IFNc/IL-10 Co-producing Cells Dominate the CD4
Response to Malaria in Highly Exposed Children
Prasanna Jagannathan1, Ijeoma Eccles-James1, Katherine Bowen1, Felistas Nankya2, Ann Auma2,
Samuel Wamala2, Charles Ebusu2, Mary K. Muhindo2, Emmanuel Arinaitwe2, Jessica Briggs1,
Bryan Greenhouse1, Jordan W. Tappero3, Moses R. Kamya4, Grant Dorsey1, Margaret E. Feeney1,5*
1Department of Medicine, San Francisco General Hospital, University of California, San Francisco, San Francisco, California, United States of America, 2Infectious Diseases
Research Collaboration, Kampala, Uganda, 3Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America,
4Department of Medicine, Makerere University College of Health Sciences, Kampala, Uganda, 5Department of Pediatrics, University of California, San Francisco, San
Francisco, California, United States of America
Although evidence suggests that T cells are critical for immunity to malaria, reliable T cell correlates of exposure to and
protection from malaria among children living in endemic areas are lacking. We used multiparameter flow cytometry to
perform a detailed functional characterization of malaria-specific T cells in 78 four-year-old children enrolled in a
longitudinal cohort study in Tororo, Uganda, a highly malaria-endemic region. More than 1800 episodes of malaria were
observed in this cohort, with no cases of severe malaria. We quantified production of IFNc, TNFa, and IL-10 (alone or in
combination) by malaria-specific T cells, and analyzed the relationship of this response to past and future malaria incidence.
CD4+T cell responses were measurable in nearly all children, with the majority of children having CD4+T cells producing
both IFNc and IL-10 in response to malaria-infected red blood cells. Frequencies of IFNc/IL10 co-producing CD4+T cells,
which express the Th1 transcription factor T-bet, were significantly higher in children with $2 prior episodes/year compared
to children with ,2 episodes/year (P,0.001) and inversely correlated with duration since malaria (Rho=20.39, P,0.001).
Notably, frequencies of IFNc/IL10 co-producing cells were not associated with protection from future malaria after
controlling for prior malaria incidence. In contrast, children with ,2 prior episodes/year were significantly more likely to
exhibit antigen-specific production of TNFa without IL-10 (P=0.003). While TNFa-producing CD4+T cells were not
independently associated with future protection, the absence of cells producing this inflammatory cytokine was associated
with the phenotype of asymptomatic infection. Together these data indicate that the functional phenotype of the malaria-
specific T cell response is heavily influenced by malaria exposure intensity, with IFNc/IL10 co-producing CD4+T cells
dominating this response among highly exposed children. These CD4+T cells may play important modulatory roles in the
development of antimalarial immunity.
Citation: Jagannathan P, Eccles-James I, Bowen K, Nankya F, Auma A, et al. (2014) IFNc/IL-10 Co-producing Cells Dominate the CD4 Response to Malaria in Highly
Exposed Children. PLoS Pathog 10(1): e1003864. doi:10.1371/journal.ppat.1003864
Editor: Jean Langhorne, National Institute for Medical Research, United Kingdom
Received August 22, 2013; Accepted November 19, 2013; Published January 9, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Centers for Disease Control and Prevention (Cooperative Agreement No U62P024421); NIH/NIAID R01AI093615 (MEF),
UCSF Centers for AIDS Research (Supplement to MEF, P30AI027763), NIH/NIAID U19AI089674 (GD), NIH/NIAID K23 AI100949 (PJ), and Burroughs Wellcome Fund/
American Society of Tropical Medicine and Hygiene (PJ). Additional support was provided by the National Center for Advancing Translational Sciences/NIH,
through UCSF-CTSI Grant Number UL1 TR000004. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript. The findings and conclusions in this paper are those of the authors and do not necessarily represent the official position of the Centers for Disease
Control and Prevention or the NIH.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Clinical immunity to malaria eventually develops in endemic
populations, but only after repeated infections with significant
morbidity to both individuals and their communities . Studies in
regions of high malaria transmission intensity have consistently
shown that the incidence of severe disease decreases considerably
after the first years of life, but sterile immunity (i.e. protection
against parasitemia) develops rarely if ever [2,3]. Moreover,
previously immune individuals may lose protection against symp-
tomatic infection in the absence of continuous exposure [4,5]. The
reasons underlying the slow acquisition of clinical immunity and the
failure to develop sterilizing immunity are unclear, but may include
responses [7–12], and/or host immunoregulatory mechanisms
induced by the parasite [13–19]. As the incidence of malaria
continues to be high in many parts of Africa despite insecticide-
treated bednets and artemisinin-based combination therapy [20–
22], there is a tremendous need to better understand mechanisms of
immunity to malaria in naturally exposed populations. The
identification of immunologic correlates of exposure and protection
in naturally exposed children would significantly help with the
rational design of vaccines and other malaria control interventions.
Both CD4+and CD8+T cells have been demonstrated to play
an important role in protective antimalarial immunity in mouse
models [23–30], and experimental challenge models in humans
and mice strongly suggest that malaria-specific T cells contribute
to protective immunity [31–36]. However, the identification of T
cell correlates of immunity in field-based studies of naturally
exposed humans has proven to be quite challenging. Prior studies
PLOS Pathogens | www.plospathogens.org1 January 2014 | Volume 10 | Issue 1 | e1003864
employing cross-sectional or prospective cohort designs have
found associations between cellular immune responses and
protection from future malaria, including IFNc responses to liver
stage [37–40] and/or merozoite stage malaria antigens [41–44].
However, such studies may be confounded by the level of exposure
to malaria-infected mosquitoes, which varies greatly within
populations, leading subjects with lower exposure to be mis-
categorized as ‘‘protected’’ [45,46]. Because naturally acquired
immunity confers relative rather than absolute protection –
manifested by a gradual decline in the incidence of clinical disease
- careful quantitative outcome measures are essential, but few
population-based studies of natural immunity have included
careful measurement of malaria incidence over time.
