scholarly journals THE KINETICS OF CELLULAR COMMITMENT DURING STIMULATION OF LYMPHOCYTES BY LECTINS

1974 ◽  
Vol 62 (2) ◽  
pp. 366-377 ◽  
Author(s):  
Gary R. Gunther ◽  
John L. Wang ◽  
Gerald M. Edelman
Keyword(s):  
Cellular Response ◽  
Thymidine Incorporation ◽  
Competitive Inhibitor ◽  
Spleen Cells ◽  
Con A ◽  
Induction Periods ◽  
Blast Cells ◽  
Staining Techniques ◽  
Kinetics Of ◽  
Stimulation Of

The kinetics of cellular commitment in the stimulation of lymphocytes by concanavalin A (Con A) has been analyzed by measurement of DNA synthesis, autoradiography, and histologic staining techniques. If the competitive inhibitor α-methyl-D-mannoside (αMM) is introduced into cultures of mouse spleen cells at various times after the addition of Con A, there is a gradual decrease in its capacity to inhibit the lectin-stimulated incorporation of [3H]thymidine. Addition of the saccharide 20 h after exposure of the cells to Con A had no effect on the level of the cellular response to the lectin. With increasing periods of contact with Con A, the percentage of blast cells and the percentage of [3H]thymidine-labeled blast cells increased in parallel with the total radioactive thymidine incorporated while the average number of autoradiographic grains per labeled blast cell remained relatively constant. These observations suggest that the rising level of [3H]thymidine incorporation results from an increase in the number of cells that respond to lectin stimulation and become refractory to inhibition with αMM. Once such cells become committed, they synthesize DNA at a rate independent of the length of exposure to the lectin. The combined results indicate that mouse splenic lymphocytes are heterogeneous in their capacities to respond to Con A and that different cells require different induction periods to be stimulated.

scholarly journals Primary generation of cytolytic T lymphocytes in the absence of DNA synthesis.

1979 ◽  
Vol 150 (1) ◽  
pp. 196-201 ◽  
Author(s):  
H R MacDonald ◽  
R K Less
Keyword(s):  
T Lymphocytes ◽  
Dna Synthesis ◽  
Cytolytic Activity ◽  
Target Cells ◽  
Cytolytic T Lymphocytes ◽  
Spleen Cells ◽  
Con A ◽  
A Cell ◽  
Stimulation Of

The requirement for DNA synthesis during the primary differentiation of cytolytic T lymphocytes (CTL) had been investigated. CTL were induced polyclonally in vitro by stimulation of normal C57BL/6 spleen cells with concanavalin A (Con A)and their cytolytic activity was tested against 51Cr-labeled target cells in the presence of Bacto Phytohemagglutinin M. With this system, CTL activity could first be detected 48 h after exposure of spleen cells to Con A. Addition of cytosine arabinoside at concentrations sufficient to reduce DNA synthesis by 95-98% in Con A-stimulated cultures did not significantly inhibit the generation of cytolytic activity on a cell-to-cell basis. These results demonstrate that derepression of the genetic information required for the expression of CTL function can occur in the absence of detectable DNA synthesis.

scholarly journals An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones.

1986 ◽  
Vol 163 (6) ◽  
pp. 1566-1582 ◽  
Author(s):  
R L Moldwin ◽  
D W Lancki ◽  
K C Herold ◽  
F W Fitch
Keyword(s):  
T Cell ◽  
Cellular Proliferation ◽  
Thymidine Incorporation ◽  
Interleukin 2 ◽  
Antigen Receptor ◽  
Inhibitory Effect ◽  
Spleen Cells ◽  
Ifn Gamma ◽  
Cytolytic T Lymphocyte ◽  
Stimulation Of

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL.

Insulin-modulated interleukin-2 production by murine splenocytes and a T-cell hybridoma

1987 ◽  
Vol 114 (1) ◽  
pp. 89-94 ◽  
Author(s):  
K. Pavelić ◽  
R. J. Bernacki ◽  
S. Vuk-Pavlović
Keyword(s):  
T Cell ◽  
Thymidine Incorporation ◽  
Interleukin 2 ◽  
Growth Stimulation ◽  
Hybridoma Cells ◽  
Con A ◽  
Murine Splenocytes ◽  
Serum Free ◽  
Free Media ◽  
Stimulation Of

ABSTRACT Murine interleukin-2 (IL-2)-producing AOFS 21 T-cell hybridoma cells and normal murine splenocytes were stimulated in serum-free media by 16 potential mitogens/growth factors. Only insulin, concanavalin A (Con A), peptidoglycan monomer and a tumour-derived insulinoid stimulated [3H]thymidine incorporation by AOFS 21 cells and splenocytes. Supernatants of these stimulated cultures were tested for IL-2 activity which generally followed the pattern of growth stimulation. Both the mitogenicity and stimulation of IL-2 secretion by insulin were second only to the effects of Con A. J. Endocr. (1987) 114, 89–94

Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes

1980 ◽  
Vol 58 (11) ◽  
pp. 1314-1317 ◽  
Author(s):  
Trevor Owens ◽  
J. G. Kaplan
Keyword(s):  
T Cells ◽  
T Cell ◽  
Concanavalin A ◽  
Thymidine Incorporation ◽  
Spleen Cells ◽  
Con A ◽  
Activated T Cell ◽  
Constant Cell ◽  
Enriched Cultures ◽  
Cell Mitogen

Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation of thymidine into DNA; the expected dilution of these two responses by nude spleen cells did not occur. However, if the nude splenocytes were added immediately prior to assay to the enriched T cells that had been precultured in presence of Con A, the expected dilution of the activated T-cell responses occurred; both 86Rb uptake and thymidine incorporation were reduced proportionally to the degree of dilution of the T cells by the nonresponding cells. These data indicate that during co-culture in presence of Con A there is interaction between the T cells, capable of responding to the mitogens, and the nude spleen cells. Attempts to demonstrate a diffusible factor in the supernatants of stimulated T cells were unsuccessful. The measured interaction is sufficient to explain our previous paradoxical findings that enrichment of T cells as measured by membrane markers did not cause a corresponding enrichment for either cation transport or for thymidine incorporation, and that depletion of T cells in the B-enriched cultures did not cause a corresponding decrease in these two Con A induced responses.

scholarly journals Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose

1971 ◽  
Vol 121 (1) ◽  
pp. 151-157 ◽  
Author(s):  
C. L. Villemez
Keyword(s):  
Reaction Rate ◽  
Initial Rate ◽  
Competitive Inhibitor ◽  
Enzyme Preparations ◽  
Polysaccharide Biosynthesis ◽  
Guanosine Diphosphate ◽  
Straight Line ◽  
Kinetics Of ◽  
Total Reaction Time ◽  
Stimulation Of

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose is not increased by additions of GDP-α-d-mannose. However, final incorporation is increased by addition of GDP-α-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is increased by all concentrations of GDP-α-d-glucose that are less than that of the GDP-α-d-[14C]mannose. Maximum stimulation of the initial rate occurs at a GDP-α-d-[14C]mannose/GDP-α-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-α-d-[14C]mannose is decreased by the addition of GDP-α-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2–3 and 50μm. Extrapolated Km values for the two linear components are 0.4–1.0 and 700–1500μm. The effect of GDP-α-d-glucose on the kinetics of 14C-labelled polysaccharide formation from GDP-α-d-[14C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. 14C-labelled polysaccharide formation from GDP-α-d-[14C]glucose also results in biphasic reciprocal plots. One component appears to have Km about 2–3μm, the other about 200–400μm. In this reaction, GDP-α-d-mannose appears to be a competitive inhibitor with Ki 20–30μm. With particulate preparations of P. aureus, GDP-α-d-[14C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-α-d-[14C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.

scholarly journals Inhibition of Ly-6A antigen expression prevents T cell activation.

1990 ◽  
Vol 172 (1) ◽  
pp. 115-120 ◽  
Author(s):  
P M Flood ◽  
J P Dougherty ◽  
Y Ron
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Antisense Oligonucleotides ◽  
Cell Activation ◽  
Cell Clone ◽  
Spleen Cells ◽  
Con A ◽  
Normal Spleen ◽  
T Cell Clone ◽  
Stimulation Of

Antisense oligonucleotides complementary to the 5' end of the mRNA encoding the Ly-6A protein were used to block the expression of that protein. Using this approach we could inhibit the expression of Ly-6A by 60-80% in antigen-primed lymph node (LN) T cells as well as in the D10 T cell clone. Inhibition of Ly-6 expression resulted in the inability to restimulate in vitro, antigen-primed T cells. It also blocked the activation of normal spleen cells by Con A, monoclonal antibody (mAb) to CD3, and mAb to Ly-6. In contrast, stimulation of normal spleen cells with the pharmacological agents PMA + ionomycin were unaffected by the inhibition of Ly-6 expression. Similar results were obtained with the D10 T cell clone; stimulation with Con A + interleukin 1 (IL-1), antigen-presenting cells (APC), or the clonotypic antibody + IL-1 was greatly reduced in the presence of antisense oligonucleotides to Ly-6. Stimulation with PMA + ionomycin was again unaffected. We also studied the effect of antisense oligonucleotides on stimulation of preactivated D10 cells. Preactivation of D10 cells with Con A + IL-1 renders them receptive to secondary stimulation by other lymphokines. In this case, antisense oligonucleotides to Ly-6 had no effect on secondary activation with IL-2, IL-4 + IL-1, or PMA + ionomycin. We conclude from these studies that Ly-6 expression is required for T cell receptor (TCR)-mediated T cell activation.

scholarly journals Concanavalin A-inducible, interleukin-2-producing T cell hybridoma.

