette formation occurs when the system is saturated, i.e., with excess SRBC, and is a measure of the total capacity of a lymphocyte population to form rosettes. In addition, the percentage of rosette formation observed at a limiting SRBC/lymphocyte ratio gives an indication of the avidity of the lymphocytes for SRBC. This interpretation may provide an explanation for the difference between the "active" and "total" rosettes. When the log of the SRBC/lymphocyte ratio is plotted against percentage of rosette formation, a straight line is obtained, suggesting that within a given normal lymphocyte sample, T cell subsets with different avidities are not detected by rosette formation at different SRBC/lymphocyte ratios.     

Proportion of lymphocytes forming E, EA, and EAC rosettes following treatment with human interferon preparations in vitro

The proportion of lymphocytes forming E, EA, and EAC rosettes after treatment with human interferon preparations in vitro was measured. While interferon increased the percentage of lymphocytes forming E rosettes, the percentage of cells forming EA rosettes was diminished. The proportion of lymphocytes forming EAC rosettes was not altered to any major extent by interferon treatment. The same effects were observed when fibroblast interferon, purified to homogeneity with regard to molecular weight, was used.     

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.     

Bioassay for thymic extracts: guinea pig spleen lymphocytes-rabbit red blood cells rosette method

An assay is described for assessing the potency of thymic extracts by the capacity of guinea pig spleen lymphocytes to form rosettes with rabbit red blood cells. Its sensitivity and reproducibility have been evaluated by statistical analysis of the data obtained testing different batches of the calf thymus extract TP-1.     

Differentiation of the palatine tonsillar tissues of the human fetus]

Differentiation of epithelial and lymphoid tissues of the palatine tonsils was studied in human embryos at the age of 8-34 weeks of development by means of histochemical, immunomorphological and morphometric methods. The anlage of the palatine tonsils appears at the age of 9 weeks of fetal development. At the age of 13-14 weeks of fetal development the tonsil suspension contains 2 subpopulations of lymphocytes possessing properties of T-cells differing in the ability of their superficial receptors to interact with sheep erythrocyte antigens forming rosettes (RFC--rosette forming cells) and with antigens of their own erithrocytes (autoRFC). The number both increases sharply by the 16th week of gestation. Simultaneously, essential alterations are noted in epithelial and lymphoid tissues. In epithelium of crypts cornified cells appear; the amount of lymphoid tissue increases sharply, primary follicles without reactive centers appear, lymphocytic infiltration of epithelium occurs. The amount of RFC does not change considerably, and the amount of autoRFC has a tendency towards some increase. From the data obtained, it is possible to suggest that human palatine tonsils already at embryonic period participate in functioning of immunogenic organs and in maintaining of immunologic homeostasis of the fetal organism.     

Ultrastructure and antigens in differentiation of thymus lymphocytes in human embryogenesis]

Thymus lymphocytes of 7--8-week human embryos have nuclei of irregular form with 1--3 distinct nucleoli characterized by absence of compact chromatin or heterchromatin. The electron-dense cytoplasm of these cells contains polysomes and an insignificant number of mitochondria. No receptors to sheep red blood cells and T antigen are revealed on their surface. In 11--12-week human embryos one can observe a decrease in the size of thymus lymphocytes, appearance of heterochromatin in their nuclei and receptors to sheep red blood cells (79%), and T antigen (60%) on the cell surface. Subsequently the quantity of compact chromatin in thymus lymphoid cells increases, and the cells acquire definitive properties and structure.     

Ia-positive macrophages bind and internalize viable lymphocytes in murine thymus

Macrophages have been shown to be present in thymus throughout its development. In the present study monoclonal and polyclonal antimacrophage reagents were used to identify, quantitate, and determine the distribution of thymic macrophages. Those studies demonstrated that significant numbers of macrophages were evenly distributed throughout the cortex and medulla, and that macrophages account for most, if not all, Ia positivity in murine thymus. Suspensions of thymic cells prepared by enzyme digestion contained 2-4% macrophage antigen-positive cells, over 95% of which were I-Ak positive in double-labeling studies. Removal of lymphocytes and macrophages left only epithelial cells and those failed to label for Ia. Subsequent to mild enzymatic digestion, up to 80% of the thymic macrophages were in the form of lymphocyte/macrophage rosettes. Morphologic evaluation of the thymocyte rosettes revealed that some of the macrophages contained internalized lymphocytes. The proportion of macrophages with internalized lymphocytes generally was less than 10%, but during the first 4 weeks of life values often approached 30%. Nurse cells, which were shown through double labeling to express both Ia and macrophage-associated antigen, were included in the population of rosetted cells which had internalized lymphocytes. The results demonstrated that there is a high level of interaction between lymphocytes and Ia-positive macrophages in the thymus which is greatest during the immediate postnatal period.     

A ganglioside receptor on lymphocytes mediates recognition of self

Lymphocytes from mouse spleen and thymus form spontaneous rosettes with autologous erythrocytes. Certain gangliosides are described here as potent inhibitors of this cell contact formation. A comparison of several gangliosides shows that lymphocytes from thymus and spleen differ significantly in their reactivity towards GM1, GM3, GD1a, GD1b and GT1b. Differences are also seen between inbred mouse strains.     

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.     

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.     

In vitro augmentation of lymphocyte sheep cell rosette formation by leukocyte dialysates.

We have studied in vitro the effects of leukocyte dialysates containing transfer factor on the formation of sheep cell rosettes by human lymphocytes. Dialysate had no effect on the toral rosettes, but increased the number of rapidly forming ("active") rosettes. This was due to an increased affinity of the lymphocytes for the sheep red cell. Trypsin-treated lymphocytes regained the ability to form rosettes more rapidly when cultured with leukocyte dialysates than with control media. These experiments suggest that leukocyte dialysate acts to increase the number or arrangement of sheep cell receptors on the lymphocyte surface.     

A Rapid Method for Determining T Lymphocyte Levels Using Acid Alpha-Naphthyl Acetate Esterase

A method for rapidly determining levels of T lymphocytes in humans is presented. The method involves staining lymphocytes in peripheral blood or buffy coat smears for acid alpha-naphthylacetate esterase for 1 hr at 37 C. After counterstaining with Wright's stain, the percentage of esterase positive cells is quantitated microscopically. The entire procedure takes less than 2 hours. Data are presented which show that the percentage of esterase-positive lymphocytes accurately reflects the percentage of lymphocytes forming E rosettes with sheep erythrocytes.