CTLA-4 control over Foxp3+ regulatory T cell function

Abstract

Naturally occurring Foxp3+CD4+ regulatory T cells (Tregs) are essential for maintaining immunological self-tolerance and immune homeostasis. Here, we show
that a specific deficiency of cytotoxic T lymphocyte antigen 4 (CTLA-4) in Tregs results in spontaneous development of systemic
lymphoproliferation, fatal T cell–mediated autoimmune disease, and hyperproduction of immunoglobulin E in mice, and it also
produces potent tumor immunity. Treg-specific CTLA-4 deficiency impairs in vivo and in vitro suppressive function of Tregs—in
particular, Treg-mediated down-regulation of CD80 and CD86 expression on dendritic cells. Thus, natural Tregs may critically
require CTLA-4 to suppress immune responses by affecting the potency of antigen-presenting cells to activate other T cells.

Full text

copy number in TG of GrB
−/−
or Pfn
−/−
mice. This
mechanism might be particularly efficient during
attempted HSV-1 reactivation events where ICP4
expression has escaped repression by viral miRNAs
and host neuron epigenetic modifications. Thus, we
propose a tripartite relation in which HSV-1 latency
is maintained through the activity of the virus, host
neuron, and contiguous CD8
+
T cells permitting
viral persistence with neuronal survival (fig. S7).
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28. We thank K. Lathrop and J. Karlsson for assistance
with microscopy and preparation of figures and
N. Zurowski for assistance with flow cytometry. We
have no conflicting financial interests. This work was
supported by NIH grants F30NS061471 (J.E.K.),
R01EY05945 (R.L.H.), R01EY015291 (P.R.K.), and
P30EY08098 (R.L.H.); a Research to Prevent Blindness
Medical Student Eye Research Fellowship (J.E.K.); and
unrestricted grants from Research to Prevent Blindness
and the Eye and Ear Foundation of Pittsburgh (R.L.H.).
Supporting Online Material
www.sciencemag.org/cgi/content/full/322/5899/268/DC1
Materials and Methods
Figs. S1 to S7
References
4 August 2008; accepted 11 September 2008
10.1126/science.1164164
CTLA-4 Control over Foxp3
+
Regulatory T Cell Function
Kajsa Wing,
1
*Yasushi Onishi,
1,2
Paz Prieto-Martin,
1
Tomoyuki Yamaguchi,
1
Makoto Miyara,
1
Zoltan Fehervari,
1
Takashi Nomura,
1
Shimon Sakaguchi
1,3,4
†
Naturally occurring Foxp3
+
CD4
+
regulatory T cells (Tregs) are essential for maintaining
immunological self-tolerance and immune homeostasis. Here, we show that a specific deficiency
of cytotoxic T lymphocyte antigen 4 (CTLA-4) in Tregs results in spontaneous development of
systemic lymphoproliferation, fatal T cell–mediated autoimmune disease, and hyperproduction of
immunoglobulin E in mice, and it also produces potent tumor immunity. Treg-specific CTLA-4
deficiency impairs in vivo and in vitro suppressive function of Tregs—in particular, Treg-mediated
down-regulation of CD80 and CD86 expression on dendritic cells. Thus, natural Tregs may critically
require CTLA-4 to suppress immune responses by affecting the potency of antigen-presenting
cells to activate other T cells.
Naturally occurring CD25
+
CD4
+
regula-
tory T cells (Tregs), which specifically
express the transcription factor Foxp3,
suppress aberrant immune responses, including
autoimmune diseases and allergy (1). Furthermore,
reduction or expansion of Tregs can be exploited to
provoke effective tumor immunity or transplantation
tolerance, respectively. Two cardinal features of
Foxp3
+
Tregs are that they constitutively express
cytotoxic T lymphocyte antigen 4 (CTLA-4), which
only happens after activation in other T cell subsets
(2–4), and that Foxp3 controls the expression of
CTLA-4 in Tregs (5–9). CTLA-4 is a potent nega-
tive regulator of T cell immune responses, as illus-
trated by CTLA-4 knockout (KO) mice, which die
prematurely from multiorgan inflammation (10,11).
