scholarly journals THE EFFECTS OF MERCAPTOETHANOL AND OF PERITONEAL MACROPHAGES ON THE ANTIBODY-FORMING CAPACITY OF NONADHERENT MOUSE SPLEEN CELLS IN VITRO

1972 ◽  
Vol 136 (3) ◽  
pp. 604-617 ◽  
Author(s):  
Chang Chen ◽  
James G. Hirsch
Keyword(s):  
Antibody Formation ◽  
Peritoneal Macrophages ◽  
Lymphoid Cells ◽  
Red Cells ◽  
Mouse Spleen ◽  
Spleen Cells ◽  
Sheep Red Cells ◽  
Low Concentrations ◽  
Nonadherent Cells

Nonadherent mouse spleen cells exhibited poor viability and little or no capacity to form antibodies to sheep red cells in the Mishell-Dutton culture system. Viability and antibody-forming capacity could be restored by addition to these cultures of low concentrations of mercaptoethanol (10–4–10–5 M), or by addition of appropriate numbers of mouse peritoneal macrophages. Macrophage concentrations lower than optimal resulted in lower lymphoid cell viability and correspondingly fewer plaque-forming cells, whereas excess macrophages resulted in marked inhibition of antibody formation despite good viability of the lymphocytes. Restoration of the nonadherent cells with mercaptoethanol was thus much simpler and more reproducible than it was with macrophages; furthermore, the number of plaque-forming cells developed in cultures restored with mercaptoethanol was approximately fourfold higher than it was in cultures restored with optimal numbers of macrophages. In the presence of mercaptoethanol, the plaque-forming capacity of the nonadherent spleen cells was not increased when small numbers of macrophages were added to the system, nor was it decreased when the few macrophages present in the nonadherent cells were further reduced or eliminated. Excess macrophages inhibited antibody formation in the cultures containing mercaptoethanol as they did in control cultures. Optimal restoration of plaque-forming capacity to the nonadherent spleen cells with mercaptoethanol required the reducing agent to be present throughout the 4 or 5 day culture period. Addition of mercaptoethanol 1 or more days after initiation of culture, or transfer of the cells to a medium free of mercaptoethanol before completion of the culture resulted in a reduction in the numbers of plaque-forming cells. The results suggest that mouse lymphoid cells do not require macrophages in order to form antibodies to sheep red cells in vitro, provided mercaptoethanol is present in the culture medium. The mechanism of action of mercaptoethanol under these conditions is not completely clear, but one of its effects is to promote the viability of lymphoid cells in the cultures.

scholarly journals REJECTION OF TUMOR ALLOGRAFTS BY MOUSE SPLEEN CELLS SENSITIZED IN VITRO

1971 ◽  
Vol 133 (4) ◽  
pp. 821-833 ◽  
Author(s):  
Irun R. Cohen ◽  
Amiela Globerson ◽  
Michael Feldman
Keyword(s):  
Inhibitory Effect ◽  
Lymphoid Cells ◽  
Mouse Spleen ◽  
Target Cells ◽  
Functional Assay ◽  
Spleen Cells ◽  
Growth Of Tumor ◽  
Selection Of

This paper reports a model system of cellular immunity in which allosensitization of mouse spleen cells is induced in vitro. Allosensitization was achieved by culturing spleen cells upon monolayers of allogeneic fibroblasts. The ability of the spleen cells to inhibit the growth of tumor allografts in vivo served as a functional assay of sensitization. We found that unsensitized spleen cells or spleen cells sensitized against unrelated fibroblast antigens had no inhibitory effect on the growth of allogeneic fibrosarcoma cells when they were injected together into irradiated recipients. In contrast, spleen cells which were specifically allosensitized in vitro were found to be highly effective in inhibiting the growth of an equal number of allogeneic tumor cells. Several times more spleen cells from mice sensitized in vivo were required to produce a similar immune effect. This confirms the findings of previous studies which indicate that sensitization in cell culture can promote the selection of specifically sensitized lymphocytes. Preincubating sensitizing fibroblasts with allo-antisera blocked the allosensitization of spleen cells. This suggests that antibodies binding to fibroblasts may inhibit the induction of sensitization by competing with lymphocytes for antigenic sites. Mouse spleen cells which were able to recognize and reject tumor allografts in vivo were unable to cause lysis of target fibroblasts in vitro. Such fibroblasts, however, were susceptible to lysis by rat lymphoid cells sensitized by a similar in vitro method. These findings indicate that the conditions required for lymphocyte-mediated lysis of target cells may not be directly related to the processes of antigen recognition and allograft rejection in vivo.

