Microbes and Infection 15 (2013) 837e843www.elsevier.com/locate/micinf

Original article

Plasmodium vivax infection induces expansion of activated naıve/memoryT cells and differentiation into a central memory profile

Ana Luiza Teixeira Silva a, Marcus Vinıcius Lacerda b, Ricardo Toshio Fujiwara a,Lilian Lacerda Bueno a, Erika Martins Braga a,*

aDepartamento de Parasitologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627,

31270-901 Belo Horizonte (MG), BrazilbFundacao de Medicina Tropical Dr. Heitor Viera Dourado, Amazonas, Manaus, Brazil

Received 22 October 2012; accepted 25 July 2013

Available online 2 August 2013

Abstract

Immunity to malaria is widely believed to wane in the absence of reinfection, but direct evidence for the presence or absence of durableimmunological memory to malaria is limited. Here, we characterized the profile of circulating naıve and memory (including central and effector)CD4þ T cells responses of individuals naturally infected by Plasmodium vivax. In the current study, we demonstrated that acute P. vivaxinfection induces a significant increase in the absolute number of both naıve and memory cells, which were responsible for the production ofanti-inflammatory (IL-10) and pro-inflammatory (IFN-g) cytokines. Finally, we described the profile of memory cell subtypes (TCM-CD45ROhighCCR7þ and TEM-CD45RO

highCCR7�), as well as the pattern of cell migration based on CD62L selectin expression, demonstratingthat P. vivax-infected donors presented with a predominantly central memory cell profile. Our results indicate that the expansion of both naıveand memory T cells, responsible for the production of both pro-inflammatory and regulatory cytokines, which might also contribute to themodulation of immune responses during P. vivax infection.� 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Plasmodium vivax; Memory response; Central memory

1. Introduction

Malaria is one of the oldest infectious diseases of mankindand still exerts a high burden on human health and society.This disease is caused by parasites of the genus Plasmodiumand is transmitted by anopheline mosquitoes, with an esti-mated 3.3 billion of the world’s population at risk [1]. Plas-modium vivax is the most common human malaria speciesoutside Africa, with 2.6 billion people at risk in Asia and inCentral and South America [2]. Despite the importance of thisdisease, which represents the most prevalent recurrent malaria[3], the immunological mechanisms associated with the

* Corresponding author. Tel.: þ55 31 34092876; fax: þ55 31 34092970.

E-mail address: embraga@icb.ufmg.br (E.M. Braga).

1286-4579/$ - see front matter � 2013 Institut Pasteur. Published by Elsevier Ma

http://dx.doi.org/10.1016/j.micinf.2013.07.009

control of parasite levels and disease severity are not fullyunderstood.

Compounding this global public health burden is the factthat primary infection does not immediately induce immunity.Nevertheless, the development of immunity to malaria inhumans living in endemic areas [4] is evident from the factthat the burden of disease falls on young children, which areparticularly susceptible. It has been estimated that a quarter ofall childhood deaths are due to malaria [5]. However, afterexposure to the parasite, older children and adults are resistantto severe morbidity and death while remaining susceptible toinfection [4]. Of note, immunity induced by P. vivax infectionleads to the recruitment of memory T cells, which are sub-sequently recruited and activated during “relapse” or “rein-fection” [6]. Indeed, CD4þ T cell responses of infectedindividuals from endemic areas to several blood-stage

sson SAS. All rights reserved.

Table 1

Description of the study population by age and hematological parameters

(mean � SD).

Individuals

a

838 A.L.T. Silva et al. / Microbes and Infection 15 (2013) 837e843

antigens have been extensively documented [7,8], whichsuggests that memory cells are induced by natural infection.However, there is a dearth of studies showing whetherpersistence of infection and/or antigen is crucial for the in-duction of the memory response and, more importantly, theextent of the memory cell contribution to the regulation orpathology of vivax malaria.

In humans, naıve T cells express high levels of the trans-membrane protein CD45RAhigh, which is an isoform of CD45,a tyrosine phosphatase [9] originally called leukocyte commonantigen [10]. During differentiation into memory T cells, naıveT cells lose surface expression of CD45RA and becomeCD45ROhigh after stimulation in vitro, and expression ofCD45RO, once acquired, is stable [11]. CD45ROþ T cellsexpand rapidly after activation by specific antigens and pro-duce large amounts of cytokines, including IFN-g [12]. Pro-tective memory is mediated by effector memory T cells (TEM)that migrate to inflamed peripheral tissues and display im-mediate effector function, whereas reactive memory celldevelopment is mediated by central memory T cells (TCM),which present little or no effector function but readily prolif-erate and differentiate into effector cells in response to anti-genic stimulation [13]. Human TCM are CD45ROhigh memorycells that constitutively express CCR7þ and CD62Lþ, tworeceptors that are also observed in naıve T cells and that arerequired for cell extravasation through high endothelial ve-nules and migration to lymphoid organs [14e16]. In contrast,human TEM are memory cells that have lost the constitutiveexpression of CCR7, are heterogeneous for CD62L expres-sion, and display characteristic sets of chemokine receptorsand adhesion molecules that are required for homing toinflamed tissues [16].

