Cell Host & Microbe

Short Article

Plasmodium falciparumErythrocyte Membrane Protein-1 SpecificallySuppresses Early Production of Host Interferon-g

Marthe C. D’Ombrain,1,2 Till S. Voss,1 Alexander G. Maier,1 J. Andrew Pearce,1 Diana S. Hansen,1

Alan F. Cowman,1 and Louis Schofield1,*1 Infection and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville,

Victoria 3050, Australia2 Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia

*Correspondence: schofield@wehi.edu.au

DOI 10.1016/j.chom.2007.06.012

SUMMARY

Plasmodium falciparum erythrocyte membraneprotein-1 (PfEMP-1) is a variable antigen ex-pressed by P. falciparum, the malarial parasite.PfEMP-1, present on the surface of infectedhost erythrocytes, mediates erythrocyte bind-ing to vascular endothelium, enabling the para-site to avoid splenic clearance. In addition,PfEMP-1 is proposed to regulate host immuneresponses via interactions with the CD36 recep-tor on antigen-presenting cells. We investigatedthe immunoregulatory function of PfEMP-1 bycomparing host cell responses to erythrocytesinfected with either wild-type parasites or trans-genic parasites lacking PfEMP-1. We showedthat PfEMP-1 suppresses the production ofthe cytokine interferon-g by human peripheralblood mononuclear cells early after exposureto P. falciparum. Suppression of this rapid proin-flammatory response was CD36 independentand specific to interferon-g production by gd-T,NK, and ab-T cells. These data demonstrate aparasite strategy for downregulating the proin-flammatory interferon-g response and further esta-blish transgenic parasites lacking PfEMP-1 aspowerful tools for elucidating PfEMP-1 functions.

INTRODUCTION

The multidomain variant antigen Plasmodium falciparum

Erythrocyte Membrane Protein-1 (PfEMP-1), expressed

on the surface of P. falciparum-infected erythrocytes,

can mediate binding to CD36, intercellular adhesion

molecule-1, vascular cell adhesion molecule, E-selectin,

P-selectin, CD31, and chondroitin sulfate A (CSA) on vas-

cular endothelial cells, thus enabling the parasite to avoid

splenic clearance (Newbold, 1999). PfEMP-1 is encoded

by the var gene family, which has been divided into three

major groups (A, B, and C) based on genomic location

130 Cell Host & Microbe 2, 130–138, August 2007 ª2007 Elsevie

and upstream sequence (Ups). Group B and C var genes

typically bind CD36, whereas group A var genes generally

do not (Robinson et al., 2003). Furthermore, group B and C

var genes tend to be associated with asymptomatic

malaria, while group A var genes have been implicated

in cerebral malaria (Kyriacou et al., 2006). The extensive

var repertoire and allelic diversity within PfEMP-1 enable

the parasite to evade humoral immunity while maintaining

cytoadherence to blood vessel walls (Hisaeda et al.,

2005). Although successful in evading antibody re-

sponses, PfEMP-1 is also proposed to modulate host im-

mune responses via CD36-dependent interactions with

antigen-presenting cells. Phagocytosis of nonopsonized

infected erythrocytes by macrophages was shown to be

dependent on CD36 ligation and was inhibited by the pro-

teolytic removal of PfEMP-1 (McGilvray et al., 2000). Fur-

thermore, infected erythrocytes inhibited DC maturation,

reducing the capacity of DC to activate T cells (Urban

et al., 1999). This was proposed to result from PfEMP-1

binding to CD36, thus implicating the CD36-binding

cysteine interdomain region-1a (CIDR-1a) domain of

PfEMP-1 as mediating this effect (Urban et al., 2001b).

In contrast, however, addition of recombinant CIDR-1a

to cultures of naive human PBMC resulted in the poly-

clonal activation of naive CD4 T cells and NK cells and

induced IFN-g production, leading to the suggestion that

this molecule may contribute to inflammatory immuno-

pathogenesis (Ndungu et al., 2006). These inconsistencies

may reflect methodological constraints that have previ-

ously restricted immunological analyses of PfEMP-1 to

use of either nonisogenic parasites selected for specific

adhesion phenotypes (Urban et al., 1999) (whereby uni-

form expression of one PfEMP-1 allele in the population

is not assured); or on recombinant PfEMP-1 subdomains

expressed in E. coli (Allsopp et al., 2002; Joergensen

et al., 2006; Ndungu et al., 2006), which may be con-

founded by misfolding, glycosylation, LPS contamination,

presentation out of cellular context, and application in

physiologically inappropriate concentrations. Clearly,

PfEMP-1 constitutes a major interface between parasite

and host, and the immunoregulatory activities of this mol-

ecule are likely to be important (Urban et al., 2005). Fur-

thermore, as PfEMP-1 domains may be developed as

r Inc.