Pathogen-specific T cells exhibit notable functional heteroge-
neity, largely dependent on the antigen and cytokine microenvi-
ronment encountered during activation, and measurement of a
single parameter of T cell function (i.e. IFNc production) may
overlook others that are more critical for protection . In other
parasitic infections such as leishmania [48,49] and toxoplasma
, the functional phenotype of the CD4+T cell response
correlates with the success or failure to clear the pathogen. Recent
observations in individuals naturally exposed to malaria suggest an
important role for CD4+T cell production of TNFa, with or
without IFNc, as a potential immunologic correlate of protection
. Conversely, CD4+T cell production of the regulatory
cytokine IL-10 has been implicated in modulating the severity of
disease [18,52] and may interfere with the development of
protective immunity [14,42,53]. The role of these inflammatory
and regulatory cytokines in mediating protective immunity in
naturally exposed children, and in determining the balance
between immunopathology and chronic repeated infection,
In this study we performed a detailed functional characteriza-
tion of malaria-specific T cell responses among four-year-old
children residing in a highly malaria-endemic region to determine
whether naturally acquired T cell responses correlate with
exposure to and/or protection from malaria. We hypothesized
that CD4+T cells producing the pro-inflammatory cytokines IFNc
and/or TNFa are associated with protection from malaria, and
that T cell production of the regulatory cytokine IL-10 may
interfere with the acquisition of protection. Our results suggest that
the functional phenotype of the malaria-specific T cell response
was heavily influenced by prior malaria exposure intensity, with
CD4+T cells co-producing IFNc and IL10 dominating this
response among highly exposed children. However, these IFNc/
IL-10 co-producing cells were not independently associated with
protection from future malaria, and may be associated with
Study population and clinical outcomes
The study cohort consisted of 78 HIV-uninfected children
followed from infancy through 5 years of age (Table 1). Blood for
this study was drawn at four years of age (range 49–51 months),
and 92% of children continued to be followed through 5 years of
age. A total of 1855 incident cases of malaria were observed in this
cohort through 5 years of age. All children were treated promptly
with artemisinin-based combination therapy, and despite the
strikingly high numbers of malaria episodes, only 4 cases of
malaria were deemed ‘‘complicated’’ (all based on a single
convulsion). No cases of severe malaria (including severe anemia)
were observed. Among children with a lower prior incidence of
malaria (,2 episodes per person year (ppy) between 1 and 4 years
of age, n=10), 90% lived in town; whereas among children with
higher prior malaria incidence (.=2 episodes ppy, n=68), only
7% of children lived in town. This suggests that children with the
lowest prior incidence had less exposure to malaria-infected
mosquitoes. Episodes of asymptomatic parasitemia were rare in
this cohort (median 1 episode per subject over the entire study
period, IQR 0–4, Table 1) and the incidence of malaria declined
only slightly in the year following the blood draw (from 5.7 to 5.1
episodes ppy), suggesting that effective clinical immunity had not
yet emerged in most children. One child had symptomatic malaria
(parasitemia with a fever requiring treatment) at the time of the
blood draw, and 17 (22%) had blood smears demonstrating
The functional phenotype of malaria-specific CD4+T cells
is influenced by prior malaria incidence
To define the frequency and function of malaria-specific T cell
responses, PBMC were stimulated with malaria-infected red blood
cells (iRBC) and analyzed by flow cytometry for production of
IFNc, IL-10, and TNFa (Fig. 1a). The median frequency of
malaria-specific CD4+T cell responses producing any of these
cytokines, alone or in combination, was 0.20% (IQR 0.12%–
0.35%). Among all children, frequencies of CD4+T cells
producing IFNc (median 0.16%) and IL-10 (median 0.14%) were
significantly higher than those producing TNFa (median 0.04%,
P,0.001, Fig. 1b). Production of these two cytokines largely
overlapped, with a median of 83% of IL-10-producing cells also
making IFNc, and a median of 71% of IFNc-producing cells also
making IL-10. Malaria-specific production of IL-2 was tested in a
subset of children (n=44), but responses were consistently of low
magnitude (median frequency 0.02%, data not shown). At the time
of the assay 17 of the 78 children had positive blood smears;
however there was no significant difference in the overall
frequency of malaria-specific IFNc+(P=0.20), TNFa+(P=0.29),
or IL-10+(P=0.21) CD4+T cells between children with or
without parasitemia. Malaria-specific CD8 T cell responses were
Despite reports of decreasing malaria morbidity across
many parts of Africa, the incidence of malaria among
children continues to be very high in Uganda, even in the
setting of insecticide-treated bednets and artemisinin-
based combination therapy. Additional control measures,
including a vaccine, are sorely needed in these settings,
but progress has been limited by our lack of understand-
ing of immunologic correlates of exposure and protection.
T cell responses to malaria are thought to be important for
protection in experimental models, but their role in
protecting against naturally acquired infection is not clear.
In this study, we performed detailed assessments of the
malaria-specific T cell response among 4-year-old children
living in Tororo, Uganda, an area of high malaria
transmission. We found that recent malaria infection
induces a malaria-specific immune response dominated
by Th1 T cells co-producing IFNc and IL-10, and that these
cells are not associated with protection from future
infection. IFNc/IL-10 co-producing cells have been de-
scribed in several parasitic infections and are hypothesized
to be important in limiting CD4-mediated pathology, but
they may also prevent the development of sterilizing
immunity. These observations have important implications
for understanding the pathophysiology of malaria in
humans and for malaria vaccine development.
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not observed in the peripheral blood of any of the 78 children,
although this does not exclude their presence in the liver and other
tissues as demonstrated by non-human primate studies .
The pattern of cytokine production by malaria-specific CD4+T
cells was noted to differ markedly based on children’s prior
incidence of malaria (Fig. 2a–c). Both IL-10-producing CD4+T
cells and IFNc-producing CD4+T cells were present at higher
frequencies among children with a higher prior incidence of
malaria ($2 episodes ppy) than among those with a lower prior
incidence (,2 episodes ppy, P,0.001 and P=0.02, respectively,
Fig. 2a). Most strikingly, CD4+T cells co-producing IFNc and IL-
10 dominated the response among children with higher prior
incidence, but were virtually absent among lower incidence
children (P,0.001, Fig. 2b). Production of TNFa followed the
opposite pattern, with higher frequencies of TNFa+/IL102CD4+
T cells observed among children with lower prior incidence than
among those with a higher prior incidence (P=0.003, Fig. 2b).
Interestingly, despite these differences in cytokine production
profiles, the overall frequency of malaria-specific CD4+T cells (i.e.
those producing any cytokine) did not statistically differ between
the higher and lower incidence groups (P=0.13).
