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CD4 T Cells from Malaria-Nonexposed Individuals Respond to
the CD36-Binding Domain of Plasmodium falciparum
Erythrocyte Membrane Protein-1 via an MHC Class
II-TCR-Independent Pathway
1
Francis M. Ndungu,*
†
Latifu Sanni,*
‡
Britta Urban,
§
Robin Stephens,*
Christopher I. Newbold,
¶
Kevin Marsh,
†
and Jean Langhorne
2
*
We have studied the human CD4 T cell response to a functionally conserved domain of Plasmodium falciparum erythrocyte
membrane protein-1, cysteine interdomain region-1
␣
(CIDR-1
␣
). Responses to CIDR-1
␣
were striking in that both exposed and
nonexposed donors responded. The IFN-
␥
response to CIDR-1
␣
in the nonexposed donors was partially independent of TCR
engagement of MHC class II and peptide. Contrastingly, CD4 T cell and IFN-
␥
responses in malaria-exposed donors were MHC
class II restricted, suggesting that the CD4 T cell response to CIDR-1
␣
in malaria semi-immune adults also has a TCR-mediated
component, which may represent a memory response. Dendritic cells isolated from human peripheral blood were activated by
CIDR-1
␣
to produce IL-12, IL-10, and IL-18. IL-12 was detectable only between 6 and 12 h of culture, whereas the IL-10
continued to increase throughout the 24-h time course. These data strengthen previous observations that P. falciparum interacts
directly with human dendritic cells, and suggests that the interaction between CIDR-1
␣
and the host cell may be responsible for
regulation of the CD4 T cell and cytokine responses to P. falciparum-infected erythrocytes reported previously. The Journal of
Immunology, 2006, 176: 5504 –5512.
There is long-standing evidence for the presence of T cells
responding to Plasmodium falciparum Ags in most adults
who have had no exposure to P. falciparum malaria (re-
viewed in Refs. 1 and 2). These T cells have been shown to re-
spond to crude extracts of P. falciparum infected RBC (iRBC)
3
(3), intact iRBC (4, 5), as well as a number of defined malarial Ags
(6 – 8). The response of these T cells, often present at high fre-
quencies (9), may be the result of mitogenic or superantigenic
activity in the malaria parasite (10 –14), or to previous exposure to
environmental organisms that share common epitopes with P. fal-
ciparum (1–3, 15, 16). MHC class II-restricted T cells from non-
exposed individuals responding to P. falciparum proteins or ex-
tracts have been shown to express the memory/activation marker
CD45RO
⫹
(4, 16 –18), or the naive cell marker CD45RA
⫹
(4, 17),
suggesting that both memory and naive T cells may be stimulated
by P. falciparum Ags, and that there may be more than one mech-
anism of T cell activation.
Because these responses occur in nonexposed individuals who
can succumb to a malaria infection, they may not be protective, but
may be preferentially expanded upon exposure to P. falciparum
(19). It has been suggested that their presence may have the po-
tential to skew the repertoire of P. falciparum-reactive T cells to-
ward the cross-reactive epitopes and therefore limit the more pro-
tective responses (19). They may also be responsible for the
initiation of the inflammatory response to P. falciparum in non-
immune individuals, which may in turn contribute to the pathology
of disease (2, 16).
The active component of the iRBC that induces proliferation in
memory CD45RO
⫹
CD4
⫹
T cells is thought to be membrane
bound (5). Candidate membrane-bound molecules are the variable
surface Ags (VSA), which include those encoded by the var mul-
tigene family, such as P. falciparum erythrocyte membrane pro-
tein-1 (PfEMP-1). Interestingly, the cysteine interdomain re-
gion-1
␣
(CIDR-1
␣
) domain of PfEMP-1 has been shown to
stimulate CD4 T cells from both exposed and nonexposed indi-
viduals (20). The CIDR-1
␣
domain of PfEMP-1, which binds to
CD36 (21), is thought to play a role in the adhesion of iRBC to the
host endothelium (22, 23). It has been suggested that this region of
PfEMP-1 could be used as an effective antiadhesive vaccine (24,
25). However, if CD4 T cells are activated nonspecifically by
CIDR-1
␣
, they may prevent the generation of specific and protec-
tive CD4 T cell responses. To determine whether pre-existing
cross-reactive CD4 T cells would influence vaccine efficacy, it is
necessary to understand how CIDR-1
␣
interacts with CD4 T cells.
In addition to cross-reactive Ag, superantigen, and mitogenic
effects of malaria parasite components on CD4 T cells, it is pos-
sible that CD4 T cells could be activated by cytokines independent
of TCR engagement (26 –28). IL-12 and IL-18 cytokines produced
*Division of Parasitology, National Institute for Medical Research, London, United
Kingdom;
†
Kenya Medical Research Institute, Centre for Geographic Medicine Re-
search, Kilifi, Kenya;
‡
Department of Pathology, Leeds General Infirmary, Leeds,
United Kingdom;
§
Nuffield Department of Clinical Medicine, Centre for Clinical
Vaccinology and Tropical Medicine, University of Oxford, Oxford, United Kingdom;
and
¶
Weatherall Institute of Molecular Medicine, University of Oxford, Oxford,
United Kingdom
Received for publication December 5, 2005. Accepted for publication February
9, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by European Union/BIOMALPAR, The Wellcome Trust,
and the Medical Research Council (MRC), U.K. J.L., L.S., and F.M.N. are supported
by the MRC; B.U. is a Wellcome Trust career development fellow. C.I.N. is funded
by the Wellcome Trust and K.M. is a Wellcome Trust senior fellow.
