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.     

Loss of net negative surface charge during MLC stimulation of human T lymphocytes: correlation to "stable" E-rosette formation and natural attachment to normal and malignant target cells.

Activation of human blood T lymphocytes in mixed lymphocyte culture (MLC) causes a reduction in the net negative surface charge, as indicated by the reduction in the electrophoretic mobility. Concomitantly, the activated cells acquire new properties, including the ability to form “stable” E rosettes, and attach to normal and malignant cells of the same species (natural attachment (NA)). These properties were found to be expressed by lymphocytes within the low electrophoretic fractions (cells with low negative charge) of the MLC populations. The formation of stable E rosettes and natural attachment capacities of human thymocytes were also found to correlate with the amount of surface negative charge. The slowly migrating (less negative charged) cortical thymocytes, reported earlier as being able to form stable E rosettes, were found also to exhibit NA activity. Medullary thymocytes carrying a high net negative surface charge lack these characteristics. We consider it likely that the reduction of negative charge during activation of peripheral T cells, facilitates cell-to-cell contacts, and thus prepares the (activated) cells to perform cooperative interactions with other cell types, and express the lytic activity of T cells.     

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.     

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.     

Human T and B lymphocyte rosette tests: Effect of enzymatic modification of sheep erythrocytes (E) and the specificity of neuraminidase-treated E

Sheep erythrocytes (E) were treated with papain or neuraminidase to evaluate what effect these enzymes would have on the E rosette test for human T lymphocytes. Few or no rosettes were detected with sheep erythrocytes treated with papain (E_P). Sensitization of E_P with rabbit antibody and mouse complement resulted in a rosette indicator (E_PAC) which could be used to detect peripheral blood complement receptor lymphocytes (CRL) and thymocyte CRL without having to perform the rosette assay at 37 °C. Neuraminidase treatment of E (E_N) enabled the detection of approximately 20% more rosette-forming cells (RFC) in human peripheral blood lymphocyte (PBL) suspensions compared to untreated E. Rosette studies on a patient with severe combined immunodeficiency disease and on human lymphoblastoid cell lines showed that the additional rosettes detected with E_N were T lymphocytes.     

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     

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.     

Rapidly expanded activated human killer cell clones have strong antitumor cell activity and have the surface phenotype of either T gamma, T-non-gamma, or null cells

Cloned lymphoid cell lines showing cytolytic activity were derived from natural killer (NK) cell-enriched cell fractions obtained by fluorescence-activated cell sorting of cells that reacted with B73 .1, an NK cell-specific monoclonal antibody (MCA). The clones were cultured for more than 30 generations (i.e., more than 10(9) descendants from a single cell). The rapid expansion was achieved by using a special culture system developed for this purpose and based on the use of two types of allogeneic feeder cells. Three phenotypically different types of cytotoxic clones were obtained. These clones showed a broad spectrum of cytolytic activity against several NK-susceptible and NK-nonsusceptible tumor target cells. One of these clones had the following binding pattern to MCA: B73 .1+, T3-, T4-, T8-, HNK1 -, and Lyt-3-. These cells formed rosettes with IgG-coated erythrocytes but not with sheep erythrocytes, and therefore might be null cell-derived. Most of the cytotoxic clones showed the following phenotype: B73 .1+, T3-, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma +. These clones were probably derived from T-gamma cells. In addition, one clone with cytolytic activity was derived from B73 .1- cells. This had the phenotype B73 .1-, T3+, T4-, T8-, HNK1 -, Lyt-3+, E+, and EA-gamma-, and may be of T-non-gamma cell origin. About 10 noncytolytic clones showed the phenotype B73 .1-, T3+, T4, or T8+, HNK1 -, Lyt-3+, Ia+, E+, and EA-gamma -. An absolute correlation was found between the presence of the B73 .1 antigen, the absence of the T3 marker, and the capacity of the cells to form EA rosettes. Furthermore, all clones except one (Lyt-3-) formed E rosettes. Although the in vitro life span varied from clone to clone, B73 .1- clones generally grew faster and for longer times (greater than or equal to 50 generations) than did B73 .1+ ones (less than or equal to 40 generations). The cytolytic activity, cell surface phenotype as determined with MCA, rosette formation, and target cell specificity spectrum remained stable over the entire culture period. We conclude that the majority of the activated MHC-nonrestricted cytolytic clones obtained in this culture system show a particular phenotype. These cells can be expanded to large numbers. Whether or not these clones might be derived from B73 .1+, HNK1 + NK cells with the morphologic appearance of large granular lymphocytes will be discussed.     

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     

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 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.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

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.     

Human lymphocyte cytotoxicity against mumps virus-infected target cells. Requirement for non-T cells.

Subpopulations of human lymphocytes were tested for their capacity to kill mumps virus-infected target cells in a 51-chromium release asaay. Using two different cell fractionation techniques, lymphocytes were fractionated into T cell-enriched (primarily T cells) and T cell-depleted (primarily B cells) subpopulations. Filtration of lymphocytes through columns coated with human immunoglobulin and rabbit anti-human-immunoglobulin (Ig-anti-Ig) rendered the resulting T-cell preparation inactive as effector cells against target cells carrying mumps virus antigens. In the second technique, lymphocytes were fractionated by centrifugation into two fractions according to their ability to form spontaneous rosettes with sheep erythrocytes (E). The E-rosette-forming population (primarily T cells) was shown to lack cytotoxic activity against mumps virus-infected target cells. This activity was present in the nonrosetting population. The results suggest that the effector cells involved in this cytotoxic system are of a non-T variety.     