1980 ◽  
Vol 152 (4) ◽  
pp. 893-904 ◽  
Author(s):  
L Harwell ◽  
B Skidmore ◽  
P Marrack ◽  
J Kappler
Keyword(s):  
T Cells ◽  
T Cell ◽  
Concanavalin A ◽  
Interleukin 2 ◽  
Hybridoma Cells ◽  
Adherent Cell ◽  
Spleen Cells ◽  
Con A ◽  
Normal Spleen ◽  
Stimulation Of

The fusion of an AKR T cell tumor line to normal B6D2F1, T cells resulted in the production of a cloned T cell hybridoma (FS6-14.13) inducible with the mitogen concanavalin A (Con A). The supernate from Con A-stimulated hybridoma cells was active both in the stimulation of an anti-sheep red blood cell response by partially T cell-depleted B cells and in the stimulation of the growth of antigen-specific T cell blasts. The active principle in both assays had a molecular weight of approximately 30-40,000. These results indicated the presence of interleukin 2 (IL2) in the hybridoma supernate. The activity of the hybridoma supernate in B cell responses was dependent on the presence of adherent cells and a few contaminating T cells. On the other hand, Con A-stimulated supernates from normal spleen cells were active after either adherent cell removal or severe T cell depletion. These results suggested that IL2 was the only active helper factor in the hybridoma supernate, but that additional helper factors were present in supernates from Con A-stimulated normal spleen cells.

scholarly journals The Qa-1 antigenic system. Relation of Qa-1 phenotypes to lymphocyte sets, mitogen responses, and immune functions.

1978 ◽  
Vol 148 (4) ◽  
pp. 963-973 ◽  
Author(s):  
T H Stanton ◽  
C E Calkins ◽  
J Jandinski ◽  
D J Schendel ◽  
O Stutman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Distinctive Feature ◽  
Thymidine Incorporation ◽  
Sheep Erythrocyte ◽  
Test System ◽  
Chromosome 17 ◽  
Target Cells ◽  
Spleen Cells ◽  
Con A ◽  
Lymphocyte Populations

The antiserum (B6 X A-Tlab) anti-A (Tlaa) defines several TL antigens expressed exclusively on thymocytes. When reacted with peripheral lymphocytes, the same antiserum defines another antigenic system, provisionally termed Qa-1. The genotypic disparity distinguishing the recipients and donors in this immunization comprises a section of chromosome 17 extending from a crossover point between H-2D and Tla to a presently unmarked point beyond Tla. Therefore although Qa-1 may constitute a single cell surface component, it is equally probable that the Qa-1 system defines two or more cell surface components determined by genes in this region, each of which may be expressed on a different cell set. Cytotoxicity assays indicate that Qa-1 antigen is expressed on Lyt-1 cells and Lyt-123 cells, and may serve to subclassify these two cell sets; it is not known whether Qa-1+ cells may occur within the small Lyt-23 set. There may be also be a cell set with the phenotype Thy-1--:Qa-1+. Another distinctive feature of the Qa-1 system is the characteristic profile of responses to mitogens exhibited by spleen cell populations from which Qa-1+ cells have been eliminated; in conventional assay of [3H]thymidine incorporation the response to lipopolysaccharide was essentially unchanged, the response to phytohemagglutinin M (PHA-M) was virtually abolished, and the response to concanavalin A (Con A) was reduced by 40%. The third distinctive feature of the Qa-1 system is the characteristic profile of changes which elimination of Qa-1+ cells produces in tests of immune function in vitro: (a) proliferation, measured by [3H]thymidine incorporation, in mixed lymphocyte culture (MLC) with major histocompatibility complex (MHC)-incompatible stimulator cells, was not affected. (b) in tests of cell-mediated cytotoxicity (CMC) of MHC-incompatible target cells, neither the generation nor the effector functions of cytotoxic lymphocytes was affected, implying that Lyt-23 prekiller and killer cells are Qa-1--. (c) primary and secondary responses to SRBC were considerably augmented, suggesting that Qa-1+ cells may be responsible for suppression in this test system. (d) accordingly the suppression of the anti-sheep erythrocyte (SRBC) response normally engendered in spleen cells by culture with SRBC was profoundly reduced by elimination of Qa-1+ cells, either before or after culture. (e) the suppression of the anti-SRBC response normally engendered in spleen cells cultured with Con A was reduced by removal of Qa-1+ cells before but not after culture with Con A. Although analysis is as yet far from complete, the Qa-1 system should already be of considerable value because it distinguishes a population of lymphocytes that is not defined by any other antigenic system, according to three criteria: (a) representation of Qa-1 cells among T-cell sets defined by Lyt phenotypes, (b) the profile of responses to mitogens exhibited by lymphocyte populations depleted of Qa-1+ cells, and (c) the profile of immune responses of lymphocyte populations depleted of Qa-1+ cells.

Differential effect of low molecular weight alcohols on the Con A stimulation of mouse spleen cells

1982 ◽  
Vol 4 (6) ◽  
pp. 305-309 ◽  
Author(s):  
Astrid Raile ◽  
Klaus P. Hammann ◽  
Otto Scheiner ◽  
Thomas Schulz ◽  
Anna Erdei ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Differential Effect ◽  
Mouse Spleen ◽  
Low Molecular Weight ◽  
Spleen Cells ◽  
Con A ◽  
Stimulation Of

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

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