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The Journal of Immunology, 1999, 163: 884-892.
Copyright © 1999 by The American Association of Immunologists

IL-12 and NK Cells Are Required for Antigen-Specific Adaptive Immunity Against Malaria Initiated by CD8+ T Cells in the Plasmodium yoelii Model1

Denise L. Doolan2,{dagger} and Stephen L. Hoffman*

* Malaria Program, Naval Medical Research Center, Bethesda, MD 20889; and {dagger} Pan American Health Organization, Regional Office of the World Health Organization, Washington, DC 20037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
CD8+ T cells have been implicated as critical effector cells in protection against preerythrocytic stage malaria, including the potent protective immunity of mice and humans induced by immunization with radiation-attenuated Plasmodium spp. sporozoites. This immunity is directed against the Plasmodium spp. parasite developing within the host hepatocyte and for a number of years has been presumed to be mediated directly by CD8+ CTL or indirectly by IFN-{gamma} released from CD8+ T cells. In this paper, in BALB/c mice, we establish that after immunization with irradiated sporozoites or DNA vaccines parasite-specific CD8+ T cells trigger a novel mechanism of adaptive immunity that is dependent on T cell- and non-T cell-derived cytokines, in particular IFN-{gamma} and IL-12, and requires NK cells but not CD4+ T cells. The absolute requirement for CD8+ T cells to initiate such an effector mechanism, and the requirement for IL-12 and NK cells in such vaccine-induced protective immunity, are unique and underscore the complexity of the immune responses that protect against malaria and other intracellular pathogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Malaria is the most important parasitic disease of humans, ranking with acute respiratory infections, diarrheal diseases, and tuberculosis as one of the major causes of mortality world wide. Recent estimates (1) attribute 950 new infections and 5 deaths every minute to malaria.

Sporozoites are the stage of the parasite’s life cycle infective to the host. Sterile protective immunity against Plasmodium spp. sporozoite challenge can be induced by immunization with radiation-attenuated sporozoites in multiple model systems and humans. This protection (reviewed in Refs. 2, 3) is effective against challenge with massive doses of infectious sporozoites, is not strain-specific, is not genetically restricted because it is efficacious in outbred and inbred mouse strains differing in genetic background as well as MHC-diverse humans, and persists for at least 9 mo in humans. An ideal vaccine against malaria would mimic the protective immunity induced by immunization with irradiated sporozoites. Such a vaccine would prevent the development of clinical symptoms and the transmission of malaria. However, the effector mechanisms of sporozoite-induced protection have not yet been fully elucidated. The study presented here was designed to address this issue.

When sporozoites are experimentally irradiated, they are able to invade hepatocytes but are unable to mature to the stage that infects erythrocytes (4, 5). The infected hepatocyte is considered the primary target of irradiated sporozoite-induced protection, immune responses against parasite-derived peptides expressed on the surface of the infected hepatocyte have been demonstrated, CD8+ T cells have been implicated as the principal effector cells, and IFN-{gamma}, and NO have been implicated as critical effector molecules (reviewed in Ref. 3). This has led to the hypothesis that CD8+ T cells induced by immunization recognize parasite-derived peptide-MHC complexes on the surface of infected hepatocytes and are activated to lyse the infected hepatocyte or release IFN-{gamma} that up-regulates NO production by the infected hepatocytes, leading to elimination of the infected hepatocyte (3, 6). In this paper we establish that CD8+ T cells play a critical role in triggering a novel mechanism of adaptive immunity which is absolutely dependent not only on IFN-{gamma} and NO, but also on IL-12 and in part on NK cells. We further demonstrate that, in BALB/c mice, parasite-specific CD8+ CTL are not sufficient, and that CD4+ T cells are not sufficient or required for irradiated sporozoite elicited protective immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice

Female 4- to 8-wk-old BALB/cByJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Outbred CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). Female 6- to 10-wk-old IFN-{gamma} gene knockout mice (IFN-{gamma} gko)3 mice with a targeted homozygous disruption of the IFN-{gamma} gene (7) were generated on the BALB/cByJ background and purchased from The Jackson Laboratory. Female and male 4- to 12-wk-old BALB/cByJ IL-12 p40 and p35 gene knockout (IL-12 gko) mice were generously supplied by Dr. Jeanne Magram (Hoffmann-LaRoche, Nutley, NJ). The generation of these mice has been described previously (8, 9). Age- and sex-matched controls were used in all experiments. Studies were approved by the Naval Medical Research Institute’s Animal Use Committee.

