of December 28, 2015.
This information is current as
Parasitemia and Promotes Pathogenesis
The Transcription Factor T-bet Regulates
Sanjai Kumar and Sheldon L. Morris
Rajdeep Banerjee, Yukiko Kozakai, Steven C. Derrick,
Nehal R. Solanki, Victoria Majam, Phuong Thao Pham,
Miranda S. Oakley, Bikash R. Sahu, Leda Lotspeich-Cole,
2013; 191:4699-4708; Prepublished online 27
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by guest on December 28, 2015
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The Journal of Immunology
The Transcription Factor T-bet Regulates Parasitemia and
Promotes Pathogenesis during Plasmodium berghei ANKA
Miranda S. Oakley,* Bikash R. Sahu,†Leda Lotspeich-Cole,* Nehal R. Solanki,*
Victoria Majam,†Phuong Thao Pham,†Rajdeep Banerjee,†Yukiko Kozakai,†
Steven C. Derrick,* Sanjai Kumar,†and Sheldon L. Morris*
The pathogenesis of experimental cerebral malaria (ECM) is an immunologic process, mediated in part by Th1 CD4+T cells.
However, the role of the Th1 CD4+T cell differentiation program on the ability to control parasitemia and susceptibility to ECM
disease during blood stage malaria has never been assessed directly. Using the Plasmodium berghei ANKA murine model of ECM
and mice deficient for the transcription factor T-bet (the master regulator of Th1 cells) on the susceptible C57BL/6 background,
we demonstrate that although T-bet plays a role in the regulation of parasite burden, it also promotes the pathogenesis of ECM.
T-bet2deficient (Tbx212/2) mice had higher parasitemia than wild type controls did during the ECM phase of disease (17.7 6
3.1% versus 10.9 6 1.5%). In addition, although 100% (10/10) of wild type mice developed ECM by day 9 after infection, only
30% (3/10) of Tbx212/2mice succumbed to disease during the cerebral phase of infection. Resistance to ECM in Tbx212/2mice
was associated with diminished numbers of IFN-g–producing CD4+T cells in the spleen and a lower accumulation of CD4+and
CD8+T cells in the brain. An augmented Th2 immune response characterized by enhanced production of activated GATA-3+
CD4+T cells and elevated levels of the eotaxin, MCP-1, and G-CSF cytokines was observed in the absence of T-bet. Our results
suggest that in virulent malarias, immune modulation or therapy resulting in an early shift toward a Th2 response may help to
ameliorate the most severe consequences of malaria immunopathogenesis and the prospect of host survival.
Immunology, 2013, 191: 4699–4708.
that contribute to the pathogenesis of CM remain unclear. Because
of limitations in studies that can be conducted in patients with CM,
researchers have relied on the Plasmodium berghei ANKA (Pb2
A) murine model of experimental cerebral malaria (ECM) to im-
prove our knowledge of the genesis and downstream biological
events that mediate the pathogenesis of CM.
During the course of a pathogen infection, the different CD4+
T cell subsets are an important component of adaptive immunity
that contribute to the resolution of acute infection and help to
establish sterilizing immunity or to suppress pathogen burden
to subpatent chronic levels, allowing host survival. However, the
proinflammatory cytokines that are generated to control rapid
pathogen replication can also cause severe immunopathogenesis,
sometimes with fatal consequences.
The Journal of
erebral malaria (CM) remains a major cause of death in
African children younger than 5 years. Despite extensive
research, the full picture of the molecular mechanisms
T cells have been shown to play an important role in the im-
munopathogenesis of ECM (1, 2). In the Pb2A C57BL/6 model,
CD4+T cells mediate the induction phase of immunopathogenesis
of ECM, whereas CD8+T cells mediate the effector phase of dis-
ease (3) by perforin and granzyme-dependent apoptosis of brain
endothelial cells (4–6). Nonetheless, compared with the role of
CD8+T cells during ECM, the immune mechanism of CD4+
T cell–mediated pathogenesis of ECM is less understood.
A pathogenic role for CD4+T cells during ECM was first
documented by Grau et al. (7), who showed that depletion of
CD4+T cells daily for 7 d and then every other day in susceptible
CBA mice during Pb2A infection results in resistance to ECM.
Subsequent studies demonstrating that CD4-deficient mice are
also resistant to ECM confirmed a role for CD4+T cells in disease
pathogenesis (3, 8). The proinflammatory cytokines IFN-g and
TNF-a have been shown to be required for the pathogenesis of
ECM (9, 10). However, the immunologic pathway that results in
the production of these cytokines during ECM has not been de-
lineated. Interestingly, it was shown recently that mice deficient
for IL-12Rb2 but not IL-12p40 or IL-12p35 are resistant to ECM,
suggesting that ECM induction through IL-12Rb2 can occur via
a novel proinflammatory pathway that acts independently of the
IL-12 ligands (11).
CD4+T cells can be classified into at least four distinct subsets
that differentiate from naive CD4+T cells (12). The Th1 subset
requires IL-12 for differentiation, produces IFN-g as its signature
cytokine, is regulated by the transcription factor T-bet, and is
important for immunity against intracellular pathogens but also
promotes autoimmunity and inflammation (13). The Th2 subset
requires IL-4 for differentiation, is regulated by the transcription
factor GATA-3, and mediates immune responses against extra-
*Division of Bacterial, Parasitic, and Allergenic Products, U.S. Food and Drug Ad-
ministration, Rockville, MD 20852; and
Transmitted Diseases, Center for Biologics Evaluation and Research, U.S. Food
and Drug Administration, Rockville, MD 20852
†Division of Emerging and Transfusion
Received for publication February 8, 2013. Accepted for publication June 17, 2013.
This work was supported by intramural grants from the U.S. Food and Drug Admin-
Address correspondence and reprint requests to Dr. Sanjai Kumar, Center for Bio-
logics Evaluation and Research, U.S. Food and Drug Administration, 5516 Nicholson
Lane, Kensington, MD 20895. E-mail address: email@example.com
Abbreviations used in this article: BSL, brain-sequestered leukocyte; CM, cerebral
malaria; ECM, experimental cerebral malaria; MFI, mean fluorescence intensity;
Pb2A, Plasmodium berghei ANKA; QRT-PCR, quantitative real time PCR; WT,
by guest on December 28, 2015
cellular pathogens but also causes asthma and allergic diseases
(14). The Th17 subset requires IL-21 for differentiation, produces
IL-17 as its signature cytokine, is regulated by the transcription
factor RORgt, and is important for immunity against extracellular
bacteria and fungi, but is also responsible for organ-specific au-
toimmune diseases (15). The regulatory T cell subset requires
TGF-b for differentiation, is regulated by the transcription factor
Foxp3, and is critical for maintenance of self-tolerance and reg-
ulation of immunity (16).
