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 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 Helper activity is required for the in vivo generation of cytotoxic T lymphocytes

1982 ◽  
Vol 155 (3) ◽  
pp. 768-782 ◽  
Author(s):  
J-A Keene ◽  
J Forman
Keyword(s):  
Immune Response ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Gene Control ◽  
Progeny Testing ◽  
Spleen Cells ◽  
Helper Activity ◽  
Ctl Activity

B6.T1a(a) (Qa-1(a)) mice that are primed in vivo and restimulated in vitro with Qa-1 congenic spleen cells from B6 (Qa-1(b)) animals are unable to generate anti-Qa-1(b) cytotoxic T lymphocytes (CTL). This nonresponsive pattern was observed regardless of the route of immunization or the time of testing in vitro. Although B6.T1a(a) mice are nonresponders to Qa-1(b) when presented on B6 cells, these mice can generate anti-Qa-1(b) CTL when primed in vivo with Qa-1 and H-Y alloantigens (females primed with B6 male cells) or Qa-1 and minor-H- alloantigens (primed with sex-matched A.BY cells). Therefore, the inability to generate anti-Qa-1(b) CTL is due to a lack of helper or accessory antigens on B6 immunizing cells obligatory during in vivo priming, rather than an absence of anti-Qa-1(b) CTL precursors (CTL-P). Demonstration that the additional determinants required during in vivo priming actually function as carrier or helper determinants was shown by the requirement for linked recognition of Qa-1 and the helper determinants (H-Y) in vivo, and the fact that H-Y was not present on susceptible target ceils. Animals primed in vivo with H-Y only could not generate anti-Qa-1 CTL activity when challenged in vitro with both Qa-1 and H-Y, indicating that recognition of the helper determinant causes in vivo priming of CTL-P rather than generating helper activity that might activate unprimed CTL-P in vitro. Whereas unprimed peripheral CTL-P require the presence of both Qa-1 (CTL) and H-Y (helper) determinants for successful in vivo priming, helper determinants were not required in vitro because primed CTL-P from B6.T1a(a) mice could be driven to CTL in vitro using sex-matched B6 stimulator cells. The generation of anti-Qa-1(b) CTL is under immune response (Ir) gene control because F(1) mice, obtained by crossing responder A/J with nonresponder B6.T1a(a) animals, generated CTL to the Qa-1(b) alloantigen when presented on B6 spleen cells. Progeny testing of backcross mice further demonstrated that the Ir gene(s) is linked to the H-2 complex. These data indicate that an H-2-linked Ir gene controls the recognition of helper determinants required for CTL priming in vivo. These helper determinants can be distinguished from CTL determinants and both must be recognized together for successful priming of CTL-P.

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.

In Vitro Generation of Cytotoxic T Lymphocytes against MAGE A1 and MAGE A3 Derived From Healthy Donors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4079-4079
Author(s):  
Lei Bao ◽  
Mindy M Stamer ◽  
Kimberly Dunham ◽  
Deepa Kolaseri Krishnadas ◽  
Kenneth G Lucas
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Lymphocytes ◽  
Cell Therapy ◽  
Cytotoxic T Lymphocytes ◽  
Cd8 T Cells ◽  
Target Cells ◽  
Ifn Gamma ◽  
Healthy Donors

