The Activation and Reactivation of Peripheral Lymphocytes in Culture

1967 ◽  
Vol 2 (1) ◽  
pp. 57-70
Author(s):  
N. R. LING ◽  
P. J. L. HOLT
Keyword(s):  
Mixed Lymphocyte Reaction ◽  
Human Lymphocytes ◽  
Maximal Activity ◽  
Cell Populations ◽  
Initial Stimulus ◽  
Purified Protein Derivative ◽  
Peripheral Lymphocytes ◽  
Lymphocyte Populations ◽  
Low Dosage

Human lymphocytes stimulated for 16 h and then cultured without stimulant showed maximal activity on days 2-3 following a stimulus of phytohaemagglutinin (PHA) and on days 3-4 following a stimulus of staphylococcal filtrate (SF). At low dosage of stimulant the response of the cells was less marked but persisted for a longer period than at high dosage. The pattern of response is discussed in relation to the mechanism of activation. After the effect of the initial stimulus had died away cell populations which had been stimulated with SF or PHA could be restimulated with either stimulant. Their response, when stimulated this second time, was quicker than that of incubated cells from the same donor which had not been previously stimulated. Prestimulated cells were also tested in two immunospecific reactions : the reaction to tuberculin-purified protein derivative (PPD), and the mixed lymphocyte reaction. Cells which had been previously exposed to SF responded more quickly to PPD than cells not previously stimulated. Cell populations which had been previously stimulated also reacted more quickly in a mixed lymphocyte reaction. It is concluded that lymphocyte populations which have been recently stimulated not only retain their capacity to react to immunospecific mitotic stimuli but also react more quickly.

scholarly journals THE UROPOD-BEARING LYMPHOCYTE OF THE GUINEA PIG

1972 ◽  
Vol 135 (5) ◽  
pp. 1037-1048 ◽  
Author(s):  
David L. Rosenstreich ◽  
Ethan Shevach ◽  
Ira Green ◽  
Alan S. Rosenthal
Keyword(s):  
Lymph Node ◽  
Guinea Pig ◽  
Human Lymphocytes ◽  
Surface Membrane ◽  
Purified Protein Derivative ◽  
Membrane Immunoglobulin ◽  
Lymph Node Cells ◽  
Lymphocyte Populations ◽  
Antigen Reactivity

In this study, the frequency of uropod formation and the type of lymphocyte bearing the uropod was investigated in various guinea pig lymphocyte populations. Without prior in vitro stimulation, almost 40% of peritoneal exudate lymphocytes (PELS) form uropods, while thymocytes and lymph node cells form far fewer. Subsequent stimulation in vitro with purified protein derivative demonstrated that there is an association between antigen reactivity and frequency of uropod formation in these populations. The ultrastructure of these uropods is identical to that described for human lymphocytes stimulated with phytohemagglutinin. In the populations studied, all the lymphocytes forming uropods lack easily detectable surface membrane immunoglobulin and are therefore most likely thymus-derived or T lymphocytes.

scholarly journals SELECTIVE TRIGGERING OF HUMAN T AND B LYMPHOCYTES IN VITRO BY POLYCLONAL MITOGENS

1974 ◽  
Vol 140 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Melvyn Greaves ◽  
George Janossy ◽  
Michael Doenhoff
Keyword(s):  
B Cells ◽  
B Lymphocytes ◽  
Human Lymphocytes ◽  
Culture Conditions ◽  
Pokeweed Mitogen ◽  
Cell Populations ◽  
T And B Cells ◽  
Activated T Cells ◽  
Lymphocyte Responses

