Constitutive activation of Wnt signaling favors generation of memory CD8 T cells.
ABSTRACT T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Ralpha and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized beta-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized beta-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.
- SourceAvailable from: Felix Schambach[show abstract] [hide abstract]
ABSTRACT: Immunity to intracellular pathogens requires dynamic balance between terminal differentiation of short-lived, cytotoxic effector CD8+ T cells and self-renewal of central-memory CD8+ T cells. We now show that T-bet represses transcription of IL-7Ralpha and drives differentiation of effector and effector-memory CD8+ T cells at the expense of central-memory cells. We also found T-bet to be overexpressed in CD8+ T cells that differentiated in the absence of CD4+ T cell help, a condition that is associated with defective central-memory formation. Finally, deletion of T-bet corrected the abnormal phenotypic and functional properties of "unhelped" memory CD8+ T cells. T-bet, thus, appears to function as a molecular switch between central- and effector-memory cell differentiation. Antagonism of T-bet may, therefore, represent a novel strategy to offset dysfunctional programming of memory CD8+ T cells.Journal of Experimental Medicine 10/2007; 204(9):2015-21. · 13.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: T cell receptor (TCR) alpha alleles undergo primary and secondary rearrangement in double-positive (DP) thymocytes. By analyzing TCRalpha rearrangement in orphan nuclear receptor RORgamma-deficient mice, in which the DP lifespan is shorter, and in Bcl-x(L)-transgenic mice, in which the DP lifespan is extended, we show that the progression of secondary V(alpha) to J(alpha) rearrangements is controlled by DP thymocyte survival. In addition, because Bcl-x(L) induces a bias towards 3' J(alpha) usage in peripheral T cells, we conclude that the programmed cell death of DP thymocytes is not simply a consequence of failed positive selection. Rather, it limits the progression of rearrangement along the J(alpha) locus and the opportunities for positive selection, thereby regulating the TCRalpha repertoire.Nature Immunology 06/2002; 3(5):469-76. · 26.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Two candidate genes for controlling thymocyte differentiation, T-cell factor-1 (Tcf-1) and lymphoid enhancer-binding factor (Lef-1), encode closely related DNA-binding HMG-box proteins. Their expression pattern is complex and largely overlapping during embryogenesis, yet restricted to lymphocytes postnatally. Here we generate two independent germline mutations in Tcf-1 and find that thymocyte development in (otherwise normal) mutant mice is blocked at the transition from the CD8+, immature single-positive to the CD4+/CD8+ double-positive stage. In contrast to wild-type mice, most of the immature single-positive cells in the mutants are not in the cell cycle and the number of immunocompetent T cells in peripheral lymphoid organs is reduced. We conclude that Tcf-1 controls an essential step in thymocyte differentiation.Nature 04/1995; 374(6517):70-4. · 38.60 Impact Factor
The Journal of Immunology
Constitutive Activation of Wnt Signaling Favors Generation
of Memory CD8 T Cells
Dong-Mei Zhao,*,1Shuyang Yu,*,1Xinyuan Zhou,* Jodie S. Haring,*,2Werner Held,†
Vladimir P. Badovinac,‡,xJohn T. Harty,*,xand Hai-Hui Xue*,x
known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell
activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Ra and CD62L, TCF-1 and
lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive
T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform
limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene
was coupled with ectopic expression of stabilized b-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced
ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared
bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia
virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45
and stabilized b-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4
T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of
the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity
upon second encounter with the same pathogen.The Journal of Immunology, 2010, 184: 1191–1199.
undergo massive clonal expansion, generating large numbers of
Ag-specific effector T cells equipped with cytokines and/or cy-
tolytic molecules to combat pathogens. The proliferative expan-
sion phase is followed by a rapid decline of Ag-specific T cells,
and such contraction of the effector T cells does not seem to be
correlated with clearance of the pathogens (4). A fraction of Ag-
specific T cells (∼5–10%) survive the contraction phase and form
a pool of memory T cells, which will respond with accelerated
expansion rate upon re-encounter with the same Ag. This immu-
rotective cellular immunity relies on Ag-specific CD4 and
CD8 T cells, which exist at very low frequencies in naive
hosts (1–3). Postinfection or immunization, naive T cells
nological memory is the basis for vaccination, and much effort has
been devoted to improve vaccine design through manipulating T
cell responses to maximize memory T cell formation (1, 5–7).
Three major classes of signals are known to be critical for T cell
activation and transition to memory T cells: signal 1 from Ag
stimulation of TCR, signal 2 derived from costimulatory mole-
cules such as CD28, and signal 3 from proinflammatory cytokines
including IL-12 and type I IFNs (1, 2). Recent studies demon-
strated that the effect of inflammatory cytokines such as IL-12 on
effector and memory CD8+T cells may be at least partly mediated
by differential expression of the T-box transcription factor, T-bet
(8, 9). It is not known whether additional signals such as mor-
phogenic Wnt signaling and its downstream transcriptional pro-
grams have a role in modulating T cell responses.