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

Figure 1. PfEMP-1 Suppresses the Innate IFN-g Response of

PBMC to P. falciparum In Vitro

uRBC, parental 3D7, and 3D7 PfEMP-1 knockdown parasites were

solubilized in 1% Triton X-100. The insoluble fraction was then solubi-

lized in 2% SDS, and the supernatant was probed in western blot with

antibodies (mAb 6H1) against the conserved intercellular region of

PfEMP-1 (A). Parental 3D7 shows a distinctive band above 250 kDa,

typical of PfEMP-1, whereas 3D7 PfEMP-1� iRBC and uRBC do not

(asterisks denote erythrocyte proteins) (A). (B) is a vector map of the

construct used to produce the 3D7 PfEMP-1� line containing the hu-

man dihydrofolate reductase (hDHFR) gene that encodes resistance

to WR99210. The expression of hDHFR is under the control of a var

upsC gene promoter. The blasticidin deaminase (BSD) gene encodes

resistance to blasticidin-S and serves to maintain stable integration

of the plasmid in the parasite chromosome. PBMC (2 3 105) from a

number of malaria-naive donors were stimulated for 24 hr with

6 3 105 3D7 parental iRBC, 3D7 PfEMP-1� iRBC, or uRBC (C), or

6 3 105 3D7 parental iRBC, trypsin-digested 3D7 parental iRBC,

uRBC, or trypsin-digested uRBC (D). Supernatants were harvested

and assayed for cytokines. The mean IFN-g detected in supernatants

is shown (C and D). Error bars are the SEM for R2 experiments. Upper-

case letters indicate donors. In 9/9 donors, the mean level of IFN-g

Cell Hos

humoral vaccines (Baruch et al., 2002), understanding

their potential role in the promotion or prevention of im-

munoregulation, immunopathology, and cell-mediated

immunity is a priority. Therefore, to investigate the immu-

nological function of endogenous, whole PfEMP-1, ex-

pressed in situ in the correct context and concentration

on the parasite surface, we used transgenic parasite lines

that lack surface expression of PfEMP-1 (Maier et al.,

2006; Voss et al., 2006). Using these parasite lines, we

demonstrate that PfEMP-1 suppresses innate IFN-g pro-

duction by naive gd-T cells and NK cells, as well as early

IFN-g production by naive ab-T cells. No evidence for

PfEMP-1-mediated polyclonal activation of these cell

populations was found. The interaction of CD36 with

CIDR-1a, proposed variously to mediate immunosuppres-

sion (Urban et al., 1999, 2001a) or polyclonal activation of

naive T cells and NK cells (Ndungu et al., 2006), was not

required for the suppression of IFN-g production. There-

fore, PfEMP-1-mediated suppression of innate IFN-g

production occurs via a CIDR-1a/CD36-independent

pathway. Furthermore, PfEMP-1 was neither a suppressor

nor an activator of malaria-induced NK cell- or T cell-

mediated cytolysis. These data demonstrate a strategy

employed by the parasite to downregulate the proinflam-

matory IFN-g response and validate the use of PfEMP-1

transgenic parasites as powerful tools for dissecting the

functional and immunological roles of this important viru-

lence protein in human malaria.

RESULTS AND DISCUSSION

PfEMP-1 Suppresses the Innate IFN-g Response

to Malaria

To assess the role of PfEMP-1 in the regulation of cellular

immune responses to malaria, transgenic parasites lack-

ing surface expression of PfEMP-1 were used (Maier

et al., 2006; Voss et al., 2006). As shown in Figure 1A,

P. falciparum 3D7 parental parasites express PfEMP-1,

whereas the 3D7 PfEMP-1 knockdown parasites do not.

This is due to the expression of a human dihydrofolate

reductase gene, which confers resistance to WR99210,

under the control of a var upsC gene promoter that allows

infiltration of the antigenic variation program, thus silenc-

ing the gene family (Voss et al., 2006) (Figure 1B). To inves-

tigate the effect of the absence and presence of PfEMP-1

on human, innate cellular immune responses, peripheral

blood mononuclear cells (PBMC) from healthy, malaria-

naive adult donors were stimulated for 24 hr with 3D7 pa-

rental, live P. falciparum schizont-infected red blood cells

(iRBC), 3D7 PfEMP-1� iRBC, or uninfected red blood cells

(uRBC), and their ability to induce proinflammatory

and anti-inflammatory cytokines was observed. Loss of

produced by PBMC following stimulation with 3D7 PfEMP-1� iRBC

exceeds that for 3D7 parental iRBC (p < 0.01; nonparametric sign

test) (C). Similarly, in 7/7 donors, the mean level of IFN-g produced

by PBMC following stimulation with trypsin-digested 3D7 parental

iRBC exceeds that for nondigested 3D7 parental iRBC (p < 0.01; non-

parametric sign test) (D).

t & Microbe 2, 130–138, August 2007 ª2007 Elsevier Inc. 131

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

PfEMP-1 resulted in substantially higher IFN-g production

in all donors compared to parental iRBC, with some do-

nors producing up to four times more IFN-g in the absence

of PfEMP-1 (Figure 1C), thus suggesting that PfEMP-1

suppresses the host innate IFN-g response to malaria. Pa-

rental 3D7 iRBC that were digested with trypsin to remove

PfEMP-1 proteolytically also induced significantly higher

IFN-g production compared to nondigested iRBC

(Figure 1D), confirming that the differences in the ability

of the parental and transgenic iRBC to induce IFN-g are

not due to differences in parasite maturation or a conse-

quence of the introduction of the plasmid. In addition,

knockdown of PfEMP-1 does not affect expression or traf-

ficking of any other known infected erythrocyte surface

proteins (T.S.V. and A.F.C., unpublished data). Further-

more, loss of PfEMP-1 had no effect on TNF-a, IL-6,

IL-10, IL-2, or IL-4 production by PBMC (Figure S1 in the

Supplemental Data available with this article online).

Therefore, the suppressive effect of PfEMP-1 appears

to be specific for pathways responsible for innate IFN-g

output.

PfEMP-1 Negatively Regulates IFN-g Production

by gd-T, NK, and ab-T Cells

We and others have previously shown that IFN-g is rapidly

produced by gd-T cells, NK cells, and ab-T cells following

stimulation of naive human PBMC with iRBC (Artavanis-

Tsakonas and Riley, 2002; D’Ombrain et al., 2007; Hen-

smann and Kwiatkowski, 2001). We have further demon-

strated that, in the majority of donors, gd-T cells are the

predominant source of early IFN-g, while NK cells and

ab-T cells make only minor contributions (D’Ombrain

et al., 2007). To identify which early IFN-g-producing cell

types are suppressed by PfEMP-1, PBMC were stimu-

lated for 24 hr with 3D7 parental iRBC, 3D7 PfEMP-1�

iRBC, or uRBC, harvested, and stained for intracellular

IFN-g in combination with CD3, gdTCR, and CD56. gd-T

cells (CD3+gdTCR+), NK cells (CD3�CD56+), and to

a lesser extent ab-T cells (CD3+gdTCR�), were each neg-

atively regulated by PfEMP-1 with respect to early IFN-g

production (Figure 2). These results contrast with previous

studies that found recombinant CIDR-1a (a subdomain of

PfEMP-1) caused the polyclonal activation of NK cells and

CD4 T cells to produce IFN-g (Allsopp et al., 2002; Ndungu

et al., 2006). Furthermore, recombinant CIDR-1a was also

found to induce IL-10 production by PBMC-derived DC

(Allsopp et al., 2002; Ndungu et al., 2006); however, the

PfEMP-1 null transgenic iRBC had no effect on IL-10 out-

put from PBMC (Figure S1). The use of recombinant pro-

teins expressed in either bacterial or mammalian cells

may be subject to effects arising from glycosylation, LPS

contamination, molar ratio/concentration dependence ef-

fects, misfolding, and expression out of appropriate cellu-

lar context. In contrast, the isogenic parasite lines used in

the present study differ only in the presence or absence of

surface PfEMP-1, which is expressed in situ in the correct

context and concentration on the iRBC surface. Further-

more, recombinant CIDR-1a was used at 5 mg/ml (Allsopp

et al., 2002; Ndungu et al., 2006), or 1.8 3 10�7 M. Assum-

132 Cell Host & Microbe 2, 130–138, August 2007 ª2007 Elsevi

ing PfEMP-1 has 5000 copies/cell, the concentration or li-

gand density of native PfEMP-1 in standard costimulation

assays is approximately 2.5 3 10�11 M, 72,000 times less

than that used in the recombinant protein studies. Impor-

tantly, the change in phenotype upon loss of PfEMP-1 in

the present study indicates bioactivity at physiological

levels of expression.

