Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells1,2,3,4. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.
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Gene Expression Omnibus
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We thank R. Karaffa and S. Durham at the Emory University School of Medicine Flow Cytometry Core Facility and R. Cross and G. Lennon in the St Jude Flow Cytometry Core Facility for FACS sorting. Whole-genome sequencing was performed by the St Jude Hartwell Sequencing facility. This work was supported by the National Institutes of Health (NIH) grant U19 AI117891 (to R.An.), R01AI030048 (to R.Ah.), U19AI057266 (to R.Ah.), R01AI114442(to B.Y.), and funds from American Lebanese Syrian Associated Charities (ALSAC) (to B.Y.).
The authors declare no competing financial interests.
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Extended data figures and tables
Extended Data Figure 1 Sell gene expression changes during effector and memory CD8 T cell differentiation are coupled to epigenetic reprogramming of the Sell promoter.
a, Cartoon of CpG positions within the Sell promoter region cloned into the CpG-free Lucia Promoter reporter construct. Putative transcription factor binding sites are indicated by coloured boxes. b, Representative methylation profiling of in vitro methylation efficiency of the reporter construct. c, Longitudinal measurement of relative light units from EL4 cells transfected with unmethylated and in vitro methylated reporter constructs. d, Real-time PCR analysis of Sell mRNA in virus-specific naive, effector, and memory P14 CD8 T cells. e, Summary of Sell proximal promoter methylation in naive, day 4 effector, day 8 effector and day 60+ memory P14 CD8 T cells. Each horizontal line represents an individual sequenced clone. Filled circles, methylated cytosine; open circles, non-methylated cytosine. f, Real-time PCR analysis of Sell mRNA expression from day 8 TE and MP P14 CD8 T cells, and day 37 L-selectinlo and L-selectinhi P14 CD8 T cells. Transcript data correspond to cell sorts used for DNA methylation measurements in Fig. 1f, h. g, h, Summary of Sell proximal promoter DNA methylation in TE and MP effector CD8 T cells and L-selectinlo and L-selectinhi memory CD8 T cells. Statistics were generated from three or more biological replicates.
a, Experimental setup for isolating MP and TE LCMV-specific CD8 T cells on days 4.5 and 8. b, Representative post-sort purity and phenotypic analysis of day 4.5 and day 8 MP and TE P14 CD8 T cells isolated from acutely infected mice used for WGBS methylation profiling.
Extended Data Figure 3 Effector-associated changes in DNA methylation occur predominantly at or near genes and are highly similar between MP and TE CD8 T cells.
Pie chart representation of newly methylated DMR genomic distribution relative to the TSS of the nearest gene.
a, Pie charts represent demethylated DMR genomic distribution relative to the TSS of the nearest gene. b, Venn diagrams of regions that undergo demethylation during differentiation of naive CD8 T cells into TE and MP subsets. c, Normalized methylation at CpG sites in the Gzmk locus from TE and MP WGBS datasets. d, Normalized differentially methylated CpG sites in the Klrg1, Prdm1 (also known as Blimp1), Runx2, and Runx3 loci from TE and MP WGBS datasets.
Extended Data Figure 5 Conditional deletion of Dnmt3a in activated CD8 T cells inhibits effector-associated de novo DNA methylation but does not impair maintenance methylation.
a, Cre recombinase expression is driven by the Gzmb promoter to initiate recombination of Dnmt3a exon 19 following T cell activation. b, Representative FACS analysis of virus-specific CD8 T cells sorted 8 days after acute viral infection of wild-type and Dnmt3a cKO mice. c, Recombination of genomic DNA from FACS-purified Dnmt3a cKO virus-specific CD8 T cells was assessed by PCR using primers that anneal to DNA outside the floxed target region. The larger PCR amplicon corresponds to the intact locus and the smaller PCR product is the amplicon of the recombined locus. d, Representative and graphical summary of Sell promoter methylation in wild-type and Dnmt3a cKO cells. Mean and s.d. were calculated from bisulfite sequencing analysis of six individually sorted populations. e, Diagram of Sell promoter CpG location proximal and distal to the TSS. f, Representative DNA methylation analysis of CpG sites distal to the Sell promoter regions in day 8 wild-type and Dnmt3a cKO antigen-specific effector CD8 T cells. Graphical summary of the average Sell distal CpG methylation in wild-type and Dnmt3a cKO cells calculated from bisulfite sequencing analysis of four individually sorted populations.