Although previous studies demonstrated the presence ofmemory T cells during P. vivax infection [6,17] and the in-duction of memory T cells after immunization [18], the patternof memory T cell subsets during vivax malaria remains poorlycharacterized, particularly with respect to the profile of TCM orTEM responses and cell migration. In the present study, weevaluated the profile of peripheral blood subsets of CD4þ Tcells expressing naıve/memory markers (CD45RAþ/ROhigh).These cells were significantly increased in individuals natu-rally infected by P. vivax compared to non-infected controls;moreover, the cells produced large amounts of IFN-g and IL-10. Furthermore, we described the profile of memory cellsubtypes (TCM-CD45RO

highCCR7þ and TEM-CD45ROhighCCR7�) and the pattern of cell migration basedon the expression of CD62L selectin.

2. Materials and methods

Malaria-infected

(n ¼ 64)

Malaria-naıve

(n ¼ 22)

2.1. Study population and blood samples

Age mean, years 56.5 � 38.8 36.4 � 12.0

Hemoglobin (g/dL) 12.8 � 1.6* 13.3 � 1.1

Platelets (cells/mm3) 115,393 � 45,300* 184,400 � 20,300

Parasitemia (parasites/mL) 5027 � 5170 e

Lymphocytes (cells/mm3) 11,212 � 5675 12,476 � 2316

*Statistically different from control group (P < 0.0001).a P. vivax infection detected by parasitological smears and PCR.

Samples from 64 patients with acute non-complicated P.vivax malaria were enrolled in this study. The patients wereunrelated outpatients who were diagnosed at the Fundacao deMedicina Tropical do Amazonas, Manaus, Amazonas State(Western Brazilian Amazon), an endemic area of low exposure

to the parasite. Twenty-two healthy age- and sex-matchedadult blood donors were recruited for this study over thecourse of several months from Belo Horizonte, Minas GeraisState, Brazil, a non-endemic area for malaria; the absence ofprevious exposure to the parasite was determined by sero-logical assays and PCR [19]. The study was approved by theEthics Committee on Research with Humans of UniversidadeFederal de Minas Gerais (Protocol# ETIC 060/07). Blood wasobtained after receiving signed informed consent.

Venous blood was collected into EDTA- and heparin-containing tubes (4 and 32 mL, respectively) immediatelybefore the initiation of antimalarial treatment and was used toprepare thick smears for microscopy, to extract parasite DNAand for PBMC isolation. Parasitological evaluation was per-formed by the examination of 200 fields at l000� magnifi-cation under oil immersion. All slides were examined by atleast two well-trained microscopists from the Brazilian Min-istry of Health. P. vivax mono-infection was confirmed byPCR as previously described [19]. Hemoglobin, hematocrit(HCT) and platelet levels were measured using an automatedblood cell counter (ABX Pentra 90; Horiba Diagnostics,Kyoto, Japan) (Table 1). The correlations between plateletcounts and the level of parasitemia and between hemoglobinlevel and parasitemia were determined for both infected andcontrol donors.

2.2. Isolation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were ob-tained as previously described [20]. Briefly, cells were isolatedfrom heparinized blood on a density gradient centrifugation(Histopaque�, SigmaeAldrich Co., USA) and were resus-pended at a final concentration of 1 � 107 cell/mL in RPMI1640 medium (Invitrogen Co., USA) supplemented with 2 mMof L-glutamine (Sigma), 5% heat-inactivated human AB serum(Sigma) and 6% Antibiotic-Antimycotic solution (Invitrogen).Purified PBMCs were fixed and frozen using 3 mL of pre-warmed (37 �C) 4% paraformaldehyde (PFA) (SigmaeAldrichCo., USA) for 5 min and then washed in cold PBS with 1%fetal bovine serum (FBS, Sigma). Finally, cells were resus-pended in 1 mL of freeze medium (90% FBS and 10% DMSO)and frozen at �196 �C. For staining, cells were thawed andresuspended in PBS 5 mM EDTA 2% FBS.