Wnt proteins are secreted, lipid-modified glycoproteins that
activate multiple signal transduction pathways to regulate a variety
and gene expression (10, 11). The canonical Wnt pathway trans-
duces signals via the intracellular mediator b-catenin. In the ab-
sence of interaction between Wnt and its Frizzled receptors,
b-catenin is kept at a low level by a multimolecular destruction
complex containing casein kinase I and glycogen synthase kinase
3. These two kinases sequentially phosphorylate a set of conserved
serine and threonine residues in the N terminus of b-catenin, and
the resulting phosphorylated footprint marks b-catenin for con-
stant degradation by the proteosome. Under this condition, the Wnt
effector transcription factors T cell factor-1 (TCF-1) and lymphoid
enhancer-binding factor 1 (LEF-1) are associated with Groucho/
transducin-like enhancer of split (TLE) corepressor proteins and
act as transcriptional repressors. When Wnt ligand binds to Friz-
zled receptors and coreceptors, glycogen synthase kinase 3 activity
is inhibited and the destruction complex is inactivated, resulting in
accumulation of b-catenin in the cytoplasm. Upon entering the
nucleus, b-catenin replaces Groucho/TLE, forms complexes with
*Department of Microbiology,
Graduate Program in Immunology, Carver College of Medicine, University of Iowa,
Iowa City, IA 52242; and†Ludwig Institute for Cancer Research, Lausanne Branch,
University of Lausanne, Epalinges, Switzerland
‡Department of Pathology, and
1D.-M.Z. and S.Y. contributed equally to this work.
2Current address: Core Biology Facility, North Dakota State University, Fargo, ND.
Received for publication April 14, 2009. Accepted for publication November 24,
This work was supported in part by internal funds to H.-H.X. from the Department of
Microbiology, University of Iowa, and in part by National Institutes of Health Grants
to H.-H.X. (AI077504 and HL095540), V.P.B. (AI083286), and J.T.H. (AI046653,
AI042767, AI050073, and AI059752).
Address correspondence and reprint requests to Dr. Hai-Hui Xue, 51 Newton Road,
BSB 3-710, Iowa City, IA 52246. E-mail address: firstname.lastname@example.org
The online version of this paper contains supplemental material.
Abbreviations used in this paper: bCat-Tg, b-catenin transgene; dTg, double trans-
genic; FLICA, fluorescent inhibitor of caspases; GABP, GA-binding protein; HSC,
hematopoietic stem cell; LCMV, lymphocytic choriomeningitis virus; LEF-1, lym-
phoid enhancer-binding factor 1; LLO, listeriolysin O; LM-OVA, L. monocytogenes
expressing OVA; LOD, limit of detection; p45-Tg, p45 transgene; TCF-1, T cell
factor-1; Tg, transgenic; TLE, transducin-like enhancer of split; VacV, vaccinia virus;
VacV-OVA, VacV expressing OVA; WT, wild-type.
TCF-1/LEF-1, and activates the transcription of Wnt target genes.
Although LEF-1 null mice did not display abnormalities in T cell
development, inactivation of TCF-1 resulted in incomplete blocks
at multiple early T cell developmental stages (12). TCF-1 and
LEF-1 double deficiency completely arrested T cell development
at the immature single-positive thymocyte stage (13), indicating
functional redundancy between TCF-1 and LEF-1 and a relatively
dominant role of TCF-1 during T cell development. Ablation of
b-catenin was initially found to perturb b selection during thy-
mocyte development (14); however, recent studies indicate that
both b-catenin and g-catenin are dispensable for thymopoiesis
(15–17). Nevertheless, deletion of phosphorylation sites in the N
terminus of b-catenin led to accumulation and constitutive acti-
vation of the canonical Wnt pathway, and, when driven by a T cell
specific promoter, the stabilized b-catenin transgene enhances
thymocyte production by extending thymocyte survival (18, 19).
Despite its critical roles during T cell development, TCF-1
appeared to be dispensable for T cell proliferation and cytotoxic
function upon activation in vitro (20), albeit the canonical Wnt
signaling has been shown to be operative in naive or activated
T cells (17). A recent study demonstrated that overexpression of
stabilized b-catenin can increase the survival of CD4+CD25+
regulatory T cells and induce an anergic phenotype in CD4+
CD252effector T cells (21). In the current study, we investigated
the effect of constitutive activation of Wnt signaling on CD4 and
CD8 T cell responses to infection.
Materials and Methods
Mice and infectious agents
The b-catenin transgenic mice (bCat-Tg) and p45 transgenic mice (p45-
Tg) were as described (19, 22). p45-Tg mice were backcrossed .10
times to C57BL/6 background, and bCat-Tg mice were backcrossed to
C57BL/6 for four generations before being crossed to p45-Tg. bCat and
p45 single transgenic (Tg) mice were used as breeders, and littermates
of all four genotypes (i.e., single Tg mice, double Tg [dTg], and no
transgene controls) were used in this study for parallel comparisons.
Listeria monocytogenes-, lymphocytic choriomeningitis virus (LCMV)-,
and vaccinia virus (VacV)-infected mice were housed in accordance with
institutional biosafety regulations. All animal experiments followed
protocols approved by the Institutional Animal Care and Use Committee
at the University of Iowa.