We also analyzed the relationship of prior malaria incidence
with the ‘‘composition’’ of the malaria-specific response (i.e. the
proportion of each cytokine combination amongst the total
malaria-specific CD4+T cell population), and found similar
results. Among children with ,2 episodes ppy, TNFa-producing
CD4+T cells (including TNFa single-producers and IFNc/TNFa
double producers) comprised a greater proportion of the malaria-
specific response than among children with $2 prior episodes ppy,
whereas in children with a higher prior malaria incidence, IL-10-
producing CD4+T cells (including IL-10 single-producers and
IFNc/IL-10 double producers) comprised a far greater fraction of
the malaria-specific response (P,0.001, Fig. 2c). There was no
significant difference in the proportion of IFNc-producing CD4+
T cells between children with higher and lower incidence. These
findings suggest that the functional phenotype of the malaria-
specific CD4+T cell response differs according to prior exposure,
and that with more prior episodes, the overall response is more
regulatory (IL-10 producing) and less inflammatory (TNFa
IFNc/IL-10 co-producing CD4+T cells correlate with
recent malaria exposure
While the data above demonstrate that there is a strong
relationship between the functional phenotype of malaria-specific
CD4+T cells and prior malaria history, we wished to determine
whether this phenotype was influenced by the time elapsed since
the most recent malaria episode, the cumulative number of prior
malaria episodes, or both, as these parameters are both logically
and statistically related (Spearman’s Rho=20.46, P,0.001). We
observed a strong inverse correlation between the frequency of
IFNc+/IL-10+/TNFa2CD4+T cells and the duration since the
last episode of malaria (Spearman’s Rho=20.39, P,0.001,
Fig. 3d), with more recent malaria associated with a higher
frequency of these co-producing cells, as well as a positive
correlation with the total cumulative number of prior episodes
(Spearman’s Rho=0.23, P=0.04, Fig. 3e). However, when
assessed in a multivariate model, the frequency of malaria-specific
IFNc/IL-10 co-producing CD4+T cells remained strongly
associated with the duration since malaria, whereas the total prior
incidence was no longer significant. Similar results were observed
for total IL-10 (Fig. 3a) and total IFNc-producing (Fig. 3b)
populations, and when assessing the duration since last episode of
parasitemia (data not shown). Interestingly, the opposite relation-
ship was observed between total TNFa+producing cells and the
Table 1. Descriptive statistics of study cohort.
Number of children enrolled78
Median age in months at time of study enrollment (IQR)5.6 (3.6–7.5)
Person-years observed from enrollment until time of blood draw 291.6
Median age in months at time of blood draw (IQR)50.9 (48.6–51.4)
Person-years observed from blood draw until end of study 60.0
Total incident episodes of malaria1855
Median incidence of malaria
Prior to blood draw (ppy (IQR)) 5.7 (3.9–7.0)
From blood draw to end of study (ppy, IQR) 5.1 (2.5–6.9)
Median days since last episode of malaria (IQR) 37 (16–66)
Median days until next episode of malaria (IQR) 47 (20–101)
Monthly period prevalence of asymptomatic parasitemia*
Prior to blood draw5%
From blood draw to end of study 11%
Symptomatic malaria at the time of blood draw, n (%) 1 (1.2%)
Parasitemia at the time of the blood draw, n (%)17 (22%)
Note: IQR, interquartile range.
*Asymptomatic parasitemia defined as positive routine blood smear in the absence of fever that was not followed by the diagnosis of malaria in the subsequent seven
days. Period prevalence calculated as the number of episodes/total months observed.
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duration since last episode of malaria, with more recent malaria
associated with a lower frequency of TNFa -producing cells
(Spearman’s Rho=0.23, P=0.041, Fig. 3c). Further, there was no
significant correlation between the number of cumulative prior
malaria episodes and TNFa+producing cells. Together these data
suggest that recency of malaria infection, rather than the total
number of past episodes, exerts a dominant influence on the
functional phenotype of malaria-specific CD4+T cells. Similar
findings were obtained when analyzing the ‘‘composition’’ (i.e. the
proportion of responding cells producing IFNc, TNFa, and/or
IL10) of the malaria-specific response and duration since last
Malaria-specific CD4+T cells are not independently
associated with protection from malaria
Protection from clinical malaria in naturally exposed individuals
can be defined using a number of outcomes, including a delayed
time to reinfection [37,38,41–43,51], a decreased incidence of
malaria over time , and/or a decreased probability of clinical
disease once parasitemic . In all cases, identification of
immune correlates of protection is challenging due to the difficulty
of distinguishing protection from a lack of exposure to malaria-
infected mosquitos [45,46]. To address this, we assessed the
relationship between malaria-specific T cell functional subsets and
protection from malaria, while adjusting for prior malaria
(duration since last episode and/or cumulative number of prior
episodes) as a surrogate measure of exposure intensity. We also
evaluated potential associations with the overall prevalence of
asymptomatic parasitemia, as clinical immunity to malaria is
normally characterized by a transition from symptomatic to
asymptomatic disease .
In univariate Cox proportional hazards analysis evaluating time
to next episode of malaria, a higher frequency of CD4+T cells
producing any IFNc or IL10, or the combinations IFNc+/IL-10+/
TNFa2and IFNc2/IL-10+/TNFa2was associated with a
significantly increased hazard of malaria (Table 2, left columns).
However following adjustment for surrogates of exposure intensity
(duration since last episode of malaria and/or cumulative prior
malaria episodes) in a multivariate model, none of these
associations remained significant. Similar relationships were
observed when we analyzed the total malaria incidence in the
year following the assay in a multivariate regression model
(Table 2, middle columns). However, in this analysis both IFNc+/
IL-10+/TNFa2(IRR 1.40 per 10 fold increase, P=0.038) and any
IL-10-producing CD4+T cells (IRR 1.41 per 10 fold increase,
P=0.039) remained independently associated with an increased
risk of malaria after controlling for duration since last malaria
infection. Nearly identical results were obtained when analyzing
the total composition of cytokine producing cells: both the fraction
of IFNc+/IL-10+/TNFa2and any IL10+cells among all cytokine-
producing cells were associated with increased malaria risk (IRR
1.47, P=0.038 and 1.40, P=0.039 per 50% increase in fraction of
responding cells, respectively). Together, these data suggest that
the dominant population of malaria-specific CD4+cells, which co-
produce IFNc and IL-10, are not associated with protection from
future malaria, and may in fact be associated with an increased
risk of malaria.
