highly purified HSC fraction identifies mitochondrial clearance by induction of mitophagosome
formation as a key mechanism in maintaining
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We thank all members of the Ito lab and Einstein Stem Cell
Institute for their comments on HSC self-renewal and M. Wolfgang,
A. Carracedo, H. You, and the Einstein Flow Cytometry and
Analytical Imaging core facilities (grant P30CA013330) for help and
materials. This work was supported by NIH (grants R01DK98263 and
R01DK100689 to Ke.I.) and NYSTEM (New York State Stem Cell
Science single-cell-core, grant C029154 to Ke.I.), Harvard Stem Cell
Institute (to C.P.L.), NIH and Ellison Medical Foundation (to R.S.),
NIH and Leukemia Lymphoma Society (to P.S.F.), and Japan Society
for the Promotion of Science (to T.S.). We declare no competing
Materials and Methods
Figs. S1 to S14
Movies S1 and S2
26 February 2016; accepted 4 October 2016
Published online 13 October 2016
T CELL EXHAUSTION
Epigenetic stability of exhausted
T cells limits durability of
reinvigoration by PD-1 blockade
Kristen E. Pauken,1 Morgan A. Sammons,2 Pamela M. Odorizzi,1 Sasikanth Manne,1
Jernej Godec,3,4 Omar Khan,1 Adam M. Drake,2 Zeyu Chen,1 Debattama R. Sen,3
Makoto Kurachi,1 R. Anthony Barnitz,3 Caroline Bartman,1 Bertram Bengsch,1
Alexander C. Huang,5 Jason M. Schenkel,6 Golnaz Vahedi,7 W. Nicholas Haining,3,8,9
Shelley L. Berger,2 E. John Wherry1*
Blocking Programmed Death–1 (PD-1) can reinvigorate exhausted CD8 Tcells (TEX) and improve
control of chronic infections and cancer. However, whether blocking PD-1 can reprogram TEX
into durable memory T cells (TMEM) is unclear. We found that reinvigoration of TEX in mice by
PD-L1 blockade caused minimal memory development. After blockade, reinvigorated TEX
became reexhausted if antigen concentration remained high and failed to become TMEM upon
antigen clearance. TEX acquired an epigenetic profile distinct from that of effector Tcells (TEFF)
and TMEM cells that was minimally remodeled after PD-L1 blockade. This finding suggests that
TEX are a distinct lineage of CD8 T cells. Nevertheless, PD-1 pathway blockade resulted in
transcriptional rewiring and reengagement of effector circuitry in the TEX epigenetic landscape.
These data indicate that epigenetic fate inflexibility may limit current immunotherapies.
Persisting antigenic stimulation during chron- ic infections and cancer can result in T cell exhaustion, a state of impaired effector func- tions, high expression of inhibitory receptors including Programmed Death–1 (PD-1, or
CD279), transcriptional reprogramming, and de-
fective immune memory (1). Collectively, these
properties prevent optimal control of persisting
pathogens and tumors. Blocking the PD-1:PD-L1
pathway can reinvigorate exhausted CD8 T cells
(TEX), improving effector functions and enhancing
viral and tumor control (1). Recently developed
inhibitors of the PD-1 and cytotoxic T lymphocyte–
associated protein 4 (CTLA-4) pathways represent
a new paradigm in cancer treatment (2–4). Although
promising, the majority of patients fail to de-
velop durable responses, and most eventually
progress (2–4). Thus, it is unclear whether blocking
PD-1 can promote long-lasting improvements
and immunological memory development in TEX.
To address this question, we analyzed the cel-
lular, transcriptional, and epigenetic changes
associated with PD-1 pathway blockade using
the mouse model of chronic lymphocytic chorio-
meningitis virus (LCMV) infection (fig. S1, A to C)
(5, 6). After treatment with antibodies against
PD-L1 (anti-PD-L1), 1080 genes were up-regulated
and 1686 genes were down-regulated [P < 0.05,
log2 fold change (LFC) ≥ 0.2] (Fig. 1A, fig. S1D,
(11), we identified two metagenes in anti-PD-L1–
treated TEX compared to control TEX; one cor-
responding to leukocyte activation and one to
cell cycle (Fig. 1E; fig. S1, E and F; and table S4).
The anti-PD-L1–treated TEX metagenes displayed
some overlap with effector T cells (TEFF), largely
driven by cell cycle pathways, but minimal overlap
with TMEM (Fig. 1E and table S4), suggesting
limited acquisition of memory potential upon
PD-1 pathway blockade can reactivate functions
in TEX, but whether reinvigoration is sustained is
unclear. There was a robust reinvigoration of TEX,
as expected (Fig. 1, F and G, and figs. S1, A and B,
and S2) (5), and expansion peaked ~3 weeks after
initiation of blockade. By 8 to 11 weeks after
treatment, however, this reinvigoration was lost,
and the quantity, proliferation, effector function,
and inhibitory receptor expression of LCMV-specific
CD8 T cells in the anti-PD-L1–treated mice were
comparable to those in control-treated mice (Fig.
1, F to H, and figs. S2 to S4). Moreover, although
anti-PD-L1 treatment reduced viral load immediately after treatment, 4 months later, viral load
was similar to that in control-treated mice (Fig. 1I).
Lastly, 18 to 29 weeks after cessation of blockade,
the transcriptional profiles of control- and anti-
PD-L1–treated groups were similar (Fig. 1J, figs.
S5 and S6, and tables S5 and S6). Collectively,
these data indicate that when antigen concentration remains high, TEX that were reinvigorated
by PD-1 pathway blockade become “reexhausted.”
One possible reason the effects of PD-L1 blockade
were not sustained is that the infection persisted.
We hypothesized that if the infection were cleared,
anti-PD-L1 might induce differentiation into TMEM.
1160 2 DECEMBER 2016 • VOL 354 ISSUE 6316
1Department of Microbiology and Institute for Immunology,
Perelman School of Medicine, University of Pennsylvania,
Philadelphia, PA, USA. 2Departments of Cell and Developmental
Biology, Genetics, and Biology, Penn Epigenetics Program,
University of Pennsylvania, Philadelphia, PA, USA. 3Department
of Pediatric Oncology, Dana-Farber Cancer Institute, Boston,
MA, USA. 4Department of Microbiology and Immunobiology,
Harvard Medical School, Boston, MA, USA. 5Department of
Medicine and Institute for Immunology, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, PA, USA.
6Department of Microbiology and Immunology, University of
Minnesota, Minneapolis, MN, USA. 7Department of Genetics and
Institute for Immunology, University of Pennsylvania,
Philadelphia, PA, USA. 8Broad Institute of MIT and Harvard,
Cambridge, MA, USA. 9Division of Hematology/Oncology,
Children’s Hospital, Harvard Medical School, Boston, MA, USA.
*Corresponding author. Email: firstname.lastname@example.org