PfEMP-1 Modulates Innate IFN-g Production

via a CD36-Independent Pathway

The simultaneous negative regulation of IFN-g production

by gd-T cells, NK cells, and ab-T cells (Figure 2) suggests

Figure 2. PfEMP-1 Negatively Regulates Early IFN-g Produc-

tion by gd-T, NK, and ab-T Cells

PBMC (2 3 105) from eight malaria-naive donors were stimulated

for 24 hr with 6 3 105 3D7 parental iRBC, 3D7 PfEMP-1� iRBC, or

uRBC. PBMC were harvested and stained for intracellular IFN-g,

gdTCR, CD3, and CD56. IFN-g+ cells were gated (A) and identified

as CD3�CD56+ NK cells (B), CD3+gdTCR+ gd-T cells (C), or

CD3+gdTCR� ab-T cells (C). FACS plots in (A)–(C) are from one repre-

sentative donor. FACS data from all eight donors is displayed graphi-

cally in (D) (uppercase letters indicate donors).

er Inc.

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

that PfEMP-1 might signal via a common mechanism.

Binding of the CIDR-1a domain of PfEMP-1 to CD36 has

been proposed to mediate the reduction in the capacity

of DC to stimulate secondary T cell responses following

parasite stimulation (Urban et al., 1999, 2001b). Further-

more, phagocytosis of nonopsonized infected erythro-

cytes by macrophages was shown to be dependent on

CD36 (McGilvray et al., 2000). Thus, we sought to deter-

mine whether the PfEMP-1-mediated negative regulation

of IFN-g production by gd-T cells, NK cells, and ab-T cells

requires the CIDR-1a/CD36 receptor-ligand interaction.

This hypothesis was tested using the CS2 strain of P. fal-

ciparum, which, unlike 3D7, expresses the var2CSA

PfEMP-1, which lacks a CIDR-1a domain and binds to

CSA but not CD36 (Figure 3). Consequently, CD36-depen-

dent pathways are not initiated by CS2 parasites. PBMC

were stimulated as previously with CS2 parental iRBC,

CS2 Skeletal Binding Protein-1 (SBP-1) KO iRBC (which

lack expression of PfEMP-1 on the infected erythrocyte

surface) (Maier et al., 2006), or uRBC. As shown in Figure 3,

the CS2 parental and SBP-1 KO PfEMP-1� iRBC recapit-

ulate the results obtained with the 3D7 parental and 3D7

PfEMP-1� iRBC (Figure 1), with one donor producing

19.6-fold more IFN-g in the absence of PfEMP-1 (Figure 3).

Clearly, suppression of IFN-g production by PfEMP-1 oc-

curs via a pathway independent of CIDR-1a/CD36 inter-

actions. The PfEMP-1 domain that mediates suppression

of IFN-g is likely to be conserved between 3D7 and CS2

and thus may involve a Duffy binding-like (DBL) domain.

We are currently creating PfEMP-1 null parasites express-

ing defined PfEMP-1 domains to identify the molecular

basis of PfEMP-1 immunoregulatory activity and to iden-

tify the corresponding host receptor.

PfEMP-1 Is Not a Ligand for NK Cell

or T Cell Cytotoxicity

In addition to cytokine production, NK cells, CD8+CD4�

gd-T cells, and CD8+ ab-T cells also possess cytotoxic ca-

pabilities. gd-T cells have been shown to be cytotoxic for

iRBC, while CD8 T cells are not, due to the lack of MHC

class I expression on iRBC (Troye-Blomberg et al.,

1999). LAMP-1 (CD107a) appears on the surface of effec-

tor cells following degranulation and can be used as

a marker of cytotoxic activation (Alter et al., 2004). NK cells

have previously been reported to lyse iRBC (Chaicumpa

et al., 1983; Orago and Facer, 1991) and induce expres-

sion of LAMP-1 following exposure to iRBC (Korbel

et al., 2005). Slight changes in NK cell activation status

were detected after incubation with peptidic DBL-1a (a

PfEMP-1 subdomain) which was proposed to bind the

Natural Cytotoxicity Receptors NKp30 and NKp46 and in-

duce NK cell cytotoxicity (Mavoungou et al., 2007). In that

study, DBL-1a peptides were used at 40 mg/well, or

around 2 3 10�4�2 3 10�5 M, assuming a peptide molec-

ular mass of approximately 2200 Daltons. This is approx-

imately 2–10 million times greater than the ligand density

of native PfEMP-1 on the surface of iRBC. Therefore, we

sought to test the role of PfEMP-1 in NK cell cytotoxicity

further using isogenic parasites differing only in the pres-

Cell Ho

ence or absence of PfEMP-1 expressed in situ in the cor-

rect context and concentration on the iRBC surface. A role

for PfEMP-1 in gd-T cell cytotoxicity was also explored.

Accordingly, we compared the ability of 3D7 parental

iRBC or 3D7 PfEMP-1� iRBC to induce LAMP-1 expres-

sion on NK cells and T cells following 6 hr of coculture

with PBMC from healthy, malaria-naive adult donors.

