A Critical Role for Granzymes in 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

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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 of -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 by influencing the mode of tumor and indicate that granzymes contribute to the efficient generation of immune effector pathways in addition to their well-known role in 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 , and uptake of necrotic cells is associated with in- death. The induction of immunogenic cell death is studied using duction of an (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). * 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, ; 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). Purity of the OT-I population previously (19). Expression of the transgenic TCR on in-house–bred was determined using anti-CD8a and anti-Va2 TCR Abs and was rou- C57BL/6.OT-I (WT.OT-I), GrAB.OT-I, and Pfp.OT-I was confirmed by tinely $90%. Subsequently, naive OT-I CTL were labeled with 5 mM staining for CD8a and Va2-TCR by FACS analysis. Mice were used at 6– CFSE and used for in vitro or in vivo cross-presentation assays. 10 wk of age and were handled according to the ethical guidelines of the

Peter MacCallum Cancer Centre and the University of Melbourne. Purification of splenic DC Downloaded from Complete RPMI 1640 medium containing 2 mM glutamine, 50 mM2- mercaptoethanol, 100 mM nonessential amino acids, 100 U/ml penicillin, Nycodenz (Axis-Shield) was made up to a density of 1.077 g/cm3 and 310 100 mg/ml streptomycin, 1 mM sodium pyruvate, and 10 mM HEPES mOsm according to previously published protocols (22). Briefly, spleens supplemented with 10% FBS was used in all experiments with primary were chopped and digested using 1 mg/ml collagenase (Worthington) and cells. The Ab clones used in this study were 53-6.7 (CD8a), N418 0.01% DNase and stopped after 20 min by addition of 0.1 M EDTA so- (CD11c), PC61.5 (CD25), MEL-14 (CD62L), H1.2F3 (CD69), GL1 lution. Splenocytes were loaded onto Nycodenz gradients and centrifuged (CD86), eBio25-D1.16 (SIINFEKL on H-2kb) (all eBioscience), 3/23 at 1700 3 g for 10 min at 4˚C. Light density fraction was collected, and (CD40), 16-10A1 (CD80), 1D4B (CD107a), AF6-88.5 (H-2kb), 2G9 (I- CD11c+ cells were enriched using a commercial DC isolation kit (Invi- http://www.jimmunol.org/ A/I-E), and B20.1 (Va2-TCR) (all BD Pharmingen). Viability of cells was trogen). After bead enrichment, preparations that contained plasmacytoid determined by the addition of PI during analysis on CantoII or LSRII and conventional DC populations were further purified by FACS (FACS analyzers (Becton Dickinson). Data analysis was performed by FlowJo Vantage–Diva or Aria II; BD) into total CD11chigh. Purity of final DC software (Tree Star). preparation was routinely .98%. Target cell culture In vitro cross-presentation assays All tumor lines were maintained in complete DMEM medium containing For cytotoxic assays, 2 3 106 effector cells in complete RPMI medium was 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, added onto the MC38ova target cell monolayer in 12-well plates with 6 and 10 mM HEPES. The different MC38 lines expressing membrane- comparable confluence (70–80%, equivalent of ∼10 tumor cells) for 4 and bound OVA (MC38ova) or the matching empty vector control (MC38ev) 18 h to ensure comparable amount of Ag present in all cytotoxic cultures. by guest on September 24, 2021 Freshly sorted CD11chigh DC were added at a DC to tumor cell ratio of 1:1 were generated using retroviral transfection (20). For functional assays, the 2 different MC38 lines were seeded in 12- or 24-well plates at least 24 h to these cultures for 3 h, after which CD8a as well as CD8a+ DC sub- before starting cytotoxic assays to ensure a stable monolayer before ad- populations were re-sorted subsequently. Labeling of DC was performed dition of effector cells. Cells of MC38 lines were seeded in increasing using CD11c–allophycocyanin and CD8a–PE–Cy7 Abs, and purity of the concentrations to allow selection of monolayer with comparable conflu- final DC subpopulations was routinely .98%. Finally, 2.5 3 104 DC were 4 ence at the time of effector cell addition. incubated with 5 3 10 CFSE-labeled, naive OT-I T cells in 96-well plates over 3 d, and numbers of proliferating OT-I cells were determined as Activation of effector cells previously published (23). Briefly, 2.5 3 104 blank Sphero beads (BD Pharmingen) were added to each tube, and stopping gate was set to 5 3 Total splenocytes from WT.OT-I, GrAB.OT-I, and Pfp.OT-I mice were 103 beads consistently for all samples. Numbers of proliferating OT-I cells 2 activated in vitro after red cell lysis in 0.83% NH4Cl. C57BL/6 splenocytes were determined from PI CD8+CFSElow cells. Activation markers on vi- were pulsed with 1 mg/ml SIINFEKL peptide (Auspep), irradiated (15 Gy), able, total CFSE+ OT-I populations