We next assessed the relationship of TNFa2producing CD4+T
cells with protection. In RTS/S vaccine recipients, malaria-
specific CD4+T cells producing TNFa in the absence of IFNc or
IL-2 have recently been shown to correlate with protection from
malaria infection . In our cohort, a greater frequency of
malaria-specific CD4+T cells producing TNFa alone (IFNc2/IL-
102/TNFa+) was associated with a significantly reduced hazard of
developing malaria (HR 0.31, P=0.015 per 10 fold increase) and
lower prospective incidence (IRR 0.44, P=0.004 per 10 fold
increase) in univariate analysis, but in multivariate models
controlling for duration since malaria and/or cumulative prior
malaria episodes, these associations were no longer significant
(Table 2). Interestingly, however, the frequency of malaria-specific
CD4+T cells producing any TNFa was inversely associated with
the monthly prevalence of asymptomatic parasitemia, even after
controlling for duration since last episode of malaria and/or
cumulative prior malaria episodes (PRR 0.41 per 10 fold increase,
P=0.011). Thus, the absence of malaria-specific CD4+T cells
producing TNFa may be associated with the phenotype of
IFNc/IL-10 co-producing cells express T-bet and are of an
effector memory phenotype
Although IL10 production by T cells was initially believed to
occur predominantly within Th2 and FoxP3+TregCD4+T cell
subsets, it is now known that additional subsets, including cells
expressing the Th1 master regulator T-bet, produce IL-10 under
conditions of continuous antigen exposure [56,57]. We assessed
transcription factor expression within the dominant population of
malaria-specific IFNc/IL-10 co-producing cells (Fig. 4a) and
found that these cells uniformly were TBet+and FoxP32(Fig. 4b–
c). These IFNc/IL-10 co-producing CD4+T cells were predom-
inantly of an early effector memory phenotype (CD45RA2,
CCR72 CD27+; Fig. 4d–e).
CD4+T cell IFNc/IL-10 responses to the polyclonal mitogen
PMA/Io have previously been shown to correlate with relative
protection against severe malaria . We therefore compared the
response to iRBC and PMA/Io stimulation, and found a strong
correlation between the frequency of IFNc/IL-10 double produc-
ing CD4+T cells following iRBC or PMA stimulation (Spearman’s
Rho=0.88, P,0.001, Supplemental Fig. S1). As PMA/Io stimu-
lation is thought to induce cytokine production by recently
activated cells, these data suggest that this mitogen stimulates
cytokine production by malaria-specific T cells that have recently
seen their cognate antigen.
Plasma IL-10 levels are elevated during malaria infection
but do not correlate with the frequency of IL-10
producing CD4+T cells
IL-10 levels measured concurrently in plasma were significantly
higher among children with parasitemia at the time of the blood
draw compared with children with no parasitemia (median
30.4 pg/ml vs 11.4 pg/ml, P=0.0035), consistent with prior
Figure 1. T cell responses to malaria-infected red blood cells using multiparameter flow cytometry. A. Gating strategy to identify live
CD3+cd2T cells. B. Intracellular cytokine assay demonstrating the T cell response of one representative malaria-exposed child to Pf-infected RBC
(iRBC; bottom row), with negative controls (uRBC and media) and positive control (PMA/Io) shown in rows above. Shown are CD8 (first column) and
CD4 (right 3 columns) production of IFNc (y-axis, columns 1–3), TNFa (x-axis, columns 1–2; y-axis, column 4), and IL-10 (x-axis, column 3–4). C. The
overall malaria-specific CD4+T cell response (left column) is followed by the overall frequency of CD4+T cells producing IFNc, IL-10, and TNFa in all
participants (n=78, horizontal black lines indicate the median response for each group, *** P,0.001, Wilcoxon Rank-Sum).
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Figure 2. Prior malaria incidence influences function of malaria-specific CD4+T cell response. A. The overall malaria-specific CD4+T cell
response (left column) is followed by the overall frequency of CD4+T cells producing IFNc, IL-10, and TNFa stratified by prior malaria incidence. Blue
dots represent responses from children with lower prior malaria incidence (,2 episodes ppy, n=10) and red dots represent responses from children
with higher prior malaria incidence ($2 episodes ppy, n=68,* P,0.05, *** P,0.001, Wilcoxon Rank-Sum. Horizontal black lines indicate the median
response for each group). Median frequencies of cytokine producing cells were similar in children with $2–5 and .5 episodes ppy (data not shown).
B–C. Boolean gating of malaria-specific CD4+T cells reveals 7 distinct cytokine-producing populations. Shown are the absolute frequency (B) and the
relative proportion (C) of each individual combination of IFNc, IL-10, or TNF-producing cells. Blue dots again represent responses from children with
,2 prior episodes ppy, and red dots represent responses from children with $2 episodes ppy (* P,0.05, ** P,0.01, *** P,0.001, Wilcoxon Rank-Sum.
Horizontal black lines indicate the median response for each group). For pie charts, blue arcs represent total proportion of CD4+T cells producing
TNFa; red arcs represent total proportion of CD4+T cells producing IL-10; and green arcs represent total proportion of CD4+T cells producing IFNc.
The proportion of IFNc2/IL-10+/TNFa2(population 3) producing cells is ,0.01% of the total malaria-specific response, and thus does not have a
visible corresponding pie slice.
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reports [58–61]. Similar to IL-10 producing CD4+T cells, plasma
IL-10 strongly correlated with recent malaria (Spearman’s
Rho=0.30, P=0.009, Supplemental Fig. S2a). However plasma
IL-10 levels did not correlate with the frequency of total IL-10
producing CD4+T cells (Spearman’s Rho =0.11, P=0.35,
Supplemental Fig. S2b), suggesting that additional cell types,
including cells of the myeloid lineage, may contribute to plasma
IL-10 levels during malaria infection .
