Blockade of Programmed Death-1 Ligands on Dendritic Cells Enhances T Cell Activation and Cytokine Production This information is current as Julia A. Brown, David M. Dorfman, Feng-Rong Ma, of October 5, 2021. Elizabeth L. Sullivan, Oliver Munoz, Clive R. Wood, Edward A. Greenfield and Gordon J. Freeman J Immunol 2003; 170:1257-1266; ; doi: 10.4049/jimmunol.170.3.1257 http://www.jimmunol.org/content/170/3/1257 Downloaded from References This article cites 40 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/170/3/1257.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on October 5, 2021 *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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Blockade of Programmed Death-1 Ligands on Dendritic Cells Enhances T Cell Activation and Cytokine Production1 Julia A. Brown,* David M. Dorfman,† Feng-Rong Ma,* Elizabeth L. Sullivan,* Oliver Munoz,* Clive R. Wood,‡ Edward A. Greenfield,* and Gordon J. Freeman2* Programmed death-1 ligand (PD-L)1 and PD-L2 are ligands for programmed death-1 (PD-1), a member of the CD28/CTLA4 family expressed on activated lymphoid cells. PD-1 contains an immunoreceptor tyrosine-based inhibitory motif and mice deficient in PD-1 develop autoimmune disorders suggesting a defect in peripheral tolerance. Human PD-L1 and PD-L2 are expressed on immature dendritic cells (iDC) and mature dendritic cells (mDC), IFN-␥-treated monocytes, and follicular dendritic cells. Using mAbs, we show that blockade of PD-L2 on dendritic cells results in enhanced T cell proliferation and cytokine production, including that of IFN-␥ and IL-10, while blockade of PD-L1 results in similar, more modest, effects. Blockade of both PD-L1 and PD-L2 showed an additive effect. Both whole mAb and Fab enhanced T cell activation, showing that PD-L1 and PD-L2 function Downloaded from to inhibit T cell activation. Enhancement of T cell activation was most pronounced with weak APC, such as iDCs and IL-10- pretreated mDCs, and less pronounced with strong APC such as mDCs. These data are consistent with the hypothesis that iDC have a balance of stimulatory vs inhibitory molecules that favors inhibition, and indicate that PD-L1 and PD-L2 contribute to the poor stimulatory capacity of iDC. PD-L1 expression differs from PD-L2 in that PD-L1 is expressed on activated T cells, placental trophoblasts, myocardial endothelium, and cortical thymic epithelial cells. In contrast, PD-L2 is expressed on placental endothe- lium and medullary thymic epithelial cells. PD-L1 is also highly expressed on most carcinomas but minimally expressed on http://www.jimmunol.org/ adjacent normal tissue suggesting a role in attenuating antitumor immune responses. The Journal of Immunology, 2003, 170: 1257–1266. ature dendritic cells (mDCs3) express high levels of The ability of three members of the B7 gene family, B7-1, B7-2, MHC and costimulatory molecules and are powerful and inducible costimulator ligand, to provide costimulatory signals M APC that initiate T cell immune responses (1). Sur- during the process of T cell activation has been well characterized prisingly, recent data have revealed that Ag presentation by im- (8, 9). Two additional B7 family members, programmed death-1 mature dendritic cells (iDCs) has a role in establishing and main- ligand (PD-L)1 (B7-H1) and PD-L2 (B7-DC), have recently been taining T cell tolerance (2–5). Despite expressing moderate levels shown to down-regulate T cell activation through their mutual re- by guest on October 5, 2021 of MHC and costimulatory molecules, presentation by iDCs stim- ceptor, programmed death-1 (PD-1) (10–12). PD-1, a CD28 ho- ulates the development of regulatory T cell populations (2, 3). mologue, contains two immunoreceptor tyrosine-based motifs that Furthermore, IL-10-pretreated mDCs express lower but detectable are phosphorylated upon receptor engagement and recruit Src ho- amounts of MHC and costimulatory molecules and induce T cell mology 2-domain-containing tyrosine phosphatase 2 (11, 13–15). anergy in vitro (3, 6, 7). Stimulatory and inhibitory signals pre- Although initially isolated from two cell lines undergoing pro- sented by dendritic cells (DCs) during Ag presentation are inte- grammed cell death (16), neither cross-linking of PD-1 by mAb or grated by the T cell and determine the final outcome of T cell PD-L results in programmed cell death. Rather, engagement