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Essential complicity of perforin-granzyme and FAS-L mechanisms to achieve tumor rejection following treatment with anti-CD137 mAb.

Morales-Kastresana A, Catalán E, Hervás-Stubbs S, Palazón A, Azpilikueta A, Bolaños E, Anel A, Pardo J, Melero I - J Immunother Cancer (2013)

Bottom Line: Rejection of EG7-derived thymomas has been shown to be CTL-dependent but not NK-dependent.In this therapeutic setting, we show that both the perforin-granzyme and FasL effector systems are readily expressed by CD8(+) T lymphocytes infiltrating the EG7 lymphomas which are undergoing rejection.Using knock-out mice, we demonstrate that both effector cytolytic systems are involved in the execution of complete immune rejections against EG7 established tumors.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMA, Gene therapy and Hepatology Unit, University of Navarra, Pamplona, Navarra, Spain.

ABSTRACT

Background: Treatment with agonist anti-CD137 (4-1BB) immunostimulatory monoclonal antibodies elicits complete tumor regressions in a number of transplanted hematological and solid malignancies in mice. Rejection is mainly dependent on cytotoxic T lymphocytes (CTL) and IFNγ, although a role for NK cells and dendritic cells has been observed in some tumor models. Rejection of EG7-derived thymomas has been shown to be CTL-dependent but not NK-dependent.

Findings: In this therapeutic setting, we show that both the perforin-granzyme and FasL effector systems are readily expressed by CD8(+) T lymphocytes infiltrating the EG7 lymphomas which are undergoing rejection. Using knock-out mice, we demonstrate that both effector cytolytic systems are involved in the execution of complete immune rejections against EG7 established tumors. In accordance, EG7 tumor cells were susceptible in vitro to both killing mechanisms acting in a synergistic fashion.

Conclusions: CD137-elicited rejection of EG7-derived tumors involves the interplay of at least two final effector cytolytic mechanisms that act in cooperation.

No MeSH data available.


Related in: MedlinePlus

Fas-FasL pathway and perforin-granzyme machineries synergistically kill EG7 cell line. 5 × 104 EG7 cells were pre-pulsed with LCMV gp33 peptide and cocultured in vitro for 4 h with 5 × 105 CD8+ T cells from LCMV immunized wild type (WT), perforin and granzymes A and B knockout (PAB-/-) or gld mice in the presence or absence of 1 μg/ml of FasL blocking antibody (MFL3). Cell death was determined by annexin V staining in CD8 negative population by flow cytometry. Ctr, control with target cells without gp33 peptide. Data in the graphs are represented as mean±SEM of three independent experiments. Statistical comparisons were performed using Student’s t test with GraphPad software. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant. P<0.05 were considered significant.
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Figure 3: Fas-FasL pathway and perforin-granzyme machineries synergistically kill EG7 cell line. 5 × 104 EG7 cells were pre-pulsed with LCMV gp33 peptide and cocultured in vitro for 4 h with 5 × 105 CD8+ T cells from LCMV immunized wild type (WT), perforin and granzymes A and B knockout (PAB-/-) or gld mice in the presence or absence of 1 μg/ml of FasL blocking antibody (MFL3). Cell death was determined by annexin V staining in CD8 negative population by flow cytometry. Ctr, control with target cells without gp33 peptide. Data in the graphs are represented as mean±SEM of three independent experiments. Statistical comparisons were performed using Student’s t test with GraphPad software. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant. P<0.05 were considered significant.

Mentions: EG7 cells express surface Fas (Additional file 2: Figure S2A inset) and hence anti-Fas agonistic antibody kills these cells in 18 h as happens in Fas-transfected L1210 cells but not in control untransfected cells. To assess the involvement of these pathways in CTL killing of EG7 cells, anti-lymphocytic choriomeningitis virus (LCMV) derived gp33 CTLs that are directed to a peptide presented by H-2Kb were elicited by immunization with LCMV-WE virus. Recovered CD8+ splenocytes from immunized wild type (WT) mice 8 days after infections showed 71% killing at 10:1 effector:target ratio on EG7 cells pulsed with the gp33 synthetic peptide (Figure 3A). Under these experimental conditions optimal immunization allows us to focus on the effector phase of CTL activity. This activity was only reduced by about one half when immunized mice were deficient in PAB. Conversely, FasL neutralization with MFL3 mAb during the 4 h cytotoxicity experiment did not hamper killing. Interestingly, this anti-FasL mAb completely abolished cytotoxicity when used on effector CTL from PAB-/- mice. CTLs from gld mice were as effective as WT mice in accordance with the results with FasL single blockade and, as expected, the MFL3 mAb did not have any effect under these conditions, further confirming the selectivity of the blocking mAb.