Figure 3. CD4+T cell functions and relationship with recent and cumulative malaria infection. The frequencies of CD4+T cells producing
any IL-10 (A) and any IFNc (B) are inversely associated with days since last malaria episode (Spearman’s Rho=20.39, P,0.001; Rho=20.23, P=0.046,
respectively). Frequencies of CD4+T cells producing any TNFa (C) are positively correlated with days since last malaria episode infection (Spearman’s
Rho=0.23, P=0.041). Frequencies of IFNc+/IL-10+/TNFa2CD4+T cells are inversely associated with days since last malaria episode (D, Spearman’s
Rho=20.39, P,0.001) and positively associated with the cumulative number of episodes in the prior 3 years (E, Spearman’s Rho=0.26, P=0.023).
Table 2. Magnitude of malaria-specific CD4+T cell responses and protection from symptomatic malaria.
Time until malariaFuture incidence of malaria Prevalence of asymptomatic parasitemia
UnivariateMultivariate* UnivariateMultivariate*Univariate Multivariate*
% CD4+T cells (Log10) HRP HRPIRRPIRRPPRRPPRRP
1.79 0.051 1.140.6711.420.0851.29 0.1340.51 0.1110.430.037
2.220.001 1.73 0.0831.65 0.0021.400.038 0.860.7030.74 0.482
n/an/an/an/an/a n/an/a n/an/an/a n/an/a
1.730.047 1.530.1981.43 0.0591.24 0.2161.630.247 1.940.130
1.32 0.3651.24 0.5321.020.917 1.040.8151.23 0.6491.30 0.567
0.650.249 1.520.2860.89 0.6491.32 0.1990.390.052 0.480.152
0.310.015 1.250.7250.440.004 0.730.2970.430.070 0.610.422
1.840.039 1.740.0911.440.076 1.410.0620.60 0.28.600.283
2.260.0011.640.117 1.700.0011.410.039 1.000.99.88.787
Note: HR: Hazard Ratio; IRR: incidence rate ratio; PRR: prevalence rate ratio. Numbered rows refer to cell populations described in Figure 2. Associations in row 3 are not
applicable because these responses were undetectable.
*Multivariate models controlled for duration since last malaria infection. Similar results were obtained when controlling for cumulative episodes over prior 3 years and
for the presence or absence of parasitemia.
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Impaired malaria-specific CD4+T cell proliferation in
heavily exposed children is partially reversed by IL-10
Immunomodulation through downregulation of antigen-specific
CD4+T cell proliferative responses has been described in the
context of several chronic parasitic infections [62–65], as well as
chronic viral infections that result in persistent antigenemia
[66,67]. We assessed proliferation of malaria-specific CD4+T
cells by measuring CFSE dilution following stimulation with
schizont extract (PfSE) in a subset of children (n=42). A significant
inverse correlation was observed between malaria-specific CD4+T
cell proliferation and cumulative prior incidence (Spearman’s
Rho=20.39, P=0.011; Fig. 5a), suggesting that heavy antigen
exposure may result in a proliferative defect in malaria-specific
CD4+T cells. We also observed an inverse correlation between
CD4+T cell proliferation following PfSE stimulation and the
frequency of IFNc/IL-10 co-producing CD4+T cells (Spearman’s
Rho=20.31, P=0.049). It has previously been suggested that
IFNc/IL-10 co-producing CD4+T cells may play an autoregu-
latory role through suppression of proliferative responses in an IL-
10 mediated manner . We therefore assessed whether in vitro
IL10 blockade would reverse the observed proliferative defect.
The ability of CD4+T cells to proliferate in response to PfSE was
partially restored in 8 of 9 subjects upon blockade of IL-10
receptor alpha (fold change 1.7, P=0.01, Fig. 5b–c), suggesting
that the CD4+T cell proliferative defect observed in heavily
exposed children may be in part due to IL-10 mediated
In this cohort of young children living in an area of very high
transmission intensity in Uganda, very little evidence of clinical
immunity had emerged by five years of age. In this setting, the
functional phenotype of the malaria-specific CD4+T cell response
was significantly influenced by prior malaria exposure; with less
prior malaria, the overall malaria-specific CD4+T cell response
was more inflammatory (TNFa-producing), but with heavier
Figure 4. CD4+T cells co-producing IFNc and IL-10 express T-bet and are of an early effector memory phenotype. CD4+T cells were
analyzed for transcription factor expression and maturational phenotype. Panel 4A shows the proportion of CD4+T cells co-producing IFNc/IL-10 in
response to iRBC stimulation for one representative child (upper right quadrant)) and panel 4B shows intranuclear transcription factor staining with T-
bet and FoxP3 of all CD4+T cells (grey) with IFNc/IL-10 CD4+T cells overlayed (4B, blue dots). Panel 4C shows the percentage of iRBC-stimulated
IFNc+, IL-10+, and IFNc/IL-10 co-producing CD4+T cells staining for intranuclear T-bet (n=10). CD4+T cells were also analyzed for cell surface
maturation markers CD45RA (x axis, panels 4D–E), CCR7 (4D), and CD27 (4E). The total CD4+T cell population is shown in grey, with IFNc/IL-10 co-
producing CD4+T cells overlayed as blue dots.
CD4 Response to Malaria in Highly Exposed Children
PLOS Pathogens | www.plospathogens.org8 January 2014 | Volume 10 | Issue 1 | e1003864
exposure, the overall malaria-specific response was more regula-
tory (IL-10 producing). To our knowledge, this is the first study to
show that Th1 IFNc/IL-10 co-producing cells constitute the
dominant population of CD4+T cells responding to malaria in
heavily exposed children. Moreover, we found no evidence that
these IFNc/IL-10 co-producing cells were associated with
protection from future malaria.
Interest in IFNc/IL-10 co-producing Th1 cells has increased in
recent years as these cells have been found to be important
regulators of the immune response to several infectious, allergic,
and autoimmune diseases [18,49,50,52,56,69,70]. In a murine
model of Toxoplasma gondii, IFNc produced by these cells was
shown to be required for pathogen eradication, and concomitant
production of IL-10 was vital for the resolution of the inflamma-
tory response and to prevent tissue pathology . However, in a
murine model of Leishmania major, co-production of IL-10 by Th1
cells prevented pathogen eradication, contributing to chronic
infection . These data suggest that IL-10 co-production by
Th1 T cells may help prevent immunopathology, but this may
come at the cost of chronic pathogen persistence .
