Essential complicity of perforin-granzyme and FAS-L mechanisms to achieve tumor rejection following treatment with anti-CD137 mAb
- Aizea Morales-Kastresana1,
- Elena Catalán2,
- Sandra Hervás-Stubbs1,
- Asis Palazón1,
- Arantza Azpilikueta1,
- Elixabet Bolaños1,
- Alberto Anel2,
- Julián Pardo†3 and
- Ignacio Melero†1Email author
© Morales-Kastresana et al.; licensee BioMed Central Ltd. 2013
Received: 6 November 2012
Accepted: 5 February 2013
Published: 29 May 2013
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.
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.
CD137-elicited rejection of EG7-derived tumors involves the interplay of at least two final effector cytolytic mechanisms that act in cooperation.
CD137 agonists hold promise to augment antitumor immune responses in a clinically significant fashion  and two fully human monoclonal antibodies (mAbs) are currently undergoing clinical development (BMS-663513 and PFZ-05082566). Hematological malignancies are not exception to the therapeutic effects of anti-CD137 mAbs and activity has been reported on experimental models of lymphoma, myeloma and mastocytomas [2–4]. The mechanism of action depends mainly on cytolytic T lymphocytes (CTLs) since depletion of CD8β T cells completely abrogates the therapeutic effect . The train of events is complex and needs antigen priming by dendritic cells  and in some tumor models the participation of natural killer (NK) lymphocytes as observed in selective depletion experiments . More recently, evidence has been published in the sense that anti-CD137 mAb enhances NK-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) [7, 8], in a way that can be exploited to enhance the antitumor activity of Herceptin and Rituximab.
Evidence has been reported showing that activated CD8+ tumor infiltrating lymphocytes (TILs) express CD137  and therefore are amenable to receive artificial costimulation by agonist anti-CD137 mAbs within the malignant tissue microenvironment. The execution of tumor rejection requires production of interferon (IFN) γ by CTLs as demonstrated by neutralizing mAbs  and with T cells derived from IFNγ-/- mice . However, little is known about the final effector mechanisms that mediate tumor cell killing. CTLs and NK cells may kill using perforin-granzyme, FasL and TNF-related apoptosis inducing ligand (TRAIL) as the executioner molecules [11–14]. Experiments performed in the EG7 tumor model whose successful treatment does not require NK cells  clearly show that both the cytolytic granule and the FasL-mediated killing mechanisms were synergistically involved in achieving complete rejections of these lymphomas.
Results and discussion
Perforin, granzymes A and B and FasL are involved in tumor rejection elicited by anti-CD137 mAbs
Experiments performed in perforin and granzyme A and B triple knockout mice (PAB-/-) indicated that although the therapeutic activity was reduced, a residual beneficial effect remained, resulting in two out of six complete rejections (Figure 1B). Conceivably, the FasL-Fas route could also be involved in the execution of rejection by CTLs. Indeed, performing the experiment in mice deficient for FasL (gld mice) also resulted in partial loss of the immunotherapeutic activity of anti-CD137 mAb (Figure 1C). These results are interpreted in the sense that pore-forming and granzyme entrance to malignant cells need to be complemented by FasL-mediated induction of apoptosis in order to optimally achieve tumor rejection.
Previously published evidence had shown that CTL production of IFNγ was required for tumor rejection  and we observed that 15.7 ± 3.1% of CD8+ TILs intracellularly express this cytokine without need for any ex vivo restimulation of the intratumoral lymphocytes (Figure 2C).
CD107a (LAMP.1) is a cytolytic granule transmembrane protein which emerges at killing synapses and remains transiently on the plasma membrane of effector cells. 7.8 ± 1.6% of CD8+ TILs were caught expressing surface CD107a, thus demonstrating that they were actively degranulating at the time of tumor harvest (Figure 2D). The fact that they are holding this “smoking gun” tells of their active participation in cytotoxicity and tumor rejection. In a separate experiment we were able to gate onto T lymphocytes stained with CD8+ and H–2 Kb-SIINFEKL tetramers and analyze the expression of effector molecules in this subset. As can be seen in Additional file 1: Figure S1, these CTLs co-express Granzyme B, IFNγ and CD107a.