The polymorphism of the CTLA-4 gene contributes
substantially to the genetic susceptibility to autoim-
mune diseases such as type 1 diabetes (12). More-
over, autoimmunity, inflammatory bowel disease,
and tumor immunity can be elicited by blocking
CTLA-4 with a specific antibody (3,4,13–15). Yet
the manner in which CTLA-4 negatively controls
immune responses is controversial (16). CTLA-4
expressed by activated effector T cells may mediate
a negative signal that attenuates their activation.
Alternatively, but not exclusively, Foxp3
+
Tregs
may require CTLA-4 for their suppressive function.
By specifically deleting the CTLA-4 gene in Foxp3
+
Tregs in mice, we have attempted to determine
the role of CTLA-4 for the maintenance of self-
tolerance and immune homeostasis.
We generated BALB/c mice expressing Cre
under the control of the Foxp3 promoter—hereafter
called FIC (Fox-IRES-Cre) mice—and BALB/c
mice expressing a floxed CTLA-4 gene (CTLA-
4
fl/fl
) [supporting online material (SOM) text and
fig. S1] (17). Compared with BALB/c wild-type
(WT) mice, FIC mice expressed Foxp3 protein at
slightly lower levels whereas CTLA-4
fl/fl
mice
expressed equivalent levels of CTLA-4 (Fig. 1A).
To assess the specificity of Cre expression, FIC
mice were crossed with Cre reporter mice (CAG
mice), which express enhanced green fluorescent
protein (EGFP) only in Cre
+
cells (18). EGFP
expression was confined to ~15% of CD4
+
T
cells and ~1.5% of CD8
+
T cells (Fig. 1B). The
vast majority of EGFP
+
CD4
+
T cells in adult
FIC
+/+
CAG mice were Foxp3
+
(97.1 T1.2%, n=4
mice), indicating that Foxp3 expression is stable
once the gene is turned on and Cre expression is not
leakyinFoxp3
–
cells (Fig. 1C). On the basis of this
specific expression of Cre in Foxp3
+
Tregs, we
generated CTLA-4 conditional KO (CKO) mice
by crossing FIC and CTLA-4
fl/fl
mice. CTLA-4
was specifically deleted in CD4
+
Foxp3
+
T cells, as
compared with FIC
+/+
WT or full CTLA-4 KO
mice (Fig. 1D). CKO mice even harbored a higher
frequency of CTLA-4–expressing CD4
+
Foxp3
–
cells than did WT littermates (Fig. 1E). Whereas
KO mice became moribund at ~20 days of age
(10,11), CKO mice remained apparently unaf-
fected until ~7 weeks of age, when they rapidly
became inactive and began to develop general
edema that was frequently accompanied by ascites
(Fig. 1F). Thus, CTLA-4 deficiency in Tregs alone
suffices to cause fatal disease, whereas the addition-
al CTLA-4 deficiency in non-Treg cells enhances
the disease. Yet, CTLA-4 expression in activated
effector T cells per se is insufficient to prevent it.
Pathological analysis of CKO mice revealed
splenomegaly and lymphadenopathy, which was
reflected in increased cell numbers (Fig. 2, A and
B). The proportion of CD4
+
T cells was unaltered,
whereas CD8
+
T cells were decreased (Fig. 2C).
Cardiomegaly and congestion of the liver was
macroscopically evident in the terminal stage of
every case. In affected hearts, mononuclear cells
densely infiltrated into the myocardium and de-
stroyed myocytes (Fig. 2, D to G), indicating that
the plausible cause of sudden death in CKO mice is
1
Department of Experimental Pathology, Institute for Frontier
Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
2
Department of Rheumatology and Haematology, Tohoku Uni-
versity Graduate School of Medicine, Sendai 980-8574, Japan.
3
Core Research for Evolutional Science and Technology, Japan
Science and Technology Agency, Kawaguchi 332-0012, Ja-
pan.
4
Laboratory of Experimental Immunology, World Premier
International Immunology Frontier Research Center, Osaka
University, Suita 565-0871, Japan.
*Present address: Department of Medical Inflammation
Research,KarolinskaInstitute,Stockholm17177,Sweden.
†To whom correspondence should be addressed. E-mail:
shimon@frontier.kyoto-u.ac.jp
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heart failure due to severe myocarditis (19). In
addition, CKO mice possessed focal lymphocyte
infiltrations in lung and salivary gland and suffered
from gastritis with various degrees of destruction of
gastric parietal cells and chief cells. Antiparietal
autoantibodies were readily detected in the sera of
CKO mice and a proportion of FIC
+/+
mice, in
which the lower expression of Foxp3 in Tregs (Fig.