The K562 cell line, a well-defined target for NK cell cyto-

toxicity, was used as a positive control, while uRBC

served as the negative control. LAMP-1 was expressed

at high levels on NK cells and at modest levels on T cells,

Figure 3. PfEMP-1 Modulates Innate IFN-g Production via

a CD36-Independent Pathway

The western blot (prepared as for Figure 1A) confirms that parental

CS2 expresses PfEMP-1 on the infected erythrocyte surface, whereas

DSBP-1KO PfEMP-1� does not (A). The typical PfEMP-1 extracellular

domain from a CD36-binding 3D7 parental parasite is shown in (B)

(Robinson et al., 2003). The CIDR-1a domain of PfEMP-1 mediates

binding to CD36 (Baruch et al., 1999). The extracellular domain of var2-

CSA, the predominant PfEMP-1 protein expressed by CS2 parental

parasites that mediates CSA binding, is illustrated in (C) (Gardner

et al., 2002). Note the absence of a CIDR-1a domain in var2CSA

PfEMP-1 (C), rendering this parasite unable to bind CD36 (Gardner

et al., 2002). PBMC (2 3 105) from ten malaria-naive donors were stim-

ulated for 24 hr with 6 3 105 CS2 parental iRBC, DSBP-1KO PfEMP-1�

iRBC, or uRBC. Supernatants were harvested and assayed for IFN-g.

The mean IFN-g detected in supernatants from ten donors is shown (D).

Error bars are the SEM for R2 experiments. In 10/10 donors, the mean

level of IFN-g produced by PBMC following stimulation with DSBP-

1KO PfEMP-1� iRBC exceeds that for CS2 parental iRBC (p < 0.001;

nonparametric sign test). Upper case letters indicate donors.

st & Microbe 2, 130–138, August 2007 ª2007 Elsevier Inc. 133

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

Figure 4. PfEMP-1 Is Not a Ligand for Malaria-Induced NK Cell or T Cell Cytotoxicity

PBMC (2 3 105) were cultured for 6 hr in the presence of 6 3 105 K562 cells, 3D7 parental iRBC, 3D7 PfEMP-1� iRBC, or uRBC. PBMC were harvested

and stained for CD3, CD56, and the cytotoxicity marker LAMP-1. CD3�CD56+ NK cells and CD3+ T cells were gated (A) and analyzed for LAMP-1

expression in response to each treatment (B). FACS plots in (A) and (B) are from one representative donor. Mean LAMP-1 expression on NK cells

and T cells from ten donors is shown in (C) and (D), respectively. Error bars are the SEM for ten donors. p values from Student’s t tests show no

statistically significant differences between LAMP-1 expression on NK cells or T cells stimulated with 3D7 parental iRBC or 3D7 PfEMP-1� iRBC

(C and D).

following incubation with K562 target cells (Figure 4). Fur-

thermore, LAMP-1 expression was detected above back-

ground levels on both NK cells and T cells following incu-

bation with 3D7 parental iRBC and 3D7 PfEMP-1� iRBC

(Figure 4). Importantly, no statistically significant differ-

ences between LAMP-1 expression levels on NK cells or

T cells stimulated with 3D7 parental or 3D7 PfEMP-1�

iRBC were detected (Figure 4), thus demonstrating that

PfEMP-1, inclusive of a DBL-1a subdomain and ex-

pressed under physiologically relevant conditions, is not

a ligand for NK cell-mediated or T cell-mediated cytotox-

icity of iRBC.

Conclusions

Plasmodium falciparum causes rapid activation of macro-

phages and DC through the production of pathogen-

associated molecular patterns, such as glycosylphospha-

tidylinositol (Schofield and Hackett, 1993) and malarial

134 Cell Host & Microbe 2, 130–138, August 2007 ª2007 Elsev

DNA (associated with haemozoin) (Parroche et al., 2007),

which act through Toll-like receptors (Krishegowda

et al., 2005; Parroche et al., 2007; Zhu et al., 2005). Sub-

sequently, NK receptor- and gdTCR-dependent innate

and ‘‘intermediate’’ adaptive pathways become activated,

resulting in rapid production of the proinflammatory cyto-

kine IFN-g (Schofield and Grau, 2005). Following 24 hr

stimulation of human PBMC with live P. falciparum-

infected erythrocytes, gd-T cells expressing NK receptors

represent the major cellular source of innate IFN-g, with

minor contributions from NK cells and ab-T cells

(D’Ombrain et al., 2007). The activating ligand for NK cells

remains to be identified, while gd-T cells are known to be

activated by malarial phosphoantigens (Behr et al., 1996;

Pichyangkul et al., 1997) and rapid activation of naive

ab-T cells by P. falciparum is thought to be due to the

presence of cross-reactive antigens that are expressed

on a wide range of common microbes (Currier et al.,

ier Inc.

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

1992; Zevering et al., 1992). Here we have identified an im-

munoregulatory mechanism of P. falciparum whereby the

early IFN-g response of gd-T cells, NK cells, and ab-T cells

is strongly suppressed by the presence of PfEMP-1.

The host receptor(s) utilized by PfEMP-1 to suppress

IFN-g production by gd-T, NK, and ab-T cells remains to

be identified; however, this pathway is independent of

CIDR-1a/CD36 interactions and thus occurs via a different

mechanism than that proposed for PfEMP-1 interactions

with DC and macrophages (McGilvray et al., 2000; Urban

et al., 1999, 2001b). As plasmacytoid DC promote early

gd-T cell activation (Pichyangkul et al., 2004), myeloid

DC stimulate malaria-specific ab-T cells (Urban et al.,

1999), and both macrophages and myeloid DC are re-

quired for NK cell activation (Baratin et al., 2005; Newman

et al., 2006), it is unlikely that PfEMP-1 suppresses early

IFN-g production via a common antigen-presenting cell.

Furthermore, the effect of PfEMP-1 is specific to IFN-g,

with no effect on a range of proinflammatory and anti-

inflammatory cytokines, including those typically asso-

ciated with macrophage and DC activation. Therefore, it

is plausible that PfEMP-1 may signal directly to gd-T cells,

NK cells, and ab-T cells via a common receptor.

Interestingly, in humans, malaria-responsive gd-T and

NK cells more frequently express the inhibitory NK recep-

tor NKG2A (Artavanis-Tsakonas et al., 2003; D’Ombrain

et al., 2007). Thus, PfEMP-1 may engage NKG2A (and/

or other inhibitory NK receptors), resulting in suppression

of IFN-g production. Alternatively, the host receptor may

be a more general, non-NK receptor that negatively regu-

lates leukocyte activation. The suppression of IFN-g by

PfEMP-1 appears to be due to a PfEMP-1 domain that

is conserved between 3D7 and CS2 parasite lines and

thus may involve a DBL domain. We are currently creating

PfEMP-1 null parasites expressing defined PfEMP-1 do-

mains to identify both the PfEMP-1 subdomain and the

corresponding host receptor that mediates innate IFN-g

suppression.

