Differential effects of STAT5 and PI3K/AKT signaling on effector and memory CD8 T-cell survival.
ABSTRACT During viral infection, effector CD8 T cells contract to form a population of protective memory cells that is maintained by IL-7 and IL-15. The mechanisms that control effector cell death during infection are poorly understood. We investigated how short- and long-lived antiviral CD8 T cells differentially used the survival and cell growth pathways PI3K/AKT and JAK/STAT5. In response to IL-15, long-lived memory precursor cells activated AKT significantly better than short-lived effector cells. However, constitutive AKT activation did not enhance memory CD8 T-cell survival but rather repressed IL-7 and IL-15 receptor expression, STAT5 phosphorylation, and BCL2 expression. Conversely, constitutive STAT5 activation profoundly enhanced effector and memory CD8 T-cell survival and augmented homeostatic proliferation, AKT activation, and BCL2 expression. Taken together, these data illustrate that effector and memory cell viability depends on properly balanced PI3K/AKT signaling and the maintenance of STAT5 signaling.
- SourceAvailable from: Nicholas P Restifo[Show abstract] [Hide abstract]
ABSTRACT: Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) can result in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy might be limited by poor persistence of TIL after adoptive-transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in viral-specific murine models, but whether this approach may enhance features of memory (e.g. long-term persistence) in TIL which are characteristically exhausted and senescent is not established. Here we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments anti-tumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of anti-tumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer. Copyright © 2014, American Association for Cancer Research.Cancer Research 11/2014; · 9.28 Impact Factor
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ABSTRACT: Protein kinase B (also known as AKT) and the mechanistic target of rapamycin (mTOR) are central regulators of T cell differentiation, proliferation, metabolism, and survival. Here, we show that during chronic murine lymphocytic choriomeningitis virus infection, activation of AKT and mTOR are impaired in antiviral cytotoxic T lymphocytes (CTLs), resulting in enhanced activity of the transcription factor FoxO1. Blockade of inhibitory receptor programmed cell death protein 1 (PD-1) in vivo increased mTOR activity in virus-specific CTLs, and its therapeutic effects were abrogated by the mTOR inhibitor rapamycin. FoxO1 functioned as a transcriptional activator of PD-1 that promoted the differentiation of terminally exhausted CTLs. Importantly, FoxO1-null CTLs failed to persist and control chronic viral infection. Collectively, this study shows that CTLs adapt to persistent infection through a positive feedback pathway (PD-1→FoxO1→PD-1) that functions to both desensitize virus-specific CTLs to antigen and support their survival during chronic viral infection. Copyright © 2014 Elsevier Inc. All rights reserved.Immunity 11/2014; 41(5):802-814. · 19.75 Impact Factor
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ABSTRACT: Activated CD8(+) T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8(+) T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.Nature Immunology 10/2014; · 24.97 Impact Factor
Differential effects of STAT5 and PI3K/AKT signaling
on effector and memory CD8 T-cell survival
Timothy W. Handa, Weiguo Cuia, Yong Woo Junga, Esen Sefika, Nikhil S. Joshia, Anmol Chandelea, Ying Liua,
and Susan M. Kaecha,b,1
aDepartment of Immunobiology andbHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520
Edited by Thomas R. Malek, Miller School of Medicine, University of Miami, Miami, FL, and accepted by the Editorial Board August 10, 2010 (received for
review March 16, 2010)
During viral infection, effector CD8 T cells contract to form a popula-
tion of protective memory cells that is maintained by IL-7 and IL-15.
The mechanisms that control effector cell death during infection are
poorly understood. We investigated how short- and long-lived
antiviral CD8 T cells differentially used the survival and cell growth
pathways PI3K/AKT and JAK/STAT5. In response to IL-15, long-lived
lived effector cells. However, constitutive AKT activation did not
enhance memory CD8 T-cell survival but rather repressed IL-7 and IL-
Conversely, constitutive STAT5 activation profoundly enhanced ef-
fector and memory CD8 T-cell survival and augmented homeostatic
proliferation, AKT activation, and BCL2 expression. Taken together,
these data illustrate that effector and memory cell viability depends
on properly balanced PI3K/AKT signaling and the maintenance of
apoptosis|cytokine signaling|interleukin 15|interleukin 7|lymphocytic
infections via rapid reactivation. The exact biochemical mecha-
nism governing the survival of memory cells and apoptosis of the
role in their differentiation and survival. IL-2, IL-7, and IL-15
promote the expansion and survival of activated T cells (2). As
memory CD8 T cells form following acute viral infection, two of
these cytokines, IL-15 and IL-7, direct memory T cell survival and
homeostasis (3, 4). Downstream of their specific receptors, IL-7
and IL-15 activate two primary pathways: janus kinase/signal
transducer and activator of transcription 5 (JAK/STAT5) and
phosphoinositide 3-kinase (PI3K)/AK-transforming (AKT). Acti-
vation of these two pathways results in decreased levels of proa-
as BCL2, and activation of cellular growth and proliferation
pathways regulated by mTOR and cyclins (5). The balance be-
tween these pro- and antiapoptotic factors controls the survival of
CD8 T cells after immunization (6).
