A Critical Role for Granzymes in Antigen Cross-Presentation through Regulating Phagocytosis of Killed Tumor Cells This information is current as Sabine Hoves, Vivien R. Sutton, Nicole M. Haynes, Edwin of September 24, 2021. D. Hawkins, Daniel Fernández Ruiz, Nikola Baschuk, Karin A. Sedelies, Maximilian Schnurr, John Stagg, Daniel M. Andrews, Jose A. Villadangos and Joseph A. Trapani J Immunol 2011; 187:1166-1175; Prepublished online 27 June 2011; Downloaded from doi: 10.4049/jimmunol.1001670 http://www.jimmunol.org/content/187/3/1166 Supplementary http://www.jimmunol.org/content/suppl/2011/06/27/jimmunol.100167 http://www.jimmunol.org/ Material 0.DC1 References This article cites 42 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/187/3/1166.full#ref-list-1 Why The JI? Submit online. by guest on September 24, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Critical Role for Granzymes in Antigen Cross-Presentation through Regulating Phagocytosis of Killed Tumor Cells Sabine Hoves,*,† Vivien R. Sutton,* Nicole M. Haynes,‡ Edwin D. Hawkins,x Daniel Ferna´ndez Ruiz,{ Nikola Baschuk,‡ Karin A. Sedelies,* Maximilian Schnurr,† John Stagg,‡ Daniel M. Andrews,‡ Jose A. Villadangos,‖ and Joseph A. Trapani*,# Granzymes A and B (GrAB) are known principally for their role in mediating perforin-dependent death of virus-infected or ma- lignant cells targeted by CTL. In this study, we show that granzymes also play a critical role as inducers of Ag cross-presentation by dendritic cells (DC). This was demonstrated by the markedly reduced priming of naive CD8+ T cells specific for the model Ag OVA both in vitro and in vivo in response to tumor cells killed in the absence of granzymes. Reduced cross-priming was due to impairment of phagocytosis of tumor cell corpses by CD8a+ DC but not CD8a2 DC, demonstrating the importance of granzymes in inducing the exposure of prophagocytic “eat-me” signals on the dying target cell. Our data reveal a critical and previously Downloaded from unsuspected role for granzymes A and B in dictating immunogenicity by influencing the mode of tumor cell death and indicate that granzymes contribute to the efficient generation of immune effector pathways in addition to their well-known role in apoptosis induction. The Journal of Immunology, 2011, 187: 1166–1175. umor cell death induced by various agents can activate inflammation due to secretion of anti-inflammatory cytokines such http://www.jimmunol.org/ the immune system and generate an Ag-specific response as TGF-b or IL-10 and transcriptional repression of IL-12p35 in T against the tumor, a concept known as immunogenic cell phagocytes, and uptake of necrotic cells is associated with in- death. The induction of immunogenic cell death is studied using duction of an immune response (6, 7). However, immunogenic cell chemotherapeutic drugs, Ab treatment, irradiation, or repeated death has been described to occur as a result of preapoptotic freeze–thaw cycles (1). The in vivo clearance of dead cells is events in response to drug treatment or irradiation (8, 9). Simi- mainly mediated by macrophages, but their engulfment by den- larly, the engulfment of primary or secondary necrotic material dritic cells (DC) and subsequent Ag processing is critical for can lead to an immune response against cellular Ags in some but cross-presentation of cellular Ags. In vitro and in vivo studies not all experimental settings (5, 10–12). revealed a central role for the CD8a+ DC subset in phagocytosis Various laboratories have shown that granzyme A (GrA) and by guest on September 24, 2021 and cross-presentation of cell-associated Ags to prime a specific granzyme B (GrB) activate important and independent cell death CTL response (2–5). The molecular signals associated with vari- pathways and are instrumental in perforin-dependent killing of ous forms of tumor cell death are known to influence the sub- virus-infected and malignant cells (13, 14). The induction of cell sequent immune response: uptake of apoptotic cells minimizes death by CTL from GrB-deficient mice is marked by reduced DNA fragmentation accompanying target cell apoptosis, but this defect can be compensated by increasing the incubation time (15). *Cancer Cell Death Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne 3002, Victoria, Australia; †Medical Clinic, Ludwig Max- In contrast, apoptosis is not defective in GrA-deficient mice (16). imilian University, 80336 Munich, Germany; ‡Cellular Immunity, Cancer Immunol- We recently showed that both NK cells and CTL from mice de- ogy Program, Peter MacCallum Cancer Centre, Melbourne 3002, Victoria, Australia; x