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The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation Shannon A The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation Shannon A. Carty, Mercy Gohil, Lauren B. Banks, Renee M. Cotton, Matthew E. Johnson, Erietta Stelekati, Andrew D. This information is current as Wells, E. John Wherry, Gary A. Koretzky and Martha S. of September 29, 2021. Jordan J Immunol published online 17 November 2017 http://www.jimmunol.org/content/early/2017/11/17/jimmun ol.1700559 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/11/17/jimmunol.170055 Material 9.DCSupplemental 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 29, 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 17, 2017, doi:10.4049/jimmunol.1700559 The Journal of Immunology The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation Shannon A. Carty,*,1 Mercy Gohil,† Lauren B. Banks,† Renee M. Cotton,‡ Matthew E. Johnson,x Erietta Stelekati,{,‖ Andrew D. Wells,‡,x,‖ E. John Wherry,{,‖ Gary A. Koretzky,†,# and Martha S. Jordan‡,‖ T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild- type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; Downloaded from rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions. The Journal of Immunology, 2018, 200: 000–000. n response to infection, naive CD8+ T cells proliferate and Several cell surface proteins can be used to identify cells with differentiate into a heterogeneous pool of Ag-specific cells differing memory potential. Ag-specific CD8+ T cells that are http://www.jimmunol.org/ having divergent cell fates. Following pathogen clearance, most CD127hi and KLRG1lo preferentially differentiate into long-lived I + Ag-specific CD8 T cells die, but a subset persists to become long- memory cells, whereas CD127lo and KLRG1hi cells are largely lived memory cells, which are able to rapidly respond to rechallenge. short-lived terminally differentiated effector cells (1–3). This program of CD8+ T cell differentiation is regulated through the integration of signals from the TCR, costimulatory/coinhibitory *Department of Medicine, Perelman School of Medicine, University of Pennsylvania, receptors, and inflammatory cytokines, which direct transcrip- Philadelphia, PA 19104; †Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; tional changes that control cell fate. Although it is evident that ‡ Department of Pathology and Laboratory Medicine, Perelman School of Medicine, particular transcription factors, such as T-bet, eomesodermin by guest on September 29, 2021 University of Pennsylvania, Philadelphia, PA 19104; xThe Children’s Hospital of { (Eomes), Blimp-1, Bcl-6, IRF4, and Runx3, are important in de- Philadelphia, Philadelphia, PA 19104; Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; ‖Institute termining the fate of activated cells (4–11), it is also becoming for Immunology, Perelman School of Medicine, University of Pennsylvania, clear that epigenetic programming plays a crucial role in T cell Philadelphia, PA 19104; and #Department of Medicine, Weill Cornell Medicine, New York, NY 10065 fate determination. DNA methylation is one epigenetic mechanism by which T cell 1Current address: Department of Medicine and University of Michigan Comprehen- sive Cancer Center, University of Michigan, Ann Arbor, MI. differentiation is regulated, and recent genome-wide studies have ORCIDs: 0000-0002-8727-9723 (M.G.); 0000-0003-4521-8911 (R.M.C.); 0000- identified coordinated epigenetic changes associated with tran- 0002-8684-5359 (M.E.J.); 0000-0003-4155-3202 (G.A.K.); 0000-0003-2359- scriptional programs during CD4+ or CD8+ T cell differentiation 3227 (M.S.J.). (12–19). It is now appreciated that DNA undergoes regulated Received for publication April 19, 2017. Accepted for publication October 10, 2017. demethylation. Recently, the ten-eleven translocation (TET) This work was supported by National Institutes of Health Grants K08 AI101008 (to family of methylcytosine dioxygenases was shown to mediate this S.A.C.), R37 GM053256 (to G.A.K.), AI105343, AI112521, AI082630, and AI115712 (to E.J.W.) and other support from R01AI082292. process by catalyzing the conversion of 5-methylcytosine (5mC) The sequencing data presented in this article have been submitted to the National to 5-hydroxymethylcytosine (5hmC) and, subsequently, to 5-for- Center for Biotechnology Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/ mylcytosine and 5-carboxylcytosine, critical enzymatic steps geo/query/acc.cgi?acc=GSE105176) under accession number GSE105176. necessary for generating unmodified cytosines (20–22). The TET Address correspondence and reprint requests to Dr. Martha S. Jordan or Dr. Gary A. family member TET2 is widely expressed in the hematopoietic Koretzky, University of Pennsylvania, BRB II/III, Room 507, 421 Curie Boulevard, Philadelphia, PA 19104 (M.S.J.) or 1300 York Avenue, A125, New York, NY 10065 system, and murine models reveal that TET2 loss leads to ex- (G.A.K.). E-mail addresses: [email protected] (M.S.J.) or pansion of hematopoietic stem cells (HSCs) and myeloid com- [email protected] (G.A.K.) partments (23–26). In T cells, TET2 contributes to CD4+ Th cell The online version of this article contains supplemental material. differentiation (27) and cooperates with TET3 to stabilize Foxp3 Abbreviations used in this article: AF, Alexa Fluor; DMC, differentially methylated expression in regulatory T cells (28). cytosine; DMR, differentially methylated region; Eomes, eomesodermin; ERRBS, + enhanced reduced representation bisulfite sequencing; 5hmC, 5-hydroxymethylcyto- However, the function of TET2 in CD8 T cell differentiation is sine; HSC, hematopoietic stem cell; IPA, Ingenuity Pathway Analysis; LCMV, lym- unknown. In this study, we investigated TET2’s role in directing phocytic choriomeningitis virus; Lm-gp33, Listeria monocytogenes that expresses the + LCMV gp33 epitope; 5mC, 5-methylcytosine; MPEC, memory precursor effector CD8 T cell fate following acute lymphocytic choriomeningitis cell; p.i., postinfection; SLEC, short-lived effector cell; TCM, central memory virus (LCMV) infection. We found that TCR signaling rapidly CD8+ T cell; TET, ten-eleven translocation; TET2cKO, TET2 conditional knockout; and dynamically regulates TET2 expression and TET activity. WT, wild-type. Although mice with selective loss of TET2 in T cells have no overt Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 thymic or peripheral T cell phenotypes at steady-state, following www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700559 2 TET2 REGULATES CD8+ T CELL MEMORY acute viral infection, CD8+ T cells preferentially adopt a memory performed using a Cytofix/Cytoperm kit (BD Biosciences) or a Foxp3/ phenotype in a cell-intrinsic manner and demonstrate superior Transcription Factor Staining Buffer Set (eBioscience), according to the pathogen control upon rechallenge. Methylation analysis of LCMV- manufacturer’s instructions. Discrimination of live cell populations was per- + formed using LIVE/DEAD Aqua stain (Invitrogen), according to the manu- specific CD8 T cells identified genomic loci that gained 5mC/5hmC facturer’s instructions. in TET2-deficient cells, including several transcriptional regulators For experiments involving measurement of intracellular 5hmC, T cells known to direct CD8+ T cell effector versus memory differentiation. were surface stained prior to fixation/permeabilization with a Cytofix/ Together, these data demonstrate a novel role for TET2 in directing Cytoperm kit (BD Biosciences), treated with DNase I (300 mg/ml in + PBS) at 37˚C for 1 h, and intracellularly stained with isotype or anti–5hmC CD8 T cell fates. (catalog number 39791; 1 mg/ml; Active Motif) Ab for 30 min, followed by fluorochrome-conjugated goat anti-rabbit secondary Ab (Invitrogen). Materials and Methods For experiments involving ex vivo stimulation,
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