Early models predicted that effector CD8 T-cellcontraction was
a result of deprivation of T-cell growth factor cytokines that wane
However, effector CD8 T cells are a heterogeneous population
composed of cells with varying intrinsic potential for survival and
differentiation into memory cells (3, 7, 8). During several types of
infections [such as lymphocytic choriomeningitis virus (LCMV),
vesicular stomatitis virus (VSV), Listeria monocytogenes, and
Toxoplasma gondii], most of the effector CD8 T cells terminally
differentiate and become short-lived, but a minority maintain
memory potential and are capable of self-renewal (9–14). Separa-
tion of these subsets is possible because most memory precursor
cells express higher amounts of IL-7Rα, CD27, and CXCR3 and
lower amounts of KLRG1, whereas shorter-lived effector cells
emory CD8 T cells form from a much larger effector pop-
ulation and provide long-term immunity against subsequent
findings indicate that the process of contraction is not stochastic
and that effector CD8 T cells have acquired distinct cell fates;
however, it does not discard the possible role of cytokine with-
drawal in the contraction of effector cells. IL-7Rα is functionally
required for memory precursor survival and optimal memory CD8
T cell generation (3, 4, 16). However, forced IL-7Rα expression
does not enhance memory CD8 T cell formation (17–19). IL-15
also helps to sustain memory CD8 T cells and KLRG1hiIL-7Rαlo
effector CD8 T cells are acutely dependent on it (2, 15). Fur-
thermore, the common γ-chain cytokine-induced proliferative
responses of short-lived (KLRG1hi) effector T cells are subdued in
comparison with memory precursor cells, suggesting that intrinsic
differences exist between these two cell populations that modulate
their cytokine responsiveness and survival (20).
To better understand the role of cytokine-derived signals in the
process of memory cell development and effector cell contraction,
we interrogated the regulation of JAK/STAT5 and PI3K/AKT
signaling pathways in viral-specific CD8+ T cells in vivo. We dis-
covered that terminally differentiated effector CD8 T cells are
impaired in the activation of the PI3K/AKT pathway but, surpris-
ingly, constitutive AKT activation did not increase their survival.
Rather, it caused feedback inhibition on the expression of IL-7Rα
and IL-2/15Rβ chains, which in turn reduced STAT5 signaling,
BCL2 expression, and memory cell formation. Conversely, con-
stitutive STAT5 activation dramatically enhanced effector CD8
T cell survival and memory CD8 T cell formation after infection.
These data highlight that key survival and mitogenic pathways are
modulated in activated T cells by their differentiation state, and
effector and memory T-cell longevity is regulated by an optimal
balance of STAT5 and AKT activity.
KLRG1loEffector CD8 T Cells Activate the PI3K/AKT Pathway in
Response to IL-15 More Robustly than KLRG1hiEffector Cells. Given
that IL-15 is an important cytokine for memory CD8 T-cell ho-
meostasis and survival, we compared the ability of KLRG1hi
memory precursor effector cells (MPECs) to respond to IL-15.
IL-7Rαhiand IL-7Rαloeffector cells express similar levels of IL-
15Rα and IL-2/15Rβ chains, whereas neither subset expresses the
high-affinity IL-2Rα chain (Fig. S1A). Both KLRG1hiand
KLRG1loCD8T cellsrapidlyphosphorylatedSTAT5 and ERK1/2
in response to IL-15, indicating that the receptor is functional on
Author contributions: T.W.H. andS.M.K. designed research; T.W.H., W.C., Y.W.J., E.S., N.S.J.,
A.C., and Y.L. performed research; T.W.H., W.C., and S.M.K. analyzed data; and T.W.H. and
S.M.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. T.R.M. is a guest editor invited by the Editorial
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 21, 2010
| vol. 107
| no. 38
ability of effector CD8 T cells to activate the PI3K/AKT pathway,
antigen-specific KLRG1loMPECs and KLRG1hiSLECs were pu-
rifiedfrommiceinfected8–10dearlier withLCMV andstimulated
had significantly higher amounts of phosphorylated AKT at both
the serine 473 and threonine 308 residues in comparison with the
KLRG1hieffector T cells (Fig. 1 A and B). This difference in
AKT473phosphorylation was observed as early as 40 min post–IL-
stimulation (Fig. 1; Fig. S2 A and B). The difference in phosphor-
ylation could not be attributed to differences in the levels of total
AKT protein in the two cell populations (Fig. 1C). Concomitant
with increased AKT phosphorylation, the KLRG1loeffector cells
also demonstrated a greater ability to phosphorylate downstream
AKT targets GSK-3β, FOXO1, and FOXO3a (Fig. 1 A and B).
