Functional Role for IκBNS in T Cell Cytokine Regulation As Revealed by Targeted Gene Disruption This information is current as Maki Touma, Valeria Antonini, Manoj Kumar, Stephanie L. of October 2, 2021. Osborn, April M. Bobenchik, Derin B. Keskin, John E. Connolly, Michael J. Grusby, Ellis L. Reinherz and Linda K. Clayton J Immunol 2007; 179:1681-1692; ; doi: 10.4049/jimmunol.179.3.1681 http://www.jimmunol.org/content/179/3/1681 Downloaded from References This article cites 61 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/179/3/1681.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 2, 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Functional Role for I␬BNS in T Cell Cytokine Regulation As Revealed by Targeted Gene Disruption1 Maki Touma,*† Valeria Antonini,*† Manoj Kumar,2*† Stephanie L. Osborn,3* April M. Bobenchik,4* Derin B. Keskin,*† John E. Connolly,‡ Michael J. Grusby,§ Ellis L. Reinherz,*† and Linda K. Clayton5*† Triggering of the TCR by cognate peptide/MHC ligands induces expression of I␬BNS, a member of the I␬B family of NF-␬B inhibitors whose expression is associated with apoptosis of immature thymocytes. To understand the role of I␬BNS in TCR triggering, we created a targeted disruption of the I␬BNS gene. Surprisingly, mice lacking I␬BNS show normal thymic progression but both thymocytes and T cells manifest reduced TCR-stimulated proliferation. Moreover, I␬BNS knockout thymocytes and T cells produce significantly less IL-2 and IFN-␥ than wild-type cells. Transfection analysis demonstrates ␬ ␬ that I BNS and c-Rel individually increase IL-2 promoter activity. The effect of I BNS on the IL-2 promoter, unlike c-Rel, Downloaded from is dependent on the NF-␬B rather than the CD28RE site; mutation of the NF-␬B site extinguishes the induction of tran- scription by I␬BNS in transfectants and prevents association of I␬BNS with IL-2 promoter DNA. Microarray analyses confirm the reduction in IL-2 production and some IFN-␥-linked transcripts in I␬BNS knockout T cells. Collectively, our findings demonstrate that I␬BNS regulates production of IL-2 and other cytokines induced via “strong” TCR ligation. The Journal of Immunology, 2007, 179: 1681–1692. http://www.jimmunol.org/ ctivation of the TCR upon Ag recognition initiates a nals emanating from the same molecule lead to two such opposite complex signaling cascade the outcome of which is af- outcomes remains a major question in immunology. A fected by a variety of factors. These include the devel- pMHC engagement of the TCR induces multiple posttransla- opmental stage of the T cell, costimulation by other cell surface tional biochemical modifications and new gene transcription (3) receptors on both the T cell and APC and the strength of the TCR- and the ubiquitous transcription factor, NF-␬B, has been impli- peptide/MHC (pMHC)6 interaction. In double-positive (DP) thy- cated in both survival and death of thymocytes (4–6). Previously, mocytes, a relatively strong signal induces negative selection and our analysis of genes induced in DP thymocytes undergoing cog- results in death of the thymocyte via apoptosis while a weak TCR nate peptide-induced apoptosis identified a novel member of the signal, by comparison, induces positive selection leading to DP I␬B family of NF-␬B inhibitors, I␬BNS (7). Induction of I␬BNS by guest on October 2, 2021 thymocyte differentiation and survival (reviewed in Refs. 1 and 2). expression in the thymus correlated with TCR signal strength and The same pMHC ligand that induces apoptosis in DP thymocytes relatively avid pMHC ligands triggered expression of I␬BNS activates peripheral T cells to proliferate. Elucidation of how sig- whereas weaker ligands did not. In fetal thymic organ cultures (FTOC) retrovirally infected to overexpress I␬BNS, development of double-negative (DN) to DP thymocytes was reduced and ad- *Laboratory of Immunobiology, Department of Medical Oncology, Dana Farber Can- dition of anti-CD3␧ mAb had a much more dramatic apoptotic cer Institute, and †Department of Medicine, Harvard Medical School, Boston, MA effect on I␬BNS-expressing than control DP thymocytes. Thus, 02115; ‡Baylor Institute for Immunology Research, Dallas, TX 75204; and §Depart- ␬ ment of Immunology and Infectious Diseases, Harvard School of Public Health, Bos- expression of I BNS alone is not sufficient for DP thymocyte ton, MA 02115 death, rather