Low affinity E-rosette formation by the human K cell.

Human T lymphocytes were separated into two subsets on the basis of relative affinity for sheep red blood cells (E), and these T cell fractions were examined for cytotoxic reactivity against antibody-sensitized Change liver cells (ADCC). High affinity E-rosette-forming cells (E-RFC) (55 +/- 6% of peripheral blood mononuclear cells), capable of rosette formation despite elevated temperatures of incubation (29 degrees C) and a limited concentration of E, contained few antibody-dependent cytotoxic cells (K cells). In contrast, low affinity E-RFC (23+/-7% of mononuclear cell suspensions) requiring cool temperatures of incubation (4 degrees C) and an excess of E to form rosettes, were highly enriched for ADCC activity. The majority of K cells exhibited low affinity interactions with E. T cells in thymus, tonsil, and lymph node formed high affinity E-rosettes and exhibited little reactivity in ADCC. Only peripheral blood and spleen contained easily identified low affinity E-RFC and anti-body-dependent cytotoxic cells. The proportion of low affinity E-RFC in the peripheral blood of normal subjects correlated closely with reactivity in ADCC, making it possible to predict cytotoxic potential for the E-rosette pattern. These data indicate that the human K cell may belong to a previously unappreciated but functionally important subset of thymic dependent mononuclear cells.     

Spontaneous adherence and rosette formation of lymphocytes to Leydig cells: an in vitro technique

A spontaneous rosette phenomenon occurs in vitro when suspensions of mouse testicular cells are mixed with suspensions of lymphocytes. The core cell of these rosettes are the Leydig cells, and the coronas are formed by lymphocytes. No other testicular cell, either seminal or Sertoli, participates in lymphocyte binding.Autogeneic, syngeneic, allogeneic, and xenogeneic lymphocytes from thymus, spleen, and peripheral blood similarly react with mouse Leydig cells. Lymphocytes from spleens of athymic nude mice and from chicken bursa also give Leydig rosettes. It is suggested that mouse Leydig cells carry membrane receptors spontaneously recognized by lymphocytes.     

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.     

Separation of human lymphocyte subpopulations. I. Transformation characteristics of rosette-forming cells

Lymphocytes were isolated from human peripheral blood by carbonyl-iron treatment and Ficoll-Isopaque centrifugation. The lymphocytes were allowed to form rosettes with sheep erythrocytes, either uncoated (E) or coated with antibody and complement (EAC).In 32 experiments E rosettes were separated from free lymphocytes on a Ficoll density gradient. Thus, depleted (non-E) and enriched (E) fractions were obtained. It was found that in the original suspension 24 ± 7.2% of the lymphocytes formed rosettes with EAC and 56 ± 8% with E. In fraction non-E these values were 56 ± 11.4 and 22 ± 8.9%, respectively; in fraction E 8 ± 3.8 and 79 ± 8.8%. Moreover, the percentages of Ig-bearing cells among the fractions were found to follow closely those of CRL.In a series of lymphocyte culture experiments these fractions were compared with the original suspension and a control suspension (rosetted, not separated), as well as with a recombined population (non-E + E). It was found that fraction non-E showed an increased response to PHA and PWM, and an enhanced MLC stimulatory capacity, whereas fraction E was decreased in these respects. Moreover, fraction E displayed significantly reduced spontaneous DNA synthesis.It is concluded that the responses to PHA or PWM, as well as the capacity to stimulate allogeneic cells, are not solely dependent on the cells forming rosettes with E.     

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.     

Erythrocyte rosettes provide an analogue for Schiff base formation in specific T cell activation

Human T cells spontaneously bind sheep E and this reflects physiologic interactions between specific adhesion molecules, principally T cell CD2, and the sheep equivalent of LFA-3. This interaction is important in T cell adhesion and in transmission of accessory activational signals. In this respect, E rosettes provide a partial analogue for T cell:accessory cell interaction and rosetting induces functional alterations in T cells. In studies of Ag-dependent T cell activation, we have obtained evidence that the formation of covalent Schiff bases between ligands on APC and T cell is an essential element. In our study, the specific chemical criteria defining Schiff base formation were applied to T cell E rosettes formed at room temperature, as follows: 1) Prior formation of Schiff bases on T cell epsilon-amino groups by glutaraldehyde inhibited E rosette formation. 2) Rosette formation was inhibited in the presence of exogenous lysine. 3) Reduction of constitutive T cell aldehydes by NaBH4 inhibited subsequent E rosette formation. In response to these chemical modifications of cellular ligands, T cell E rosette formation and T cell inductive interaction with APC were affected in the same way. 4) Oxidation of NaBH4-treated T cells by NaIO4 or galactose oxidase to regenerate cell-surface aldehydes on N-acetylneuraminic acid or galactose residues respectively, consistently restored E rosette formation. 5) Conversion of reversible Schiff bases to irreversible secondary amines by NaCNBH3 stabilized E rosettes against mechanical disruption. Together, these data demonstrate that E rosettes provide an analogue for the Schiff base-forming reactions that are essential in specific T cell activation.