1A) might somehow affect Treg function (20)(Fig.
2, H to N, and SOM text). Myocarditis and gastritis
in CKO mice (and gastritis in FIC mice) could be
adoptively transferred with splenocytes and purified
CD4
+
T cells into T cell–deficient BALB/c athymic
nude (nu/nu) mice, indicating that these auto-
immune conditions were both T cell–mediated
(Fig. 2O and fig. S2). Furthermore, CKO mice
developed several hundred–fold and threefold
higher levels of serum immunoglobulin E (IgE)
and immunoglobulin G (IgG), respectively, than
the levels in FIC or WT mice (Fig. 2, P and Q).
Costaining of intracellular cytokines and Foxp3
revealed an increased frequency of interleukin-2
(IL-2)–,IL-4–,andIFN-g–producing Foxp3
–
CD4
+
cells in both the spleen and lymph node (LN) of
diseased CKO and KO mice (Fig. 2R and fig. S3).
IL-17–secreting (Th17) cells increased in KO but
not CKO mice, suggesting that Th17 cells might
contribute to the rapid disease progression in the
former.Thus,CTLA-4–deficient Tregs fail to
control the spontaneous activation of other T cells
and their differentiation into Th1 and Th2 lineage
cells that mediate autoimmune disease and allergy.
We next tested whether Treg-specific CTLA-
4 deficiency also influenced the potency of tumor
immunity. BALB/c nu/nu mice were reconsti-
tuted with splenocytes from CKO or control FIC
mice containing equivalent numbers of T cells and
inoculated with BALB/c-derived RL♂1leukemia
cells (21). All recipients of FIC splenocytes died
of tumor progression within a month. In contrast,
recipients of CKO splenocytes halted the tumor
growth, with the majority surviving the 6-week
observation period, during which 60% of them
completely rejected the tumor (Fig. 3A). As pre-
viously shown (21), transfer of BALB/c spleno-
cytes after depletion of CD25
+
Tcellsledtothe
rejection of RL♂1 leukemia cells in nu/nu mice.
In this setting, FIC Tregs cotransferred with CD25
–
T cells suppressed tumor rejection, whereas CKO
Tregs did not (Fig. 3B). Thus, Treg-specific CTLA-
4 deficiency affects in vivo Treg suppressive func-
tion, leading to enhanced tumor immunity.
We next explored the possibility that CTLA-4
deficiency might impair the generation, survival,
or suppressive function of Foxp3
+
Tregs. CKO
mice exhibited no significant alteration in number
or composition of CD4
+
and CD8
+
thymocytes
(Fig. 4A). The majority of Foxp3
+
WT thymo-
cytes expressed CTLA-4, whereas Foxp3
+
CKO
thymocytes contained a mix of CTLA-4
+
and
CTLA-4
–
cells in both the CD4–single positive
and CD4/CD8–double positive compartments
(Fig. 4A). Because the CTLA-4 gene is deleted
only after Foxp3 is expressed, CTLA-4 is either
up-regulated before Foxp3 expression in CKO
mice or it may take some time for the Cre protein
to accumulate in Foxp3
+
cells, meanwhile allow-
ing the expression of CTLA-4. The frequency of
Foxp3
+
thymocytes was not significantly changed
between CKO and WT mice, whereas the number
of Foxp3
+
and Foxp3
–
T cells in the spleen and
LNs increased enormously by active proliferation
(Fig.4B,figs.S4andS5,andSOMtext).Thus,
Foxp3-inducible CTLA-4 deficiency minimally
alters thymic selection of Tregs and probably
triggers immunological diseases through affect-
ing Treg function in the periphery.
Because Foxp3 is encoded by the X chromo-
some, female nonautoimmune FIC
+/−
CTLA-4
fl/fl
mice are a mosaic for CTLA-4–intact and –deficient
Tregs. They harbored equal numbers of CTLA-4
+
and CTLA-4
–
Foxp3
+
T cells, indicating that
both populations equally survive in physiological
non-inflammatory conditions (Fig. 4C). Further-
more, when CTLA-4–deficient or –intact Tregs
were transferred to nu/nu mice, both populations
showed a similar degree of homeostatic prolifer-
ation, and neither one caused autoimmunity (fig.
