Human suppressor T cells induced by concanavalin A: suppressor T cells belong to distinctive T cell subclasses.

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A. Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

2H1--a novel antigen involved in T lymphocyte triggering

A mAb anti-2H1, was produced against PHA-activated T cells from a lower primate, Aotus trivirgatus. Anti-2H1 reacted with 90% of peripheral T cells but was found to react with only 10% of thymocytes and some, but not all, leukemic T cell lines. 2H1 expression was dramatically increased when thymic cells were activated by Con A plus PMA. In contrast, anti-2H1 did not react with B cells, macrophages, null cells, or hematopoietic stem cell lines. More importantly, anti-2H1 antibody can induce T cell activation and proliferation in synergy with PMA or anti-T11(3). SDS-PAGE analysis of polypeptides immunoprecipitated with anti-2H1 showed two major polypeptides of 140 and 105 kDa. Thus, the 2H1 Ag can be distinguished from T3, T11, and 9.3 Ag. These results indicate that the 2H1 Ag appears to be involved in the activation of T lymphocytes.     

Fc-receptor heterogeneity of human suppressor T cells.

Concanavalin A (Con A) stimulated the IgM-binding subpopulation of human T cells (TM) to suppress the pokeweed mitogen-induced differentiation of B lymphocytes to plasma cells. Control TM cells that had not been Con A activated did not suppress. The degree of suppression was related to the number of Con A-TM cells added to the cultures and it was abolished by irradiation of the T lymphocytes either before or after the 24-hr culture period with Con A. Suppression did not require the presence of TG cells, whose suppressor potential has been previously established. These findings indicate that suppressor activity is not confined to the TG subpopulation but may be expressed by TM cells also.     

Biosynthesis and surface expression of T8 by peripheral blood T4+ cells in vitro

Biosynthetic labeling with 35S-methionine and 35S-cysteine of isolated T4+ cells from Con A-activated T cells demonstrated that the T8 antigen was synthesized by activated T4+ cells. Two-color fluorescence analysis of the activated T cell population from which the T4+ fraction was obtained showed that both T4+T8- and T4+T8+ cells were present. The T8 antigen that was immunoprecipitated by monoclonal anti-T8 from activated T4+ cells migrated with an electrophoretic mobility corresponding to an m.w. of approximately 33,000, a previously reported m.w. value for T8 antigen. Con A activation of highly purified peripheral T4+T8- and T8+T4- subsets indicated that both T4+T8- and T8+T4- cells can give rise to T4+T8+ cells. However, substantial T4, T8 coexpression by T4+T8- cells required a signal from T8+T4- cells which could be supplied by incubating T4+T8- cells with irradiated T8+ cells or the supernatant from Con A-activated T8+T4- cells. The generation of T4+T8+ cells from a subset of T4+T8- T cells may be an important mechanism in immune activation and/or the further differentiation of peripheral T4+ cells.     

Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus.

The association of vesicular stomatitis virus proteins with intracellular and plasma membranes was examined by pulse and pulse-chase labeling of virus-infected HeLa cells with [35S]methionine and separation of cell homogenates into three major membrane fractions in discontinuous sucrose gradients. The glycoprotein G was primarily associated with rough endoplasmic reticulum-like membranes after short radioactive pulses (2 to 4 min) but accumulated in the plasma membrane-enriched fraction and the smooth internal membrane fraction with longer pulse or chase periods. The nucleocapsid protein N and the matrix protein M accumulated in the rough endoplasmic reticulum and plasma membrane-like fractions but not in the smooth internal membrane fraction. Only a fraction (35 to 40%) of the viral protein synthesized during a short pulse in the mid-cycle of infection was apparently utilized in released virus. The newly synthesized virus proteins first appeared in released virus in the order: M, N and L, and G.     

Protein Composition of the Cell Wall and Cytoplasmic Membrane of Escherichia coli

Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions. The fraction with the higher density was enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes, and the similar morphology of the fragments and intact cell walls. The less-dense fraction was enriched in vesicles derived from the cytoplasmic membrane, as indicated by the enrichment of cytochromes, the enzymes lactic and succinic dehydrogenase and nitrate reductase, and the morphological similarity of the vesicles to intact cytoplasmic membrane. Both fractions were rich in phospholipid. The protein composition was compared by mixing the cytoplasmic membrane-enriched fraction from a (3)H-labeled culture with the cell wall-enriched fraction from a (14)C-labeled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved; of these, 27 were increased two- to fourfold in the cytoplasmic membrane-enriched fraction, whereas 6 were similarly increased in the cell wall-enriched fraction. One of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope. This protein accounted for 70% of the total protein of the cell wall, and its occurrence in the envelope from spheroplasts suggests that it is a structural protein of the outer membranous component of the cell wall.     

