Preserved Dendritic Cell HLA-DR Expression and Reduced RegulatoryT Cell Activation in Asymptomatic Plasmodium falciparum and P.vivax Infection

Steven Kho,a Jutta Marfurt,a Rintis Noviyanti,b Andreas Kusuma,b Kim A. Piera,a Faustina H. Burdam,c Enny Kenangalem,c,d

Daniel A. Lampah,c Christian R. Engwerda,e Jeanne R. Poespoprodjo,c,d,f Ric N. Price,a,g Nicholas M. Anstey,a Gabriela Minigo,a

Tonia Woodberrya

Global and Tropical Health Division, Menzies School of Health Research and Charles Darwin University, Darwin, Australiaa; Eijkman Institute for Molecular Biology, Jakarta,Indonesiab; Timika Malaria Research Programme, Papuan Health and Community Development Foundation, Timika, Papua, Indonesiac; Rumah Sakit Umum DaerahKabupaten Mimika, Timika, Papua, Indonesiad; QIMR Berghofer Medical Research Institute, Brisbane, Australiae; Department of Paediatrics, University of Gadjah Mada,Yogyakarta, Indonesiaf; Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdomg

Clinical illness with Plasmodium falciparum or Plasmodium vivax compromises the function of dendritic cells (DC) and ex-pands regulatory T (Treg) cells. Individuals with asymptomatic parasitemia have clinical immunity, restricting parasite expan-sion and preventing clinical disease. The role of DC and Treg cells during asymptomatic Plasmodium infection is unclear. Dur-ing a cross-sectional household survey in Papua, Indonesia, we examined the number and activation of blood plasmacytoid DC(pDC), CD141�, and CD1c� myeloid DC (mDC) subsets and Treg cells using flow cytometry in 168 afebrile children (of whom15 had P. falciparum and 36 had P. vivax infections) and 162 afebrile adults (of whom 20 had P. falciparum and 20 had P. vivaxinfections), alongside samples from 16 patients hospitalized with uncomplicated malaria. Unlike DC from malaria patients, DCfrom children and adults with asymptomatic, microscopy-positive P. vivax or P. falciparum infection increased or retainedHLA-DR expression. Treg cells in asymptomatic adults and children exhibited reduced activation, suggesting increased immuneresponsiveness. The pDC and mDC subsets varied according to clinical immunity (asymptomatic or symptomatic Plasmodiuminfection) and, in asymptomatic infection, according to host age and parasite species. In conclusion, active control of asymptom-atic infection was associated with and likely contingent upon functional DC and reduced Treg cell activation.

Plasmodium falciparum and Plasmodium vivax are both majorcauses of malaria morbidity and mortality in Southeast Asia

(1, 2). Clinical disease from these infections, malaria, is character-ized by the presence of fever together with detectable Plasmodiumparasites in the blood. Asymptomatic parasitemia, the carriage ofPlasmodium parasites without clinical disease, is also prevalent inboth high- and low-transmission areas, with estimates of 11%in children and adults in Timika, Papua, Indonesia (3) and 47% inPapua New Guinea school children (4). Indeed, in most areaswhere malaria is endemic, the majority of parasite carriers areasymptomatic (5), and those with gametocytes are a major reser-voir for transmission by mosquitoes, contributing to malariatransmission within a population (6, 7).

During acute P. falciparum and P. vivax malaria (8) and duringprimary prepatent P. falciparum infection (9), blood dendriticcells (DC) are functionally compromised, with an inability to ap-propriately stimulate cellular immunity. This is paralleled by re-duced HLA-DR expression (8, 10). The relative increase in regu-latory T (Treg) cells during acute malaria (11, 12) is also thoughtto inhibit host immunity. Together, Plasmodium modulation ofDC and Treg cell function appears to foster an immune suppres-sive environment in malaria that assists parasite growth and par-asite transmission (13). However, in individuals living in areas ofmalaria endemicity who have asymptomatic parasitemia and clin-ical immunity, it remains to be determined whether or how DCand Treg cells are modulated by Plasmodium.

