T cells but not NK cells are essential for cell-mediated immunity against Plasmodium chabaudi...

INFECTION AND IMMUNITY, Oct. 2010, p. 4331–4340 Vol. 78, No. 100019-9567/10/$12.00 doi:10.1128/IAI.00539-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

�� T Cells but Not NK Cells Are Essential for Cell-MediatedImmunity against Plasmodium chabaudi Malaria�

William P. Weidanz,1* GayeLyn LaFleur,1 Andrew Brown,1 James M. Burns, Jr.,2Irene Gramaglia,3,4 and Henri C. van der Heyde3,4

Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin1; Department of Microbiology andImmunology, Drexel University College of Medicine, Philadelphia, Pennsylvania2; La Jolla Bioengineering Institute,

La Jolla, California3; and La Jolla Infectious Disease Institute, San Diego, California4

Received 20 May 2010/Returned for modification 12 June 2010/Accepted 19 July 2010

Blood-stage Plasmodium chabaudi infections are suppressed by antibody-mediated immunity and/or cell-mediated immunity (CMI). To determine the contributions of NK cells and �� T cells to protective immunity,C57BL/6 (wild-type [WT]) mice and B-cell-deficient (JH�/�) mice were infected with P. chabaudi and depletedof NK cells or �� T cells with monoclonal antibody. The time courses of parasitemia in NK-cell-depleted WTmice and JH�/� mice were similar to those of control mice, indicating that deficiencies in NK cells, NKT cells,or CD8� T cells had little effect on parasitemia. In contrast, high levels of noncuring parasitemia occurred inJH�/� mice depleted of �� T cells. Depletion of �� T cells during chronic parasitemia in B-cell-deficient JH�/�

mice resulted in an immediate and marked exacerbation of parasitemia, suggesting that �� T cells have a directkilling effect in vivo on blood-stage parasites. Cytokine analyses revealed that levels of interleukin-10, gammainterferon (IFN-�), and macrophage chemoattractant protein 1 (MCP-1) in the sera of �� T-cell-depleted micewere significantly (P < 0.05) decreased compared to hamster immunoglobulin-injected controls, but thesecytokine levels were similar in NK-cell-depleted mice and their controls. The time courses of parasitemia inCCR2�/� and JH�/� � CCR2�/� mice and in their controls were nearly identical, indicating that MCP-1 is notrequired for the control of parasitemia. Collectively, these data indicate that the suppression of acute P.chabaudi infection by CMI is �� T cell dependent, is independent of NK cells, and may be attributed to thedeficient IFN-� response seen early in �� T-cell-depleted mice.

Malaria remains a leading cause of morbidity and mortality,annually killing about 2 million people worldwide (32, 33).Despite decades of research, malaria is a reemerging diseasebecause of increasing drug resistance by malarial parasites andinsecticide resistance by the mosquito vector. Most infectedindividuals do not succumb to malaria but develop clinicalimmunity where parasite replication is controlled to some de-gree by the immune system without eliciting clinical disease orsterile immunity (14, 38).

Understanding the immunologic pathways leading to thecontrol of blood-stage parasite replication is important fordefining the mechanisms of disease pathogenesis and improv-ing vaccines currently in development. The early events of theimmune response depend upon activation of the innate im-mune system, which regulates the downstream adaptive im-mune response needed to control or cure (44). Natural killer(NK) and �� T cells function early in the immune response topathogens as components of the innate immune system. Bothcell types have been proposed to play significant roles in thesubsequent clearance of blood-stage malarial parasites by ac-tivating the adaptive immune system (35, 43, 44). The mecha-nism by which they accomplish this appears to be mediated viatheir secretion of gamma interferon (IFN-�) induced by cyto-kines such as interleukin-12 (IL-12), tumor necrosis factor

alpha (TNF-�), and IL-6 produced by other components of theinnate immune system, including macrophages and dendriticcells (17, 25, 26, 37, 49).

Blood-stage malaria parasites are cleared by mature isotypesof antibodies and/or by antibody-independent but T-cell-de-pendent mechanisms of immunity (2, 15, 22). Both responsesrequire CD4� �� T cells; in addition, the expression of cell-mediated immunity (CMI) during both acute and chronic ma-laria is dependent on �� T cells activated by CD4� �� T cells(29, 47, 49, 50). Wild-type (WT) mice depleted of �� T cells byantibody treatment or gene knockout suppress P. chabaudiparasitemia by antibody-mediated immunity (AMI) (21, 52).Mice depleted of B cells by the same procedures also cure theiracute infections in the same timeframe as intact control micebut then develop chronic low-grade parasitemia of long-lastingduration, indicating that B cells and their antibodies areneeded to sterilize the infection as we originally reported (15,48) and has since been confirmed by others (51). B-cell-defi-cient mice depleted of �� T cells cannot suppress P. chabaudiparasitemia (49, 50, 52).