2
Address correspondence and reprint requests to Dr. Jean Langhorne, Division of
Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, Lon-
don, NW7 1AA, U.K. E-mail address: jlangho@nimr.mrc.ac.uk
3
Abbreviations used in this paper: iRBC, infected RBC; PfEMP-1, P. falciparum
erythrocyte membrane protein-1; CIDR-1
␣
, cysteine-rich interdomain region 1
␣
; DC,
dendritic cell; SEB, staphylococcal enterotoxin B; BFA, brefeldin A; PPD, purified
protein derivative; PRR, pattern recognition receptor.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
by dendritic cells (DCs) are able to induce IFN-
␥
production in
CD4 T cells. These two cytokines are produced by DCs that could
interact with P. falciparum through pattern recognition receptors
(PRRs) like TLRs or the scavenger receptor, CD36 (29). With this
study presented here, we examined the nature of CIDR-1
␣
-specific
responses in malaria nonexposed and exposed individuals. We
were able to show that in contrast to malaria nonexposed adults,
exposed adults had MHC class II-restricted CD4 T cell and IFN-
␥
in vitro responses to CIDR-1
␣
, and that CIDR-1
␣
directly acti-
vated DCs to produce IL-12, IL-10, and IL-18.
Materials and Methods
Donors and blood sampling
Nonexposed donors. Sixty healthy adult volunteers working at the Na-
tional Institute for Medical Research (London, U.K.) were recruited. A
detailed travel history was taken from each of these donors to confirm that
they had never been exposed to P. falciparum (or never suffered from
malaria). All experimental procedures followed the guidelines of good con-
duct in clinical research of the U.K. government, and were performed with
the informed consent obtained from the volunteers. Ethical clearance was
granted by the Barnet Ethical Committee in North London. From each of
these donors, 20 ml of blood were collected in heparinized vaccutainers.
Exposed donors. Nineteen healthy adult volunteers, between 25 and 40
years of age, living in Ngerenya, a malaria endemic area on the Kenyan
coast, were recruited. A detailed history was taken from each of these
donors to ascertain that they had lived in Ngerenya most of their lives and
that they were healthy at the time of blood donation. The area has pro-
longed seasonal P. falciparum malaria transmission following the short and
long rains in the months of October to November and March through July,
respectively. The Anopheles gambie sensu stricto mosquito complex is the
main vector contributing to ⬃10 infective bites per person per annum (30).
All experiments followed the guidelines of good conduct in clinical re-
search of the Kenyan government, and were performed with informed con-
sent from the volunteers. Ethical clearance was granted by the Kenyan
national ethical committee. From each of these donors, 15 ml of blood were
collected in heparinized vaccutainers.
Preparation and purification of PfEMP-1 fragments.
Two recombinant protein fragments of PfEMP-1 were expressed and pu-
rified as described elsewhere (20). CIDR1-
␣
was obtained from a PfEMP-1
gene of the Malayan Camp laboratory isolate of P. falciparum (GenBank
accession no. U27338), and a relatively conserved fragment of the intra-
cellular exon 2 region of a PfEMP-1 gene was isolated from the A4 lab-
oratory isolate (GenBank accession no. AJ413950). Before use, CIDR-1
␣
and the negative control protein, exon 2, were taken through a thorough
purification process involving gel filtration. To remove any residual LPS
contamination, fractions corresponding to the recombinant protein of in-
terest in the gel filtration elution profile were further purified by polymyxin
B chromatography (Endo-trap; Profos Ag). The column was washed se-
quentially with 1% sodium deoxycholate (Sigma-Aldrich), water, and PBS
before loading with the protein. The flow-through was collected and sub-
jected to two additional rounds of polymyxin B affinity chromatography.
Finally, PBS was replaced with endotoxin-free tissue culture-grade water
using PD columns (Amersham Biosciences). The protein solutions were
sterilized through a 0.2-
m filter, and stored at ⫺80
o
C. The amount of
endotoxin in each protein preparation was determined in a semiquantitative
Limulus amoebocyte assay (E-Toxate; Sigma-Aldrich) using an endotoxin
standard (Sigma-Aldrich), following manufacturer’s instructions. The en-
dotoxin level was found to be ⬍0.10 endotoxin units/mg (5 pg/mg protein),
which is too low to induce cytokine production by human PBMC (at least
100 pg of LPS was required to induce cytokine production by human
monocytes (31)).
P. falciparum cultures
The Malayan Camp P. falciparum parasites used for DC stimulations were
cultured according to standard methods (32) using RPMI 1640 with albu-
max (Invitrogen Life Technologies) supplemented with L-glutamine until
they reached the late trophozoite stage. All parasites were grown in the
presence of 0.5
g/ml mycoplasma removal agent (Serotec).
Isolation of PBMC from whole blood
PBMC were isolated as described previously (20). Blood was diluted 1/2
in RPMI 1640 without additives, and purified over Lymphoprep (Ny-
comed) by centrifugation at 800 ⫻gfor 20 min. The PBMC layer was then
collected from the interphase, washed three times in RPMI 1640, and re-
suspended at the appropriate concentration in complete RPMI 1640 me-
dium (RPMI 1640 containing 10% heat-inactivated human AB serum, 2
mM L-glutamine, 100
g/ml streptomycin, 100
g/ml penicillin, and 10
mM HEPES (Invitrogen Life Technologies)).