IL-10 levels are increased during malaria infection [58,59,61]
and this regulatory cytokine is thought to play a key role in
dampening proinflammatory responses and preventing the devel-
opment of severe malarial anemia and cerebral malaria . In
mice, Th1 cells were elegantly shown to be the major producer of
IL-10 and were critical for limiting the pathology associated with
malaria infection . T cell production of IL-10 has also been
described in reports of human malaria infection [14,52,73–77].
Plebanski and colleagues described a switch in production from
IFNc to IL-10 in CD4+T cells from Gambian adults stimulated
with altered peptide ligands of the circumsporozoite protein, with
an associated suppression of proliferative responses in vitro . T
cells co-producing IFNc/IL-10 following nonspecific PMA/
ionomycin stimulation were described in the context of acute
malaria infection , and were also more abundant among
children with uncomplicated rather than severe malaria ,
consistent with a role in modulating inflammation. More recently,
Gitau and colleagues described malaria-specific co-production of
IFNc and IL-10 following stimulation of CD4+T cells with a
variety of expressed PfEMP variants, although these co-producing
cells represented a minor fraction of the total antigen-specific
CD4+T cell response . The potential role that malaria-specific
IFNc/IL-10 co-producing CD4+T cell cells play in mediating or
inhibiting protective immunity in humans has not thus far been
We observed that CD4+T cells co-producing IFNc/IL-10
dominate the T cell response to malaria in heavily exposed
children, and that the overall frequency and proportion of these
cells among malaria-specific T cells was strongly correlated with
recent exposure to malaria, more so than cumulative prior
exposure. These IFNc/IL-10 co-producing cells express T-bet,
indicating that they have differentiated along the Th1 pathway.
The dominance of this functional phenotype among malaria-
specific T cells has not previously been reported, and may be
related to the unusually high malaria exposure intensity of our
cohort, as this cell population was of much lower frequency among
children with ,2 malaria episodes per year. Further, frequencies
of IL-10–producing and IFNc/IL10 co-producing cells were not
associated with protection from future malaria after controlling for
recent and/or cumulative prior malaria, but were instead
associated with an increased risk of cumulative malaria in the
year following the assay, although this may be due to the inability
to fully adjust for the level of environmental exposure to malaria
using clinical surrogates such as prior malaria incidence.
We further observed that heavy malaria exposure was
associated with a decreased ability of CD4+T cells to proliferate
in response to malaria antigens, and that this impaired prolifer-
ation is partially reversed by IL-10 blockade. These data are
consistent with in vitro studies of recently activated IL7R2,
CD252, CD4+T cells which co-produce IFNc and IL-10 and
T cell proliferation through IL-10 dependent
mechanisms . In addition, prior studies have shown that IL-
10 blockade increases malaria-specific IFNc cytokine production
in filaria-coinfected individuals  and in cord blood mononu-
clear cells from neonates born to mothers exposed to malaria .
A similar IL10-dependent functional impairment of CD4+T cells
has been described in other infections such as HIV that are
characterized by chronic high-level antigen stimulation [80,81].
Together, these data are consistent with the hypothesis that
IFNc/IL-10 co-producing CD4+T cells primarily function to limit
Figure 5. CD4+T cell proliferation impaired in setting of heavy prior exposure. A. The proliferation fold change (fraction of CFSE-lo cells
following PfSE stimulation vs uRBC stimulation) is significantly reduced in children with higher prior malaria exposure ($2 episodes ppy, n=33) vs
children with low malaria exposure (,2 episodes ppy, n=9, P=0.007, Wilcoxon Rank Sum. Horizontal lines show medians for each group with 95%
CI). B. Impact of IL-10 blockade on CD4+T cell proliferation following PfSE stimulation in one representative subject. The left panel shows CFSE
dilution following PfSE stimulation with addition of isotype control, and the right panel shows CFSE dilution following PfSE stimulation with addition
of anti IL-10 receptor a blocking antibody. C. Change in the percent of CD4+T cells divided following isotype control vs anti IL-10 receptor a blocking
antibody in a subset of 9 children from whom additional cells were available (fold change 1.7, P=0.01).
CD4 Response to Malaria in Highly Exposed Children
PLOS Pathogens | www.plospathogens.org9January 2014 | Volume 10 | Issue 1 | e1003864
the immunopathology associated with malaria infection – includ-
ing cerebral malaria, anemia, and death - through autoregulation
of CD4+T cell proliferation and cytokine production. A similar
role has been attributed to IL-10-producing Th1 cells in other
parasitic diseases characterized by heavy continuous antigen
exposure [49,50], with evidence that IL-10 produced by Th1
effector cells acts through a negative feedback loop to regulate
CD4+T cell responsiveness, limiting inflammation and tissue
pathology at the cost of impaired pathogen clearance [56,71]. It is
possible that unmeasured confounders, such as helminthic co-
infections, may have been unequally represented in the high and
low-incidence groups, particularly as the lower incidence children
were more likely to reside in town. However routine deworming
was performed in all study subjects every 3–6 months, lessening
the likelihood that co-infection with helminths explains our
findings. Further studies are needed to determine if IL-10-
producing Th1 cells contribute to pathogen persistence, and to
the failure of humans to develop sterile protective immunity to
In addition, we found that children with the fewest prior
episodes of malaria were significantly more likely to have malaria-
specific production of TNFa without IL-10, and that the absence
of this inflammatory cytokine was associated with the phenotype of
asymptomatic infection. Studies in murine models have shown
that TNFa plays an important role in inhibiting the development
of hepatic stages of malaria [82,83]. Importantly, a recent study of
RTS/S vaccine recipients identified antigen-specific CD4+T cell
production of TNFa as a correlate of protection in vaccinees .
In contrast to that study, we found no evidence of protection after
controlling for prior malaria, though we did observe that
asymptomatic infection was inversely associated with the frequen-
cy of TNFa producing CD4+T cells, independent of prior
malaria. Together our data suggest that production of this
inflammatory cytokine may decrease with increasing cumulative
malaria exposure, enabling a transition to asymptomatic infec-
A notable strength of this study was the availability of
comprehensive malaria clinical histories spanning from early
infancy to the time of the immunologic assessment, plus one
additional year thereafter, which enabled us to assess for T cell
correlates of both exposure to and protection from malaria.