Recently, efforts have been made to assess the contribution of
each CD4+T cell subset to the pathogenesis of ECM. Regulatory
T cells can be beneficial or detrimental to the induction of ECM
depending on the method and timing of regulatory T cell depletion
and the genetic background of the host (17–24). Th17 cells, which
induce tissue inflammation and are capable of disrupting the blood
brain barrier (25), do not appear to contribute to the pathogenesis
of ECM (26).
in the immunopathogenesis of ECM using C57BL/6 mice deficient
for the Tbx21 gene that encodes the T-bet transcription factor, the
master regulator of Th1 cells. T-bet controls the Th1 genetic pro-
gram in naive CD4+T cells, directly activates Ifng; it is expressed in
a variety of immune cells including dendritic cells, B cells, CD8+
T cells, and NK cells as well as Th1 cells; and it is essential for
immunopathology and autoimmunity in numerous disease models
(13). We demonstrate that T-bet regulates parasitemia and promotes
pathogenesis during Pb2A infection and that ECM resistance in
Tbx212/2mice is associated with a diminished number of IFN-g–
expressing CD4+T cells during the induction phase, a reduction of
brain sequestered CD4+and CD8+T cells during the effector phase
and amplification of a Th2 immune response characterized by an
expansion of an activated GATA-3+CD4+T cell population and
overproduction of the eotaxin, MCP-1, and G-CSF cytokines.
Materials and Methods
Mice and parasite infections
Six- to eight-week-old female wild type (WT) and Tbx212/2mice on the
C57BL/6 background were purchased from The Jackson Laboratory (Bar
Harbor, ME). Importantly, Tbx212/2mice were previously backcrossed on
the C57BL/6 genetic background for eight generations. All mice were
maintained at the U.S. Food and Drug Administration animal care facility
and treated in accordance with the guidelines of the Animal Care and Use
Committee. An uncloned parasite line of Pb2A parasites was used for all
infections in this study. Infection was initiated in a donor mouse by in-
jection of thawed Pb2A parasites. Once parasitemia reached ∼5% in the
donor mouse, blood was collected and diluted in PBS. Infection was then
induced in experimental mice by i.p. injection of 106Pb2A parasites and
mice were monitored for clinical systems of ECM as described previously
(27–29). Parasitemia (Parasitized RBCs / Total RBCs 3 100) was deter-
mined by examining Giesma-stained thin blood films.
CD4+and CD8+T cells as well as the phenotype of these CD8+T cells in
the brain on day 6 after infection and to enumerate the number of 1) CD4+
T cells, CD8+T cells, and T-bet+, TNF2a+and IFN2g+T cell subsets on
days 0, 3, and 6 after infection and 2) CD69+GATA-3+CD4+T cells on day
6 after infection in the spleen. Splenocytes were prepared as described
previously (30), and brain-sequestered leukocytes (BSLs) were made from
perfused tissue as follows: a single-cell suspension was first prepared by
treatment with DNase (3 U/ml) and collagenase (0.5 mg/ml) under fre-
quent agitation and trituration in a volume of 3 ml for 1 h at room tem-
perature (Roche Applied Science, Indianapolis, IN). Leukocytes were then
purified by centrifugation at 515 3 g for 30 min at 21˚C on 33% Percoll
(Sigma-Aldrich, St. Louis, MO).
Single-cell suspensions of splenocytes and BSLs were stained with eFluor
(BD Biosciences, San Jose, CA), stained with the following Abs (purchased
from BD Biosciences, BioLegend, or eBiosciences) specific for FITC–anti-
TCR-ab, PerCP–anti-CD4, Pacific blue–anti-CD4, APC/Cy7–anti-CD8, PE/
Cy7–anti-CD69, PerCP–anti-CD69, Pacific blue–anti-CD69, PE–anti-CD44,
PE/Cy7–anti-CD62L, PE/Cy7–anti-CXCR3, PE/Cy7–anti-IFNAR1, PE/Cy7–
anti-IFN-g, PE–anti-TNF-a, PE–anti-T-bet, PE–anti-GATA-3 in HBSS con-
taining 1% BSA for 30 min at 4˚C, washed three times in HBSS containing
0.1% BSA, fixed, and then analyzed on an LSR II flow cytometer using FACS
Diva (BD Biosciences) and Flowjo (Tree Star, Ashland, OR) software. For
intracellular staining, a 4-h incubation with brefeldin A (BD Biosciences) was
included and cells were permeabilized prior to intracellular staining. Lastly,
isotype controls were used for analysis of CD69, IFN-g, TNF-a, T-bet, and
Fresh spleen tissue was collected simultaneously from WT and Tbx212/2
mice and stored at 280˚C until use. For preparation of high-quality RNA,
tissue was resuspended in Tri-Reagent (Molecular Research Center, Cin-
cinnati, OH) and pulsed-homogenized to create a suspension. RNAwas then
purified by performing two chloroform extractions, an isopropanol precip-
itation, a wash with 70% ethanol, and a final resuspension in nuclease-free
water. RNAwas then treated with 8 U of Turbo DNA-free (Ambion, Austin,
TX) for 30 min at 37˚C, and cDNA was then synthesized from 1 mg of
DNase-treated RNA in a 20-ml reaction volume containing iScript reverse
transcriptase, random primers, deoxynucleoside triphosphates (dNTPs), and
magnesium chloride (Bio-Rad Laboratories, Hercules, CA) at 42˚C for 30
min (31). Quantitative real-time PCR (QRT-PCR) was performed in a 20-ml
reaction volume containing 2 ml cDNA, 10 ml SsoFast EvaGreen Supermix
(Bio-Rad), and 500 nM commercially available primers specific for mouse
T-bet, GATA-3, Foxp3, or RORgt gene fragments (Qiagen, Valencia, CA).