were characterized using FACS anal- 3 6 and used as stimulator cells. Effector cells (2.5 10 /ml) were seeded at ysis by simultaneous staining with CD8, CD69, CD25, and CD62L Abs. a 1:1 ratio with stimulators in 6-well plates for 3 d in presence of 200 ng/ ml LPS (Escherichia coli). For functional assays, cells were harvested and In vivo cross-presentation assays viable cells obtained by Ficoll density gradient centrifugation. The re- sulting CTL population contained 70–90% blasted CD8a+Va2+ cells as After washing in PBS, cellular content of cytotoxic assays was injected i.p. determined by FACS analysis. into C57BL/6 recipient mice (three mice per group). Animals had received 106 enriched CFSE-labeled, naive OT-I i.v. shortly prior to being chal- Cytotoxic assays lenged. Spleens were harvested after 3 d, and numbers of proliferating OT-I cells per spleen were obtained using blank beads as described earlier. 51 Cytotoxic assays using monochromate ([ Cr]) with activated effector For in vivo titration experiments, indicated numbers of tumor cells killed cells were performed under standard conditions using EL-4/E.G7ova or by GrAB.OT-I or WT.OT.I (incubation over 18 or 4 h, respectively) were MC38ev/MC38ova target cells. Briefly, target cells were labeled with 150 injected i.p., and CFSE dilution of CFSE-labeled naive OT-I T cells was 51 6 mCi [ Cr]/10 cells in serum-free complete DMEM for 60–75 min. Total determined after 3 d. and spontaneous releases for [51Cr] as well as harvesting procedure at the time points indicated were performed as previously described (21). Phagocytosis assays For FACS-based killing assays, E.G7ova target cells were labeled with 5 mM CFSE. Target and effector cells were seeded into 24-well plates at Staining of MC38ova target cells with 5 mM CFSE was performed 24 h prior to starting the assays with different OT-I effectors to allow optimal a target to effector cell ratio of 1:2. Cytotoxic cultures were harvested into 6 chilled PBS to attenuate CTL killing, and morphology was analyzed by adherence of tumor cells in 24-well plates. For cytotoxic assays, 10 ef- flow cytometry. fector cells were added in complete RPMI medium for the indicated times onto MC38ova monolayer selected for comparable confluence at the time high Degranulation of effector cell addition (70–80%). Subsequently, total CD11c sorted DC were added for 3 h to allow phagocytosis. Combined cytotoxic and EL-4 or E.G7ova target cells (2 3 104) were incubated at a target to ef- phagocytosis assays were harvested, stained with CD8a Ab, and analyzed fector ratio of 1:2 in the presence of 2.5 mg/ml FITC-conjugated anti- by flow cytometry. Gating was performed on the CD8a2 or CD8a+ sub- CD107a/LAMP-1 and GolgiStop (BD Pharmingen) for 5 h in 96-well populations to assess CFSE uptake within the different subpopulations. 1168 GRANZYMES REGULATE PHAGOCYTOSIS OF TUMOR CELLS

For PS-blocking experiments with purified, recombinant annexin V (final 25 mg/ml; BD Biosciences), cytotoxic cultures were performed in complete DMEM medium to provide sufficient Ca2+ to allow annexin V binding. Expression of activation markers and cytokine production by DC DC were isolated from FLT3 ligand tumor-bearing mice (24) and finally retrieved from cytotoxic cultures of WT.OT-I or GrAB.OT-I (over 4 or 18 h, respectively) by FACS as described for cross-presentation assays. DC (20,000) were cultivated overnight in complete RPMI medium supple- mented with 10 ng/ml recombinant GM-CSF (MBL). Staining for SIIN- FEKL on H-2Kb and activation markers CD40, CD80, CD86, and I-A/E (all biotinylated, together with CD11c-allophycocyanin and CD8a–PE– Cy7 to define DC subpopulations) was performed after Fc receptor blocking with 2.4G2 Ab clone. Finally, DC were incubated with eFluor 450 conjugated to streptavidin (eBioscience) for analysis. Geometric mean of eFluor 450 fluorescence was assessed from duplicate (CD8a2 DC) or triplicate (CD8a+ DC) values for each Ag. Supernatants from DC cultures were analyzed for IL-12p70 and TNF-a by Cytokine Bead Array (BD Biosciences) according to the manufacturer’s instructions.

Confocal microscopy Downloaded from The CD8a+ DC were re-sorted via FACS from cytotoxic cultures of CFSE- labeled MC38ova target cells incubated either with GrAB.OT-I or WT.OT- I target cells over 4 or 18 h. Cells were attached to slides by centrifugation (250 rpm, 10 min) and then fixed with 3.7% (w/v) paraformaldehyde in 100 mM PIPES, 5 mM MgSO4, 10 mM EGTA, and 2 mM DTT (10 min, room temperature) (25). All DC were imaged in Prolong Gold anti-fade http://www.jimmunol.org/ reagent (Invitrogen). Cell samples were examined using an Olympus Fluoview FV10-ASW1.7 viewer attached to an MRC-1024 Bio-Rad laser- scanning confocal microscope or a Zeiss Axioskop2 microscope at room temperature. All data shown are raw and unaltered. Statistical analysis Statistical analysis was performed using GraphPad Prism software version 5.0a (GraphPad Software).