The suppression of early IFN-g production by PfEMP-1

was quantitatively highly significant (up to 20-fold). Given

the dual role of IFN-g in malaria protection and pathogen-

esis, these data raise the possibility that this process

might impact either IFN-g-dependent antiparasite immu-

nity or IFN-g-dependent immunopathogenesis. For exam-

ple, protection against reinfection with P. falciparum ma-

laria has been associated with higher plasma levels of

IFN-g (Luty et al., 1999), possibly resulting from increased

macrophage activation and enhanced parasite clearance

(Hisaeda et al., 2005). Furthermore, in the P. chabaudi mu-

rine model of malaria, ifn-g�/� and ifn-g receptor�/� mice

exhibit higher mortality rates (Favre et al., 1997; Su and

Stevenson, 2000), while in the P. berghei-K173 murine

model of malaria, a 24 hr IFN-g peak, not observed in

the closely related P. berghei-ANKA cerebral malaria

model, was associated with protection from the cerebral

syndrome (Mitchell et al., 2005). The suppression of early

IFN-g production by PfEMP-1, therefore, may represent

an immune evasion strategy employed by P. falciparum,

which serves to downregulate the antiparasitic IFN-g

Cell Hos

response. Furthermore, IFN-g inhibits the infectivity of

P. berghei sporozoites for hepatocytes in mice and rats

and is a critical effector mechanism in immunity to liver

stage infection (Schofield et al., 1987a, 1987b). It is thus

plausible that the suppression of IFN-g by PfEMP-1 may

also favor reinfection upon subsequent sporozoite chal-

lenge in humans. However, although critical in protective

antimalarial immunity, IFN-g may also play an important

role in pathogenesis. In humans, polymorphisms in both

IFN-g and IFN-g Receptor 1 genes are associated with

severe malaria (Kwiatkowski, 2005), and high levels of

circulating IFN-g were found to be indicators of poor

prognostic outcome (Hunt and Grau, 2003). Similarly, in

the P. berghei-ANKA model of cerebral malaria, neutrali-

zation of IFN-g in vivo prevents fatalities (Grau et al.,

1986), and ifn-g�/� and ifn-g receptor�/� mice fail to de-

velop the cerebral syndrome (Amani et al., 2000). The sup-

pression of IFN-g by PfEMP-1 may thus also be beneficial

for the host, possibly serving to reduce the severity of clin-

ical symptoms and immunopathogenesis. Clearly, the role

of IFN-g in malaria protection and pathogenesis is com-

plex and needs to be addressed in the context of ade-

quate epidemiological study designs. Therefore, to allow

the impact of PfEMP-1-mediated immune regulation to

be assessed in relation to relevant parasitological and clin-

ical outcome variables, we are currently conducting longi-

tudinal cohort field studies in Papua New Guinea (Michon

et al., 2007).

EXPERIMENTAL PROCEDURES

PBMC Preparation

Blood from healthy nonimmune adults was obtained from the Austra-

lian Red Cross Blood Service under appropriate ethical approval.

Blood was diluted 1:1 in PBS, and PBMC were separated by density

centrifugation with Ficoll-Paque PLUS (Amersham Biosciences, Swe-

den). PBMC were washed and resuspended in complete medium

(RPMI 1640, 5% heat-inactivated FBS, 2 mM L-glutamine, 25 mM

HEPES, 50 mM 2-ME, 100 U/ml penicillin, and 100 mg/ml streptomycin

sulfate) for use in cytokine induction and LAMP-1 cytotoxicity assays.

Cultivation of P. falciparum

3D7, CS2, and transgenic P. falciparum parasites were cultivated at

37�C with 5% CO2, 1% O2, and 1% N2 in 150 cm2 flasks at 4% hemat-

ocrit using O+ human erythrocytes (Australian Red Cross Blood

Service [ARCBS]) in RPMI 1640 buffered with 25 mM HEPES and sup-

plemented with 0.5% Albumax II, 2 mg/ml glucose, 28 mM sodium

bicarbonate, 25 mg/ml gentamycin, and 100 mg/ml hypoxanthine.

Parasite development was monitored by light microscopy using

methanol-fixed, Giemsa-stained thin blood films. The construct used

to produce the transgenic 3D7 PfEMP-1� parasite line was made by

transfecting 3D7 with pHBupsCR (Voss et al., 2006). The blasticidin de-

aminase gene serves to maintain stable integration of the plasmid in

the parasite chromosome. 3D7 PfEMP-1� parasites were cultured

with 4 mg/ml blasticidin-S and 4 nM WR99210 (Voss et al., 2006). Tryp-

sin-digested 3D7 parental iRBC were included as an additional control

for the absence of PfEMP-1. Trypsin digestion of live iRBC was carried

out as previously described (Beeson et al., 2000). Parasites were

sorbitol synchronized, knob selected, and screened for mycoplasma

negativity. iRBC were purified by magnetic cell sorting CS columns

(Miltenyi Biotec, CA) and resuspended in complete medium for use

in cytokine induction and LAMP-1 cytotoxicity assays.

t & Microbe 2, 130–138, August 2007 ª2007 Elsevier Inc. 135

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

Western Blot

Immunoblot analysis was performed using iRBC purified by magnetic

cell sorting CS columns (Miltenyi Biotech, CA). iRBC (1 3 107) were ex-

tracted with 150 ml of 1% (v/v) Triton X-100 in PBS and protease inhib-

itors (Roche). The Triton X-100 insoluble pellet was then extracted with

2% SDS (w/v) and loaded onto the gel in reducing SDS sample buffer

(Baruch et al., 2002).

Cytokine Induction Assay

In 96-well plates, 2 3 105 PBMC were stimulated with 6 3 105 live pa-

rental, trypsin-digested parental, or transgenic iRBC, or 6 3 105 uRBC

per well. Cultures were incubated for 24 hr (37�C, 5% CO2). Golgistop

(BD PharMingen) was added at 20 hr. Cells were harvested and ana-

lyzed by flow cytometry. Culture supernatants were analyzed for cyto-

kine content by ELISA and Cytometric Bead Array (BD PharMingen).