Determination of CD4 T cell and NK cell activation by CD69
expression and intracellular cytokine detection
PBMC (10
6
) were resuspended in 250
l of complete RPMI 1640 and
plated out in 24-well microtiter plates. Costimulatory mAbs against CD28
and CD49d (BD Biosciences) were added to a final concentration of 1
g/ml each, and either CIDR-1
␣
, exon 2, or staphylococcal enterotoxin B
(SEB; Sigma-Aldrich) was added at optimal concentrations (5
g/ml for
both CIDR-
␣
and EXON2 and 1
g/ml for SEB). The tubes were incubated
in a humidified 37
o
C, 5% CO
2
incubator for a total of 7 h. Brefeldin A
(BFA; 10
g/ml) was included for the last 4 h of culture. After 7 h, the cells
were harvested and incubated with fluorochrome-labeled mAbs to CD4
(BD Biosciences clone RPA-T4), NK cells (anti-CD56-allophycocyanin
Immunotec clone PNIM2474), CD69 (BD Pharmingen; clone FN50), and
IFN-
␥
(BD Pharmingen; clone B27), and IL-10 (BD Pharmingen; clone
JES3-19F1). Four-color flow cytometric analyses were performed on the
FACSCalibur flow cytometer (BD Immunocytometry Systems). Data were
acquired using CellQuest (BD Immunocytometry Systems), collecting 5 ⫻
10
4
-gated CD4
⫹
events. Data were displayed in two-color dot plots using
CellQuest. Side scatter and FL3 (CD4 PerCP) gating were used to exclude
any CD4
⫹
monocytes during data analysis. Donors were considered pos-
itive if the proportion of CD4
⫹
T or NK cells expressing CD69, or CD69
and cytokine production (separately) was at least 3-fold above the medium
control.
PBMC proliferation assays
PBMC were labeled with CFSE (Molecular Probes) as described previ-
ously (33). Cells were washed three times with complete RPMI 1640, and
dye incorporation was assessed by flow cytometry. CFSE-labeled PBMC
were plated out at 2 ⫻10
5
cells/well in a 96-well U-bottom plate (Nunclon;
Invitrogen Life Technologies), and cultured in a final volume of 200
lof
complete RPMI 1640. The CIDR-1
␣
and exon 2 fragments of PfEMP-1
were used at a final concentration of 0.25
g/ml. Purified protein deriva-
tives (PPD) at 10
g/ml were used as a positive control.
Each condition was set out in duplicate or triplicate. Plates were incu-
bated at 37°C and 5% CO
2
in a humidified atmosphere for 7 days. Super-
natants were removed for the measurement of cytokines before cytometric
analysis. The proliferative responses of the CFSE-labeled CD4 T cells were
determined by flow cytometry. Cells were stained with the appropriate
combinations of allophycocyanin-, PE-, and PerCP-conjugated Abs. Flow
cytometry was performed either on a FACSCalibur using Cell Quest soft-
ware (BD Biosciences) or on a Coulter EPICS XL with XL system II in
London and Kilifi, respectively. Cell division was determined from the
proportion of cells with reduced fluorescence intensity of CFSE as described
previously (34) (see Fig. 2). The flow cytometer was set to count events for a
fixed length of time (1 min) to permit the determination of the relative numbers
of recovered viable cells within each well (a measure of the magnitude of the
response to Ag) (34). Analysis using the allophycocyanin-, PE-, PerCP-con-
jugated Abs allowed the proportions of the different cell subsets within the
dividing and nondividing populations to be determined. Data are presented as
mean proportions or relative total numbers of dividing CD4 T cells. CD4 T cell
proliferation was considered positive if the mean number of dividing cells in
triplicate wells containing an Ag exceeded two times the mean value of trip-
licate wells without Ag by more than two stimulation indices.
Inhibition assay with anti-MHC class II-blocking Ab
PBMC from normal healthy donors were incubated with PPD, and either
the anti-MHC class II Ab L243 (directed against monomorphic determi-
nants of HLA-DR; American Type Culture Collection), or the mouse
IgG2a isotype control, at different concentrations; 0, 5 and 10
g/ml, be-
fore determination of cytokine secretion or T cell proliferation as described
above. For each positive response, the ability of the anti-MHC class II mAb
to inhibit either CD4 T cell proliferation or IFN-
␥
production in response
to CIDR-1
␣
was expressed as percentage inhibition (based on a corre-
sponding response in the presence of an isotype control Ab at the same
concentration as L243).
5505The Journal of Immunology
DC cytokine assays with whole PBMC
PBMC (10
6
) were incubated with medium only, exon 2 (5
g/ml), CIDR-
␣
(5
g/ml), or LPS (1
g/ml) in a final volume of 500
l in 48-well mi-
crotiter plates. BFA (10
g/ml) was added 4 h before staining the cells for
intracellular cytokine detection by flow cytometry. Two different subpopu-
lations of DCs were identified in whole PBMC by surface staining of the
cells with either anti-BDCA-1 (blood DC Ag-1) FITC and CD19 PerCP, or
anti-BDCA-2 and CD123 PerCP (35, 36). The average percentages of
BDCA-1- and BDCA-2-positive cells identified in PBMC were 0.43%
(SD ⫽0.9) for seven individuals and 0.05% (SD, 0.046) for five individ-
uals, respectively. Cells were fixed and then permeabilized for intracellular
staining by using the Fix and Perm kit (Caltag Laboratories) according to
the manufacturer’s instructions. Intracellular staining for IL-10 and IL-12
was done using allophycocyanin anti-human IL-10, and PE anti-human
IL-12p70, respectively. A total of 4 ⫻10
5
cells were acquired per sample
on the FACSCalibur.
