The spleen CD4+ T cell response to blood-stage Plasmodium chabaudi malaria develops in two phases characterized by different properties.

Abstract

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-A(b-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-α and IFN-γ production depend mostly on conventional CD4(+) T cells. IFN-γ is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-γ and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

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The Spleen CD4+ +T Cell Response to Blood-Stage
Plasmodium chabaudi Malaria Develops in Two Phases
Characterized by Different Properties
Sandra Marcia Muxel1*, Ana Paula Freitas do Rosa ´rio1, Cla ´udia Augusta Zago1, Sheyla Ine ´s Castillo-
Me ´ndez1, Luiz Roberto Sardinha1,2, Se ´rgio Marcelo Rodriguez-Ma ´laga1, Niels Olsen Saraiva Ca ˆmara1,
Jose ´ Maria A´lvarez1, Maria Regina D’Impe ´rio Lima1
1Departamento de Imunologia, Instituto de Cie ˆncias Biome ´dicas, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Sa ˜o Paulo, Brazil, 2Instituto Israelita de Ensino e Pesquisa Albert
Einstein, Sa ˜o Paulo, Sa ˜o Paulo, Brazil
Abstract
The pivotal role of spleen CD4+T cells in the development of both malaria pathogenesis and protective immunity makes
necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium
infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4+T cells during P.
chabaudi malaria. We took advantage of the fact that a great proportion of CD4+T cells generated in CD1d-/-mice are I-Ab-
restricted (conventional cells), while their counterparts in I-Ab-/-mice are restricted by CD1d and other class IB major
histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4+T cells are the main
protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and
chronic parasitaemias. The early phase of the conventional CD4+T cell response is intense and short lasting, rapidly
providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal
immunoglobulin. Both TNF-a and IFN-c production depend mostly on conventional CD4+T cells. IFN-c is produced
simultaneously by non-conventional and conventional CD4+T cells. The early phase of the response finishes after a week of
infection, with the elimination of a large proportion of CD4+T cells, which then gives opportunity to the development of
acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4+T cells occurs at the beginning of the
second phase of the response, but not earlier, helping both IFN-c and parasite-specific antibody production. We concluded
that conventional CD4+T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-
conventional CD4+T cells as a link between innate and acquired immunity. This study contributes to the understanding of
malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind
immune responses to Plasmodium infection.
Citation: Muxel SM, Freitas do Rosa ´rio AP, Zago CA, Castillo-Me ´ndez SI, Sardinha LR, et al. (2011) The Spleen CD4+T Cell Response to Blood-Stage Plasmodium
chabaudi Malaria Develops in Two Phases Characterized by Different Properties. PLoS ONE 6(7): e22434. doi:10.1371/journal.pone.0022434
Editor: Ben L. Kelly, Louisiana State University, United States of America
Received September 30, 2010; Accepted June 28, 2011; Published July 21, 2011
Copyright: ? 2011 Muxel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from FAPESP (www.fapesp.br), CAPES (www.capes.gov.br), and CNPq (www.cnpq.br). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: sandrammuxel@gmail.com
Introduction
Malaria, the infectious disease caused by Plasmodium parasites, is
a major global health problem that is responsible for the death of
over a million people every year [1]. Humans with no previous
experience of malaria almost invariably develop a febrile illness
that may become severe and lead to death. The asexual blood-
stage of the parasite is responsible for the clinical symptoms of the
disease. Three overlapping syndromes, severe anaemia, cerebral
malaria and respiratory distress, account for most of the severe
cases and deaths [2]. Because many of the features of severe
malaria are similar to those of sepsis [3], over-vigorous responses
to parasites have been implicated in the aetiology of these
syndromes [4,5]. Thus, although TNF-a and IFN-c appear to be
protective against the parasite, very high serum concentrations of
proinflammatory cytokines are associated with great morbidity
[6,7]. Yet, exposure to one or two malaria infections appears to be
sufficient to induce complete protection from severe illness and
death [8], while sterile immunity to malaria parasites is probably
never achieved.