IFN-g ELISA

IFN-g was detected in culture supernatants by standard sandwich

ELISA. Purified mouse anti-human IFN-g (NIB42) mAb was used for

capture, and biotinylated mouse anti-human IFN-g (4S.B3) was used

for detection (BD Biosciences, CA).

Detection of TNF-a, IL-6, IL-10, IL-2, and IL-4

TNF-a, IL-6, IL-10, IL-2, and IL-4 were detected in supernatants from

the cytokine induction assays of five donors using the BD Cytometric

Bead Array Human Th1/Th2 Cytokine Kit II (BD Biosciences, CA)

according to the manufacturer’s instructions. Samples were analyzed

in a FACSCalibur flow cytometer (BD Biosciences, CA). Data analysis

was performed with the BD Cytometric Bead Array Software (BD

Biosciences, CA).

LAMP-1 Cytotoxicity Assay

K562 cells (ATCC, Manassas, VA) were maintained at 37�C and 5%

CO2 in IMDM supplemented with 4 mM L-glutamine, 1.5g/l sodium bi-

carbonate, and 10% FBS. In 96-well plates, 2 3 105 PBMC were stim-

ulated with 6 3 105 parental or transgenic iRBC, K562, or uRBC cells

per well. Cultures were incubated for 6 hr (37�C, 5% CO2). Golgistop

(BD PharMingen) was added after 1 hr. Cells were harvested and ana-

lyzed for LAMP-1 expression by flow cytometry.

Flow Cytometry

Anti-TCR-g/d FITC, CD3-APC, CD56-biotin (conjugated with Strepta-

vidin-PerCP-Cy5.5), IFN-g-PE, IgG1l isotype control-PE, CD107a

(LAMP-1)-biotin (conjugated with Streptavidin-PerCP-Cy5.5), and

CD56-PE (BD Biosciences, CA) were used for surface, intracellular,

or isotype control staining according to the manufacturer’s recom-

mendations. PBMC were surface stained for 40 min with appropriate

combinations of labeled mAbs. For biotin-labeled antibodies, there

was a second 40 min incubation for conjugation with fluorescently la-

beled streptavidin. PBMC from cytokine induction assays were stained

for intracellular IFN-g using the BD Cytofix/Cytoperm Kit (BD Biosci-

ences, CA) according to the manufacturer’s instructions, with appro-

priate isotype-matched controls. Finally, cells were resuspended in

200 ml PBS and analyzed in a FACSCalibur flow cytometer (BD Biosci-

ences, CA). Dead cells were gated out by forward and side scatter, and

100,000 live cell events were collected. Data analysis was performed

with Weasel v2.2.3 software.

Statistical Analyses

Nonparametric sign tests were used to determine statistically signifi-

cant differences in IFN-g production between parental and transgenic

iRBC as well as between nontreated parental and trypsin-digested

parental iRBC at the population level (Lehmann, 1975). As the Central

Limit Theorem applied, Student’s t tests were used to determine sta-

tistically significant differences in LAMP-1 expression between paren-

tal and transgenic iRBC.

136 Cell Host & Microbe 2, 130–138, August 2007 ª2007 Elsevi

Supplemental Data

The Supplemental Data include one supplemental figure and can be

found with this article online at http://www.cellhostandmicrobe.com/

cgi/content/full/2/2/130/DC1/.

ACKNOWLEDGMENTS

We thank anonymous donors and the Australian Red Cross Blood Ser-

vice for providing blood samples. This study was supported by grants

from the National Health and Medical Research Council of Australia

and from the National Institutes of Health. A.F.C. and L.S. are Interna-

tional Research Scholars of the Howard Hughes Medical Institute.

Received: April 2, 2007

Revised: June 12, 2007

Accepted: June 28, 2007

Published: August 15, 2007

REFERENCES

Allsopp, C.E., Sanni, L.A., Reubsaet, L., Ndungu, F., Newbold, C.,

Mwangi, T., Marsh, K., and Langhorne, J. (2002). CD4 T cell responses

to a variant antigen of the malaria parasite Plasmodium falciparum,

erythrocyte membrane protein-1, in individuals living in malaria-

endemic areas. J. Infect. Dis. 185, 812–819.

Alter, G., Malenfant, J.M., and Altfield, M. (2004). CD107a as a func-

tional marker for the identification of natural killer cell activity. J. Immu-

nol. Methods 294, 15–22.

Amani, V., Vigario, A.M., Belnoue, E., Marussig, M., Fonseca, L., Maz-

ier, D., and Renia, L. (2000). Involvement of IFN-gamma receptor-med-

icated signaling in pathology and anti-malarial immunity induced by

Plasmodium berghei infection. Eur. J. Immunol. 30, 1646–1655.

Artavanis-Tsakonas, K., and Riley, E.M. (2002). Innate immune re-

sponse to malaria: Rapid induction of IFN-gamma from human NK

cells by live Plasmodium falciparum-infected erythrocytes. J. Immunol.

169, 2956–2963.

Artavanis-Tsakonas, K., Eleme, K., McQueen, K.L., Cheng, N.W., Par-

ham, P., Davis, D.M., and Riley, E.M. (2003). Activation of a subset of

human NK cells upon contact with Plasmodium falciparum-infected

erythrocytes. J. Immunol. 171, 5396–5405.

Baratin, M., Roetynck, S., Lepolard, C., Falk, C., Sawadogo, S.,

Uematsu, S., Akira, S., Ryffel, B., Tiraby, J.-G., Alexopoulou, L., et al.

(2005). Natural killer cell and macrophage cooperation in MyD88-

dependent innate responses to Plasmodium falciparum. Proc. Natl.

Acad. Sci. USA 102, 14747–14752.

Baruch, D.I., Ma, X.C., Pasloske, B., Howard, R.J., and Miller, L.H.

(1999). CD36 peptides that block cytoadherence define the CD36

binding region for Plasmodium falciparum-infected erythrocytes.

Blood 94, 2121–2127.

Baruch, D.I., Gamain, B., Barnwell, J.W., Sullivan, J.S., Stowers, A.,

Galland, G.G., Miller, L.H., and Collins, W.E. (2002). Immunization of

Aotus monkeys with a functional domain of the Plasmodium falcipa-

rum variant antigen induces protection against a lethal parasite line.

Proc. Natl. Acad. Sci. USA 99, 3860–3865.