Human lymphocytes from spleen and tonsils have been cultured with a variety of polyclonal mitogens. Cultures consisted of either unseparated T and B cells or alternatively purified T or B lymphocytes. The purity of the starting cell populations and the origin of activated lymphoblasts was analyzed with a panel of seven markers which discriminate between T and B cells. The selectivity of the lymphocyte responses was influenced by cell populations in a given culture, the mitogen used, and to a limited extent on culture conditions. Purified T lymphocytes from tonsil and spleen responded to phytohemagglutinin (PHA), pokeweed mitogen (PWM), and staphylococcal enterotoxin B (SEB). Purified B cells from spleen responded well to PWM, weakly to SEB and lipopolysaccharide, but not at all to PHA. Tonsil B cells responded weakly to PWM and SEB but not to PHA. Some B lymphocytes do respond to PHA in the presence of activated T cells. These results are discussed in relation to previously reported selective responses of human cells and parallel studies in animal species.

scholarly journals CONTACT SENSITIVITY IN THE MOUSE

1970 ◽  
Vol 132 (1) ◽  
pp. 1-15 ◽  
Author(s):  
G. L. Asherson ◽  
M. Zembala
Keyword(s):  
Time Course ◽  
Peritoneal Macrophages ◽  
Peritoneal Exudate ◽  
Cell Populations ◽  
Contact Sensitivity ◽  
Skin Reactions ◽  
Peritoneal Exudate Cells ◽  
Lymphocyte Populations ◽  
Circulating Antibody ◽  
Cytophilic Antibody

Contact sensitivity skin reactions were produced in mice by immunization with 2-phenyl-4-ethoxymethylene oxazolone (oxazolone) and detected by the increase in ear thickness after challenging the ears with 2% oxazolone. These skin reactions can be transferred from immunized donors to irradiated recipients by peritoneal exudate cells induced by thioglycollate. The peritoneal exudate cells were separated into purified macrophage and purified lymphocyte populations. Both cell populations transferred skin reactions. However, their time course was different. The reactions produced by lymphocytes were greater at 24 hr than at 12 hr while the reactions produced by macrophages declined slightly between 12 and 24 hr. The working hypothesis was formed that the peritoneal lymphocytes conveyed a factor (presumptive cytophilic antibody) to peritoneal macrophages which enabled them to transfer ear reactions. Experiment showed that peritoneal and lymph node lymphocytes from sensitized donors within a Millipore chamber conveyed a factor to macrophages outside the chamber which enabled them to transfer ear reactions. In contrast, peritoneal macrophages (from sensitized donors) within the chamber and peritoneal lymphocytes outside the chamber were inactive. These findings suggested that there are three modes of immunological tissue damage: hypersensitivity mediated by lymphocytes (classical delayed hypersensitivity), hypersensitivity mediated by circulating antibody (classical immediate type hypersensitivity), and hypersensitivity mediated by macrophages which have passively acquired a factor (macrophage-mediated hypersensitivity).

Inhibition of Lymphocyte Transformation: Effect of Soy Bean Trypsin Inhibitor and Synthetic Anti-Proteases

1975 ◽  
Vol 53 (12) ◽  
pp. 1337-1341 ◽  
Author(s):  
Pierre Moreau ◽  
Jacques Dornand ◽  
J. G. Kaplan
Keyword(s):  
Trypsin Inhibitor ◽  
Mixed Lymphocyte Reaction ◽  
Human Lymphocytes ◽  
Lymphocyte Transformation ◽  
Aminocaproic Acid ◽  
Early Event ◽  
Mitogen Stimulation ◽  
Bean Trypsin Inhibitor ◽  
A Cell ◽  
Protease Action

Soybean trypsin inhibitor (SBI) was found to inhibit transformation of human lymphocytes induced by mitogens (leucoagglutinin, concanavalin A, NaIO4) or in mixed lymphocyte reaction (MLR). SBI covalently cross-linked to Sepharose beads inhibited the MLR and mitogen stimulation virtually completely. We have confirmed the work of others which showed that the synthetic anti-proteases e-aminocaproic acid and tosyl-L-lysyl-chloromethane (TLCK) also inhibited mitogen-induced blastogenesis and we have shown that phenylmethylsulfonylfluoride was effective also; all of these agents were found to inhibit the MLR as well. SBI and TLCK were most inhibitory when added along with mitogen or when mixing allogeneic cells in a MLR; significant decrease in inhibition was noted when TLCK was added 1 h after mitogen. These data support the hypothesis that protease action at a cell surface is an essential early event common to all types of lymphocyte transformation.