L. monocytogenes expressing OVA (LM-OVA) was a gift from Dr. Hao
Shen (University of Pennsylvania, Philadelphia, PA) and Dr. Leo Le-
francois (University of Connecticut, Storrs, CT) (23). Attenuated LM-OVA
was created by introducing an in-frame deletion in the actA gene as de-
scribed (referred to as actA2LM-OVA) (24). Naive Tg mice and littermate
controls were first i.v. infected with 5 3 106CFU actA2LM-OVA. Some of
the mice were challenged with 7 3 105CFU virulent LM-OVA via i.v.
injection on day 45 after the primary infection. Bacteria were grown and
quantified as previously described (25). The numbers of LM-OVA (CFUs)
present in the livers and spleens were determined on day 3 after the sec-
ondary infection as previously described (25). In experiments evaluating
CD8 T cell responses to viral infection, mice were infected via i.p. in-
jection with 3 3 106PFU of VacV expressing OVA (VacV-OVA) (26) or
2 3 105PFU of LCMV-Armstrong (27).
Detection of Ag-specific T cells
Splenocytes were harvested on indicated days after primary or secondary
infection and stimulated with the following peptides for 5 to 6 h in the
presence of brefeldin A (BD Biosciences, San Jose, CA): 5 mM listeriolysin
O (LLO)190–201peptide (NEKYAQAYPNVS) for CD4 responses after LM-
OVA infection, 200 nM of OVA257–264peptide (SIINFEKL) for CD8 re-
sponses after LM-OVA or VacV-OVA infection, 200 nM each of GP33–41
(KAVYNFATC), NP396–404(FQPQNGQLI), NP205–212(YTVKYPNL), or
GP276–286 (SGVENPGGYCL) peptide for CD8 responses after LCMV
infection. The stimulated cells were then surfaced stained, fixed and per-
meabilized, and intracellularly stained for IFN-g and, in some cases, IL-2
following standard protocols (28). For detection of CD62L, cells were
pretreated with 0.1 mM TNF-a protease inhibitor 2 (Peptides International,
Louisville, KY) for 30 min before peptide stimulation to prevent cleavage
of surface CD62L (29). All fluorochrome-conjugated Abs were from either
eBioscience (San Diego, CA) or BD Biosciences. The stained cells were
analyzed on an FACSCalibur flow cytometer (BD Biosciences) followed
by data analysis using FlowJo software (Tree Star, Ashland, OR). For
calculation of the absolute numbers of Ag-specific CD4 or CD8 T cells per
spleen, total numbers of splenocytes were multiplied with the frequency of
CD4+or CD8+Thy1.2+IFN-g+cells after stimulation with the specific
peptide. The number of cells producing IFN-g in the absence of peptide
was subtracted. It should be noted that b-catenin transgene was coupled
with a GFP expression indicator via an internal ribosome entry site (19). In
the bCat-Tg mice, GFP was highly expressed in developing thymocytes
but substantially attenuated in mature CD4 or CD8 T cells (19). We have
confirmed this finding and that the GFP fluorescence channel can be used
to detect highly expressed Thy1 marker or TCR Vb subtypes on total or
Ag-specific CD8 T cells (data not shown and Supplemental Fig. 1).
CD8 T cells
phase, the mice were i.p. injected with 1 mg BrdU on day 7 postinfection
with actA2LM-OVA and given 0.8 mg/ml BrdU in drinking water for
additional 2 d. Splenocytes were isolated, incubated with OVA257–264
peptide, and surface-stained as above, followed by fixation and per-
meabilization procedures as recommended in the BrdU Flow Kit (BD
Biosciences). Anti-BrdU and anti–IFN-g Abs were used simultaneously
for intracellular staining to detect BrdU uptake in IFN-g+CD8 T cells. For
detection of activated caspase-3 and -7 in Ag-specific T cells, splenocytes
were peptide-stimulated and surface-stained as above, and then incubated
with the fluorescent inhibitor of caspases reagent at 37˚C for 60 min as
recommended in the Vybrant FAM caspase-3 and -7 assay kit (Invitrogen,
Carlsbad, CA). The cells were then washed and used in intracellular
staining for IFN-g.
Isolation of Ag-specific T cells and quantitative RT-PCR
Naive CD8 T cells were purified from uninfected OT-I Tg micevia negative
selection to .95% purity (StemCell Technologies, Vancouver, British
Columbia, Canada). To obtain Ag-specific effector and memory T cells,
naive C57BL/6 (Thy1.2+) mice were adoptively transferred with 500 CD8
T cells from OT-I TCR Tg mice (Thy1.1+), and 1 d later infected with
actA2LM-OVA. Ag-specific T cells were purified on day 5 (as effector
T cells) and day 135 (as memory T cells) postinfection by labeling sple-
nocytes with PE-conjugated Thy1.1 Ab followed by anti–PE-coated
magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manu-
facturer’s directions. Labeled Thy1.1+OT-1 Tg T cells were then posi-
tively enriched to .90% purity using the autoMACS (Miltenyi Biotec).