Results by guest on September 24, 2021 Target cell death mediated by CTL in the absence of GrAB Our experiments aimed to determine whether tumor cells un- dergoing cell death in response to either granzyme-deficient or -sufficient CTL would impact on the induction of an immune response to a tumor Ag. To enable the study of MHC-restricted responses, we generated C57BL/6 TCR-transgenic mice (WT. b OT-I, responding to the OVA peptide257–264 H-2K -SIINFEKL) that also lacked expression of GrAB (GrAB.OT-I). Consistent with previous reports (13, 14), there was minimal killing of OVA- expressing EL-4 (E.G7ova) target cells by GrAB-deficient CTL over a standard 4-h assay; however, the deficiency was partly rescued by extending the incubation time (Fig. 1A). Cytotoxicity was Ag-restricted as no lysis occurred in target cells devoid of peptide (EL-4). For subsequent experiments, it was important to identify assay conditions where equivalent numbers of dead target FIGURE 1. Target cell death induced by GrAB.OT-I. A, Cytotoxicity of 51 cells could be generated in response to WT or GrAB-deficient GrAB.OT-I is delayed. Cr-labeled E.G7ova or Ag-negative EL-4 target CTL. For example, the experiment of Fig. 1A carried out with cells were incubated with varying numbers of WT.OT-I or GrAB.OT-I effector cells incubated for 4 h (short term) or 22 h (long term). Graphs E.G7ova cells showed that equivalent release of [51Cr] could be show means 6 SEM of triplicates of one representative experiment. B, achieved at an E:T ratio of 10 if the incubation time was extended Delayed killing phenotype induced by GrAB-deficient effector cells is from 4 up to 22 h. The results of many (n = 7–19) cytotoxic assays highly reproducible. Summary of all [51Cr] release assays with E.G7ova or are summarized in Fig. 1B, demonstrating the strong repro- SIINFEKL-pulsed EL-4 at a E:T ratio of 10:1 given as means + SEM. ducibility of these killing kinetics. Black bars, WT.OT-I; white bars, GrAB.OT-I. Statistical analysis by one- To ensure that genuine target cell death (as observed with [51Cr] way ANOVA including Bonferroni posttests. C, Long-term killing assay release) was induced by GrAB-deficient CTL over the extended with GrAB.OT-I results in comparable morphology of dead target cells as incubation time, CFSE-labeled E.G7ova were cocultured with that of short-term killing assay with WT.OT-I. CFSE-labeled E.G7ova either WT or GrAB-deficient CTL and analyzed for their scatter target cells were incubated with WT.OT-I or GrAB.OT-I at an E:T ratio of profile by flow cytometry (Fig. 1C). Unlike cells that undergo 2:1 for 4 or 22 h. Morphology of target cells was analyzed by categorizing morphology as live cells (FSChighSSClow), dead/dying cells (FSClow necrosis, cells killed through the granule pathway characteristi- SSChigh), and debris (FSClowSSClow). *p , 0.05, ***p , 0.001. n.s., not cally undergo both a reduction in size (reduced forward scatter) significant. and increased granularity (increased side scatter) as they die (26). The Journal of Immunology 1169

As seen with [51Cr] release assays, few dead target cells were (MC38ova) was also delayed in response to GrAB.OT-I CTL (Fig. observed at 4 h in response to GrAB.OT-I CTL. With prolonged 3A). Following similar kinetics to dying E.G7ova cells, incubation incubation time, however, the extent of target cell death ap- of MC38ova with GrAB.OT-I over 18 h resulted in comparable proached that seen with WT.OT-I CTL. Minimal killing was ob- cytotoxicity to incubation with WT.OT-I over 4 h and was con- served using OT-I CTL deficient in perforin (Pfp.OT-I) even under firmed in several similar assays (n = 4–5; summarized in Fig. 3B). prolonged incubation (Supplemental Fig. 1), showing that killing To assess cross-presentation of the OVA Ag, purified CD11chigh through other death pathways was minor in this setting. DC were added to simultaneously completed cytotoxic cultures To show that GrAB-deficient cells exhibit the same level of comprising MC38ova cells together with either WT or GrAB-deficient activation as WT CTL, we compared the phenotype of both effector TCR transgenic CTL (for overview of the experimental sche- populations. Stimulation with syngeneic SIINFEKL-pulsed spleno- dule, see Supplemental Fig. 2A). After 3-h incubation, CD8a2 cytes resulted in similar expression patterns of the activation and CD8a+ DC subpopulations were reisolated via FACS, incu- markers CD69, CD25, and CD62L by GrAB.OT-I and WT.OT-I bated with CFSE-labeled, naive WT.OT-I lymphocytes, and T cell cells over the activation period (Fig. 2A). Reduced cytotoxicity proliferation was assessed by quantifying the number of respond- was not due to impairment of granule exocytosis, as both CTL ing T cells showing CFSE dilution (Fig. 3C for individual histo- populations showed exposure of the lysosomal marker CD107a in grams and quantified in Fig. 3D). Marked T cell proliferation was response to cognate target cells (exemplified in Fig. 2B, and data seen in response to the CD8a+ DC exposed to target cells killed by pooled over four independent assays in Fig. 2C). Both CTL WT CTL in cytotoxic assays. However, cross-presentation was populations also showed equivalent secretion of IFN-g upon en- greatly reduced when CD8a+ DC were exposed to target cells countering EG.7ova target cells (Fig. 2D). Differences in target killed by GrAB.OT-I from