Encouragingly,someaspectsinmousemodelsofmalariaappearto
mirror the human disease with reasonable accuracy. In acutely
infected mice, the type 1 responses from CD4+and CD8+T cells are
the main participants in the development of both malaria
pathogenesis and protective immunity [9,10,11]. While TNF-a and
IFN-c are associated with the development of clinical manifestations
of the disease [12,13,14], the control of acute infection also depends
on proinflammatory cytokines [15,16,17], which together with acute-
phase antibodies [18] may promote parasite clearance by macro-
phages [19,20]. CD4+T cellsare thought to be essential for complete
elimination of the parasite during the late phase of the disease
[10,21,22], which may also hold true for humans because these cells
are critically required to help B cells produce parasite-specific high-
affinity immunoglobulin G (IgG) antibodies. However, it is not yet
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apparent how much of the pathology is dependent on a contribution
from the acquired immune response [4].
The pivotal role of CD4+T cells in the development of both
malaria pathogenesis and protective immunity makes them putative
targets for new strategies to improve the outcome of the disease. On
top of being important sources of IFN-c and helping B cells to
secrete antibodies, these cells play a key role in the regulation of
immune responses. The acquisition of clinical immunity is likely to
be coordinated by CD4+T cells, as clinical immunity remains
relatively robust over long periods after being established. The
possibility that CD4+T cells are implicated in this process is
suggested by the fact that IL-10 or TGF-b deficiency intensifies the
clinical signs in mice suffering from malaria [13,23]. Therefore, a
profound comprehension of the mechanisms involved in CD4+T
cell activation and regulation during Plasmodium infection may
improve the chances of developing effective vaccines and other
potential immunotherapies to prevent severe malaria syndromes.
CD4+T cells from the spleen, the main lymphoid organ for
protection against blood-borne infectious diseases, can be divided
into two categories based on how they recognise antigens.
Conventional CD4+T cells recognise peptides associated with
class II molecules of the major histocompatibility complex (MHC),
whereas non-conventional CD4+T cells are a heterogeneous
population mostly restricted by class IB MHC molecules [24],
which includes CD1d-restricted natural killer T (NKT) cells that
recognise lipids [25]. It is generally accepted that non-conven-
tional CD4+T cells are particularly shaped for early immune
responses to infections while conventional CD4+T cells are
responsible for acquired immunity. Because CD4+T cells from
these two categories have been implicated in the immune response
to Plasmodium parasites [2], we sought to examine in detail the
behaviour of non-conventional and conventional splenic CD4+T
cells during blood-stage P. chabaudi AS strain malaria, a suitable
model for the human disease caused by Plasmodium falciparum [26].
The information provided here is fundamental for unravelling the
molecular mechanisms behind the early phase of the CD4+T cell
response to Plasmodium infection, knowledge that could help
explain why people develop severe malaria and how they get
immune against the symptoms of the disease.
Materials and Methods
Mice, parasite and infection
Six-to-eight-wk-old C57BL/6 (wild type; WT) (originally from
The Jackson Laboratory), CD1d-/-[27] and I-Ab-/-(ABBN12) [28]
male mice were bred under specific pathogen-free conditions at the
Isogenic Mice Facility (Department of Immunology/University of
Sa ˜o Paulo, Brazil). CD1d-/-and I-Ab-/-mice were used after nine
backcrosses onto the C57BL/6 background. P. chabaudi AS strain
was maintained as described elsewhere [15]. Mice were inoculated
intraperitoneally (i.p.) with 16106infected red blood cells (iRBC).
WT and CD1d-/-mice were challenged i.p. with 16108iRBC on
day 30 postinfection (p.i.). Parasitaemias were monitored by
microscopic examination of Giemsa-stained thin tail blood smears.
Ethics statement
All procedures were in accordance with national regulations of
ethical guidelines for mice experimentation and welfare of the
Conselho Nacional de Sau ´de and Cole ´gio Brasileiro em
Experimentac ¸a ˜o Animal (COBEA) - Brazil, and the protocols
were approved by the Health Animal Committee (Comissa ˜o de
E´tica no Uso de Animais – CEUA – ICB/USP) of the Instituto de
Cie ˆncias Biome ´dicas of the Universidade de Sa ˜o Paulo, Sa ˜o Paulo,
Brazil (permit number 0019/2005 and 0036/2007).
Spleen cell suspension
Spleen cells were washed and maintained in cold RPMI 1640,
supplemented with penicillin (100 U/mL), streptomycin (100 mg/
mL), 2-mercaptoethanol (50 mM), L-glutamine (2 mM), sodium
pyruvate (1 mM), and 3% heat-inactivated foetal calf serum. All
supplements were purchased from Invitrogen Life Technologies.
