A human lymphoid cell line with receptors for both sheep red blood cells and complement

The human lymphoid cell line MOLT 4, from a patient with acute lymphocytic leukemia, was initially considered to be derived from T lymphocytes, on the basis of rosette formation with sheep erythrocytes (E). This cell line has now also been found to form rosettes with sheep erythrocytes sensitized with rabbit antibody and mouse complement (EAC). Evidence is presented that the formation of both E and EAC rosettes is due to two separate receptors on the MOLT cells: (a) EAC rosettes were formed more rapidly and were more stable than E rosettes; (b) preincubation of MOLT with an EAC membrane preparation inhibited resetting with EAC and not with E; (c) MOLT formed rosettes with EAC prepared from trypsinized E, but did not bind to trypsin-treated E alone. The implications of this finding, in regard to the derivation of this cell line, are discussed.     

Immunoglobulin-positive mononuclear cells in human peripheral blood: detection by mixed anti-globulin and direct anti-globulin-rosetting reactions.

Ig-bearing mononuclear cells were identified in Ficoll-Hypaque preparations of human peripheral blood by using mixed anti-globulin (MAG) and direct anti-globulin rosettes; indicator cells consisted of sheep erythrocytes coated with human F(ab')2 or anti-F(ab')2 antibody, respectively. Of the cell population isolated from 10 normal subjects, a mean of 68% was lymphocytes. However, fewer than 50% of the cells with detectable surface Ig were lymphocytes. On viable cell preparations using chromic chloride-treated sheep erythrocytes (CrCl3SRBC) coated with anti-F(ab')2 antibody, a mean of 20.1% of the lymphocytes formed rosettes, i.e., were B. Up to 6% of peripheral blood lymphocytes formed mixed Ig-rosettes and E-rosettes. On viable lymphocytes using F(ab')2-coated CrCl3SRBC, MAG rosettes were insensitive in detection of B lymphocytes. Formaldehyde treatment of lymphocytes increased the number of B cells detectable to 25.5% of the lymphocyte population. Study of T-enriched and B-enriched populations showed that the observed increase in B cell reactivity was real and not due to MAG-rosetting T cells. A one-stage procedure for T and B lymphocyte separation is described.     

Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes     

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.     

Morphological differences in the interactions between human mononuclear cells and coated or uncoated sheep red blood cells

Summary Enriched preparations of E, EA and EAC rosettes formed by human peripheral blood mononuclear cells were freeze-etched and examined electron-microscopically. In E rosettes only lymphocytes were involved, whereas in EA and EAC rosettes lymphocytes and mononuclear phagocytes participated as rosette-forming cells. In EA and EAC rosettes, cytoplasmic extensions of the rosette forming cell were seen to penetrate the sheep red blood cell, whereas E rosettes showed a broad zone of adherence without penetration. None of the three types of rosettes showed an interspace between the membranes. Unlike E rosettes, EA and EAC rosettes showed polarity in the adherence of sheep red blood cells. These observations made by freeze-etch electron microscopy indicate distinct morphological differences between rosettes formed with coated or uncoated erythrocytes.The authors wish to thank Prof. Dr. A. Cats, Dr. P.C.J. van Breda Vriesman and Dr. J.C.H. de Man for helpful discussion and criticism; the assistance of Miss R. Kleinjan and Mrs. A.C. Scheurkogel-van Efferen is gratefully acknowledged. This work was supported in part by a grant of the Praeventiefonds     

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.     

Interleukin 1 activity in normal human urine

Human leukocyte dialysates contain components capable of amplifying cutaneous delayed-type hypersensitivity (DTH) reactions. In the present study, two such amplifiers, both less than 3500 m.w., were partially purified from human leukocyte dialysates by gel filtration on Sephadex G-10 followed by high pressure reverse-phase liquid chromatography. These amplifiers of DTH were examined for their effects on production of the migration inhibitory lymphokines leukocyte migration inhibition factor (LIF) and macrophage migration inhibition factor (MIF). The amplifiers were found to increase LIF and MIF production by antigen- or alloantigen-stimulated human peripheral blood lymphocytes in a dose-dependent fashion. Further analysis demonstrated that although antigen-stimulated T4 and T8 cell subpopulations could produce LIF activity under the assay conditions employed, amplification of lymphokine production by modulator was only observed with the T4 subset.     

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.     

Lymphocyte populations of Callithrix jacchus marmosets.

A lymphocyte population of common marmosets (Callithrix jacchus) was identified by rosette formation with African green monkey erythrocytes; the rosette-forming cells appeared to be T lymphocytes, as approximately 62% of circulating lymphocytes and 85% of thymus cells formed rosettes with African green monkey erythrocytes. In addition, common marmoset lymphoid cells carrying T-lymphotropic Herpesvirus saimiri or Herpesvirus ateles formed rosettes with African green monkey erythrocytes and treatment of common marmoset circulating lymphocytes with an anti-T cell serum and complement (C′) eliminated rosette-forming cells. Common marmoset T lymphocytes apparently carry a surface receptor for African green monkey erythrocytes, but unlike humans and other closely related nonhuman primates, T lymphocytes of common marmosets fail to form rosettes with sheep erythrocytes.     

Numbers of rosette formine cells in human peripheral blood.