Isolation of BDCA-1 (CD1C) positive myeloid DCs from whole
PBMC
Positive selection of BDCA-1-positive myeloid DCs was done using a
magnetic bead isolation system according to the manufacturer’s protocol
(Miltenyi Biotec). Briefly, BDCA-1-expressing B cells were magnetically
labeled with CD19 microbeads and subsequently depleted on a MACS
column (Miltenyi Biotec), followed by positive selection of BDCA-1-pos-
itive blood DCs in the B cell-depleted fraction. This positive selection step
was repeated to increase the purity of the BDCA-1 DCs collected. The
purity of the isolated DCs was assessed by flow cytometry and was always
⬃95%. The enriched BDCA-1
⫹
DCs contained ⬍1% CD19
⫹
B cells and
⬍1.5% CD14
⫹
monocytes (mean of 1.4% (SD, 0.4).
BDCA-1-DCs cytokine assays
A total of 10
4
-10
5
purified BDCA-1 DCs were incubated with medium
only, exon 2 (5
g/ml), CIDR-
␣
(5
g/ml), Malayan camp P. falciparum
schizonts (30 schizonts/DC), and LPS (1
g/ml) in a final volume of 500
l in 48-well microtiter plates. When cytokine production was assessed by
flow cytometry, BFA (10
g/ml) was added 4 h before harvesting and
staining of cells. In this case, cells were examined for IL-12 and IL-10
production after 6- or 12-h stimulation, using a FACSCalibur as described
above. Otherwise in cultures without BFA, supernatants for cytokine mea-
surement by ELISA were harvested after 12 or 24 h poststimulation.
Determination of cytokine concentrations by ELISA
Supernatants from the PBMC or isolated DC cultures were tested for the
presence of cytokines using the OTEIA sets (BD Pharmingen) for IFN-
␥
,
IL-10, and IL-12p70, and a human IL-18 ELISA kit (Medical and Biolog-
ical Laboratories) following the manufacturer’s instructions. The concen-
tration of the cytokines was determined from the averages of duplicate/
triplicate wells. Donor responses were considered positive if the cytokine
concentration was 2-fold over the medium control.
Data analysis
Data were stored, formatted, and analyzed with Microsoft Excel. Graphs
were plotted in Prism-Graphics (GraphPad Software) and Stata version 8.0
FIGURE 1. CIDR-1
␣
induce CD69 up-regulation and IFN-
␥
production in CD4 T and NK cells from malaria nonexposed individuals. a, CIDR-1
␣
-
specific CD4 T and CD56 NK cells were detected directly from PBMC 7 h after stimulation with Ag (and controls) by flow cytometry. Cells were gated
on CD4 (row 1) and CD56 (row 2) -positive cells. Specifically activated cells were identified as being CD69
⫹
. Some of the activated cells produced IFN-
␥
.
The percentages of the total number of cells responding by CD69 expression and IFN-
␥
are shown in the upper left and right quadrants, respectively. b,
Proportions of malaria nonexposed donors responding by CD4 T and NK cells to CIDR-1
␣
by CD69 up-regulation and production of IFN-
␥
. A response
was considered positive when the proportion of CD69 or IFN-
␥
-positive cells was 3-fold above the medium control (a 3-fold cutoff was preferred for use
in this assay because of clustering around the 2-fold cutoff used with the 7-day proliferation assay). Each of the dots represents a single donor (n⫽23).
Twenty and 13 of 23 donors responded by CD69 up-regulation and IFN-
␥
production in CD4 T cells, respectively. All of the 10 donors tested for NK cell
activation responded by CD69 up-regulation, and 9 of them also responded by IFN-
␥
production.
5506 T CELL RESPONSES IN MALARIA NONIMMUNE INDIVIDUALS
computer softwares. Differences between groups were tested by Mann-
Whitney Utest.
Results
CIDR-1
␣
activates CD4 T and NK cells from malaria nonexposed
individuals to induce CD69 expression and IFN-
␥
production
PBMC from healthy individuals were stimulated in vitro for 7 h
with 5
g/ml CIDR-1
␣
protein. During this time, a proportion of
CD4 T and NK cells up-regulated expression of CD69, and some
of these cells produced IFN-
␥
. The proportion of responding CD4
T and NK cells was determined by the combined flow cytometric
analysis of CD69 expression, and intracellular staining of IFN-
␥
or
IL-10 in CD4 T and CD56 NK cells. Representative examples of
typical responders are shown in Fig. 1afor CD4 T and NK cells. In
these donors, 45% of CD4 T cells expressed CD69, and 4% produced
IFN-
␥
in response to CIDR-1
␣
(⬃10% of the CD69
⫹
cells). Simi-
larly, CIDR-1
␣
also activated over 60% of CD56
⫹
-positive NK cells
and 12.3% (⬃20% of the CD69
⫹
cells) produced IFN-
␥
.
Results of CD69 and IFN-
␥
expression on CD4 T and NK cells
from 23 and 10 malaria nonexposed individuals, respectively, are
summarized in Fig. 1b. PBMC from the majority of donors (89%)
responded by up-regulating expression of CD69, and approxi-
mately half (56%) by both CD69 expression and IFN-
␥
production
on CD4 T cells. NK cells in PBMC from all of the 10 nonexposed
individuals tested also expressed CD69 and produced IFN-
␥
. All
IFN-
␥
-producing CD4 T and NK cells were CD69 positive. Intra-
cellular IL-10 was not detectable in CD4 T cells stimulated by
either CIDR-1
␣
or SEB after7hofculture. Stimulation with exon
2 or SEB were conducted as negative and positive controls, re-
spectively. Exon 2 did not induce CD69 expression or any signif-
icant cytokine induction, whereas the superantigen (SEB) readily
stimulated both cell types. Taken together, these data demonstrate
the presence of CD4 T and CD56 NK cells able to respond to
CIDR-1
␣
and produce IFN-
␥
to this malaria protein in peripheral
blood of P. falciparum nonexposed individuals.
