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 Two distinct mechanisms regulate the in vivo generation of cytotoxic T cells.

1982 ◽  
Vol 156 (3) ◽  
pp. 918-923 ◽  
Author(s):  
M S Sy ◽  
S H Lee ◽  
M Tsurufuji ◽  
K L Rock ◽  
B Benacerraf ◽  
...  
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Concanavalin A ◽  
Cytotoxic T Cells ◽  
Cytolytic T Lymphocytes ◽  
Spleen Cells ◽  
Suppressor T Cells ◽  
Con A

Treatment of responder cells with monoclonal anti-Ly-1,2 antibodies plus complement in vitro completely eliminated their ability to generate azobenzenearsonate (ABA)-specific cytolytic T lymphocytes (CTL). However, addition of the concanavalin A-stimulated supernatants of rat spleen cells (Con A-Sup) can fully reconstitute the response. Therefore, Lyt-1,2-bearing T cells are required for the generation of ABA-specific CTL, and such requirement can be replaced by factors present in the Con A- sup. Suppressor T cells (Ts), when adoptively transferred into naive recipients, will inhibit the in vivo priming of CTL. This inhibition can also be reversed by in vitro addition of Con A-Sup. furthermore, mice serving as donors of Ts also show profound unresponsiveness when primed and restimulated in vitro. In contrast to the Ts-mediated inhibition, in vitro addition of Con A-Sup was unable to abolish the unresponsiveness observed in these cultures. Thus, we identified two unresponsive states in a hapten-specific killing system that differ in their ability to be reconstituted by Con A-Sup.

scholarly journals Effector T lymphocytes in lymphocytic choriomeningitis virus-infected mice. Cytolytic activity of Lyt-23 spleen cells in vitro does not correlate with elimination of infectious virus from spleens.

1981 ◽  
Vol 153 (4) ◽  
pp. 992-997 ◽  
Author(s):  
M Varho ◽  
F Lehmann Grube ◽  
M M Simon
Keyword(s):  
T Lymphocytes ◽  
Infectious Virus ◽  
Lymphocytic Choriomeningitis ◽  
Cytolytic Activity ◽  
Lymphocytic Choriomeningitis Virus ◽  
Target Cells ◽  
Spleen Cells ◽  
Marginal Effects ◽  
Infected Cells

The T lymphocytes from mice recovering from infection with lymphocytic choriomeningitis virus were selected for subclasses by treatment with anti-Lyt antisera and complement. Lyt-23 cells and mixtures of lyt-1 and Lyt-23 cells caused up to one-half the destruction of cultivated target cells as compared with untreated T lymphocytes; Lyt-1 cells alone were not cytotoxic. Selected and unselected spleen T cells were also inoculated intravenously into previously infected mice. Whereas unselected cells reduced infectious virus in the spleens of the recipients approximately 100-fold, only marginal effects, which were not preferentially associated with one particular subclass, were seen with selected LYT-23 or Lyt-1 lymphocytes or a mixture of both. Apparently the Lyt-23 cells, shown to by cytolytic for infected cells in vitro, did not cause elimination of a measurable quantity of the virus from mice.

scholarly journals GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO

1974 ◽  
Vol 140 (3) ◽  
pp. 718-730 ◽  
Author(s):  
H. Robson MacDonald ◽  
Howard D. Engers ◽  
Jean-Charles Cerottini ◽  
K. Theodor Brunner
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Primary Response ◽  
Target Cells ◽  
Spleen Cells ◽  
Response Curves ◽  
Specific Cytotoxicity ◽  
Allogeneic Stimulation ◽  
Ctl Activity