Asymptomatic individuals in areas of malaria endemicity haveclinical immunity that restricts parasite expansion and preventsclinical disease. Such immunity is not sterilizing, but instead, a

state of host tolerance exists whereby parasites persist in the bloodwithout the occurrence of clinical symptoms (14, 15). The devel-opment of clinical immunity requires repeated parasite infections(14), and thus, the transmission intensity and parasite diversity inregions of endemicity determine age associations in the time todevelopment of clinical immunity (16).

It is not fully understood which immune cells mediate clinicalimmunity or whether children and adults respond similarly (14,15, 17). Research suggests a protective effect both for antibod-ies (18–21) and effector T cell cytokine responses (particularlygamma interferon [IFN-�]) (21–24). As the induction and main-tenance of both effective B cell (antibody) and T cell (cytokine)

Received 19 February 2015 Returned for modification 21 April 2015Accepted 24 May 2015

Accepted manuscript posted online 1 June 2015

Citation Kho S, Marfurt J, Noviyanti R, Kusuma A, Piera KA, Burdam FH,Kenangalem E, Lampah DA, Engwerda CR, Poespoprodjo JR, Price RN, Anstey NM,Minigo G, Woodberry T. 2015. Preserved dendritic cell HLA-DR expression andreduced regulatory T cell activation in asymptomatic Plasmodium falciparum andP. vivax infection. Infect Immun 83:3224 –3232. doi:10.1128/IAI.00226-15.

Editor: J. H. Adams

Address correspondence to Tonia Woodberry, Tonia.Woodberry@menzies.edu.au.

G.M. and T.W. made equal contributions to this work.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00226-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00226-15

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responses require functional DC (25) and each may be modulatedby Treg cells (26), we sought to examine these cells in both chil-dren and adults with patent asymptomatic P. falciparum or P.vivax infection and to compare their responses to those seen withacute uncomplicated P. falciparum or P. vivax malaria. We hy-pothesized that the appropriate activation of DC and Treg cellswould characterize asymptomatic Plasmodium infection.

(This work was presented in part as a poster at the 13thInternational Symposium on Dendritic Cells [DC2014], Tours,France.)

MATERIALS AND METHODSStudy participants and sample collection. This study was conducted inTimika, in Papua, Indonesia, which has equal prevalence of P. falciparumand P. vivax, unstable malaria transmission, and asymptomatic para-sitemia and acute uncomplicated malaria occurring in both children andadults (3). Timika thus represents a unique location to evaluate DC andTreg cell activation in clinical immunity (asymptomatic infection) andimmunopathology (acute malaria) across different ages. As part of ahousehold survey conducted between April and July in 2013, 168 children(2 to 10 years) and 162 adults (16 to 40 years) were evaluated using field-based flow cytometry of peripheral blood. All participants were residentsin the Timika district, with similar proportions of Papuans (49% of chil-dren and 40% of adults) and non-Papuan migrants (51% of children and60% of adults) from surrounding provinces. The participants wereasymptomatic, with neither fever nor other symptoms of malaria at re-cruitment or in the 24 h prior to recruitment.

Ten milliliters of venous blood was collected from a single adult perhousehold (i.e., 162 households), and finger prick capillary blood wascollected from children into lithium heparin tubes. Among the 168 chil-dren (2 to 10 years) who provided blood samples for this immunologystudy, there was an average of 1.24 children per household. Blood sampleswere transported to the laboratory at room temperature for microscopicparasite detection and flow cytometry, which were performed within 6 hof blood collection. Thick and thin blood smears stained with 5% Giemsasolution were examined by trained microscopists to identify Plasmodiumspecies and parasitemia. Individuals found to be parasitemic were treatedaccording to standard local antimalarial treatment protocols, comprisingdihydroartemisinin-piperaquine for 3 days for each species plus, if notG6PD deficient, a 14-day course of primaquine for P. vivax.