long-term cultures. OT-I T cell pro- Downloaded from cell death in response to GrAB.OT-I or WT.OT-I CTL could liferation represented genuine DC-dependent cross-presentation, as therefore not be put down to inadvertent changes in activation CD8a2 DC failed to induce OT-I proliferation under the same status, exocytosis of granule contents, or induction of effector conditions. Live tumor cells cocultured with DC induced minimal cytokines. cross-presentation of the OVA Ag (Fig. 3C,3D), most likely due to the small number of dead cells in those populations. Further, irra- Cell death mediated by GrAB-deficient CTL causes impaired diated MC38ova target cells alone did not induce proliferation of http://www.jimmunol.org/ cross-presentation in vitro naive OT-I cells (see Supplemental Fig. 2B), excluding the possi- Our next aim was to determine whether our observations with bility that SIINFEKL presented by tumor cells could directly ac- E.G7ova could be generalized to other OVA-expressing tumor tar- tivate OT-I T cells. get cells. Consistent with our results on E.G7ova target cells, kill- The reduction in cross-presentation in response to target cells ing of OVA-expressing MC38 mouse adenocarcinoma target cells killed by GrAB.OT-I CTL did not occur simply as a result of by guest on September 24, 2021

FIGURE 2. Activation status of WT.OT-I and GrAB.OT-I. A, Expression of activation markers on GrAB.OT-I and WT.OT-I is comparable. Repre- sentative profiling of the indicated activation markers on WT.OT-I and GrAB.OT-I was performed daily over 3 d. To measure activation markers on viable (PI2) OT-I T cells, CD8a–Pacific blue+ cells were analyzed simultaneously for expression of CD25–Alexa Fluor 750, CD62L–allophycocyanin, and biotinylated CD69 detected by streptavidin–PE–Cy7. Histograms of one experiment of six independent measurements are depicted. B, Degranulation of WT.OT-I and GrAB.OT-I in response to OVA Ag is comparable. GrAB.OT-I or WT.OT-I were incubated either without target cells or with EL-4 or E.G7ova targets for 5 h in the presence of FITC-conjugated anti-CD107a and GolgiStop. CD107a/LAMP-1 exposure was determined by flow cytometry from 2500 PI2CD8a–PE–Cy7+ events. Representative dot plots of one out of four independent experiments are depicted. C, CD107a exposure on GrAB.OT-I or WT. OT-I upon contact with EL-4 (white bars) or E.G7ova (black bars) targets was measured as described for B. Means + SEM summarize frequencies of CD107a+CD8a+ CTL from four independent degranulation assays (duplicate values), which were corrected by subtracting the background of the matching CTL in the absence of tumor cells. Statistical analysis using Mann–Whitney U test. D, GrAB.OT-I and WT.OT-I secrete comparable amounts of IFN-g after cognate target cell contact. GrAB.OT-I or WT.OT-I were incubated for 3 h with either EL-4 or E.G7ova target cells, and IFN-g secretion of viable CD8a– PE–Cy7+ CTL was measured using commercial bispecific Ab technology (allophycocyanin-conjugated anti–IFN-g). Representative histograms of three independent experiments are depicted. n.s., not significant. 1170 GRANZYMES REGULATE PHAGOCYTOSIS OF TUMOR CELLS Downloaded from http://www.jimmunol.org/

FIGURE 3. Cross-presentation of target cells killed by GrAB-deficient CTL is reduced in vitro. A, Delayed killing of MC38ova by GrAB.OT-I. [51Cr] 1 release of MC38ova incubated with increasing numbers of WT.OT-I or GrAB.OT-I over 4 and 18 h (short or long term, respectively). Means SEM are by guest on September 24, 2021 shown from triplicates of one representative experiment. B, Summary of all [51Cr] release assays with MC38ova at an E:T ratio of 10:1 given as means + SEM. Statistical analysis by one-way ANOVA including Bonferroni posttests. C and D, Target cell death induced by WT.OT-I results in cross-presentation. Cytotoxic cultures of MC38ova with WT.OT-I or GrAB.OT-I were established over 4 and 18 h, respectively, and subsequently total CD11chigh DC were added for 3 h. In addition, CD11chigh DC were also added to wells for 3 h with MC38ova without any CTL to assess influence of tumor cells alone. CD8a+ and CD8a2 DC were re-sorted thereafter and subsequently cultured with CFSE-labeled, naive WT.OT-I for 3 d. Representative histograms are shown in C and quantification showing means + SEM, performed using beads for calibration gated on PI2CD8+CFSElow cells, is depicted in D. Graphs shown are representative from four independent repeats, and statistical analysis was performed using t test. *p , 0.05, **p , 0.01, ***p , 0.001. n.s., not significant. the prolonged incubation, as extending the length of the cytotoxic- presentation was clearly dependent on target cell death, as pro- ity assay performed with WT.OT-I to 18 h did not diminish the longed incubation of MC38ova target cells with Pfp.OT-I CTL proliferation of naive responder T cells (Fig. 4A). Also, cross- also greatly impaired subsequent cross-presentation (Fig. 4B),

