Cutting Edge: IL-12 Inversely Regulates T-bet and
Eomesodermin Expression during Pathogen-Induced
CD8?T Cell Differentiation1
Naofumi Takemoto,* Andrew M. Intlekofer,* John T. Northrup,* E. John Wherry,†and
Steven L. Reiner2*
Cytokines are critical determinants for specification of
sets. Little is known, however, about how cytokines regu-
late expression of T-bet and eomesodermin (Eomes) in
effector and memory CD8?T cells. We now report that
IL-12, a signature of cell-mediated immunity, represses
Eomes while positively regulating T-bet in effector CD8?
ter resolution of infection and abatement of IL-12 signal-
ing, Eomes expression rises whereas T-bet expression de-
clines in memory CD8?T cells. Eomes becomes
derepressed in effector cells by ablation of IL-12 signaling.
In the absence of IL-12, the dynamics of clonal expansion
reveal how a pathogen-associated signal, such as IL-12,
could act as a switch, regulating appropriate clonal
growth and decline while, in parallel, shaping a unique
pattern of fate-determining transcription factors. The
Journal of Immunology, 2006, 177: 7515–7519.
and Eomes appear to cooperate in regulating differentiation,
function, and homeostasis of cytotoxic cells (1–8). During the
immune response to viral infection, Eomes and T-bet expres-
sion are dynamically regulated (8). Eomes expression progres-
sively increases from the effector to memory phases, after clear-
ance of the pathogen. In contrast, T-bet expression is maximal
during acute infection and declines after resolution (8).
The signals that regulate induction and repression of T-bet
and Eomes during a CD8?T cell-mediated immune response
have not yet been defined. In CD4?T cells, a paradigm has
tors, including T-bet, can be heavily regulated by the cytokine
environment. T-bet expression is primarily regulated in a pos-
oth CD8?T cells and NK cells play a critical role in
host defense against infection with intracellular bacte-
ria and viruses. The T-box transcription factors T-bet
itive manner by IFN-? signaling in Th cells, whereas IL-12 sig-
naling acts in a selective or instructive manner to “lock-in” T-
bet expression in Th1 cells (2, 9, 10). In contrast, IL-12, a
signature of intracellular infection, promotes Th1 polarization
by repressing GATA-3, the critical transcription factor for Th2
differentiation (11, 12). IL-4 negatively regulates T-bet expres-
sion and Th1 differentiation while promoting GATA-3 induc-
tion and Th2 differentiation (2, 11, 12).
sion during the immune response to Listeria monocytogenes.
IL-12 repressed Eomes expression in Ag-specific CD8?T cells
during infection. In contrast, maximal induction of T-bet in
CD8?T cells required IL-12 signaling. We propose that IL-12
is a key signal determining the dominance of T-bet or Eomes
expression at discrete stages of the immune response.
Materials and Methods
Mice and cell culture
All animal work was in accordance with the guidelines of the University of
Pennsylvania (Philadelphia, PA). C57BL/6 and Il12a?/?(B6.129Cg-
Il12atm1Jm) mice were obtained from The Jackson Laboratory. P14 [B6.D2Cg-
phocytic choriomeningitis virus (LCMV)3-derived peptide GP33–41were used
for adoptive transfer. Tbx21?/?(B6.129Cg-Tbx21tm1.Yang) mice were gener-
ated by targeted deletion of exons 2–6, and were phenotypically identical to
published Tbx21?/?(B6.129S6-Tbx21tm1Glm) mice (3). Mice were infected
CFU. P14 cells and Db-GP33?CD8?T cells were sorted based on Thy1.1
clonal CD8?T cell stimulation using Ab ligation, and P14 T cell activation
using peptide and T cell-depleted splenocytes as APC were done as previously
described (8). FACS analyses, chromatin immunoprecipitation (ChIP) assay,
and real-time PCR were performed as previously described (8).
Results and Discussion
Limited induction of Eomes in the effector CD8?T cell response to L.
vivo, we first examined Eomes expression over the course of L.
monocytogenes infection. At 7 days after infection, effector
*Abramson Family Cancer Research Institute and Department of Medicine, University of
Received for publication August 21, 2006. Accepted for publication September 26, 2006.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants AI061699 and
AI42370 (to S.L.R.) and AI071309-01 (to E.J.W.), the Commonwealth Universal Re-
search Enhancement Program, Pennsylvania Department of Health (to E.J.W.), and the
2Address correspondence and reprint requests to Dr. Steven L. Reiner, Abramson Family
Cancer Research Institute, University of Pennsylvania, BRB II/III, Rm. 414, 421 Curie
Blvd., Philadelphia, PA 19104. E-mail address: email@example.com
3Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; ChIP,
chromatin immunoprecipitation; CNS, conserved non-coding sequence; LMgp33, rListe-
ria monocytogenes expressing GP33–41.
