CTLA-4 Overexpression Inhibits T Cell Responses through a CD28-B7-Dependent Mechanism This information is current as John J. Engelhardt, Timothy J. Sullivan and James P. Allison of October 2, 2021. J Immunol 2006; 177:1052-1061; ; doi: 10.4049/jimmunol.177.2.1052 http://www.jimmunol.org/content/177/2/1052 Downloaded from References This article cites 51 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/177/2/1052.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • 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 *average by guest on October 2, 2021 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology CTLA-4 Overexpression Inhibits T Cell Responses through a CD28-B7-Dependent Mechanism1 John J. Engelhardt,*† Timothy J. Sullivan,† and James P. Allison2* CTLA-4 has been shown to be an important negative regulator of T cell activation. To better understand its inhibitory action, we constructed CTLA-4 transgenic mice that display constitutive cell surface expression of CTLA-4 on CD4 and CD8 T cells. In both in vivo and in vitro T cell responses, CTLA-4 overexpression inhibits T cell activation. This inhibition is dependent on B7 and CD28, suggesting that overexpressed CTLA-4 inhibits responses by competing with CD28 for B7 binding or by interfering with CD28 signaling. In addition, expression of the transgene decreases the number of CD25؉Foxp3؉ T cells in these mice, but does not affect their suppressive ability. Our data confirm the activity of CTLA-4 as a negative regulator of T cell activation and that its action may be by multiple mechanisms. The Journal of Immunology, 2006, 177: 1052–1061. cell activation occurs through the interaction of T cells lation of this tyrosine interferes with binding to the medium chain Downloaded from and APCs. The specificity of this response is determined of the clathrin coated pit adaptor protein, AP-2, and leads to in- T by the binding of the TCR to peptide-MHC complexes on creased surface expression of CTLA-4 (13–16). CTLA-4 has also the APC. In most circumstances, TCR signal is not sufficient for been shown to have a relatively short half-life when compared optimal T cell activation and a costimulatory signal is also required with CD28 or transferrin receptor, which again suggests that sur- for activation. In primary T cell responses, CD28 is the primary face expression of CTLA-4 is tightly regulated (17). During Ag positive costimulator of T cell activation, whereas CTLA-4, a recognition, CTLA-4 translocates from this endosomal compart- http://www.jimmunol.org/ structural homolog for CD28, provides an inhibitory signal. ment to the immunological synapse. In this way, access of CTLA-4-mediated inhibition was most strikingly demonstrated by CTLA-4 to its ligands is regulated both spatially and temporally. the production of CTLA-4-deficient mice, which die of a massive CTLA-4 localization to the immunological synapse has been lymphoproliferative disorder leading to tissue destruction and ram- shown to be regulated by TCR signal strength, leading to the pos- pant autoimmunity (1–3). Although the exact nature of this inhi- sibility that only a high-affinity peptide MHC-TCR interaction will bition is still unclear, CTLA-4 has been shown to reduce IL-2 lead to CTLA-4 surface expression and subsequent T cell inhibi- production after ligation (4, 5) and can inhibit T cell expansion by tion (17). decreasing levels of cyclin D3, cyclin-dependent kinase 4 (cdk4), and cdk6 (6, 7). The rampant autoimmunity in CTLA-4-deficient Although it is clear that CTLA-4 is a critical negative regulator by guest on October 2, 2021 mice suggests a critical role for CTLA-4 in limiting T cell of T cell activation and effector function, it is difficult to determine responses. the role of CTLA-4 in the primary activation of T cells. To address Despite their homology, CTLA-4 and CD28 are fundamentally this question, we have created CTLA-4 transgenic mice that over- different in their effects on T cell activation. Although these mol- express CTLA-4 on naive T cells. Overexpression of CTLA-4 may ecules share the same ligands, B7.1 and B7.2, CTLA-4 binds to increase the effects caused by endogenous CTLA-4 and allow both with a significantly higher affinity (8, 9). Recent data have these events to be studied more effectively. Previous studies have also shown that B7.1 is the preferred ligand for CTLA-4, whereas used CTLA-4 transgenics to investigate mechanisms of CTLA-4- B7.2 