EG7 tumor cells are synergistically killed by the perforin-granzyme and the FasL pathways
Therefore at least under these conditions, FasL and the cytolytic granule machinery seem to synergistically operate to bring about death of these lymphoma cells. TRAIL engagement of its death promoting receptors could have also been involved, but this was ruled out since EG7 is not killed by 1μg of a recombinant form of TRAIL that readily killed Jurkat cells as a positive control (Additional file 2: Figure S2B).
Materials and methods
Mice and cell lines
C57BL/6 wild type mice (6-8 weeks old) were purchased from Harlan Laboratories. Mice deficient for perforin and granzymes A and B (PAB-/-) or with a mutation in FasL (gld mice) were a kind gift of Markus Simon (Max-Planck Institute of Immunobiology, Freiburg, Germany). PAB-/- and gld mice were bred into the BL/6 background. Animal experimentation followed FELASA guidelines and approval of the Ethics Committee for Animal Experimentation from CITA (2011-01) and University of Navarra (study number 066/10). The murine thymoma cell line EG7 was a kind gift from Dr. Claude Leclerc (Institut Pasteur, Paris, France) and was authenticated using the master cell banks by RADIL (Case number: 6592-2012). Jurkat cells, L1210 and Fas-transfected L1210 have been described [17, 18].
In vivo tumor growth
5 × 105 EG7 cells were subcutaneously injected into the flank of WT, PAB-/- or gld mice. Mice were intraperitoneally treated with 100 μg/dose of anti-CD137 or control Rat IgG on days 8, 10, 12 and 14 following tumor cell inoculation. Rat IgG antibody was purchased from Sigma-Aldrich and anti-CD137 clon 1D8  was from Bristol-Myers Squibb (Lawrenceville, NJ). Mice and tumor size were monitored twice a week and mice were sacrificed when the tumor size reached 300 mm2.
Preparation of cell suspensions from EG7 tumors
Tumors were excised and incubated for 15 minutes at 37°C in a solution containing Collagenase-D+DNase-I (Roche) in RPMI. Afterwards, tumors were disrupted mechanically and passed through a 70-μm cell strainer (BD Falcon). Erythrocytes from cell suspensions were lysed with potassium ammonium chloride lysing buffer.
Single-cell suspensions were pretreated with FcR-Block (anti-CD16/32 clone 2.4G2). Anti-CD3ϵ, anti-CD8α, anti-CD4, anti-FasL, anti-IFNγ, anti-granzymeB and isotype Rat IgG and Hamster IgG isotype controls were all purchased from Biolegend; anti-CD107a, anti-Fas, mouse IgG isotype control and Apoptosis Detection Kit-I were from BD Pharmingen; and anti-TRAIL mAb was from eBioscience. H-2Kb-SIINFEKL tetramers were purchased from Beckman Coulter. For intracellular stainings, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences). Cells were studied with FACSCanto II or FACSCalibur and were analyzed using FlowJo (TreeStar software). Specific mean fluorescense intensity (MFI) and percentage values in graphs are represented after subtraction of the control isotype-matched signal.
Fas and TRAIL induced cell death assays
105 cells were cultured with human recombinant TRAIL, purified anti-Fas antibody (clone Jo2) or isotype control (both from BD Biosciences) for 18 h. Human recombinant TRAIL functional on human and mouse receptors was produced in E.coli and purified as previously described . Cell death was analyzed by Annexin V and 7-aminoactinomycin D (7-AAD) double staining.
Generation of ex vivo gp33-specific CD8+ cells
Mice were infected with 105 pfu LCMV-WE i.p. as described . On day eight after infection, CD8+ cells were positively selected from spleen using anti-CD8-MicroBeads (Miltenyi Biotec). Purity of selected CD8+ cells was higher than 95%.