Mouse cytotoxic T lymphocytes (CTL) were generated in unidirectional mixed leukocyte cultures (MLC) using normal C57BL/6 spleen cells as responding cells and irradiated DBA/2 spleen cells as stimulating cells. Cytotoxicity was assayed on 51Cr-labeled P-815 (DBA/2) target cells, and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Upon inclusion of 2-mercaptoethanol in the culture medium, it was found that significant CTL activity could be detected for as long as 3 wk in primary MLC. Reexposure of MLC cells to the original stimulating alloantigens after 14–41 days in culture resulted in significant cell proliferation and rapid regeneration of high levels of immunologically specific cytotoxicity. CTL activity in these secondary cultures increased dramatically within the first 24 h and reached higher peak levels than those found at the peak of the primary response. Furthermore, proliferation and reappearance of CTL activity could be demonstrated following each of as many as four sequential alloantigenic stimulations of the same initial cell population at 20-day intervals. Interestingly, cells recovered from MLC at the peak of the primary response on day 4 were insensitive to further allogeneic stimulation. Taken together, these results are consistent with the hypothesis that CTL differentiate in MLC to become long-lived memory cells which gradually lose their cytotoxic activity. Upon reexposure to specific alloantigen, such memory CTL rapidly regain their functional activity and proliferate to generate an expanded CTL population.

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.

Targeting of «T» Lymphocytes against Human Hepatoma Cells by a Bispecific Monoclonal Antibody: Role of Different Lymphocyte Subsets

1992 ◽  
Vol 78 (2) ◽  
pp. 79-86 ◽  
Author(s):  
Qi Chen ◽  
Peinan Sun ◽  
Ignazia Prigione ◽  
Hong Xie ◽  
Silvano Ferrini
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Hepatoma Cells ◽  
Peripheral Blood Lymphocytes ◽  
Cytolytic Activity ◽  
Cytotoxic T Cell ◽  
Target Cells ◽  
Human Hepatoma ◽  
Human Hepatoma Cells

In an attempt to construct bispecific monoclonal antibodies (bimAbs) able to target cytotoxic T lymphocytes against human hepatoma cells, an HGPRT-deficient mutant of the Hepama-6 hybridoma, which produces an antihuman-hepatoma mAb, was directly fused with splenocytes from Balb/C mice immunized by a polyclonal cytotoxic T-cell line. Hybrid hybridomas were selected in HAT medium, and their supernatants were directly screened for the ability to induce IL-2-cultured cytotoxic T lymphocytes to kill hepatoma cells in a 51Cr-release assay. The selected hybrid hybridoma, termed DQ-33, secretes a bimAb, which reacts with a CD3-associated determinant. When resting peripheral-blood lymphocytes were used as effector cells, virtually no cytolytic activity could be induced by DQ-33, whereas phytohemagglutinin-activated lymphocytes that had been expanded in vitro in IL-2-containing medium could be efficiently targeted against hepatoma cells. Targeting by DQ-33 bimAb was analyzed on different subsets of IL-2-cultured lymphocytes. It was evident that CD+4–8+ TCRα/β+ and CD3+4–8-TCRγ/δ+ lymphocytes were efficiently induced by bimAb to lyse human hepatoma cells, whereas no induction of cytolysis could be observed when CD3 + 4+8-TCRα/β+ cells were used as effectors. DQ-33 bimAb was also able to induce lymphokine secretion (IL-2, GM-CSF and TNF-α) by all the different subsets of lymphocytes analyzed in the presence of target cells expressing the relevant antigen, independent of the expression of cytolytic activity.

scholarly journals Cross-reactive lysis of trinitrophenyl (TNP)-derivatized H-2 incompatible target cells by cytolytic T lymphocytes generated against syngeneic TNP spleen cells.

