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

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.

Figure 1

Both perforin-granzyme and FasL pathways contribute to rejection of EG7 tumors upon treatment with anti-CD137 mAbs. Wild type (A), perforin and granzyme A and B knockout (PAB^-/-) (B) and FasL-mutant gld (C) mice were injected s.c. with 5 × 10⁵ EG7 tumor cells and treated i.p. with 100 μg of control Rat IgG or anti-CD137 mAb on days 8, 10, 12 and 14 after tumor cell challenge. Mean tumor diameters were sequentially measured 2-3 times per week. 6 mice per group were included. Statistical comparisons were performed using a nonlinear regression statistical method (Y= (MaxVol * exp(X-TimeO))/( 1 + exp((X-TimeO)/RateGrowth)) with GraphPad software. ***, P<0.001 were considered statistically significant.

Figure 2

The cytotoxic mechanisms responsible for EG7 tumor rejection are present in TILs after treatment with anti-CD137 mAb. Mice were challenged s.c. with 5 × 10⁵ EG7 cells and treated i.p. with anti-CD137 mAb on days 9 and 11. Two days later, tumors were removed and TILs were analyzed by flow cytometry for intracellular granzyme B (A left) and percentage of H-2K^b-SIINFEKL tetramer⁺ cells (A right), surface FasL (B), intracellular IFNγ (C) and surface CD107a (D) expression on gated CD8⁺ and CD4⁺ T lymphocytes. Histograms show CD8⁺ gated T cells that come from a representative experiment. Grey histograms represent isotype-matched control antibody stainings and open histograms protein-specific surface or intracellular stainings from representative cases. The insets in each histogram show the mean fluorescense intensity (MFI) after subtracting the background staining or the percentage of CD107a positive cells (D) for the indicated immunostainings on CD8⁺ and CD4⁺ T cells from individual tumors. The experiment was performed with 7-8 mice per group and mean±SEM is included in each histogram.

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.