Assessment of CD4 T cell division and cytokine production in
response to CIDR-1
␣
in nonexposed individuals
CFSE-labeled PBMC from healthy nonexposed adults were stim-
ulated with CIDR-1
␣
and control Ags for 7 days after which CD4
FIGURE 2. CD4 T cell proliferation, IFN-
␥
, and
IL-10 production in response to CIDR-1
␣
in malaria
nonexposed individuals. a, A representative example of
CIDR-1
␣
-specific CD4 T cell proliferative responses in
nonexposed PBMC donors. Divided CD4 T cells were
identified as those with reduced CFSE fluorescence in-
tensity on the x-axis of similar histograms to those
shown above. The numbers shown are the percentages
of CD4 T cells in the respective gates. b, Proportions of
malaria nonexposed donors responding to CIDR-1
␣
by
CD4 T cell proliferation (n⫽34), IFN-
␥
(n⫽33), and
IL-10 (n⫽33) production. Exon 2 was used as a neg-
ative control and in all cases; the medium control value
was subtracted from the Ag-specific value. Each dot
represents data from a single individual. Cytokine re-
sponses that were 2-fold above the medium control, and
proliferative responses above 2 stimulation indices
were considered positive. Eight of 34 individuals had
proliferative stimulation indices of over 2, 18 of 33 and
16 of 33 individuals had concentrations above 2-fold of
the medium control for IFN-
␥
and IL-10, respectively.
c, Relationship between the numbers of divided CD4 T
cells and the percentage of CD69
⫹
CD4 T cells after
incubation of PBMC with CIDR-1
␣
for 7 days and 7 h,
respectively (n⫽14). Generally, all those that re-
sponded by cell division also responded by CD69 up-
regulation. However, more donors responded by CD69
up-regulation than cell division.
5507The Journal of Immunology
T cell proliferation was assessed by flow cytometry. A represen-
tative example of a positive CD4 T cell response is shown in Fig.
2a. The response to CIDR-1
␣
was similar to that achieved with the
positive control Ag (PPD), whereas little cell division took place in
cultures of PBMC and the negative control Ag (exon 2). Fig. 2b
summarizes the results of PBMC from 34 malaria nonexposed do-
nors. Of these, 8 gave a positive proliferative response. The
amounts of IFN-
␥
and IL-10 were determined from PBMC culture
supernatants harvested from the 7-day proliferation assays (Fig.
2b). Seventeen and 16 of 33 donors were positive for IFN-
␥
and
IL-10 production, respectively. As described previously (20), there
was generally an inverse or no association between IL-10 produc-
tion and CD4 T cell proliferation. Interestingly, some individuals
responded by IFN-
␥
production and no cell division, and we ob-
served an unexpected positive association between IFN-
␥
and
IL-10 concentrations (Spearman’s
coefficient ⫽0.62, p⫽0001,
data not shown). In donors (n⫽14) where CD4 T cell responses
were measured by both the CFSE-based proliferation and the 7-h
activation assays, it was clear that while most of the individuals (10 of
14) responded by CD69 expression, only 4 of the 14 responded by cell
division (Fig. 2c). Thus, up-regulation of expression of CD69 in re-
sponse to CIDR-1
␣
did not always lead to cell division.
IFN-
␥
produced in response to CIDR-1
␣
is not MHC class
II-restricted in malaria nonexposed individuals
To determine whether the response to CIDR-1
␣
in malaria non-
exposed individuals is the result of an MHC class II/TCR-medi-
ated interaction, PBMC and CIDR-1
␣
were cultured in the pres-
ence of Ag and different concentrations of the anti-MHC class II
Ab (L243). L243 is directed against HLA-DR, and inhibits clas-
sical MHC class II-restricted anti-PPD CD4 T cell proliferative
and IFN-
␥
production responses in PBMC obtained from individ-
uals previously immunized with bacillus Calmette-Gue´rin (BCG)
as shown in Fig. 3, left panel.
FIGURE 3. Median percent inhibitions for the CD4 T cell and IFN-
␥
responses to CIDR-1
␣
by the anti-MHC class II mAb. PBMC from malaria
nonexposed and exposed adults were incubated in the presence of CIDR-1
␣
or PPD, and different concentrations of either the anti-MHC class II mAb, L243,
or the mouse IgG2a isotype control; 0, 5, and 10
g/ml. The cells were cultured for 7 days and thereafter analyzed for CD4 cell division by flow cytometry
as described in Fig. 2. IFN-
␥
concentrations were determined in culture supernatants by ELISA. Percent inhibitions were calculated by subtracting the L243
values from the corresponding values associated with the respective concentrations of the isotype control Ab. Each dot represents an individual donor and
data is shown for 7 and 11 donors, for CD4 T cell division and IFN-
␥
production, respectively. There was a significant dose dependent effect between 5
and 10
g/ml for IFN-
␥
(p⫽0.002 Mann-Whitney Utest) but not for the CD4 T cell response (p⫽0.4).