Amplification and detection of specific product were performed using the
CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with the fol-
lowing cycle profile: 1 cycle at 95˚C for 30 s and 40 cycles with 1 cycle
consisting of 5 s of denaturation at 95˚C and 5 s of annealing and extension
at 61˚C. The relative concentrations of RNA were determined using a
standard curve derived from the PCR products of 10-fold serial dilutions of
plasmid containing a mouse b-actin gene fragment. Real-time PCR was
performed on four mice per group in duplicate reactions.
Detection of serum cytokines
Serum cytokine profiles were assessed using the Bio-Plex Pro Mouse Cyto-
kine 23-plex assay (Bio-Rad) specific for the IL-1a, IL-1b, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, eotaxin, G-CSF,
GM-CSF, IFN-g, KC, MCP-1, MIP-1a, MIP-1b, RANTES, and TNF-a
cytokines. Serum samples were incubated sequentially with beads coated
with capture Ab, biotinylated detection Ab, and streptavidin-PE conjugate
with three washes performed between each incubation step. Analytes were
then assayed in a final volume of 125 ml, and data were acquired using the
Bio-Plex 200 reader and analyzed using Bio-Plex Manager software ver-
The log-ranktestwasused to determine differencesin survivalbetween WT
and Tbx212/2mice. Differences in parasitemia were analyzed by applying
pairwise comparisons. Differences in cell counts and serum cytokine levels
were determined using the Student t test when parametric assumptions
were met. Otherwise, the Mann–Whitney U test was applied. Lastly, dif-
ferential expression of transcription factor RNA was analyzed using the
T-bet dependent immunity regulates parasite growth and
induces ECM pathogenesis
To determine a role for T-bet in the pathogenesis of ECM, we
measured the susceptibility of mice deficient for the Tbx21 gene
encoding the T-bet transcription factor to ECM. Simultaneously, we
assessed the role of T-bet in the regulation of parasite burden by
comparing the parasitemia of Tbx212/2mice with WT controls.
Following infection with Pb2A parasites, 10 of 10 (100%) WT
mice developed ECM by day 9 after infection. In contrast, only 3 of
10 (30%) Tbx212/2mice succumbed to malaria during the cerebral
phase of infection indicating that Tbx212/2mice are significantly
protected from ECM disease (p , 0.01, log-rank; Fig. 1A). Tbx212/2
mice that were resistant to ECM developed severe anemia and
hyperparasitemia by day 14 after infection and were sacrificed.
4700EFFECT OF T-BET ON ECM
by guest on December 28, 2015
The absence ofT-betalso hadaneffectonparasitegrowthduring
a Pb2A infection. On day 6 after infection, when the majority of
WT mice become moribund, Tbx212/2mice (17.7 6 3.1%) had
a 1.6-fold higher parasite burden than WT mice did (10.9 6 1.5%).
This difference was even more dramatic on day 7 after infection,
when Tbx212/2mice (19.70 6 2.0) had a 4.5-fold higher para-
sitemia (p = 0.03, Mann2Whitney test) than WT mice did (4.34 6
2.44; Fig. 1B). These results indicate that T-bet also plays an im-
portant role in the regulation of Pb2A parasitemia.
Within the group of Pb2A-infected Tbx212/2mice, we noted
a positive correlation between parasite burden and susceptibility
to disease. On day 6 after infection, Tbx212/2mice with ECM
(28.96 6 3.27; n = 3) had significantly higher parasitemia (2.3-
fold) than Tbx212/2mice without ECM (12.82 6 2.53; n = 7; p ,
0.01, Student t test; Fig. 1C). Parasitemia was also markedly
higher in Tbx212/2mice with ECM compared with WT mice with
ECM (10.86 6 1.49, n = 10; p , 0.01). These results suggest that
the parasitemia threshold for disease induction may be higher in
Tbx212/2mice compared with WT mice.
Measurement of pathogenic T cells during Pb2A infection
To determine the relative contribution of pathogenic T cells in
T-bet–mediated susceptibility to ECM, we quantitated CD4+and
CD8+T cells in the spleens of naive mice (day 0) and during the
induction (day 3) and symptomatic (day 6) phase of ECM by flow
cytometric analysis. There was no significant difference in CD4+
or CD8+T cell counts in WT versus Tbx212/2-infected mice over
the course of infection (Fig. 2A, 2B).
We also compared brain-sequestered CD4+and CD8+T cells in
WT versus Tbx212/2mice on day 6 after infection to determine
whether ECM resistance caused by the absence of T-bet is asso-
ciated with a decrease in the induction or migration of pathogenic
immune cell subsets to the brain. Remarkably, there was a 5.8-fold
reduction in the number of BSLs in Tbx212/2(125 6 35 3 103
BSLs) compared with WT (725 6 152 3 103BSLs; Fig. 3A) mice,
indicating that T-bet is important for the accumulation of BSLs in
ECM susceptible mice. This reduction in BSLs correlated with a
significant decrease in brain-sequestered CD4+(Fig. 3B, 3C) and
CD8+(Fig. 3E, 3F) T cells in the absence of T-bet. Tbx212/2mice
(3.5 6 1.3 3 103) had a 9.5-fold reduction (p , 0.04, Mann2
Whitney test) in brain-sequestered CD4+T cells (Fig. 3D) com-
pared with WT mice (32.7 6 7.8 3 103). Furthermore, Tbx212/2
mice (19.8 6 6.0 3 103) had an 18-fold reduction (p , 0.04,
Mann2Whitney test) in brain-sequestered CD8+T cells (Fig. 3G)
compared with WT mice (356 6 77 3 103).
We next measured the expression of biomarkers of brain-
sequestered CD8+T cells that have previously been shown to cor-
relate with ECM pathogenesis. Previous studies have demonstrated
that brain-sequestered CD8+T cells during the cerebral phase of
ECM are differentiated and activated (4) and express the chemokine
receptor CXCR3 (32) and the IFN (a, b) receptor 1 (IFNAR1) (33).
