Cutting Edge: Chromatin Accessibility Programs CD8 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

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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- 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 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 . 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, ; 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 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 changes in accessibility were observed from naive to mem- ory cells. This indicated that most memory-related DARs 1e6 -fragLength 1 –hist 1 -size 100.” All other data display and analyses were Downloaded from performed using custom R/Bioconductor scripts. ATAC-seq data are detailed were programmed early and shared with effector cells, whereas in Supplemental Table I and are available under accession number GSE83081 only a small subset (module 4) was reprogrammed during in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/ geo/). Raw microarray data were downloaded from accession number differentiation from effector to memory. Modules 3 and 5 GSE30341 (6). Data processing was performed using the affy R/Bioconductor showed inverse relationships between accessibility and gene package (20). Mapping of ATAC-seq peaks to genes was performed by anno- expression. These loci are hypothesized to represent cis-elements tating each peak to the nearest gene transcription start site and microarray data

that function as negative regulators of gene expression. http://www.jimmunol.org/ annotatedusingtheEntrezID. Hierarchal clustering and GO annotation partitioned the modules into two distinct groups, with modules 1, 3, 4, and 7 Quantitative RT-PCR defining functions of effector T cells: cell division components, cDNA was prepared from total RNA using SuperScript II reverse transcriptase immune effector processes, and cell surface receptors (Fig. 2B). (Invitrogen). Relative cDNA quantities for each gene listed were quantitated Modules 2, 5, and 6 contained genes with roles in tran- using real-time PCR and calculated as a percentage of Actb expression within each sample. All primer sequences are available on request. scriptional regulation, chromatin organization, and negative regulators of metabolism. For example, loci in module 2 lost accessibility and expression between naive and day 8 and Results and Discussion continued to decrease to day 30. GO terms in module 2 by guest on October 1, 2021 Chromatin accessibility is remodeled during acute LCMV infection consisted of negative regulators of metabolism (Eif2ak3 and Mice were infected with LCMV Armstrong to induce an acute Id3) and DNA binding (Tcf7 and Ikzf1), possibly represent- infection resulting in functional memory, or with LCMV clone ing regulators that maintain a naive CD8 T cell fate until 13 to induce a chronic infection and T cell exhaustion (21). activation signals are encountered. These analyses highlight Virus-specific CD8 T cells were isolated at time points rep- the kinetic coordination of chromatin reprogramming and resenting distinct phases of the CD8 T cell response gene expression associated with CD8 T cell function. (Supplemental Fig. 1A): LCMV Armstrong on day (aD)4 and aD8, acute effector; aD30, memory; and LCMV clone 13 on Transcription factor networks coordinate accessibility changes day (clD)8 and clD28, early and late stages of T cell ex- Enriched, transcription factor motifs in the DARs of the above haustion, respectively. ATAC-seq (13, 16) was employed to modules were classified into eight categories (I–VIII) and generate an unbiased chromatin accessibility map at each time sorted based on those shared between modules (I and II) or point. Principal component analysis (PCA) of all accessible unique to a specific module (III–VIII) (Fig. 2C, Supplemental sites (89,746) indicated a progressive linear shift across the Table IV). Modules containing genes that were repressed in PC1 axis from the naive to the differentiated time points that activated CD8 T cells were surrounded by loci enriched for correlated with cell activation (Fig. 1A). Compared to naive developmental transcription factor binding sites, including cells, all time points demonstrated large gains in accessibility those for Tcf3 (category VI; module 2) and GATA (category that peaked at day 8 (Fig. 1B, Supplemental Fig. 1B, V; module 6) factors. Supplemental Table II). The PC2 axis separated effector (day Category I motifs were enriched for AP-1 and NFAT family 8) and late differentiation time points (aD30/clD28). More transcription factor binding sites and were shared across regions lost than gained accessibility between the effector multiple modules associated with T cell activation. Interest- (aD8) and memory (aD30) phases (Fig. 1B). For example, the ingly, the complexity of NFAT motifs correlated with the Pdcd1 underwent gains in accessibility at each phase, distinct functions of modules 1 and 7. GO analysis of module including a region that was unique to the chronic phase (Fig. 1, which revealed a strong enrichment for cell surface receptors 1C) (22). In contrast, Sell contained cis-elements that tran- (e.g., Ctla4 and Fasl), contained only NFAT monomeric siently lost accessibility at aD4 and aD8 compared with naive motifs and displayed maximal changes in chromatin accessi- and aD30 (Fig. 1D). Overall, accessibility changes correlated bility and gene expression at day 8. In contrast, module 7 with mRNA expression (6, 10) (Supplemental Fig. 1C), in- genes (Tbx21 and Zbtb32), which function in dicating a functional relationship between the datasets. activation and immune effector processes, were enriched for 2240 CUTTING EDGE: CD8 T CELL ATAC-seq Downloaded from FIGURE 1. CD8 T cell chromatin accessibility is dynamically remodeled during viral infection. Following infection of wild-type mice with LCMV Armstrong or clone 13, Ag-specific CD8 T cells (gp33, gp276, np396) were isolated and analyzed at the indicated time points. (A) PCA of 89,746 accessible loci as determined by ATAC-seq of naive (N) CD8 T cells and LCMV Armstrong–derived (aD4, aD8, and aD30) or clone 13–derived (clD8 and clD28) CD8 T cells. (B) Scatter plots showing changes in chromatin accessibility between the indicated cell types. The numbers of significant differently accessible loci (false discovery rate , 0.05, fold change . 2) are shown. Genome plots of the (C) Pdcd1 and (D) Sell loci showing changes in accessibility. ATAC-seq data are representative of three or two (aD4) independent mice. rpm, reads per million. http://www.jimmunol.org/