In parallel with the household survey, 14 adults and 2 children beingtreated for acute uncomplicated malaria in Rumah Sakit Mitra Masyara-kat Hospital (RSMM) in Timika were enrolled as a comparator group.Malaria was defined by a temperature of �37°C or history of fever withclinical malaria symptoms, a blood film positive for Plasmodium, and noalternative clinical explanation. Venous blood was collected and pro-cessed identically as in the household survey samples.

Flow cytometric analysis. To examine DC, 100 to 300 �l of wholeblood was stained with different flow cytometry panels, comprising lin-eage markers anti-CD3 antibody (clone HIT3a), anti-CD14 antibody(clone HCD14), anti-CD19 antibody (clone H1H9), and anti-CD56 an-tibody (clone HCD56) conjugated to Alexa Fluor 488 (AF488), anti-CD1cantibody (clone L161) conjugated to phycoerythrin (PE), anti-CD303antibody (clone 201A) conjugated to peridinin chlorophyll a protein-cyanine 5.5 (PerCP-Cy5.5), anti-HLA-DR antibody (clone L243) conju-gated to PerCP, anti-CD141 antibody (clone M80) conjugated to allophy-cocyanin (APC), and anti-CD34 antibody (clone 581) conjugated toAF488. Whole blood was stained with antibody master mixes (preparedweekly) for 15 min at room temperature. Red blood cells were lysed withfluorescence-activated cell sorting (FACS) lysing solution (BD Biosci-ences) and washed with phosphate-buffered saline (PBS). Cells were fixedin 1% (wt/vol) paraformaldehyde in PBS and then stored at 4°C in thedark and read within 3 h of staining.

For the phenotypic evaluation of Treg cells, 50 �l of whole blood wasstained in TruCount tubes (BD Biosciences) containing 52,345 beads and

using a lyse-no-wash protocol to allow accurate calculation of absolutecell numbers (27). Whole blood was stained with anti-CD45RA antibody(H100) conjugated to fluorescein isothiocyanate (FITC), anti-CD25 an-tibody (BC 96) conjugated to PE, anti-CD4 antibody (RPA-T4) conju-gated to PerCP, and anti-CD127 antibody (A019D5) conjugated to AlexaFluor 647 (AF647). Red blood cells were lysed by adding 450 �l of FACSlysing solution (BD Biosciences). Samples were read within 1 h of staining.

All samples were acquired on a portable BD Accuri C6 4-color flowcytometer using CFlow Sampler software (BD Biosciences). All antibodieswere purchased from BioLegend (San Diego, CA).

Data analysis. Flow cytometric data were analyzed using FlowJo soft-ware (TreeStar, Ashland, OR). The absolute number of DC was calculatedby multiplying the proportion of DC events over white blood cell (WBC)events from flow cytometric data, with the absolute number of WBCbeing derived from automated blood counts (in units of 109 cells per liter),then multiplying by 1,000 to give the number of DC per microliter ofblood. Automated blood counts were performed on all blood samplestested for DC using an MS9-5sH cell count analyzer (Melet SchloesingLaboratories, Osny, France). Absolute numbers of Treg cells were calcu-lated using TruCount tubes (BD Biosciences) according to the manufac-turer’s instructions to give the number of Treg cells per microliter ofblood. Samples with less than 80 events in the DC gate (n � 8) or Treg cellgate (n � 1) were excluded. All statistical analyses were performed usingGraphPad Prism 6 (GraphPad Software, La Jolla, CA). The Mann-Whit-ney U test was used for comparison between groups. Spearman’s rankcorrelation coefficient was used to measure statistical associations be-tween cell variables and parasitemia. Exact P values are presented but werenot corrected for multiple comparisons.

Ethical approval. The study was approved by the Human ResearchEthics Committees of Gadjah Mada University, Yogyakarta, Indonesia,the Eijkman Institute Research Ethics Commission, Jakarta, Indonesia,and the NT Department of Health and Families and Menzies School ofHealth Research, Darwin, Australia. Written informed consent was ob-tained from all participants (or the primary caregiver or relative) prior toblood sampling.