The prominent role played by IFN-� in immunity to malariais generally accepted by most researchers. P. chabaudi malariais more severe in WT mice treated with neutralizing antibodyand in IFN-��/� mice, as indicated by the increased magnitudeand duration of parasitemia and mortality in mice deficient inIFN-� versus intact controls (24, 39, 46). In B-cell-deficientanimals, the similar neutralization of IFN-� by treatment withanti-IFN-� monoclonal antibody (MAb) or gene knockout ofIFN-� has an even greater effect on the time course of para-sitemia, which remains at high levels and fails to cure (1, 46),

* Corresponding author. Mailing address: Department of MedicalMicrobiology and Immunology, University of Wisconsin School ofMedicine and Public Health, Madison, WI 53706. Phone: (608) 262-9027. Fax: (608) 262-8418. E-mail: [email protected].

� Published ahead of print on 26 July 2010.

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indicating that IFN-� is essential for the expression of anti-parasite CMI and contributes to AMI in this model system.

The early source of IFN-� remains controversial, with bothNK cells and �� T cells being proposed to produce this criticalcytokine necessary for the activation of the adaptive immuneresponse and the development of protective immunity (9). Theresults of earlier genetic studies failed to correlate susceptibil-ity to P. chabaudi infection with NK activity (31, 44). Subse-quently, Mohan et al. (25) reported that NK cell activityagainst tumor cell targets correlates with protection against P.chabaudi; anti-asialo GM1 polyclonal antibody depletion ofNK cells results in significantly increased levels of peak para-sitemia and a prolonged duration of infection compared tocontrols. The mode of action by which NK cells function ap-pears to be via the secretion of cytokines (25) rather thandirect cytotoxicity against the blood-stage parasites. The sur-face expression of lysosome-associated membrane protein 1(LAMP-1) by subsets of human NK cells exposed to Plasmo-dium falciparum-infected erythrocytes may suggest otherwise(20). NK cells in collaboration with dendritic cells are respon-sible for optimal IFN-� production dependent upon IL-12 (17,36, 39, 40). In contrast to the findings of Mohan et al., otherstudies indicate similar P. chabaudi parasitemia in depletedmice and intact controls after NK1.1 MAb depletion of NKcells (19, 41, 53). Using microarray analysis of blood cells fromP. chabaudi-infected mice, Kim et al. (18) reported a rapidproduction of IFN-� and activation of IFN-�-mediated signal-ing pathways as early as 8 h after infection; however, NK cellsdid not express IFN-� or exhibit IFN-�-mediated pathways intheir analysis. At this time, NK cells are replicating and mi-grating from the spleen to the blood. In humans with P. fal-ciparum malaria, increased production of IFN-� by PBMC inresponse to parasitized RBCs correlates with protection fromhigh-density parasitemia and clinical malaria (10, 11); earlyIFN-� production by PBMC obtained from malaria naive do-nors is primarily by �� T cells and not by NK cells (26). Animalmodels by definition do not exactly mimic the human condi-tion, and the experimental malaria in mice uses distinct speciesfrom those that infect humans. Nevertheless, analysis of pro-tective immunity provides important information on how aprotective immune response to Plasmodium may be elicited.

Whether both NK cells and �� T cells have essential rolesduring the early stages of the immune response to blood-stagemalaria remains to be determined. Likewise, whether thesecells function early in CMI to malaria parasites is unknown. Toaddress these issues, we infected NK-cell- or ��-T-cell-de-pleted JH�/� mice with blood-stage P. chabaudi. The resultingtime course of parasitemia was monitored and compared tocontrol mice. In addition, spleen cells from depleted and con-trol mice were profiled by cytofluorimetry, and the serum levelsof inflammatory cytokines were measured.

MATERIALS AND METHODS

Parasites and infections of mice. CCR2�/� mice, �2m�/� mice, and CD1d�/�

mice of both sexes on a C57BL/6 (WT) background were purchased from Jack-son Laboratories (Bar Harbor, ME). B-cell-deficient JH�/� mice, which weregenerated by deletion of the JH region in embryonic stem cells, fail to produceimmunoglobulin (Ig) and are devoid of B cells because B-cell differentiation isblocked at the large CD43� precursor stage (8). JH�/� mice originally obtainedfrom Dennis Huszar were bred and maintained in our laboratory. JH�/� mice,tenth generation backcrossed to C57BL/6 (WT) mice, were crossed with

CCR2�/� mice, which lack the receptor for the chemokine, macrophage che-moattractant protein 1 (MCP-1). �2m�/� mice and CD1d�/� mice were alsobred with JH�/� mice to produce double-knockout (KO) mice, which were phe-notyped by standard PCR techniques, using gene-specific primers. �2m�/� micedefective in major histocompatibility complex (MHC) class I expression aredeficient in NK cells, NKT cells, and CD8� T cells. CD1d�/� mice are deficientin NKT cells. All mice were bred and maintained at the AAALAC-accreditedUniversity of Wisconsin-Madison vivarium and infected between 8 and 12 weeksof age. Animal studies have been reviewed and approved by the University ofWisconsin Animal Care Committee.

P. chabaudi adami 556KA, which produces nonlethal infections and sterilizingimmunity in immunologically intact mice, was maintained and used as describedpreviously (6). Unless stated otherwise, age- and sex-matched experimental andcontrol mice were injected intravenously (i.v.) with 105 P. chabaudi-parasitizederythrocytes obtained from a source mouse. The injections of both antibodiesand parasites into the peritoneal cavity causes the activation of resident macro-phages and results in a reduced inoculum size. This is prevented by injectingantibodies and parasites by different routes. Previous experience indicated thatthe kinetics of parasitemia following the injection of 105 parasitized erythrocytesi.v. were equivalent to those following an injection with 106 parasitized erythro-cytes given intraperitoneally (i.p.).