FIGURE 4. In vitro cross-presentation of target cells killed by GrAB-deficient CTL is dependent on cell death. A, Prolonged tumor cell incubation with WT. OT-I in cytotoxic cultures does not impair cross-presentation. The experimental setup as described for Fig. 3C with cytotoxic cultures over 4 and 18 h for both CTL types. B, Cell death mediated in the presence of perforin and granzymes is necessary for the induction of cross-presentation. Cytotoxic cultures, including Pfp.OT-I as effector cells (long term), were set up as described for Fig. 3B.GraphsdepictedinA and B are representative of at least three independent experiments (black bars, CD8a+ DC; white bars, CD8a2 DC) and represent means + SEM. C, Long-term incubation with perforin-deficient CTL does not result in cell death. Cytotoxic cultures were established with MC38ova tumor cells and various effector cells over 18 h. Specific [51Cr] release was determined as means + SD from triplicates of one representative of three independent experiments. Statistical analysis using t test: *p , 0.05, **p , 0.01. n.s., not significant. The Journal of Immunology 1171 as a result of reduced target cell death in the absence of perforin in long-term cytotoxic assays. Again, the impact of other cell death pathways induced by CTL could be excluded, as killing of MC38ova tumor cells was minimal upon incubation with perforin- deficient CTL (Fig. 4C). Despite the marked reduction in T cell proliferation in the absence of perforin or GrAB, the expression of activation markers CD69, CD25, and CD62L on the few T cells that did undergo cell division was similar to T cells responding to MC38ova target cells killed by WT CTL (Supplemental Fig. 3A). Reduced in vivo cross-priming in response to target cells killed by GrAB.OT-I CTL Subsequently, we wanted to examine whether in vivo priming of the OVA response was also abnormal when tumor cells were killed in the absence of GrAB. For this purpose, the cellular content of in vitro cytotoxic assays with MC38ova exposed to either WT.OT-I (short term) or GrAB.OT-I (long term) was injected i.p. into naive C57BL/6 mice that had also received naive, CFSE-labeled OT-I

T cells i.v. prior to Ag challenge. The number of proliferating Downloaded from OT-I responder cells in the spleen was determined after 3 d (Fig. 5A). Consistent with our previous in vitro results, the num- ber of proliferating T cells was significantly reduced in recipi- ents of MC38ova target cells killed by GrAB.OT-I (Fig. 5B). Al- though differing quantitatively, the quality of the T cell response

within both groups was comparable as indicated by similar http://www.jimmunol.org/ numbers of cell divisions and expression of activation markers (Supplemental Fig. 3B). Administration of increasing numbers of MC38ova cells killed FIGURE 5. In vivo cross-presentation of tumor cells killed in the ab- by GrAB.OT-I (long term) or WT.OT-I (short term) revealed a sence of GrAB is impaired. A and B, In vivo proliferation induced by strong dose dependence of T cell activation in vivo (Fig. 5C). This MC38ova target cells killed in the absence of granzymes is markedly re- effect was only apparent using MC38ova killed by WT.OT-I; duced. MC38ova target cells were incubated in vitro with either GrAB.OT- however, priming efficacy of tumor cells killed in the absence of I for 18 h (long term) or WT.OT-I for 4 h (short term), and cellular content granzymes was strongly impaired even at high doses of MC38ova. was subsequently injected i.p. into WT C57BL/6 mice after i.v. transfer of 6 10 CFSE-labeled, naive WT.OT-I cells. Spleens were obtained after 3 d, by guest on September 24, 2021 Tumor cell death in the absence of granzymes results in and OT-I response was analyzed by flow cytometry. A, Representative impaired phagocytosis by CD8a+ DC gating examples from mice having received cytotoxic cultures with WT.OT-I or GrAB.OT-I effector cells are given. B, Quantification by A critical event in Ag cross-presentation is the acquisition of tu- gating on PI2CD8a+CFSElow cells from total splenocytes using calibration mor Ag through phagocytosis of the cell-associated Ag by DC. beads as shown in A. Means + SEM from one experiment with three mice + 2 Therefore, we determined the capacity of CD8a and CD8a DC per group of four independent repeats with similar results are shown. C, to phagocytose dead/dying, CFSE-labeled MC38ova target cells In vivo titration of WT.OT-I killed tumor cells induces dose-dependent after their incubation with either GrAB.OT-I or WT.OT-I CTL. proliferation of naive OT-I T cells. MC38ova target cells were incubated Purified CD11chigh DC were added to simultaneously finished in vitro with either GrAB.OT-I for 18 h (long term) or WT.OT-I for 4 h cytotoxic cultures and analyzed for CFSE content 3 h later (Fig. (short term), and indicated numbers of tumor cells were administered i.p. 6A,6B). Bright CFSE+ CD8a+ DC became evident when the DC to WT C57BL/6 that had received CFSE-labeled, naive WT.OT-I cells. were added to MC38ova killed by WT.OT-I CTL over 4 h and Readout was performed as described after 3 d. Graph shows means + SEM of three animals per group of one of three independent experiments. Sta- were even more pronounced after 18 h, indicating phagocytic tistical analysis using t test: *p , 0.05. uptake, but not for CD8a2 DC (see Supplemental Fig. 4A). How- ever, far less phagocytosis was seen when MC38ova were incu- bated with GrAB.OT-I CTL or with no CTL at all. Despite the fact similar in CD8a+ DC exposed to short-term cytotoxic cultures of that similar levels of target cell death occurred in response to target cells and WT.OT-I (Fig. 6E,6F). This indicated that even GrAB.OT-I CTL after 18 h, no significant phagocytosis of the when CD8a+ DC were incubated with equal amounts of tumor Ag, dead cells was observed on prolonged