Abstract Abstract 4079 Poster Board III-1014 MAGE A1 and MAGE A3 are cancer testis antigens that are expressed on a number of malignant tumor cells, but not by normal cells, except for male germ cells which lack HLA expression. Therefore, MAGE cytotoxic T lymphocytes are strictly tumor-specific. Adoptive transfer of antigen specific cytotoxic T lymphocytes (CTL) provides immediate graft-versus tumor effects while minimizing risk for graft-versus-host disease. The aim of the current study was to find ideal conditions for expansion of CTL targeting tumor-associated antigens from peripheral blood mononuclear cells (PBMCs) of healthy donors to be used in allogenic cell therapy. In this study we investigated the ability to generate MAGE A1 and MAGE A3 specific cytotoxic T cells using autologous dendritic cells (DC) loaded with MAGE A1 and MAGE A3 overlapping peptides. CTL lines specific for MAGE A1 and MAGE A3 were established by stimulating CD8 T cells from healthy donors with autologous dendritic cells loaded with MAGE A1 or MAGE A3 overlapping pooled peptides in round-bottomed, 96-well plates. CD8+ T cells were restimulated with the same ratio of peptide pulsed DC on days 7 and 14 in the presence of IL-2 (50 U/ml), IL-7 and IL-15 (5 ng/ml). These microcultures were screened 10 days after the third stimulation for their capacity to produce interferon-gamma (IFN-gamma) when stimulated with autologous EBV-transformed B lymphocytes (BLCL) transduced with lentivirus(LV) encoding MAGE A1 or MAGE A3 and autologous BLCL transduced with LV encoding GFP. MAGE A1 and MAGE-A3 specific IFN-gamma producing cells were rapidly expanded in OKT3 and IL2. The specificity of the rapidly expanded MAGE A1 and MAGE A3 specific T cells was confirmed by IFN-gamma production as measured by intracellular cytokine staining and ELISA as well as antigen specific cytotoxicity by a standard 51chromium (51Cr) release assay. We successfully generated MAGE A1 and MAGE A3 specific CTL lines from healthy donors using this method. Specific CTL lines showed cytotoxicity in vitro not only to target cells pulsed with MAGE A1 or MAGE A3 peptides but also to target cells transduced with LV-MAGE A1 or LV-MAGE A3. Specific cytolytic activity was accompanied by IFN-gamma secretion. These data indicate that tumor antigen specific CTL can be expanded using overlapping peptides regardless of an individual's HLA specificity. The ability to generate tumor specific CTL from donors of various HLA backgrounds provide a rationale for utilizing MAGE A1 and MAGE A3 overlapping peptides for expansion of antigen specific T cells for adoptive T-cell therapy against MAGE A1 or MAGE A3 expressing tumors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Enumeration of immune interferon-producing cells induced by allogeneic stimulation

1980 ◽  
Vol 28 (2) ◽  
pp. 542-545
Author(s):  
Y Ito ◽  
H Aoki ◽  
Y Kimura ◽  
M Takano ◽  
K Maeno ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Lymphocytes ◽  
Peak Level ◽  
Spleen Cells ◽  
Antigen Stimulation ◽  
Immune Interferon ◽  
Allogeneic Stimulation

We succeeded in enumerating interferon-producing cells induced by allogeneic stimulation, and proved that they were indeed T lymphocytes. The peak level of these cells in spleen was attained on day 5 after immunication, was maintained for about 2 days, and declined thereafter. Titers of interferon produced in vitro by sensitized spleen cells were maximum on day 7. This suggests that the maturation of immune interferon-producing cells follows cell proliferation after antigen stimulation.

ENUCLEATED CYTOTOXIC T LYMPHOCYTES BIND SPECIFICALLY TO TARGET CELLS IN VITRO

1980 ◽  
Vol 29 (5) ◽  
pp. 374-378 ◽  
Author(s):  
YAEL KAUFMANN ◽  
GIDEON BERKE
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Target Cells

scholarly journals Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo.

1996 ◽  
Vol 183 (1) ◽  
pp. 317-322 ◽  
Author(s):  
P Paglia ◽  
C Chiodoni ◽  
M Rodolfo ◽  
M P Colombo
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Soluble Protein ◽  
Target Cells ◽  
Gm Csf ◽  
Dc Line

The priming of an immune response against a major histocompatibility complex class I-restricted antigen expressed by nonhematopoietic cells involves the transfer of that antigen to a host bone marrow-derived antigen presenting cell (APC) for presentation to CD8+ T lymphocytes. Dendritic cells (DC), as bone marrow-derived APC, are first candidates for presentation of tumor-associated antigens (TAA). The aim of this study was to see whether DC are able to prime in vivo antigen-specific cytotoxic T lymphocytes after exposure to a soluble protein antigen in vitro. Lacking a well-defined murine TAA, we took advantage of beta-galactosidase (beta-gal)-transduced tumor cell lines as a model in which beta-gal operationally functions as TAA. For in vivo priming both a DC line, transduced or not transduced with the gene coding for murine GM-CSF, and fresh bone marrow-derived DC (bm-DC), loaded in vitro with soluble beta-gal, were used. Priming with either granulocyte macrophage colony-stimulating factor-transduced DC line or fresh bm-DC but not with untransduced DC line generated CTL able to lyse beta-gal-transfected target cells. Furthermore, GM-CSF was necessary for the DC line to efficiently present soluble beta-gal as an H-2Ld-restricted peptide to a beta-gal-specific CTL clone. Data also show that a long-lasting immunity against tumor challenge can be induced using beta-gal-pulsed bm-DC as vaccine. These results indicate that effector cells can be recruited and activated in vivo by antigen-pulsed DC, providing an efficient immune reaction against tumors.

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.

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