Additionally, AKT phosphorylation could beinhibited by the PI3K
inhibitor Ly294002, indicating that cytokine-induced AKT phos-
phorylation was PI3K-dependent (Fig. S2C). This reduction in
results were obtained following both IL-2 and IL-7 stimulation (in
which IL-7Rα expression was made equivalent via transgenic IL-
7Rα expression) (Fig. S2D). Furthermore, this difference was not
mediated by the binding and activation of the inhibitory receptor
KLRG1 (21) during sorting because similar results were observed
when the cells were separated based on CD27, which inversely
correlates with KLRG1 expression (Fig. S2C). Last, the impaired
AKT phosphorylation was not specific to the P14 T cell receptor
(TCR) transgenic T cells analyzed above because similar results
were observed in subsets of KLRG1hiand KLRG1locells isolated
from polyclonal populations of CD44hieffector cells after LCMV
infection (Fig. 2A, compare Wt KLRG1hiand KLRG1lolanes).
Increased PI3K/AKT Signaling Does Not Enhance Memory CD8 T-Cell
Development. To determine whether the reduced PI3K/AKT sig-
naling in the KLRG1hieffector CD8 T-cell subsets contributed to
their shortened lifespan, we tried to “rescue” this defect in the
and tensin homolog (PTEN) deficiency and overexpression of
constitutively active AKT by retroviral transduction. We first ex-
amined PTEN protein levels by Western blotting, but found no
significant differences between the KLRG1hiand KLRG1loef-
fector cell subsets, implying that relative PTEN levels are not re-
sponsible for the differences in AKT phosphorylation (Fig. S3A).
To analyzetheeffects ofPTEN deficiency invirus-specific effector
and memory CD8 T cells, we crossed mice carrying a floxed allele
of Pten onto mice that express Cre recombinase under control of
theGranzyme B promoter(Ptenf/f; gzb-Cre+; referred toas Pten−/−
mice) and then infected Pten−/−mice with LCMV (22, 23). Ptenf/f;
gzb-Cre−littermate animals (referred to as Wt) were used as con-
trols. In this system, Pten is expressed normally during thymic de-
velopment and in naïve T cells, but upon T-cell activation Cre
recombinase is expressed and thus the Pten locus is deleted from
>90% of the activated CD8 T cells during LCMV infection (23).
Ptenf/f;Gzb-Cre+LCMV tetramer-specific T cellsandthat Pten−/−
T cells from LCMV-infected mice had constitutive AKT phos-
phorylation (Fig. 2A and Fig. S3B). Notably, the Pten−/−KLRG1lo
effector cells still retained a better ability to phosphorylate AKT
than the Pten−/−KLRG1hicells (Fig. 2A), which implied that
PTEN-independent mechanisms were involved in the differential
induced better survival of effector CD8 T cells under serum-
starvation conditions in vitro as previously described (Fig. S4A)
(24) but, surprisingly, there were only modest effects on the ex-
pansion and contraction of effector CD8 T cells in response to
LCMV infection (Fig. 2B). In general, the clonal burst of Pten−/−
effector CD8 T cells was approximately one half that of the Wt
littermate controls, and there was a slight, but not significant, re-
duction in the number of memory Pten−/−T cells that formed in
lymphoid and nonlymphoid tissues compared with WT controls
(Fig. 2B). These results demonstrated that increased activation of
AKT induced by PTEN deficiency was insufficient to prevent ap-
optosis of effector CD8 T cells or increase memory CD8 T cell
formation following viral infection.
e f f i d
d l o
c i f i c
( l a
g i s
tivate the PI3K/AKT pathway in response to IL-15. Small numbers (∼2,500) of
subsequently infected with LCMV. (A) KLRG1hior KLRG1loCD8+ Thy1.1+ P14
CD8 T cells were isolated by FACS from mice 8–10 d p.i. and ∼1 × 106cells were
are representative examples of two to six experiments, and densitometric
quantification of phosphorylation experiments is shown in B.
KLRG1loeffector CD8 T cells preferentially maintain the ability to ac-
Ly294002 (10μ μM)
IL-15 - + + +
- + + +
# of Thy1.1+(RV+)
CD8+ T cells
Time (Days) Time (Days)
formation. (A) Ptenfl/flGzb-Cre+(Pten−/−) or Ptenfl/flGzb-Cre−(Wt) were
infected with LCMV, and at day 8 p.i., CD44hiCD8 T cells from Pten−/−or Wt
controls were isolated by FACS based on KLRG1hior KLRG1loexpression and
cultured overnight with or without IL-15. Approximately 1 × 106cells per
sample were assayed via Western blot for phosphorylated AKT. (B) Line graph
T cells in the spleens of Pten−/−(green line) and Wt (black line) mice at the
indicated times p.i. (C) Small numbers (∼2,500 cells) of P14 Ly5.1+ T cells
transduced with either CA-AKT or CTRL RV that expresses Thy1.1 were trans-
ferred into congenic Ly5.2+ C57BL/6 mice that were infected with LCMV. Line
graph shows the number of CA-AKT (blue line) and CTRL RV+ (Thy1.1+; black
line) P14CD8T cells per 106peripheral blood mononuclear cells(PBMCs) at the
indicated times p.i. All data points show the mean (±SEM) of more than five
mice over separate experiments. All statistics were calculated by Student’s t
test. ***P < 0.0005, **P < 0.005, *P < 0.05.