Among brain-sequestered CD8+T cells in WT mice, 46.6 6 5.3%
(158 6 20 3 103) and 50.9 6 4.6% (188 6 55 3 103) are central
(CD44+CD62L+) and effector (CD44+CD62L2) memory cells, re-
spectively (Fig. 4A), 31.9 6 3.4% (118 6 34 3 103) are activated
(CD69+; Fig. 4C), 74.1 6 5.1% (258 6 41 3 103) express CXCR3
(Fig. 4E), and 8.5 6 0.9% (30 6 6 3 103) express IFNAR1 (Fig.
4G). Although Tbx212/2mice have an 18-fold reduction in brain-
sequestered CD8+T cells compared with WT mice, the phenotype
of brain-infiltrating CD8+T cells in WT (Fig. 4I) versus Tbx212/2
(Fig. 4B, 4D, 4F, 4H, 4J) mice is similar. Thus, lower cell counts
of brain sequestered CD8+T cells rather than a difference in any
identifiable CD8+T cell phenotype can be attributed to resistance to
ECM in Tbx212/2mice.
Expression of T-bet in CD4+and CD8+T cells during Pb2A
We measured the expression of T-bet in splenic CD4+T cells over
the course of Pb2A infection by flow cytometry (Fig. 5A). T-bet
is not expressed in naive CD4+T cells; we find that T-bet was
Pb2A infection of C57BL/6 mice. WT and Tbx212/2mice were infected
with 106Pb2A parasites, and susceptibility to ECM and parasitemia was
measured over the course of infection. (A) Tbx212/2mice were signifi-
cantly protected from ECM; 100% of WT mice versus 30% of Tbx212/2
mice exhibited symptoms of ECM (p , 0.01, log-rank). (B) Parasite
burden was greater in Tbx212/2mice during the cerebral phase of infec-
tion. (C) Among Tbx212/2mice, mice with ECM had significantly higher
parasitemia than mice without ECM did (p = 0.03, two-way ANOVA),
indicating that a threshold parasitemia was required to trigger ECM.
T-bet regulates parasite burden but promotes ECM during
CD8+T cells during a Pb2A infection. WT and Tbx212/2mice were
infected with 106Pb2A parasites, and splenocytes were purified and stained
with Abs specific to TCR-ab+, CD4+and CD8+T cells. Lymphocyte pop-
ulations were then enumerated over the course of infection. There was no
significant difference in the absolute number of (A) CD4+and (B) CD8+
T cells over the course of Pb2A infection in WT versus Tbx212/2mice.
Effect of T-bet on the production of pathogenic CD4+and
The Journal of Immunology4701
by guest on December 28, 2015
constitutively expressed in only 0.90 6 0.24% of CD4+T cells in
noninfected mice. In comparison, T-bet expression was upregu-
lated in 3.46 6 1.44% of CD4+T cells (12.62 6 5.90 3 105cells)
on day 3 after infection and peaked at 15.13 6 0.90% (24.14 6
3.73 3 105cells) on day 6 after infection (Fig. 5C, 5D). T-bet has
also been shown to be important for the generation of cytotoxic
duction in brain-sequestered CD4+and CD8+T
cells during the symptomatic phase of Pb2A in-
fection. WT and Tbx212/2mice were infected with
106Pb2A parasites, and BSLs were isolated from
perfused brain tissue on day 6 after infection and
then stained with fluorescent-labeled Abs specific
for TCR, CD4, and CD8 for flow cytometry. (A)
Significantly fewer BSLs were recruited to the brain
in Tbx212/2mice. Comparison of brain sequestered
(B, C) CD4+and (E, F) CD8+T cells in WT and
Tbx212/2mice by flow cytometry demonstrated
a (D) 9.5-fold reduction in CD4+T cells and an (G)
18-fold reduction in CD8+T cells in the absence of
T-bet. *p # 0.05.
Tbx212/2mice have a significant re-
Tbx212/2mice on day 6 after infection with Pb2A parasites were analyzed for expression of (A, B) CD44 and CD62L, (C, D) CD69, (E, F) CXCR3, and
(G, H) IFNAR1. (I) In WT mice, the majority of brain-sequestered CD8+T cells are central memory (CM) or effector (EM) memory cells and express the
CXCR3 chemokine receptor, and a subset (31.9 6 3.4%) express the CD69 activation marker. (J) Although Tbx212/2mice have significantly fewer brain-
sequestered CD8+T cells, these CD8+T cells express known biomarkers.
Phenotypic analysis of known biomarkers of brain-sequestered CD8+T cells during ECM. Gated CD8+T cells purified from WT and
4702EFFECT OF T-BET ON ECM
by guest on December 28, 2015
effector CD8+T cells (34). Therefore, we also assessed the ki-
netics of T-bet expression by CD8+T cells over the course of
infection (Fig. 5B). T-bet was expressed in 6.22 6 1.03% of CD8+
T cells in noninfected mice. On day 3 after infection, the pro-
portion (6.04 6 2.36%) of CD8+T cells expressing T-bet was
similar to basal expression in naive mice. However, on day 6
after infection, 22.32 6 3.17% of CD8+T cells expressed T-bet
(25.74 6 5.27 3 105cells; Fig. 5C, 5D). Remarkably, although
there was no significant difference between the percentage or ab-
solute number of T-bet+expressing CD4+versus CD8+T cells, the
mean fluorescence intensity (MFI), a measure of the protein
quantity per cell, of T-bet was 4.17-fold higher in CD8+T cells
(2.35 6 0.47 3 103) than in CD4+T cells (0.56 6 0.26 3 103) on
day 6 after infection (p , 0.02, Mann2Whitney test; Fig. 5E)
indicating that T-bet is likely important for the generation of
pathogenic CD8+T cells as well as the differentiation of proin-
flammatory Th1 CD4+T cells that produce the ECM-inducing
cytokines in susceptible strains of mice.
Tbx212/2mice produce more GATA-3+CD4+T cells
We next measured the splenic levels of the T-bet, GATA-3, RORgt,
and Foxp3 transcription factors that regulate Th1, Th2, Th17, and
Treg CD4+T cell differentiation, respectively, by QRT-PCR (Fig.
6A, 6B, 6C, 6D). There was no significant difference in tran-
scription factor levels on day 3 after infection. However, there was
a substantial increase (5.2-fold) in the production of T-bet tran-
scripts from day 3 (13.58 6 2.04 3 103) to day 6 (70.83 6 8.25 3
103) in WT mice (Fig. 6A). Furthermore, there was a significant
difference (1.8-fold; p = 0.05, mixed model) in splenic mRNA
levels of GATA-3 on day 6 after infection in WT (22.38 6 1.56 3
103) versus Tbx212/2(40.86 6 11.97 3 103) mice, suggesting that
ECM-resistant Tbx212/2mice can produce more GATA-3+CD4+
T cells during the symptomatic phase of infection (Fig. 6B).