The parasitized erythrocytes were washed prior to inoculation to prevent thecarryover of cytokines and chemokines in donor blood. The resulting parasitemiawas assessed by counting the number of parasitized erythrocytes in 500 to 1,000erythrocytes in Giemsa-stained thin blood films.

Antibody depletion. Purified anti-NK1.1 MAb (hybridoma PKI36) was a giftfrom Charles Czyprinski (University of Wisconsin, Madison) and anti-TCR�(GL3) was kindly provided by Francesca Neethling and Jon Weidanz (ReceptorLogic, Abilene, TX). Purified mouse Ig and hamster Ig were purchased fromThermo Scientific (Rockford, IL) and used as a control for the depleting MAbs.Depletion of NK cells was accomplished by using the following treatment regi-men: 250 �g of anti-NK1.1 in phosphate-buffered saline was injected i.p. intoeach mouse on days �1, 0, 1, 3, 5, 10, and 15 postinoculation (p.i.) with para-sitized erythrocytes. Depletion of �� T cells was achieved by the i.p. injection of500 �g of anti-GL3 on days �1, 0, 1, and 15 p.i. Normal mouse Ig and hamsterIg were injected i.p. into control mice according to the same regimen used for thedepleting MAbs.

Flow cytometry. Four-color flow cytometry was performed as described pre-viously (16). Briefly, the spleen was dissected from a euthanized animal, disag-gregated by passing the spleen through nylon mesh, and the erythrocytes werelysed by hypotonic shock. Washed cells were incubated with Fc block (BectonDickinson, San Diego, CA) and then stained with the following MAbs labeledwith fluorescein isothiocyanate or phycoerythrin purchased from Pharmingen:anti-CD3e (145-2C11), anti-CD4 (GK1.5), anti-CD8 (Ly-2), anti-TCR�� (H57),anti-TCR� (GL3), anti-NK1.1 (PK136), anti-F4/80 (M5-2C11), and anti-CD11b(M1/70). Propidium iodide was added prior to flow cytometric analysis to excludedead cells. An equal number of cells (10, 000) from MAb-treated test mice orIg-treated control mice was analyzed. The number of cells examined was suffi-cient to accurately assess the percentages of different subsets of spleen cells. Thispercentage was multiplied by the total number of live cells in the spleen (mea-sured as trypan blue-negative cells by using a hemocytometer) to obtain thenumber of cells within selected subsets of splenocytes. More than 10,000 eventscalculates the percentage to 0.01% accuracy, which is beyond the machine errorand interanimal variation.

Cytokine measurement by bead array. The levels of selected cytokines (IL-6,IL-10, IL-12p70, IFN-�, TNF-�, and MCP-1) in the sera of experimental animalsand their controls obtained days 4 and 8 p.i. were assayed by using a mouseinflammation kit from BD Biosciences (La Jolla, CA) according to the manu-facturer’s instructions. The limit of detection of this assay is 20 pg of cytokine/mland is indicated by a dashed line in Fig. 1C, 2C, 4C, and 6C.

IgG isotype ELISA. The isotypic profile of the antigen-specific antibodies inthe sera of infected mice depleted of NK or �� T cells and their controls wasdetermined by enzyme-linked immunosorbent assay (ELISA) using P. chabaudiapical membrane antigen-1 (rPCAMA-1)- or P. chabaudi merozoite surfaceprotein-142 (rPCMSP-1)-coated wells as described previously (5). Briefly, serumfrom each animal harvested day 19 p.i. (NK cell-depleted and mouse Ig-treatedcontrols) and day 21 p.i. (�� T-cell-depleted and hamster Ig-treated controls) wasassayed in duplicate on antigen-coated wells at dilutions that ranged from 1:100to 1:1,000. Antigen-specific antibodies were detected with horseradish peroxi-dase-conjugated rabbit antibody specific for mouse IgM, IgG1, IgG2b, or IgG3(Zymed Laboratories) or with horseradish peroxidase-conjugated goat anti-mouse IgG2c (IgG2a b allotype; Southern Biotechnology Associates, Inc., Bir-mingham, AL) (23) and ABTS [2,2�azinobis(3-ethylbenzthiazolinesulfonic acid)]

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as the substrate. In each assay, wells were coated with purified IgM (eBioscience,San Diego, CA) or with IgG1, IgG2b, or IgG3 (Zymed Laboratories). Isotypecontrol antibodies were used to generate standard curves (16 ng/ml to 2 �g/ml).The IgG2c standard curve was generated by using a purified monoclonal IgG2c(IgG2a b allotype) antibody (BD Biosciences Pharmingen, San Jose, CA). Theconcentration of each Ig isotype was expressed in units per milliliter, where 1U/ml was equivalent to 1 �g of myeloma standard/ml.

Statistical analysis. Analysis of variance with the StatView program (SASInstitute, Cary, NC) was performed to statistically compare parasitemia, cellnumbers, and percentages in different groups of mice obtained from depletionstudies. The statistical significance of all other parasitemia data was determinedby using a Student t test. A P value of 0.05 was considered significant.