Constitutive AKT activation is not sufficient for memory CD8 T-cell
| www.pnas.org/cgi/doi/10.1073/pnas.1003457107Hand et al.
pathways and the Granzyme B Cre conditional knockout system
also examined the role of AKT signaling in memory cell de-
velopment by expressing a constitutively active form of murine
AKT (referred to as CA-AKT) that is myristoylated and constitu-
tively phosphorylated at Ser473 and Thr308 (25). In these experi-
ments, P14 TCR transgenic T cells (Ly5.1+) were transduced with
a retrovirus(RV) expressing CA-AKTand the markerThy1.1, and
then transferred to congenic (Ly5.2+) LCMV-infected animals
(26). P14 CD8 T cells transduced with empty control (CTRL) RV
were used for comparison. Similar to Pten−/−cells, CA-AKT RV
effector CD8 T cells harvested from day 8 LCMV-infected mice
survived better under serum starvation in vitro compared with
CTRL RV cells (Fig. S4B). When analyzed in vivo during LCMV
infection, the CA-AKT P14 CD8 T-cell expansion was slightly in-
creased, but the death of the CA-AKT RV P14 CD8 T cells during
the contraction phase, between days 8 and 75 postinfection (p.i.),
was considerably greater than CTRL RV cells (Fig. 2C). These
results indicate that increased and prolonged AKT activation does
not improve the viability of effector CD8 T cells and can actu-
ally be detrimental to their subsequent survival and memory CD8
AKT Signaling Is Associated with Down-Regulation of Cytokine
Receptors and STAT5 Signaling. Although constitutive activation of
PI3K/AKT had insignificant (Pten−/−) or detrimental (CA-AKT)
test whether increasing AKT activity modified the composition and
function of the effector and memory CD8 T-cell populations. First,
the subsets of surviving CD8 T cells that formed with constitutive
AKT activity were examined. The percentage of KLRG1hiLCMV-
specific CD8 T cells was not increased by constitutive AKT activity
and, in fact, declined more than control cells over the weeks fol-
lowing infection(Fig. 3AandFig.S5). This indicatedthatincreased
AKT activity alone could not rescue the death of the KLRG1hi
AKT activity. Additionally, the expression of both IL-7Rα and
CD62L were considerably down-regulated in PTEN-deficient and
CA-AKT–expressing LCMV-specific memory CD8 T cells, consis-
tent with previous findings (Fig. 3A and Fig. S5) (27, 28). In addi-
receptor CD27 and IL-2/15Rβ chain, a hitherto undescribed effect
IL-15–dependent KLRG1hieffector cells in the CA-AKT cell
population (Fig. 3A) (10, 29). This effect on IL-2/15Rβ expression
was not observed in Pten−/−CD8 T cells (Fig. S5), perhaps because
the steady-state levels of AKT activity were lower in these cells
compared with CA-AKT cells based on ribosomal protein S6
phosphorylation (Fig. S6). Maintenance of IL-2/15Rβ expression
may allow for better survival of Pten−/−CD8 T cells following in-
fection compared with CA-AKT-expressing cells (Fig. 2 B and C).
We tested whether reduced expression of cytokine receptors in
CA-AKT–expressing CD8 T cells prevented the phosphorylation
STAT5 phosphorylation was inhibited ∼2- to 3-fold (Fig. 3B).
Likewise, the key antiapoptotic protein BCL2, which is also
a STAT5 target gene, was almost undetectable in both the Pten−/−
on BCL2 expression likely contributed to the inability of increased
AKT activity to enhance effector CD8 T cell survival and memory
development (Fig. 2 B and C).
Last, we examined how constitutive AKT activity affected
several important attributes of functional, long-lived memory
CD8 T cells. The Pten−/−and CA-AKT memory CD8 T cells
were able to produce IFNγ, TNFα, and Granzyme B, but the
production of IL-2 was diminished (Fig. 3A and Figs. S5 and S7).
Thus, increased AKT activity impaired formation of “polyfunc-
tional” memory CD8 T cells (30). The reduced responsiveness of
the CA-AKT memory CD8 T cells to IL-15 also greatly reduced
their ability to self-renew, as they proliferated significantly less
after transfer into congenic lymphoreplete hosts (Fig. 3C) (31).
Likewise, when challenged with recombinant Listeria expressing
the LCMV peptide GP33–41(Listeria-GP33), CA-AKT effector
CD8 T cells had weakened secondary responses relative to the
control T-cell populations (Fig. 3D). Together, these findings
suggest that defective AKT signaling alone is not the primary
cause of terminal effector cell apoptosis following infection,
because constitutive AKT activation did not hinder cell death.