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
CD8?T cells did not show substantial Eomes induction rela-
tive to naive cells (Fig. 1A). Eomes expression did, however, in-
crease in Ag-specific memory cells (6 wk postinfection). T-bet
expression reached its maximal induction at the effector phase
and partially declined in the memory phase (Fig. 1A). The lim-
ited induction of Eomes during effector differentiation to L.
monocytogenes infection was unexpected, because Eomes is in-
duced in effector CD8?T cells during LCMV and vaccinia vi-
rus infection (8).
IL-12 reciprocally regulates Eomes and T-bet expression in vivo
One major difference between the immune responses to L.
monocytogenes and LCMV is the relative induction of and de-
pendence on IL-12. In response to intracellular bacterial infec-
tion, there is substantial induction of IL-12, which, in turn,
plays an essential role in host resistance (13, 14). In some viral
infections, such as LCMV, IL-12 induction is limited and is
dispensable for effective anti-viral immunity (15). We, there-
fore, wished to test whether robust IL-12 signaling during the
acute response to L. monocytogenes could account for the ab-
sence of Eomes induction in CD8?T cells.
We first examined Eomes mRNA level during L. monocyto-
prietary subunit of IL-12 (p35). Although IL-12 is generally es-
sential for host resistance to L. monocytogenes infection,
Il12a?/?mice can survive low-doses (up to 1000 CFU) of in-
fection (16). After clearance of a primary low-dose challenge,
Il12a?/?mice are fully immune to rechallenge with an other-
wise lethal dose of bacteria. This experimental system, thus, al-
lowed us to examine effector and memory CD8?T cell devel-
opment against Listeria in the absence of IL-12.
developed into effector and memory CD8?T cells in Il12a?/?
recipients exhibited elevated expression of Eomes compared
with those that developed in wild-type recipients (Fig. 1B). In
addition, low-dose infection caused detectable Eomes induc-
tion at the effector phase in wild-type recipients (Fig. 1B), con-
bacteria (Fig. 1A). The elevated expression of Eomes in an IL-
12-deficient background was also observed in endogenous Db-
GP33?CD8?T cells at the effector phase of infection (Fig.
1C). Because naive CD8?T cells from Il12a?/?mice did not
show higher Eomes expression compared with wild-type cells,
the relative increase in Eomes expression in Il12a?/?effector
infection. Maximal T-bet mRNA and protein induction was
diminished in the IL-12-deficient background, both in trans-
ferred P14 cells (Fig. 1B) and endogenous Db-GP33?CD8?T
cells (Fig. 1C). The relative difference in Eomes and T-bet ex-
pression between Il12a?/?and wild-type CD8?background
was more pronounced at the effector phase than the memory
during the acute phase of infection (17).
Further support that IL-12 negatively regulates Eomes in
sistently exhibited Eomes induction in effector P14 cells com-
pared with naive cells using low-dose (1000 CFU) infection
(Fig. 1B, left). In contrast, Eomes was not induced in wild-type
effector cells with 5000 CFU infection (Fig. 1A, left). This dis-
parity was verified by directly comparing effector cells trans-
ferred into recipients that received different doses from the
same culture. As anticipated (18), varying the bacterial dose re-
sulted in proportionate induction of IL-12, with low-dose re-
cipients exhibiting significantly reduced splenic IL-12p40
mRNA level at 24 h after infection (Fig. 1D). As noted previ-
ously (17), IL-12p35 mRNA was present constitutively in
vivo. A, P14 cells (Thy1.1?) transferred (1 ? 105throughout the study) into
wild-type mice (Thy1.2?/?) were sorted on days 7 and 42 after infection.
transferred into wild-type or Il12a?/?mice were sorted on days 7 and 64 after
infection. Eomes and T-bet mRNA levels were determined by real-time PCR.