The numbers of cells per spleen were counted using a Neubauer
chamber (Sigma-Aldrich).
Phenotypic analysis of spleen cells
Spleen cells (16106) were stained with allophycocyanin (APC)-,
peridin-clorophyll-protein (PercP)-,
(FITC)-, phycoerythrin (PE)- or CyChrome (Cy)-labelled mono-
clonal antibodies (mAb) (BD Pharmingen) to CD4 (H129.19),
CD8 (53-6.7), CD45R–B220 (RA3-6B2), CD69 (H1.2F3), CD21
(7G6), CD23 (B3B4) and CD1d (1B1) and with the PE-labelled
CD1d-a-GalCer tetramer from the NIH (National Institutes of
Health - USA) Tetramer Facility. Stained cells were analysed by
flow cytometry using a FACSCalibur device with CellQuest
software (BD Biosciences).
fluorescein-isothiocyanate
IFN-c intracellular detection
Spleen cells (16106) were cultured with GolgiStop according to
the manufacturer’s instructions in the presence of 5 mg/mL plate-
bound purified anti-CD3 mAb (145-2C11) or medium alone for
10 h at 37uC in a 5% CO2atmosphere. After washing, cells were
surface stained with FITC- or APC-labelled mAb to CD4 and
CD8. Cells were then fixed with Cytofix/Cytoperm buffer, stained
with PE-labelled mAb to IFN-c (XMG-1.2) diluted in Perm/Wash
buffer, and analysed by flow cytometry. All reagents were
purchased from BD Pharmingen.
CFSE proliferation assay
The proliferative T cell response was measured as previously
described [29]. Briefly, spleen cells (36107) were incubated for
30 min at 37uC with carboxyfluorescein succinimidyl ester (CFSE)
(Molecular Probes) (5 mM) in phosphate-buffered saline (PBS)
supplemented with 0.1% bovine serum albumin (BSA). Cells
(16106) were then cultured in 96-well plates (Costar) with iRBC
(36106) or medium alone for 72 h at 37uC in a 5% CO2
atmosphere. In some experiments, blocking mAb to I-Ab(AF6-
120.1) and IgG2a isotypic control (G155-178) were added to the
cultures (both mAb used at 2.5 mg/mL concentrations). After
incubation, cells were stained with PE or APC-labelled mAb to
CD4 and CD8 and analysed by flow cytometry.
Multicytokine assessment
Several cytokines (IFN-c, TNF-a, IL-2, IL-4 and IL-5) were
quantified simultaneously, by the cytometric bead array (CBA; BD
Pharmingen), using supernatants obtained from the same cultures
used in the CSFE proliferation assay. This technique uses flow
cytometry to measure soluble analytes in a particle-based
immunoassay and was conducted according to the manufacturer’s
instructions. The lower limit of detection for all cytokines in this
assay was 20 pg/mL.
Purification of cell subpopulations
Spleen cells (16108) from CD1d-/-mice were incubated with
PE-labelled mAb to CD4 and with anti-PE microbeads diluted in
PBS with 0.5% BSA and 2 mM ethylenediaminetetraacetic acid
(EDTA) (Invitrogen Life Technologies). Cells were then purified
using LS columns (Midi MACS; Miltenyi Biotec). The positive
fraction showed . 85% CD4+cells. To obtain spleen cell
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preparations depleted of dendritic cells (DC), negative selection
with anti-Pan DC microbeads (Miltenyi Biotec) was performed,
and the negative fraction showed , 0.5% DC (CD11c+I-Ab+cells).
Adoptive cell transfers of conventional CD4+cells
WT and I-Ab-/-mice received i.p. 46106purified CFSE-
labelled CD4+cells obtained from CD1d-/-mice. These mice were
infected i.p. with 16106iRBC on the day of transfer. Proliferative
CD4+cell response in vivo was analysed on days 4 and 7 of
infection. Non-infected mice were used as controls.
