Cutting Edge: Chromatin Accessibility Programs CD8 T Cell Memory Christopher D. Scharer, Alexander P. R. Bally, Bhanu Gandham and Jeremy M. Boss This information is current as of October 1, 2021. J Immunol 2017; 198:2238-2243; Prepublished online 8 February 2017; doi: 10.4049/jimmunol.1602086 http://www.jimmunol.org/content/198/6/2238 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/02/07/jimmunol.160208 Material 6.DCSupplemental References This article cites 30 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/198/6/2238.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 by guest on October 1, 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 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Th eJournal of Cutting Edge Immunology Cutting Edge: Chromatin Accessibility Programs CD8 T Cell Memory Christopher D. Scharer,1 Alexander P. R. Bally,1 Bhanu Gandham, and Jeremy M. Boss CD8 T cell memory is characterized by rapid recall of been studied (1–4), it is still unclear how these properties are effector function, increased proliferation, and reduced molecularly programmed following Ag encounter and main- activation requirements. Despite the extensive func- tained during homeostasis. The functional fate of T cells in- tional characterization, the molecular mechanisms that volves the integration of external signals through distinct facilitate these enhanced properties are not well charac- transcription factor complexes, such as Nfatc1 (5), at cis- terized. In this study, the assay for transposase- regulatory sites; however, the catalog of cis-regulatory sites that participate in CD8 T cell differentiation is not known. accessible chromatin sequencing was employed to Downloaded from map the cis-regulatory elements in CD8 T cells The dynamic alterations in transcriptional and epigenetic responding to acute and chronic lymphocytic chorio- programs, such as DNA methylation and histone modifica- tions, that are enacted during the different stages of the CD8 meningitis virus infections. Integration of chromatin T cell response to infection have been profiled in multiple accessibility profiles with gene expression data identi- systems (6–12); however, these datasets have not been inte- fied unique regulatory modules that were enriched for grated. The consequences of epigenetic mechanisms and http://www.jimmunol.org/ distinct combinations of transcription factor–binding programs can be observed through the changes in chromatin motifs. Memory CD8 T cells displayed a chromatin accessibility. In this study, the assay for transposase-accessible accessibility structure that was absent from other acute chromatin sequencing (ATAC-seq) (13) was used to define and exhausted cells types and included key effector and distinct epigenetic profiles in T cells during different stages of proliferative genes. Stimulation of memory cells acute and chronic viral infections. These profiles reveal a revealed enhanced transcription of “memory-primed” unique epigenetic state of memory CD8 T cells that may genes compared with naive cells. Thus, memory CD8 provide a mechanism for enhanced function. T cells display a preprogrammed chromatin accessibil- ity profile and maintain a molecular history of cis- Materials and Methods by guest on October 1, 2021 element usage, thereby reducing the steps necessary to Mice and lymphocytic choriomeningitis virus infection revive effector functions. The Journal of Immunology, Wild-type C57BL/6J mice were obtained from The Jackson Laboratory and 2017, 198: 2238–2243. bred on-site. Thy1.1+ P14 mice and viral stocks of lymphocytic choriomen- ingitis virus (LCMV) strains Armstrong and clone 13 were provided by Dr. Rafi Ahmed (Emory University). Infections were performed on 6- to 8-wk- old mice as previously described (14), and viral titers were confirmed by pon recognition of cognate Ag and given the proper plaque assay (15). All animal protocols were approved by the Emory Uni- costimulatory signals, CD8 T cells proliferate and versity Institutional Animal Care and Use Committee. U differentiate into short-lived effector cells capable of killing infected cells. Following Ag clearance, a small pop- Cell isolation and analysis ulation of long-lived memory cells emerges that has unique CD8 T cells were enriched from splenocytes by MACS (Miltenyi Biotec) in functional properties from their