Monoclonal antibodies, L-35 and L-36, define novel T cell activation antigens

Two novel T cell specific activation antigens were characterized and were defined by monoclonal antibodies developed against mitogen-stimulated human T cells. These antigens, designated as L-35 and L-36 were expressed on both the CD 4(Leu-3) and the CD 8(Leu-2) subsets of activated but not resting T cells. Moreover these antigens were not expressed on a number of T, B, and myeloid tumor cell lines. L-35 and L-36 were expressed on interleukin 2 (IL 2)-dependent cloned T cell lines, and were weakly expressed on the T cell tumor line, HSB-2. L-35 was expressed on granulocytes and a small subset of thymocytes. SDS-PAGE analysis of 125I-labeled lysates from phytohemagglutinin (PHA)-activated T cells demonstrated that L-35 existed as a complex of 32,000 and 97,000 dalton polypeptides under reducing and nonreducing conditions. Anti-L-36 immunoprecipitated a 90,000 dalton structure from PHA-activated cell lysates prepared with CHAPS detergent. When immunoprecipitates were analyzed from [35S]methionine labeled lysates, anti-L-35 precipitated only the 97,000 dalton component, suggesting that the 32,000 dalton subunit of L-35 complex was not synthesized by the activated cell population. Furthermore anti-L-35 did not immunoprecipitate a 32,000 dalton component from 125I-labeled lysates of anti-Leu-4 or Con A-activated cells, suggesting that the 32,000 dalton component of the L-35 complex may represent a subunit of PHA. The 32,000 dalton protein could not, however, be precipitated from cells incubated with PHA for less than 1 day. These results suggested that anti-L-35 recognizes a 97,000 dalton structure expressed on activated T cells, and that upon activation by PHA, becomes associated with a subunit of this mitogen.     

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 μU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristics responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.     

A novel glycoprotein expressed on sheep T and B lymphocytes is involved in a T cell activation pathway

We have produced a new mouse mAb that identifies a sheep T cell activation Ag. The mAb B5-5 is specific for low m.w. components on nearly all sheep thymocytes and peripheral T and B lymphocytes but does not label immature B cells in Peyer's patches or germinal centers. After cross-linking of target structures either directly by plastic-bound mAb or indirectly using anti-Ig reagents, peripheral T cells, but not thymocytes or peripheral B cells, were activated. IL-2 was secreted by T cells after cross-linking and activation was strongly augmented in the presence of PMA. The addition of soluble B5-5 mAb to mitogen-stimulated cultures of sheep lymphocytes resulted in a suppression of PHA responses and augmentation of PWM responses and had a variable effect on Con A responses but had no effect on LPS- or protein A-induced proliferation. When added to alloantigen-stimulated cultures, B5-5 augmented the proliferative response. The B5-5 membrane component consists of 14- to 19-kDa glycoproteins but the banding patterns obtained during SDS-PAGE analysis of 125I-labeled Ag differed between thymocytes, peripheral T cells, and peripheral B cells. On the basis of its range of expression on lymphoid cells and known biochemical and functional properties, we conclude that the B5-5 component on sheep lymphocytes is different from T cell activation Ag in other species.     

Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured 3T3 fibroblasts. In the present study, rabbit antibodies directed against CBP35 were used to analyze the subcellular distribution of CBP35 in 3T3 cells. Several lines of evidence indicate that CBP35 is found externally exposed at the cell surface: immunofluorescent staining of live 3T3 cells; agglutination of suspension of 3T3 fibroblasts by specific antibodies; and isolation, by immunoaffinity chromatography, of a Mr 35,000 component from cells surface-labeled with 125I. In addition to the plasma membrane, CBP35 could also be found intracellularly, as revealed by immunofluorescence studies of fixed and permeabilized 3T3 cells. The staining pattern showed the presence of CBP35 on the nucleus and in the cytoplasm. These results are consistent with the finding that among several subcellular fractions, CBP35 can be found by immunoblotting procedures in the nuclear pellet, the soluble fraction, and the plasma membrane fraction of the postnuclear supernatant.     