incubation. No phagocy- their reduced capacity to phagocytose tumor cells killed in the tosis whatever occurred when Ag-negative MC38ev cells were absence of granzymes resulted in reduced Ag cross-presentation used as targets in the cytotoxicity assays (Fig. 6B and Supple- but comparable expression of activation markers. mental Fig. 4A). Fluorescence microscopy also confirmed that When we first described membrane changes during tumor cell the flow cytometry findings represented authentic target cell en- killing in the absence of GrAB using live cell microscopy, the most gulfment rather than nonspecific cell adhesion (Fig. 6C; single- obvious difference in cell death was the lack of early PS exposure channel images and histograms of the CD8a+ DC population can on the dying target cell (17), a hallmark of generic apoptosis but be found in Supplemental Fig. 4B). also a key event for engulfment by phagocytes (27). Therefore, we Analysis of CD8a+ DC upon engulfment of MC38ova killed in aimed to investigate phagocytosis in the presence of PS blockage the presence or absence of granzymes revealed a marked reduction by annexin V and cross-presentation of target cell from cytotoxic of SIINFEKL peptide reaching the cell surface bound to H-2Kb assays with WT.OT-I (which induce early PS exposure). Cytotoxic (Fig. 6D) when tumor cells were killed by GrAB.OT-I. However, cultures were established with CFSE-labeled MC38ova target expression of activation markers and cytokine production were cells and activated WT.OT-I effector cells over 4 h, and recom- 1172 GRANZYMES REGULATE PHAGOCYTOSIS OF TUMOR CELLS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. Impaired phagocytosis of tumor cells killed in the absence of GrAB. CD11c+ DC were added to cytotoxic cultures of CFSE-labeled MC38ova tumor cells and GrAB-sufficient and -deficient effectors to allow phagocytic uptake. After 3 h, DC were identified by CD11c–allophycocyanin staining, and DC subpopulations were defined by CD8a–PE–Cy7 expression by flow cytometry. A, CFSE uptake was analyzed for each subpopulation as depicted schematically. B, Tumor cell death in the presence of GrAB leads to phagocytosis by CD8a+ DC. CFSE-labeled MC38ova or MC38ev target cells were incubated with either GrAB.OT-I or WT.OT-I in cytotoxic assays for 4 or 18 h (short term and long term). Gray, DC alone; orange, tumor cells without CTL; red, GrAB.OT-I; green, WT.OT-I. Histograms are shown from one representative of four independent experiments. CFSE histograms of CD8a2 DC are shown in Supplemental Fig. 4A. C, CD8a+ DC incubated with tumor cells from cytotoxic assays with WT.OT-I show uptake of CFSE-labeled particles. DC from the above-described experiment were re-sorted and fixed on slides for confocal microscopy (360 oil immersion); scale bars, 20 mm. Images are shown from one of two independent experiments; single-channel images can be found in Supplemental Fig. 4B. D, Impaired phagocytosis results in reduced presentation of SIINFEKL on H-2Kb molecules of CD8a+ DC. CD8a2 and CD8a+ DC were re-sorted from cytotoxic cultures of CFSE-labeled MC38ova tumor cells together with GrAB.OT-I or WT.OT-I (over 18 or 4 h, respectively). DC (20,000) were seeded overnight and labeled with CD11c–allophy- cocyanin, CD8a–PE–Cy7, and biotinylated eBio25-D1.16, recognizing SIINFEKL on H-2kb visualized by eFluor 450 bound to streptavidin. Gray, tumor cells without CTL; red, GrAB.OT-I; green, WT.OT-I. Histograms are representative of five experiments, and graph summarizes results from the corre- sponding experiment with triplicate values. E and F, Activation status of CD8a+ DC from cytotoxic cultures with GrAB.OT-I and WT.OT-I is similar. In parallel to H-2Kb expression as shown in D, we analyzed activation markers CD40, CD80, CD86, and MHC class II (I-A/E) expression and the production of IL-12p70 and TNF-a from supernatants by CD8a2 and CD8a+ DC using CBA. One representative experiment of three independent repeats is depicted as means + SEM in D–F. Data were analyzed using t test: **p , 0.01. The Journal of Immunology 1173 binant annexin V (25 mg/ml) was added to these cultures shortly Despite DC being exposed to equal numbers of dead/dying before addition of DC. The addition of soluble annexin V had tumor cells (Fig. 3A,3B), the cross-presentation of OVA Ag was no effect on phagocytosis by CD11chighCD8a+ DC and subsequent markedly impaired when MC38ova cells were killed by GrAB- cross-presentation (Fig. 7A,7B). By comparison, this amount of deficient effectors (Fig. 3C,3D). Our in vitro studies focused on annexin V was sufficient to inhibit macrophage-mediated uptake the ability to prime a specific CTL response by CD8a+ DC, the of MC38ova tumor cells killed by WT.OT-I under the same con- most effective cross-presenting cells within the immune system. ditions (Supplemental Fig. 4C). The data indicate that other As an internal control, we used CD8a2 DC reisolated from the phagocytic signals in addition to PS exposure are important for same cytotoxic cultures and exposed to the same amount of Ag as target cell uptake by DC and that these signals are lacking in target stimulators of naive OT-I T cells. This allowed us to determine cells killed in the absence of GrAB. genuine cross-presentation rather than nonspecific carryover of tumor cell Ag from the cytotoxic cultures. In addition, MC38ova Discussion cells alone