RESULTS

Effect of NK cell versus �� T-cell depletion on the timecourse of P. chabaudi parasitemia in WT mice. To determinethe effects of treatment with anti-NK1.1 versus treatment withanti-GL3, four parameters of P. chabaudi malaria in WT micewere compared; these parameters included the time course ofparasitemia, the profile of splenic mononuclear cells, the se-rum levels of selected cytokines, and the antibody isotype re-sponses to recombinant P. chabaudi antigenic constructs.

Two groups of WT mice (n 4/group) were individuallyinoculated i.v. with. 105 P. chabaudi-parasitized erythrocytes,followed by 250 �g i.p. of either anti-NK1.1 MAb or mouse Igon days �1, 0, 1, 3, 5, 10, and 15 p.i. The time course of acuteparasitemia in both test and control groups of mice was similar(Fig. 1A), with peak parasitemia occurring on day 9 and clear-ance of parasites from the blood to 0.01% occurring on days15 and 19, respectively. This regimen of anti-NK1.1 treatmentdepleted NK cells and NKT cells from the spleen; indeed, fewNK1.1 cells and NKT cells were detected by flow cytometric

analysis of splenocytes obtained from antibody-treated mice onday 19 p.i. (Fig. 1B). The number of cells in each splenic cellpopulation other than NK cells and NKT cells did not differsignificantly (P � 0.05) between the anti-NK1.1 MAb-treatedmice and the mouse Ig control groups. Likewise, the numbersof �� T cells and activated (B220�) �� T cells, as well as thenumber of splenic macrophages (F4/80�, CD11d�), were notdiminished by the antibody depletion of NK cells. The differ-ence in numbers observed between test and control groups wasnot significant. To determine whether NK cell depletion af-fected the levels of selected cytokines (IL-6, IL-10, IL-12,TNF-�, and IFN-�) and the chemokine, MCP-1, we quantifiedthese proteins in the sera of test and control mice bled on days4 and 8 p.i. Day 4 corresponds to the period of ascendingparasitemia when the protective immune response is beingactivated by the innate immune system to control parasitereplication. By day 8 p.i., the activated immune system appearsto be functioning as indicated by the decrease in the slope ofparasitemia (Fig. 1A). The levels of all cytokines and chemo-kines were below the limits of resolution (20 pg/ml) in un-infected animals. The concentration of IFN-� increased signif-icantly (P 0.05) in both groups of mice on days 4 and 8 p.i.,with the greatest increase seen on day 4 p.i. (Fig. 1C). Thelevels of serum MCP-1 also increased markedly on day 4 p.i.and then declined by day 8 p.i. IL-10 was elevated on day 8 p.i.but not day 4. The remaining cytokines (IL-12, TNF-�, andIL-6) were not detected above baseline. There were no signif-icant differences (P � 0.05) in IL-10, IFN-�, or MCP-1 con-centrations between NK-depleted and control groups. Similarresults were obtained upon repeating the experiment.

FIG. 1. P. chabaudi infection in C57BL/6 mice depleted of NK cells and mouse Ig-treated controls (n 4/group). (A) Time course ofparasitemia after the inoculation of 105 parasitized erythrocytes i.v. in C57BL/6 mice treated with anti-NK 1.1 or mouse Ig (250 �g) on days �1,0, 1, 3, 5, 10, and 15 p.i. injected i.p. (B) Cytofluorimetric analysis of spleen cells from NK cell-depleted mice and controls 19 days p.i. (C) Serumcytokine levels in NK cell-depleted mice and controls on days 4 and 8 p.i. *, P 0.05.

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Depletion of �� T cells from WT mice decreases the levelsIFN-� and MCP-1 early during the course of P. chabaudimalaria. Previously, we and others have reported that the timecourse of P. chabaudi parasitemia was prolonged for severaldays in WT mice depleted of �� T cells by MAb treatment orgene knockout compared to infected controls (21, 52). Todetermine the role of �� T cells in the control of acute P.chabaudi parasitemia, we inoculated two groups of WT mice(n 4 animals/group) i.v., with each mouse receiving 105 P.chabaudi parasitized erythrocytes, followed by the i.p. injectionof 500 �g of either anti-TCR �� MAb or hamster Ig on days�1, 0, 1, and 15 p.i. The time course of parasitemia was similarin both groups of mice, with peak parasitemia occurring on day9 p.i. (Fig. 2A). However, the mice injected with anti-GL3showed significantly (P 0.05) increased parasitemia on days13 and 15 p.i. compared to control mice but exhibited modestdifferences in suppressing the duration of patent parasitemia(day 15 p.i. for hamster Ig-treated controls and day 19 p.i. for�� T-cell-depleted mice), a result reminiscent of the publishedfindings from previous studies.

Flow cytometric analysis of spleen cell populations fromanti-GL3-treated mice and controls revealed a statistically sig-nificant (P 0.05) depletion of �� T cells in the antibody-treated mice compared to hamster Ig-treated controls. Thenumber of cells in each �� T-cell subset (CD4� and CD8�)was greater in the �� T-cell-depleted animals, but the differ-ences did not reach significance (Fig. 2B). Likewise, the num-ber of splenic macrophages increased in �� T-cell-depletedanimals compared to controls (Fig. 2B), whereas they wereapproximately the same in NK-depleted animals compared totheir corresponding controls (Fig. 1B).