Total RNA was then extracted and reverse-transcribed as previously de-
scribed (30). Gene-specific probes and primer sets were from the Taqman
Gene Expression Assay system (Applied Biosystems, Foster City, CA),
and quantitative PCR was performed on an ABI 7300 Real Time PCR
System (Applied Biosystems). b-actin was used to normalize the expres-
sion of other genes of interest. For each individual gene, its relative ex-
pression in naive T cells was arbitrarily set to one, and its expression
changes in effector and memory T cells were calculated as fold repression
Dynamic gene expression changes of TCF-1 and LEF-1 during
T cell responses
To explore if the Wnt signaling pathway has a role in modulating
levels in naive, effector,and memoryT cells.Effector and memory
T cells were obtained 5 and 135 d postinfection from mice that had
were then infected with an attenuated strain of L. monocytogenes
gene expression changes during T cell responses, being expressed
higher levels in memory T cells (32, 33). Consistent with previous
observations, IL-7Ra transcripts were considerably lower in day 5
effector T cells than those in naive T cells, whereas day 135
memory T cells showed higher IL-7Ra expression than naive
T cells (Fig. 1). We previously demonstrated that GA-binding
1192 ACTIVE Wnt PATHWAY FAVORS MEMORY CD8 T CELL FORMATION
protein (GABP) is required for transcriptional activation of the IL-
7Ra gene (34); however, the expression of GABPa (the DNA
binding subunit) and GABPb1 (the transactivation subunit) were
relatively stable during T cell responses (Fig. 1). In contrast, both
TCF-1 and LEF-1 were dramatically downregulated in day 5 ef-
fector T cells compared with naive T cells, but were partially re-
stored in day 135 memory T cells. The expression levels of both
transcription factors in memory T cells were still lower than naive
T cells, consistent with previous findings that Ag-experienced
human CD8 T cells expressed less TCF-1 and LEF-1 than naive
cells (35). The corepressor Groucho/TLE family consists of four
proteins of similar molecular weight and structure in mammals
(36), and another corepressor C-terminal binding protein 1 has
been shown to interact with TCF family transcription factors (37).
Interestingly, all four TLE and C-terminal binding protein 1 tran-
scripts were decreased in effector T cells and partially upregulated
LEF-1–mediated activation and repression of Wnt downstream
genes are both attenuated in effectors and restored in memory
T cells, albeit to a lesser extent compared with naive T cells.
Constitutively active Wnt signaling limits CD8 T cell
expansion and contraction but favors formation of memory
CD8 T cells
7Ra are suggestive of their potential functional importance during
T cell responses. It has been shown that enforced expression of IL-
7Ra alone was not sufficient to protect effector T cells from
TCF-1 extended thymocyte survival (19, 22), we hypothesized that
to effector T cells and thus result in a larger memory T cell pool. To
test this, we used two lines of Tg mice, expressing a stabilized
b-catenin and a p45 isoform of TCF-1, respectively (19, 22). The
critical phosphorylation sites, preventing proteosome-mediated
degradation. The stabilized b-catenin transgene (referred as bCat-
Tg) is driven by the CD4 promoter, which allows transgene ex-
pression in both CD4 and CD8 T cells (19, 39). Among multiple
TCF-1 isoforms, the p45 TCF-1 protein contains an N-terminal
thymic cellularity and thymocyte development in TCF-1–deficient
mice (22). The p45 transgene was driven by an H-2Kbpromoter
coupled with IgH enhancer, resulting in persistent expression in
lymphocytes including T lineage cells (referred to as p45-Tg) (22).
of both transgenes may help ensure constitutively active Wnt sig-
naling in effector T cells in which the endogenous TCF-1 proteins
Vb subtypes on splenic CD8 and CD4 T cells and observed no
not shown for CD4 T cells). It was shown previously that the ex-
pression of constitutive active b-catenin extended thymocyte sur-
To determine if bCat-Tg functionally altered the repertoire of Ag-
specific T cells, we measured their functional avidity, which would
likely be affected by skewed TCR-a subtypes (41, 42). We infected
the attenuated actA2LM-OVA (28). On day 7 postinfection, we
OVA257–264peptide, and measured the fraction of cells producing
IFN-g. As shown in Supplemental Fig. 1C, no apparent differences
in T cell functional avidity were observed among the various Tg
strains, suggesting that the TCR repertoire was not detectably
skewed by the bCat-Tg and/or p45-Tg.
To measure T cell responses in vivo, we infected WT, bCat-Tg,
the OVA257–264-specific CD8 T cell responses using intracellular
cytokine staining for peptide-stimulated IFN-g on days 5, 7, 9, 14,
and 42 postinfection. During the clonal expansion phase (5–9
d postinfection), all groups of mice showed expansion of OVA-
specificCD8Tcells,asmanifested byIFN-g productionuponOVA
mouse strains (Fig. 2A, 2G). Effector CD8 T cells in each group
showed similarly diminished expression of CD62L and IL-7Ra,
similar CD27highsubsets and IL-2 production, except that the fre-
quency of IL-2+IFN-g+CD8 T cells in bCat-Tg mice appeared
smaller (Fig. 2A). The T cell expansion, in terms of frequency and
by ectopic expression of stabilized b-catenin alone but was limited
by the forced expression of p45 TCF-1 isoform, especially in the
presence of both transgenes (Fig. 2B). The limited T cell expansion
was also reflected in decreased total CD8 T cell frequency in sple-
nocytes (Fig. 2C). These results suggest that downregulation of
TCF-1 might be necessary for optimal T cell expansion.
During the contraction phase (day 14 postinfection), ∼90% of
the Ag-specific CD8 T cells was eliminated in WT mice as ex-
pected. When normalized to their respective peak responses,
a similar contraction was seen in bCat-Tg, whereas a higher
percentage of cells survived in p45-Tg (25%) and dTg (60%) mice
infection) OT-I CD8 T cells were separated as detailed inMaterials and Methods. Total RNAwas extractedand analyzed for expression ofindicated transcripts using
graphs. The data are means 6SEM of triplicate measurements of a total of three samples from two independent experiments. Ctbp1, C terminal binding protein 1.