S6). CTLA-4–deficient Foxp3
+
Tregs were as poor
at producing pro-inflammatory cytokines as were
their WT or FIC counterparts (fig. S3). Taken to-
gether, CTLA-4 deficiency, per se, does not affect
the survival of Tregs or render them pathogenic.
Phenotypically, CTLA-4–deficient naive Tregs
in FIC
+/−
CTLA-4
fl/fl
females normally expressed
typical Treg markers including CD44, CD103,
glucocorticoid-induced tumor necrosis factor re-
ceptor, latency-associated peptide, and intracellular
IL-10 (Fig. 4D and fig. S7). The comparatively
higher expression of these molecules by Tregs from
CKO mice is presumably secondary to ongoing
inflammation in CKO mice, as illustrated by an
activated phenotype of their Foxp3
–
non-Treg cells.
CTLA-4–deficient Tregs, whether naive from
FIC
+/−
CTLA-4
fl/fl
CAG females or activated from
CKO mice, had diminished suppressive capacity
compared with CTLA-4–intact Tregs in cultures
of carboxyfluorescein diacetate succinimidyl ester
(CFSE)–labeled responder T cells (Tresp) in the
presence of splenic CD11c
+
dendritic cells (DCs)
and anti-CD3 monoclonal antibody (mAb), as as-
sessed by the percentage and number of CFSE-
diluting (i.e., divided) Tresp (Fig. 4E, figs. S8 and
S9, and SOM text). Moreover, CKO Tregs clearly
failed to suppress allo-reactive Tresp proliferation,
even at high Treg/Tresp ratios (Fig. 4F). FIC or
WT Tregs, whether cultured alone or together with
Tresp cells, specifically hampered up-regulation
of the expression of CD80 and CD86, but not
CD40 and major histocompatability complex
class II, in DCs (22–26). In contrast, CKO Tregs
failed to exert this effect (Fig. 4G, figs. S10 to
S13, and SOM text). Activated FIC Tregs (but
Fig. 1. Specific deletion
of CTLA-4 expression in
Foxp3
+
Tcellsresultsin
fataldisease.(A)Flowcy-
tometric analysis of intra-
cellular Foxp3 (left) and
CTLA-4 (right) in freshly
isolated LN CD4
+
T cells
from male FIC, CTLA-4
fl/fl
,
or BALB/c WT mice. (B)
EGFP expression in CD4
+
or CD8
+
Tcellsderived
from male FIC-CAG mice.
(C) Sorted CD4
+
EGFP
+
cells in FIC-CAG mice were
stained for Foxp3. (D)
CTLA-4 and Foxp3 expres-
sion in LN CD4
+
T cells
fromBALB/cWT,CKO,or
KO mice. (E) Frequency
of CTLA-4–expressing
CD4
+
Foxp3
–
T cells in
CKO and normal litter-
mates (n=5).(F)Sur-
vival of KO and CKO mice
as compared with normal
littermates. Data represent
three or more independent
experiments. Vertical bars
indicate SEM.
Foxp3
CTLA-4
A
5.8 11.2
2.4
13.3 0.4
44.4
CTLA-4 CKO
FIC/Y CTLA-4 KO
0.2 0.1
47.1
D
E
B
Foxp3 CTLA-4
C
% of Max
WT
FIC/Y
WT
CTLA-4fl/fl
EGFP
Counts
CD4+CD8+
14.8% 1.4%
EGFP Foxp3
Sorted EGFP+ cells
Spleen
0
10
20
30
40
CKO
WT
010 20 30 40 50 60 70 80
0
20
40
60
80
100
CKO
n=11
WT
n=17
KO
n=6
Days
Percent survival
F
%CD4+Foxp3-CTLA-4+
Foxp3 Foxp3
CD4+
Counts
LN
80.6 41.9 52.5
p<0.01p<0.01
98.0%
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not activated CKO Tregs) also reduced the ex-
pression of CD86-GFP fusion protein retrovirally
expressed in L-cells, a fibroblast cell line (Fig.
4H). This indicates that Treg-dependant modula-
tion of CD86 expression on DCs is at least partly
due to down-regulation of the expression and not
masking of the molecule by soluble CTLA-4.