Instead, it induced a negative feedback loop that repressed the
expression of IL-7 and IL-15 cytokine receptors and impaired
STAT5 signaling and BCL2 expression (6). These results do not
necessarily rule out that the basal levels of AKT activity are
important for memory T-cell survival but rather underscore that
CD8 T cells
CA-AKT RV CTRL RV
to diminished cytokine receptor expression, STAT5 signaling, homeostatic
turnover, and recall responses. As in Fig. 2, P14 CD8 T cells were transduced
with CA-AKT or CTRL RVs, and ∼2,500 cells were transferred into C57BL/6 mice
that were infected with LCMV. (A) Histogram plots show expression of in-
dicated proteins in CA-AKT (blue) and CTRL (black line) RV+ (Thy1.1+) P14
memory CD8 T cells from mice infected 60 d previously with LCMV. In the top
two rows, the percentage of cells expressing “high” levels of each protein and
in the bottom row the mean fluorescence intensity (MFI) of the protein are
shown. The dotted line indicates staining of BCL2 isotype control antibody. (B)
Day 30 CA-AKT (blue) and CTRL (black line) RV+ (Thy1.1+) P14 effector CD8
T cells were stimulated with or without the cytokines IL-2, IL-7, or IL-15 for
30 min, fixed, permeabilized, and stained for intracellular STAT5Tyr694phos-
phorylation. Overlapping histograms show levels of phosphorylated STAT5
memory CD8 T cells were isolated 30 d p.i., labeled with carboxyfluoroscein
succinimidyl ester (CFSE), and transferred into naïve (Ly5.2+) C57BL/6 hosts.
Approximately 45 d later the donor cells were assayed for cell division by flow
transferred into naïve C57BL/6 mice that were subsequently infected with
Listeria-GP33 and analyzed 7 d later. The fold expansion (normalized to the
number of donor RV+ CD8 T cells transferred) is shown in the bar graph. All
data shown are representative of two or three experiments with more than
two mice per experimental group.
Hand et al.PNAS
| September 21, 2010
| vol. 107
| no. 38
the proper balance of AKT signaling is critical for optimal
memory CD8 T-cell formation and function.
STAT5 Signals Are Sufficient for the Survival of CD8 Effector and
Memory T Cells. Next, we investigated the role of STAT5 signaling
in effector CD8 T-cell survival because it is the other primary
signaling pathway downstream of IL-7 and IL-15. To do so, P14
CD8 T cells were transduced with constitutively active (CA-
STAT5) or dominant-negative (DN-STAT5) RVs and transferred
into LCMV-infected mice. The percent and number of RV+
(Thy1.1+) effector CD8 T cells were measured over the course of
LCMV infection, and we found that the different forms of STAT5
had striking effects on effector cell expansion and memory cell
formation (Fig. 4A). By day 8 p.i., the CA-STAT5 effector P14
CD8 T cells expanded ∼2- to 3-fold more than CTRL RV cells.
and did not contract thereafter, leading to the formation of ≈30–
40 × 106memory CD8 T cells in the spleen of each animal (Fig.
4B). The CTRL RV population declined normally, and therefore
the expression of CA-STAT5 resulted in an ∼280-fold increase in
thenumber of memoryCD8 T cells that formed 75 d p.i. (Fig. 4B).
The preferential survival of the CA-STAT5 CD8 T cells was also
P14 CD8 T-cell population (Fig. 4A). Conversely, DN-STAT5
expression substantially reduced expansion and memory cell for-
mation compared with the CTRL RV P14 CD8 T cells (Fig. 4A).
Thus, constitutive STAT5 activity profoundly enhances virus-
specific effector and memory T-cell survival after infection.
We next inspected how CA-STAT5 affected the survival of the
different effector and memory CD8 T cell subsets, based on
KLRG1 and IL-7Rα expression, following LCMV infection. As
expected, the KLRG1hiIL-7RαloCTRL RV population con-
tracted nearly 100-fold between days 8 and 75 p.i. (Fig. 4C). In
contrast, this same cell population expressing CA-STAT5 only
declined ∼2-fold during this time period. Thus, unlike CA-AKT,
the constitutive activation of STAT5 could rescue the survival of
terminal effector cells considerably. Rather than contracting, CA-
STAT5 actually caused the other three subsets of P14 CD8 T cells
(KLRG1hiIL-7Rαhi, KLRG1loIL-7Rαlo, and KLRG1loIL-7Rαhi)
The effect of CA-STAT5 was greatest on the KLRG1loIL-7Rhi
LCMV-specific CD8 T cells, as this population increased in
number ∼18-fold during this time period and was the most abun-
dant in the memory CD8 T-cell population (Fig. 4C).
To determine whether CA-STAT5 affected other phenotypes
and functions of the memory CD8 T cells, we performed a similar
analysis as that described above for CA-AKT transduced cells.
Unlike the effects of CA-AKT, we found less profound alterations
in the expression of proteins such as IL-7Rα, IL-2/15Rβ, CD27,
and CD62L (Fig. S8). In contrast to CA-AKT transduced T cells,
IL-2/15Rβ and CD27 were slightly up-regulated in the CA-STAT5
memory CD8 T-cell population. As expected, because it is a target
of STAT5, BCL2 was substantially increased in the CA-STAT5
effector and memory CD8 T cells (Fig. 5A and Fig. S8) (32). Next,
we examined the recall responses of CA-STAT5 CD8 T cells to
secondary LM-GP33 infection, but did not see any substantial
beneficial effect of increased STAT5 activity on either subset of
effector CD8 T cells (Fig. 5B).
