PRDM1/Blimp-1 Controls Effector Cytokine Production in Human NK Cells Matthew A. Smith, Michelle Maurin, Hyun Il Cho, Brian Becknell, Aharon G. Freud, Jianhua Yu, Sheng Wei, Julie This information is current as Djeu, Esteban Celis, Michael A. Caligiuri and Kenneth L. of September 27, 2021. Wright J Immunol published online 13 October 2010 http://www.jimmunol.org/content/early/2010/10/13/jimmun ol.1001682 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2010/10/14/jimmunol.100168 Material 2.DC1 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 September 27, 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 13, 2010, doi:10.4049/jimmunol.1001682 The Journal of Immunology PRDM1/Blimp-1 Controls Effector Cytokine Production in Human NK Cells Matthew A. Smith,*,† Michelle Maurin,* Hyun Il Cho,* Brian Becknell,‡ Aharon G. Freud,‡ Jianhua Yu,‡ Sheng Wei,* Julie Djeu,* Esteban Celis,* Michael A. Caligiuri,‡ and Kenneth L. Wright*,† NK cells are major effectors of the innate immune response through cytolysis and bridge to the adaptive immune response through cytokine release. The mediators of activation are well studied; however, little is known about the mechanisms that re- strain activation. In this report, we demonstrate that the transcriptional repressor PRDM1 (also known as Blimp-1 or PRDI-BF1) is a critical negative regulator of NK function. Three distinct PRDM1 isoforms are selectively induced in the CD56dim NK population in response to activation. PRDM1 coordinately suppresses the release of IFN-g, TNF-a, and TNF-b through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFN-g and Downloaded from TNF-a but does not alter cytotoxicity, whereas overexpression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci. Collectively, these data demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/ atural killer cells play critical functions in innate and IFN-g production and increased cytotoxicity (9). IL-18 induces nu- adaptive immunity. Although these lymphocytes were clear localization of NF-kB p50/p65 which, cooperatively with N initially identified by their ability to lyse leukemia cells AP-1, increases IFN-g and cytotoxicity (10). Furthermore, NFAT in a non-MHC–restricted manner, subsequent studies highlighted induces transcription of GM-CSF and TNF-a in NK cells (11). their role in cytokine production. In response to activating stimuli, Conversely, relatively few negative regulators of activation-induced NK cells proliferate, increase cytotoxicity, and produce cytokines, transcription have been identified in NK cells. ATF3 was recently such as IFN-g, TNF-a, and GM-CSF (1). IL-2 upregulates the ex- shown to downregulate IFN-g levels, and ATF2/2 mice exhibit in- pression of effector molecules and enhances natural cytotoxicity creased resistance to murine CMV infection (12). The transcription against a variety of targets. Furthermore, IL-2 and IL-15 signal factor H2.0-like homeobox negatively regulates IFN-g production, by guest on September 27, 2021 through the common gcR to control proliferation, with IL-15 be- primarily through degradation of phosphorylated STAT4 not ing uniquely required for survival in vivo (2). IL-12 and IL-18 direct DNA-binding activity (13). signal through distinct heterodimeric receptor complexes to elicit PRDM1 (also known as Blimp-1 or PRDI-BF1) is a transcrip- increases in IFN-g via several mechanisms, including increased tional repressor encoded by the PRDM1 gene on chromosome transcription, message stability, and nuclear retention (3–5). Syn- 6q21. It was originally identified as a postinduction suppressor of ergistic increases in cytotoxicity and IFN-g production are ob- IFNB in virally infected osteosarcoma cells (14). Subsequent served in response to costimulation with IL-12 and IL-18 (6, 7). work revealed a pivotal role in the terminal differentiation of Ab- Cytokine-mediated activation of NK cells proceeds through producing plasma cells (15). We and other investigators previously several well-characterized nuclear transcription factors, many of showed that PRDM1 exerts its repressive functions through re- which are functionally conserved between T and NK lineages (8). cruitment of histone-modifying enzymes (HDAC2, G9a, PRMT5, STAT4 is induced