The Journal of Immunology Defining CD4 T Cell Memory by the Epigenetic Landscape of CpG DNA Methylation H. Kiyomi Komori, Traver Hart, Sarah A. LaMere, Pamela V. Chew, and Daniel R. Salomon Memory T cells are primed for rapid responses to Ag; however, the molecular mechanisms responsible for priming remain incom- pletely defined. CpG methylation in promoters is an epigenetic modification, which regulates gene transcription. Using targeted bisulfite sequencing, we examined methylation of 2100 genes (56,000 CpGs) mapped by deep sequencing of T cell activation in human naive and memory CD4 T cells. Four hundred sixty-six CpGs (132 genes) displayed differential methylation between naive and memory cells. Twenty-one genes exhibited both differential methylation and gene expression before activation, linking pro- moter DNA methylation states to gene regulation; 6 of 21 genes encode proteins closely studied in T cells, whereas 15 genes represent novel targets for further study. Eighty-four genes demonstrated differential methylation between memory and naive cells that cor- related to differential gene expression following activation, of which 39 exhibited reduced methylation in memory cells coupled with increased gene expression upon activation compared with naive cells. These reveal a class of primed genes more rapidly expressed in memory compared with naive cells and putatively regulated by DNA methylation. These findings define a DNA methylation sig- nature unique to memory CD4 T cells that correlates with activation-induced gene expression. The Journal of Immunology, 2015, 194: 1565–1579. ifferentiation into fast-acting memory cells following Ag have a demethylated IL-4 gene promoter (10), do not express IFN-g, recognition is a central tenet of T cell immunology. and have a methylated IFN-g promoter (11, 12). Similarly, Foxp3 D Memory T cells are often described as being “primed” (13–15), IL-2 (6, 16–18), IL-17A (19), and other immune genes have for rapid responses to Ag; however, the molecular mechanisms been shown to be regulated by DNA methylation. responsible for this priming remain incompletely defined. Meth- Although these studies have been performed to look at the impact ylation of CpG dinucleotides in promoter regions is one mecha- of DNA methylation on the expression of single genes, few have nism of epigenetic regulation of gene transcription (1–3). CpG employed a more global examination of DNA methylation in CD4 methylation is maintained during cell division, but it can be al- T cells (20). Indeed, many candidate genes have been screened for tered by aging or environmental stimuli such as disease (1, 4) and promoter CpG methylation in CD4 T cells profiled at rest and T cell activation (5–7). Many genes corresponding to immune following activation (9, 17, 18, 21), during development (3, 22), or function have been identified as being regulated by CpG meth- comparing conventional T cells to regulatory T cells (23). CD4 ylation, implicating a role for CpG methylation in T cell function T cells have also been studied in disease contexts such as latent and differentiation. Clear examples of this phenomenon include autoimmune diabetes in adults (24), bronchial asthma (8), or the patterns of DNA methylation and gene expression for IFN-g systemic lupus erythematosus (25). Recently, Hashimoto et al. (7) and IL-4 in the development of Th1 and Th2 lineages. Differen- conducted a global DNA methylation analysis in murine naive, tiated Th1 cells express IFN-g and exhibit a demethylated IFN-g effector, and memory CD4 T cells. The authors found that most promoter (8, 9). These same cells do not express IL-4 and have differential methylation between naive and memory cells occurred a methylated IL-4 promoter. Conversely, Th2 cells express IL-4, in introns and intergenic regions. Interestingly, the methylation changes occurring following activation of memory CD4 T cells localized to enhancer regions. Department of Molecular and Experimental Medicine, The Scripps Research Insti- To fully appreciate the impact of epigenetic changes in disease tute, La Jolla, CA 92037 states it is important to understand how CpG methylation regulates Received for publication May 6, 2014. Accepted for publication December 8, 2014. function and activation-dependent lineage commitments of human This work was supported by National Institutes of Health Grants U19 A1063603 (to naive and memory CD4 T cells in healthy individuals. We used D.R.S. and T.H.), TL1 TR001113-01 (to S.A.L.), T32DK007022-30, and a postdoc- toral Juvenile Diabetes Research Foundation fellowship (to H.K.K.), and by the deep RNA sequencing (RNA-seq) of naive and memory cells Molly Baber Research Fund and the Verna Harrah Research Fund, supporting the