RESULTSStudy participants. Whole-blood capillary samples were ob-tained from 168 children, of whom 15 had asymptomatic P. fal-ciparum infection, 36 had asymptomatic P. vivax infection, and117 had no parasites detectable by microscopy and were consid-ered controls. Venous blood was collected from 176 adults, ofwhom 20 had asymptomatic P. falciparum infection, 20 hadasymptomatic P. vivax infection, 122 were aparasitemic controls,and 14 had acute uncomplicated malaria. Ninety-four percent(153/162) of asymptomatic adults had lived in Timika for morethan 2 years. The self-reported durations of residency were similarbetween the 40 asymptomatic adults, for whom the median resi-dency was 10 years and the interquartile range (IQR) 5 to 15 years,and the 122 aparasitemic control adults, for whom the medianresidency was 10 years and the IQR 5 to 20 years. Participantcharacteristics are summarized in Table 1. Groups were for themost part gender balanced. Parasitemia was significantly higher inadults with uncomplicated malaria than in the species-matchedadult asymptomatic group. Adults with uncomplicated malariahad significantly lower lymphocyte counts and significantlyhigher monocyte counts than the adult controls. There were nosignificant differences in age or lymphocyte or monocyte countbetween healthy controls and children and adults with asymptom-atic parasitemia.

Flow cytometry was constrained by the amount of blood avail-able for testing, and hence, it was not always possible to conductboth DC and Treg cell stains for each participant. In 12% (91/784)

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of stains, flow cytometry data were deemed unreliable due to in-sufficient lysis of red blood cells.

HLA-DR expression of circulating DC during asymptomaticpatent Plasmodium infection. Peripheral blood total DC wereidentified by flow cytometry as lineage marker-negative HLA-DR� cells (Fig. 1B). The activation/maturation of circulatingblood DC was examined by measuring the median fluorescenceintensity of HLA-DR expression. There was increased HLA-DRexpression on total DC in adults with asymptomatic P. vivax in-fection (P � 0.05) (Fig. 2A), while the HLA-DR expression inthose with asymptomatic P. falciparum infection was comparableto that in controls. In contrast, during acute uncomplicated P.falciparum or P. vivax malaria, DC HLA-DR expression was sig-nificantly lower (P � 0.0001 and P � 0.0001, respectively) (Fig.2A). In children, HLA-DR expression was increased significantlyin asymptomatic P. vivax infections (P � 0.02) (Fig. 2D).

In a limited number of samples, additional staining was possi-ble and HLA-DR expression could be examined on two myeloidDC (mDC) subsets, CD1c� (blood dendritic cell antigen 1[BDCA-1] positive) mDC (CD1c� mDC) and CD141� (BDCA-3positive) mDC (CD141� mDC). In both mDC subsets, HLA-DRwas preserved during asymptomatic infection, but in 3 adults withuncomplicated malaria, HLA-DR expression appeared reduced(Fig. 2B, C, E, and F). Additional data are required to determinethe significance of the apparent reduction.

Age- and species-specific variations in DC subset numbers.Blood DC subsets showed marked variations according to clinicalimmunity (asymptomatic or symptomatic Plasmodium infec-tion), host age, and parasite species. Three DC subsets were iden-tified by flow cytometry (Fig. 1A) and are reported as the absolutenumber of CD303� (BDCA-2 positive) plasmacytoid DC (pDC),CD1c� mDC, and CD141� mDC (see Table S1 in the supplemen-tal material).

Plasmacytoid DC expanded in children with asymptomatic P.vivax infection (P � 0.002, n � 34) and were retained in adultswith asymptomatic infection (Fig. 3). The expansion/retentionappeared specific to asymptomatic infection, because pDC de-clined significantly in adults with acute uncomplicated malariafrom either P. falciparum or P. vivax (P � 0.015, n � 4) (Fig. 3).

CD1c� mDC declined significantly in children with asymp-tomatic infection with P. falciparum (P � 0.012, n � 10) or P.vivax (P � 0.006, n � 33) (Fig. 3). CD1c� mDC were retained byadults with asymptomatic infection but declined in adults withacute uncomplicated malaria from either P. falciparum or P. vivax(P � 0.001, n � 8) (Fig. 3). Adult malaria patients were evaluatedas a single group because the low number of patients with either P.falciparum or P. vivax precluded analysis for each species.