naive precursors, including accordance with the manufacturer’s instructions, stained in FACS buffer lower activation requirements, enhanced proliferative capacity, (PBS, 1% BSA, 1 mM EDTA), and sorted on a BD FACSAria II. Abs used for sorting were from Tonbo Biosciences (CD8 FITC, CD4 PerCP-Cy5.5, and more rapid induction of effector genes (1–4). Although CD44 allophycocyanin-Cy7) or BioLegend (CD62L Alexa Fluor 700, the functional enhancements of memory CD8 T cells have Thy1.1 Pacific Blue, and PD-1 PE). Mouse MHC class I (H-2Db) tetramers Department of Microbiology and Immunology, Emory University School of Medicine, Address correspondence and reprint requests to Dr. Jeremy M. Boss, Emory University, Atlanta, GA 30322; and Emory Vaccine Center, Emory University School of Medicine, 1510 Clifton Road, Room 3001, Atlanta, GA 30322. E-mail address: jmboss@emory. Atlanta, GA 30322 edu 1C.D.S. and A.P.R.B. contributed equally to this work. The online version of this article contains supplemental material. ORCIDs: 0000-0001-7716-8504 (C.D.S.); 0000-0003-4494-5033 (A.P.R.B.); 0000- Abbreviations used in this article: aD, LCMV Armstrong on day; ATAC-seq, assay for 0002-4027-1062 (B.G.); 0000-0002-2432-1840 (J.M.B.). transposase-accessible chromatin sequencing; clD, LCMV clone 13 on day; DAR, dif- ferentially accessible region; DEG, differentially expressed gene; GO, Gene Ontology; Received for publication December 21, 2016. Accepted for publication January 18, LCMV, lymphocytic choriomeningitis virus; PC, principal component; PCA, principal 2017. component analysis. This work was supported by National Institutes of Health Grant R01 AI113021 (to J.M.B.). A.P.R.B. was supported by National Institutes of Health Grant T32 AI007610. Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 The ATAC-sequencing data presented in this article have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus database (http://www. ncbi.nlm.nih.gov/geo/) under accession number GSE83081. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602086 The Journal of Immunology 2239 corresponding to LCMV peptides gp33 var C41M (KAVYNFATM), gp276 Chromatin accessibility and gene expression patterns are linked to (SGVENPGGYCL), and np396 (FQPQNGQFI) were obtained from the T cell function National Institutes of Health Tetramer Core facility at Emory University. For ex vivo experiments, 104 P14 CD8 T cells were adoptively transferred Chromatin accessibility data from naive, day 8, and day 30 into wild-type hosts. After 1 d, mice were infected with LCMV Armstrong. At were integrated with their respective gene expression data (6). day 28, splenocytes from immune mice or naive P14 mice were stimulated with 4 mM LCMV gp33 var C41M peptide for the times indicated. Cells DARs (n = 8239) were mapped to a differentially expressed were fixed in 1% paraformaldehyde, and virus-specific P14 cells were sorted gene (DEG), and a normalized Euclidean distance metric by FACS using markers described above. RNA was prepared using the Pin- followed by k-means clustering was used to categorize the point slide RNA isolation system (Zymo Research). patterns of gene expression and accessibility changes at all loci relative to each other (23). Seven distinct k-means mod- ATAC-seq and data analyses ules were identified (Fig. 2A, Supplemental Fig. 1D, ATAC-seq libraries were generated from 104 cells for each sample, sequenced, Supplemental Table III). Some modules demonstrated coor- and data processed as detailed previously (16). Differential accessibility was dinate increases (1, 7) or decreases (2, 6) in accessibility and . determined using edgeR (17), and loci with accessibility changes 2-fold and gene expression from naive to day 30 CD8 T cells. In module a false discovery rate of ,0.05 were called significant. Gene Ontology (GO) term enrichment was determined using DAVID (18) and motifs identified 4, both accessibility and gene expression increased to day 8, with HOMER (19) using all significant acute time point peaks as back- followed by a subsequent decrease in both parameters at ground. For transcription factor footprinting analysis, a bed file of motif aD30. With the exception of module 4, progressive/linear occurrences in the differentially accessible region (DAR) was used as input for the HOMER (19) annotatePeaks.pl script with the following options: “-norm