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. 2017 Dec 21;552(7685):404-409.
doi: 10.1038/nature25144. Epub 2017 Dec 13.

Effector CD8 T cells dedifferentiate into long-lived memory cells

Ben Youngblood  1   2   3 , J Scott Hale  1   2 , Haydn T Kissick  4 , Eunseon Ahn  1   2 , Xiaojin Xu  1   2 , Andreas Wieland  1   2 , Koichi Araki  1   2 , Erin E West  1   2 , Hazem E Ghoneim  3 , Yiping Fan  5 , Pranay Dogra  3 , Carl W Davis  1   2 , Bogumila T Konieczny  1   2 , Rustom Antia  6 , Xiaodong Cheng  7 , Rafi Ahmed  1   2
Affiliations

Effector CD8 T cells dedifferentiate into long-lived memory cells

Ben Youngblood et al. Nature. .

Abstract

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 cells. 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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Conflict of interest statement

The authors report no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. CD62L (L-selectin) gene expression changes during effector and memory CD8 T cell differentiation are coupled to epigenetic reprogramming of the CD62L promoter
A) Real-time PCR analysis of CD62L mRNA in virus-specific naïve, effector, and memory P14 CD8 T cells. B) Cartoon diagram of CpG positions within the CD62L promoter region cloned into the CpG free Lucia Promoter reporter construct. Putative transcription factor binding sites are indicated by colored boxes. C) Representative methylation profiling of in vitro methylation efficiency of the reporter construct. D) Longitudinal measurement of Relative light units from EL4 cells transfected with unmethylated and in vitro methylated reporter constructs. E) Summary graph of CD62L proximal promoter methylation of naïve, day 4 effector, day 8 effector, >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 CD62L mRNA expression from day 8 terminal-effector and memory-precursor P14 CD8 T cells, and day 37 effector-memory and central-memory P14 CD8 T cells. Transcript data correspond to cell sorts used for DNA methylation measurements in figure 1f and 1h. G & H) Summary graph of the CD62L proximal promoter DNA methylation in TE and MP effector CD8 T cells and CD62Llo and CD62Lhi memory CD8 T cells.
Extended Data Figure 2
Extended Data Figure 2. Isolation of memory precursor (MP) and terminal effector (TE) CD8 T cells for whole-genome methylation profiling
A) Experimental setup for isolating day 4.5 and day 8 MP and TE LCMV-specific CD8 T cells. 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 whole-genome bisulfite sequencing (WGBS) methylation profiling.
Extended Data Figure 3
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 cell subsets
A) Pie chart representation of newly methylated DMR genomic distribution relative to the transcriptional start site of the nearest gene.
Extended Data Figure 4
Extended Data Figure 4. Both MP and TE CD8 T cells acquire demethylated effector loci
A) Pie chart representation demethylated DMR genomic distribution relative to the transcriptional start site of the nearest gene. B) Venn diagram of regions that undergo demethylation during differentiation of naïve CD8 T cells into TE and MP subsets. C) Normalized graph of methylation at CpG sites in the Gzmk locus of TE and MP WGBS data sets. D) Normalized graph of differentially methylated CpG sites in the Klrg1, Prdm1 (Blimp1), Runx2, and Runx3 loci from TE and MP WGBS data sets.
Extended Data Figure 5
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 granzyme b promoter to initiate recombination of Dnmt3a exon 19 following T cell activation. B) Representative FACS analysis of virus-specific CD8 T cells sorted at 8 days post acute viral infection of WT and Dnmt3a cKO mice. C) Recombination of genomic DNA from FACS purified Dnmt3a cKO cells virus-specific CD8 T cells was assessed by PCR using primers that anneal to DNA outside of 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 CD62L promoter methylation in WT and Dnmt3a cKO cells. Average and standard deviation were calculated from bisulfite sequencing analysis of 6 individually sorted populations. E) Cartoon diagram of CD62L promoter CpG location proximal and distal to the transcriptional start site. F) Representative DNA methylation analysis of CpG sites distal to the CD62L promoter regions in Day 8 WT and Dnmt3a cKO antigen-specific effector CD8 T cells. Graphical summary of the average CD62L distal CpG methylation in WT and Dnmt3a cKO cells calculated from bisulfite sequencing analysis of 4 individually sorted populations.