Taken together, Treg-mediated CD80/CD86 down-
regulation may limit the activation of naive T cells
via CD28, resulting in specific immune suppres-
sion and tolerance.
Thus, CTLA-4 expressed in Foxp3
+
Tregs is
critically required for their in vivo and in vitro
suppression, which is mediated at least in part by
Fig. 2. Autoimmune disease and
hyperproduction of IgE in CKO mice.
(A) Splenomegaly and lymphad-
enopathy in a CKO and a WT litter-
mate. Lymphocyte numbers (B)and
frequencies of T cell subsets (C)in
spleens of 6- to 10-week-old CKO
and WT littermates (n= 11 to 13).
(D) The heart of a CKO (left) and a
FIC
+/−
CTLA-4
fl/fl
mouse (right). His-
tology (hematoxylin and eosin stain-
ing) of the heart of a CKO [(Eand F)
×50 and ×200, respectively] and a
FIC mouse [(G) ×50; inset, ×200].
Histology of the stomach [(Hand I)
×100], lung [(Jand K) ×100], and
salivary gland [(Land M)×50]ofa
CKO [(H), (J), and (L)] and a FIC
mouse [(I), (K), and (M)]. Serological
and histological development of
gastritis in WT, FIC
+/+
,andCKO
mice (N), and BALB/c nu/nu mice 7
weeks after cell transfer from CKO
or FIC
+/+
mice (O). Gastric lesions
were histologically graded as 2
(black circle), 1 (gray circle), and 0
(open circle) (19). Serum concen-
trations of IgE (P)andIgG(Q)in
indicated groups of mice. (R)Fre-
quencies of cytokine-producing cells
among CD4
+
Foxp3
–
splenocytes of
6- to 9-week-old CKO, 16- to 20-
day-old KO, or normal littermates
(n= 5 to 6). Error bars indicate SEM.
CKOFIC
D
B A
CKO WT CKO WT
Spleen LN
E
WT
(n=8)
FIC
(n=10)
0.01
0.1
1
10
100
1000
IgE (µg/ml)
Spleen
LN
0
10
20
30
40
50
Cell number (x107)
WT
(n=10)
FIC
(n=10)
1
10
100
1000
10000
WT
(n=13)
FIC
(n=11)
CKO
(n=15)
0
5
10
15
20
0
5
10
15
20
FIC CKO
Anti-parietal cell Ab
(unit)
Ctrl
CKO
Ctrl
KO
0
10
20
30
40
% IL-2+ cells
Ctrl
CKO
Ctrl
KO
0
10
20
30
40
% IFN-γ+ cells
Ctrl
CKO
Ctrl
KO
0
5
10
15
20
25
% IL-4+ cells
Ctrl
CKO
Ctrl
KO
0
5
10
15
% IL-17+ cells
CD3
CD4 CD8
0
10
20
30
40
% of splenocytes
C
J
H
N
L
I
K
M
FG
Q
O
P
R
p<0.05
CKO
WT
Anti-parietal cell Ab
(unit)
IgG (µg/ml)
CKO
(n=10)
CKO
(n=10)
p<0.05 p<0.05
p<0.05
p<0.001
p<0.001
p<0.001
p<0.01
p<0.01
p<0.01
p<0.05
p<0.05
p<0.05
p<0.001
p<0.01
CKO FIC
Fig. 3. Treg-specific CTLA-4
deficiency promotes tumor
immunity. (A)BALB/cnu/nu
mice received 3 × 10
7
spleno-
cytes from FIC or CKO mice,
followed by intradermal inocu-
lation of 1.5 × 10
5
RL♂1
leukemia cells. Crosses indicate
death due to tumor growth.
(B)BALB/cCD25
–
cells (1.5 ×
10
7
) were cotransferred with
3.8 × 10
5
CD25
high
CD4
+
T cells from CKO or FIC mice and inoculated with 1.5 × 10
5
RL♂1cells(n=3).
Tumor diameters were measured every other day for 6 weeks. Mice were euthanized when tumor diameters
exceeded 20 mm. Error bars indicate SEM.