The CD141� mDC subset appeared to be retained by childrenand adults with asymptomatic infection (Fig. 3).

Reduced Treg cell activation in asymptomatic patent Plas-modium infection. Regulatory T cells were assessed in 94 childrenand 149 adults by flow cytometry as CD4� CD25� CD127low lym-phocytes and subdivided into activated and resting Treg cells(aTregs and rTregs, respectively) based on their CD45RA expres-sion (28) (Fig. 1C).

Treg cell numbers were significantly lower in children withasymptomatic P. falciparum infection (P � 0.0004, n � 6), whilein children with asymptomatic P. vivax infection, Treg cell num-bers were similar to the numbers in aparasitemic controls.

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tion, there was no change in the absolute or relative number ofTreg cells (Table 2). In contrast, there was a significant decrease inthe absolute number of Treg cells during acute uncomplicatedmalaria from each species (P � 0.0001) (Table 2)

Importantly, in both adults and children with asymptomatic P.falciparum or P. vivax infection, significantly smaller proportionsof circulating Treg cells were activated (CD25high CD45RA�) thanin aparasitemic controls, resulting in significantly increased ratiosof rTregs to aTregs in asymptomatic Plasmodium infection, whilethe opposite was observed in acute uncomplicated malaria (Table2). These observations suggest that reduction in the proportion ofactivated Treg cells is associated with clinical immunity.

Treg cell and DC activation and parasitemia. There was noevidence of a relationship between Treg cell activation and para-sitemia within each of the asymptomatic adult or asymptomatic

child (Spearman’s rank correlation coefficient [rs] � �0.05, P �0.8, n � 32, and rs � �0.05, P � 0.8, n � 16, respectively) or adultmalaria (rs � 0.2, P � 0.5, n � 14) groups. There was no overlap inparasitemia between the asymptomatic adults and malaria pa-tients (Table 1). Total DC HLA-DR expression was inversely cor-related with parasitemia in adults with asymptomatic P. falcipa-rum infection (rs � �0.54, P � 0.02, n � 18) and positivelycorrelated in children with asymptomatic P. vivax infection (rs �0.80, P � 0.01, n � 9).

DISCUSSION

This study represents the first description of appropriate DC mat-uration and Treg cell activation in asymptomatic Plasmodium in-fection, characterized by increased or retained HLA-DR expres-sion on DC and reduced activation of Treg cells in both children

FIG 1 Representative staining of DC and T cells in fresh whole blood from children and adults. (A) DC subsets were identified by gating on lineage-negative WBC(CD3, -14, -19, and -56) and then on expression of CD303, CD1c, and CD141 surface markers to identify CD303� plasmacytoid DC, CD1c� myeloid DC, andCD141� myeloid DC, respectively. (B) Total DC were identified as lineage marker-negative (CD3, -14, -19, -34, and -56) and HLA-DR-positive peripheral bloodmononuclear cells. (C) Total Treg cells were identified as CD4� lymphocytes with high expression of CD25 and low expression of CD127 surface markers. TotalTreg cells were then subdivided into active and resting Treg cells based on expression of CD45RA.

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and adults with asymptomatic P. falciparum or asymptomatic P.vivax parasitemia. In contrast, in adults with acute malaria, DChave reduced HLA-DR expression and an increased proportion ofTreg cells are activated. These data provide evidence of appropri-ate immune activation in asymptomatic infection and immunedysfunction in acute clinical malaria. The active control of asymp-tomatic infection is associated with and is likely contingent uponfunctional DC and reduced Treg cell activation.

The evaluation of adults and children highlighted similaritiesin DC and Treg cell activation in the two age groups, and notwith-standing some differences depending on parasite species (P. fal-ciparum and P. vivax), our results suggest some common DC- andTreg cell-related immunological mechanisms contribute to thecontrol of Plasmodium parasitemia in children and adults. In ma-laria, a similar panspecies effect has been noted with DC dysfunc-tion in both acute P. falciparum and P. vivax malaria (8) and Tregcell activation in acute P. falciparum (12) and P. vivax (29, 30)malaria. It remains an open question whether there are age-asso-ciated differences in DC and Treg cell activation during acutemalaria, because the low number of uncomplicated malaria sam-ples examined (particularly in children) prevented a comprehen-sive comparison of DC and Treg cell activation between childrenand adults with acute malaria.