Extended Data Figure 6
Extended Data Figure 6. Effector-stage de novo DNA methylation is enriched at genes that regulate effector and memory T cell differentiation
A) Normalized graph of Dnmt3a-mediated de novo methylation at CpG sites in the Lef1 and IL6ST loci from WGBS data sets. B) Summary graph of maintenance methylated regions in WT and Dnmt3a cKO effector WGBS datasets. C) Connectivity plot showing IPA predicted interactions of ID2 and ID3 with Dnmt3a-targeted loci.
Extended Data Figure 7
Extended Data Figure 7. Dnmt3a deficient CD8 T cells undergo effector differentiation
A) Summary graphs of gp33-specific CD8 T cell quantity at effector and memory time points in lymphoid and nonlymphoid tissues. Summary graphs of viral titers in spleens (B) and day 5 lung and liver (C) of acutely infected WT and Dnmt3a cKO mice. D) Quantitative-PCR analysis of Dnmt3a exon 19 recombination using a primer set that binds to DNA internal of the floxed target region. The average and standard deviation of intact (non-recombined) floxed Dnmt3a alleles were determined by quantitative PCR from 4 individually sorted gp33-specific effector and memory CD8 T cell populations. E) Real-time PCR analysis of CD62L mRNA expression of naïve and tetramer+ WT and Dnmt3a cKO effector CD8 T cells. F) Representative FACS analysis of Klrg1, CD127, CD27, and CD62L expression on WT and Dnmt3a cKO effector and memory gp33-specific CD8 T cell splenocytes. G) Summary graph showing the percent of CD62L positive LCMV-specific CD8 T cell splenocytes recognizing the LCMV dominate epitopes gp276, and np296.
Extended Data Figure 8
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 3000 demethylated regions among WT and cKO effector CD8 T cell WGBS data sets relative to the naïve WGBS data set. B) Normalized graph of effector loci methylation at CpG sites in the IFNg, Prf1, and GzmK loci from WT and cKO WGBS data sets. 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 hours of ex vivo gp33 peptide stimulation.
Extended Data Figure 9
Extended Data Figure 9. CD62L low MP effector CD8 T cells develop into Tcm CD8 T cells
A) Representative FACS analysis of CD62L expression on Thy1.1+ CFSE+ MP and TE CD8 T cells 1 day post transfer into naïve recipient mice. The limit of our detection was ~10-20 CD62L+ cells in each of the lymphoid and nonlymphoid tissues at 1 day post-transfer. B) Summary graph of number of transferred TE and MP CD8 T cells in the spleen, blood, lymph node, IEL, lung, and liver of the recipient mice 1 day post-transfer. C) Summary graph of percent undivided (undiluted CFSE) CD62L positive virus-specific memory CD8 T cells arising from adoptively transferred MP vs. TE cells. Data are from 3 independent experiments. D) Representative post-sort purity FACS analysis of undivided CD62Lhi and CD62Llo MP P14 cells 28 days after adoptive transfer.
Extended Data Figure 10
Extended Data Figure 10. Memory CD8 T cells retain demethylated effector loci
Representative analysis and summary graph for loci-specific methylation profiling of (A) Gzmb and (B) Perforin DMRs in naïve, effector (day 8 gp33 tetramer+) and memory (>day 40 gp33 tetramer+) CD8 T cells (Standard deviation is calculated from 3 independently sorted samples).
Figure 1
Figure 1. Dynamic changes in DNA methylation during effector and memory CD8 T cell differentiation