839A.L.T. Silva et al. / Microbes and Infection 15 (2013) 837e843

2.3. Cell phenotyping by flow cytometry andintracellular staining

Fig. 1. Flow cytometric analysis of naıve and memory CD4þ T cells. Results

were expressed in absolute numbers (cells/mm3) of (A) CD4þCD45RAþ and

(B) CD4þCD45ROhigh T cells from malaria-naıve and P. vivax-infected donors

PBMCs were stained using monoclonal antibodies todetermine the surface expression of memory markers(CD45RA, CD45RO, CCR7 and CD62L) on CD4þ T cells, aswell as the co-expression of IFN-g and IL-10. The followingmonoclonal antibodies conjugated either with fluoresceinisothiocyanate (FITC), phycoerythrin (PE), or phycoerythrin-cyanine 5 (PE-Cy5) were used in the phenotypic analysis:FITC anti-human CD4 (clone RPA-T4) and CD45RA (cloneHI100), PE anti-human IL-10 (clone JES3-19F1), IFN-g(clone B27), CCR7 (clone 150,503) and CD62L (cloneDREG-56), PE-Cy5 anti-human CD4 (clone RPA-T4), andCD45RO (clone UCHL1) (all from BD Pharmingen, USA).Cells were incubated with 2 mL of undiluted monoclonal an-tibodies in the dark for 30 min at room temperature. Intra-cellular staining for cytokines was performed using theeBioscience fixation/permeabilization buffer kit followingmanufacturer’s instructions. After incubation, PBMCs werewashed twice with 2 mL of phosphate-buffered saline con-taining 0.01% sodium azide followed by fixation in 200 mL offixative solution (10 g/L paraformaldehyde, 1% cacodylicacid, and 6.65 g/L sodium chloride). Phenotypic analyses wereperformed using a Becton Dickinson FACScan flow cytometer.Data on 5 � 104 lymphocytes (gated by forward and sidescatter properties) were collected, and the analysis was per-formed using the CellQuest software (BD Biosciences, USA).

(n ¼ 22 and 64, respectively). Lines represent mean average for the observed

data. Statistical differences were detected using Student t test and are indicated

2.4. Statistical analysis on the graphs with significant P values.

The one-sample KolmogoroveSmirnoff test was used todetermine whether variability followed a normal distributionpattern. P values were determined by two-tailed Student ttests. Correlation analysis was performed using Pearson cor-relation. A P value <0.05 was considered significant. Allstatistics were determined using the Prism 5.0 software forWindows (GraphPad Software, Inc.).

3. Results

3.1. Absolute counts of both naıve and memory CD4þ Tcells are increased in donors infected with P. vivax

To determine the phenotype of CD4þ T cells in peripheralblood, we characterized the absolute counts of naıve(CD45RAþ) and memory (CD45ROhigh) cells during P. vivaxinfection. Our data show that the numbers of both naıve(Fig. 1A) and memory (Fig. 1B) cells increased in P. vivax-infected subjects (mean ¼ 1979 � 175.5 cells/mm3 and2443 � 258.3 cells/mm3, respectively) compared to malaria-naıve donors (mean ¼ 1309 � 125.5 cells/mm3 and1214 � 95.2 cells/mm3, respectively) (P ¼ 0.0231 andP ¼ 0.0040, respectively). When the frequencies of naıve andmemory cells were evaluated, similar differences between P.vivax-infected donors and control donors were observed(Supplementary Fig. S1).

3.2. Naıve and memory CD4þ T cells produce IFN-g orIL-10 during natural infection with P. vivax

Following the increase in the absolute number of naıve andmemory CD4þ T cells in P. vivax-infected donors, we showedthat individuals naturally infected by P. vivax also presented asignificant increase in the absolute number of IFN-g- (Fig. 2Aand B, respectively) and IL-10-producing cells (Fig. 2C andD) compared to non-infected individuals (for gating strategysee Supplementary Fig. S2); which was also observed whenfrequencies were determined (Supplementary Fig. S3). Inter-estingly, the production of these cytokines in infected in-dividuals was more predominant in memory cells(CD4þCD45ROhigh) (Fig. 2).