Dynamic regulation of Wnt pathway-related genes during CD8 T cell responses. Naive, effector (day 5 postinfection), and memory (day 135 post-
The Journal of Immunology 1193
(Fig. 2G, 2H). When the mice were examined at the memory
phase (day 42 postinfection), dTg mice had higher frequencies and
increased numbers of Ag-specific CD8 cells compared with WTor
single Tg mice (Fig. 2D, 2E, 2G), despite a similar CD8 frequency
in splenocytes among all Tg and control strains (Fig. 2F). When
normalized to the peak response, more Ag-specific CD8 T cells
survived and became memory cells in dTg mice (29% in dTg
versus 3–6% in WT and single Tg mice; Fig. 2H). The memory
CD8 T cells in all of the mouse strains examined exhibited similar
CD27highsubsets and similar upregulation of CD62L and IL-7Ra
(Fig. 2D). These observations collectively indicate that constitu-
tive activation of Wnt signaling in T cells favors the generation of
memory CD8 T cells.
Heightened secondary expansion and bacteria clearance in
double Tg mice
Effective immunological memory is characterized by an acceler-
ated expansion of memory cells upon re-encounter of the same Ag.
Interestingly, the Ag-specific memory CD8 T cells showed in-
creased capacity to produce IL-2 upon in vitro restimulation with
OVA peptide in bCat-Tg or p45-Tg mice (Figs. 2D, 3A), and IL-2+
IFN-g+CD8 T cells in dTg mice were significantly elevated in
absolute numbers (Fig. 3B). To further determine if the increased
number and IL-2 production by dTg memory CD8 T cells confers
functional advantages, we challenged the immune mice (45 d after
the primary infection) with virulent LM-OVA. After the secondary
infection, OVA-specific CD8 T cells in all mouse strains expanded
WT, bCat-Tg, p45-Tg, and dTg mice were infected
with actA2LM-OVA and the CD8 T cell response to
OVA257–264was monitored in the spleen at day 7 (A)
and day 42 (D) postinfection using intracellular cy-
tokine staining for IFN-g. The percentages of IFN-g+
cells were further analyzed for IL-2+,
CD62Lhigh, IL-7Ra+, or CD27highsubsets, and their
percentages are shown. Staining with isotype control
for CD62L and IL-7Ra is displayed as a dotted line in
histograms showing IL-7Ra staining. Data are repre-
three mice examined in each experiment. Frequency
in splenocytes (C) on day 7 postinfection. Frequency
in splenocytes (F) on day 42 postinfection. Fold
changes of mean values for each Tg strain versus WT
specific CD8 T cell responses shown as total numbers
of the two independent experiments with similar re-
sults (n = 3). H, Survival rate of OVA-specific CD8
T cells at contraction and memory phases. The mean
value of OVA-specific CD8 T cell numbers at days 14
and 42 (as in G) was normalized to respective peak
response on day 7. The percentage of survived cells is
group of Tg mice versus WT controls.
Constitutive activation of Wnt signal-
1194 ACTIVE Wnt PATHWAY FAVORS MEMORY CD8 T CELL FORMATION
rapidly, and those in dTg mice were detected at higher frequencies
and absolute numbers (Fig. 3C, 3D) as compared with WT con-
trols (1.5- and 2.4-fold increase in cell number in dTg mice
compared with controls at days 3 and 5, respectively), albeit the
increase did not reach a statistical significance. It is noteworthy
that the secondary expansion of Ag-specific CD8 T cells was not
limited by the enforced expression of the p45 TCF-1 protein, which
is different from the primary CD8 response. The contraction fol-
lowing the secondary CD8 response was more moderate as com-
pared with that after the primary response (1), and dTg mice
over WT mice (Fig. 3D). Significantly more secondary memory
CD8 T cells were detected on day 42 after secondary infection in
dTg (3.6-fold increase over WT littermates; p , 0.05) (Fig. 3D).
Thus, in the presence of constitutively active Wnt signaling, the
increased primary memory CD8 T cells, when rechallenged, gave
rise to larger numbers of secondary effector and memory T cells.
An important feature of memory T cells is to provide protection
upon encounter with the same pathogen. We next determined
protection from virulent LM-OVA by measuring bacterial numbers
(CFUs) in livers and spleens on day 3 after rechallenging naive or
immunized mice. Whereas the bacteria expanded drastically in
controlled the infection, as many fewer CFUs were seen in WT,
single Tg, and dTg mice, especially in the spleen (Fig. 3E, 3F).
Interestingly, virulent LM-OVA was almost completely cleared in
the liver of dTg mice, which is likely accounted for by a larger
expansion of Ag-specific CD8 T cells observed in these mice.