A) Analysis of on-off-on gene expression from published naïve, and LCMV-specific effector, and memory CD8 T cell microarray data sets. B) Histogram analysis of CD62L protein expression on naïve, effector and memory P14 CD8 T cells following acute LCMV infection. C) Experimental setup and bisulfite sequencing DNA methylation analysis of the CD62L promoter from P14 CD8 T cell purified (>95% purity) at the indicated stages of differentiation. Each horizontal line represents an individual sequenced clone. Filled circles = methylated cytosine. Open circles = non-methylated cytosine. Representative post-sort purity and DNA methylation analysis of the CD62L proximal promoter CpG sites among D) terminal effector (TE) and memory precursor (MP) P14 CD8 T cells FACS purified 8 days after acute LCMV infection, or E) effector-memory (Tem) and central memory (Tcm) P14 CD8 T cells FACS purified 37 days after acute LCMV infection.
Figure 2
Figure 2. Memory-precursor CD8 T cells acquire genome-wide effector-associated DNA methylation programs
A) Summary graph of the number of newly methylated DMRs in TE and MP subsets relative to naïve cells identified from WGBS analyses. B) Normalized graph of CpG methylation in the Ccr7, Tcf7, and CD62L loci from WGBS data sets. Each vertical line indicates a CpG site and the ratio of red to blue indicate the % of methylated versus unmethylated CpGs at these sites. C) Summary graph of the number of demethylation DMRs between the effector subsets and naïve cells. D) Normalized graph of methylation at CpG sites Gzmb, IFNg, and Prf1 loci from TE and MP WGBS data sets. E) Heat-map representation of top 3000 newly methylated regions (relative to naïve CD8 T cell methylation) from WGBS analysis of tetramer+ WT and Dnmt3a cKO effector CD8 T cell. F) Summary graph of de novo methylated regions in WT and Dnmt3a cKO effector. G) Normalized graph of Dnmt3a-mediated de novo methylation at CpG sites in the CD62L, Ccr7, and Tcf7 loci. H) Top Canonical Pathways and Upstream Regulators from Ingenuity Pathway Analysis of gene-associated Dnmt3a-mediated DMRs.
Figure 3
Figure 3. Dnmt3a mediated de novo DNA methylation regulates the kinetics of gene re-expression during the effector to memory CD8 T cell transition
A) Longitudinal measurement of WT and Dnmt3a cKO gp33-specific CD8 T cell numbers among PBMC during acute LCMV infection. B) Longitudinal analysis of central-memory (CD62Lhi) and C) memory-precursor (Klrg1lo CD127hi) phenotypes of gp33-specific WT and Dnmt3 cKO CD8 T cells in PBMCs (n ≥ 5). D) Graphs of the %CD62L positive WT and Dnmt3a cKO gp33-specific memory CD8 T cells in the spleen and lymph node at 44 dpi. E) Representative histogram analysis of CD62L expression on WT (blue line) and Dnmt3a cKO (red line) P14 cells from the PBMC acutely infected chimeric animals at 8, 16, 30, and 44 days post infection. F) Paired analysis of Klrg1lo CD127hi and CD27hi phenotypes on WT and Dnmt3a cKO P14 splenocytes at 35 days post infection from co-transfer experiments (N= 5).
Figure 4
Figure 4. Effector CD8 T cells erase Dnmt3a-mediated DNA methylation programs during their development into memory CD8 T cells
A) Experimental setup for obtaining CFSE labeled TE and MP CD8 T cells at day 1 and 28 post-transfer into naïve mice. B) Representative FACS analysis of CFSE label and CD62L expression of transferred TE and MP populations. C) Graph of the absolute number of transferred MP cells that were undivided and CD62L positive detected in the spleen on day 1 (input) and 28 days post-transfer. D) Real-time PCR analysis of the CD62L transcript from the undivided CD62hi versus CD62Llo memory populations. E) Bisulfite sequencing DNA methylation analysis of the CD62L promoter from the undivided CD62hi versus CD62Llo memory populations.
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Comment in

  • The origins of memory T cells.
    Omilusik KD, Goldrath AW. Omilusik KD, et al. Nature. 2017 Dec 21;552(7685):337-339. doi: 10.1038/d41586-017-08280-8. Nature. 2017. PMID: 29293221 No abstract available.

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