Continued proliferation could contribute to the increase in
examine this point, we treated animals containing CA-STAT5 or
CTRL RV P14 memory CD8 T cells with BrdU from day 65 to 75
p.i. and found CA-STAT5 expression induced a significant in-
crease in proliferation compared with controls (Fig. 5C). In addi-
tion, the KLRG1locells incorporated BrdU to a greater degree
than the KLRG1hicells, indicating that sustained proliferation
contributes to the profound rise in the number CA-STAT5–
expressing KLRG1lomemory CD8 T cells (Fig. 5C).
that STAT5 signaling increases cell viability by potentiating PI3K/
AKT signaling (33, 34). To test whether a similar mechanism was
occurring in the CA-STAT5 RV-expressing T cells, we examined
the ability of these cells to phosphorylate AKT in response to IL-
15. We found that CA-STAT5 expression increased the ability of
both KLRG1hiand KLRG1loT cells to induce pAKT473both
unstimulated and in response to IL-15 (Fig. S9). Thus, the pro-
found increase in effector and memory CD8 T-cell survival and
proliferation by CA-STAT5 may not solely stem from increased
expression of STAT5 target genes, but may partially rely on aug-
mented AKT signaling.
Terminally differentiatedeffectorandmemoryCD8T cells,which
can be distinguished by a KLRG1hiCD27loIL-7Rαlophenotype,
generally have a shorter lifespan and reduced proliferative
responses compared with KLRG1loCD27hiIL-7Rαhicells (10, 13,
IL-7Rαlocells has been attributed to increased expression of the
cell cycle inhibitor p27kipand decreased expression of BMI1 rel-
ative to the IL-7Rαhisubset (17, 36). In addition, the terminally
differentiated KLRG1hiCD27loIL-7RαloT cells show poor AKT
activation in response to both cytokine and TCR stimulation (21,
37, 38). Given that AKT activity is necessary for cell survival in
a variety of cell types, we hypothesized that the reduced AKT ac-
tivity in KLRG1hiIL-7Rαloterminal effector cells was the basis of
their shortened lifespan (39). However, our results showed that
elevating AKT activity in virus-specific CD8 T cells could increase
effector T-cell survival under cytokine deprivation conditions
in vitro, but could not reduce effector CD8 T cell death during the
contraction phase in vivo and, in the case of CA-AKT expression,
even exacerbated their contraction. Persistent AKT activity in the
virus-specific CD8 T cells induced a negative feedback mechanism
that repressed expression of IL-7Rα and IL-2/15Rβ chains and the
signals they transduce, such as STAT5. This effect correlated with
Ly5.1 (TCR Tg+)
Day 6 Day 60
DN-STAT5 RV CA-STAT5 RV CTRL RV
# of Thy1.1+(RV+)
# CD8+ T cells/
# CD8+ T cells/
# CD8+ T cells/
# CD8+ T cells/
cells during and after infection. Activated P14 Ly5.1+ CD8 T cells were trans-
duced with CA-STAT5, DN-STAT5, or CTRL RVs that express Thy1.1. Small
numbers (∼2,500) of RV+ (Thy1.1+) P14 CD8 T cells were transferred into con-
genic Ly5.2+ C57BL/6 mice that were infected with LCMV. (A) Contour plots
at days 6 and 30 p.i. (B) Line graphs show the number of CA-STAT5 (red) and
CTRL RV+ (black) (Thy1.1+) P14 CD8 T cells recovered from the spleen at dif-
ferent times postinfection. (C) As in B, total numbers of splenic KLRG1hi
IL-7Rαlo, KLRG1loIL-7Rαhi, KLRG1hiIL-7Rαhi, and KLRG1loIL-7RαloCA-STAT5
in the line graphs. Data shown are representative of the mean (±SEM) of 5–20
animals from five separate experiments. All statistics were calculated with
Student’s t test. ***P < 0.0005, **P < 0.005, *P < 0.05.
STAT5 signals profoundly augment survival of all LCMV-specific CD8 T
| www.pnas.org/cgi/doi/10.1073/pnas.1003457107Hand et al.
impaired homeostatic turnover of memory CD8 T cells and sec-
ondary responses to reinfection. Both approaches we took to ac-
AKT signaling. Presumably, more subtle increases in AKT acti-
vation that do not affect cytokine receptor expression and down-
stream STAT5 activity could enhance the survival of effector CD8
T cells. Although it is likely that defective AKT signaling in the
KLRG1hieffector CD8 T cells contributes to their reduced fitness
and longevity, our data suggest that activating AKT alone is not
of this molecule is needed for optimal effector cell viability and
memory CD8 T-cell function and homeostasis.