Depletion of �� T cells affected cytokine production. Theconcentrations of serum IFN-� and MCP-1 were significantly(P 0.05) lower on day 4 p.i. in �� T-cell-depleted animalscompared to controls; on day 8 p.i., the level of IL-10 was lowerin antibody-treated animals than in controls (Fig. 2C). Al-though IL-12 was decreased in the sera of the �� T-cell-de-pleted animals compared to controls, the levels were too low tomake any conclusions with certainty. The other cytokines(TNF-� and IL-6) were not detected above baseline. We re-peated the experiment with nearly identical results.

Depletion of NK cells or �� T cells from WT mice increasesselected antibody isotype responses to malaria antigens. An-tibody isotypes specific for rPCAMA-1 and rPCMSP-1 weremeasured in serum samples from the same mice harvested onday 19 or day 22 p.i. The results indicate that both NK celldepletion and �� T-cell depletion caused marked increases(P 0.05) in the levels of IgG2c and IgG3 antibodies to bothantigens compared to infected Ig-treated controls (Fig. 3).

NK cells are not essential for expression of CMI against P.chabaudi malaria. To determine whether NK cells play anessential role in the activation of CMI that suppresses acuteparasitemia in B-cell-deficient mice, we inoculated two groupsof JH�/� mice (n 4 animals/group), with each mouse receiving105 P. chabaudi-parasitized erythrocytes i.v., followed by eitheranti-NK1.1 MAb or mouse Ig i.p., using the regimen describedabove. The time course of acute parasitemia was similar inboth groups of mice (Fig. 4A), with peak parasitemia occurringon day 9 p.i. and the suppression of parasitemia to levels of0.01% occurring in NK-cell-depleted mice by day 15 p.i. andby day 17 p.i. in control mice. There were few splenic NK1.1

FIG. 2. P. chabaudi infection in C57BL/6 mice depleted of �� T cells and hamster-Ig treated controls (n 4 animals/group). (A) Time courseof parasitemia after the inoculation of 105 parasitized erythrocytes i.v. in C57BL/6 mice treated with anti-GL3 or hamster Ig (500 �g) on days �1,0, 1, and 15 p.i. injected i.p. (B) Cytofluorimetric analysis of spleen cells from �� T-cell-depleted mice and controls 21 days p.i.. (C) Serum cytokinelevels in �� T-cell-depleted mice and controls on days 4 and 8 p.i. *, P 0.05.



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cells in anti-NK1.1-treated mice compared to control mice, asdetected by flow cytometry on day 21 p.i. (Fig. 4B).

To determine the effects of NK cell depletion on othersplenic cell populations, we assessed the number of CD4�,CD8�, ��, and �� T cells on day 21 p.i. by flow cytometry. Thenumber of cells in each population was similar in the anti-NK1.1-treated and mouse Ig control groups (Fig. 4B). Al-though NK cell depletion did not alter �� T-cell activation, adecrease in the number of splenic macrophages (F4/80�,CD11d� cells) as a result of NK cell depletion was observed(Fig. 4B). The differences in macrophage numbers in test andcontrol mice did not reach significance.

We also determined whether NK cell depletion in JH�/� miceaffected the levels of selected cytokines (IL-6, IL-10, IL-12,TNF-�, and IFN-�) and the chemokine MCP-1 on days 4 and8 p.i. Similar to the results observed in WT mice, the concen-trations of IFN-� increased significantly on both day 4 p.i. andday 8 p.i.; the greatest concentration was on day 4 p.i. (Fig.4C). The levels of serum MCP-1 also increased markedly onday 4 p.i. and then declined by day 8 p.i. IL-10 was elevated

only on day 8 p.i.. The other cytokines (IL-12, TNF-�, andIL-6) were not detected above baseline levels. There were nosignificant differences in IL-10, IFN-�, or MCP-1 concentra-tion between NK-depleted and control groups.

CMI against P. chabaudi is fully functional in knockout micedeficient in NK cells or NKT cells. To determine the contri-bution of NK, NKT, and CD8� T cells to control of P.chabaudi parasitemia, we injected 106 parasitized erythrocytesinto groups of B-cell-deficient JH�/� � �2m�/�, JH�/� �CD1d�/� double KO mice and their B-cell-deficient, heterozy-gous controls (JH�/� � �2m�/�, JH�/� � CD1d�/�). �2m�/�

lack MHC class I expression and exhibit severely diminishednumbers of NK cells, NKT cells, and CD8� T cells (30, 45).CD1d�/� mice lack NKT cells (3). The parasitemia was similarin the B-cell-deficient mice lacking �2m or CD1d and theircontrols; both groups suppressed their acute parasitemia witha similar time course (Fig. 5).