Ex vivo cytotoxicity assay
Target cells were pre-incubated with the LCMV-immunodominant peptide gp33 (acquired from Neosystem Laboratory) and effector CD8+ cells were stained with CellTracker Green (Invitrogen). Effector and target cells were incubated at a ratio of 10:1 (effector:target) during 4 hours. In certain experiment anti-FasL blocking antibody (clone MFL3) (BD Biosciences) was added during the 4 h cytotoxicity assay. Gated CellTracker Green-negative targets were analyzed for annexin V and 7-AAD double staining.
As a whole, we have demonstrated using a tumor model amenable to successful immunotherapy with anti-CD137 mAb that the FasL and perforin-granzyme killing machineries act non-redundantly and synergistically to execute complete tumor rejections upon therapy with agonist anti-CD137 mAb.
Cytotoxic T lymphocyte
Antibody-dependent cell-mediated cytotoxixity
Tumor infiltrating lymphocyte
TNF-related apoptosis inducing ligand
Perforin and granzyme A and B
Mean fluorescense intensity
7- Aminoactinomycin D.
The authors would like to thank to Luis Martinez Lostao and Diego de Miguel for providing recombinant human TRAIL protein. We are grateful to Dr. Maria Jure-Kunkel (Bristol Myers Squibb) for kindly providing 1D8 monoclonal antibody.
This work was supported by grants from MEC/MICINN of Spain (SAF2008-03294; SAF2011-22831; SAF2011-25390), Departamento de Educación and Departamento de Salud del Gobierno de Navarra, Redes temáticas de investigación cooperativa RETIC (RD06/0020/0065), European commission VII framework program (ENCITE), SUDOE-IMMUNONET and “UTE for project FIMA”. JP is supported by Aragón I+D (ARAID) and Gobierno de Aragón/Fondo Social Europeo. AMK and EC receive an FPI scholarship from MEC (Spain), AP a scholarship from FIS from MSC (Spain) and SHS is supported by a Ramon y Cajal contract from MICINN (Spain).
- Ascierto PA, Simeone E, Sznol M, Fu YX, Melero I: Clinical experiences with anti-CD137 and anti-PD1 therapeutic antibodies. Semin Oncol. 2010, 37: 508-516. 10.1053/j.seminoncol.2010.09.008.View ArticlePubMedGoogle Scholar
- Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA, Hellstrom KE, Mittler RS, Chen L: Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med. 1997, 3: 682-685. 10.1038/nm0697-682.View ArticlePubMedGoogle Scholar
- Wilcox RA, Flies DB, Zhu G, Johnson AJ, Tamada K, Chapoval AI, Strome SE, Pease LR, Chen L: Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors. J Clin Invest. 2002, 109: 651-659.PubMed CentralView ArticlePubMedGoogle Scholar
- Murillo O, Arina A, Hervas-Stubbs S, Gupta A, McCluskey B, Dubrot J, Palazon A, Azpilikueta A, Ochoa MC, Alfaro C: Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma. Clin Cancer Res. 2008, 14: 6895-6906. 10.1158/1078-0432.CCR-08-0285.PubMed CentralView ArticlePubMedGoogle Scholar
- Murillo O, Dubrot J, Palazon A, Arina A, Azpilikueta A, Alfaro C, Solano S, Ochoa MC, Berasain C, Gabari I: In vivo depletion of DC impairs the anti-tumor effect of agonistic anti-CD137 mAb. Eur J Immunol. 2009, 39: 2424-2436. 10.1002/eji.200838958.View ArticlePubMedGoogle Scholar
- Melero I, Johnston JV, Shufford WW, Mittler RS, Chen L: NK1.1 cells express 4-1BB (CDw137) costimulatory molecule and are required for tumor immunity elicited by anti-4-1BB monoclonal antibodies. Cell Immunol. 1998, 190: 167-172. 10.1006/cimm.1998.1396.View ArticlePubMedGoogle Scholar