Tumor diameter (mm)
0
010
10
20 30 40
30
20
Days
FIC (n=6)
CKO (n=7)
A
Tumor diameter (mm)
0
010
10
20 30
30
20
Days
B
CD25- (n=4)
CD25- + FIC Treg (n=3)
CD25- + CKO Treg (n=3)
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CTLA-4–dependent down-regulation of CD80
and CD86 on antigen presenting cells. Tregs prob-
ably use multiple suppressive mechanisms, and
the importance of each one may vary depending
on the environment and the context of immune
responses (1). However, if the CTLA-4–mediated
mechanism of suppression is defective, Tregs can-
not sustain self-tolerance and immune homeosta-
sis, even if other suppressive mechanisms become
more active to compensate for the deficiency.
Thus, CTLA-4 is a key molecular target for con-
trolling Treg-suppressive function in both physiolog-
ical and pathological immune responses including
autoimmunity, allergy, and tumor immunity.
References and Notes
1. S. Sakaguchi et al., Cell 133, 775 (2008).
2. B. Salomon et al., Immunity 12, 431 (2000).
3. T. Takahashi et al., J. Exp. Med. 192, 303 (2000).
4. S.Read,V.Malmstrom,F.Powrie,J. Exp. Med. 192, 295 (2000).
5. S. Hori, T. Nomura, S. Sakaguchi, Science 299,
1057 (2003), published online 9 January 2003;
10.1126/science.1079490.
Fig. 4. CTLA-4–deficient Tregs
develop and survive normally
but have defective function.
(A) Thymocyte expression of
Foxp3 and CTLA-4 in a 2.5-
week-old CKO or a WT litter-
mate. SP, single positive; DP,
double positive. (B)Frequency
and number of CD4
+
Foxp3
+
T cells in spleens and LNs of 6-
to 8-week-old CKO or WT litter-
mates (n=7).(C)Foxp3and
CTLA-4 expression in splenic
CD4
+
TcellsfromaFIC
+/−
CTLA-4
fl/fl
female mouse (left).
Percentages of Foxp3
+
CTLA-4
+
and Foxp3
+
CTLA-4
–
Tcellsin
each mouse (5 to 8 weeks of
age) are connected (n=8)
(right). ns, not significant. (D)Ex-
pression of cell surface mole-
cules on CD4
+
LN cells from
CKO, FIC, or FIC
+/−
CTLA-4
fl/fl
mice.
(E)CD25
high
EGFP
+
cells (naive
CTLA-4
–
Treg) and CD25
high
EGFP
–
cells (naive CTLA-4
+
Treg) from
FIC
+/−
CTLA-4
fl/fl
CAG female
mice and CD25
high
CD4
+
Tcells
from FIC mice (FIC Treg) were
cocultured with CD25
–
CD4
+
T cells (Tresp), anti-CD3 mAb,
and live splenic DCs for 3 days.
Percentages and numbers (in
parentheses) of CFSE-diluting
Tresp cultured at a 1:2 Treg-
to-Tresp ratio (left). Numbers
of CFSE-diluting Tresp cultured
at graded ratios of Treg:Tresp
(right). (F) Percentage and num-
bers of CFSE-labeled BALB/c
Tresp cocultured with CKO or
FIC Tregs and X-irradiated
C57BL/6 splenocytes for 4 days
at 1:1 Treg:Tresp ratio (left)
and numbers at graded ratios
(right). (G) CD80 and CD86
expression of live splenic DCs
after a 2-day culture with Tresp,
CD4
+
EGFP
+
Tregs, or a mix
thereof, and anti-CD3 mAb.
Histograms show mean fluo-
rescence intensity (MFI). (H)
L-cells, expressing the Fc recep-
tor, were retrovirally transduced
to express CD86-EGFP fusion
protein, cocultured with indi-
catedTregsandanti-CD3mAb
for 2 days, and assessed for GFP
level. Data in (A) and (D) to (H) represent three or more independent experiments. Error bars indicate SEM.