Beeson, J.G., Rogerson, S.J., Cooke, B.M., Reeder, J.C., Chai, W.,

Lawson, A.M., Molyneux, M.E., and Brown, G.V. (2000). Adhesion of

Plasmodium falciparum-infected erythrocytes to hyaluronic acid in

placental malaria. Nat. Med. 6, 86–90.

Behr, C., Poupot, R., Peyrat, M.A., Poquet, Y., Constant, P., Dubois,

P., Bonneville, M., and Fournie, J.J. (1996). Plasmodium falciparum

stimuli for human gammadelta T cells are related to phosphorylated

antigens of mycobacteria. Infect. Immun. 64, 2892–2896.

Chaicumpa, W., Chatkaeomorakot, A., Patarapotikul, J., and Looaree-

suwan, S. (1983). Effects of human natural killer (NK) cells on Plasmo-

dium falciparum infected red cells. Asian Pac. J. Allergy Immunol. 1,

135–139.

er Inc.

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

Currier, J., Sattabongkot, J., and Good, M.F. (1992). ‘Natural’ T cells

responsive to malaria: Evidence implicating immunological cross-

reactivity in the maintenance of TCR alpha beta+ malaria-specific

responses from non-exposed donors. Int. Immunol. 4, 985–994.

D’Ombrain, M.C., Hansen, D.S., Simpson, K.M., and Schofield, L.

(2007). gd-T cells expressing NK receptors predominate over NK cells

and conventional T cells in the innate IFN-g response to Plasmodium

falciparum malaria. Eur. J. Immunol. 37, 1864–1873.

Favre, N., Ryffel, B., Bordmann, G., and Rudin, W. (1997). The course

of Plasmodium chabaudi chabaudi infections in interferon-g receptor

deficient mice. Parasite Immunol. 19, 375–383.

Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., Hyman,

R.W., Carlton, J.M., Pain, A., Nelson, K.E., Bowman, S., et al. (2002).

Genome sequence of the human malaria parasite Plasmodium falcipa-

rum. Nature 419, 498–511.

Grau, G.E., Piguet, P.F., Engers, H.D., Louis, J.A., Vassalli, P., and

Lambert, P.H. (1986). L3T4+ T lymphocytes play a major role in the

pathogenesis of murine cerebral malaria. J. Immunol. 137, 2348–2354.

Hensmann, M., and Kwiatkowski, D. (2001). Cellular basis of early

cytokine response to Plasmodium falcipaum. Infect. Immun. 69,

2364–2371.

Hisaeda, H., Yasutomo, K., and Himeno, K. (2005). Malaria: Immune

evasion by parasites. Int. J. Biochem. Cell Biol. 37, 700–706.

Hunt, N.H., and Grau, G.E. (2003). Cytokines: Accelerators and brakes

in the pathogenesis of cerebral malaria. Trends Immunol. 24, 491–499.

Joergensen, L., Turner, L., Magistrado, P., Dahlback, M.A., Vester-

gaard, L.S., Lusingu, J.P., Lemnge, M., Salanti, A., Theander, T.G.,

and Jensen, A.T. (2006). Limited cross-reactivity among domains of

the Plasmodium falciparum clone 3D7 erythrocyte membrane protein

1 family. Infect. Immun. 74, 6778–6784.

Korbel, D.S., Newman, K.C., Almeida, C.R., Davis, D.M., and Riley,

E.M. (2005). Heterogeneous human NK cell responses to Plasmodium

falciparum-infected erythrocytes. J. Immunol. 175, 7466–7473.

Krishegowda, G., Hajjar, A.M., Zhu, J., Douglass, E.J., Uematsu, S.,

Akira, S., Woods, S.A., and Gowda, D.C. (2005). Induction of pro-

inflammatory responses in macrophages by the glycosylphosphati-

dylinositols of Plasmodium falciparum. J. Biol. Chem. 280, 8606–8616.

Kwiatkowski, D.P. (2005). How malaria has affected the human ge-

nome and what human genetics can teach us about malaria. Am. J.

Hum. Genet. 77, 171–192.

Kyriacou, H.M., Stone, G.N., Challis, R.J., Raza, A., Lyke, K.E., Thera,

M.A., Kone, A.K., Doumbo, O.K., Plowe, C.V., and Rowe, J.A. (2006).

Differential var gene transcription in Plasmodium falciparum isolates

from patients with cerebral malaria compared to hyperparasitaemia.

Mol. Biochem. Parasitol. 150, 211–218.

Lehmann, E.L. (1975). Nonparametrics: Statistical Methods Based on

Ranks (San Francisco: McGraw-Hill International Book Company).

Luty, A.J., Lell, B., Schmidt-Ott, R., Lehman, L.G., Luckner, D., Greve,

B., Matsusek, P., Herbich, K., Schmid, D., Migot-Nabias, F., et al.

(1999). Interferon-g responses are associated with resistance to rein-

fection with Plasmodium falciparum in young African children. J. Infect.

Dis. 179, 980–988.

Maier, A.G., Rug, M., O’Neill, M.T., Beeson, J.G., Marti, M., Reeder, J.,

and Cowman, A. (2006). Skeleton Binding Protein 1 functions at the

parasitophorous vacuole membrane to traffic PfEMP1 to the Plasmo-

dium falciparum-infected erythrocyte surface. Blood 109, 1289–1297.

Mavoungou, E., Held, J., Mewono, L., and Kremsner, P.G. (2007). A

Duffy binding-like domain is involved in the NKp30-mediated recogni-

tion of Plasmodium falciparum-parasitized erythrocytes by natural

killer cells. J. Infect. Dis. 195, 1521–1531.

McGilvray, I.D., Serghides, L., Kapus, A., Rotstein, O.D., and Kain, K.C.

(2000). Nonopsonic monocyte/macrophage phagocytosis of Plasmo-

dium falciparum-parasitized erythrocytes: A role for CD36 in malarial

clearance. Blood 96, 3231–3240.

Cell Ho

Michon, P., Cole-Tobian, J.L., Dabod, E., Schoepflin, S., Igu, J.,

Susapu, M., Tarongka, N., Zimmerman, P.A., Reeder, J.C., Beeson,

J.G., et al. (2007). The risk of malarial infections and disease in Papua

New Guinean children. Am. J. Trop. Med. Hyg. 76, 997–1008.