Constitutively active Wnt signaling suppresses caspase-3/7
Our observations above indicate that forced expression of the p45
TCF-1 isoform and a constitutively active b-catenin limited the
contraction of Ag-specific CD8 effectors and resulted in increased
to a more effective reduction in bacterial burden. The reduced
contraction of effector T cells in dTg mice may be a result of in-
creased proliferation and/or survival of Ag-specific T cells. To
distinguish these possibilities, we i.p. injected BrdU into dTg and
WT littermate controls on day 7 postinfection with actA2LM-OVA
and measured BrdU incorporation in OVA-specific CD8 T cells on
day 9, the early stage ofcontraction phase. As shown in Fig. 4A,the
WT and dTg mice. We next determined the expression levels of
prosurvival Bcl-2 family members in OVA-specific T cells. Con-
sistent with previous findings that Bcl-2 expression is down-
regulated in Ag-specific effector T cells in response to LCMV
infection (43), the OVA-specific T cells in both WT and dTg mice
expressed similarly low levels of Bcl-2 (Supplemental Fig. 2A). In
thymocytes, activation of b-catenin upregulates Bcl-XLand in-
creases their survival (19, 22). We purified OVA-specific CD8
T cells by cell sorting of MHC-I/SIINFEKL tetramer-positive cells
from both WTand dTg mice and by quantitativeRT-PCR measured
transcript levels of Bcl-XLand Mcl-1, other prosurvival Bcl-2
family members. Both genes showed a minimal increase in ex-
pression (,1.5-fold on average) in dTg OVA-specific T cells
compared with those in WT littermates (Supplemental Fig. 2B),
consistent withpreviousobservations thatbCat-Tg didnotincrease
Bcl-XLexpression in mature T cells (19).
Apoptotic signals converge on activation of effector caspases,
via either intrinsic (such as withdrawal of growth factors) or ex-
trinsic pathways (such as Fas engagement by Fas ligand) (44, 45).
To further discern the apoptotic status of Ag-specific CD8 T cells
during the contraction phase, we detected activated caspase-3 and
-7 using a fluorescent inhibitor of caspases (FLICA) methodology,
presence of constitutively active Wnt signaling
manifested enhanced functionality. Frequency (A)
and numbers (B) of IL-2 producing OVA-specific
memory CD8 T cells. The numbers were calculated
from the frequency of IL-2+IFN-g+cells (A) and the
absolute number of OVA-specific CD8 T cells on
day 42 postinfection as in Fig. 2G. C and D, Sec-
ondary Ag-specific CD8 T cell responses. Mice of
indicated genotypes were first immunized with ac-
tA2LM-OVA as in Fig. 2, and detection of OVA-
specific CD8 T cells on day 42 postimmunization
was confirmed in periphery blood leukocytes by
intracellular staining for IFN-g (data not shown).
The immunized mice were then infected with viru-
lent LM-OVA, and CD8 responses to OVA were
determined. OVA257–264-specific CD8 T cells at day
as Thy1.2+IFN-g+CD8 cells, with the percentages
shown in C. Total numbers of Ag-specific CD8
T cells per spleen in each group are shown in D as
means 6 SEM. E and F, Clearance of secondary
bacterialinfectionby primaryCD8 memoryT cells.
Naive or immunized mice were infected with viru-
lent LM-OVA, and 3 d later, livers and spleens were
harvested and CFUs were determined. Data are re-
ported as CFU numbers per gram of liver (E) or per
spleen (F). LOD, limit of detection. Each symbol
represents one mouse. All data are from one of two
independent experiments with similar results. pp ,
0.05; ppp , 0.01 by Student t test for each group of
Tg mice versus WT controls.
The Journal of Immunology1195
in which the active center of activated caspases is bound by the
fluoromethyl ketone moiety of fluorochrome-labeled FLICA (46).
As shown in Fig. 4B, a smaller portion of OVA-specific CD8 ef-
fector cells from dTg mice had active caspase-3/7 compared with
those from WT controls. Collectively, the reduced contraction of
effector T cells in the presence of active Wnt signaling is at least
partly explained by reduced caspase activation and hence allevi-
ated apoptosis, which is probably independent of Bcl-2 and other
prosurvival Bcl-2 family members.
Constitutively active Wnt signaling favors memory CD8 T cell
formation in response to viral infection
We next investigated if constitutively active Wnt signaling can
promote memory CD8 T cell formation in response to other forms
of infectious agents in addition to bacterial infection. VacV and
LCMV are known to elicit cellular immune responses mainly
mediated by CD8 T cells. We first infected WTand dTg mice with
VacV-OVA (26, 47). On day 42 postinfection, we measured OVA-
specific memory CD8 T cells in the spleen by intracellular
staining for IFN-g after the OVA peptide stimulation. The OVA-
specific memory CD8 T cells in dTg mice exhibited on average
a ∼2.5-fold increase compared with WT littermate controls (Fig.
5A). We next infected the mice with LCMV-Armstrong and de-
termined memory phase CD8 responses in the spleen to multiple
epitopes including the immunodominant epitopes located in gly-
coproteins GP33–41and GP276–286, nucleoprotein NP396–404, and
subdominant epitopes NP205–212 (48). By measuring peptide-
stimulated IFN-g production, dTg mice were found to have more
memory CD8 T cells to GP33–41, NP396–404, and NP205–212epi-
topes than the WT control mice (Fig. 5B), and all these increases
are statistically significant. On the other hand, GP276–286-specific
memory CD8 T cells in dTg mice showed only a small increase
(Fig. 5B). Taken together, these results suggested that activation of
Wnt signaling positively impacted the generation of memory CD8
T cells in response to both bacterial and viral infections.
Constitutively active Wnt signaling favors memory CD4 T cell
formation but does not improve secondary expansion
affected CD4 T cell responses by detecting LLO190–201peptide-
specific CD4 cells at various stages after actA2LM-OVA infection.
