CD70 Is Downregulated by Interaction with CD27 Mirela Kuka, Ivana Munitic, Maria Letizia Giardino Torchia and Jonathan D. Ashwell This information is current as of September 25, 2021. J Immunol 2013; 191:2282-2289; Prepublished online 2 August 2013; doi: 10.4049/jimmunol.1300868 http://www.jimmunol.org/content/191/5/2282 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/08/06/jimmunol.130086 Material 8.DC1 References This article cites 35 articles, 19 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/191/5/2282.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology CD70 Is Downregulated by Interaction with CD27 Mirela Kuka,1,2 Ivana Munitic,1,3 Maria Letizia Giardino Torchia, and Jonathan D. Ashwell Engagement of the receptor CD27 by CD70 affects the magnitude and quality of T cell responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic cells (DCs), T cells, and B cells only upon activation. In this article, we show that a second level of regulation also is present. First, although undetectable on the cell surface by flow cytom- etry, immature DCs have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DCs and T cells were higher in mice deficient in CD27, or on DCs for which the interaction between CD70 and CD27 was precluded by blocking Abs. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor. The Journal of Immunology, 2013, 191: 2282–2289. Downloaded from nteraction between the costimulatory receptor CD27 and its restricted, and is barely detectable on the cell surface at steady state, ligand CD70 is required for optimal T cell activation (1–3). and even then only rare cells in the thymic medulla and the lamina I Studies using CD27- and CD70-deficient mice or anti-CD70 propria are CD70+ (17–20). Transient transcriptional upregulation of blocking Abs have found defects in primary and/or secondary T cell CD70 occurs in dendritic cells (DCs) activated via TLR- or CD40- responses in a variety of infectious models (4–8). Furthermore, mediated stimulation and in Ag-activated T and B cells (6, 20). In manipulations that increase CD27–CD70 interactions have been DCs, where its expression seems to be most relevant, CD70 is http://www.jimmunol.org/ successfully used in experimental vaccination protocols (9, 10). It is transported by the invariant chain to late endocytic structures, where notable that a fine line exists between beneficial and deleterious it colocalizes with MHC-II molecules (21, 22). Upon interaction of CD70-mediated effects. For example, whereas efficient clearance activated DCs with cognate CD4 T cells, CD70 is codelivered to the of acute lymphocytic choriomeningitis virus (LCMV) strains requires immune synapse with MHC-II, ensuring optimal T cell stimulation. CD27 occupancy by CD70, this interaction precludes clearance of Uncontrolled CD27–CD70 interactions have detrimental effects. the chronic LCMV strain (7, 8, 11). Therefore, the existence of In mouse models where CD70 was constitutively expressed on regulatory mechanisms for the CD70–CD27 pathway ensures ef- B cells, DCs, or T cells, a continuous generation of effector T cells fective and prevents deleterious immune responses. was observed, which in B cell and DC CD70 transgenics resulted Normally, tight control of CD27 and CD70 expression avoids in an autoimmune disease and death (23–25). In contrast, consti- by guest on September 25, 2021 excessive T cell activation. CD27, a member of the TNFR family, is tutive CD70 expression on DCs was sufficient to break peripheral constitutively expressed by T cells as a membrane-bound homodimer, tolerance and, among other things, generate tumor-specific respon- and its surface levels change during T cell activation (3). The baseline ses to peptide immunization without the need for adjuvants (24). In level in resting naive and memory T cells is upregulated during the addition to these observations made in transgenic mice, the im- first days after TCR engagement because of increased transcription portance of excessive CD27–CD70 interactions has been demon- (12–14). Notably, surface levels of CD27 are downregulated during strated in a chronic LCMV infection model (11). Continuous CD27 T cell effector differentiation by shedding and/or decreased tran- engagement, likely mediated by a subset of CD70-expressing scription, and some terminally differentiated