Several prior studies have reported correlations between T cell
responses or IL-10 production and protection from malaria in
naturally exposed children [37,42,53], but such studies have
generally been unable to adequately account for prior malaria
exposure. While we did observe associations, both positive and
negative, between malaria-specific CD4+T cells of varying
functional phenotypes and the risk of future malaria, most of
these associations were not significant after adjusting for recent or
cumulative prior episodes of malaria, surrogates for the level of
ongoing exposure to malaria-infected mosquitos. Hence the failure
to account for malaria exposure intensity may lead to spurious
associations with protection. Although we did not identify T cell
phenotypes that were associated with protection from future
malaria, this may be related to the young age of children in this
cohort, as there was little evidence that clinical immunity had
developed prior to 5 years of age. Future longitudinal studies
examining responses in older children and adults, incorporating
more precise entomological measurements of malaria exposure,
In conclusion, among naturally exposed children living in a high
endemicity setting, malaria-specific CD4+T cells were present in
the vast majority of children, and their functional phenotype
differed greatly based on the level of prior exposure to malaria, in
particular the duration of time since last infection. IFNc/IL-10 co-
producing Th1 cells dominated the CD4+T cell response to
malaria in these heavily exposed children, but were not associated
with protection from future infection. These CD4+T cells may
play important immunomodulatory roles in the pathogenesis of
malaria in childhood.
Study site, participants, and follow-up procedures
Samples for this study were obtained from children enrolled in
the Tororo Child Cohort (TCC) in Tororo, Uganda, a rural
district in south-eastern Uganda with an entomological inoculation
rate (EIR) estimated at 379 infective bites per person year (PPY) in
2012 . Details of this cohort have been described elsewhere,
and the sub-study described in this report includes only HIV-
uninfected children born to HIV-uninfected mothers [20,84–87].
Briefly, children in the TCC were enrolled at infancy (median 5.5
months of age) and followed for all medical problems at a
dedicated study clinic open seven days a week. Monthly
assessments were done to ensure compliance with study protocols
and perform routine blood smears. All children were prophylac-
tically dewormed with mebendazole every 3–6 months per
Ugandan Ministry of Health guidelines . Children who
presented with a documented fever (tympanic temperature
$38.0uC) or history of fever in the previous 24 hours had blood
obtained by finger prick for a thick smear. If the thick smear was
positive for malaria parasites, the patient was diagnosed with
malaria regardless of parasite density, and given artemisinin-based
combination therapy for treatment of uncomplicated malaria.
Children were followed until 5 years of age unless prematurely
Incident episodes of malaria were defined as all febrile episodes
accompanied by any parasitemia requiring treatment, but not
preceded by another treatment in the prior 14 days . The
incidence of malaria was calculated as the number of episodes per
person years (ppy) at risk. Asymptomatic parasitemia was defined
as a positive routine blood smear in the absence of fever that was
not followed by the diagnosis of malaria in the subsequent seven
days, and was reported as a count outcome as it was measured via
monthly surveillance. The period prevalence of asymptomatic
parasitemia was calculated as the number of episodes/total
Written informed consent was obtained from the parent or
guardian of all study participants. The study protocol was
approved by the Uganda National Council of Science and
Technology and the institutional review boards of the University
of California, San Francisco, Makerere University and the Centers
for Disease Control and Prevention.
Sample collection and processing
At approximately 4 years of age, ,6–10 mls of whole blood was
obtained from each subject in acid citrate dextrose tubes. Plasma
was collected, and peripheral blood mononuclear cells (PBMC)
were isolated by density gradient centrifugation (Ficoll-Histopa-
que; GE Life Sciences). PBMC were cryopreserved in liquid
nitrogen and shipped to our laboratory in San Francisco for
Plasmodium falciparum blood-stage 3D7 parasites were grown by
standard methods and harvested at 5–10% parasitemia. Red blood
CD4 Response to Malaria in Highly Exposed Children
PLOS Pathogens | www.plospathogens.org10 January 2014 | Volume 10 | Issue 1 | e1003864
cells infected with mature asexual stages were purified magneti-
cally, washed, and cryopreserved in glycerolyte prior to use
(iRBC). Uninfected RBCs (uRBC) were used as controls. To assess
the impact of parasite diversity on T cell responses, responses to
iRBCs prepared from 4 distinct Tororo field strains were
compared to iRBC prepared from 3D7. Responses to the 4 field
strains were very similar, indicating that parasite diversity does not
significantly influence the T cell response magnitude (Supplemen-
tal Fig. S3). Schizont extracts (PfSE) for use in proliferation assays
 were prepared by 3 freeze-thaw cycles of iRBC in liquid N2
for freezing and 37uC water bath for thawing, then resuspended in
R10 media and stored at 220uC until use.
Intracellular cytokine staining
Thawed PBMC were rested overnight in 10% fetal bovine
serum (Gibco) and counted prior to stimulation with uRBC,
iRBC, media, or phorbol miristate acetate/calcium ionophore
(PMA/Io) at 16106cells/condition. An E:T ratio of 1:3 was used
with uRBC and iRBC . Anti-CD28 and –CD49d were added
for costimulation (0.5 mg/ml, BD Pharmingen). Brefeldin-A and
Monensin (BD Pharmingen) were added at 6 hours of incubation
at a final concentration of 10 mg/ml to inhibit cytokine secretion.
