Type I IFNs Control Antigen Retention and Survival of CD8 α+ Dendritic Cells after Uptake of Tumor Apoptotic Cells Leading to Cross-Priming This information is current as of September 24, 2021. Silvia Lorenzi, Fabrizio Mattei, Antonella Sistigu, Laura Bracci, Francesca Spadaro, Massimo Sanchez, Massimo Spada, Filippo Belardelli, Lucia Gabriele and Giovanna Schiavoni J Immunol 2011; 186:5142-5150; Prepublished online 25 Downloaded from March 2011; doi: 10.4049/jimmunol.1004163 http://www.jimmunol.org/content/186/9/5142 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/03/25/jimmunol.100416 Material 3.DC1 References This article cites 42 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/186/9/5142.full#ref-list-1 by guest on September 24, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Type I IFNs Control Antigen Retention and Survival of CD8a+ Dendritic Cells after Uptake of Tumor Apoptotic Cells Leading to Cross-Priming Silvia Lorenzi,1 Fabrizio Mattei, Antonella Sistigu, Laura Bracci, Francesca Spadaro, Massimo Sanchez, Massimo Spada, Filippo Belardelli, Lucia Gabriele, and Giovanna Schiavoni Cross-presentation is a crucial mechanism for generating CD8 T cell responses against exogenous Ags, such as dead cell-derived Ag, and is mainly fulfilled by CD8a+ dendritic cells (DC). Apoptotic cell death occurring in steady-state conditions is largely tolero- genic, thus hampering the onset of effector CD8 T cell responses. Type I IFNs (IFN-I) have been shown to promote cross-priming of CD8 T cells against soluble or viral Ags, partly through stimulation of DC. By using UV-irradiated OVA-expressing mouse EG7 Downloaded from thymoma cells, we show that IFN-I promote intracellular Ag persistence in CD8a+ DC that have engulfed apoptotic EG7 cells, regulating intracellular pH, thus enhancing cross-presentation of apoptotic EG7-derived OVA Ag by CD8a+ DC. Notably, IFN-I also sustain the survival of Ag-bearing CD8a+ DC by selective upmodulation of antiapoptotic genes and stimulate the activation of cross-presenting DC. The ensemble of these effects results in the induction of CD8 T cell effector response in vitro and in vivo. Overall, our data indicate that IFN-I cross-prime CD8 T cells against apoptotic cell-derived Ag both by licensing DC and by enhancing cross-presentation. The Journal of Immunology, 2011, 186: 5142–5150. http://www.jimmunol.org/ ross-presentation of cell-associated Ag, such as dead cell- nals by the DC (5). It has been reported that triggering of TLR4 derived Ag, is a crucial process for generating CD8 T cell increases the efficiency of peptide presentation on MHC-I mole- C responses to Ag that are not expressed by APC, such as cules by DC, suggesting that the enhancement of cross-presentation viruses that do not infect APC or tumors of nonhematopoietic may represent another mechanism promoting cross-priming (6). origin (1). Among APC, dendritic cells (DC) are specialized for With regard to the mechanisms regulating cross-presentation, it is cross-presentation, and accumulating literature indicates that known that presentation of particulate Ag, such as cellular Ag, is in vivo this process is mainly fulfilled by the CD8a+ DC subset critically dependent on the timing of persistence within phag- (2). In the steady state, CD8a+ DC constitutively cross-present osomal compartment, a process governed by intraphagosomal pH by guest on September 24, 2021 self Ag, such as material derived from apoptotic cells as a result (6). Studies from Amigorena laboratory have established a strict of constitutive cell turnover, leading to self-tolerance (3). How- correlation between phagosomal alkalinization, which delays the ever, in the context of infection or pathological distress, signals proteolytic activity of lysosomal enzymes, and the efficiency of consisting of microbial compounds or of inflammatory stimuli cross-presentation (7). released by cells of innate immunity act as danger signals that Type I IFNs (IFN-I) are a family of inflammatory cytokines induce DC activation, in a process referred to as licensing of DC produced by innate cells upon pathogenic challenge, playing to cross-priming (4). The underlying molecular mechanisms that multiple roles in the stimulation of immune responses, including result from such licensing are still under investigation and include DC activation and CD8 T cell effector function in vitro and in vivo. enhanced costimulatory signals and diminished proapoptotic sig- Of interest, IFN-I have been shown to promote cross-priming against viral or protein Ag, partly through the stimulation of DC (8–10). Furthermore, cross-priming stimulated