did not activate OT-I T cells directly, either when Many studies during the past years have addressed the question of presented on live cells or in response to irradiation (Fig. 3 and the immunogenicity of target cell death associated with various Supplemental Fig. 2B). The observed effect was also independent death stimuli such as gamma- or UV-irradiation, treatment with on the DC to T cell ratio used in our assay system (Supplemental agonistic Abs or chemotherapeutic drugs, or repeated freeze–thaw Fig. 2C). Finally we showed that on CD8a+ DC, far fewer H-2Kb cycles. However, cell death mediated by granule exocytosis via molecules were occupied by SIINFEKL peptide after being ex- perforin-dependent granzyme delivery has not been studied to posed to tumor cells killed in the absence granzymes (Fig. 6D), date. In this study, we used GrAB-sufficient and -deficient CTL to whereas levels of activation signals were comparable with those Downloaded from kill tumor cells expressing the model Ag OVA to examine the role DC exposed to tumor cells killed by WT.OT-I (Fig. 6E,6F). of granzymes in cross-presentation. Our results clearly demon- Therefore, we could demonstrate that granzyme-mediated cell strate in vitro and in vivo that granzymes induce an immunogenic death was necessary for phagocytosis and subsequent cross- form of cell death by influencing the phagocytic uptake of cell- presentation of cell-associated Ag. associated Ag. There is strong consensus that GrB potently induces cell death

through direct activation of procaspases and/or proteolytic pro- http://www.jimmunol.org/ cessing of Bid (26). Although GrA has been shown to kill target cells when applied with perforin in vitro (28), it was also recently shown to induce the secretion of proinflammatory cytokines such as IL-1b from activated macrophages in a perforin-dependent manner (29). Of interest, IL-1b has lately been shown to be critical in tumor Ag presentation in vivo (30). Our findings are therefore consistent with an emerging body of work identifying previously unsuspected functions of granzymes in the induction of an immune response in addition to their well-described role in by guest on September 24, 2021 cytotoxicity. The intriguing mechanistic observation of our study is that the reduced Ag cross-priming was brought about by defective Ag uptake by CD8a+ DC. We found that only tumor cell death in- duced by granzyme-sufficient CTL resulted in strong phagocyto- sis, as confirmed by both flow cytometry and confocal microscopy (Fig. 6). By marked contrast, CTL-mediated killing in the absence of granzymes (GrAB.OT-I) resulted in only minor engulfment of tumor cells. As described previously (17), the lack of PS exposure on dying cells characterized the altered mode of tumor cell death induced by GrAB-deficient CTL compared with the “classic” apoptotic morphology induced by WT CTL, which features early exteriorization of PS. Exposure of PS is the major “eat-me” signal for uptake of apoptotic cells by phagocytes (31). However, studies using blockade of PS exposure by recombinant annexin V on apoptotic RMA lymphoma cells demonstrated the impact of PS on FIGURE 7. Inhibition of PS exposure does not inhibit phagocytosis and engulfment by macrophages but not DC, indicating that other cross-presentation of tumor cells killed by WT.OT-I. A, Blockage of PS by molecular signals are required for uptake by DC (32). Using + annexin V does not inhibit engulfment of WT.OT-I killed tumor cells. annexin V to block PS-mediated recognition or uptake by CD8a CFSE-labeled MC38ova tumor cells were incubated over 4 h with either DC, we could confirm that PS is not critical for uptake and sub- GrAB.OT-I or WT.OT-I. FACS-sorted CD11chigh total DC were added for sequent cross-presentation by CD8a+ DC (Fig. 7A,7B). In turn, 3 h, and additionally, phagocytosis in WT.OT-I cytotoxic cultures was this would suggest that during cell death induced by GrAB- allowed in presence or absence of 25 mg/ml recombinant, purified annexin high + deficient CTL, factors other than PS exposure on the outer leaf- V. Histograms display uptake by CD11c CD8a as analyzed by flow let of the plasma membrane are necessary to initiate uptake cytometry. B, Block of PS by annexin V does not effect cross-presentation and eventual cross-presentation by CD8a+ DC. Various receptors of WT.OT-I killed tumor cells by CD8a+ DC. PS-blocking cultures were set up as described above, and CD11chighCD8a2 or CD11chighCD8a+ are described that may be involved in phagocytosis and cross- subpopulations were re-sorted and used in cross-presentation assays. One presentation of Ags by DC, which include Tim-3 and Tim-4, representative experiment of three independent repeats is depicted in A and various integrins and complement receptors; however, this topic B. Bars represent means 1 SEM of triplicate values, analyzed by t test: is still quite controversial and debated (33–36). In this context, one *p , 0.05, ***p , 0.001. n.s., not significant. of the potentially interesting signals might be the unidentified 1174 GRANZYMES REGULATE PHAGOCYTOSIS OF TUMOR CELLS ligand for Clec9A, a C-type lectin-like receptor expressed on 5. Sancho, D., O. P. Joffre, A. M. Keller, N. C. Rogers, D. Martı´nez, P. Hernanz- a+ Falco´n, I. Rosewell, and C. Reis e Sousa. 