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.     

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.     

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     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.

A two-step centrifugation procedure has been developed to isolate greater quantities of highly purified sheep erythrocyte antigen-binding cells (ABC) than previously possible. The first step involves partially separating sheep erythrocyte rosettes from unrosetted lymphocytes by their difference in buoyant density on Ficoll-Hypaque. Subsequent passage through a linear 5 to 10% Ficoll gradient produces further purification of rosettes by sedimentation velocity. Approximately 4.5 X 10(6) ABC can be obtained at 50 to 100% purity from 10(9) immune spleen cells (5 days post-immunization) and 1 X 10(5) ABC at 20 to 40% purity from 10(9) nonimmune spleen cells. The purified ABC from 5-day immune animals are 80 to 90% B cells and 10 to 20% T cells, and represent between 30 and 40% of the original ABC in the spleen cell population. Less than 0.2% of the purified ABC are plaque-forming cells (PFC) and less than 2% have intracellular immunoglobulin (Ig) or J chain. The quantities of ABC obtained are sufficient for investigating biochemical parameters of antigen-induced lymphocyte activation and for direct analysis of the surface isotypes found on antigen-binding cells after immunization.     

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.     

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.     

IgM complex receptors on subpopulations of murine lymphocytes.

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5mu but not by Fab mu. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.     

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.     

Characteristics of spontaneous rosette formation by mouse lymphocytes with autologous erythrocytes

A substantial proportion of mouse spleen and thymus cells form spontaneous rosettes with syngeneic and allogeneic erythrocytes. Rosette formation is independent of the metabolic state of the cell: Inhibitors of ATP formation (NaCN, NaN₃) and of cell motility (cytochalasin B, colchicine, vinblastine) have little or no effect. Erythrocytes can be bound by living or dead lymphocytes. The stability of rosettes against high temperature and shearing forces is weaker than that of immune rosettes. The increase or decrease of electrostatic repulsion between cells will enhance or reduce rosette formation. Membrane proteins are involved in cell-to-cell contacts, since several proteases destroy relevant structures on the cell surfaces of lymphocytes, erythrocytes, 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.     

DIFFERENTIATION OF ANTIBODY-FORMING CELLS IN TOAD SPLEEN : A Study Using Density and Sedimentation Velocity Cell Separation

Antibody-forming cells (AFC), developing in toad spleen after stimulation with polymerized flagellin, were studied with an immune adherence assay. Differentiation was followed by several parameters: thymidine uptake to monitor dividing cells; equilibrium density centrifugation in albumin gradients to monitor cell density; microscopic measurements and sedimentation velocity separation to monitor cell size; stained preparations to follow cell morphology. Almost all AFC observed early in the response were dividing cells; the proportion of dividing AFC dropped to 4% 2 wk after stimulation. The earliest AFC detected (3 days) formed a relatively homogeneous light density population, and were purified 17-fold by equilibrium density centrifugation. As the response developed, additional denser peaks were found, so that late in the response dense AFC predominated. Dividing AFC were confined to the light density region throughout the response. Cell diameter measurements revealed that the earliest AFC were all very large cells. In a manner analogous to the density changes, smaller AFC appeared as the response developed until they finally comprised the majority of the AFC population. Dividing AFC were always relatively large, but encompassed a wide range of sizes. Sedimentation velocity separation was employed in a closer study of the immature AFC; they were purified 140-fold by this procedure. The earliest AFC consisted of several readily separable size populations in the range 9–18 µ diameter. The presence of separate peaks related by factors of two in volume suggested that the largest cells undergo a series of halving divisions before entering a division growth cycle. The results suggest an AFC differentiation sequence from a very large, light density, dividing "blast" cell to a nondividing cell with the size, density, and morphological appearance of a small lymphocyte. Stages of this sequence can be defined and selected out for investigation, using sedimentation velocity and equilibrium density centrifugation as complementary techniques.     

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.     

Control of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony-forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells

An enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll-Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS-D-chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies. The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D⁺ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D⁺ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non-rosette-forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D⁺ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.     

A separation chamber to sort cells,nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm² and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.     

Enumeration and isolation of rabbit T and B lymphocytes by using antibody-coated erythrocytes.