1976 ◽  
Vol 144 (6) ◽  
pp. 1609-1620 ◽  
Author(s):  
S J Burakoff ◽  
R N Germain ◽  
B Benacerraf
Keyword(s):  
T Lymphocytes ◽  
Red Blood Cells ◽  
Target Cell ◽  
Blood Cells ◽  
Surface Antigens ◽  
Lymphoid Cells ◽  
Target Cells ◽  
Cytolytic T Lymphocytes ◽  
Spleen Cells ◽  
Normal Spleen

Normal spleen cells, when cultured with irradiated trinitrophenyl (TNP)-derivatized syngeneic spleen cells, develop cytotoxic effectors that lyse most effectiviely a TNP-derivatized target that is H-2 compatible with the effector. However, these effectors also lyse to a lesser extent TNP tumor and TNP spleen targets that are H-2 incompatible. This cross-reactive lysis correlates with the degree of cytolysis seen on the TNP-derivatized syngeneic target; it appears to be medicated by Thy 1.2-bearing cells and is inhibited by antisera to the K and/or D loci of the target cell and not by antisera to non-K or non-D surface antigens. Nonradiolabeled TNP-derivatized lymphoid cells syngeneic to either the stimulator or the target are able to competitively inhibit cross-reactive lysis, while TNP chicken red blood cells are unable to specifically inhibit lysis. These data on cross-reactive lysis of TNP-conjugated targets are most consistent with the altered-self hypothesis.

scholarly journals CELLS MEDIATING SPECIFIC IN VITRO CYTOTOXICITY

1972 ◽  
Vol 135 (4) ◽  
pp. 890-906 ◽  
Author(s):  
Pierre Golstein ◽  
Hans Wigzell ◽  
Henric Blomgren ◽  
Erik A. J. Svedmyr
Keyword(s):  
T Cells ◽  
In Vitro Cytotoxicity ◽  
Cell Populations ◽  
Target Cells ◽  
Present System ◽  
Spleen Cells ◽  
A Cell ◽  
Thymus Cells ◽  
Immune Spleen Cells

In order to investigate whether only T cells are involved in a cell-mediated cytotoxic system in vitro, we tested the cytotoxicity of immune killing cell populations as deprived as possible of B cells. Educated thymus cells, immune spleen cells purified by filtration through a column of beads coated with antimouse Ig antiserum, and finally educated thymus cells further purified by filtration through such a column fully retained their specific cytotoxic activity. This very strongly suggests that only T cells are involved in the killing of target cells by allogeneic immune cells in vitro, in this system. Receptor-bearing cells involved in killing in the present system are thus very probably T cells. This point was further strengthened by the demonstration of specific adsorption, on the relevant monolayers, of each of the three above mentioned killing cell populations.

scholarly journals GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO

1974 ◽  
Vol 140 (3) ◽  
pp. 703-717 ◽  
Author(s):  
Jean-Charles Cerottini ◽  
Howard D. Engers ◽  
H. Robson MacDonald ◽  
K. Theodor Brunner
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Relative Number ◽  
Fold Increase ◽  
Culture Conditions ◽  
Target Cells ◽  
Spleen Cells ◽  
Response Curves ◽  
Cytotoxic Response

Mouse cytotoxic T lymphocytes (CTL) were generated in mixed leukocyte cultures (MLC) using spleen cells as responding cells and irradiated allogeneic spleen cells as stimulating cells. Cytotoxicity was assessed by a quantitative 51Cr assay system and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Inclusion of 2-mercaptoethanol in the MLC medium resulted in a 20–40-fold increase in the relative number of CTL generated at the peak of the response. Under these culture conditions, cell-mediated cytotoxic activity was detectable in MLC populations as early as 48 h after the onset of the cultures. When spleen cells from mice immunized with allogeneic tumor cells 2–4 mo previously were cultured with irradiated spleen cells of the same alloantigenic specificity (MLC-Imm), it was found that the cell-mediated cytotoxic response was detectable earlier and reached higher levels than that observed in a primary MLC. At the peak of the response, MLC-Imm populations were observed to lyse up to 50% of the target cells within 3 h at a lymphocyte: target cell ratio of 0.3:1. Immunological and physical characterization of the effector cells generated in MLC-Imm indicated that they were medium to large-sized T lymphocytes. Altogether, these studies suggested the existence of an anamnestic cell-mediated cytotoxic response in MLC-Imm.

scholarly journals The modulation of lymphocyte functions by molecules secreted by macrophages. II. Conditions leading to increased secretion.