2009. Identification of a dendritic cell CD8 DC shown to be critical for Ag cross-presentation of ne- receptor that couples sensing of necrosis to immunity. Nature 458: 899–903. crotic cells (5). Furthermore, another intriguing aspect is the im- 6. Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, and pact of granzymes on various intrinsic danger signals such as P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine , high-mobility group box 1 molecules, heat shock mechanisms involving TGF-beta, PGE2, and PAF. J. Clin. Invest. 101: 890– proteins, ATP, or uric acid exposed or released during cell death 898. that are known to enhance immunogenicity (8, 9, 30, 37, 38). 7. Kim, S., K. B. Elkon, and X. Ma. 2004. Transcriptional suppression of interleukin-12 gene expression following phagocytosis of apoptotic cells. Im- There have been publications to date showing that tumor cells munity 21: 643–653. killed by CTL can release high-mobility group box 1 molecules or 8. Obeid, M., T. Panaretakis, N. Joza, R. Tufi, A. Tesniere, P. van Endert, are able to induce calreticulin exposure on tumor cells (39, 40); L. Zitvogel, and G. Kroemer. 2007. Calreticulin exposure is required for the immunogenicity of gamma-irradiation and UVC light-induced apoptosis. Cell however, the impact of granzymes in these particular reports is Death Differ. 14: 1848–1850. still unclear. 9. Obeid, M., A. Tesniere, F. Ghiringhelli, G. M. Fimia, L. Apetoh, J. L. Perfettini, Our findings are likely to be particularly relevant where tumor M. Castedo, G. Mignot, T. Panaretakis, N. Casares, et al. 2007. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat. Med. 13: 54–61. cells are resistant to receptor-mediated (Fas, TNF, TRAIL) cell 10. Brusa, D., S. Garetto, G. Chiorino, M. Scatolini, E. Migliore, G. Camussi, and death, resulting in a reliance on the granzyme/perforin pathway to L. Matera. 2008. Post-apoptotic tumors are more palatable to dendritic cells and enhance their antigen cross-presentation activity. Vaccine 26: 6422–6432. kill the cell. It is well known that cell death through Fas, TNF, or 11.Sauter,B.,M.L.Albert,L.Francisco,M.Larsson,S.Somersan,and TRAIL results in classic apoptosis, with early PS exposure leading N. Bhardwaj. 2000. Consequences of cell death: exposure to necrotic tumor to efficient phagocytosis. It is also well recognized that tumors can cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J. Exp. Med. 191: 423–434. reduce their sensitivity to these death mechanisms, for example by 12. Hirt, U. A., and M. Leist. 2003. Rapid, noninflammatory and PS-dependent Downloaded from downregulating receptor levels, or through the expression of cy- phagocytic clearance of necrotic cells. Cell Death Differ. 10: 1156–1164. toplasmic inhibitors such as dominant-negative Fas-associated 13. Pardo, J., S. Balkow, A. Anel, and M. M. Simon. 2002. The differential con- tribution of granzyme A and granzyme B in cytotoxic T lymphocyte-mediated death domain or cellular FLIP (41). In such circumstances, the apoptosis is determined by the quality of target cells. Eur. J. Immunol. 32: 1980– lack of expression of GrAB by CTLs or the additional expression 1985. of intrinsic granzyme inhibitors (serpins) by the tumor would be 14. Simon, M. M., M. Hausmann, T. Tran, K. Ebnet, J. Tschopp, R. ThaHla, and A. Mu¨llbacher. 1997. In vitro- and ex vivo-derived cytolytic leukocytes from predicted to result in a marked deficiency in phagocytosis and granzyme A x B double knockout mice are defective in granule-mediated apo- http://www.jimmunol.org/ therefore reduced cross-presentation of tumor Ag. Together with ptosis but not lysis of target cells. J. Exp. Med. 186: 1781–1786. 15. Heusel, J. W., R. L. Wesselschmidt, S. Shresta, J. H. Russell, and T. J. Ley. 1994. our previous studies (17, 42), we have now shown that PS fails to Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA be displayed on a variety of cells attacked by GrAB-deficient fragmentation and apoptosis in allogeneic target cells. Cell 76: 977–987. CTLs, including RMA-S and MS9II, and that reduced phagocy- 16. Ebnet, K., M. Hausmann, F. Lehmann-Grube, A. Mu¨llbacher, M. Kopf, M. Lamers, and M. M. Simon. 1995. Granzyme A-deficient mice retain potent tosis of dead/dying cells can be demonstrated for both E.G7ova cell-mediated cytotoxicity. EMBO J. 14: 4230–4239. and MC38ova (this study). Our studies extend to both class I- 17. Waterhouse, N. J., V. R. Sutton, K. A. Sedelies, A. Ciccone, M. Jenkins, restricted CTLs and NK cells, suggesting our observations will S. J. Turner, P. I. Bird, and J. A. Trapani. 2006. Cytotoxic T lymphocyte-induced killing in the absence of granzymes A and B is unique and distinct from both have impact when either the innate or the adaptive immune system apoptosis and perforin-dependent lysis. J. Cell Biol. 173: 133–144. is engaged to respond to a potentially immunogenic tumor. Fi- 18. Pham, C. T. N., D. M. MacIvor, B. A. Hug, J. W. Heusel, and T. J. Ley. 1996. by guest on September 24, 2021 nally, the magnitude of the immunogenicity induced by GrAB in Long-range disruption of gene expression by a selectable marker cassette. Proc. Natl. Acad. Sci. USA 93: 13090–13095. comparison with other types of cell death via TNFR superfamily 19. Voskoboinik, I., V. R. Sutton, A. Ciccone, C. M. House, J. Chia, P. K. Darcy, members or chemotherapeutic drugs remains to be determined. H. Yagita, and J. A. Trapani. 2007. 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