Role of phosphatidylinositol-anchored proteins in T cell activation

A novel class of cell surface proteins are attached to the plasma membrane via a phosphatidylinositol (PI)-glycan anchoring structure, and these proteins can be selectively removed from the cell surface by the enzyme PI-specific phospholipase C (PI-PLC). Enzyme treatment led to a prolonged reduction in cell surface expression of several PI-anchored proteins. Activation of T cells led to a marked decrease in the ability of PI-PLC to remove PI-anchored surface proteins from the activated T cells. This decrease in PI-PLC sensitivity may reflect an alteration in the PI-glycan anchoring structures, or in a general membrane property, which renders the PI-anchored proteins inaccessible to the enzyme. When murine T lymphocytes were treated with PI-PLC and then stimulated with either Con A, the calcium ionophore A23187 and PMA, or an anti-CD3 mAb, the response to Con A stimulation was inhibited by 90%, whereas the responses to ionophore and PMA or anti-CD3 were not affected. Removal of PI-anchored proteins inhibited an early event in the activation process in response to Con A because both IL-2 production and IL-2R expression were inhibited by the PI-PLC treatment. Inhibition of the Con A response was secondary to removal of a PI-linked protein from the responder T cell population because PI-PLC treatment of T-depleted spleen cells did not alter their ability to act as a source of accessory cells. It is unlikely that removal of the known PI-linked proteins on murine T cells, Thy-1 and Ly-6, can fully account for the inhibition of Con A response because the cell line M2B3, that lacks these surface proteins, responded normally to Con A stimulation. These studies demonstrate that one or more PI-anchored T cell proteins play an important role in an early step of Con A activation, perhaps involving T cell-accessory cell interactions. In contrast, the ability to stimulate T cells by direct cross-linking of TCR/CD3 complex is not dependent on the presence of these PI-anchored proteins.     

Evidence for plasma membrane-associated forms of sucrose synthase in maize

Plasma membrane fractions were isolated from maize (Zea mays L.) endosperms and etiolated kernels to investigate the possible membrane location of the sucrose synthase (SS) protein. Endosperms from seedlings at both 12 and 21 days after pollination (DAP), representing early and mid-developmental stages, were used, in addition to etiolated leaf and elongation zones from seedlings. Plasma membrane fractions were isolated from this material using differential centrifugation and aqueous two-phase partitioning. The plasma membrane-enriched fraction obtained was then analyzed for the presence of sucrose synthase using protein blots and activity measurements. Both isozymes SS1 and SS2, encoded by the lociSh1 andSus1, respectively, were detected in the plasma membrane-enriched fraction using polyclonal and monoclonal antisera to SS1 and SS2 isozymes. In addition, measurements of sucrose synthase activity in plasma membrane fractions of endosperm revealed high levels of specific activity. The sucrose synthase enzyme is tightly associated with the membrane, as shown by Triton X-100 treatment of the plasma membrane-enriched fraction. It is noteworthy that the gene products of bothSh1 andSus1 were detectable as both soluble and plasma membrane-associated forms.     

Down-regulation of homing receptors after T cell activation

The specific pattern of lymphocyte localization and recirculation is important for the induction and expression of normal immune responses. In order to home to lymph nodes (LN), lymphocytes must first recognize and bind to specific high endothelial venules (HEV) in the LN. Binding to LN HEV is mediated by specific lymphocyte receptors, termed homing receptors, which are recognized by the mAb MEL-14. We examined the changes that occur in homing receptor expression after activation of murine T lymphocytes in vitro. Cells activated in MLC or by Con A undergo a 75% loss in their ability to recognize HEV, as demonstrated by a decrease in binding to HEV in vitro. Large, activated cells isolated from a primary MLC by elutriator centrifugation were completely unable to recognize HEV, whereas the small cells in the same culture continued to bind well. Flow cytometric analysis with MEL-14 showed that the activated fraction had lost expression of gp90MEL-14, the homing receptor Ag, whereas the inactivated cells remained MEL-14+. Concomitant with the loss of homing receptor expression, most of the activated cells became strongly peanut agglutinin (PNA)-positive, demonstrating a marked change in surface glycosylation. Thus, these MLC consist of two major populations of T cells--small, inactivated lymphocytes that are MEL-14+PNAlo and large, activated blast cells that are MEL-14-PNAhi. Purified MEL-14+ T cells activated by Con A gave rise to MEL-14- progeny, showing that gp90MEL-14 is lost from gp90MEL-14-positive precursors, rather than from the selective growth of MEL-14- cells. Furthermore, the loss of Ag expression on at least some activated cells is reversible in resting culture, with almost half of the cells reverting to MEL-14+ after the cessation of stimulation. These experiments show that activation of T cells results in down-regulation of surface homing receptors, resulting in their inability to recognize and bind to the endothelial surface of HEV. This suggests that the activation of T cells in vivo would result in a dramatic and physiologically significant change in their migration and localization properties which would be important during a normal immune response.     