in response to IL-12 and is required for optimal and LSD1) and Groucho corepressors (16–18). Through silencing of direct (cMyc, CIITA, Pax5) and indirect targets, PRDM1 is a master regulator of terminal differentiation of B lymphocytes, me- *Immunology Program, H. Lee Moffitt Cancer Center and Research Institute; †Department of Molecular Medicine, University of South Florida, Tampa, FL diating cell cycle exit, repression of early B cell factors, and in- 33612; and ‡Ohio State Comprehensive Cancer Center, Columbus, OH 43210 duction of Ig secretion (19, 20). Received for publication May 20, 2010. Accepted for publication September 11, More recently, a role for PRDM1 in T lymphocytes has emerged. 2010. PRDM1 is expressed in CD4 and CD8 T cell lineages and is critical This work was supported by the James and Ester King Biomedical Research Program for maintenance of homeostasis. Conditional knockout in T lym- (09KT-03). phocytes leads to increased effector populations, resulting in severe The sequences presented in this article have been submitted to the Gene Expression Omnibus under accession number GE22919. colitis (21, 22). Upon activation, an autoregulatory loop exists, whereby IL-2 induces PRDM1 expression, which, in turn, nega- Address correspondence and reprint requests to Dr. Kenneth L. Wright, H. Lee Moffitt Cancer Center, MRC4E, 12902 Magnolia Drive, Tampa, FL 33612. E-mail tively regulates IL-2 transcription (23, 24). During CD4 polariza- address: Ken.Wright@moffitt.org tion, PRDM1 is preferentially expressed in Th2 cells and reinforces The online version of this article contains supplemental material. commitment to this lineage through repression of Ifng, cfos, and Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; Ct, threshold tbx21 (24, 25). Within the CD8 lineage, PRDM1 is expressed at cycle; KD, knockdown; NT, nontargeting; PARP, poly(ADP-ribose) polymerase; higher levels in exhausted subsets and promotes acquisition of poly-IC, polyinosinic-polycytidylic acid; siRNA, small interfering RNA. the effector phenotype through suppression of memory potential Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 (26–28). Thus, in addition to well-characterized B cell-specific www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001682 2 PRDM1 REGULATES NK EFFECTOR CYTOKINE PRODUCTION functions, PRDM1 is a critical regulator of T lymphocytes. In this and counts per minute were determined using a PerkinElmer 1470 auto- report, we provide a functional description of PRDM1 in NK cells. matic gamma counter (PerkinElmer, Wellesley, MA). The percentage of cytotoxicity was calculated using the following formula: (experimental 2 spontaneous release)/(total 2 spontaneous release) 3 100. Materials and Methods Chromatin immunoprecipitation Cells and cytokines A total of 20 3 106 purified NK cells were stimulated for 24 h with IL-2 Primary human NK cells were isolated via negative selection using the (100 U/ml), IL-12 (10 ng/ml), and IL-18 (100 ng/ml). Chromatin EZSep kit (StemCell Technologies, Vancouver, BC, Canada), according to was prepared, as previously described (30). A total of 4.5 3 106 cell the manufacturer’s instructions. Purity was verified by flow cytometry, 2 + + equivalents were used for each immunoprecipitation reaction. Primary and cells were routinely found to be 90–95% CD3 CD56 CD16 . Cells Abs were used at 0.5 mgineach900-ml reaction and were incubated over- were maintained in RPMI 1640 (Life Technologies, Carlsbad, CA), sup- night. Abs used included PRDM1 (PRDI-BF1) (Cell Signaling Tech- plemented with 10% FBS and 1% penicillin-streptomycin. For small in- nology) and normal rabbit IgG (Upstate Biotechnology, Lake Placid, NY). terfering RNA (siRNA) experiments, cells were grown in Accell Delivery Immune complexes were captured with protein A/G beads (Santa Cruz Media (Dharmacon, Chicago, IL), supplemented with 2% FBS. For Biotechnology, Santa Cruz, CA) and washed, as described. Eluted DNA stimulations, the following recombinant human cytokines were used: IL-2 was column purified (Qiagen) after reversal of cross-links (4 h at 65˚C) (100 U/ml; PeproTech, Rocky Hill, NJ), IL-12 (10 ng/ml; PeproTech), IL- and RNase treatment and proteinase K treatment. PCR was performed 18 (100