Suppression of CMI against P. chabaudi is mediated by ��T cells. To confirm that �� T-cell function in the activation ofCMI that suppresses acute parasitemia as we reported previ-ously (49, 50, 52), we inoculated B-cell-deficient mice i.v. with105 P. chabaudi-parasitized erythrocytes and then injected themice i.p. with either anti-GL3 or hamster Ig as describedabove. The parasitemia was similar throughout the period ofascending parasitemia (Fig. 6A), with both test and controlgroups exhibiting similar magnitudes of peak parasitemia, Sub-sequently, the time course of parasitemia diverged significantlywith �� T-cell-depleted animals exhibiting significantly greaterparasitemia (P 0.05) that did not resolve. There were sig-nificantly fewer �� T cells detected by flow cytometry in thespleens of anti-GL3-treated mice compared to controls (Fig. 6B).

To determine whether the depletion of �� T cells affectedthe profile of other cell populations that might contribute to thesuppression of CMI to P. chabaudi infection, we assessed thenumbers of splenic CD4� T cells, CD8� T cells, NK cells, NKTcells, and dendritic cells on day 21 p.i. by flow cytometry. Thenumber of cells in each �� T-cell subset (CD4� and CD8�)was greater in the �� T-cell-depleted compared to controls(Fig. 6B). We also observed an increase in the numbers ofsplenic macrophages in �� T-cell-depleted animals. In contrast,the numbers of NK cells and NKT cells were approximately thesame in the absence of �� T cells compared to controls. Exceptfor the depletion of �� T cells in test versus control mice, thesechanges in spleen cell numbers of the different cell populationsassayed in test and control mice were modest and did not reachstatistical significance.

To determine whether �� T-cell depletion affected the levelsof selected cytokines (IL-6, IL-10, IL-12, TNF-�, and IFN-�)and the chemokine MCP-1, we measured the levels of theseproteins in serum on days 4 and 8 p.i. in groups of miceinjected i.p. with either anti- -cell �� MAb or hamster Ig. Theconcentration of IFN-� was significantly (P 0.05) lower onday 4 p.i. in �� T-cell-depleted animals than in controls (Fig.6C); on days 4 and 8 p.i., the levels of MCP-1 and IL-10 werealso depressed in �� T-cell-depleted animals compared to con-trols. The concentration of IL-10 was too low on day 4 p.i. todraw a firm conclusion. However, the other cytokines assayed(IL-12, TNF-�, and IL-6) were not detected above baseline.

The MCP-1 receptor, CCR2, does not play an essential pro-tective role in immunity to P. chabaudi malaria. Although the

FIG. 3. Antibody isotype responses to rPCAMA-1 (top panel) andrPCMSP-1 (bottom panel) determined by ELISA in P. chabaudi-in-fected C57BL/6 mice depleted of NK cells or �� T cells and theircontrols using serum harvested at days 19 and 21 p.i., respectively.*, P 0.05.



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role of IFN-� in suppression of P. chabaudi parasitemia isestablished, the observation that the levels of MCP-1 in thesera of �� T-cell-depleted mice were also depressed suggeststhat this chemokine, which has been reported to play an im-portant role in monocyte migration and polarization of thetype 1 cytokine response (7), may have an important role incuring acute parasitemia. To test this supposition, we injected

both CCR2�/� mice and heterozygous controls i.p. with 106 P.chabaudi-parasitized erythrocytes and monitored parasitemiaover time; the time course of parasitemia was similar in bothCCR2�/� mice and controls (Fig. 7A).

We also bred JH�/� � CCR2�/� double KO mice to deter-mine whether MCP-1 was essential for the expression of CMIagainst the parasite. Double KO mice and JH�/� � CCR2�/�

FIG. 4. P. chabaudi infection in B-cell-deficient mice depleted of NK cells and mouse Ig-treated controls (n 4 animals/group). (A) Timecourse of parasitemia after the inoculation of 105 parasitized erythrocytes i.v. in B-cell-deficient mice treated with anti-NK 1.1 or mouse Ig (250 �g)on days �1, 0, 1, 3, 5, 10, and 15 p.i. injected i.p. (B) Cytofluorimetric analysis of spleen cells from NK cell-depleted mice and controls at 19 dayp.i. (C) Serum cytokine levels in NK cell-depleted mice and controls on days 4 and 8 p.i. *, P 0.05.

FIG. 5. Time course of P. chabaudi parasitemia in double KO mice lacking NK cells, NKT cells, or CD8� T cells. (A) Time course ofparasitemia in JH�/� � �2m�/� double KO mice (n 7) and their JH�/� � �2m�/� controls (n 4) inoculated i.p. with 106 P. chabaudi-parasitizederythrocytes. (B) Time course of parasitemia in JH�/� � CD1�/� double KO mice (n 5) and their JH�/� � CD1d�/� controls (n 5) inoculatedi.p. with 106 P. chabaudi-parasitized erythrocytes.



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controls inoculated i.p. with 106 parasitized erythrocytes hadsimilar time courses of parasitemia (Fig. 7B).