C
8.6 5.2
7.4
FIC+/-CTLA-4fl/fl
Foxp3
CTLA-4
A
84.9
2.3
4.7 1.4
2.1
0.8 0.07
0.2
7.5 0.1
0.3
83.7
2.6
4.3 2.2
0.8
0.8 0.1
0.1
4.9 0.1
0.5
CD4
SP
CD4/8
DP
CD8
SP
CTLA-4 CKO
WT littermate
CD4
CD8
Foxp3
8.7
8.3
CTLA-4
+
CTLA-4
-
0
2
4
6
8
10
12
Foxp3+
% of CD4+ cells
D
CD103 GITR
Foxp3-
Foxp3+
CD44
% of Max
100
0
100
0
ns
Tresp alone
+ FIC Treg
+ CTLA-4- naive Treg
+ CTLA-4+ naive Treg
0
100
200
300
400
500
MFI (CD80)
Tresp
FIC Treg
CKO Tr eg
DC
-
-
-
+
+
-
-
+
+
+
-
+
+
-
+
+
-
-
+
+
-
+
-
+
CD80 CD80
CD86 CD86 0
100
200
300
400
500
600
MFI (CD86)
DC alone
DC+ FIC Treg
DC+ CKO Treg
isotype control
DC+Tres p
DC+Tresp+FIC Treg
DC+Tresp+CKO Treg
isotype control
DC+Treg DC+Tresp+ Tr eg
% of Max
G
p<0.01p<0.01
p<0.001 p<0.05
B
0
40
0
60
CKO
WT
%Foxp3+
of
CD4+
cells
30
20
40
50
10
Foxp3+CD4+
cells
(x106)
30
20
10
Spl LN Spl LN
p<0.001 p<0.01
CD86-GFP
% of Max
+FIC Treg
+CKO Treg
CD86-GFP L-cells
H
CTLA-4
1:8 1:4
1:2
0
10
15
20
25
Treg/Tresp ratio
Cell number (x103)
5
1:1
1:16
1:9
1:3
0
4
6
8
10
Treg/Tresp ratio
Cell number (x102)
1:1
2
Tresp alone
+ FIC Treg
+ CKO Treg
CFSE
ETreg:Tresp=1:2
96.6% (13,614)
70.0% (2,996)
25.6% (253)
20.7% (181)
CFSE
% of Max
100
0
CKO
FIC/Y FIC+/-CTLA-4 fl/fl
F
19.5% (787)
28.5% (706)
5.1% (107)
% of Max
20
0
Treg:Tresp=1:1
100
0
% of Max
100
0
% of Max
50
50
100
0
10 OCTOBER 2008 VOL 322 SCIENCE www.sciencemag.org274
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fellowship by Astra-Zeneca, Loughborough, UK.
Supporting Online Material
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Materials and Methods
SOM Text
Figs. S1 to S13
References
5 May 2008; accepted 15 August 2008
10.1126/science.1160062
Environmental Genomics
Reveals a Single-Species Ecosystem
Deep Within Earth
Dylan Chivian,
1,2
*Eoin L. Brodie,
2,3
Eric J. Alm,
2,4
David E. Culley,
5
Paramvir S. Dehal,
1,2
Todd Z. DeSantis,
2,3
Thomas M. Gihring,
6
Alla Lapidus,
7
Li-Hung Lin,
8
Stephen R. Lowry,
7
Duane P. Moser,
9
Paul M. Richardson,
7
Gordon Southam,
10
Greg Wanger,
10
Lisa M. Pratt,
11,12
Gary L. Andersen,
2,3
Terry C. Hazen,
2,3,12
Fred J. Brockman,
13
Adam P. Arkin,
1,2,14
Tullis C. Onstott
12,15
DNA from low-biodiversity fracture water collected at 2.8-kilometer depth in a South African
gold mine was sequenced and assembled into a single, complete genome. This bacterium,
Candidatus Desulforudis audaxviator, composes >99.9% of the microorganisms inhabiting the
fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate-reducing,
chemoautotrophic thermophile that can fix its own nitrogen and carbon by using machinery
shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent life-style
well suited to long-term isolation from the photosphere deep within Earth’s crust and offers an
example of a natural ecosystem that appears to have its biological component entirely encoded
within a single genome.
Amore complete picture of life on, and
even in, Earth has recently become
possible by extracting and sequencing
DNA from an environmental sample, a process
called environmental genomics or metagenomics
(1–8). This approach allows us to identify mem-
bers of microbial communities and to character-
ize the abilities of the dominant members even
when isolation of those organisms has proven
intractable. However, with a few exceptions (5,7),
assembling complete or even near-complete ge-
nomes for a substantial portion of the member
species is usually hampered by the complexity of
natural microbial communities.