Therefore, we next compared the number of GATA-3+CD4+
T cells and their activation status by flow cytometry in WT versus
Tbx212/2mice on day 6 after infection (Fig. 7A, 7B). Similar to
results determined by QRT-PCR, Tbx212/2mice (2.95 6 1.25 3
105) had substantially more (5.2-fold) GATA-3+CD4+T cells than
WT mice did (0.57 6 0.19 3 105; p , 0.01, Mann2Whitney
test). Furthermore, a larger portion of GATA-3+CD4+T cells were
activated as determined by CD69 expression in Tbx212/2(54.5%)
compared with WT (37.3%) mice, resulting in a 7.3-fold differ-
ence in activated GATA-3+CD4+T cells between the two groups
of mice (p , 0.01, Mann2Whitney test; Fig. 7C). These results
indicate that the CD4+T cell helper response is Th2 skewed in the
absence of T-bet.
The absence of T-bet is associated with a reduction in the
number of IFN-g–producing CD4+T cells during the induction
phase of ECM
It is well established that T-bet is essential for the regulation of
IFN-g expression (35), and murine malaria studies demonstrating
that IFN-gR2/2mice (36, 37) are resistant to ECM indicate that
IFN-g is required for the development of ECM. We therefore
compared the expression of IFN-g in CD4+and CD8+T cells in
WT versus Tbx212/2mice. Loss of T-bet did not alter the per-
centage or number of IFN-g–producing CD8+T cells over the
course of infection. However, there was a significant difference in
IFN-g–producing CD4+T cells during the induction phase (day 3;
Fig. 8A, 8B), but not the effector phase (day 6; data not shown)
of disease. There was a 3.11-fold decrease (p , 0.01, Mann2
Whitney test) in the percentage of IFN-g+CD4+T cells in the
absence of T-bet (Fig. 8C) that translated into a 2.90-fold reduc-
tion (p = 0.05, Mann2Whitney test) in the number of CD4+
T cells that produce IFN-g+in Tbx212/2mice (2.80 3 105cells)
compared with WT mice (8.13 3 105cells; Fig. 8D). Despite this
decrease in IFN-g–producing CD4+T cells in Tbx212/2mice on
day 3 after infection, there was no difference in the MFI of IFN-g
in CD4+T cells between the two groups of mice (Fig. 8E). These
results suggest that resistance to ECM in Tbx212/2mice is caused
by diminished production of IFN-g+CD4+T cells during the early
phase of disease pathogenesis. Consistent with this reduction in
IFN-g+CD4+T cells during the induction phase of disease, we
observed a 1.8-fold reduction (p , 0.02, Mann2Whitney test) in
serum IFN-g on day 3 after infection in Tbx212/2compared with
WT mice (data not shown).
Tbx212/2mice produce diminished numbers of TNF-a+CD8+
T cells during the effector phase of disease
Because TNF-a is an important proinflammatory cytokine, we
also compared the expression of TNF-a in CD4+and CD8+T cells
over the course of Pb2A infection in WT versus Tbx212/2mice
(Fig. 9A, 9B, 9C, 9D, 9E, 9F). Although there was no difference in
the production of TNF-a+CD4+T cells, there was a significant
reduction in the percentage of CD8+T cells that express TNF-a
during the effector phase (day 6) of disease (Fig. 9D). Loss of T-
bet resulted in a 1.80-fold reduction in the percentage of TNF-a–
producing CD8+T cells (p , 0.01, Student t test); 5.26 6 0.35%
T cells over the course of a Pb2A infection. WT mice
were infected with 106Pb2A parasites, and T-bet ex-
pression was measured in CD4+TCRab+and CD8+
TCRab+cells by flow cytometry. T-bet expression is
shown in (A) CD4+and (B) CD8+T cells on gated
TCR-ab+cells. T-bet was quantitated by subtracting
the percentage of PE2anti-mouse IgG1+cells (blue)
from PE2anti-T-bet+cells (red). The (C) percentage
and (D) absolute number of T cells that express T-bet
and the (E) MFI of T-bet are shown during the induc-
tion (day 3) and effector (day 6) phase of Pb2A in-
fection. Expression of T-bet in CD4+T cells is induced
on day 3 and peaks on day 6. In contrast, expression of
T-bet in CD8+T cells at day 3 does not differ from
baseline levels in naive mice (data not shown). How-
ever, on day 6 after infection, when CD8+T cells are
known to be pathogenic, T-bet is highly expressed in
T-bet expression in CD4+and CD8+
The Journal of Immunology4703
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versus 2.93 6 0.25% of CD8+T cells produced TNF-a in WTand
Tbx212/2mice, respectively. This difference translated into a 1.82-
fold decrease in the number of TNF-a+CD8+T cells in Tbx212/2
mice (2.42 6 0.54 3 105cells) compared with WT mice (4.41 6
0.70 3 105cells) (Fig. 9F).
Cytokine profile is Th2 skewed in Tbx212/2mice
To delineate the mechanism of ECM resistance in Tbx212/2mice,
we compared the serum cytokine profiles of WT versus Tbx212/2
mice. Of the 23 cytokines examined, Tbx212/2mice had signifi-
cantly higher levels of cytokines associated with the Th2 response
on day 6 after infection. In addition to elevated levels of IL-4 and
IL-5 (Fig. 10A, 10B), Tbx212/2mice had 6.9-fold more eotaxin
(p , 0.01, Mann2Whitney test), 5.2-fold more MCP-1 (p , 0.01,
Mann2Whitney test), and 4.1-fold more G-CSF (p , 0.01, Mann2
Whitney test) than WT mice (Fig. 10C, 10D, 10E). Interestingly,
there was no significant difference in serum levels of the Th1-
associated cytokine IFN-g, the anti-inflammatory cytokine IL-10,
or the Th17-associated cytokine IL-17 between WT and Tbx212/2
mice on day 6 after infection.