Asymptomatic parasitemia in areas of endemicity is associatedwith reduced parasite burdens compared to results for those withclinical illness (31). While we hypothesize that activation of DCand reduced activation of Treg cells may contribute to the controlof parasitemia during clinical immunity, it is unlikely that lowparasitemia per se results in these measures of immune activation.In contrast to the increased/retained HLA-DR expression wefound in asymptomatic parasitemia, our observations from ex-perimental P. falciparum blood stage infections of malaria-naivevolunteers indicate that DC HLA-DR expression levels on pDCdecrease prior to the onset of symptoms (9), and at levels of par-asitemia much lower than those seen in patent asymptomatic in-fections in the current and other studies in areas of malaria ende-micity (32, 33). While we speculate that the retention/increase inDC HLA-DR expression, which is required for antigen presenta-tion, and the reduction in Treg cell activation observed in individ-uals from area of endemicity with asymptomatic parasitemia con-tribute to their clinical immunity, the relative contribution toreduced parasite growth and/or reduced inflammatory responsesremains to be determined. Our DC HLA-DR data support theearlier description of HLA-DR retention by circulating blood DCin Fulani children, which is understood to contribute to protectiveimmunity against malaria (34).

FIG 2 HLA-DR median fluorescence intensity (MFI) of total DC was determined in adult controls (n � 48), asymptomatic P. falciparum-infected adults (n �18), asymptomatic P. vivax-infected adults (n � 19), uncomplicated P. falciparum-infected adults (n � 6), and uncomplicated P. vivax-infected adults (n � 8)(A), as well as in child controls (n � 37), asymptomatic P. falciparum-infected children (n � 10), asymptomatic P. vivax-infected children (n � 9), anduncomplicated P. vivax-infected children (n � 2) (D). In a subset of samples, HLA-DR MFI of CD1c� myeloid DC (B, E) and CD141� myeloid DC (C, F) wasdetermined in adult controls (n � 4), adults with asymptomatic malaria (n � 4), and adults with uncomplicated malaria (n � 3), as well as in child controls (n� 2) and children with asymptomatic malaria (n � 8). Bars and whiskers show median values and interquartile ranges. Mann-Whitney U test was used forcomparisons between groups (P � 0.05 was considered significantly different). Data were obtained by analysis of fresh whole blood using the 4-color flowcytometry panels shown in Fig. 1. AS, asymptomatic malaria; UM, uncomplicated malaria; Pf, P. falciparum; Pv, P. vivax; HLA-DR, human leukocyte antigen(MHC class II).

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Treg cells are efficient suppressors of immune responses. Weobserved a significant drop in circulating Treg cell numbers inacute malaria, which coincided with a significant drop in lympho-cytes and CD4 T cell counts. Lymphopenia is common in clinicalmalaria (35, 36), occurring already at subpatent parasitemia dur-ing a first P. falciparum infection (32), which suggests that theabsence of lymphopenia in asymptomatic Plasmodium carriers isnotable and potentially reflects or contributes to clinical immu-nity. Transient T cell migration from the peripheral blood (37) totissues such as the spleen (38) or to sites of inflammation duringacute malaria contributes to lymphopenia, and T cells reemergefollowing antimalarial treatment (36). We observed no significantchange in the proportion of Treg cells within the CD4 T cell com-partment, suggesting that Treg cells leave the peripheral bloodtogether with other CD4 T cells to travel to sites of inflammation(39). However, in clinical malaria, more of the Treg cells remain-ing in the circulation displayed a recently activated CD25high

CD45RA� phenotype, which represents highly suppressive Tregcells (28). In contrast, in asymptomatic Plasmodium infection, thecirculating Treg cells were of a less activated phenotype than thosein uninfected controls.