ELISPOT assay for Ig-producing cells
Ig-producing cells were quantified by the ELISPOT assay as
previously described [30]. In brief, 96-well flat-bottom microtest
plates (Costar) coated overnight (4uC) with goat anti-mouse total Ig
(10 mg/mL) were saturated with 1% gelatine (Merck) in PBS for
120 min. Titrated numbers of spleen cells (16106to 56102cells/
well) were cultured for 6 h at 37uC in a 5% CO2atmosphere. The
spots were developed by adding goat anti-mouse IgM, IgG2a or
IgG1 biotinylated antibodies followed by the addition of a
phosphatase alkaline-avidin conjugate (all antibodies and conju-
gates were purchased from Southern Biotechnology Associates). 5-
Bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma-Aldrich) di-
luted in 2-amino-2-methyl-1-propanol (AMP) (Merck) was used as
the substrate. From the titration plots (number of cells plated vs.
spots) and the total number of splenocytes, the number of total Ig-
producing cells per spleen was calculated.
ELISA test for parasite-specific antibodies
Anti-P. chabaudi antibodies were quantified by ELISA as
previously described [31]. In brief, 96-well flat-bottom microtest
plates (Costar) were coated overnight (4uC) with a total P. chabaudi
extract (8 mg/mL). Plates were saturated with 1% BSA for 1 h.
After washing, 100 mL of mouse serum samples (diluted from 1/2)
was added and left for 90 min at room temperature or overnight
(4uC), followed by goat anti-mouse IgG2a and IgG1 peroxidase-
conjugated antibodies (Southern Biotechnology Associates) for 1 h.
After washing, 100 mL of tetramethylbenzidine (TMB) (Zymed)
was added to each well and 15 min later, the absorbance values
were quantified by a Spectra Max 190 spectrophotometer
(Molecular Devices) with a 650-nm wavelength filter.
Statistical analysis
Statistical significance was analysed in Graph Pad Prism 4
software using the Mann-Whitney U and Tukey’s multiple
comparison tests. Differences between groups were considered
significant when p,0.05 (5%).
Results
I-Ab-restricted CD4+T cells are required for the control of
parasitaemia during the early and late P. chabaudi
infection
To evaluate the protective role of CD1d- and I-Ab-restricted
CD4+T cells during early and late P. chabaudi malaria, we
determined the parasitaemia curves in WT, CD1d-/-and I-Ab-/-
mice infected with 16106iRBC. WT and CD1d-/-mice developed
comparable first parasitaemia peaks, but in CD1d-/-mice the
second peak was higher and occurred earlier (on days 13 to 15 p.i.)
when compared to WT mice (on day 17 p.i.) (Figure 1). Despite
these differences, mice from both groups showed efficient control
of parasite growth and survived the infection. Contrarily, I-Ab-/-
mice developed a significantly higher first peak and prominent
recrudescent peaks compared to WT mice. Furthermore, these
mice were unable to eliminate the parasite and around 50% were
dead one month after infection. These results indicate that parasite
control during early and late P. chabaudi malaria is dependent on I-
Ab-restricted CD4+T cells, whereas the contribution of CD1d-
restricted CD4+T cells is modest and limited to the second
parasitaemia peak.
The majority of spleen CD4+T cells activated during the
first days of P. chabaudi infection are restricted by class II
MHC molecules
As the previous results suggest a protective role for I-Ab-
restricted CD4+T cells in early P. chabaudi malaria, we set out to
evaluate the kinetics of the CD4+T cell response to infection in
mice lacking CD1d and I-Abmolecules using as parameters spleen
cellularity and expression of the early activation marker CD69
[32]. The aim of this analysis was to compare non-conventional
and conventional CD4+T cells, which are thought to be primarily
involved in innate and acquired immunity, respectively. We took
advantage of the fact that a great proportion of CD4+T cells
generated in CD1d-/-mice are restricted by I-Abmolecules
(conventional CD4+T cells), whereas their counterparts in I-Ab-/-
mice are mostly restricted by CD1d molecules (non-conventional
CD4+T cells). According to our data, the lack of CD1d molecules
led to reduced numbers of CD4+cells per spleen in infected mice
(Figure 2A). Although the CD4+cell population was notably
smaller in I-Ab-/-mice compared to WT mice, it also expanded
from day 0 to 7 p.i. when maximum cellularity was attained in WT
mice. Regarding the CD69 expression, the mean fluorescence
intensity (MFI) was considerably higher in CD4+cells in all groups
of infected mice compared to controls (Figure 2B). However, for
CD4+cells generated in the absence of I-Abmolecules, a two-fold
Figure 1. Parasitaemia curves in P. chabaudi-infected WT, CD1d-/-and I-Ab-/-mice. Mice were infected with 16106iRBC. Each curve
corresponds to the means 6 SD (n=5–10). *, p,0.05, CD1d-/-or I-Ab-/-mice compared with WT mice. Data are representative of three experiments.