scholarly journals THE EFFECTS OF ANTIGENS AND OF PHYTOHEMAGGLUTININ ON RABBIT SPLEEN CELL SUSPENSIONS

1966 ◽  
Vol 124 (4) ◽  
pp. 621-634 ◽  
Author(s):  
G. Harris ◽  
R. J. Littleton
Keyword(s):  
Dna Synthesis ◽  
Spleen Cell ◽  
Cell Suspensions ◽  
Red Cells ◽  
Specific Response ◽  
Spleen Cells ◽  
Sheep Red Cells ◽  
Stimulation Of

Phytohemagglutinin (PHA) stimulated the rate of DNA synthesis in rabbit spleen cell suspensions. Unlike antigens, previous immunization to PHA was not necessary and the specific response could not be transferred by macrophages, although lymphocytes primed by incubation in PHA were able to stimulate other spleen cells not directly exposed to PHA. When rabbits were stimulated by in vivo immunization with antigens, spleen cells proliferating in response to antigen were stimulated to divide by in vitro contact with PHA. Using the technique of specific hemolytic plaque formation by individual cells synthesizing γM-antibody to sheep red cells (plaque-forming cells), no evidence was obtained that stimulation of cell division by PHA resulted in specific antibody formation, although the presence of antigen resulted both in stimulation of cell proliferation and the production of plaque-forming cells. The presence of both sheep red cells and PHA in the medium of the same cell suspensions did not enhance the production of plaque-forming cells although there was a summative effect on DNA synthesis.

scholarly journals IMMUNOLOGICAL FUNCTIONS OF HUMAN T-LYMPHOID CELL LINE (MOLT)

1974 ◽  
Vol 140 (2) ◽  
pp. 538-548 ◽  
Author(s):  
Akikazu Takada ◽  
Yumiko Takada ◽  
Jun Minowada
Keyword(s):  
Red Blood Cells ◽  
Cell Line ◽  
Blood Cells ◽  
Lymphoid Cells ◽  
Mouse Spleen ◽  
Spleen Cells ◽  
Short Term ◽  
Molt Cell ◽  
Human B Cell

A short term incubation of the mixture of established human T-lymphoid cells (MOLT) and sheep red blood cells (SRBC) resulted in the release of factors which nonspecifically suppressed the response of mouse spleen cells against heterologous erythrocytes in vitro. Neither human B-cell line (RPMI 1788), nor the supernate of MOLT cell suspension in the absence of SRBC had such suppressive effects. The supernate of the mixture of MOLT cells with chicken red blood cells (CRBC) did not suppress either anti-CRBC or anti-SRBC responses of mouse spleen cells. Since CRBC did not form rosettes with MOLT cells, it is suspected that the origin of the production of these factors might be MOLT cells forming SRBC rosettes. Some of these factors are dialysable.

scholarly journals INCREASED REACTIVITY OF MOUSE SPLEEN CELLS SENSITIZED IN VITRO AGAINST SYNGENEIC TUMOR CELLS IN THE PRESENCE OF A THYMIC HUMORAL FACTOR

1973 ◽  
Vol 138 (6) ◽  
pp. 1521-1532 ◽  
Author(s):  
Claude Carnaud ◽  
David Ilfeld ◽  
Itzhak Brook ◽  
Nathan Trainin
Keyword(s):  
Tumor Cells ◽  
Culture Media ◽  
Lymphoid Cells ◽  
Humoral Factor ◽  
Mouse Spleen ◽  
Spleen Cells ◽  
Effector Phase ◽  
Syngeneic Tumor ◽  
Mediate Cytotoxicity

