but also points to key opportunities for improving
the long-term durability of these effects.
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We thank the Wherry lab for discussions and critically reading the
manuscript. We thank C. Surh for providing the antibody against IL-7
(anti-IL-7). The anti-IL-7 antibody is available from La Jolla Institute of
Allergy and Immunology, and the IL-7 used is available from the
National Cancer Institute, both under material transfer agreements
with the University of Pennsylvania. The data presented in this
manuscript are tabulated in the main paper and in the supplementary
materials. Sequencing data are available at Gene Expression
Omnibus [accession numbers GSE86796 (microarray), GSE86881
(RNA seq), and GSE86797 (ATAC seq)]. This study was supported by
a Robertson Foundation–Cancer Research Institute Irvington
Fellowship (K. E.P.), an American Cancer Society Postdoctoral
Fellowship (M. A.S.), National Institutes of Health grant F30DK100159
(J. M.S.), German Research Foundation Fellowship BE5496/1-1 (B.B.),
and National Institutes of Health grant T32 2T32CA009615-26 (A.C.H.).
This work was funded by the National Institutes of Health (grant
CA78831 to S.L.B. and grants AI105343, AI112521, AI082630, AI115712,
AI117950, and AI108545 to E.J. W.). This research was also supported
by the Parker Institute for Cancer Immunotherapy. E.J. W. has a patent
licensing agreement on the PD-1 pathway. The authors declare no
additional conflicts of interest.
Materials and Methods
Figs. S1 to S18
Tables S1 to S12
18 January 2016; accepted 19 September 2016
Published online 27 October 2016
T CELL EXHAUSTION
The epigenetic landscape of
T cell exhaustion
Debattama R. Sen,1,2 James Kaminski,3 R. Anthony Barnitz,1 Makoto Kurachi,4,5
Ulrike Gerdemann,1 Kathleen B. Yates,1 Hsiao-Wei Tsao,1 Jernej Godec,1,2
Martin W. LaFleur,1,2 Flavian D. Brown,1,2 Pierre Tonnerre,6 Raymond T. Chung,6
Damien C. Tully,7 Todd M. Allen,7 Nicole Frahm,8 Georg M. Lauer,6 E. John Wherry,4,5
Nir Yosef,3,7,9†‡ W. Nicholas Haining1,10,11†‡
Exhausted T cells in cancer and chronic viral infection express distinctive patterns
of genes, including sustained expression of programmed cell death protein 1 (PD-1).
However, the regulation of gene expression in exhausted T cells is poorly understood.
Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and
show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells
in humans and a mouse model of chronic viral infection acquire a state-specific
epigenetic landscape organized into functional modules of enhancers. Genome editing
shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that
contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer
targets for genome editing that alter gene expression preferentially in exhausted
CD8+ T cells.
Tcell exhaustion—an acquired stateof T cell dysfunction—is a hallmark of cancer and chronic viral infection (1, 2), and clinical trials of checkpoint blockade, which aim to reverse T cell exhaustion in cancer,
have proven strikingly effective (3, 4). Chimeric
antigen receptor (CAR)–T cell therapy has also
proven highly effective for hematologic malignancies (5), but the development of exhaustion
in T cells engineered to treat solid tumors remains a substantial barrier to its broader use
(6). The identification of mechanisms that regulate exhausted T cells is therefore a major goal
in cancer immunotherapy.
To identify regulatory regions in the genome
of exhausted CD8+ T cells, we used an assay
for transposase-accessible chromatin with high-
throughput sequencing (ATAC-seq) (7) to demar-
cate areas of accessible chromatin in mouse
antigen-specific CD8+ T cells differentiating in
response to lymphocytic choriomeningitis virus
(LCMV) infection (fig. S1A and table S1). Acute
LCMV infection elicits highly functional effec-
tor CD8+ T cells, whereas chronic LCMV infection
gives rise to exhausted CD8+ T cells (1–3, 8, 9).
Analysis of high-quality ATAC-seq profiles (fig.
S1, B to H) from naïve CD8+ T cells and those
at day 8 and day 27 postinfection (p.i.) (d8 and
d27, respectively) revealed that naïve CD8+
T cells underwent large-scale remodeling (Fig.
1A and fig. S2A) during differentiation [as de-
tected by DESeq2, with a false discovery rate
(FDR) < 0.05]. The majority (71%) (fig. S2A) of
chromatin-accessible regions (ChARs) either
emerged (e.g., those at the Ifng locus) or disap-
peared (e.g., Ccr7) (Fig. 1A) as naïve CD8+ T cells
underwent differentiation. The gain and loss of
ChARs were not balanced; a much larger frac-
tion of regions emerged at d8 p.i. and persisted
or emerged only at d27 than were either tran-
siently detected at d8 p.i. or lost from naïve cells
(Fig. 1B). Thus, differentiation from a naïve CD8+
T cell state is associated with a net increase,
rather than decrease, in chromatin accessibility
Comparison of ChARs from exhausted CD8+
T cells with those found in functional effector
or memory CD8+ T cells revealed marked differences in the pattern of regulatory regions. Differential regulatory regions between acute and
chronic infection (Fig. 1C and fig. S2C) showed
features of enhancers: They tended to be depleted
of transcription start sites (TSSs) and enriched
for intergenic and intronic areas (Fig. 1D), and
found distal to gene promoters (fig. S2D). The
magnitude of difference in the profile of regulatory regions between exhausted and functional
CD8+ T cells was greater than that seen in gene
expression. We found that 44.48% of all ChARs
1Department of Pediatric Oncology, Dana-Farber Cancer
Institute, Boston, MA 02115, USA. 2Division of Medical
Sciences, Harvard Medical School, Boston, MA 02115, USA.
3Center for Computational Biology, University of California,
Berkeley, Berkeley, CA 94720, USA. 4Institute of
Immunology, University of Pennsylvania, Philadelphia, PA
19104, USA. 5Department of Microbiology, University of
Pennsylvania, Philadelphia, PA 19104, USA. 6Gastrointestinal
Unit and Liver Center, Massachusetts General Hospital,
Harvard Medical School, Boston, MA 02115, USA. 7Ragon
Institute of Massachusetts General Hospital, Massachusetts
Institute of Technology, and Harvard University, Boston, MA
02139, USA. 8Vaccine and Infectious Disease Division, Fred
Hutchinson Cancer Research Center, Seattle, WA 98109,
USA. 9Department of Electrical Engineering and Computer
Science, University of California, Berkeley, Berkeley, CA
94720, USA. 10Division of Pediatric Hematology and
Oncology, Children’s Hospital, Boston, MA 02115, USA.
11Broad Institute of Harvard and Massachusetts Institute of
Technology, Cambridge, MA 02142, USA.
*These authors contributed equally to this work. †These authors
contributed equally to this work. ‡Corresponding author. Email:
firstname.lastname@example.org (N. Y.); email@example.com.