5508 T CELL RESPONSES IN MALARIA NONIMMUNE INDIVIDUALS
PBMC from seven nonexposed individuals tested in this inhi-
bition assay gave positive IFN-
␥
responses but no CD4 T cell
division in response to CIDR-1
␣
, in contrast to the anti-
PPD-specific CD4 T cell and IFN-
␥
responses, which were inhib-
ited by the anti-MHC class II Ab. The IFN-
␥
response to CIDR-1
␣
was not inhibited by anti HLA-DR Abs in five of seven nonex-
posed individuals, suggesting that the anti-CIDR-1
␣
IFN-
␥
re-
sponse of the majority of nonexposed donors, unlike their response
to PPD, does not require Ag presentation to the CD4 T-TCR in the
context of the MHC class II-peptide complex (Fig. 3, middle
panel).
Cross-reactivity between CIDR-1
␣
epitopes with other Ags may
explain the inhibition of IFN-
␥
production when MHC class II
molecules were blocked in two individuals. In this case, the anti-
CIDR-1
␣
response would present a classical memory response.
The majority of CD4 T cell and IFN-
␥
responses to CIDR-1
␣
in
malaria-exposed individuals are MHC class II restricted
Despite the response of CD4 T cells to CIDR-1
␣
and its apparent
independence of HLA-DR presentation, it is possible that repeated
exposure to malaria infection would result in the generation of an
additional MHC class II-restricted response. If this is the case, it
might be possible to use MHC class II-blocking Abs to distinguish
memory responses from TCR-independent responses to CIDR-1
␣
and possibly other malarial Ags.
Therefore, the dependence of CD4 T cells responding to
CIDR-1
␣
on MHC class II presentation was tested in 19 exposed
adults. All responses (n⫽7) where we observed proliferation to
CIDR-1
␣
(stimulation indices: 2 to 6.3) were inhibited by anti
HLA-DR Abs, and those responses (n⫽10) which resulted in
IFN-
␥
(54.80 and 422.40 pg/ml measured by ELISA) were also
inhibited by the anti-HLA-DR Abs as shown in Fig. 3, right panel.
The median percentage inhibitions of CD4 T cell proliferation in
response to CIDR-1
␣
were 37 and 45%, for 5 and 10
g/ml, re-
spectively, and were significantly different from the L243-negative
control (0% inhibition) ( p⫽0.001 and p⫽0.001 (Mann-Whitney
Test)).
The median percentage inhibition of the IFN-
␥
responses was
19% for 5
g/ml and 38% for 10
g/ml Ab, and were significantly
different from 0% ( p⫽0.002, p⫽0.01, Mann-Whitney Test),
suggesting that the IFN-
␥
response to CIDR-1
␣
in exposed indi-
viduals is, at least in part, MHC class II restricted. Together, these
data suggest that both the CD4 T cell proliferative and IFN-
␥
re-
sponses in exposed adults are MHC class II restricted and are
therefore different from those observed in malaria nonexposed in-
dividuals, which may be mediated by various mechanisms includ-
ing bystander T cell activation. In contrast, inhibition of both the
CD4 and IFN-
␥
responses of nonexposed and exposed individuals
to PPD by the anti-MHC class II Ab was of similar magnitude.
Myeloid DCs from malaria nonexposed individuals produce
IL-10, IL-12p70, and IL-18 in response to CIDR-1
␣
It is possible that DCs may directly interact with CIDR-1
␣
through
CD36 or PRRs such as TLRs (reviewed in Ref. 37) resulting in the
production of IL-18 and IL-12p70, which in turn may activate and
induce IFN-
␥
transcription in the responding CD4 T and NK cells
without engaging the TCR via the MHC class II peptide complex.
It has been shown that the presence of these two cytokines is suf-
ficient to induce IFN-
␥
production in Th-1 T cells in mice (27, 28).
Therefore, we investigated the cytokine response (IL-10, IL-12,
and IL-18) of DCs to CIDR-1
␣
from BDCA-1
⫹
(myeloid DC) and
BDCA-2
⫹
(plasmacytoid DC) subpopulations in both whole
PBMC and the isolated DC populations.
Whole PBMC were cultured with CIDR-1
␣
, exon 2, LPS (pos-
itive control), or medium only (control). DCs from these cultures
were then analyzed for intracellular IL-10 and IL-12p70 produc-
tion by flow cytometry at 3, 6, 18, and 24 h poststimulation.
CIDR-1
␣
and LPS, but not exon 2 and the medium controls in-
duced IL-10 and IL-12 production in the BDCA-1 subpopulation
in all the individuals tested (Fig. 4). However, kinetics of IL-12
and IL10 production were different. Although the proportion of
cells positive for IL-10 continued to increase throughout the 24-h
culture period, those producing IL-12p70 reached a peak between
6 and 12 h. At 24 h, there were nearly no IL-12-positive cells.
Clearly, these results suggest that similar to the response to LPS
(Fig. 4), CIDR-1
␣
induces IL-12p70 and IL-10 responses in DCs.
By contrast, these cytokines were not detected in the plasmacytoid
FIGURE 4. Kinetics of IL-10 and IL-12p70 production by BDCA-1
⫹
DCs stimulated by CIDR-1
␣
, LPS, and exon 2 (negative control protein).
䡺,f, and 3represent the percentages of cells positive for the cytokine
after PBMC from malaria nonexposed were stimulated with exon 2, CIDR-
1
␣
, and LPS, respectively. The error bars are SEs of the means of five
donors. Cells were harvested and stained for flow cytometry at 3, 6, 12, and
24 h. In each case, 4 ⫻10
5
cells were acquired and the percentage of
BDCA-1
⫹
cells positive cytokines determined.
5509The Journal of Immunology
(BDCA-2
⫹
cells) in response to CIDR-1
␣
and LPS (data not
shown).
To determine whether the BDCA-1 DC cytokine response to
CIDR-1
␣
was the result of a direct interaction between the DCs
and CIDR-1
␣
without the involvement of other cells, BDCA-1
⫹
DCs were isolated using magnetic beads. A representative isola-
tion is shown in Fig. 5a.