3.3. Absolute number of IFN-g-producing naıve andmemory CD4þ T cells correlates with IL-10 productionin P. vivax-infected donors

Once the production of both pro- and anti-inflammatorycytokines was detected within memory CD4þ T cells, wefurther assessed the association between the production ofIFN-g and IL-10. In infected patients, the absolute numbers ofIFN-g-producing naıve and memory T cells were directlycorrelated with IL-10 production by the same subsets

Fig. 2. Cytokine production in naıve and memory T cells. Expression of surface markers of naıve and memory CD4þ T cells co-expressing inflammatory or

regulating cytokine (IFN-g and IL-10, respectively) in malaria-naıve and P. vivax-infected donors (n ¼ 22 and 64, respectively). Results were expressed in absolute

numbers (cells/mm3) and the lines represent mean for (A) CD45RAþIFN-g, (B) CD45ROhighIFN-g, (C) CD45RAþIL-10 and (D) CD45ROhighIL-10 in CD4þ T

cells. Statistical differences were detected using Student t test and are indicated on the graphs with significant P values.

840 A.L.T. Silva et al. / Microbes and Infection 15 (2013) 837e843

(r ¼ 0.9034 and r ¼ 0.7223, respectively, P < 0.0001 for both)(Fig. 3A and B). No association between IFN-g and IL-10production was observed in samples from healthy controlindividuals.

3.4. P. vivax infection induces the development of centralmemory cells

Fig. 3. Correlation of absolute numbers of circulating IL-10 and IFN-g-pro-

ducing T helper among 64 patients with Plasmodium vivax malaria. Statistical

significance was determined by Pearson correlation.

The pattern of activation and migration of memory cellsduring malaria infection was determined by flow cytometricanalysis in which the co-expression of CD45RO, CCR7and CD62L cell surface markers was evaluated todistinguish central (CD45ROhighCCR7þCD62Lþ) andeffector (CD45ROhighCCR7�CD62L�) memory T cells(Supplementary Fig. S4). Our data show that P. vivax-infecteddonors presented a significant increase in the circulatingnumber of central memory CD4þ T cells, determined by thesignificant expression of CCR7 and CD62L (P ¼ 0.0147 andP ¼ 0.0002, respectively) (Fig. 4). Moreover, absolute countsof memory CD4þ T cells lacking the expression of CCR7 andCD62L were significantly reduced in infected individuals(P ¼ 0.0234 and P ¼ 0.0422, respectively) compared tohealthy control donors (Fig. 4). When the frequency of cellswas evaluated, we also detected that P. vivax-infected donorspresented a significant increase in the number of circulatingcentral memory CD4þ T cells, determined by the significantexpression of CCR7 (44.74 � 30.3%) and CD62L(44.44 � 24.4%), compared to control individuals(32.38 � 19.1%) (P ¼ 0.0040 and P ¼ 0.0374, respectively).The frequency of cells with no expression of CCR7 andCD62L by memory CD4þ T cells was also reduced in infectedindividuals (15.92 � 17.1% and 17.33 � 16.3%, respectively)compared to healthy control donors (22.95 � 11.1% and

Fig. 4. Flow cytometric analysis of central and memory CD4þ T cells in malaria-naıve and P. vivax-infected donors (n ¼ 22 and 64, respectively). Expression of

CCR7 and/or CD62L distinguishes two subsets of T cell: central memory (CD45ROhighCCR7þ/CD45ROhighCD62Lþ) or effector memory (CD45ROhighCCR7�/CD45ROhighCD62L�). Data are represented by mean average (bars) and standard error. Statistical differences were detected using Student t test and are indicated

on the graphs with significant P values.

841A.L.T. Silva et al. / Microbes and Infection 15 (2013) 837e843

27.30 � 14.5%, respectively) (P ¼ 0.0093 and P ¼ 0.0034,respectively).

4. Discussion

Memory T cells represent a dynamic repository of antigen-experienced T lymphocytes that have accumulated over anindividual’s lifetime [16]. Most mature peripheral T cells areat rest and can be divided into naıve and memory cells basedon their response to antigens during a secondary response, thepresence of pre-formed RNAs and the expression of specificsurface cell markers [21]. Presumably, the importance ofmemory T cells relies on the reduction of morbidity and theenhanced survival of the host after previous exposure to apathogen, either by ensuring the production of effector cells orby further enhancing antibody specificity [22]. In malaria, thedevelopment of immune memory responses requires acontinuous exposure to parasite antigens for generation andlong-term maintenance after its establishment [4]. While therole of effector CD4þ T cells in blood-stage malaria immunityhas been extensively studied in both murine and humaninfection, the characterization of the memory CD4þ T cellresponse is still poorly defined, particularly for P. vivaxinfection.