Mitchell, A.J., Hansen, A.M., Hee, L., Ball, H.J., Potter, S.M., Walker,

J.C., and Hunt, N.H. (2005). Early cytokine production is associated

with protection from murine cerebral malaria. Infect. Immun. 73,

5645–5653.

Ndungu, F.M., Sanni, L., Urban, B., Stephens, R., Newbold, C.I.,

Marsh, K., and Langhorne, J. (2006). 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. J. Immunol. 176, 5504–5512.

Newbold, C.I. (1999). Antigenic variation in Plasmodium falciparum:

Mechanisms and consequences. Curr. Opin. Microbiol. 2, 420–425.

Newman, K.C., Korbel, D.S., Hafalla, J.C., and Riley, E.M. (2006).

Cross-talk with myeloid accessory cells regulates human natural killer

cell interferon-g responses to malaria. PLoS Pathogens 2, e118.

10.1371/journal.ppat.0020118.

Orago, A.S., and Facer, C.A. (1991). Cytotoxicity of human natural killer

(NK) cell subsets for Plasmodium falciparum erythrocytic schizonts:

Stimulation by cytokines and inhibition by neomycin. Clin. Exp. Immu-

nol. 86, 22–29.

Parroche, P., Lauw, F.N., Goutagny, N., Latz, E., Monks, B.G., Visintin,

A., Halmen, K.A., Lamphier, M., Olivier, M., Bartholomeu, B.C., et al.

(2007). Malaria hemozoin is immunologically inert but radically en-

hances innate responses by presenting malaria DNA to Toll-like recep-

tor 9. Proc. Natl. Acad. Sci. USA 104, 1919–1924.

Pichyangkul, S., Saengkrai, P., Yongvanitchit, K., Stewart, A., and

Heppner, D.G. (1997). Activation of gammadelta T cells in malaria:

Interaction of cytokines and a schizont-associated Plasmodium falci-

parum antigen. J. Infect. Dis. 176, 233–241.

Pichyangkul, S., Yongvanitchit, K., Kum-arb, U., Hemmi, H., Akira, S.,

Krieg, A.M., Heppner, D.G., Stewart, V.A., Hasegawa, H., Looareesu-

wan, S., et al. (2004). Malaria blood stage parasites activate human

plasmacytoid dendritic cells and murine dendritic cells through a toll-

like receptor 9-dependent pathway. J. Immunol. 172, 4926–4933.

Robinson, B.A., Welch, T.L., and Smith, J.D. (2003). Widespread func-

tional specialization of Plasmodium falciparum erythrocyte membrane

protein 1 family members to bind CD36 analysed across a parasite ge-

nome. Mol. Microbiol. 47, 1265–1278.

Schofield, L., Ferreira, A., Altszuler, R., Nussenzweig, V., and Nussenz-

weig, R.S. (1987a). Interferon-gamma inhibits the intrahepatocytic de-

velopment of malaria parasites in vitro. J. Immunol. 139, 2020–2025.

Schofield, L., Villaquiran, J., Ferreira, A., Schellekens, H., Nussenz-

weig, R., and Nussenzweig, V. (1987b). Gamma interferon, CD8+ T

cells and antibodies required for immunity to malaria sporozoites.

Nature 330, 664–666.

Schofield, L., and Hackett, F. (1993). Signal transduction in host cells

by a glycosylphosphatidylinositol toxin of malaria parasites. J. Exp.

Med. 177, 145–153.

Schofield, L., and Grau, G.E. (2005). Immunological processed in ma-

laria pathogenesis. Nat. Rev. Immunol. 5, 722–735.

Su, Z., and Stevenson, M.M. (2000). The central role of endogenous

IFN-g in protective immunity against acute blood-stage Plasmodium

chabaudi AS infection. Infect. Immun. 68, 4399–4406.

Troye-Blomberg, M., Worku, S., Tangteerawatana, P., Jamshaid, R.,

Soderstrom, K., Elghazali, G., Moretta, L., Hammarstrom, M., and Min-

cheva-Nilsson, L. (1999). Human gamma delta T cells that inhibit the in

vitro growth of the asexual blood stages of the Plasmodium falciparum

parasite express cytolytic and proinflammatory molecules. Scand. J.

Immunol. 50, 642–650.

Urban, B.C., Ferguson, D.J., Pain, A., Willcox, N., Plebanski, M., Aus-

tyn, J.M., and Roberts, D.J. (1999). Plasmodium falciparum-infected

st & Microbe 2, 130–138, August 2007 ª2007 Elsevier Inc. 137

Cell Host & Microbe

PfEMP-1 Suppresses Early IFN-g Production

erythrocytes modulate the maturation of dendritic cells. Nature 400,

73–77.

Urban, B.C., Mwangi, T., Ross, A., Kinyanjui, S., Mosobo, M., Kai, O.,

Lowe, B., Marsh, K., and Roberts, D.J. (2001a). Peripheral blood den-

dritic cells in children with acute Plasmodium falciparum malaria.

Blood 98, 2859–2861.

Urban, B.C., Willcox, N., and Roberts, D.J. (2001b). A role for CD36 in

the regulation of dendritic cell function. Proc. Natl. Acad. Sci. USA 98,

8750–8755.

Urban, B.C., Ing, R., and Stevenson, M.M. (2005). Early interactions

between blood-stage plasmodium parasites and the immune system.

Curr. Top. Microbiol. Immunol. 297, 25–70.

138 Cell Host & Microbe 2, 130–138, August 2007 ª2007 Elsev

Voss, T.S., Healer, J., Marty, A.J., Duffy, M.F., Thompson, J.K., Bee-

son, J.G., Reeder, J.C., Crabb, B.S., and Cowman, A.F. (2006). A var

gene promoter controls allelic exclusion of virulence genes in Plasmo-

dium falciparum malaria. Nature 439, 1004–1008.

Zevering, Y., Amante, F., Smillie, A., Currier, J., Smith, G., Houghten,

R.A., and Good, M.F. (1992). High frequency of malaria-specific T cells

in non-exposed humans. Eur. J. Immunol. 22, 689–696.

Zhu, J., Krishegowda, G., and Gowda, D.C. (2005). Induction of

proinflammatory responses in macrophages by the glycosylphos-

phatidylinositols of Plasmodium falciparum. J. Biol. Chem. 280,

8617–8627.

ier Inc.