Thymus-derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood were quantitated by assaying percentages of cells forming erythrocyte rosettes. T-cell rosettes were formed with neuraminidase treated sheep erythrocytes. B-cell rosettes were formed with complement coated sheep erythrocytes. Large differences in the percentages of T-rosette forming cells were noted depending on the method used to assay these cells. When rosette forming cells (RFC) and non-RFC were counted concurrently the percentage of T-cell rosettes were 53–75% whereas methods involving the separate counting of RFC and total cells gave T-cell RFC percentages of 23–40%. These differences were due to the “co-rosetting” of non-RFC into the T-cell rosette clusters. This occurred because of the gentleness required to resuspend the fragile T-cell rosettes. “Co-rosetting” was demonstrated by forming stable complement receptor rosettes with complement-coated human erythrocytes and resuspending them either gently or vigorously. Significantly higher percentages of rosettes were noted with gentle cell suspension than with vigorous resuspension. The percentages of rosette forming T-cells in human peripheral blood are therefore lower than previously estimated.     

The ligand of the erythrocyte receptor of T lymphocytes: expression on white blood cells and possible involvement in T cell activation

We have recently described a monoclonal antibody (mAb) to sheep erythrocytes, termed L180/1, that blocks the formation of E rosettes between human or sheep T lymphocytes and sheep red blood cells. The cell surface glycoprotein (GP) of 42,000 apparent m.w. recognized by mAb L180/1 was given the preliminary name T11 target structure or T11TS. In the present report, it is shown that T11TS is also expressed on sheep white blood cells, notably on activated T lymphocytes, that are shown to actively synthesize this cell surface GP. In addition, the mixed lymphocyte reaction between outbred sheep is inhibited by mAb L180/1 at an early stage of the response. Together with the known involvement of the E receptor in T lymphocyte activation, these results are taken to suggest that T11 and T11TS are complementary cell interaction molecules involved in regulating T cell proliferation.     

Acute lymphoblastic leukaemia: a heterogenous disease.

By using several techniques to detect surface markers on T and B lymphocytes, 11 cases of acute lymphoblastic leukaemia (A.L.L.) were studied. In four cases an insignificant number of markers were detected on the lymphoblast populations. In one case a significant number of blasts formed both sheep red blood cell rosettes and Fc rosettes, suggesting a T-cell origin for the neoplastic cells, and in another case the presence of Fc and C3 receptors on the lymphoblast population indicated a B-cell origin. In a further five cases 14-43% of the blasts had detectable surface immunoglobulin. It is concluded that A.L.L. is a heterogeneous disorder, some cases failing to express surface markers and others having either a T-or a B-lymphocyte origin or both.     

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.     

Peripheral blood lymphocyte subpopulations in sarcoidosis.

We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sarcoidosis and 15 healthy controls. T cells were estimated from the number of lymphocytes forming rosettes in vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreased cellular immunity and increased reactivity of the antibody-forming system as commonly seen in sarcoidosis.     

Rapid magnetic purification of rosette-forming lymphocytes.

High gradient magnetic separation, which as previously been shown effective in extracting erythrocytes from a flowing cell suspension, has been used to separate rosetted and unrosetted human peripheral blood lymphocytes. The hemoglobin in the sheep red cells used to form rosettes was first oxidizied to the paramagnetic methemoglobin form. Samples of 50 x 10(6) lymphocytes could be processed in 10 min under sterile conditions with greater than 90% purity of the rosetted cell fraction and maintenance of T cell function in mixed lymphocyte cultures.     

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.     

Early stages of human marrow lymphocyte differentiation: induction in vitro by thymopoietin and ubiquitin.

To study early stages of human lymphocyte differentiation, bone marrow cells were physically separated according to their density and size by gradient centrifugation and then velocity sedimentation. The isolated cell fractions were incubated with putative inducing agents and then assayed for their expression of an array of surface differentiation markers. The inducing agents used were two polypeptides, thymopoietin (Tp) and ubiquitin (Ub), and the cyclic nucleotide, dibutyril cyclic 3'5' adenosine monophosphate (cAMP). Tp, Ub, and cAMP each induced the ability to form sheep erythrocyte rosettes by small lymphocytes, which may thus represent T cell precursors. Ub and Tp induced rosette formation with mouse erythrocytes on lymphocytes of more heterogenous size, which may be "early" B cell precursors. Ub alone could induce surface IgM expression on small lymphocytes, which might be "late" B cell precursors. Both Tp and Ub induced Fc receptors on small lymphocytes. Complement receptors could not be induced on marrow lymphocytes by Tp, Ub, or cAMP. A number of lymphocyte precursors can thus be identified by their physical characteristics and their ability to respond to particular soluble factors with the expression of new differentiation markers.     

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.     

A method for simultaneous isolation and cryopreservation of bovine lymphocytes

A technique for the simultaneous isolation and cryopreservation of bovine lymphocytes was presented. Whole blood was slowly diluted 1:2 with RPMI-Hepes containing 15% Me₂SO to yield final concentrations of 50% whole blood and 7.5% Me₂SO. Aliquots were cooled to at least ?80 °C at a rate of 2.5 °C/min and subsequently immersed in liquid nitrogen for storage. Samples were thawed rapidly by agitation in a 37 °C water bath and diluted rapidly with warm RPMI-Hepes. After centrifugation, the lymphocyte pellet was washed and suspended in medium for cell identification and lymphocyte stimulation assays. Few red blood cells, granulocytes, and monocytes survived this freezing and thawing procedure. Recovery of lymphocytes was 69–75%, as compared to a recovery of 58–68% using isolation on density gradients. Only small differences in the numbers of lymphocytes that (i) form spontaneous rosettes with sheep red blood cells and (ii) bind anti-IgG were found between the two isolation procedures. Cell proliferation in response to PPD-B or PHA and the enhancement of amino acid transport in response to PPD-B were the same for lymphocytes isolated by both methods.     

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.