In the current study, we showed that individuals naturallyinfected by P. vivax present significant increases in circulatingnaıve and memory CD4þ T cells in the peripheral blood whencompared to non-exposed donors, corroborating a previousstudy that demonstrated the presence of memory CD4þ T cellsin acute P. vivax infection [6]. Of note, we observed significantincreases in the absolute numbers of CD45RAþ andCD45ROþ lymphocytes in infected patients, which may reflecta possible co-expression of both CD45 isoforms, expectedduring T cell hyperactivation or, more likely, in the naturaltransition from naıve to memory phenotype [23,24]. Becausethe concomitant expression of CD45RA and CD45RO was notassessed in the evaluated individuals, we cannot exclude thepossibility that CD45RAþ cells may also represent suchtransient cells, which contribute to the production of both IFN-g and IL-10 along with true memory (CD45ROhigh) CD4þ Tcells. We hypothesized that the inherent production of thesecytokines may be associated with natural exposure to theparasite, promoting the generation of long-term memory cells,

which would further facilitate the secretion of several immu-noregulators during infection. Notably, a recent study deter-mined that IFN-g-producing CD45ROþCD4þ T cells have anestimated half-life of 3 years, whereas IL-10-producingCD45ROþCD4þ T cells are stably maintained for at least 6years after the last-documented malaria infection [17]. Inter-estingly, the expression of IFN-g and IL-10 by memory cellsdiffers from the pattern of cytokine production observed forCD4þ cells [25] and CD45ROþCD4þ cells after antigenrestimulation [17] during malaria, in which IL-10 productionis reduced in infected individuals. Although in our study it wasnot possible to assure that same cells were producing IFN-gand IL-10, the dual expression of these cytokines weredescribed in experimental infections with Trypanosoma cruzi[26] or Toxoplasma gondii [27], may be explained as anegative autoregulatory feedback loop via the co-induction ofIL-10 in addition to the pre-existent IFN-g in the same cells[28], resulting in IL-10-producing Th1 cells [29,30]. Whereasthe IFN-g produced by IFN-gþIL-10þ Th1 cells would berequired for the eradication of pathogens, the concomitantproduction of IL-10 by these cells may direct for the resolutionof the inflammatory response and the prevention of immunepathology [27]. Indeed, the type of memory CD4þ T cell isconsidered important for the exacerbation or regulation ofpathology [4].

According to a model proposed by Sallusto et al., memoryT lymphocytes could be characterized as central (classic type)or effector memory cells based on their distinct homing ca-pacities and effector functions [16]. In this study, we reportedfor the first time that patients with acute P. vivax infectionpresented a significant augmentation of the number ofCD4þCD45ROhigh T cells expressing CCR7 and CD62L and asignificant decrease in CD4þCD45ROhigh T cells, whencompared to non-exposed individuals, suggesting a profile ofcentral rather than effector memory cells during infection.Whereas a more conventional idea is that effector cells are theprecursors of memory cells [31], it has been proposed thatnaıve T cells may differentiate into central memory T cellsand, finally, to effector memory cells [16]. Whether the sig-nificant increase in circulating central memory cells duringvivax malaria would represent a consequent increase ineffector cells remains to be determined. Nonetheless, our datasuggest that the majority of memory CD4þ T cells display a

842 A.L.T. Silva et al. / Microbes and Infection 15 (2013) 837e843

central memory phenotype and thus are not expected to pro-duce any of the prototypic cytokines of the effector cell line-age (IFN-g, IL-4 and IL-5) after TCR stimulation, althoughthey do proliferate extensively and acquire effector lympho-kine production later [32]. Moreover, the apparent lack ofexpression of homing receptors on memory CD4þ cells ininfected individuals would not necessarily affect the efficacyof the immune response during natural infection by P. vivaxdue to the systemic immunity required in malaria. Alternately,the decline of memory CD4þ cells with an ability to migrate toinflammatory sites (effector memory cells) could result in adecreased response to the liver stages of the parasite. However,the mechanisms involved in the development of P. vivaxparasite control by memory cells at the liver and blood stagesare still poorly elucidated; such studies are required for anunderstanding of protective immunity and crucial for vaccinedevelopment. Moreover, further studies are also necessary todetermine the influence of high or low exposure to P. vivax inendemic areas and, more importantly, whether the induction ofa memory response would be associated with the naturalrelapse expected during the P. vivax life cycle.

Acknowledgment

This work was financially supported by Fundacao deAmparo a Pesquisa do Estado de Minas Gerais/FAPEMIG(Grant # CBB APQ-0997-4.0 1/07), Pronex Malaria, DECIT/MS (555646/2009-2) and Pro-Reitoria de Pesquisa of Uni-versidade Federal de Minas Gerais. Ricardo Fujiwara andErika Braga are supported by Brazilian National ResearchCouncil (CNPq) fellowships.

Appendix A. Supplementary data

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.micinf.2013.07.009.

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