Studies on Treg cell activation in asymptomatic Plasmodiuminfection are limited. A study from Peru, where malaria is hypoen-demic, reports similar CD25� Foxp3� CD127low Treg cell countsbetween symptomatic and asymptomatic P. falciparum infections

and uninfected controls (40). In contrast, Indonesian school-agedchildren with asymptomatic patent or subpatent Plasmodium in-fection show a trend to reduced CD25� Foxp3� CD127low Tregcell proportions among CD4 T cells compared to results for un-infected children (41), similar to our observation in asymptomaticP. falciparum-infected children. These asymptomatic children,however, display increased Treg cell TNFR2 expression and sup-pression of Th2 responses (41). We were unfortunately unable toassess TNFR2 expression in this study due to limited blood vol-umes and limited channels on our field flow cytometer, whichprecludes direct comparison of these studies.

Self-resolving murine models of malaria consistently show atransient expansion of Treg cells in the spleen shortly after infec-tion with P. yoelli or P. chabaudi, followed by a drop in Treg cellnumbers due to apoptosis (42). Continued Treg cell expansionnegatively impacts immune control of the infection (42), withTreg cell activation resulting in increased suppression of T cellresponses (43). In humanized mice expressing HLA-DR0401,Treg cell activation has been associated with suppression of pro-tective anti-parasitic antibody responses (44) and failure to con-trol parasite growth.

Resting CD25� CD45RA� Treg cells (rTregs) provide a reser-voir for recently activated CD25high CD45RA� Treg cells (aTregs),which are highly suppressive, highly proliferative cells with a shortlife span (28). The increased frequency of rTreg-to-aTreg ratio in

FIG 3 Top: absolute number of CD303� plasmacytoid DC (A), CD1c� myeloid DC (B), and CD141� myeloid DC (C) in peripheral blood of adult controls (A,n � 48; B, n � 52; C, n � 39), asymptomatic P. falciparum-infected adults (A, n � 17; B, n � 20; C, n � 14), asymptomatic P. vivax-infected adults (A, n � 18;B, n � 20; C, n � 17), and adults with uncomplicated malaria (A, n � 4; B, n � 8; C, n � 7). Bottom: absolute numbers of CD303� plasmacytoid DC (D), CD1c�

myeloid DC (E), and CD141� myeloid DC (F) in peripheral blood of child controls (D, n � 74; E, n � 77; F, n � 54), asymptomatic P. falciparum-infectedchildren (D, n � 12; E, n � 10; F, n � 9), and asymptomatic P. vivax-infected children (D, n � 34; E, n � 33; F, n � 30). Bars and whiskers show median valuesand interquartile ranges. Mann-Whitney U test was used for comparisons between groups (P � 0.05 was considered significantly different). Data were obtainedby analysis of fresh whole blood using the 4-color flow cytometry panels shown in Fig. 1A.

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asymptomatic P. falciparum and P. vivax infection, which wasparticularly prominent in children, contrasts strikingly with thesignificant drop in rTreg-to-aTreg frequency found in acute ma-laria. In malaria, a relative increase in aTregs occurred alongside arelative decrease in rTregs, while in asymptomatic infection, theproportion of aTregs decreased and the rTreg proportion re-mained largely unchanged. These findings suggest that while acutemalaria is accompanied by Treg cell overactivation, potentiallyleading to exhaustion of the Treg cell reservoir, asymptomaticPlasmodium infection is characterized by a dampened Treg cellresponse, which may indicate that a less suppressive environmentfor antiparasitic immune responses is contributing to control ofparasitemia.

While it is unclear how Treg cell overactivation may beavoided, one study suggests that immature blood DC contributeto Treg cell differentiation via surface binding of the immuno-modulatory cytokine transforming growth factor beta (TGF-) tolatency-associated peptide (LAP) (45). Curiously, downregula-tion of LAP on blood DC coincides with DC maturation, charac-terized by upregulation of HLA-DR and costimulatory molecules(45), suggesting that overcoming the maturation defect observedin acute malaria may be linked to reducing Treg cell overactiva-tion. Whether LAP downregulation on DC is impaired in malariaremains to be established.