Essential complicity of perforin-granzyme and FAS-L mechanisms to achieve tumor rejection following treatment with anti-CD137 mAb.

Morales-Kastresana A, Catalán E, Hervás-Stubbs S, Palazón A, Azpilikueta A, Bolaños E, Anel A, Pardo J, Melero I - J Immunother Cancer (2013)

Fas-FasL pathway and perforin-granzyme machineries synergistically kill EG7 cell line. 5 × 104 EG7 cells were pre-pulsed with LCMV gp33 peptide and cocultured in vitro for 4 h with 5 × 105 CD8+ T cells from LCMV immunized wild type (WT), perforin and granzymes A and B knockout (PAB-/-) or gld mice in the presence or absence of 1 μg/ml of FasL blocking antibody (MFL3). Cell death was determined by annexin V staining in CD8 negative population by flow cytometry. Ctr, control with target cells without gp33 peptide. Data in the graphs are represented as mean±SEM of three independent experiments. Statistical comparisons were performed using Student’s t test with GraphPad software. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant. P<0.05 were considered significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3987045&req=5

Figure 3: Fas-FasL pathway and perforin-granzyme machineries synergistically kill EG7 cell line. 5 × 104 EG7 cells were pre-pulsed with LCMV gp33 peptide and cocultured in vitro for 4 h with 5 × 105 CD8+ T cells from LCMV immunized wild type (WT), perforin and granzymes A and B knockout (PAB-/-) or gld mice in the presence or absence of 1 μg/ml of FasL blocking antibody (MFL3). Cell death was determined by annexin V staining in CD8 negative population by flow cytometry. Ctr, control with target cells without gp33 peptide. Data in the graphs are represented as mean±SEM of three independent experiments. Statistical comparisons were performed using Student’s t test with GraphPad software. *, P<0.05; **, P<0.01; ***, P<0.001; ns, no significant. P<0.05 were considered significant.
Mentions: EG7 cells express surface Fas (Additional file 2: Figure S2A inset) and hence anti-Fas agonistic antibody kills these cells in 18 h as happens in Fas-transfected L1210 cells but not in control untransfected cells. To assess the involvement of these pathways in CTL killing of EG7 cells, anti-lymphocytic choriomeningitis virus (LCMV) derived gp33 CTLs that are directed to a peptide presented by H-2Kb were elicited by immunization with LCMV-WE virus. Recovered CD8+ splenocytes from immunized wild type (WT) mice 8 days after infections showed 71% killing at 10:1 effector:target ratio on EG7 cells pulsed with the gp33 synthetic peptide (Figure 3A). Under these experimental conditions optimal immunization allows us to focus on the effector phase of CTL activity. This activity was only reduced by about one half when immunized mice were deficient in PAB. Conversely, FasL neutralization with MFL3 mAb during the 4 h cytotoxicity experiment did not hamper killing. Interestingly, this anti-FasL mAb completely abolished cytotoxicity when used on effector CTL from PAB-/- mice. CTLs from gld mice were as effective as WT mice in accordance with the results with FasL single blockade and, as expected, the MFL3 mAb did not have any effect under these conditions, further confirming the selectivity of the blocking mAb.

Bottom Line: Rejection of EG7-derived thymomas has been shown to be CTL-dependent but not NK-dependent.In this therapeutic setting, we show that both the perforin-granzyme and FasL effector systems are readily expressed by CD8(+) T lymphocytes infiltrating the EG7 lymphomas which are undergoing rejection.Using knock-out mice, we demonstrate that both effector cytolytic systems are involved in the execution of complete immune rejections against EG7 established tumors.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMA, Gene therapy and Hepatology Unit, University of Navarra, Pamplona, Navarra, Spain.

ABSTRACT

Background: Treatment with agonist anti-CD137 (4-1BB) immunostimulatory monoclonal antibodies elicits complete tumor regressions in a number of transplanted hematological and solid malignancies in mice. Rejection is mainly dependent on cytotoxic T lymphocytes (CTL) and IFNγ, although a role for NK cells and dendritic cells has been observed in some tumor models. Rejection of EG7-derived thymomas has been shown to be CTL-dependent but not NK-dependent.

Findings: In this therapeutic setting, we show that both the perforin-granzyme and FasL effector systems are readily expressed by CD8(+) T lymphocytes infiltrating the EG7 lymphomas which are undergoing rejection. Using knock-out mice, we demonstrate that both effector cytolytic systems are involved in the execution of complete immune rejections against EG7 established tumors. In accordance, EG7 tumor cells were susceptible in vitro to both killing mechanisms acting in a synergistic fashion.

Conclusions: CD137-elicited rejection of EG7-derived tumors involves the interplay of at least two final effector cytolytic mechanisms that act in cooperation.

No MeSH data available.


Related in: MedlinePlus