NFAT/AP-1 composite motifs, and they displayed stepwise pression. The Fas ligand (Fasl) gene locus, which is induced increases in accessibility and expression through aD30. These following CD8 T cell activation (Fig. 3C, Supplemental Fig. data correlate with the distinct regulatory functions of the 1C), contains enhancers representing modules 1 and 3 with different NFAT DNA binding modes (5), with NFAT/AP-1 NFAT and SOX family motifs, respectively. The NFAT heterodimers regulating later cell response events and NFAT motif gained accessibility during activation, but the SOX monomeric sites signaling directly from TCR stimulation. family motifs lost accessibility as Fasl gained expression, The enriched transcription factor motifs in modules 3 and 5 suggesting that these elements may be involved in activating by guest on October 1, 2021 suggest that these loci may function as negative regulatory and silencing expression, respectively. In contrast, the ex- elements. Module 3, which gained expression but lost acces- pression of the T cell developmental factor Tcf7 (27) is sibility over time, was enriched for the SOX family of tran- downregulated during the effector phase and is upregulated scription factors (category IV), which are essential for T cell following Ag clearance (Supplemental Fig. 1C). Accessibility development and memory formation (24). Similarly, al- was lost in two modules (2, 6), which were enriched for the though module 5 denoted genes that lost expression, DNA developmental transcription factor motifs for Tcf3 and sequence analysis identified AP-1 and NFAT consensus mo- GATA. A gain in accessibility at a repressive module 5 peak, tifs (category I) and the transcriptional Tbet (25) which contained an AP-1 family motif, highlighted multiple (category II). Depending on the dimerization partner, AP-1 roles for consensus elements (Fig. 3D). Thus, T cell response transcription factors can function as activators or genes employ multiple modules consisting of both positive (26). Taken together, the presence of positive and negative and negative regulatory control signals to govern gene ex- regulatory elements provides a mechanism to promote a new pression during distinct phases of CD8 T cell responses. cell fate while actively repressing the previous state. High-resolution transcription factor footprints were deter- Memory CD8 T cells contain a unique subset of accessible gene mined for AP-1, Tbet, and NFATc1. Compared to AP-1 and promoters Tbet, the specific NFATc1 motif demonstrated little to no change PCA (Fig. 1A) showed that memory CD8 T cells possessed a in accessibility across all differentiation stages analyzed (Fig. 3A). distinct accessibility profile from naive and effector cells. This For all three factors, the surrounding sequences gained accessi- is exemplified by the fact that none of the expression/ bility, with AP-1 demonstrating the largest gains (Fig. 3A, 3B). accessibility k-means modules displayed a full reset in These data suggest that NFATc1 may initiate the opening of comparingmemorytonaive.Tofurthercharacterizethe chromatin, thereby facilitating the binding of other factors such differentiation-specific programming steps, the changes in as AP-1, which have been shown to favor nucleosome-depleted promoter accessibility of DEGs were determined during the sites (13). In contrast, Tcf3 accessibility was decreased following transitions from naive to effector (aD8) to memory (aD30) the initial activation (Fig. 3B). CTCF motifs were not enriched during acute LCMV infection. Compared to naive, both aD8 in any module and demonstrated no changes in accessibility effector and aD30 memory cells demonstrated a positive genome wide, suggesting that the above changes are specific. correlation, with upregulated genes gaining and downregu- Two loci were chosen to highlight the complex interplay of lated genes losing promoter accessibility (Fig. 4A). Analysis coordinated changes in chromatin accessibility and gene ex- between effector (aD8) and memory (aD30) cells revealed, as The Journal of Immunology 2241 Downloaded from