Parasites

Plasmodium yoelii (17X NL nonlethal strain, clone 1.1) was maintained by alternating passage of the parasites in Anopheles stephensi mosquitoes and CD-1 mice. For irradiated sporozoite immunizations, P. yoelii 17X NL-infected mosquitoes taken 14 days after an infectious blood meal were subjected to 10,000 rads of gamma radiation from a 137Cs source, and sporozoites were isolated using a modification of the technique described by Ozaki et al. (10). Briefly, infected mosquitoes were anesthetized at -20°C, washed in 80% ethanol, M199 (Quality Biologicals, Gaithersburg, MD) containing fungizone (50 µg/ml), M199 containing penicillin (100 U/ml), and streptomycin (100 µg/ml), and then placed on a glass slide. The thorax of each mosquito was cut immediately anterior to the wings, the proboscis was separated from the upper body, and both segments from groups of 50 mosquitoes were suspended in a volume of 50 µl M199 and centrifuged through a sterile column of glass wool in a microcentrifuge tube for 2 min at 10,000 rpm. Each column was rinsed with 50 µl M199. Sporozoite pellets were harvested after each centrifugation, and combined. Irradiated sporozoites were counted using a hemocytometer and diluted to a final concentration of 100,000 sporozoites per 0.2 ml volume in M199 containing penicillin (100 U/ml) and streptomycin (100 µg/ml) without serum.

For challenge of irradiated sporozoite-immunized mice, sporozoites were harvested from nonirradiated P. yoelii 17X NL-infected mosquitoes 14 days after an infectious blood meal using the modified Ozaki technique, and diluted to a final concentration of 5000 infectious sporozoites per 0.2 ml volume in M199 containing 5% FCS. For challenge of DNA-immunized mice, sporozoites were obtained 14 days after an infectious blood meal by hand-dissection of P. yoelii 17X NL-infected mosquito glands in M199 medium containing 5% FCS and diluted to a final concentration of 50 infectious sporozoites per 0.2 ml volume in M199 containing 5% FCS.

DNA plasmids

The DNA vaccines encoding the P. yoelii circumsporozoite protein (PyCSP) and P. yoelii hepatocyte erythrocyte protein 17 kDa (PyHEP17) genes have been described previously (6, 11, 12). Briefly, the full-length PyCSP or PyHEP17 genes were cloned into the VR1012 vector, with expression of the encoded gene being driven by a CMV immediate/early gene promoter.

Immunizations and challenges

For irradiated sporozoite immunizations, mice were immunized three times at 2-wk intervals i.v. in the tail vein with 100,000 irradiated sporozoites in a total volume of 200 µl M199 without serum. For DNA immunizations, mice were immunized three times at 3-wk intervals i.m. in each tibialis anterior muscle with 50 µg of each plasmid DNA construct in a total volume of 50 µl saline, or unmodified VR1012 plasmid. Two weeks after the third immunization, irradiated sporozoite-immunized mice were challenged with 5000 infectious sporozoites, and DNA-immunized mice were challenged with 50 infectious sporozoites by tail-vein injection. Different challenge doses were used for the different vaccines because the challenge dose was selected so as to ensure that all naive control mice were infected but that vaccine-induced sterile protection was not completely overwhelmed. Giemsa-stained thin blood films were examined on days 5–14 postchallenge, up to 50 oil-immersion fields being examined for parasites. Protection was defined as the complete absence of blood-stage parasitemia. Statistical analysis was performed using the {chi}2 test (uncorrected) or Fisher’s Exact test (two-tailed) (if the expected cell value was less than five) (Epi Info Version 6.04b, Centers for Disease Control, Atlanta, GA).