activated by CD3/CD28 crosslinking to identify a set of high-value Salomon Laboratory. This is manuscript 23060 from The Scripps Research Institute. candidate genes for epigenetic regulation. Then, using high-throughput The sequences presented in this article have been submitted to the National Center targeted microdroplet PCR (26), we successfully mapped 57,706 for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm. nih.gov/geo/) under accession number GSE59860. CpGs across 1946 selected genes. This study shows an inverse association between promoter CpG Address correspondence and reprint requests to Dr. Daniel R. Salomon, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 methylation and RNA expression, particularly in genes without North Torrey Pines Road, Mail Code MEM-241, La Jolla, CA 92037. E-mail address: a promoter CpG island (CGI). We identified 132 genes that were [email protected] differentially methylated between CD4 naive and memory subsets. The online version of this article contains supplemental material. In contrast, 48 h following activation, there was surprisingly little Abbreviations used in this article: AIM2, absent in melanoma 2; CGI, CpG island; variation in CpG methylation from resting to activated cells. These FDR, false discovery rate; RNA-seq, RNA sequencing; TSS, transcriptional start site. 132 genes mapped to pathways involved in cellular migration, Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 hematological system development and function, and inflammatory www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401162 1566 DNA METHYLATION OF MEMORY CD4 T CELLS responses, consistent with the conclusion that DNA methylation Preparation of bisulfite-converted DNA is important for regulation of CD4 T cell function. Moreover, Bisulfite-converted DNAwas prepared as previously described (26). Briefly, 21 genes exhibited differential methylation between naive and DNA was bisulfite treated using the EpiTect bisulfite kit (Qiagen) fol- memory CD4 T cells that correlated with differential gene ex- lowing the manufacturer’s protocol. Converted DNA was concentrated on pression at rest, and 84 genes demonstrated differential methylation Agencourt AMPure XP beads and quantitated using the Quant-iT ssDNA between memory and naive cells that correlated to differential gene assay kit (Invitrogen) and a Qubit fluorimeter (Invitrogen). expression following activation. Ultimately, we mapped most of RainDance Technologies microdroplet PCR these genes to three pathways involved in delivering a number of Microdroplet PCR was conducted as previously described (26). Briefly, 2 mg critical inflammatory cytokine signals, many with established bi- bisulfite-converted DNA was merged with the CD4-specific 2100 gene ological significance in CD4 T cell activation and innate immunity droplet library and amplified with 55 cycles of PCR. Following amplifica- and many as novel candidates for additional research. tion, the droplet emulsion was broken and the amplified DNA was purified using a MinElute PCR purification kit (Qiagen) following standard protocols. Materials and Methods Preparation of sequencing libraries and deep bisulfite Ethics statement sequencing All of the present studies were covered by Human Subjects Research RainDance Technologies PCR products were concatenated as previously Protocols approved by the Institutional Review Board of The Scripps described (26). Briefly, 400 ng microdroplet PCR product was end-repaired Research Institute. Informed written consent was obtained from all study and concatenated. Concatenated products were then fragmented to 200 bp subjects in the study. using a Covaris S2. Fragmentation was confirmed on an Agilent Tech- nologies Bioanalyzer using an HS DNA chip. Ten to 100 ng fragmented Isolation and activation of human lymphocytes PCR product was end-repaired and A-tailed. Indexed adapters were li- gated, and ligation product was purified on Agencourt AMPure XP beads Peripheral blood was collected from healthy donors, and PBMC were followed by size selection from 2% agarose. Purified product was ampli- collected by centrifugation through a Histopaque (Sigma-Aldrich) gradient. fied with 18 cycles of PCR followed by size selection from 2% agarose. CD4 T cells were negatively selected from PBMC using the naive CD4 Libraries were assessed on an Agilent Technologies Bioanalyzer using T cell isolation kit II (Miltenyi Biotec) or the memory CD4 T cell isolation a DNA chip and quantitated using the Quant-iT dsDNA BR assay kit and kit (Miltenyi Biotec) from six donors. CD8 T cells and B cells were a Qubit fluorimeter. Cluster generation and sequencing on an Illumina positively selected from PBMC using CD8 or CD19 microbeads (Miltenyi HiSequation 2000