scholarly journals STIMULUS-RESPONSE IN THE MIXED LYMPHOCYTE REACTION

1973 ◽  
Vol 138 (6) ◽  
pp. 1602-1607 ◽  
Author(s):  
Michael R. Harrison ◽  
William E. Paul
Keyword(s):  
T Lymphocytes ◽  
Spleen Cell ◽  
Mixed Lymphocyte Reaction ◽  
Cell Populations ◽  
Target Cells ◽  
Mouse Spleen Cell ◽  
F1 Hybrid ◽  
Stimulus Response ◽  
X Irradiation ◽  
Mixed Lymphocyte Reactions

Mixed lymphocyte reactions occur when mouse spleen cell populations depleted of thymus-derived (T) lymphocytes are cultured with allogeneic target cells inactivated by mitomycin C or X irradiation, and when F1 hybrid responder cells are cultured with inactivated parental target cells. These responses might be interpreted as indicating that T lymphocytes are not required for responsiveness and that F1 lymphocytes recognize parental alloantigens. Data reported here indicate that the more likely explanation for these surprising results is that inactivated target cells recognize the "responding" cells and this recognition leads to the response observed.

Simultaneous Reconstitution of Multiple CMV-Specific CD8+ T-Cell Populations with Divergent Functionality in Hematopoietic Stem Cell Transplant Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 190-190 ◽  
Author(s):  
Simon F. Lacey ◽  
Joy Martinez ◽  
Ghislaine Gallez-Hawkins ◽  
Lia Thao ◽  
Jeff Longmate ◽  
...  
Keyword(s):  
T Cell ◽  
T Lymphocytes ◽  
Hematopoietic Stem Cell Transplant ◽  
Cell Populations ◽  
Cell Transplant ◽  
Transplant Recipients ◽  
Median Percentage ◽  
Hematopoietic Stem ◽  
Cd8 T Lymphocytes ◽  
Lymphocyte Populations

Abstract Recent studies using direct stimulation of PBMC from CMV-positive individuals with optimal CTL epitope peptides have identified new antigens that are targets of the host immune response. The growing number of targets of the cellular immune response has prompted an evaluation of which antigens are potentially associated with protective immunity. A panel of seven human cytomegalovirus (CMV) epitope peptides and the corresponding MHC-I tetramers was used to evaluate cellular immunity in six healthy seropositive donors and in six hematopoietic stem cell transplant recipients (HSCT). Broad CMV-specific responses were found to epitopes within several CMV polypeptides that were restricted by multiple HLA alleles in several individuals. The results document the simultaneous expansion of several of these CD8+ T-lymphocyte populations following allogeneic HSCT. The combined levels of CMV epitope-specific T-cells exceeded 20% of CD8+ T-lymphocytes in some individuals. The cytotoxic functionality of 26 different populations of CMV-specific T-lymphocytes detected within this group of 12 individuals was addressed by utilizing a recently described assay that measures transient surface levels of the lysosomal membrane proteins LAMP-1 (CD107a) and LAMP 2 (CD107b) after peptide stimulation. This degranulation/mobilization assay can be combined with tetramer staining of antigen-specific CD8+ T-lymphocytes, and has potential as a surrogate marker for cytotoxic function. We found that a significant proportion of CD8+ T-lymphocytes specific for epitopes within the CMV pp65 and pp50 gene products had functional potential as measured by this assay (median percentage of cells within 14 T-cell populations staining with pp65 or pp50 tetramers that degranulated on stimulation with cognate peptide = 26.0% and 19.8%). By contrast, CD8+ T-lymphocytes specific for epitopes within the CMV IE-1 gene product had markedly reduced functionality (median percentage of cells within 12 T-cell populations staining with IE-1 tetramers that degranulated on stimulation with cognate peptide = 5.6%). This difference was significant (p= 0.003 by an F-test after adjusting for HLA and using interaction of subjects and epitopes as the error). This reduced degranulation efficiency of IE-1-specific T cells is consistent with their inefficient cytotoxic recognition of CMV-infected autologous fibroblasts. Further characterization of this functional dichotomy includes comparison of cell surface marker phenotype and cytokine release in response to antigenic presentation. These functional differences between T-lymphocyte populations within the same individual have implications for choosing antigens that are both protective and necessary to include in a CMV vaccine for transplant patients at risk for infectious complications after viral reactivation.