by TLR3, TLR4, Dipartimento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanita`, and TLR9 ligands was shown to be critically dependent on IFN-I Rome, Italy signaling, implying these cytokines as important mediators in 1 Current address: Laboratorio di Immunoncologia, Dipartimento di Oncoematologia infection-stimulated cross-priming (9). Recent work from our lab- Pediatrica, Ospedale Pediatrico Bambino Gesu`, Rome, Italy. oratory has shown that human DC generated in the presence of Received for publication December 22, 2010. Accepted for publication February 28, 2011. IFN-a exhibit enhanced cross-presentation of allogeneic apoptotic This work was supported by grants from ACC 2006 National Program 2 (ACC2/ cell Ag to autologous CD8 T cells, suggesting a role for IFN-I in WP5.4) and from the Italian Society for Cancer Research. DC cross-presentation of cell-associated Ag (11). In this study, we Address correspondence and reprint requests to Dr. Giovanna Schiavoni, Diparti- demonstrate that IFN-I promote cross-priming in vivo against cell- mento di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanita`, Viale associated Ag derived from tumor apoptotic cells through multiple Regina Elena, 299, 00166 Rome, Italy. E-mail address: [email protected] actions on CD8a+ DC, as follows: 1) by enhancing Ag persistence The online version of this article contains supplemental material. and, thus, cross-presentation; 2) by sustaining the survival of Ag- Abbreviations used in this article: apoEG7, apoptotic EG7; CLSM, confocal laser- bearing DC selectively; and 3) by activating DC. scanning microscopy; CT, threshold cycle; DC, dendritic cells; DPI, diphenylene iodonium; IFN-DC, IFN-treated DCs; IRF, IFN regulatory factor; LN, lymph node; MFI, mean fluorescence intensity; MHC-OVAp, MHC class I molecule Kb bound to Materials and Methods the peptide SIINFEKL of OVA; NOX2, NADPH oxidase 2; NT-DC, untreated DC; Mice PI, propidium iodide; WT, wild-type. Female C57BL/6 mice (5–7 wk old) and OT-I TCR-transgenic mice were Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 purchased from Charles River Laboratories. IFN regulatory factor (IRF)- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1004163 The Journal of Immunology 5143 82/2 mice were generated and bred, as described (12). All mice were footpads with 5 3 105 apoEG7-DC alone or containing IFN-I (2.5 3 104 manipulated in accordance with the local Ethical Committee guidelines. U/mouse). Three days later, the popliteal LN excised and the resulting cell suspensions were stained with anti-CD8, CD25, and CD69 mAbs and Reagents analyzed by FACS. In vivo priming was also assessed by injecting i.v. 20 3 106 apoEG7 cells alone or plus IFN-I (106 U/mouse) into IRF-82/2 High-titer IFN-I was prepared from the C243-3 cell line, as described in or control recipients, adoptively transferred with CFSE-labeled OT-I cells. detail elsewhere (13). Anti-mouse IFN-I sheep Ig was used at 1000 neu- OT-I proliferative response was measured 3 d later in the spleen. Ag- tralizing units (14). For flow cytometry, the following mAbs were used: specific IFN-g production by CD8 T cells was assessed by ELISPOT as- anti-CD11c, which was used either in FITC, PE, or allophycocyanin form; say using reagents and methods, as recommended by the manufacturer PE anti-CD8, CD25, and CD69; biotin anti-CD40, CD86, and I-A (all from (Mabtech AB). IFN-g spot-forming cells were analyzed by ImageJ soft- BD Pharmingen); tricolor anti-CD8 (Caltag Laboratories); and biotin anti- ware. mouse MHC class I molecule Kb bound to the peptide SIINFEKL of OVA (MHC-OVAp; clone 25-D1.16; eBioscience). Biotinylated mAbs were Quantitative RT-PCR detected with streptavidin PerCP (BD Pharmingen) or streptavidin tricolor (Caltag Laboratories). Lysosensor green DND-189 (Invitrogen) was used Quantitative RT-PCR in sorted populations of splenic DC was performed at 5 mM. Diphenylene iodonium (DPI; Sigma-Aldrich) was used at 5 mM. using Sensimix Plus SYBR kit containing the fluorescent dye SYBR Green (Quantace). Forward and reverse primers (Supplemental Table I) were Isolation of DC and OT-I lymphocytes purchased from Primm. Quality and specificity of amplicons in each sample were detected by dissociation curve analysis. Triplicates were performed The procedure of splenic DC isolation has been described in detail else- for each experimental point. For quantization, threshold cycle (CT) values where (15). Briefly, total splenocytes were subjected to density-gradient were determined by the Sequence Detection System software (Applied centrifugation in Nycodenz solution (1077 g/ml; Life Technologies).