To determine whether these enriched BDCA-1 DCs could make
IL-18, IL-12, and IL-10 in response to CIDR-1
␣
and P. falciparum
schizont iRBC, DCs were cultured with CIDR-1
␣
, iRBC, RBC,
and control Ags for up to 24 h. Culture supernatants were har-
vested at 12 and 24 h and tested for the presence of IL-18 by
ELISA (no appropriately labeled anti-IL-18 mAbs for intracellular
staining were available). At these time points, IL-18 was present in
the supernatants of DCs cultured with CIDR-1
␣
or iRBC as shown
in Fig. 5. To confirm that the isolated DCs also made IL-12 in
response to CIDR-1
␣
, cells were harvested after 6 and 12 h post-
stimulation and tested for IL-10 and IL-12p70 production by in-
tracellular staining and flow cytometry. The purified BDCA-1 DCs
produced both IL-10 and IL-12 in response to CIDR-1
␣
and LPS,
but not to medium or exon 2 controls (Fig. 5). However, the per-
centage of cytokine-positive cells from the isolated DCs were
lower than those seen with the same BDCA-1
⫹
DCs within whole
PBMC in Fig. 4.
Discussion
In this study, we show that the CD4 T cell response of P. falci-
parum nonexposed individuals to CIDR-1
␣
(the CD36-binding do-
main of the variant Ag PfEMP-1 (21)), described earlier by us
(20), is not dependent on engagement of the TCR with MHC class
II. This contrasts strongly with the CD4 T cell response of immu-
nized individuals to PPD, and more importantly is different from
the response of exposed donors to CIDR-1
␣
, where both responses
are dependent on MHC class II (HLA-DR). It is highly unlikely
that the difference between the response of the nonexposed and the
exposed donors to CIDR-1
␣
could be explained by racial differ-
ences, because the extents to which the PPD responses were in-
hibited in both groups were similar and the nonexposed groups
were from a wide mix ethnic groups.
Activation of CD4 T cells from nonexposed donors by the
CIDR-1
␣
domain was unusual in that the IFN-
␥
response was
rapid and could be detected within7hofculture, and before cell
division took place. Indeed, up-regulation of CD69 and IFN-
␥
pro-
duction were able to take place in the absence of subsequent cell
division. Although CD69 is an accepted marker for a positive T
cell response, our data would suggest that not all CD69
⫹
CD4 T
cells become fully activated, and go on to proliferate. It may be
that some of these T cells are activated in the absence of IL-2
FIGURE 5. Cytokine responses to CIDR-1
␣
and control Ags in isolated DCs. a, Represen-
tative plots showing isolated BDCA-1
⫹
DCs.
DCs were isolated from PBMC obtained from
buffy coats purchased from the National blood
bank (U.K.) in two steps; depletion of CD19-
positive B cells followed by positive selection
of BDCA-1
⫹
DCs. The data shown in these
plots are gated on a live gate to exclude dead
cells. A total of 1 ⫻10
5
cells were analyzed in
each case. B cells were depleted before
BDCA-1
⫹
DCs were positively isolated from
the flow-through fraction. Counterstaining with
CD14 FITC shows that some of the isolated
BDCA-1
⫹
blood DCs were also CD14 positive.
b,Left panel, IL-12p70 and IL-10 responses in
BDCA-1
⫹
isolated DCs at 6 (䡺) and 12 (3)
hours after stimulation with Ag determined by
flow cytometry. Isolated BDCA-1
⫹
DCs were
cultured at 1 ⫻10
5
cells/well in the presence of
medium only, exon 2 (negative control), CIDR-
1
␣
, and LPS (positive control). A total of 5 ⫻
10
4
cells were acquired on the flow cytometer
and analyzed for cytokine production. The er-
ror bars represent the SEM for six donors. b,
Right panel, Kinetics of IL-18 production by
isolated BDCA-1-positive DCs stimulated by
medium only, exon 2 (negative control),
CIDR1-
␣
, schizont-iRBC, RBC (negative con-
trol for iRBC), and LPS. Isolated BDCA-1
⫹
cells were cultured at 100,000 cells/well and
culture supernatants were harvested after 12
and 24 h of antigenic stimulation. IL-18 con-
centrations in the culture supernatants were de-
termined by ELISA (appropriately labeled Abs
for intracellular staining were unavailable). The
concentrations for the medium controls were
subtracted from those of the respective Ags.
The error bars represent the SEMs of six
donors.
5510 T CELL RESPONSES IN MALARIA NONIMMUNE INDIVIDUALS
and/or other costimulatory molecules. The early response of non-
exposed individuals to CIDR-1
␣
contrasts with conventional acti-
vation of CD4 T cells, where T cells first respond to their specific
peptide-MHC class II complexes by making IL-2 and proliferating
before differentiating into either Th1 or Th2 cells (reviewed in Ref.
38). CD4 Th1 cells then go on to produce IFN-
␥
.
The possibility that the response of nonexposed donors to
CIDR-1
␣
may not be a classical CD4 T cell/MHC class II response
is supported by the observation that in the majority of cases the
response was not inhibited by blocking HLA-DR, in contrast to the
response of these donors to PPD. The lack of inhibition by anti-
class II Abs also rules out a superantigen-like response, which
would involve binding to nonpolymorphic regions of MHC class II
and TCR (39, 40).
CIDR-1
␣
did not appear to stimulate CD4 T cells directly, and
therefore was unlikely to act as a mitogen. CD4 T cell activation,
proliferation, or cytokine production only took place when whole
PBMC were cultured with CIDR-1
␣
, suggesting that the effect on
nonexposed CD4 T cells may be indirect.