Antibodies

Purified control rat Ig was purchased from Rockland Company (Gilbertsville, PA). The anti-CD4+ mAb (mAb) GK1.5 (rat IgG2a) (13) was obtained from American Type Culture Collection (Manassas, VA) (TIB207). The anti-CD4+ mAb YTA3.1.2 (rat IgG2b) (14) was provided by Dr. H. Waldmann (Sir William Dunn School of Pathology, Oxford, UK.) The anti-CD8+ mAb 2.43 (mouse IgG2a) (15) was obtained from ATCC (TIB210). The anti-IFN-{gamma} mAb XMG-6 (rat IgG1) (16) was provided by Dr. F. Finkelman (University of Cincinnati Medical Center, Cincinnati, OH). The anti-IL-12 mAb C17.8 (rat IgG2a) (17) was kindly provided by Drs. M. Wysocka and G. Trinchieri (Wistar Institute, Philadelphia, PA). All Igs were purified from ascites (Harlam Bioproducts for Science, Indianapolis, IN) by 50% ammonium sulfate precipitation and final Ab concentrations determined by optical density. Anti-asialo GM1 antiserum (rabbit) was purchased from Wako Bioproducts (Richmond, VA).

In vivo depletions

In vivo depletion regimes were designed so as to ensure that the treatments were effective and reproducible (data not presented). Immunized mice were treated as follows.

Undepleted. On days -7, -6, -5, -4, -3, -2, and 0 (relative to challenge with P. yoelii sporozoites on day 0), mice received a single i.p. dose of 1.0 mg purified rat Ig.

CD4+ T cell depletion. On days -7, -6, -5, -4, -3, -2, 0, and +2, mice received a single i.p. dose of 1.0 mg of the anti-CD4+ mAb GK1.5.

CD8+ T cell depletion. On days -5, -4, -3, -2, and 0, mice received a single i.p. dose of 0.5 mg of the anti-CD8+ mAb 2.43.

IFN-{gamma} depletion. On days -3, -2, -1, and +2, mice received a single i.p. dose of 1.0 mg of the anti-IFN-{gamma} mAb XMG-6.

IL-12 depletion. At 12 h before and 3 h after challenge, mice received a single i.p. dose of 1.0 mg of the anti-IL-12 mAb C17.8.

NO depletion. Twice daily, commencing 24 h before sporozoite challenge and for 72 h postchallenge, mice were administered 50 mg aminoguanidine (Sigma, St. Louis, MO)/kg body weight in 0.5 ml PBS via gastric lavage.

NK cell depletion. On days -2, 0, +2, and +4, mice received a single i.v. dose of 200 µl of anti-asialo GM1 antiserum diluted 1:8 in 0.5x PBS (25 µl stock; ~675 µg purified Ab).

FACS analysis

The efficiency of anti-CD4+ and anti-CD8+ Ab depletion in vivo was determined by performing single-color fluorescent activated cell sorting using the FACScan (FAX 4000 Royal, Becton Dickinson Immunocytometry Systems, San Jose, CA). Spleen cells and/or PBMCs from Ab-treated and untreated mice were examined either at the time of challenge or when parasites were first detected in the peripheral blood. Approximately 1 x 106 cells of the population to be analyzed were stained with either anti-CD8+ FITC or anti-CD4+ FITC (PharMingen, San Diego, CA) for 1 h at 4°C. Unstained and FITC controls were included for each sample. Stained cells were washed three times, resuspended in paraformaldehyde (0.5%, v/v) and stored at 4°C until analyzed.

CTL analysis

Spleen cells from immunized mice, harvested after the third immunization, were incubated at a concentration of 5 x 106 cells in 2 ml of RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 2 mM L-glutamine, 50 µM 2-ME, 50 U/ml penicillin, and 50 U/ml streptomycin (cRPMI) in a 24-well plate in the presence of 2.5 µM of the 16-mer PyCSP synthetic peptide (residues 280–296) containing the previously defined 9-mer CTL epitope (residues 280–288, SYVPSAEQI) (18). Rat T-stim (2.5% v/v) (Collaborative Biomedical Products, Bedford, MD) was added at 48 h as a source of IL-2. At 7 days, these effector cells were used in a conventional 6-h chromium release assay. Target cells were MHC-matched (H-2d) P815 mastocytoma cells (ATCC TIB 64) pulsed overnight with the 9-mer CTL epitope or no peptide and labeled with 100 Ci 51Cr (sodium chromate solution). Percent lysis was determined as: [(experimental release - medium control release)/(maximum release - medium control)] x 100.