The peak expansion of LLO190–201-specific effector CD4 T cells in
of the Ag-specific CD4 cells (Fig. 6B). On the other hand, the fre-
quency of Ag-specific CD4 T cells in p45-Tg and dTg mice was
similar to the WT controls (Fig. 6B). The frequency of CD4 T cells
in the spleens was also similar among WT, bCat-Tg, and p45-Tg
mice, but slightly decreased in dTg mice (Fig. 6C). The absolute
numbers of Ag-specific CD4 T cells were similar among all Tg
strains and WT littermate controls on day 5 postinfection; however,
postinfection (Fig. 6G). Despite the change in kinetics, Ag-specific
CD4 T cells in all mouse strains showed similar capacity of IL-2
production, similar downregulation of CD62L and IL-7Ra, and
comparable portions of CD27highsubsets (Fig. 6A).
Although p45-Tg and dTg mice exhibited an early decline in the
201-specific CD4 T cells in WTand single Tg mice further declined
by day 42 postinfection, those in dTg mice persisted at a higher
frequency and total number (Fig. 6D, 6E, 6G). The splenic CD4
6F). In contrast to their numbers, memory CD4 T cells in all Tg
strains and WT controls exhibited similar re-expression of CD62L
and IL-7Ra on the cell surface. Unlike memory CD8 T cells, dTg
memory CD4 T cells from dTg mice did not show an improved
specific effector CD8 T cells. A, Proliferation rate of effector CD8 T cells
at early contraction phase. WT and dTg mice were infected with actA2
LM-OVA and given BrdU on day 7 via i.p. injection and in drinking water
for 2 d. Percentages of BrdU+cells in IFN-g+CD8 effectors are shown. B,
Activation of caspase-3/7 in effector CD8 T cells at early contraction
phase. Splenocytes were isolated from WT and dTg mice on day 9 post-
infection, and activated caspase-3/7 were detected using the FLICA
methodology. Percentages of cells having activated caspase-3/7 are shown.
Representative flow cytometric profiles are shown on upper panels, and
accumulative data from 2 independent experiments are shown in lower
panels. ppp , 0.01 by Student t test.
Detection of BrdU uptake and caspase activation in Ag-
CD8 T cells after VacV-OVA infection. WTand dTg micewere i.p. infected
with VacV-OVA, and the OVA-specific CD8 T cells on day 42 postinfection
were determinedby intracellular stainingfor IFN-g. B, Ag-specificmemory
epitopes GP33–41, NP396–404, and GP276–286 and subdominant epitopes
NP205–212were determined by ICS for IFN-g. Fold changes of mean values
were shown. pp , 0.05; ppp , 0.01 by Student t test.
Constitutive activation of Wnt signaling enhances memory
1196 ACTIVE Wnt PATHWAY FAVORS MEMORY CD8 T CELL FORMATION
ability to produce IL-2 upon peptide stimulation (Fig. 6D). These
data suggest that enforced expression of p45 and a stabilized
b-catenin enhances the generation of memory CD4 T cells, which
does not seem to be accompanied by obvious functional changes.
after rechallenge with virulent LM-OVA. The Ag-specific CD4
T cells in all mouse strains expanded rapidly (Fig. 6H and Supple-
mental Fig. 3). Although there were more primary memory CD4
ceiling level as seen in WTand single Tg mice (Fig. 6H). The LLO-
specific CD4 cells contracted similarly and stabilized on a similar
level as secondary memory CD4 cells in WTand dTg mice. On the
other hand, if any, fewer LLO-specific CD4 cells were detected on
days 14 and 42 after secondary infection in bCat-Tg and p45-Tg
mice (Fig. 6H and Supplemental Fig. 3). Collectively, these data
suggest that activation of the Wnt pathway does not improve the
secondary response of CD4 cells, even though more memory CD4
cells were generated during the initial immunization.
The canonical Wnt pathway, with TCF-1 and LEF-1 as the effector
transcription factors, is known to play critical roles in T cell de-
velopment, but its function in peripheral T cells remains relatively
unknown. In this study, we report that constitutive activation of the
Wnt pathway, by the enforced expression of a stabilized b-catenin
together with a b-catenin receptive p45 TCF-1 isoform, resulted in
accompanied by an increased ability to produce IL-2. Moreover,
upon rechallenge, the enlarged memory CD8 T cell pool expanded
cleared faster. Future studies should determine if primary memory
dTg CD8 cells have enhanced per cell protective capacity. Never-
theless, our results indicated that constitutive activation of the ca-
nonical Wnt pathway favors formation of memory CD8 T cells,
leading to a “win by numbers” after re-encountering the same
pathogen. On the other hand, although CD4 memory T cells were
also increased, enforced Wnt signaling did not improve the
creases primary but not secondary memory CD4
T cells. Mice were infected as in Fig. 2, and the CD4
responses to LLO190–201were determined on various
days postinfection. A and D, Detection and character-
ization of LLO1902201-specific CD4 T cells at day 7 (A)
and day 42 (D) postinfection. The percentages of
Thy1.2+IFN-g+CD4 cells are shown in the contour
plots in the absence (shown for A) or presence of LLO
peptide stimulation. IFN-g+CD4 cells were further
analyzed for IL-2+, CD62Lhigh, IL-7Ra+, or CD27high
subsets in memory CD4 cells, and their percentages are
shown. Staining with isotype control for CD62L and
IL-7Ra is displayed as a dotted line in histograms
showing IL-7Ra staining. Data are representative of
two independent experiments with at least three mice
examined in each experiment. Frequency of IFN-g+
cells in CD4 (B) and frequency of CD4 cells (C) in
splenocytes on day 7 postinfection. Frequency of IFN-
g+cells in CD4 (E) and frequency of CD4 cells (F) in
splenocytes on day 42 postinfection. Fold changes of
mean values for each Tg strain versus WT mice are
shown for B, C, E, and F. G, Kinetics of LLO-specific
CD4 T cell responses are shown as total numbers at
indicated time points. H, Secondary Ag-specific CD4 T
cell responses. Mice were first infected with actA2LM-
OVA and 45 d later rechallenged with virulent LM-
OVA as in Fig. 3C. CD4 responses to LLO190–201were
determined on days 3, 5, 14, and 42 after the secondary
infection. Total numbers of Ag-specific CD4 T cells
per spleen in each group are shown as means 6 SEM.