C, Db-GP33?CD8?T cells from wild-type or Il12a?/?mice were sorted on
day 7 after infection and compared with sorted CD44lowCD8?(naive) T cells
from uninfected mice of the respective genotypes. Eomes and T-bet mRNA
levels were determined by real-time PCR, and T-bet protein levels were deter-
mined by intracellular staining. Mean fluorescence intensity of Db-GP33?ef-
fector cells is indicated. D, 24 h after infection of either 1000 or 5000 CFU
LMgp33, splenic IL-12p40 mRNA levels were analyzed. ?, Non-immune. E,
P14 cells transferred into wild-type mice were sorted on day 7 after infection
real-time PCR throughout represent mean ? SEM of triplicate determinations
of pooled specimens from within a single experiment, normalized to values for
hypoxanthine guanine phosphoribosyl transferase. All results are representative
of at least two separate experiments.
IL-12 reciprocally regulates T-bet and Eomes expression in
7516CUTTING EDGE: IL-12-MEDIATED REPRESSION OF EOMES IN CYTOTOXIC T CELLS
spleens from uninfected mice and its level did not change sig-
nificantly after either dose of infection (data not shown). Effec-
tor P14 cells (7 days postinfection) that developed in response
to low-dose bacteria (and lower IL-12p40 mRNA) exhibited
higher Eomes expression than those that developed in response
to high-dose infection (Fig. 1E). It is possible that expression of
expansion that ensues from higher doses of bacteria also con-
tribute to the modulation of Eomes levels.
IL-12 induces T-bet and represses Eomes in vitro
IL-12 also substantially reduced Eomes expression in CD8?T
IL-4 up-regulated Eomes expression in developing effector
CD8?T cells. These differences were accentuated when cells
polarizing conditions) compared with rIL-4 plus anti-IL-12 Ab
(Tc2-polarizing conditions). By contrast, T-bet expression,
both at the mRNA and protein levels, was positively influenced
by rIL-12 (Fig. 2A). The differential effects of IL-12 on Eomes
and T-bet expression were also observed in splenic NK cells
when directly examined ex vivo (data not shown). Together,
these results suggest that the expression of Eomes and T-bet are
inversely regulated by IL-12, in vitro and in vivo.
Histone acetylation often correlates with a transcriptionally
poised status of a gene (19). We, therefore, examined the his-
tone acetylation status of the Eomes locus by ChIP assay in
CD8?T cells activated under type 1 or type 2 cytokine condi-
tions. To detect potential regulatory regions, we searched for
evolutionarily conserved non-coding sequences (CNS), which
often correspond to functional cis-elements (20). Comparison
of 10 kb of upstream sequences from human and mouse Eomes
type 1 cytokine conditions were associated with increased acet-
ylation of histone H3 (Fig. 2B) and H4 (data not shown) at the
proximal Eomes promoter (CNS4-7).
IL-12-mediated repression of Eomes in the absence of T-bet
declined after elimination of bacteria, while Eomes induction
was observed most prominently after recovery from primary L.
monocytogenes infection (Fig. 1A). In addition, T-bet is thought
to play a role in enhancing the expression of IL-12R?2 in
CD4?T cells (2, 10). Together, these findings raise the possi-
bility that T-bet may contribute to repression of Eomes by reg-
ulating IL-12R?2 expression in CD8?T cells. In such a sce-
nario, IL-12 responsiveness, conferred by T-bet, might cause
cells exposed to IL-12 to limit their expression of Eomes.
wild-type mice (Ref. 21 and data not shown). We found that
IL-12R?2 expression was substantially but not fully abrogated
in Tbx21?/?effector CD8?T cells compared with wild-type
effector cells during infection (Fig. 3A). Similar reduction of
IL-12R?2 expression was also observed in Tbx21?/?CD8?T
cells activated in vitro (Fig. 3B).
cells stimulated with peptide/APC in the presence of described cytokines, or
under type 1 (Tc1) or type 2 (Tc2) conditions in vitro, were analyzed at day 5
intensity of wild-type cells is indicated. B, Top, Sequence alignment in VISTA
format as the percentage of identity (vertical axis, range 50–100%) between
mouse and human relative to position in the mouse sequence (horizontal axis).
Sequences with ?75% identity ?100 bp are regarded as CNS. A schematic
diagram of the mouse Eomes locus is aligned above the VISTA plot. Bottom,
Histone acetylation status of the Eomes locus in CD8?T cells developed under
Tc1- or Tc2-polarizing conditions for 6 days was examined by ChIP assay.
