Apoptotic Dendritic Cells Induce Tolerance in Mice through Suppression of Dendritic Cell Maturation and Induction of Antigen-Specific Regulatory T Cells This information is current as of September 27, 2021. Rahul Kushwah, Jordan R. Oliver, Jinyi Zhang, Katherine A. Siminovitch and Jim Hu J Immunol 2009; 183:7104-7118; Prepublished online 16 November 2009; doi: 10.4049/jimmunol.0900824 Downloaded from http://www.jimmunol.org/content/183/11/7104 Supplementary http://www.jimmunol.org/content/suppl/2009/11/16/jimmunol.090082 http://www.jimmunol.org/ Material 4.DC1 References This article cites 42 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/183/11/7104.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 27, 2021 • 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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Apoptotic Dendritic Cells Induce Tolerance in Mice through Suppression of Dendritic Cell Maturation and Induction of Antigen-Specific Regulatory T Cells1 Rahul Kushwah,*† Jordan R. Oliver,*† Jinyi Zhang,‡¶ Katherine A. Siminovitch,‡¶ and Jim Hu2*† Dendritic cell (DC) apoptosis has been shown to play a role in maintaining a balance between tolerance and immunity. However, the mechanisms of how DC apoptosis affects the immune response are unclear. We have shown that in vitro culture of apoptotic DCs with immature DCs, results in their uptake by immature DCs, which subsequently turn into tolerogenic DCs, which then ؉ ␤ secrete TGF- 1 and induce Foxp3 regulatory T cells (Tregs). In this study we looked at the effects of apoptotic DCs in vivo. Here we show that apoptotic DCs are taken up by viable DCs in vivo, which suppresses the ability of viable DCs to undergo maturation Downloaded from and subsequent migration to the lymph nodes in response to LPS. Additionally, delivery of apoptotic DCs to LPS inflamed lungs results in resolution of inflammation, which is mediated by the ability of apoptotic DCs to suppress response of viable DCs to LPS. Additionally, apoptotic DCs also induce TGF-␤1 secretion in the mediastinal lymph nodes, which results in expansion of Foxp3؉ Tregs. Most importantly, we show that delivery of apoptotic DCs followed by OVA in CFA to mice suppresses T cell response to OVA and instead induces de novo generation of OVA-specific Tregs. Furthermore, delivery of apoptotic DCs followed by OVA in CFA results in expansion of Tregs in TCR transgenic (OT-II) mice. These findings demonstrate that apoptotic DCs are taken up http://www.jimmunol.org/ by viable DCs in vivo, which promotes tolerance through suppression of DC maturation and induction of Tregs. The Journal of Immunology, 2009, 183: 7104–7118. endritic cells (DCs)3 are potent APCs with the ability to cells, analogous to necrotic cells provide danger signals which initiate T cell responses. DCs are present in the periph- induce activation of DCs and subsequent induction of T cells (4). D eral tissues where they are constantly engulfing Ags In contrast to necrotic cells, apoptotic cells are thought to be along with dying cells. However, the ability to initiate a T cell cleared rapidly without any immunological response (3, 5). Studies response depends on the ability of DC to undergo maturation and have identified necrotic cells acting as adjuvants whereas apoptotic by guest on September 27, 2021 subsequently migrate to draining lymph nodes, where in the pres- cells have been reported as immunogenic (6–8) or immunosup- ence of appropriate cytokines, T cells can be differentiated into a pressive (9, 10). particular lineage in an Ag-specific manner. In addition to priming Though studies have been conducted to look at the effects of effector T cell responses, DCs also play a role in induction of apoptotic or necrotic cells on DCs, there is lack of information on tolerance. Due to the ability of DCs to engulf dead cells, they are the effects of apoptotic or necrotic DCs on viable DCs. DC apop- affected by the death of other cells in close proximity (1–3). Ac- tosis in itself is thought to be an important event for maintenance cording to the danger hypothesis, it has been suggested that injured of tolerance. Transgenic mice with DC-specific apoptosis defects go on to develop a systemic autoimmune response, which is not *Physiology and Experimental Medicine Research Program, Hospital for Sick Chil- observed in transgenic mice harboring apoptosis defects in T and dren, Toronto, ON, Canada; †Department of Laboratory Medicine and Pathobiology, B cells (11–14). Additionally, selective apoptosis of DCs is ob- University of Toronto, Toronto, ON, Canada; ‡The