In mice, Pb2A parasites cause a highly virulent infection that is
uniformly fatal. Depending on the genetic background of the host
mouse strain, the cause of death is attributed to clinical symptoms
of ECM (susceptible strain) or severe anemia (resistant strain).
Earlier studies have demonstrated that in susceptible C57BL/6
mice, T cells play a key role in the pathogenesis of ECM. Al-
though extensive studies have indicated that CD8+T cells and
IFN-g are the important mediators of ECM, CD8+T cells are not
attributed as the primary source of IFN-g in mice undergoing the
pathogenesis of ECM (38). On the other hand, how CD4+T cells
contribute toward the clinical syndrome of ECM remain poorly
understood. In this study, we examined the role of proinflam-
matory CD4+Th1-type responses in the development of ECM
by comparing the immunopathogenesis of WT C57BL/6 mice to
Tbx212/2mice that bear a genetic deletion in the Tbx21 gene.
Depending on their cytokine milieu during TCR activation, naive
CD4+T cells can differentiate into several lineages of helper
T cells that are defined based on their cytokine production and ef-
fector function (39). T-bet is a transcription factor that is expressed
on a variety of immune cells. However, this molecule is best rec-
ognized for its critical requirement for the differentiation of naive
Th0 cells into effector Th1 CD4+T cells.
We find that Tbx212/2C57BL/6 mice are highly resistant to
ECM disease (100% WT versus only 30% Tbx212/2mice de-
veloped ECM; Fig. 1A). Interestingly, this protection against ECM
in Tbx212/2mice is only partial, indicating that T-bet–indepen-
dent mechanisms of stimulating a proinflammatory response (pos-
sibly mediated by CD8+T cells) that can induce the pathogenesis
of ECM exist. In addition to playing a role in the induction of
ECM, we also found that presence of T-bet was detrimental for
parasite growth. On day 7 after infection, Tbx212/2mice had 4.5-
fold higher parasitemia than WT C57BL/6 mice did (Fig. 1B).
Lastly, our results indicate that the parasitemia threshold for dis-
ease induction could be higher in the absence of T-bet. On day 6
after infection, Tbx212/2mice with ECM (28.96 6 3.27, n = 3)
had 2.3-fold higher parasitemia than Tbx212/2mice without ECM
did (12.82 6 2.53, n = 7; Fig. 1C) and 2.67-fold higher para-
sitemia than WT mice with ECM did (10.86 6 1.49, n = 10).
Thus, although Th1-mediated proinflammatory cytokines might
be necessary to control the acute phase of malaria infection, such
responses can also promote the pathogenesis of disease in the
CD4+and CD8+T cells are known to have a pathogenic role
during ECM. In our studies, the numbers of CD4+and CD8+
T cells were not distinguishable in the spleen between the two
groups of mice (Fig. 2A, 2B). We enumerated brain-sequestered
CD4+and CD8+T cells and found that WT mice had 32.7 6 7.8 3
103brain-sequestered CD4+T cells and 356 6 77 3 103brain
CD4+T cell differentiation as detected by QRT-PCR. WT (n = 4) and
Tbx212/2(n = 4) mice were infected with 106Pb2A parasites, and ex-
pression of (A) T-bet (B) GATA-3 (C) Foxp3 and (D) RORgt was determined
by QRT-PCR of cDNA synthesized from RNA isolated from spleen tissue of
infected mice on days 3 and 6 after infection. Similar to quantitation by flow
cytometry, QRT-PCR results demonstrate that T-bet is maximally expressed
(70.83 6 8.25 3 103transcripts) on day 6 after infection in spleen tissue
from WT mice. Although there is no significant difference in expression of
Foxp3 and RORgt between WT versus Tbx212/2mice, Tbx212/2mice
express 1.8-fold more GATA-3 transcripts compared with WT mice. *p #
0.05, ***p , 0.001.
Relative copy number of transcription factors required for
in the absence of T-bet. WT (n = 4) and Tbx212/2(n = 4) mice were
infected with 106Pb2A parasites, and flow cytometry was performed on
splenocytes to determine GATA-3 expression and activation status (CD69)
of CD4+T cells. On day 6 after infection, splenocytes were harvested and
stained with Abs specific for TCR, CD4, GATA-3, and CD69. (A) A sig-
nificantly larger proportion of gated CD4+T cells express both CD69 and
GATA-3 in Tbx212/2(red) compared with WT (blue) mice. (B) For gating
purposes, CD4+T cells were also stained with the PerCP2anti-Armenian
Hamster IgG and PE2anti-rat IgG2b isotype control Abs for CD69 and
GATA-3, respectively. (C) Tbx212/2mice have 5.2-fold more GATA-3+
(Th2) and 7.3-fold more CD69+GATA-3+(activated Th2) CD4+T cells
than WT mice on day 6 after infection, indicating that the absence of
T-bet skews the CD4+T cell repertoire during Pb–A infection. Values are
expressed as absolute cell numbers rather than percentages to normalize
for differences in splenic cell numbers between the two groups of mice.
**p , 0.01.
Expansion of activated Th2 (CD69+GATA-3+) CD4+T cells
4704EFFECT OF T-BET ON ECM
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sequestered CD8+T cells. In contrast, Tbx212/2mice had 9.5-fold
fewer brain-sequestered CD4+T cells and 18-fold fewer brain-
sequestered CD8+T cells compared with susceptible WT mice.
Thus, Th1 CD4+T cells might also contribute to disease by pro-
moting the recruitment and retention of pathogenic CD8+T cells
at the site of pathogenesis in brain tissue. These results are rem-
iniscent of the murine diabetes model in which loss of T-bet ex-
pression in CD4+T cells impaired cellular migration and sub-
sequent infiltration into the pancreas, which is needed to promote
Recent studies have identified several phenotypic characteristics
of brain-sequestered CD8+T cells during ECM. Because the
majority of BSLs are CD8+T cells during ECM and because
CD8+T cells are known to exert a pathogenic effect in the brain
during the effector phase of disease, we compared the phenotype
of brain-infiltrating CD8+T cells in WT versus Tbx212/2mice to
determine whether the absence of T-bet is associated with the loss
of a particular CD8+T cell phenotype. The majority of brain-
sequestered CD8+T cells have been shown to be activated and
differentiated memory T cells (4). In addition, 90% of brain-
sequestered CD8+T cells express the CXCR3 chemokine receptor
(32), and this chemokine receptor has been shown to be essential
for susceptibility to ECM (41). Furthermore, a recent study has
demonstrated that expression of IFNAR1 by CD8+T cells is re-
quired for ECM pathogenesis (33). Consistent with these studies,
46.6 6 5.3% (158 6 20 3 103) and 50.9 6 4.6% (188 6 55 3 103
) of brain-sequestered CD8+T cells in WT mice were central
(CD44+CD62L+) and effector (CD44+CD62L2) memory cells,
respectively, 31.9 6 3.4% (118 6 34 3 103) expressed the CD69
activation marker, 74.1 6 5.1% (258 6 41 3 103) expressed
CXCR3, and a small subset (8.5 6 0.9%) expressed IFNAR1 (Fig.