Unprimed mouse spleen cells cultured in vitro on syngeneic tumor cell monolayers have been previously shown to become specifically sensitized and to mediate cytotoxicity against the same type of tumor cells. This complete in vitro system of cell-mediated response has been presently used to test the effect of a thymic humoral factor (THF) upon the differentiation process leading to the generation of specifically committed lymphocytes. Culture media were supplemented with 2% THF during either the sensitization or effector phase, or both phases of the reaction. Whereas the addition of THF during both phases or during sensitization only resulted in a significant increase in the cytotoxicity index, THF added during the effector phase was ineffective. The behavior of unsensitized spleen cells and of spleen cells sensitized against nonrelated transplantation antigens remained unmodified by THF. After showing that the entire reaction is mediated by lymphocytes of thymic origin, THF was directly tested on T or B spleen cells. It was found that only T cells reacted to THF by an increased cytotoxic capacity, while B cells remained inactive after addition of THF. It was therefore concluded that THF activates a postthymic population of lymphoid cells, transforming them into fully competent lymphocytes.

scholarly journals IMMUNE RESPONSES IN VITRO

1969 ◽  
Vol 130 (2) ◽  
pp. 365-379 ◽  
Author(s):  
Carl W. Pierce
Keyword(s):  
Immune Responses ◽  
Culture System ◽  
Sheep Erythrocyte ◽  
Lymphoid Cells ◽  
Mouse Spleen ◽  
Suppressive Activity ◽  
Spleen Cells ◽  
High Concentrations ◽  
Antigen Antibody

The effects of hyperimmune anti-sheep erythrocyte (SRBC) antibody on the plaque-forming cell (PFC) response to SRBC by mouse spleen cells in vitro were studied. Anti-SRBC antibody specifically suppressed the PFC response against SRBC. The degree of suppression was directly related to the amount of antibody added and was overcome by large amounts of antigen. Suppressive activity was absorbed from the sera by SRBC and could be partially eluted from the antigen by heat. The PFC response in cultures stimulated with antigen-antibody complexes prepared with high concentrations of antibody were suppressed; however, some complexes prepared at lower antibody concentrations stimulated greater responses than SRBC alone. Antibodies collected after four immunizations had greater suppressive ability than those collected after two immunizations. The degree of suppression was as great whether antibody was added at the initiation of the cultures or 24 hr later, suggesting that during the first 24 hr the culture system was antigen-dependent. Incubation of separated lymphoid cells with antibody did not impair their ability to develop a PFC response in vitro. However, if macrophages were incubated with antibody either before or after incubation with SRBC, the subsequent PFC response by lymphoid cells was suppressed. The data are consistent with the conclusion that antibody suppresses the PFC response in vitro by neutralizing the antigenic stimulus at the macrophage-dependent phase of the response.

scholarly journals Specific elimination of cytotoxic effector cells. I. Adsorptive behavior of effectors and their precursors and spleen cell monolayers.

1976 ◽  
Vol 144 (4) ◽  
pp. 996-1008 ◽  
Author(s):  
J R Neefe ◽  
D H Sachs
Keyword(s):  
Mouse Spleen ◽  
Spleen Cells ◽  
Effector Cells ◽  
Cell Monolayers ◽  
Specific Suppression ◽  
Normal Spleen ◽  
Adsorptive Behavior ◽  
Cytotoxic Effector ◽  
Primed Mouse

Monolayers formed of normal mouse spleen cells attached to polystyrene coated with poly-L-lysine were tested for their ability to bind specifically antigen-reactive cells in normal or primed mouse spleen. 88 to greater than 98% of the activity of cytotoxic populations was removed by a single adsorption. However, normal spleen cells or spleen cells previously primed in vitro could not be depleted of their capacity to be sensitized, even when adsorption effectively removed all residual cytotoxic activity from the same previously primed population. In fact, exposure to an immunoadsorbent augmented the ultimate cytotoxicity generated in a nonspecific fashion. This augmentation was especially dramatic in the case of a previously primed population and may have reflected the removal of a nonspecific suppressor. If antigen-reactive precursors cannot be removed efficiently by adsorption, other approaches to the generation of tolerant lymphoid populations, such as specific suppression of precursor differentiation must be sought.

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