additional signaling via the TCR complex is required Received for publication April 12, 2007. Accepted for publication May 18, 2007. to regulate the response outcome. The costs of publication of this article were defrayed in part by the payment of page The NF-␬B protein family plays a key role in the overall reg- charges. This article must therefore be hereby marked advertisement in accordance ulation of the immune system. Activation through the TCR is de- with 18 U.S.C. Section 1734 solely to indicate this fact. pendent on NF-␬B for proliferation and cytokine production (8– 1 This work was supported by National Institutes of Health Grants AI19807 (to 10). The pathway leading from the TCR to NF-␬B activation E.L.R.) and AI51779 (to L.K.C.). involves multiple components culminating in the ubiquitination 2 Current address: Beth Israel Deaconess Medical Center Genomic Center, Depart- ment of Medicine, Harvard Medical School, Boston, MA 02115. and degradation of I␬B inhibitors, thereby releasing NF-␬Btoen- 3 Current address: Department of Molecular and Cell Biology, Division of Immunol- ter the nucleus and direct gene transcription. This process is com- ogy and Cancer Research Laboratory, University of California, Berkeley, CA 94720. plicated by the presence of five NF-␬B family members, each ca- 4 Current address: Center for Vascular Biology, University of Connecticut Health pable of forming multiple hetero- and homodimers (11). The Center, Farmington, CT 06030. complexity is amplified by the fact that while specific NF-␬B- 5 Address correspondence and reprint requests to Dr. Linda K. Clayton, Dana Farber binding DNA sites are preferentially bound by particular dimers, Cancer Institute, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: [email protected] multiple varieties of dimers can also bind with comparable affin- 6 Abbreviations used in this paper: pMHC, peptide-MHC complex; DP, double pos- ities to the same DNA sequence (12–14). Functions unique to one itive; DN, double negative; LN, lymph node; WT, wild type; KO, knockout; BM, NF-␬B dimer may result from the range of DNA sequences bound bone marrow; FC, fold change; rh, recombinant human; SAP, SLAM-associated at high affinity by that protein dimer (15). Thus, gene regulation at protein. a specific NF-␬B site is dependent on the identities and amounts of Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 dimers expressed in the cell; this can also vary over time as the www.jimmunol.org 1682 I␬BNS REGULATION OF T CELL CYTOKINES accumulation or destruction of some dimers results in NF-␬B VSV8 peptide for 2 h. After 24 h, 37 Bq/well [3H]thymidine were added dimer exchange on DNA (16). and incorporated radioactivity measured 18 h later. ␬ For MLRs, LN T cells purified as described above (2 ϫ 105/well) were Until recently, seven NF- B inhibitor proteins were known: ϫ 5 ␬ ␣ ␬ ␤ ␬ ␧ ␬ ␥ cultured with irradiated spleen cells (2 10 /well) from syngeneic WT I B , I B ,I B ,I B , Bcl-3, p100, and p105 (reviewed in Ref. mice or allogeneic BALB/c mice for 48 h and [3H]thymidine incorporation 17). This family expanded with the identification of I␬BNS (7) and determined as above. I␬B␨ (18–20). I␬BNS and I␬B␨ show more sequence homology to Measurement of cytokine production each other and to Bcl-3 than to I␬B␣ and, like Bcl-3, these proteins appear to be nuclear rather than cytoplasmic. I␬B␨ (21), Bcl-3 (22, Cytokine production was induced as above except 5 ϫ 105 LN T cells were 23), and I␬BNS (24, 25) have been shown to regulate promoters of used/well. After 24 h, supernatants were collected for multiplex cytokine cytokines controlled by NF-␬B and to be involved in inflammatory analysis (Luminex) as described (29) but using an anti-Mouse MultiCyto- kine Reporter. Concentrations were calculated using Bio-Plex Manager 3.0 responses. Their mechanisms of action are different from that of software with a five-parameter curve-fitting algorithm applied for standard I␬B␣ because their expression is transcriptionally regulated and curve calculations. more restricted in cell expression patterns (7, 18–21, 26, 27) unlike NK cell function and cytokine production the more ubiquitous NF-␬B and other I␬B family members. These I␬BNS, Bcl-3, and I␬B␨ properties can narrow the action of Splenic NK cells (purity Ͼ75% as determined by anti-mouse CD49b/ NF-␬B providing an additional layer of specificity control.