4A, 4C, 4E, 4G, and 4I). Although resistance to ECM in Tbx212/2
mice is associated with a dramatic reduction in the number of
CD8+T cells in the brain, the phenotype of brain-sequestered
CD8+T cells in Tbx212/2mice appears to be similar to that of
T cells contribute to ECM pathogenesis by secreting ECM-
inducing cytokines. Th1 CD4+T cells and CD8+T cells can gen-
erate a panel of proinflammatory cytokines of overlapping charac-
teristics and functions. Thus, the differential contributions of CD4+
and CD8+T cells in the pathogenesis of ECM are difficult to dis-
cern. Although naive CD4+T cells do not express T-bet, our data
is diminished in the absence of T-bet on day 3 after
infection. WT (n = 5) and Tbx212/2(n = 5) mice
were infected with 106Pb2A parasites, and IFN-g
was quantitated in CD4+TCRab+and CD8+TCRab+
cells in infected mice. Representative dot plots of
expression of (A) IFN-g and (B) rat IgG1 isotype
control in CD4+T cells on gated T cells in WT (red)
and Tbx212/2(blue) mice are shown. (C) There was
a 3.11-fold reduction in the percentage and a (D)
2.90-fold reduction in the absolute number of CD4+
T cells that express IFN-g. (E) IFN-g–specific MFIs
did not differ between the two groups of mice. *p #
0.05, **p , 0.01
Production of IFN-g by CD4+T cells
CD8+T cells on day 6 after infection. WT (n = 5)
and Tbx212/2(n = 5) mice were infected with 106
Pb2A parasites, and TNF-a expression on CD4+
and CD8+T cells on days 3 and 6 after infection
was measured by flow cytometry. (A) TNF-a and
(B) rat IgG1 isotype control expression on CD8+
T cells is shown on gated T cells on day 6 after
infection in WT (red) and Tbx212/2(blue) mice.
On day 3 after infection, there is no difference in
the (C) percentage or (E) absolute number of CD4+
and CD8+T cells that express TNF-a. However,
on day 6 after infection, there is (D) a 1.80-fold
reduction (p , 0.01, Mann2Whitney test) in the
percentage and (F) a 1.82-fold reduction in the ab-
solute number of CD8+T cells that express TNF-a.
***p , 0.001.
A reduction in TNF-a production by
The Journal of Immunology4705
by guest on December 28, 2015
showed an enhanced percentage of T-bet–expressing CD4+
T cells, but not CD8+cells on day 3 after infection (Fig. 5C, 5D).
In contrast, expansion of T-bet–expressing CD8+T cells was not
observed until day 6 after infection. These data support previous
T cell subset depletion studies that demonstrate the pathogenic
role of CD4+T cells during the induction (day 3 onward) phase
versus CD8+T cells during the effector (day 6) phase of ECM (3).
T-bet expression in CD8+T cells promotes the generation of cy-
totoxic effector T cells. Thus, in addition to inducing the differ-
entiation of proinflammatory Th1 CD4+T cells, T-bet might also
play a crucial role in ECM pathogenesis by promoting the ex-
pansion and migration of pathogenic CD8+T cells. A potential
role for T-bet in the generation of cytotoxic CD8+T cells that are
pathogenic during ECM is supported by the 4.9-fold increase in
the MFI of T-bet expression in CD8+T cells from day 3 (0.48 6
0.86 3 103) to day 6 (2.35 6 0.47 3 103) after infection (Fig. 5E).
We further assessed the effect of T-bet on the CD4+T cell dif-
ferentiation program by comparing the expression of the transcrip-
tion factors that regulate Th1, Th2, Th17, and Treg differentiation
in WT versus Tbx212/2C57BL/6 mouse spleen tissue over the
course of a Pb2A infection by QRT-PCR. We found that Tbx212/2
mice expressed 1.8-fold more mRNA that encodes the GATA-3
transcription factor required for Th2 differentiation compared
with WT mice on day 6 after infection (Fig. 6B). In accordance
with these results, we also demonstrated a 7.3-fold increase in
activated GATA-3+CD4+T cells in Tbx212/2mice compared with
WT mice by flow cytometry (Fig. 7A, 7B, 7C). These results
suggest a shift toward a Th2 response that might confer protection
from severe disease. These results are consistent with recent reports
documenting higher GATA-3 expression by CD4+T cells and
a Th2-skewed response in the absence T-bet (39).
Data from the murine experimental model of ECM and cyto-
kine profiling of samples from field studies have long suggested
that proinflammatory cytokines are the major contributors to CM
pathogenesis (42). Studies depleting IFN-g (10) or using IFN-g2
deficient mice (43) have demonstrated that IFN-g is a critical
mediator of ECM in susceptible strains of mice. In addition, IFN-
gR2deficient mice have been shown to be resistant to ECM, and
this resistance is associated with reduced levels of CD8+T cells in
the brain (37). Importantly, a recent study indicates that the major
source of IFN-g that modulates ECM pathogenesis is CD4+
T cells (38). The frequency of IFN-g expressing CD4+T cells has
been found to be inherently lower in Tbx212/2mice (44). In ac-
cordance, we report a 2.9-fold reduction in the number of CD4+
T cells that express IFN-g+in Tbx212/2mice during the induction
phase (day 3) of disease (Fig. 8D), when CD4+T cells are known
to exert a pathogenic effect (2). In contrast, the frequency of IFN-g+
CD8+T cells was not reduced in Tbx212/2mice. It is likely that
the reduced frequency of IFN-g–expressing CD4+T cells during the
induction phase of disease in Tbx212/2mice contributes to the
ECM-resistant phenotype observed in these mice.