is preferred by CD28 (10). The localization and expression mediated inhibition, and have demonstrated that CTLA-4 overex- pattern of these two molecules also differ. CD28 is constitutively pression can rescue the phenotype of CTLA-4-deficient mice. A expressed on the cell surface of naive and activated T cells (11), transgenic construct lacking the cytoplasmic tail of CTLA-4 only whereas CTLA-4 is not detectable on naive T cells and is induced partially rescues this phenotype, suggesting that CTLA-4 signal upon T cell activation (12). Once expressed, CTLA-4 localizes to transduction is important for its inhibitory function (18, 19). How- an endosomal compartment due to a tyrosine-based intracellular ever, an extensive functional analysis of the consequences of localization motif in its cytoplasmic tail. Mutation or phosphory- CTLA-4 overexpression on T cells has not been reported. In this study, we demonstrate that constitutive overexpression of CTLA-4 leads to inhibition of T cell responses only when CD28/B7 inter- *Division of Immunology, Howard Hughes Medical Institute, Memorial Sloan-Ket- actions occur. tering Cancer Center, New York, NY 10021; and †Division of Immunology, Depart- ment of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Received for publication November 23, 2005. Accepted for publication May 1, 2006. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance Media and cell culture with 18 U.S.C. Section 1734 solely to indicate this fact. 1 All cell culture was performed in RPMI 1640 medium supplemented with This work was supported in part by a grant from the National Cancer Institute. J.P.A. ␮ is an investigator of the Howard Hughes Medical Institute and also holds the David 10% FCS, 2 M L-glutamine, and 100 U/ml penicillin and streptomycin H. Koch Chair in Immunologic Studies. (all from BioWhittaker), and 2 ␮M 2-ME (Sigma-Aldrich). All cells were 2 Address correspondence and reprint requests to Dr. James P. Allison, Immunology cultured in a 37°C humidified incubator at 5% CO2. CHO cells expressing k Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, I-E were obtained from Dr. N. Shastri (University of California, Berkeley, NY 10021. CA), and CA36.1 L cells expressing I-Ek and B7.1 were described earlier. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 1053 Mice The p value for survival data was calculated using the Fisher exact test. The p values for proliferation data were calculated using an unpaired t test. CTLA-4 transgenic mice were generated by injection of the CTLA-4 trans- ϫ genic construct into (C57BL/6 CBA/J)F1 fertilized eggs. Mice were backcrossed at least 10 times onto the C57BL/6 background for additional Results Ϫ Ϫ experiments. 5C.C7 TCR transgenic RAG2 / were obtained from Tac- Generation of CTLA-4 transgenic mice onic Farms. CTLA-4 transgenic 5C.C7 TCR transgenic RAG2Ϫ/Ϫ were obtained by breeding onto the 5C.C7 background and backcrossed six To investigate the role of CTLA-4 in regulating naive T cell re- times. CD28Ϫ/ϪRAG2Ϫ/Ϫ 5C.C7 TCR transgenic mice were described ear- sponses, we generated transgenic animals overexpressing full- Ϫ/Ϫ Ϫ/Ϫ lier, and RAG2 CD28 CTLA-4 transgenic 5C.C7 TCR transgenic length CTLA-4 under the control of the murine H-2Kb promoter were obtained by crossing 5C.C7 TCRϩ RAG2Ϫ/Ϫ CTLA-4 transgenic ϩ Ϫ Ϫ Ϫ Ϫ and the 3Ј Ig enhancer (Fig. 1A), a combination previously shown onto the 5C.C7 TCR RAG2 / CD28 / background, and backcrossing to homozygosity. All mice were bred and maintained in microisolator to direct expression primarily to the lymphoid lineage (20). cages in accordance with the animal care and use regulations of University Flow cytometric analysis of the CTLA-4 transgenic line with an of California (Berkeley, CA) and Memorial Sloan-Kettering Cancer anti-CTLA-4 Ab demonstrated increased expression of CTLA-4 Center. on both the surface of naive T cells and in the cytoplasm. This Abs and flow cytometry increased surface expression is apparent in splenic (data not shown) and lymph node T cell populations (Fig. 1B). These The following Abs were used for flow cytometry: TriColor anti-CD4 animals provide a useful tool for studying the consequence of (RM4-5), Alexa-488 anti-CD8␣ (53-6.7) (Caltag), PE anti-CTLA-4 (UC10-4F10), PE anti-CD8␣, FITC anti-CD69 (H1.2F3), FITC anti- CTLA-4 overexpression on T cell development, homeostasis, CD62L (MEL-14), biotin anti-CD28 (37.51), PE anti-CD25 (PC61), PerCP and activation.