AcH3, acetylated histone H3.
IL-12 induces T-bet and represses Eomes in vitro. A, CD8?T
day 7 after LMgp33 infection. B, CD8?T cells developed under type 1 (Tc1)
conditions in vitro were sorted on day 5. IL-12R?2 and Eomes mRNA levels
were determined by real-time PCR.
7517The Journal of Immunology
Despite the apparent role for T-bet in enhancing IL-12R?2
be the sole mechanism underlying IL-12-mediated repression
expression of Eomes than wild-type cells (Fig. 3A), a finding
distinct from Il12a?/?naive cells. Following infection, how-
regulate Eomes expression (Fig. 3A). Likewise, type 1 condi-
tions caused substantial repression of Eomes in Tbx21?/?
CD8?T cells activated in vitro (Fig. 3B). IL-12-mediated re-
pression of Eomes, therefore, is not solely reliant on T-bet-me-
diated regulation of IL-12R?2. The observation that
than wild-type naive CD8?T cells may point to a compensa-
tory mechanism to offset T-bet deficiency.
Diminished expansion and contraction of CD8?T cells in the absence
Eomes is highly expressed in memory CD8?T cells and seems
to play a causal role in regulating their homeostasis (8). In ad-
dition, Eomes expression in effector cells arising in the absence
of IL-12 approximates that of normal memory cells (Fig. 1B).
We, therefore, wondered whether the enhanced induction of
Eomes in effector cells in the absence of IL-12 (Fig. 1, B and C)
would influence the subsequent development of memory cells.
T cells that developed in the absence of IL-12 exhibited
enhanced expression of IL-7R? (Fig. 4A), which has been
suggested to mark memory precursors during acute infection
When the dynamics of clonal expansion and contraction
were evaluated, however, we found that IL-12 was not simply a
negative regulator of memory formation, but that it dually pro-
moted expansion and contraction of the clonal burst. During
listeriosis, there was diminished expansion of effector cells in
IL-12-deficient recipients as judged by the total number of P14
T cells per spleen (Fig. 4B). After resolution of infection, how-
ever, there was diminished contraction in the absence of IL-12
as judged by the higher proportion of memory cells relative to
the peak of expansion (Fig. 4C). These results suggest that, in
the absence of IL-12 signaling, CD8?T cells seem to be less
prone to expand and less prone to contract.
At present, we are uncertain whether the observed effects of
IL-12 on expansion and contraction are due directly to IL-12
signaling or through another inflammatory mediator that is di-
onstrated that numerous inflammatory cytokines and effector
molecules during acute infection regulate the dynamics of
stead of IL-12 signaling, secretion of IFN-?, which is regulated
by IL-12, may play a critical downstream role in mediating
clonal expansion and contraction. Treatment with CpG oligo-
nucleotides, for example, reverses defects in clonal contraction
caused by antibiotic treatment, but in a manner that is strictly
mice also express elevated levels of IL-7R? (25), which further
suggest that the effects of IL-12 on expansion and contraction
may be acting through IFN-?.
T-bet and Eomes seem to act as functional homologues in
several respects (7, 8). Nonetheless, T-bet and Eomes exhibit
inverse patterns of expression in effector and memory CD8?T
cells, respectively. In addition, our preliminary analyses suggest
appear to be predominantly regulated by T-bet, while some
sis are regulated by Eomes (unpublished results). The apparent
differences in patterns of T-bet and Eomes expression in differ-
ent subsets (effector and memory) of CD8?T cells can now be
partially explained by the opposing role of IL-12 on their ex-
pression. This finding is reminiscent of the opposing role of
IL-12 in the balance of type 1 and type 2 subsets of CD4?ef-
fector cells. In parallel, IL-12 may regulate expansion and con-
traction of effector CD8?T cells, perhaps via its regulation of
IFN-?. Agonism and antagonism of the IL-12 signaling path-
way may, thus, provide novel strategies to boost or attenuate
CD8?T cell-mediated immune responses through parallel ef-
fects on transcription factor expression and on inflammatory
mediators that influence clonal dynamics.
members of the Reiner laboratory for helpful discussions, and Dr. H. Shen for
The authors have no financial conflict of interest.
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7518 CUTTING EDGE: IL-12-MEDIATED REPRESSION OF EOMES IN CYTOTOXIC T CELLS
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7519The Journal of Immunology