ficient in granzymes A and B (GrAB) induce a unique and phe- Immune Signaling Laboratories, Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne 3002, Victoria, Australia; {Department of Microbiology notypically distinct form of target cell death (17). The cell death and Immunology, University of Melbourne, Melbourne 3010, Victoria, Australia; morphology induced by GrAB-deficient CTL appeared similar to ‖Immunology Department, Walter and Eliza Hall Institute of Medical Research, Melbourne 3050, Victoria, Australia; and #Department of Microbiology and Immu- that induced by wild-type (WT) CTL. However, quantitative and nology, University of Melbourne, Melbourne 3052, Victoria, Australia kinetic live cell microscopy showed that cells dying in response to Received for publication May 19, 2010. Accepted for publication May 24, 2011. GrAB-deficient CTL failed to display phosphatidylserine (PS) on S.H. was supported by a fellowship of the Deutsche Forschungsgemeinschaft (Ho their surfaces. Accordingly, dying cells did not bind annexin V 4007/1-1). J.A.T. and V.R.S. were supported by a program grant from the National until their cell membranes had become permeable to propidium Health and Medical Research Council of Australia (454569). J.A.T. received a senior iodide (PI), indicative of secondary necrosis (17). However, the fellowship from the National Health and Medical Research Council of Australia (288999). E.D.H. and D.M.A. were supported by a Peter Doherty fellowship from functional significance of this finding was not further explored. the National Health and Medical Research Council of Australia. J.S. was supported Studies addressing the role of granzymes in priming an immune by a Canadian Institutes of Health Research fellowship. response have not previously been reported. In this study, we used Address correspondence and reprint requests to Dr. Sabine Hoves and Prof. Ag-specific killing of tumor cells by WT or granzyme-deficient Joseph A. Trapani, Cancer Cell Death Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew’s Place, East Melbourne, VIC 3002, CTL to examine the downstream effects of GrAB on generation Australia. E-mail addresses: [email protected] and [email protected] of an immune response. For the first time to our knowledge, we The online version of this article contains supplemental material. show that cell death occurring in the absence of GrAB leads to Abbreviations used in this article: DC, dendritic cell; GrA, granzyme A; GrAB, reduced cross-presentation of a model tumor Ag in vitro and granzymes A and B; GrB, granzyme B; PI, propidium iodide; PS, phosphatidylserine; in vivo. In terms of mechanism, we demonstrate that engulfment of WT, wild-type. tumor cells by DC is critically dependent on the presence of gran- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 zymes. These data define a new and important role of gran- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001670 The Journal of Immunology 1167 zymes in influencing the quality and magnitude of an immune V-bottom plates. Effector cells were subsequently counterstained with + 2 response. This shows that the mode of CTL-mediated cell death CD8a–PE–Cy7 Ab, and finally a total of 2500 CD8a PI events were influences subsequent target cell phagocytosis and Ag cross- analyzed for CD107a/LAMP-1 exposure. presentation and thus has far more profound effects on immu- Cytokine secretion assay nity than previously recognized. Active secretion of IFN-g from activated GrAB.OT-I or WT.OT-I CTL was determined by commercial cytokine secretion assay (Miltenyi Biotech). Materials and Methods For activation, 5 3 104 CTL were incubated in a target to effector ratio of Mice and reagents 1:5 with either EL-4 or E.G7ova target cells for 3 h, and IFN-g cytokine secretion assay was performed according to the manufacturer’s instruc- C57BL/6 and C57BL/6.bm1 mice were obtained from the Walter and Eliza tions thereafter. CTL were additionally stained with CD8a Ab, and a total 2 + Hall Institute of Medical Research (Melbourne, VIC, Australia). C57BL/6. of 5000 PI CD8a events were analyzed for IFN-g–allophycocyanin se- GrAB mice were originally obtained from M. Simon [Max Planck Institut cretion. fu¨r Immunbiologie, Freiburg, Germany (18)] and were maintained at the Peter MacCallum Cancer Centre and crossed with C57BL/6.OT-I mice OT-I enrichment and CFSE labeling (GrAB.OT-I). Genotype of GrAB.OT-I was confirmed by PCR. Perforin- Naive WT.OT-I CTL were enriched after red cell lysis by depletion using deficient C57BL/6.OT-I mice (Pfp.OT-I) were generated as described CD8+ isolation kit (Miltenyi Biotech).