FIGURE 2. Chromatin accessibility is correlated with gene expression. (A) The change in accessibility (y-axis) versus the change in gene expression (x-axis) for each of the seven k-means modules. The number of loci in each module is indicated. (B) Heat map displaying the top five enriched GO terms identified for each module organized by hierarchical clustering. (C) Transcription factor DNA binding motifs enriched in each module. Modules are organized as in (B). Motifs are grouped (I–VIII) according to those that are shared between, or are specific to, individual modules. http://www.jimmunol.org/ expected, that upregulated active genes gained promoter ac- clD8 and clD28 were distinct from their acute infection cessibility (Fig. 4A). However, in sharp contrast, most of the counterparts. This was exemplified by examination of the downregulated genes still maintained open, accessible pro- “memory-primed” gene subset from above, which in clD28 moters (Fig. 4A, Supplemental Table V), suggesting that these cells showed a 2-fold loss in overall accessibility and did genes were primed for expression. GO analysis revealed that not reduce expression to the same level as aD30 cells the primed genes were enriched for effector functions, such as (Supplemental Fig. 2A–C), suggesting differential program- proliferation and replication, cell activation, and response to ming for these sites in exhausted CD8 T cells. At non- stress (Fig. 4B). promoter peaks for primed genes, 140 loci showed changes in by guest on October 1, 2021 Analyses of the accessibility and gene expression data from accessibility between aD30 and clD28 cell types (Fig. 4C, CD8 T cells generated in this study (Fig. 1A) and elsewhere Supplemental Table VI). For example, the transcription factor (22, 28, 29) from a chronic LCMV infection revealed that Irf8 and the peptidase Ctsc have elements that were more

FIGURE 3. The cis-regulatory sites coordinate gene expression. (A) Chromatin accessibility footprints of transcription factor motifs within all accessible loci for each cell type following an acute infection were plotted at resolution. (B) Total ATAC-seq reads occurring within 200 bp of selected transcription factor motifs plotted in (A). Genome plot of the Fasl (C) and Tcf7 (D) loci showing the different k-means modules of accessible peaks from Fig. 2A. 2242 CUTTING EDGE: CD8 T CELL ATAC-seq

FIGURE 4. Memory CD8 T cells exhibit a unique accessibility structure. (A) Change in mRNA expression for each DEG versus the change in promoter Downloaded from accessibility for the indicated cell types following LCMV Armstrong infection. (B) REVIGO (30) summary of enriched GO biological process terms for primed genes identified in (A) (right plot). (C) Volcano plot displaying significant nonpromoter peaks for the primed gene set. (D) Quantitative RT-PCR time course of the indicated genes from memory or naive P14 CD8 T cells activated with gp33 peptide. Data are normalized to expression of Actb, and error bars represent SD. Each time point was performed in triplicate from cells isolated from independent memory or naive mice. All genes were significantly increased across the time course. *p , 0.05 between specific time points as measured by two-way ANOVA with a Bonferroni post test. http://www.jimmunol.org/ accessible in clD28, whereas the kinetochore component epitopes, add a unique integrative analysis of gene expression Kntc1 and the mitotic kinesin Kif23 have elements that were and accessibility, and specifically identify a set of primed silenced (Supplemental Fig. 2D, 2E). These data indicate that promoters/genes associated with the memory differentiation memory CD8 T cells have a unique chromatin structure that state. Compared to naive cells, memory CD8 T cells are is distinct from exhausted cells and may contribute to their preprogrammed to respond to environmental cues and stim- unique functional properties. uli, thereby reducing the molecular/epigenetic steps necessary to induce effector function. The function of the primed loci Increased transcription and rapid induction of effector functions in in memory CD8 T cells may serve as an epigenetic history of memory CD8 T cells by guest on October 1, 2021 functional elements that program the effector response. The presence of accessible promoters in memory cells suggests that such genes would be expressed at higher levels or allow for more rapid induction during a recall response. The expression Acknowledgments levels of representative primed genes from P14 aD28 memory We thank members of the Boss laboratory and Dr. J. Kohlmeier for insightful CD8 T cells were compared with naive precursors by quan- comments, the New York University Genome Technology Center for sequenc- ing, the Emory Flow Cytometry Core for FACS sorting, the National Institutes titative real-time PCR. All of the genes chosen, except the of Health Tetramer Core at Yerkes for tetramers, and the Emory Integrated transcriptional repressor Zbtb32, including key effector mol- Genomics Core for sequencing library quality control. ecules (Gzma, Gzmb, IFN-g), the transcription factor Eomes, and the adhesion molecule Itgal, demonstrated significantly higher levels of expression in resting memory cells compared Disclosures with naive (Fig. 4D). To determine reactivation kinetics of The authors have no financial conflicts of interest. the primed genes, aD28 memory and naive P14 CD8 T cells were activated ex vivo with gp33 peptide over a time course References and gene expression levels were measured. All genes chosen 1. Veiga-Fernandes, H., U. Walter, C. Bourgeois, A. McLean, and B. Rocha. 2000. Response demonstrated increased levels of transcription but with dis- of naı¨ve and memory CD8+ T cells to antigen stimulation in vivo. Nat. Immunol. 1: 47–53. 2. Pihlgren, M., C. Arpin, T. Walzer, M. 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grative analysis of large gene lists using DAVID bioinformatics resources. Nat. 30. Supek, F., M. Boˇsnjak,N. Skunca,ˇ and T. Smuc.ˇ 2011. REVIGO summarizes and http://www.jimmunol.org/ Protoc. 4: 44–57. visualizes long lists of gene ontology terms. PLoS One 6: e21800. by guest on October 1, 2021