In addition to elevated temperatures and a
lack of O
2
, conditions within Earth’s crust at
depths >1 km are fundamentally different from
those of the surface and deep ocean environ-
ments. Severe nutrient limitation is believed to
result in cell doubling times ranging from 100s
to 1000s of years (9–11), and as a result sub-
surface microorganisms might be expected to
reduce their reproductive burden and exhibit the
streamlined genomes of specialists or spend
most of their time in a state of semi-senescence,
waiting for the return of favorable conditions.
Such microorganisms are of particular interest
because they permit insight into a mode of life
independent of the photosphere.
One bacterium belonging to the Firmicutes
phylum (Fig. 1A), which we herein name Can-
didatus Desulforudis audaxviator, is prominent
in small subunit (SSU or 16S) ribosomal RNA
(rRNA) gene clone libraries (11–14) from almost
all fracture fluids sampled to date from depths
greater than 1.5 km across the Witwatersrand basin
(covering 150 km by 300 km near Johannesburg,
South Africa). This bacterium was shown in a
previous geochemical and 16SrRNA gene study
(11) to dominate the indigenous microorga-
nisms found in a fracture zone at 2.8 km below
land surface at level 104 of the Mponeng mine
(MP104). Although Lin et al.(11 ) discovered
that this fracture zone contained the least-diverse
natural free-living microbial community reported
at that time, exceeding the ~80% dominance by
the methanogenic archaeon IUA5/6 of a com-
paratively shallow subsurface community in Idaho
(15), we were nonetheless surprised when the cur-
rent environmental genomics study revealed only
one species was actually present within the frac-
ture fluid. Furthermore, we found that the
genome of this organism appeared to possess
all of the metabolic capabilities necessary for
an independent life-style. This gene complement
was consistent with the previous geochemical
and thermodynamic analyses at the ambient
~60°C temperature and pH of 9.3, which indi-
cated radiolytically generated chemical species as
providing the energy and nutrients to the system
(11), with formate and H
2
as possessing the
greatest potential among candidate electron
donors, and sulfate (SO
4
2–
) reduction as the
dominant electron-accepting process (11).
DNA was extracted from ~5600 liters of fil-
tered fracture water by using a protocol that has
been demonstrated to be effective on a broad
range of bacterial and archaeal species, includ-
ing recalcitrant organisms (16). A single, com-
plete, 2.35–megabase pair (Mbp) genome was
assembled with a combination of shotgun Sanger
sequencing and 454 pyrosequencing (16). Sim-
ilar to other studies that obtained near-complete
consensus genomes from environmental sam-
ples (5,17), heterogeneity in the population of
the dominant species as measured with single-
nucleotide polymorphisms (SNP) was quite low,
showing only 32 positions with a SNP observed
1
Physical Biosciences Division, Lawrence Berkeley National
Laboratory, Berkeley, CA 94720, USA.
2
Virtual Institute for
Microbial Stress and Survival, Berkeley, CA 94720, USA.
3
Earth Sciences Division, Lawrence Berkeley National Lab-
oratory, Berkeley, CA 94720, USA.
4
Departments of Biological
and Civil and Environmental Engineering, Massachusetts
Institute of Technology, Cambridge, MA 02139, USA.
5
Energy
and Efficiency Technology Division, Pacific Northwest National
Laboratory, Richland, WA 99352, USA.
6
Department of
Oceanography, Florida State University, Tallahassee, FL
32306, USA.
7
Genomic Technology Program, U.S. Depart-
ment of Energy (DOE) Joint Genomics Institute, Berkeley, CA
94598, USA.
8
Department of Geosciences, National Taiwan
University, Taipei 106, Taiwan.
9
Division of Earth and
Ecosystem Sciences, Desert Research Institute, Las Vegas, NV
89119, USA.
10
Department of Earth Sciences, University of
Western Ontario, London, ON N6A 5B7, Canada
11
Department
of Geological Sciences, Indiana University, Bloomington, IN
47405, USA.
12
Indiana Princeton Tennessee Astrobiology
Initiative (IPTAI), NASA Astrobiology Institute, Bloomington, IN
47405, USA.
13
Biological Sciences Division, Pacific Northwest
National Laboratory, Richland, WA 99352, USA.
14
Department
of Bioengineering, University of California, Berkeley, CA 94720,
USA.
15
Department of Geosciences, Princeton University,
Princeton, NJ 08544, USA.
*To whom correspondence should be addressed. E-mail:
DCChivian@lbl.gov
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