doi:10.1371/journal.pone.0022434.g001
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increase in CD69 expression was already observed in non-infected
mice (compared to non-infected WT mice), which is a known
feature of NKT cells [25], and infection led to additional
augmentations of these values. It is worth noting that the absence
of ab T cells did not result in compensatory increase of cd T cells
since less than 1% of CD4+cells in I-Ab-/-mice expressed TCRcd,
a proportion similar to that of WT mice, and these percentages
were not modified by the infection (data not shown).
To further characterise CD4+T cells responding to P. chabaudi
infection in CD1d-/-, I-Ab-/-and WT mice, these cells were
analysed in relation to their ability to recognise the CD1d-a-
GalCer tetramer. As expected, CD1d-/-mice were deficient in
CD4+cells that recognised the tetramer (Figure 2C). In addition,
the majority of CD4+cells expressing CD69 in 7-day infected WT
mice were not specific for CD1d-a-GalCer molecules. Confirming
the notion that a large proportion of CD4+cells generated in I-
Ab-/-mice were restricted by CD1d molecules, 31.5% of CD4+
cells recognised CD1d-a-GalCer molecules, compared to 3.5% in
WT mice. However, the percentages of CD1d-a-GalCer-restricted
CD4+cells expressing CD69 in both WT and I-Ab-/-mice were
not affected by infection. When total numbers of CD4+cells per
spleen were considered, on day 7 p.i., there was a 2-fold increase
in the Tetramer+and Tetramer-cell populations of WT and I-
Ab-/-mice (Figure 2D). Tetramer-CD4+cells in I-Ab-/-mice may
comprise CD1d-restricted CD4+T cells with specificities other
than a-GalCer and also, CD4+T cells restricted by class IB MHC
molecules other than CD1d [24]. It should be noted, however,
that the absolute increase was more than 10-fold greater for the
Tetramer-cell population in WT mice (14.86106cells) compared
to the other cell populations (which ranged from 4.7–13.26105
cells). The fact that Tetramer+CD4+cell numbers per spleen are
comparable in I-Ab-/-and WT mice, showing a similar increase
following infection, suggests that the antigen presenting cell (APC)
function is preserved in the absence of conventional CD4+T cells.
We concluded that both non-conventional and conventional
CD4+T cells promptly respond to P. chabaudi malaria, but the
majority of splenic CD4+T cells activated during the first days of
infection are restricted by class II MHC molecules.
I-Ab-restricted CD4+T cells proliferate vigorously during
the first days of P. chabaudi infection
As the previous data showed that non-conventional and
conventional CD4+T cell populations in the spleen increased
with similar kinetics during acute P. chabaudi malaria, experiments
Figure 2. CD4+ +cell activation in the spleen of P. chabaudi-infected WT, CD1d-/-and I-Ab-/-mice. (A) Numbers of CD4+cells per spleen on
days 0, 4 and 7 of infection. Data represent the means 6 SD (n=6–9). (B) Histograms showing CD69 expression in gated CD4+cells on days 0 and 7 of
infection. Numbers in histograms represent the means 6 SD (n=4–6) of MFI values. (C) Dot plots showing gated CD4+cells that recognise CD1d-a-
GalCer tetramers and express CD69 molecules on days 0 and 7 of infection. Numbers in dot plots represent the means 6 SD (n=4–6) of the
percentages of each subpopulation. (D) Numbers of Tetramer+CD4+cells and Tetramer-CD4+cells per spleen on days 0 and 7 of infection. Data
represent the means 6 SD (n=6). In A–D, *, p,0.05, infected mice compared with non-infected mice; e, p,0.05, CD1d-/-or I-Ab-/-mice compared with
WT mice. Data are representative of three experiments.