Enumeration of three major blood DC subsets in asymptom-atic infection revealed notable differences according to Plasmo-dium species. Plasmacytoid DC expanded in asymptomatic P.vivax infection in children, suggesting that pDC may contribute tocontrol of parasitemia and/or disease in asymptomatic infection.In contrast, there was no expansion of myeloid DC, with reducedCD1c� mDC in children with asymptomatic infection and adultswith malaria. CD141� mDC were stable in children and adultswith asymptomatic infection. Urban and colleagues have de-scribed increased CD141� mDC in African children with severe P.falciparum malaria (46), but they did not investigate asymptom-atic or uncomplicated malaria. The DC counts we report in con-trol microscopy-negative Indonesian children were similar to thepDC, CD1c� mDC, and CD141� mDC counts reported in Ken-yan control children (46).

The expansion or retention of blood DC in asymptomatic P.falciparum or P. vivax parasitemia contrasts with that seen in acutemalaria, where P. falciparum and P. vivax parasitemias are associ-ated with reductions in pDC and mDC numbers (8). In a limitednumber of samples where additional field flow cytometry was un-dertaken, HLA-DR expression appeared to be retained by CD1cand CD141 mDC during asymptomatic parasitemia but lost inmalaria. These data support the notion that DC remain capable ofantigen presentation in asymptomatic parasite carriers; however,further studies are required to comprehensively evaluate HLA-DRexpression and the function of blood DC in asymptomatic infec-tion.

Our study had a number of limitations. We evaluated para-sitemia by microscopy only, and it is possible that a significantproportion of microscopy-negative aparasitemic controls hadsubpatent PCR-level parasitemia (47). Another limitation is thatsubjects were not followed longitudinally and those with asymp-tomatic parasitemia were treated, preventing assessment ofwhether people would remain asymptomatic, clear the infection,or develop malaria (48). Notwithstanding these limitations, at thetime of blood collection, we can identify clear differences in DCT

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and Treg cell activation between the groups. Future longitudinalstudies of DC and Treg cell activation, including submicroscopicinfection, will further enhance the understanding of immune ac-tivation in asymptomatic infection.

Asymptomatic parasitemia is largely ignored by immunizationprograms in countries where malaria is endemic, meaning a pro-portion of children and adults with asymptomatic parasitemia areroutinely vaccinated with little understanding of the effectsasymptomatic parasitemia has on the efficacy of current child-hood vaccines (49). As all vaccines (standard childhood vaccinesand new immunizations being developed) need functional DC tostimulate protective immune responses, our study begins to ad-dresses this current knowledge gap. While more vaccine immuno-genicity and efficacy studies are required, the data suggest inade-quate immune responsiveness during acute malaria but thatvaccinating asymptomatic carriers of Plasmodium may be poten-tially acceptable, at least in areas of low-to-medium transmission.

ACKNOWLEDGMENTS

We thank the participants from the household survey for taking part inthis study, field and laboratory staff at Gedung Penelitian, RSMM, fortheir collective effort in acquiring samples, Ferryanto Chalfein andPrayoga for their microscopy expertise, Leily Triyanti, Ella Curry, Gren-nady Wirjanata, and Irene Handayuni for their support in the laboratoryand assistance with logistics, RSMM hospital staff for their permission touse the hematological analyser, and Yati Soenarto, Yodi Mahendradhata,and Franciscus Thio for facilitation of this study. We thank Mark Chat-field for assistance with data analysis.

This study was funded by the National Health and Medical ResearchCouncil of Australia (project grants 1021198 and 1021121, program grant1037304, and fellowships to N.M.A., C.R.E., and G.M.). The field workwas funded by Wellcome Trust (Senior Fellowship in Clinical Science091625 to R.N.P. and Training Fellowship 062058 to J.R.P.). The fundingbodies had no input into the design, collection, analysis, or interpretationof data and no input into the writing of the manuscript or submission forpublication.

We declare no conflicts of interest.

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