In contrast, our data clearly show that constitutive STAT5 sig-
in the CA-STAT5 memory CD8 T cells, especially in the KLRG1lo
cells, which naturally have a higher proliferative capacity relative to
not augment the proliferative capacity of CD8 T cells to second-
ary infection, suggesting that increasing STAT5 activity does not
STAT5 augmented the expression of CD27, IL-2/15Rβ chains, and
BCL2. Thus, elevated STAT5 activity appears to greatly enhance
the survival and homeostasis of all antigen-specific effector and
memory CD8 T cells, which is consistent with the observation that
mice made transgenic for STAT5 have significantly increased num-
bers of memory cells (40–42).
The implications of these results are that STAT5 activity is
this contributes to their death. In support of this idea, bona fide
antiapoptotic STAT5 target genes, such as Bcl2, Pim2, and Serpi-
na3g (Spi2a), are up-regulated in KLRG1loIL-7Rhieffector CD8
T cells compared with KLRG1hiIL-7Rlocells (3, 10, 43). Addi-
human CD27hiCCR7hi“central memory” cells (TCM) compared
with CD27loCCR7lo“effector memory” T cells (TEM) (38). Al-
though KLRG1hiand KLRG1loeffector CD8 T cells can phos-
(17, 18), it remains to be determined whether STAT5 signals are
to their relative short-term and long-term fates. It is possible that
the KLRG1loIL-7RhiMPECs contain more STAT5 activity at
steady state in vivo simply because they can see more IL-7 and
IL-15. For example, KLRG1hiIL-7Rαlocells have slightly reduced
expression of IL-15Rα and IL-2/15Rβ compared with KLRG1lo
IL-7Rαhicells, which may become important at physiological IL-15
levels in vivo. Conversely, KLRG1hiIL-7Rαloand KLRG1loIL-
differentially with IL-15- and IL-7-producing cell types. In support
of this idea, exogenous administration of IL-2 and IL-15 can keep
dependent on continuous cytokine treatment (20, 44). Further-
more,under steady-state conditionsinvivo, theKLRG1hiIL-7Rαlo
cells contain considerably less BCL2 than the KLRG1loIL-7Rαhi
cells, but the KLRG1hiIL-7Rαlocells up-regulate BCL2 when ex-
cytokines islimitedinvivo.Itisalso possiblethat STAT5 activation
is relatively similar between KLRG1hiand KLRG1loeffector CD8
T cells during the contraction phase in vivo but that differential
expression of other transcription factors or microRNAs modulates
STAT5-dependent gene expression, leading to differential gene
expression and cell survival. Finally, in a T-cell line, it has been
shown that STAT5 cannot act alone, and requires PI3K/AKT sig-
naling, to activate ccnd2 (CYCLIN D2) and bcl2l1 (BCLXL)
genes requiring both STAT5 and PI3K/AKT activity. Our studies
with CA-AKT were unable to test this possibility because of the
down-regulation of the cytokine receptors and STAT5 signaling.
The cause of the defect in AKT phosphorylation in terminally
differentiated KLRG1hiCD27loIL-7Rαloeffector and memory
CD8 T cells is not known. Our data showed that CA-STAT5 could
potentiate AKT signaling and complement AKT activation in
KLRG1hicells, indicating cross-talk between the two pathways.
Perhaps a paucity of STAT5 activity in the KLRG1hieffector CD8
T cells in vivo leads to the observed defect in AKT activity, which,
in turn, reduces the long-term fitness and survival of this cell
population. Expression of KLRG1 may also contribute to reduced
AKT activation. A recent report showed that blocking the in-
hibitory receptor KLRG1 from engaging its ligands, E- and N-
phorylation and proliferation of human KLRG1hiCD27loCD8
T cells (21). All of our biochemical studies were performed on
FACS-purified CD8 T cells, which do not express high levels of E-
cadherin, and therefore the role of KLRG1 was not directly in-
vestigated here (21).
As effector CD8 T cells transition to memory cells, they switch
from glycolysis to fatty acid oxidation due to the down-regulation
TORC1 in T cells during viral infection increases formation of
memory CD8 T cells. Thus, the poor memory CD8 T cell de-
velopment and function observed with CA-AKT in our experi-
ments may be linked to this mechanism as well. PI3K/AKT/
TORC1 signaling can also regulate the expression of L-selectin
(CD62L), which distinguishes the TEMand TCMmemory T-cell
TCMpopulations, whereas the fraction of terminally differentiated
KLRG1hiIL-7Rlocells that persists into the memory stage is
overwhelmingly composed of CD62LloTEMcells (10, 13, 47).
However, terminally differentiated TEM (KLRG1hi
CD62Llo) display impaired AKT phosphorylation compared with
their KLRG1loCD27hicounterparts, which suggests that an ad-
ditional AKT-independent mechanism for CD62L repression
exists inCD8Tcells(21,37).Wewouldpostulatethat PI3K/AKT-
dependent regulation of CD62L expression predominantly occurs
in the TCMsubset, which has increased AKT signaling.