We also found that there was a significant reduction in the
percentage of CD8+T cells that express TNF-a in Tbx212/2
compared with WT mice during the effector phase of ECM (Fig.
9D). TNF-a has long been considered a critical mediator of ECM
pathogenesis (9). However, a subsequent study indicates that the
related cytokine lymphotoxin-a rather than TNF-a is essential to
the development of ECM (45). Despite this discrepancy in the role
of TNF-a in the pathogenesis of ECM, TNF-a is also important
for optimal immunoregulation of pathogen clearance by the host.
It is plausible that diminished production of TNF-a by CD8+
T cells can contribute to the hindered parasite clearance observed
in Tbx212/2mice during the effector phase (day 6) of disease
(Fig. 1B). Thus, T-bet might exert multifactorial effects during a
Pb2A infection, which lies beyond the CD4+T cell differentiation
In addition to elevated serum levels of the classical Th2 cyto-
kines IL-4 and IL-5 (Fig. 10A, 10B), Tbx212/2mice also produced
significantly higher serum levels of the cytokines eotaxin, G-CSF,
and MCP-1 on day 6 after infection with Pb2A. Tbx212/2mice
(1547 6 260.8) had 6.93-fold more serum eotaxin compared with
WT mice (223.1 6 59.5) on day 6 after infection (Fig. 10C).
Eotaxin is well recognized as a potent chemoattractant for eosi-
nophils (46, 47). Although eosinophil accumulation was not
compared in WT versus Tbx212/2mice, it has previously been
reported that Ghanian pediatric patients with CM had uniformly
low eosinophil counts because of tissue sequestration and de-
struction rather than decreased production during acute illness
followed by eosinophilia 30 d after cure (48). Importantly, the
eotaxin receptor is expressed by Th2 CD4+T cells, and eotaxin is
critical for the generation and maintenance of Th2 cells at aller-
genic sites and promotes the production of IL-4 and IL-5 (49, 50).
Therefore, it is likely that eotaxin participates in the amplification
of the Th2 response observed in Tbx212/2mice.
Expression of serum G-CSF was increased by 4.07-fold in
Tbx212/2(1384 6 286.1) compared with WT mice (339.7 6 92.8)
on day 6 after infection (Fig. 10D). G-CSF was first identified as
a growth factor for neutrophils (51). Indeed, administration of
recombinant human G-CSF to attenuated P. berghei XAT–infected
CBA mice resulted in a 5-fold elevation in the peripheral blood
neutrophil count and a significantly lower peak parasitemia com-
pared with control mice (52), and elevated G-CSF levels in pregnant
serum of Tbx212/2(n = 3, day 0 and n = 8, day 6) compared with WT (n =
3, day 0 and n = 8, day 6) mice. Serum levels of 23 cytokines were
measured in naive (day 0) and Pb2A infected (day 6) mice using the Bio-
Plex Pro Mouse Cytokine 23-plex assay. Only cytokines with a statistically
significant differential expression in WT versus Tbx212/2mice are shown.
Tbx212/2mice have significantly elevated levels of (A) IL-4, (B) IL-5, (C)
eotaxin, (D) G-CSF, and (E) MCP-1 on day 6, during the cerebral phase of
infection. **p , 0.01, ***p , 0.001.
A panel of Th2-associated cytokines are elevated in the
4706EFFECT OF T-BET ON ECM
by guest on December 28, 2015
women with asymptomatic malaria may be involved in main-
taining low parasitemia levels (53). However, G-CSF also plays an
important role in adaptive immunity and has been shown to induce
Th2 polarization in CD4+T cells. CD4+T cells treated with G-
CSF display diminished IFN-g and enhanced IL-4 production and
upregulation of the GATA-3 transcription factor (54, 55). Fur-
thermore, G-CSF has also been shown to impair the generation of
cytolytic effector cells by posttranscriptional suppression of TNF-
Serum MCP-1 is elevated in Tbx212/2mice (2995 6 1102)
by 5.16-fold on day 6 after infection compared with WT mice
(580.4 6 120.5; Fig. 10E). MCP-1 has been shown to play an
important role in several neuroinflammatory diseases (57). How-
ever, in a study of 481 Thai patients with malaria, MCP-1 gene
polymorphisms were not associated with CM (58) and in the
murine model of ECM, MCP-1 expression in the brain did not
differ between susceptible versus resistant strains of mice (59).
Although MCP-1 is well recognized for its ability to attract mono-
cytes, it is also essential for Th2 polarization; MCP-1–deficient
mice are unable to mount Th2 responses (60). In summary, al-
though the eotaxin, G-CSF, and MCP-1 cytokines display diverse
immune effector functions, each of these three cytokines have been
shown to contribute significantly to Th2 immunity. Our studies
indicate that resistance to ECM in Tbx212/2mice can be attrib-
uted to a shift to a Th2 response. These findings are in general
agreement with field studies and experimental studies showing
that adults harboring helminth infections (61) and mice infected
with Schistosoma mansoni have increased resistance to CM (62).
Helminth parasites are generally known to trigger a Th2 response
in their hosts. We think that the increased serum levels of these
three cytokines observed in ECM-resistant Tbx212/2mice may be a
function of a generalized Th2 switch, and the precise association
of these individual cytokines with resistance to ECM remains to
Recently, our knowledge of the complex interplay between net-
works of cytokines and transcription factors on differentiation of
naive CD4+T cells into different subsets has expanded tremen-
dously (12). Nevertheless, how infections with different Plasmo-
dium species modulate these differentiation programs in naive
CD4+T cells in HLA-disparate hosts and thus influence the out-
come of pathogenesis of severe malaria has not been studied.
Results from this study offer an initial insight on the contribution
of CD4+T cell differentiation programs on the pathogenesis of
ECM in mice. Additional studies directed toward understanding
the influence of malarial Ags and toxins on the differentiation
program of naive CD4+T cells in young children and partially
immune adults could facilitate the design of superior antiparasitic
and antidisease vaccines against malaria.
We thank the veterinary staff at the Center for Biologics Evaluation and
Research for the care and maintenance of mice.
The authors have no financial conflicts of interest.
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