Antibody depletion of �� T cells in mice chronically infectedwith P. chabaudi causes an immediate and pronounced exac-erbation of parasitemia. �� cells are required to suppressparasitemia during acute and chronic P. chabaudi malaria inB-cell-deficient C57BL/6 mice (49, 50, 52). As indicated in Fig.4A, depletion of �� T cells prevented the suppression of acute

parasitemia, as seen in control mice. In addition, the depletionof �� T cells during chronic malaria causes the exacerbation ofparasitemia. To gain insight on how �� cells function, we in-jected chronically infected mice i.p. on day 60 p.i. with eitheranti-TCR�� MAb or hamster Ig and monitored their para-sitemia daily during the first week after injection (Fig. 8). Thetime course of acute parasitemia was similar in both groups ofmice (data not shown), and both experimental and control

FIG. 6. P. chabaudi infection in B-cell deficient mice depleted of �� T cells and hamster Ig-treated controls (n 4 animals/group). (A) Timecourse of parasitemia after the inoculation of 105 parasitized erythrocytes i.v. in B-cell-deficient mice treated with anti-GL3 or hamster Ig (500 �g)on days �1, 0, 1, and 15 p.i. injected i.p. (B) Cytofluorimetric analysis of spleen cells from �� T-cell-depleted mice and controls 21 days p.i.(C) Serum cytokine levels in �� T-cell-depleted mice and controls on days 4 and 8 p.i. *, P 0.05.

FIG. 7. Comparison of time courses of P. chabaudi parasitemia in CCR2 KO mice (n 6) and C57BL/6 controls (n 6) (A) and in JH�/� � CCR2�/�

double KO mice (n 4) and their JH�/� � CCR2�/� controls (n 4) (B) after the inoculation of 106 P. chabaudi-parasitized erythrocytes i.p.



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mice exhibited similar parasitemia prior to the injection of thedepleting antibody (Fig. 8). However, there was an immediateand marked increase in parasitemia which rose dramatically tohigh levels (i.e., �45%) in antibody-treated JH�/� mice by day5 after treatment with anti-GL3, whereas parasitemia re-mained at preinjection levels in the control mice. A repeat ofthis experiment yielded nearly identical results.

DISCUSSION

Both NK cells and �� T cells have been purported to func-tion early in the innate immune response to blood-stage ma-laria parasites (35, 43, 44). These assumptions in part are basedon the outcomes of cell depletion studies utilizing antibodiesand/or gene KO in mouse models of blood-stage malaria.Whether either cell type dominates in protection is unknown.Likewise, the function of these cells in AMI and CMI to ma-laria remains to be determined.

Previously, it was reported that the depletion of NK cellseither enhanced the severity of the AS strain of P. chabaudichabaudi infections in terms of mortality and/or parasitemia orhad little, if any, effect on these parameters of infection com-pared to intact controls (27, 44). A number of factors may havecontributed to the divergent results reported in the literature,including differences in the strains and species of Plasmodiumused, different mouse strains utilized, and differences in thetarget(s) of NK cell-depleting antibodies used. Parasitemia isof greater magnitude and longer in duration when NK cells aredepleted with anti-asialo GM1 polyclonal rabbit antibody butnearly identical to controls when NK cells are depleted withanti-NK1.1 MAb. Anti-NK1.1 MAb is specific for NK cells andNK T cells expressing the NK1.1 antigen. It has been suggestedthat anti-asialo GM1 polyclonal antibody may recognize and/or

cross-react with other carbohydrate moieties (27). This couldaffect the function of other cell types such as macrophages,dendritic cells, neutrophils, and T cells, in addition to NK cells,resulting in the enhancement of parasitemia and the severity ofinfection seen in depleted mice.

We utilized the adami subspecies of P. chabaudi in studiesreported here and observed that the depletion of NK cells fromeither WT mice or JH�/� mice failed to alter the time course ofparasitemia compared to controls given mouse Ig. Treatmentwith anti-NK1.1 MAb is reported to also deplete NKT cellsfrom liver and spleen (30). Our data indicate that both of thesecell types were depleted in the spleens of WT mice and B-cell-deficient mice treated with anti-NK1.1 MAb, suggesting a min-imal role, if any, for NK cells and NKT cells in the activationof either AMI or CMI against P. chabaudi adami. Otherwise,the profiles of splenic cell populations in depleted mice weresimilar to mouse Ig-treated controls. There were no significantdifferences in serum cytokine levels, including IFN-�, betweenMAb-treated and control mice. Our results agree with those ofothers who observed that the depletion of splenic cells withanti-NK1.1 MAb does not alter the time course of P. chabaudiparasitemia (19, 41, 53).

The conclusion that NK cell or NKT cells play little if anyrole in protective immunity against malaria was further sup-ported by the observations that the time course of P. chabaudiadami parasitemia was similar in JH�/� � �2m�/� double KOmice and JH�/� � �2m�/� controls, as well as in JH�/� �CD1d�/� mice and their JH�/� � CD1d �/� controls. NK cells,NKT cells, and CD8� T cells are lacking in JH�/� � �2m�/�

mice, and NKT cells are lacking in JH�/� � CD1�/� mice. Thedepletion of CD8� T cells from WT mice by antibody treat-ment or gene KO of �2m has been reported to have a minimaleffect on the time course of parasitemia (45). The role ofCD8� T cells in CMI to P. chabaudi in B-cell-deficient mice,lacking antibody, had not been determined previously. Thedata obtained in the present study indicate that CD8� T cellsdo not play an essential role in suppressing parasitemia bycytotoxicity or regulating the function of other cell types im-plicated in protection.