Multiple reports now show that a CD8 T cell’s differentiation
state influences its ability to survive and proliferate by a variety of
mechanisms. Our work shows that sustained STAT5 activity is
CTRL RV CA-STAT5 RV
CTRL RV CA-STAT5 RV
1 . 1
s l l e
SLEC MPEC SLEC MPEC
both KLRG1hiIL-7RαloSLECs and KLRG1loIL-7RαhiMPECs. As in Fig. 4, P14 CD8
T cells were transduced with CA-STAT5 or CTRL RVs and ∼2,500 cells were
transferred into C57BL/6 mice that were infected with LCMV. (A) Histogram
plots show expression of BCL2 in CA-STAT5 (red) and CTRL (black line) RV+
(Thy1.1+) P14 CD8 T cells 15 d post-LCMV infection. The MFIs of the proteins
are indicated and the dotted line indicates staining of BCL2 isotype control
antibody. Data are representative of five separate experiments. (B) Ap-
proximately 5,000 P14 TCR transgenic T cells containing CA-STAT5 or CTRL
RV+ (Thy1.1+) cells were isolated from mice infected 15 d previously with
LCMV and transferred into naïve C57BL/6 mice that were subsequently
infected with Listeria-GP33. Seven days later, the expansion of the donor CA-
STAT5 or CTRL RV+ (Thy1.1+) P14 CD8 T cells was analyzed in the peripheral
blood. Data are representative of three to five mice. (C) Mice containing CA-
STAT5 (red) or CTRL (black line) RV (Thy1.1+) P14 CD8 T cells were treated
with BrdU in their drinking water from day 65 to 75 p.i. and then stained in-
tracellularly for BrdU. Contour plots on the right show the percent of BrdU+
cells within the KLRG1hior KLRG1lomemory CD8 T cells. Data shown are
representative of two experiments.
STAT5 increases the survival, proliferation, and AKT activation of
Hand et al.PNAS
| September 21, 2010
| vol. 107
| no. 38
a very powerful signal for enhancing memory CD8 T-cell survival,
regardlessofdifferentiationstate,andsuggeststhat this signalmay
be lacking in terminally differentiated cells. Furthermore, STAT5
activation increased the production of memory T cells without any
obvious oncogenic effects, and therefore modulation of this mol-
chronic infections or tumors.
Materials and Methods
Mice and Viral/Bacterial Infection. C57BL/6Ncr mice were purchased from the
National Cancer Institute. B6.SJL-PtprcaPepcb/BoyJ (Ly5.1+) and Pten floxed
(Ptenfl/fl) animals were purchased from The Jackson Laboratory. Granzyme B
Cre transgenic animals were the kind gift of Joshy Jacob (Emory University,
Atlanta, GA). P14 and P14 IL-7Rα transgenic (IL-7Rαtg) mice have been pre-
viously described (17). To make P14 chimeric mice, small numbers (∼2,500) of
Wt or P14 CD8 T cells were adoptively transferred into C57BL/6 mice that were
subsequently infected with 2 × 105pfu LCMV Armstrong i.p. For secondary
infection, C57BL/6 mice containing ∼2,500 P14 CD8 T cells were infected with
2 × 104cfu recombinant Listeria monocytogenes i.v. that expresses the LCMV
GP33–41 epitope (strain XFL203, but referred to as Listeria-GP33) (3). All
experiments were done with approved Institutional Animal Care and Use
Retroviral Transfection. Activated P14 TCR transgenic T cells were transfected
with murine stem cell virus retroviruses containing CA-AKT, CA-STAT5, or DN-
STAT5 and coexpressing Thy1.1, as described previously (10, 26). CA-AKT, DN-
STAT5, and CA-STAT5 retroviruses have been described previously (25, 48).
Cell Sorting, CFSE Labeling, and Adoptive Transfer. Splenocytes containing Wt
P14CD8T cellsorCA-AKT/CA-STAT5 retrovirallytransfected cellswereisolated
or Ly5.1, and then sorted using the FACSAria (BD Biosciences) instrument. Al-
ternatively, activated Wt or Pten−/−T cells were sorted by expression of CD8,
CD44, and KLRG1. In all experiments, splenocytes were depleted of non-CD8 T
cells by magnetic separation before sorting/adoptive transfer. In homeostatic
were labeled with CFSE (Invitrogen) per the manufacturer’s instructions.
ACKNOWLEDGMENTS. We thank A. Poholek, J. Rathmell, G. Shadel, B. Su,
J. Mandl, Y. Belkaid, and the members of the Kaech laboratory for helpful
comments and discussions; Z. Zhao and G. Lyon for technical assistance;
J. Jacob for the Granzyme B Cre mice; S. Haxhinasto and C. Benoist for the
CA-AKT retrovirus; and A. Abbas and H. Dooms for the CA-STAT5 and DN-
STAT5 retroviruses. This work was supported by National Institutes of Health
Grant R01 AI066232-01 (to S.M.K.) and the Cancer Research Institute (S.M.K.)
and a Canadian Institutes of Health Research Doctoral Research Award (MDR
75905) (to T.W.H.).
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| www.pnas.org/cgi/doi/10.1073/pnas.1003457107 Hand et al.