Data are representative of two independent experi-
ments with at least three mice examined in each ex-
periment. pp , 0.05; ppp , 0.01 by Student t test for
each group of Tg mice versus WT controls.
Constitutively active Wnt signaling in-
The Journal of Immunology1197
secondary expansion of Ag-specific CD4 cells and their IL-2 pro-
and CD8 responses have been noted. Forexamples,CD4 expansion
upon activation is less pronounced than that of CD8 cells, and for-
mation of memory CD4 T cells requires prolonged Ag contact (3,
49, 50). Our observations thus suggest a specific role of the ca-
nonical Wnt signaling pathway in promoting memory CD8 T cell
formation and enhancing their functionality.
One implication of our study is the potential use of Wnt ligands
as adjuvants in vaccination to enhance memory CD8 T responses.
Current strategies to improvevaccination efficiency include the use
of epitopes with enhanced affinity of binding to the TCR and the
use of costimulatory molecules and of cytokines as adjuvants (5,
51). The addition of noncytokine factors, such as Wnt ligands,
may further improve the T cell responses. We found that p45
overexpression limited initial CD8 T cell expansion; however,
when coupled with stabilized b-catenin expression, the contrac-
tion of Ag-specific CD8 T cells was also reduced, resulting in
increased numbers of memory CD8 cells. Our observations are in
line with a recent finding that in vitro treatment of CD8 T cells
with pharmacological inhibitors of glycogen synthase kinase 3,
hence stabilizing b-catenin and activating Wnt pathway, blocked
their differentiation to effector T cells but promoted them to adopt
a CD8 memory stem cell phenotype (52). There are at least 19
known Wnt proteins in mammals and 10 known Fzds receptors for
Wnt ligands (10, 11). Using reporter assays, it has been shown that
Wnt signaling is active in both naive and activated T cells (17).
Future studies need to be directed toward identification of Wnt
ligand and Fzd receptor pairs that are critical in mature T cell
function for potential adjuvant applications. It should be noted that
the expression of stabilized b-catenin alone was not sufficient to
improve CD8 T cell memory. This may be explained by the
downregulation of TCF-1 and LEF-1 in effector T cells, which
may render them unresponsive to activation of Wnt signaling. We
previously demonstrated that downregulation of IL-7Ra in ef-
fector T cells was mediated by the PI3K and Akt pathway and that
inhibition of the PI3K/Akt pathway in activated T cells allowed
retention of higher IL-7Ra expression (53). Elucidation of the
signaling pathway(s) leading to TCF-1/LEF-1 downregulation and
intervention of this pathway may ultimately help maximize the
beneficial effects of active Wnt signaling.
Activation of the canonical Wnt signaling has diverse effects on
hematopoietic cells, and the effects are dependent on the cell
context and the way Wnt signals are delivered. For example, pu-
rified Wnt ligands can expand hematopoietic stem cells (HSCs)
in vitro and enhance their self-renewal and reconstitution activities
(54, 55); however, constitutive or induced expression of stabilized
b-catenin in mouse HSCs had adverse effects on HSC mainte-
nance and blocked multilineage differentiation (56, 57). The sta-
bilized bCat-Tg used in our study was previously shown to extend
thymocyte survival (19). In agreement with this observation, we
found that coupled with p45 expression, stabilized b-catenin
partly protected effector CD8 cells from massive apoptosis during
the contraction phase. The reduced apoptosis/contraction in dTg
mice can be at least in part explained by suppression of caspase
activation. In contrast to upregulation of Bcl-XL by stabilized
b-catenin in thymocytes, we did not find substantial upregulation
of prosurvival Bcl-2 family members including Bcl-2, Bcl-XL, and
Mcl-1, which is suggestive of differential regulatory roles of Wnt
pathway in developing and mature T cells. A recent study de-
scribed the induction of anergy in nonregulatory T cells (CD4+
CD252) following retroviral transduction with a stable b-catenin
(21). We observed a reduced expansion of Ag-specific CD4 and
CD8 cells in the presence of the p45 transgene alone or in com-
bination with the stabilized bCat-Tg during the primary responses.
We do not believe that these cells are anergic because memory
cells derived from these cells expanded robustly and cleared
bacterial infection upon secondary challenge. The discrepancy
may lie in the difference in the experimental system and how Wnt
activation is achieved. Nevertheless, it should be kept in mind that
when and how Wnt signaling is activated may determine the
outcomes of prophylactic or therapeutic interventions.
We thank Dr. Zuoming Sun (University of Illinois, Chicago, IL) for pro-
Long Pham for useful discussion, and Lecia Epping for technical assis-
The authors have no financial conflicts of interest.
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The Journal of Immunology 1199