At 24 hours of incubation, cells were washed, fixed and
Ebioscience fix/perm reagents used for nuclear transcription
Surface and/or intracellular staining of PBMC was done with
standard protocols [91,92] using the following antibodies for the
primary analysis: Brilliant violet 650-conjugated CD4 (Biole-
gend), PerCP–conjugated anti-CD3, APC-H7-conjugated CD8,
PE-Cy7-conjugated IFNc, PE-conjugated anti-IL-10, and FITC-
conjugated TNFa (BD Pharmingen). Alexa 700-conjugated
CD14 and CD19, APC-conjugated anti-cd (Biolegend), and
Live/dead aqua amine (Invitrogen) were included as exclusion
gates to reduce unwanted nonspecific antibody binding when
measuring antigen-specific T cell populations . Additional
experiments utilized Brilliant violet 421-conjugated anti-IL-2,
Brilliant violet 605-conjugated CD45RA, Brilliant violet 710-
conjugated CD27 (Biolegend), APC-conjugated CCR7 (R&D
Systems); eFluor 660-conjugated T-bet and FITC-conjugated
CFSE proliferation assay
Thawed PBMC were rested for one hour, washed in 10%
Human AB media (Gemini), and 3–66106PBMC were labeled
with 1 ml of 1.25 mM 5,6-carboxyfluorescein diacetate succinimi-
dyl ester (CFSE; Molecular Probes) for seven minutes. CFSE-
labeled PBMC were incubated in 96-well, deep-well culture plates
(Nunc, Roskilde, Denmark) at a density of 106PBMC per well at a
final volume of 1 ml for 7 days. In a subset of patients, CFSE-
labeled PBMC were incubated with antigen in the presence of
(clone 37607; R&D Systems) or IgG1 isotype control antibody at
10 mg/mL. Antigens tested included media, phytohemagglutinin
(PHA; 5 mg/mL; Sigma-Aldrich), uRBC, or PfSE at an E:T ratio
of 1:3 schizont equivalents. At day 7 cells were treated with 100
units DNase I (Invitrogen) in culture medium at 37uC for 10 min,
washed, and stained with surface antibodies (PerCP–conjugated
anti-CD3, APC-H7-conjugated CD8 (BD Pharmingen), Brilliant
and CD19, and APC-conjugated anti-cd (Biolegend)) before
(IL-10Ra) blocking antibody
Flow cytometry data analysis
Flow cytometry profiles were gated on CD3+, cd-negative
lymphocytes, and 200,000 to 300,000 events were collected.
Samples were analyzed on an LSR2 three laser flow cytometer
(Becton Dickinson) with FACSDiva software. Color com-
pensations were performed for each patient’s PBMC using
beads or samples single stained for each of the fluorochromes
used. Data were analyzed using FlowJo (Tree Star, San Carlos,
CA) and Pestle (version 1.7)/SPICE (version 5.3; M. Roederer,
Vaccine Research Center, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda,
MD). In experiments with CFSE-labeled cells, the ratio of
CFSE-lo cells following PfSE stimulation vs uRBC stimulation
was calculated and reported as the proliferation fold change.
The FlowJo Proliferation Platform provided additional informa-
tion about the division characteristics of CD4+T cells. To
examine the effect of IL-10 blockade on proliferation, these
parameters for CD4+T cells were generated in samples that had
been stimulated with PfSE plus anti-IL-10Ra and compared to
the values obtained from samples stimulated with PfSE plus
Plasma levels of IL-10 were measured by dual Ab sandwich-
ELISA kits, according to manufacturer’s instruction (R&D
Systems, Minneapolis, MN). Each sample was tested in duplicate,
and cytokine concentrations were calculated using a standard
curve generated from recombinant cytokines. Cytokine values
were expressed as picograms (pg) per milliliter.
All statistical analyses were performed using Prism 4.0
(GraphPad), STATA version 12 (College Station), or SPICE
v.5.3 (NIAID). Frequencies of malaria-specific cytokine producing
T cells (alone or in combination) are reported after background
subtraction of the frequency of the identically gated population of
cells from the same sample stimulated with control. Background-
subtracted responses were consider positive if .0.01% parent
population . Comparisons of cytokine frequencies between
prior malaria incidence groups were done using the Wilcoxon rank
sum test, and the Wilcoxon signed-rank test was used to compare
paired data. Statistical analyses of global cytokine profiles (pie
charts) were performed by partial permutation tests using the
SPICE software  . Continuous variables were compared using
Spearman correlation. For multivariate regression models, non-
normal variables were log-transformed. To allow for nonlinear
relationships between clinical exposure variables and immunologic
outcomes, we fit linear splines with knots chosen to best represent
observed relationships. Associations between immune parameters
and time to next malaria episode were evaluated using the
Kaplan-Meier product limit formula, and a multivariate cox
proportional hazards model was used to adjust for surrogates of
malaria exposure found to be associated with these parameters
(duration since last episode of malaria and/or cumulative episodes
in the prior 3 years). Negative binomial regression was used to
estimate associations between immune parameters and the
prospective incidence of malaria in the following year (incidence
rate ratios, IRR) and prevalence of asymptomatic parasitemia
during the entire study period (prevalence rate ratios, PRR),
adjusting for prior malaria as above. Two-sided p-values were
calculated for all test statistics and P,0.05 was considered
CD4 Response to Malaria in Highly Exposed Children
PLOS Pathogens | www.plospathogens.org11January 2014 | Volume 10 | Issue 1 | e1003864
stimulation. The frequency of iRBC-stimulated CD4+T cells
producing IFNc/IL-10 is strongly correlated with the frequency of
PMA/Io-stimulated CD4+ T cells co-producing these cytokines
(Spearman’s Rho=0.86, P,0.0001).
Relationship between iRBC and PMA/Io
malaria and CD4+T cells. Plasma IL-10 is inversely associated
with days since last episode of malaria (A, Spearman’s
Rho=20.30, P=0.009). There is no significant association
between plasma IL-10 levels and the frequency of IL-10 producing
CD4+ T cells (B, Spearman’s Rho=0.11, P=0.35).
Relationship of Plasma IL-10 levels with
blood cells comparing responses to 3D7 and field
isolates. Intracellular cytokine staining assay demonstrating the
CD4+T cell response of a malaria-exposed child to several strains
of field isolates, with negative control (uRBC), positive control
T cell responses to malaria-infected red
(PMA/Io), lab-adapted 3D7, and four distinct field isolates from
Tororo, Uganda (provided courtesy of Dr. Philip Rosenthal). Plots
are gated on CD4+T cells and shown are frequencies of CD4+T
cells making IFNc alone (top left quadrant), IFNc and IL-10 (top
right quadrant), and IL-10 alone (bottom right quadrant).
We are grateful to all the parents and guardians for kindly giving their
consent and to the study participants for their cooperation. We thank all
the members of the study team for their tireless effort and excellent work.
We thank Dr. Phil Rosenthal and Jenny Legac for provision of the parasite
strains used for these experiments.
Conceived and designed the experiments: PJ BG GD MEF. Performed the
experiments: PJ IEJ KB FN SW CE JB. Analyzed the data: PJ IEJ KB FN
GD BG MEF. Contributed reagents/materials/analysis tools: AA MKM
EA MRK JWT GD MEF. Wrote the paper: PJ IEJ KB FN AA SW CE
MKM EA JB BG JWT MRK GD MEF.
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