Our results confirm and extend earlier observations that ��T cells may play an important role in malaria. They are foundin elevated numbers in the blood of humans acutely infectedwith P. falciparum and remain at high levels for prolongedperiods of time (16, 28). During experimental P. chabaudimalaria, the depletion of �� T cells from WT mice results insignificantly elevated parasitemia at several time points priorto curing (21, 52). Moreover, �� T cells are required for theCMI-mediated suppression of P. chabaudi parasitemia in B-cell-deficient mice (49, 50, 52). The profiles of other splenicmononuclear cells in �� T-cell-depleted mice (WT and JH�/�

mice) infected with P. chabaudi did not differ significantly fromthat of control mice. In contrast to mice depleted of NK cells,the levels of serum IFN-� and MCP-1 were significantly de-pressed in �� T-cell-depleted WT and JH�/� mice compared tocontrol mice.

Diverse antibody isotypes specific for AMA-1 and MSP-1malaria antigenic constructs were present in the sera of NKcell- and �� T-cell-depleted mice. Both depleted modelsdisplayed multiple antibody isotypes to rPCAMA-1 andrPCMSP-1, sharing similar changes to those measured in con-

FIG. 8. Immediate exacerbation of P. chabaudi parasitemia inchronically infected B-cell-deficient mice, following the injection i.p. of1 mg of anti-GL3 versus 1 mg of hamster Ig. The mice had beeninoculated 60 days previously with 106 P. chabaudi-parasitized eryth-rocytes i.p. *, P 0.05.



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trol sera. These data indicate that mice depleted of either celltype are capable of mounting a robust antibody response thatis purported to suppress parasitemia by AMI (2, 22).

The critical role of IFN-� in immunity to malaria is uncon-tested. The neutralization of IFN-� by MAb in WT mice ordepression of IFN-� production in KO mice prolongs the du-ration of P. chabaudi parasitemia, which eventually cures (39,46), but in JH�/� mice leads to high levels of noncuring para-sitemia (1, 46). These data indicate that IFN-� plays an im-portant role in AMI immunity to malaria and an essential rolein CMI to the parasite. In contrast, the function of the che-mokine MCP-1 in immunity to malaria has only recently beendescribed (34). Chemokines and their receptors, such asMCP-1 and its receptor, CCR2, play an important role in themobilization of bone marrow cells, monocyte migration, andthe polarization of the type 1 cytokine response (4). Sponaas etal. (34) observed that peak parasitemia was similar in CCR2mice infected with the more virulent AS strain of P. chabaudichabaudi and their controls. After reaching peak parasitemia,the clearance of parasitemia in CCR2�/� mice is delayed,suggesting that “CD11bhigh cells are part of a second wave ofmechanisms controlling post-peak parasitemia, rather thanpart of the first line of defense” (34). As indicated above, thelevels of MCP-1 were severely suppressed in both WT andJH�/� mice infected with P. chabaudi adami and depleted of ��T cells by treatment with anti-GL3 (Fig. 2C). However, thetime course of parasitemia in CCR2�/� mice and heterozygouscontrols was nearly identical, and parasitemia again was similarin JH�/� mice, lacking the CCR2 receptor for MCP-1, and theircontrols. Taken together, these findings suggest little, if any,role for MCP-1, in immunity to P. chabaudi malaria.

Robinson et al. (26) suggest that �� T cells are an importantcell type for producing IFN-�. �� T cells contribute to theresolution of P. chabaudi parasitemia in immunologically in-tact animals and are essential for protective CMI. When �� Tcells were depleted from WT mice or JH�/� mice in the presentstudy, the serum levels of IFN-� were significantly depressed.We can speculate that early IFN-� produced by �� T cells isnecessary for the recruitment and activation of as-yet-uniden-tified effector cell populations that are responsible for killingblood-stage parasites or inhibiting their growth. On the otherhand, the immediate, marked and significant (P 0.05) in-crease in parasitemia seen after depleting �� T cells from JH�/�

mice chronically infected with P. chabaudi suggests a differentpossibility. Our observation of exacerbating parasitemia imme-diately after �� T-cell depletion in chronically infected micesuggests that at this stage of infection �� T cells are functioningdirectly as killer cells. Human �� T cells have been reported tokill blood-stage P. falciparum parasites in vitro through agranulysin-dependent mechanism (12, 13, 42). Previous studieshave shown that B220� �� T cells activated by CD4� T cellsallow chronically infected JH�/� mice to control their para-sitemia at low levels for extended periods, indicating that �� Tcells are continually required for control of parasitemia.Should future studies reveal the mechanisms of how these cellscontrol and/or suppress parasitemia, it may be possible toutilize these cells or their receptors for the immunotherapyof malaria. In addition, vaccines and/or immunomodulatorsaimed at activating �� T cells to proliferate and become effec-tor cells capable of killing blood-stage malaria parasites could

provide new approaches for the suppression of parasitemiaduring malaria.

ACKNOWLEDGMENTS

This study was supported by grants AI12710 (W.P.W.), AI49585(J.M.B.), and AI40667 (H.C.V.D.H.) from the National Institutes ofHealth.

We thank Carole Long and Jon Weidanz for critical discussion ofthe data. We also thank Katherine Schmiegel for technical assistancewith CCR2�/� mice.

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