Extended Data Figure 6 Effector-stage de novo DNA methylation is enriched at genes that regulate effector and memory T cell differentiation.
a, Normalized Dnmt3a-mediated de novo methylation at CpG sites in the Lef1 and Il6st loci from WGBS datasets. b, Summary of maintenance methylated regions in wild-type and Dnmt3a cKO effector WGBS datasets. c, Connectivity plot showing IPA-predicted interactions of ID2 and ID3 with Dnmt3a-targeted loci.
a, Summary of gp33-specific CD8 T cell quantities at effector and memory time points in lymphoid and nonlymphoid tissues. b, c, Summaries of viral titres in spleen (b) and day 5 lung and liver (c) of acutely infected wild-type and Dnmt3a cKO mice. d, Quantitative PCR analysis of Dnmt3a exon 19 recombination using a primer set that binds to DNA internal to the floxed target region. The mean and s.d. of intact (non-recombined) floxed Dnmt3a alleles were determined by quantitative PCR from four individually sorted gp33-specific effector and memory CD8 T cell populations. e, Real-time PCR analysis of Sell mRNA expression of naive and tetramer+ wild-type and Dnmt3a cKO effector CD8 T cells. f, Representative FACS analysis of Klrg1, CD127, CD27, and L-selectin expression on wild-type and Dnmt3a cKO effector and memory gp33-specific CD8 T cell splenocytes. g, Summary graph for the percentage of wild-type and Dnmt3a cKO L-selectin-positive gp276 and np396-specific CD8 T cells.
Extended Data Figure 8 Effector molecule loci are demethylated during differentiation of virus-specific Dnmt3a cKO CD8 T cells.
a, Heat-map representation of top 3,000 demethylated regions in wild-type and Dnmt3a cKO effector CD8 T cell WGBS datasets relative to the naive WGBS dataset. b, Normalized effector loci methylation at CpG sites in the Ifng, Prf1, and Gzmk loci from wild-type and Dnmt3a cKO WGBS datasets. c, Representative FACS analysis of Tbet, Eomes, and Ki67 expression of gp33-specific effector CD8 T cells. d, Representative FACS analysis of cytokine production from virus-specific memory CD8 T cells following 5 h of ex vivo gp33 peptide stimulation.
a, Representative FACS analysis of L-selectin expression on Thy1.1+ CFSE+ MP and TE CD8 T cells 1 day after transfer into naive recipient mice. The limit of our detection was approximately 10–20 CD62L+ cells in each of the lymphoid and nonlymphoid tissues at 1 day post-transfer. b, Summary of number of transferred TE and MP CD8 T cells in the spleen, blood, lymph node, IEL (intraepithelial lymphocytes), lung, and liver of the recipient mice 1 day post-transfer. c, Summary of per cent undivided (undiluted CFSE) L-selectin-positive virus-specific memory CD8 T cells arising from adoptively transferred MP versus TE cells. Data are from three independent experiments. d, Representative post-sort purity FACS analysis of undivided L-selectinhi and L-selectinlo MP P14 cells 28 days after adoptive transfer.
Representative analysis and summary graphs of locus-specific methylation profiling of Gzmb (a) and Prf1 (b) DMRs in naive, effector (day 8 gp33 tetramer+) and memory (day 40+ gp33 tetramer+) CD8 T cells. s.d. calculated from three independently sorted samples.
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Youngblood, B., Hale, J., Kissick, H. et al. Effector CD8 T cells dedifferentiate into long-lived memory cells. Nature 552, 404–409 (2017). https://doi.org/10.1038/nature25144
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