Rosette formation with antibody-coated erythrocytes (Ab-E) was employed for the enumeration and isolation of rabbit B cells (Ig+T-) and T cells (Ig-T+). The cells bearing surface Ig (Ig+ cells) were enumerated by a direct immunocytoadhesion technique utilizing anti-rabbit IgG antibody-coated erythrocytes (Ab-E). To enumerate cells bearing thymus cell antigen (T+ cells), an indirect rosette technique was used in which lymphocytes were first sensitized with guinea pig anti-rabbit thymus cell antiserum and then rosetted with anti-guinea pig IgG Ab-E. To demonstrate the specificity of the anti-thymus cell antiserum, a 51Cr radioimmunoassay for counting rosettes was employed along with visual counting to enumerate Ig+ and T+ cells in lymph node cell populations. When Ig+ and T+ lymph node cells were rosetted simultaneously with sheep and human erythrocytes, no mixed rosettes (less than 1%) were observed. Ficoll-Hypaque gradient centrifugation was used to obtain purified Ig+T- and Ig-T+ cells by removing rosetted T+ and Ig+ cells, respectively. The purity of isolated Ig-T+ cells was indicated by 94 to 95% indirect rosetting with anti-thymus cell antiserum and by 0 to 3% direct rosetting with anti-rabbit IgG Ab-E. The purity of isolated Ig+T- cells was indicated by 90 t0 94% direct rosetting with anti-rabbit IgG Ab-E and by 2 to 3% indirect rosetting with anti-thymus cell antiserum. The percentage of Ig+T- and Ig-T+ cells were determined in peripheral blood and in various lymphoid organs. The isolated Ig+T- and Ig-T+ cells were also characterized by their responses to mitogens. Thus, nearly pure Ig+T- and Ig-T+ cells were isolated by "negative selection," which should minimize functional changes of the cells, and thereby facilitate the study of their biologic properties, e.g., their response to mitogens.     

Melanoma-associated immunosuppression through B cell activation of suppressor T cells.

Using normal human lymphocytes isolated by sedimentation and cotton column adherence, we have developed a reliable assay of immunosuppression of PHA-induced blastogenesis by serum from selected melanoma patients. These lymphocyte cultures contained both responder cells (subpopulation x) and regulator cells (subpopulation y). Lymphocytes isolated by gradient centrifugation on sodium metrizoate-Ficoll contained responder cells (x) but no regulator cells (y). Cultures of lymphocytes isolated by this method were stimulated by PHA but were not suppressed by the addition of melanoma serum. When lymphocytes were isolated by a cotton column adherence/Lymphoprep centrifugation-double separation, subpopulations (x) and (y) were isolated. We have established that both subpopulations are necessary for suppression to occur, and that (y) operates as the regulator of (x). Finally, by manipulating B cell and T cell populations isolated by nylon column adherence or AET rosette separation, we have determined that the regulator ability of subpopulation (y) is the result of B cell activation of suppressor T cells.     

Migration inhibitory factor (MIF) and proliferative responses by human lymphocyte subpopulations separated by sheep erythrocyte rosette formation.

Human peripheral blood lymphocytes were separated by a combination of rosette formation with sheep erythrocytes and differential density centrifugation into subpopulations of rosette positive (T-enriched) cells and rosette negative (T depleted) cells. These were then tested in vitro for the production of macrophage migration inhibitory factor (MIF) and for incorporation of ³H-thymidine in response to specific antigens. Both T enriched and T depleted cell populations produced MIF but only T enriched cells exhibited significant antigen-induced ³H-thymidine incorporation. These findings using a T cell surface marker as the basis for cell separation, a technique which should not alter the B cell surface, confirm an earlier report in which human cells were separated on the basis of surface immunoglobulin, a B cell marker.     

人和猴T淋巴细胞表面TRBC受体及其配体不同于E2分子和CD2的配体

CD2 (E receptor, LFA-3 receptor) and E2 molecules (Bernard, 1988) on human T lymphocytes, CD58 (LFA-3, lymphocyte function associated antigen 3) on human erythrocytes and S14,S42,S110-220 molecules (Bernard, 1987) of sheep erythrocytes are involved in rosette formation of human T lymphocytes with human or sheep erythrocytes. Rosette formation of human and macaque pan-T lymphocytes with tree shrew (Tupaia belangeri) red blood cells (TRBC) (TRBC rosette) has shown different physicochemical properties from that of rosette formation with sheep red blood cells (E rosette) (Ben, 1985). CD2, CD3/TCR complex, CD5, CD6, and CD7 are not involved in TRBC rosette formation (Zheng, 1990). In order to know whether E2, LFA-3,S14,S42 and S110-220 molecules are involved in TRBC rosette formation or human and macaque T lymphocytes, rosette inhibition and antigenic modulation or co-modulation were performed with relevant monoclonal antibodies (McAbs), and hemolytic assay and slide agglutination were also conducted. TRBC rosette formation of human and rhesus monkey PBL was not blocked by E2 McAb (inhibition rate 2.8% and 2.1%, respectively). In contrast, human E rosette formation was obviously blocked at inhibition rate of 49.8% and macaque E rosette formation was slightly inhibited (13.3%). The modulation or co-modulation of E2 molecule with E2 McAb did not affect human TRBC rosette formation. Similar results were shown in rosette formation inhibition of Jurkat cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphometric ultrastructural evaluation of human peripheral blood T lymphocyte subpopulations isolated on the basis of their affinity for sheep red blood cells

The present investigation were undertaken to compare on the basis of ultrastructural morphometric analysis human T lymphocyte subfractions forming rosettes with sheep red blood cells (SRBC). The following T lymphocyte subfractions were obtained: ARFC (active rosettes), T1RFC (rosettes after 1 h at 4 degrees C) and T2RFC (rosettes after 2 h at 4 degrees C). Analysis of the mean cell and nuclei volumes of lymphocyte subfractions led to the separation of two cell groups differing in volume in each donor: a cell group consisting of large cells (T1RFC) and group of small cells (ARFC and T2RFC).