1976 ◽  
Vol 144 (1) ◽  
pp. 155-166 ◽  
Author(s):  
E R Unanue ◽  
J M Kiely ◽  
J Calderon
Keyword(s):  
Dna Synthesis ◽  
B Cell Differentiation ◽  
Spleen Cells ◽  
First Case ◽  
Antibody Formation In Vitro ◽  
Increased Activity ◽  
Striking Effect ◽  
Antibody Secreting Cells ◽  
Stimulation Of

Cultures of peritoneal exudate cells rich in macrophages were studied for the secretion of lymphostimulatory molecules. Two conditions produced increased secretion: (a) addition to the cultures of various agents that readily interacted with macrophages, such as latex particles, antibody-coated red cells, endotoxin, Listeria organisms, or Be salt; or (b) addition of activated lymphocytes. In the first case the increased activity was found during the first 24 or 48 h after uptake of the stimuli. Increased activity was found in normal or peptone-stimulated macrophages but not in macrophages after injection of endotoxin or thioglycollate. The addition of T lymphocytes from Listeria-infected mice to macrophage cultures increased greatly the activities. This increase was also produced by addition to antigen-primed T cells together with antigen. The lymphocytes by themselves did not secrete active factors. The lymphostimulatory activities were tested on thymocyte DNA synthesis and on antibody formation in vitro. The latter assay was done on spleen cells from immunized mice where one striking effect was the stimulation of differentiation to antibody-secreting cells. Some dissociation of both activities (thymocyte DNA synthesis and B-cell differentiation) was observed with selected culture fluids.

scholarly journals Clonal heterogeneity in the functional requirement for Lyt-2/3 molecules on cytolytic T lymphocytes: analysis by antibody blocking and selective trypsinization.

1982 ◽  
Vol 156 (6) ◽  
pp. 1711-1722 ◽  
Author(s):  
H R MacDonald ◽  
A L Glasebrook ◽  
J C Cerottini
Keyword(s):  
T Lymphocytes ◽  
Direct Evidence ◽  
Cross Reactivity ◽  
Cytolytic Activity ◽  
Antigenic Determinants ◽  
Target Cells ◽  
Cytolytic T Lymphocytes ◽  
Clonal Heterogeneity ◽  
Flow Microfluorometry

While it is well established that murine cytolytic T lymphocytes (CTL) express the Lyt-2/3 molecular complex on their surface, conflicting results have been reported concerning the role of this complex in CTL activity. In the present study this question was reinvestigated at the clonal level. Although different (H-2b anti-H-2d) CTL clones expressed comparable amounts of Lyt-2/3 molecules, as assessed by quantitative flow microfluorometry, the activity of some clones was inhibited by low doses (10 ng) of monoclonal anti-Lyt-2 or anti-Lyt-3 antibodies (in the absence of complement), whereas other clones were not inhibited by either antibody at doses as high as 5 microgram. Treatment of these clones with doses of trypsin sufficient to cleave Lyt-2/3 antigenic determinants from the cell surface resulted in a similar dissociation: clones that were inhibited by antibodies lost cytolytic activity, whereas "uninhibited" clones were unaffected by trypsin treatment. Moreover, the dissociation observed among different alloreactive clones could be demonstrated with self-H-2-restricted (H-2b anti-MSV) clones exhibiting cross-reactivity with normal H-2d products. The lytic activity of these clones against the relevant syngeneic target cells was unaffected by anti-Lyt-2 antibodies or trypsin, whereas their cross-reactivity on H-2d target cells was abolished by either treatment. These results provide direct evidence for clonal heterogeneity in the requirement for Lyt-2/3 molecules in CTL-mediated lysis. It is proposed that the function of Lyt-2/3 molecules is to stabilize the interaction between CTL receptors and the corresponding antigens on the target cells and that the requirement for such a stabilization is correlated with low number and/or affinity of CTL receptors.

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