effector memory T cells B cells, led to T cell cytokine-mediated splenic germinal center and (TEM) retain a CD27-negative phenotype (13–15). CD27 can also be marginal zone destruction, thus precluding the generation of a neu- reversibly downregulated on memory CD8 T cells that enter non- tralizing Ab response. lymphoid organs (16). In contrast, expression of CD70, a homotri- It is generally believed that the downregulation of T cell CD27 meric transmembrane member of the TNF family, is much more levels during persistent stimulation is an activation-intrinsic event. However, some evidence suggests that it is also the interaction with Laboratory of Immune Cell Biology, National Cancer Institute, National Institutes of CD70 that results in decreased CD27levelsintheabsenceofacti- Health, Bethesda, MD 20892 vation. For example, T cell coculture with B cell lines expressing CD70 1M.K. and I.M. contributed equally to this work. triggered CD27 downregulation, and even naive T cells in CD70 Tg 2Current address: Division of Immunology, Transplantation and Infectious Diseases, mice had substantially lower CD27 levels (26, 27). In the course of San Raffaele Scientific Institute, Milan, Italy. studying mice deficient in either CD27 or CD70, we made the un- 3Current address: Department of Biotechnology, University of Rijeka, Rijeka, Croatia. expected observation that in the absence of one the other was up- Received for publication March 29, 2013. Accepted for publication June 26, 2013. regulated. In this article, we show by Ab blocking and genetic This work was supported by the Intramural Research Program of the Center for manipulation that the relationship between CD27 and CD70 expres- Cancer Research, National Cancer Institute, National Institutes of Health. sion is reciprocal and mediated by direct protein–protein interactions. Address correspondence and reprint requests to Dr. Jonathan D. Ashwell, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Building 37, Room 3002C, Bethesda, MD 20892. E-mail address: [email protected] Materials and Methods Mice The online version of this article contains supplemental material. Abbreviations used in this article: CHX, cycloheximide; LCMV, lymphocytic cho- C57BL/6 (B6) mice were obtained from Frederick Cancer Research Facility 2/2 riomeningitis virus; DMFI, change in mean fluorescence intensity; poly(I:C), poly- (Frederick, MD). CD70 mice backcrossed to B6 for 13 generations 2 2 inosinic-polycytidylic acid; WT, wild-type. were described (8). CD27 / mice (4) were a gift from Jannie Borst www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300868 The Journal of Immunology 2283 (The Netherlands Cancer Institute, Amsterdam, The Netherlands) and were CD272/2, and CD702/2 mice were infected i.p. with 2 3 105 PFU maintained in our facility. The National Cancer institute Animal Care and LCMV. Use Committee approved all animal studies. Cell stimulation and flow cytometry Reagents Splenocytes were cultured at a concentration of 5 3 106 cells per milliliter Abs to CD27, TCR-b, B220, CD16/CD32 clone 2.4G2 (Fc block), iso- and stimulated with poly(I:C) (30 mg/ml) for 22 h or primed with IFN-g type controls, and fixation and permeabilization buffers were obtained (50 ng/ml) for 2 h and then stimulated with LPS (200 ng/ml) for an ad- from BD Biosciences. The unconjugated Ab to CD70 (clone FR70), the ditional 20 h. In some experiments, cells were treated for 15 min with fluorochrome-conjugated Abs to CD70 (clone FR70), CD11c and MHC anti-CD27 blocking Ab (5 mg/ml) or with isotype control prior to stim- class II, the blocking Ab to CD27 (clone LG.3A10), the isotype control, ulation. In internalization experiments, anti-CD70 PE-conjugated Ab was and recombinant IFN-g were purchased from eBioscience. LCMVArmstrong added to the cultures after stimulation at the indicated time points, at a 53b (LCMV) was a gift from Rafi Ahmed (Emory University School of concentration of 2 mg/ml. Cells were treated or not with CHX (20 mg/ml) b Medicine, Atlanta, GA). GP33-41 H-2D (GP33) tetramers were obtained 1 h before adding anti-CD70 PE-conjugated Ab. For staining of surface from the National Institutes of Health Tetramer Core Facility at Emory molecules, CD16/CD32 clone 2.4G2 was added to block Fc-receptor University. Power SYBR Green was obtained from Applied Biosystems.