Samuel Lunenfeld Research In- stitute, Mount Sinai Hospital, Toronto, ON, Canada; ¶Departments of Medicine, Im- served in sepsis patients along with breast cancer patients, with its munology, Medical Genetics, and Microbiology, University of Toronto and Toronto significance being relatively unclear (15–18). General Hospital Immature DCs are highly phagocytic and have the ability to Received for publication March 17, 2009. Accepted for publication September ingest entire cells. Previous studies have reported that ingestion of 15, 2009. necrotic cells is recognized as immunostimulatory, whereas inges- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance tion of apoptotic cells appears to be an immunologically null event with 18 U.S.C. Section 1734 solely to indicate this fact. (3, 19). Despite the importance of DC apoptosis in the immune 1 This work was supported in part by Operating Grants from the Canadian Institutes response, studies have not investigated the effects of ingestion of of Health Research, the Canadian Cystic Fibrosis Foundation, and the Foundation apoptotic or necrotic DCs by viable DCs. To the best of our knowl- Fighting Blindness-Canada (to J.H.). J.H. was a Canadian Cystic Fibrosis Foundation Scholar and a recipient of the Canadian Cystic Fibrosis Foundation Zellers Senior edge, in addition to our current study, the only other study that has Scientist Award, and holds a Premier’s Research Excellence Award of Ontario, investigated this phenomena is our other work, where we demon- Canada. R.K. is a recipient of Canadian Cystic Fibrosis Foundation Doctoral Award. strated that immature DCs efficiently uptake both apoptotic as well 2 Address correspondence and reprint requests to Dr. Jim Hu, Physiology and Exper- imental Medicine Research Program, Hospital for Sick Children, 555 University Av- as necrotic DCs in vitro (manuscript submitted). The uptake of enue, Toronto, ON, Canada M5G 1X8. E-mail address: [email protected] apoptotic DCs suppresses subsequent LPS-induced maturation and 3 ␤ ␤ Abbreviations used in this paper: DC, dendritic cell; Treg, regulatory T cell; BALF, induces secretion of TGF- 1 and up-regulation of TGF- 2 which bronchoalveolar lavage fluid; MLN, mediastinal lymph node; PLN, popliteal lymph induces differentiation of naive T cells into Foxp3ϩ T . Further- node; PI, propidium iodide; MFI, mean fluorescent intensity. regs more, we also showed that it is the uptake of only apoptotic DCs ␤ ϩ Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 that induces TGF- 1 secretion and induction of Foxp3 Tregs in www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900824 The Journal of Immunology 7105 vitro, for uptake of apoptotic splenocytes did not produce the same Induction of DC apoptosis and necrosis effects, indicating that the observed suppression is likely not phos- Dendritic cells were cultured on a 6-well dish and irradiated for 2 min with phatidylserine dependent, since even apoptotic splenocytes have a UV transilluminator, with a peak intensity of 9000 mW/cm2 at the filter phosphatidylserine exposed on the surface which can then interact surface and a peak emission of 313 nm. Induction of apoptosis was con- with its receptor on viable DCs. Moreover, previous studies have firmed using apoptosis, necrosis and healthy cell quantification kit also indicated that exposure of murine DCs to apoptotic cells does (Biotium), following the manufacturer’s instructions. Additionally, stain- ␤ ing with annexin V-FITC and PI was also used to confirm apoptosis. Ne- not induce TGF- 1 secretion (20–22). The aim of our current crosis was induced by first pelleting cells, followed by freeze and thaw. study was to study the effects of apoptotic DCs on viable DCs in vivo and to test whether they can be used for suppression of ex- CFSE labeling of DCs isting inflammation and induction of Ag-specific tolerance in mice. DCs were harvested and suspended in balanced salt solution (BSS) con- In this study, we show that delivery of apoptotic DCs in mice taining 1 ␮M CFSE at a concentration of 106 cells/ml. Subsequently, the results in their rapid uptake by viable DCs both in the spleen as cells were incubated at 37°C for 10 min. FCS was added to a concentration well as the draining lymph nodes. Though, the uptake of either of 5% and the cells were washed twice. necrotic or apoptotic DCs by viable DCs in vivo does not effect In vivo assays to assess suppression by apoptotic DCs their phenotype, albeit, it has an effect on subsequent response to Wild-type C57BL/6 mice were injected with 100 ␮l of saline alone or 4 ϫ LPS.