Partial characterization of a growth-inhibiting protein in 3T3 cell plasma membranes

A plasma membrane-enriched fraction from 3T3 cells has been detergent-solubilized, and the supernatant of this solubilization was reconstituted into liposomes using soybean lecithin. When these vesicles were added to actively growing cells, cell growth rates were inhibited to levels that were comparable to those observed with the original plasma membranes (at least 50% of maximum growth rates). Liposomes without proteins, or liposomes containing proteins from SV3T3 plasma membranes did not significantly inhibit growth of 3T3 cells. Treatment of the reconstituted vesicles with urea or high concentrations of salt did not eliminate the growth-inhibiting properties of these reconstituted membranes. These results indicate that the specific growth-inhibiting factor in 3T3 cell plasma membranes is a membrane protein that has significant non-polar interactions with the membrane bilayer.     

Production of a fibronectin-associated lymphokine by cloned mouse T cells

Azobenzenearsonate-specific cloned mouse T cells able to transfer delayed hypersensitivity reactions in vivo produced macrophage agglutination factor (MaggF) after stimulation with mitogen or antigen in vitro. Mitogen (Con A) elicited MAggF production directly from T cells. Responses to Ag were Ag-specific, required syngeneic accessory cells in addition to T cells, and were independent of T cell fine specificity for azobenzenearsonate. Mouse MAggF shared a number of biochemical and immunochemical properties with the fibronectins (FN): 1) high Mr similar to that of plasma FN; 2) binding to gelatin, heparin, and polyclonal antibodies and mAb specific for cellular and plasma FN; 3) inhibition of activity in solution by monoclonal anti-human FN directed against plasma FN gelatin-binding domain; and 4) action on peritoneal exudate macrophages mediated through a FN-receptor cross reactive with one on human monocytes. MAggF production required active protein synthesis and was associated with significant increases in gelatin-binding immunoreactive FN (Mr 440 kDa on immunoblotting) in culture supernatants and T cell lysates. Metabolically labeled peptides could be precipitated by anti-FN from culture supernatants of activated T cells. Stimulated cultures contained significantly more cells with immunohistologically demonstrable cytoplasmic FN than unstimulated control cultures. We suggest that T cell FN is a distinct species of cellular FN which may play an important role in mediating delayed hypersensitivity inflammatory reactions in vivo.     

Signal transduction in T lymphocites under simulated microgravity conditions: involvement of PKC isoforms

Previous data obtained from experiments either in space or in clinostats have shown that: a) human T lymphocytes activation is strongly inhibited; b) the distribution of protein kinase C (PKC) in human leukocytes is altered; c) expression of IL-2 and IL-2-R-alpha is altered. In this study we focus our attention on different isoforms of PKC to determine whether microgravity directly affects the activity and subcellular distribution of PKC. This work was carried out with Con A and anti-CD 28 activated human T cells in simulated microgravity conditions in the Random Positioning Machine (RPM). The cellular fractions (nuclear, cytosolic and membrane) extracted were subjected to Western blotting and RT-PCR analysis.     

Regulation of plasminogen activator in 3T3 cells: effect of phorbol myristate acetate on subcellular distribution and molecular weight

The tumor promoter, phorbol myristate acetate (PMA), stimulates plasminogen activator production and extracellular release in confluent Swiss 3T3 cells. Coordinated with the increased extracellular release is a redistribution of the activity into plasma membrane-enriched fractions and a shift in the predominant molecular weight species from 75,000 to 49,000 daltons. The evidence suggests that PMA induces the formation of the 49,000 dalton species which is preferentially located in plasma membrane-enriched fractions.     

Immunosuppressive activity of human seminal plasma. I. Inhibition of in vitro lymphocyte activation.

A high molecular weight fraction prepared from human seminal plasma by gel filtration chromatography suppresses human lymphocyte transformation and DNA synthesis induced by mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and allogenic cells. This same fraction also suppresses the stimulated response of mouse lymphocytes to allogenic cells and to various mitogens, including T cell-dependent and T cell-independent mitogens. The induction, but not the expression, of cell-mediated cytotoxicity is also suppressed. Similar high molecular weight fractions suppress the in vitro humoral response of mouse spleen cells to both a T cell-dependent (SRBC) and a T cell-independent (DNP-F) antigen. The high m.w. fraction exhibited in vitro suppressive activity at concentrations of 0.1 to 1.0 mg/ml which corresponds to a 1/50 or greater dilution of human seminal plasma. These observations support the concept that a local immune response against sperm in the female reproductive tract is actively suppressed by a component in seminal plasma.