of activation. PD-1 results in cell cycle arrest (11, 12, 17). Unlike other CD28 family members, PD-1 demonstrates a broad expression pattern and is found on activated T, B, and myeloid cells (18) and on a subset of thymocytes (19). PD-1Ϫ/Ϫ mice dis- *Department of Medical Oncology, Dana-Farber Cancer Institute, Department of play a variety of autoimmune pathologies, demonstrating the role Medicine, Harvard Medical School, and †Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and ‡Wyeth of PD-1 as a negative regulator of the immune response (15, 20). Research, Cambridge, MA 02140 Engagement of PD-1 by PD-L1-Ig or PD-L2-Ig fusion protein has Received for publication July 22, 2002. Accepted for publication November 5, 2002. an inhibitory effect on proliferation and cytokine production during The costs of publication of this article were defrayed in part by the payment of page anti-CD3-mediated stimulation (10, 11). Chinese hamster ovary charges. This article must therefore be hereby marked advertisement in accordance (CHO) cell transfectants capable of presenting OVA peptide in the with 18 U.S.C. Section 1734 solely to indicate this fact. context of I-Ad and expressing either PD-L1 or PD-L2 with or 1 This work was supported by National Institutes of Health Grants AI39671, AI41584, and CA84500 (to G.J.F.) and Congressionally Directed Medical Research Programs without B7-2 costimulation inhibit the proliferation and cytokine Postdoctoral Traineeship Grant BC011188 from the Breast Cancer Research Program production of DO11 TCR transgenic T cells, particularly at low (to F.-R.M.). concentrations of Ag (11). The PD-L:PD-1 pathway inhibits pro- 2 Address correspondence and reprint requests to Dr. Gordon J. Freeman, Department liferation by reducing the production of IL-2 and restricts the num- of Medical Oncology, Dana-Farber Cancer Institute, Department of Medicine, Har- vard Medical School, 44 Binney Street, Boston, MA 02115. E-mail address: ber of T cells that gain entry into the cell cycle as well as their [email protected] subsequent division rate (12). In contrast to these results, Dong et 3 Abbreviations used in this paper: mDC, mature dendritic cell; iDC, immature den- al. (21) have reported that PD-L1-Ig (B7-H1-Ig) will moderately dritic cell; DC, dendritic cell; FDC, follicular DC; PD-1, programmed death-1; PD-L, PD-1 ligand; CHO, Chinese hamster ovary; DN, double negative; DP, double stimulate T cell proliferation and strongly up-regulate IL-10 pro- positive. duction. Tseng et al. (22) have reported that PD-L2-Ig (B7-DC-Ig) Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 1258 BLOCKADE OF PD-L ON DC ENHANCES T CELL ACTIVATION strongly up-regulates T cell activation and IFN-␥ production. Ad- source International, Camarillo, CA) and 800 U/ml GM-CSF (BD PharM- ditional experiments with blocking mAbs and knockout mice are ingen, San Diego, CA). Maturation of iDCs was accomplished by replating ϫ 6 needed to resolve these differences. the cells for 2 days at 2 10 cells/well in 6-well plates (Falcon; BD Biosciences) in IL-4 (1000 U/ml) and GM-CSF (800 U/ml) supplemented In this study, we report the development and characterization of with IL-1␤ (10 ng/ml; US Biological, Swampscott, MA), TNF-␣ (10 ng/ mAbs that specifically recognize either human PD-L1 or PD-L2. ml; BD PharMingen), IL-6 (1000 U/ml; BD PharMingen), and PGE2 (1 We demonstrate that blockade of PD-L1 and PD-L2 on various ␮g/ml; Sigma-Aldrich). mDCs were consistently 90–95% CD11cϩDRϩ ϩ populations of monocyte-derived DCs enhances CD4ϩ T cell pro- with Ͻ2% CD14 cells. Where indicated, IL-10 (40 ng/ml; BD PharM- ingen) was added to the maturation culture in addition to the mixture of liferation and cytokine production. Both whole mAb and Fab en- inflammatory cytokines. The presence of a low number of CD14ϩ cells hance T cell activation. Additionally, we show that PD-L are ex- having a fibroblast-like appearance was noted after 2 days of culture with pressed much more broadly in normal tissues than are other IL-10. The ability of IL-10 to convert DCs into macrophage-like cells has ϩ members of the B7 gene family. A variety of epithelial-derived been previously reported (24). Before coculture with T cells, CD14 mac- tumors also express PD-L1, suggesting that these malignancies rophages were removed by incubation with anti-CD14 magnetic beads (Dynal Biotech, Great Neck, NY).