- Kohrt HE, Houot R, Goldstein MJ, Weiskopf K, Alizadeh AA, Brody J, Muller A, Pachynski R, Czerwinski D, Coutre S: CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies. Blood. 2011, 117: 2423-2432. 10.1182/blood-2010-08-301945.PubMed CentralView ArticlePubMedGoogle Scholar
- Kohrt HE, Houot R, Weiskopf K, Goldstein MJ, Scheeren F, Czerwinski D, Colevas AD, Weng WK, Clarke MF, Carlson RW: Stimulation of natural killer cells with a CD137-specific antibody enhances trastuzumab efficacy in xenotransplant models of breast cancer. J Clin Invest. 2012, 122: 1066-1075. 10.1172/JCI61226.PubMed CentralView ArticlePubMedGoogle Scholar
- Palazon A, Martinez-Forero I, Teijeira A, Morales-Kastresana A, Alfaro C, Sanmamed MF, Perez-Gracia JL, Penuelas I, Hervas-Stubbs S, Rouzaut A: The HIF-1alpha hypoxia response in tumor-infiltrating T lymphocytes induces functional CD137 (4-1BB) for immunotherapy. Cancer Discov. 2012, 2: 608-623. 10.1158/2159-8290.CD-11-0314.View ArticlePubMedGoogle Scholar
- Wilcox RA, Flies DB, Wang H, Tamada K, Johnson AJ, Pease LR, Rodriguez M, Guo Y, Chen L: Impaired infiltration of tumor-specific cytolytic T cells in the absence of interferon-gamma despite their normal maturation in lymphoid organs during CD137 monoclonal antibody therapy. Cancer Res. 2002, 62: 4413-4418.PubMedGoogle Scholar
- Lopez JA, Brennan AJ, Whisstock JC, Voskoboinik I, Trapani JA: Protecting a serial killer: pathways for perforin trafficking and self-defence ensure sequential target cell death. Trends Immunol. 2012, 33: 406-412. 10.1016/j.it.2012.04.001.View ArticlePubMedGoogle Scholar
- Pardo J, Aguilo JI, Anel A, Martin P, Joeckel L, Borner C, Wallich R, Mullbacher A, Froelich CJ, Simon MM: The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation. Microbes Infect. 2009, 11: 452-459. 10.1016/j.micinf.2009.02.004.View ArticlePubMedGoogle Scholar
- Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ: Natural innate and adaptive immunity to cancer. Annu Rev Immunol. 2011, 29: 235-271. 10.1146/annurev-immunol-031210-101324.View ArticlePubMedGoogle Scholar
- Kagi D, Vignaux F, Ledermann B, Burki K, Depraetere V, Nagata S, Hengartner H, Golstein P: Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science. 1994, 265: 528-530. 10.1126/science.7518614.View ArticlePubMedGoogle Scholar
- Brossart P, Goldrath AW, Butz EA, Martin S, Bevan MJ: Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL. J Immunol. 1997, 158: 3270-3276.PubMedGoogle Scholar
- Simon MM, Waring P, Lobigs M, Nil A, Tran T, Hla RT, Chin S, Mullbacher A: Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis. J Immunol. 2000, 165: 3663-3672.View ArticlePubMedGoogle Scholar
- Pardo J, Balkow S, Anel A, Simon MM: The differential contribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated apoptosis is determined by the quality of target cells. Eur J Immunol. 2002, 32: 1980-1985. 10.1002/1521-4141(200207)32:7<1980::AID-IMMU1980>3.0.CO;2-Z.View ArticlePubMedGoogle Scholar
- Pardo J, Perez-Galan P, Gamen S, Marzo I, Monleon I, Kaspar AA, Susin SA, Kroemer G, Krensky AM, Naval J, Anel A: A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis. J Immunol. 2001, 167: 1222-1229.View ArticlePubMedGoogle Scholar
- Martinez-Lostao L, Garcia-Alvarez F, Basanez G, Alegre-Aguaron E, Desportes P, Larrad L, Naval J, Martinez-Lorenzo MJ, Anel A: Liposome-bound APO2L/TRAIL is an effective treatment in a rabbit model of rheumatoid arthritis. Arthritis Rheum. 2010, 62: 2272-2282. 10.1002/art.27501.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.