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were then performed to compare the in vitro proliferative responses
of these cells. Both spontaneous (non-stimulated in vitro) and iRBC-
stimulated proliferations were evaluated to address the actual and
potential proliferative responses, respectively. For CD4+cells
obtained from WT and CD1d-/-mice on day 4 p.i., there was a
consistent spontaneous proliferation of 8.0% and 15.0% of the
cells after 72 h of culture, notably higher than those of non-
infected mice (0.5% and 1.0%, respectively) (Figure 3A). In vitro
stimulation with iRBC resulted in further increases of these values
up to 35.0%. Contrasting with CD4+cells from CD1d-/-mice that
proliferated as much as CD4+cells from WT mice, CD4+cells
from I-Ab-/-mice showed low spontaneous proliferation in 4-day
infected mice, with the average frequency of dividing cells
increasing from 0.5% to 10.0% following in vitro iRBC-stimulation.
Interestingly, on day 7 p.i., the spontaneous and iRBC-stimulated
proliferation of CD4+cells from WT and CD1d-/-mice was
indistinguishable from that in non-infected controls.
To investigate whether the intense proliferative response of
spleen CD4+cells from 4-day infected WT and CD1d-/-mice
requires interaction with DC and I-Abmolecules, DC-depleted
cultures and anti-I-AbmAb-treated cultures were compared with
positive controls. As shown in Figure 3B, in both cases there was
considerable reduction in the percentages of proliferating cells.
The fact that a significant proliferation is still observed following
blockade of I-Ab- and DC-mediated interactions may result from
in vivo priming in the presence of I-Abmolecules, allowing the
TCR signalling and, in consequence, a few rounds of cell
proliferation driven by cytokines and costimulatory molecules.
To extend this analysis in vivo, CFSE-labelled CD4+cells from the
spleens of CD1d-/-mice (mostly I-Ab-restricted CD4+T cells) were
adoptively transferred into WT and I-Ab-/-mice that were
subsequently infected and their proliferative responses were
determined 4 and 7 days later. When transferred into infected
WT mice, CFSE-labelled CD4+cells rapidly proliferated with
10% and 15% of dividing cells on days 4 and 7 p.i., respectively
(Figure 3C). Corroborating the notion that this response requires
interaction with class II MHC molecules, these CD4+cells failed to
respond to P. chabaudi malaria when transferred into I-Ab-/-mice.
I-Ab-restricted CD4+T cells are the major source of
IFN-c production in the spleen during the first days of
P. chabaudi infection
To evaluate the contribution of non-conventional and conven-
tional CD4+T cells to the proinflammatory cytokine response to P.
chabaudi malaria, IFN-c production by spleen cells from WT,
CD1d-/-and I-Ab-/-mice was quantified on days 0, 4 and 7 of
infection. Ex vivo production of IFN-c (intracellular staining) was
determined in both non-stimulated and anti-CD3 mAb-stimulated
cell cultures, which revealed effector cells induced during the acute
infection and the total potential of CD4+cells to secrete IFN-c,
respectively. In both cases, the maximum response was observed
on day 7 p.i., when the percentages of IFN-c-producing CD4+
cells were 3-fold higher in WT and CD1d-/-mice compared to I-
Ab-/-mice (Figure 4A). Interestingly, on day 4 p.i., no significant
Figure 3. Proliferative response of spleen CD4+cells from P. chabaudi-infected WT, CD1d-/-and I-Ab-/-mice. (A) Histograms showing
spontaneous and iRBC-stimulated in vitro proliferation of gated CD4+cells on days 0, 4 and 7 of infection. Numbers in histograms represent the
means 6 SD (n=6–8) of the percentages of replicating (CFSElow) cells. (B) Effects of DC depletion and anti-I-AbmAb treatment on the in vitro
proliferation of gated CD4+cells from WT mice on days 0 and 4 of infection. Data represent the means 6 SD (n=6) of the percentages of replicating
(CFSElow) cells. (C) CFSE-labelled CD4+cells (46106) from the spleens of CD1d-/-mice were adoptively transferred into WT and I-Ab-/-mice. The
recipient mice were then infected and the in vivo proliferative response was determined 0, 4 and 7 days later. Numbers in histograms represent the
means 6 SD (n=6) of the percentages of replicating (CFSElow) cells. In A-C, *, p,0.05, infected mice compared with non-infected mice. In A and C, e,
p,0.05, CD1d-/-or I-Ab-/-mice compared with WT mice. In A, #, p,0.05, spontaneous proliferation compared with iRBC-stimulated proliferation. In B,
F, p,0.05, DC-depleted or mAb-treated cell cultures compared with control cell cultures. Data are representative of three experiments.
doi:10.1371/journal.pone.0022434.g003
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