It has previously been shown that the cytokines IL-12 and IL-18
can activate Th1 cells independently of TCR ligation (27, 28). In
our case, the CIDR-1
␣
domain of PfEMP-1 could stimulate my-
eloid (BDCA-1
⫹
) but not plasmacytoid (BDCA-2
⫹
) isolated di-
rectly from peripheral blood of nonexposed donors to produce both
IL-12 and IL-18. In addition to these cytokines, IL-10 was also
secreted, albeit with a slower kinetic. These effects of CIDR-1
␣
on
BDCA-1
⫹
DCs could be reproduced by P. falciparum schizont-
iRBC (data not shown). Our data therefore suggest that CIDR-1
␣
can activate DCs to produce cytokines that allow a TCR-indepen-
dent response. The mechanisms by which this takes place are not
known. CIDR-1
␣
can bind CD36 (21), and ligation of CD36 on
DCs has been shown to lead to production of IL-10 (29). It is thus
possible that this is the means of DC activation. It is also possible
that ligation of PRR on DC, such as TLR, are involved (37). We
are confident that the effects of CIDR-1
␣
on DCs are not due to
contaminant LPS or other bacterial products as the negligible lev-
els of LPS measured were not able to stimulate DCs (our obser-
vation and Ref. 31), and another PfEMP-1 recombinant protein
from the cytoplasmic tail of PfEMP-1 (exon 2) expressed in the
same system and similarly purified had no stimulatory effect.
The slower and very significant production of IL-10 by the my-
eloid DCs in response to CIDR-1
␣
is similar to earlier observa-
tions of Urban et al. (41), where they showed that iRBC expressing
a PfEMP-1 on the surface, which bound CD36, inhibited the up-
regulation of costimulatory molecules on human cultured DC, in-
duced IL-10 production, and inhibited an allogeneic T cell re-
sponse. Our results suggest that it is the CIDR-1
␣
domain of
PfEMP-1, known to bind CD36 (21), that is responsible for these
down-regulatory responses of the DC, and further in our case the
IL-10 response may be initially associated with a short and tightly
regulated IL-12 response that is sufficient to activate NK cells and
TCR-independent and TCR-dependent T cell IFN-
␥
responses. It
would be of interest to determine whether there is variability of
this response among nonexposed donors, and whether those donors
that do not make NK or T cell responses to CIDR-1
␣
, do not make
an initial IL-12 burst.
In stark contrast to the IFN-
␥
response of nonexposed individ-
uals, both CD4 T cell and IFN-
␥
responses in malaria-exposed
individuals were inhibited by the MHC class II Ab. It is likely that
these differences between the CIDR-1
␣
response in nonexposed
and exposed individuals are due to the presence of memory CD4
T cells in the PBMCs from immune individuals. Because they have
been exposed to P. falciparum infections throughout their lives, it
is reasonable to expect that the malaria-exposed adults have accu-
mulated memory CD4 T cells specific to P. falciparum Ags in their
peripheral circulation. Our results raise the possibility that such
anti-MHC class II-blocking Abs may provide a way of distinguish-
ing normal Ag recall responses from the pre-existing TCR-inde-
pendent T cell responses to malaria Ags found among unexposed
donors, which would otherwise make it difficult to interpret T cells
assays in nonimmune children or in vaccine studies with nonex-
posed volunteers.
The observation that malaria-immune adults have classical
MHC class II-restricted CD4 T cell and IFN-
␥
responses to
CIDR-1
␣
is encouraging and suggest that these individuals have
CIDR-1
␣
-specific memory T cells. Due to its functional conser-
vation, surface location (accessible to Ab) and involvement with
cytoadhesion of iRBC to endothelial cells (thought to mediate pa-
thology) during P. falciparum infections, it is a potential vaccine
candidate for malaria. It is not yet known how similar the Malayan
camp var 1 CIDR-1
␣
sequence used in these studies is to the
CIDR-1
␣
variants circulating in Kilifi (Ngerenya). Work to se-
quence var genes from field isolates is ongoing, and will allow us
to compare the CIDR-1
␣
variant used here with those expressed in
field isolates. However, the fact that CD4 T cell and IFN-
␥
re-
sponses observed in a large proportion of malaria-exposed indi-
viduals reacted with a single variant of CIDR-1
␣
suggests that
there might be cross-reactivity between CIDR-1
␣
domains from
different PfEMP-1 molecules. Similar cross-reactivity between
CIDR-1
␣
variants has been observed in studies where rodents
were immunized simultaneously with several CIDR-1
␣
variants
(25, 42, 43). Together, these observations are encouraging in that
it may be possible to overcome the immense antigenic diversity of
PfEMP-1 in a CIDR-1
␣
-based vaccine.
It is not entirely clear how the presence of pre-existing T cells
will affect the development of CIDR-1
␣
-specific vaccines. How-
ever, it seems unlikely that bystander activated CD4 T cells would
protect the vaccinees from disease, because these cells may not
give cognate help to B cells to make protective Ab. More studies
will be needed to investigate whether the frequency of CIDR-1
␣
-
specific CD4 T cells is associated with pathology in nonexposed
individuals being exposed to P. falciparum for the first time.
Acknowledgments
We thank Cecile Voisine, Douglas Brown, Jane E. Blythe, and Anne-Marit
Sponaas for their helpful criticism and advice. This paper is published with
permission of the director of Kenya Medical Research Institute.
Disclosures
The authors have no financial conflict of interest.
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