scholarly journals Serum albumin is essential for in vitro growth of activated human lymphocytes.

1975 ◽  
Vol 142 (4) ◽  
pp. 949-959 ◽  
Author(s):  
H Polet ◽  
H Spieker-Polet
Keyword(s):  
Serum Albumin ◽  
Human Lymphocytes ◽  
In Vitro Growth ◽  
Con A ◽  
Promoting Effect ◽  
Purified Protein Derivative ◽  
Activated T Lymphocytes ◽  
Proliferation Of Lymphocytes ◽  
Albumin Molecule

The effect of human plasma, the plasma protein fractions of Cohn, and crystallized serum albumin on the in vitro growth of human lymphocytes activated by concanavalin A (Con A) or bacterial lipopolysaccharide was compared. It was found that fraction V or serum albumin (SA) is essential for growth of activated T and B lymphocytes. The other plasma proteins have no effect. The growth response of Con A-activated T lymphocytes to increasing concentrations of SA is similar to the response to increasing equivalent concentrations of plasma suggesting but not proving that SA is the only growth-stimulating factor in plasma when added to a protein-free culture medium. The growth-promoting effect of SA is not due to the fatty acids or hormones bound to SA but is attributed to the albumin molecule itself or to a factor tightly bound to it. SA can also effectively replace plasma to stimulate proliferation of lymphocytes activated by allogeneic lymphocytes or purified protein derivative of tuberculin.

scholarly journals TWO DIFFERENT COMPLEMENT RECEPTORS ON HUMAN LYMPHOCYTES

1973 ◽  
Vol 138 (4) ◽  
pp. 798-811 ◽  
Author(s):  
Gordon D. Ross ◽  
Margaret J. Polley ◽  
Enrique M. Rabellino ◽  
Howard M. Grey
Keyword(s):  
Peripheral Blood ◽  
Human Lymphocytes ◽  
The Other ◽  
Complement Receptors ◽  
Chronic Lymphatic Leukemia ◽  
Lymphoblastoid Cells ◽  
Normal Peripheral Blood ◽  
Peripheral Lymphocytes ◽  
Immune Adherence ◽  
Lymphatic Leukemia

In the present study it was shown that normal peripheral lymphocytes have two different complement receptors: one for C3b (the immune adherence receptor) and one for C3b subsequent to its cleavage by C3b inactivator. The two receptors are not cross-reactive and were shown by tests with various antisera to be antigenically distinct. Both the immune adherence receptor and the receptor for C3b inactivator-cleaved C3b were found on normal peripheral lymphocytes and on cultured lymphoblastoid cells. In 15 out of 18 chronic lymphatic leukemia patients, the immune adherence receptor was either partially or completely missing from the peripheral lymphocytes, while the lymphocyte receptor for C3b inactivator-cleaved C3b was retained. Normal erythrocytes, on the other hand, were found to have only the immune adherence receptor. Granulocytes from normal peripheral blood appeared to have only a receptor for C3b and did not have a receptor for C3b inactivator-cleaved C3b.

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