Cytokine analysis

Pooled sera from experimental mice were assayed for circulating murine IFN-{gamma} using a commercially available kit (Intertest-{gamma}; Genzyme, Cambridge, MA), as described by the manufacturer. Concentrations were calculated by interpolation from standard curves based on recombinant cytokine dilutions run in parallel on the same plate.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
CD8+ T cells are required for P. yoelii sporozoite-induced protective immunity

Initially, we confirmed that immunization of BALB/c mice with radiation-attenuated P. yoelii sporozoites conferred solid protective immunity against challenge with 5000 infectious P. yoelii sporozoites, as assessed by the complete absence of blood-stage parasitemia (Table IGo). We further established that in vivo depletion of CD8+ T cells completely eliminated protective immunity (Table IGo). Depletion of CD4+ T cells had no effect. These data emphasize the critical role of CD8+ T cells, but not CD4+ T cells, in the effector arm of preerythrocytic stage protection in BALB/c mice, as reported previously (19).


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  		Table I. Sporozoite-induced protective immunity of BALB/c mice is dependent on CD8+ T cells and IFN-{gamma}, but not CD4+, T cells

 
IFN-{gamma} is required for sporozoite-induced protective immunity

In previous studies in the Plasmodium berghei model, sporozoite-induced protective immunity of A/J (20) and BALB/c mice (21) was abrogated by in vivo depletion of IFN-{gamma}. However, one study (22) in the P. yoelii model reported that IFN-{gamma} receptor knockout (IFN-{gamma}R-/-) mice on a (C57BL/6 x 129) background failed to develop protective immunity after a single immunization with irradiated sporozoites but were protected after multiple immunizations. We elected to further study the role of IFN-{gamma} in the P. yoelii BALB/c model. Accordingly, sporozoite-immune BALB/c mice were depleted in vivo of IFN-{gamma} using the anti-IFN-{gamma} mAb XMG-6. Results (Table IGo) showed that IFN-{gamma} was absolutely required for the protective immunity induced by immunization with irradiated P. yoelii sporozoites.

The critical role for IFN-{gamma} was confirmed using IFN-{gamma} gko on a BALB/c background. These mice have a targeted disruption of the IFN-{gamma} gene and are therefore unable to mount an IFN-{gamma} response (7). In these experiments, wild-type mice and IFN-{gamma} gko mice were immunized with irradiated sporozoites and challenged with infectious sporozoites in parallel. Before challenge, wild-type mice were treated with a mAb against IFN-{gamma} or a control Ab, or left untreated. Wild-type mice (untreated and control) were solidly protected against challenge with 5000 sporozoites, whereas wild-type mice depleted of IFN-{gamma} and IFN-{gamma} gko mice were not protected (Table IIGo). Therefore, in BALB/c mice immunized with P. yoelii sporozoites, as with BALB/c and other inbred strains immunized with P. berghei sporozoites, CD8+ T cells are critical effector cells and IFN-{gamma} is a critical mediator of sporozoite-induced protective immunity.


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  		Table II. Irradiated sporozoite induced immunity in IFN-{gamma} gko mice1

 
CD4+ T cell secreted IFN-{gamma} is not required for protection against sporozoite challenge

The absolute requirement for both CD8+ T cells and IFN-{gamma} in the effector mechanism of sporozoite-induced protective immunity dictates that the IFN-{gamma} is secreted by CD8+ T cells, and that the interaction of CD8+ T cells with the peptide-MHC complex on the surface of the infected hepatocyte (18, 23) is critical. Data here establish that CD4+ cells are not adequate or required for the effector arm of protective immunity, at least in BALB/c mice (Table IGo). However, IFN-{gamma} can be produced by CD4+ and CD8+ T cells as well as by NK cells in response to bacterial or parasitic infection (24). Furthermore, it is known that class II as well as class I MHC molecules are expressed on the surface of virally infected human hepatocytes (25, 26), that both CD4+ T cells and CD8+ T cells can recognize parasite-derived peptides presented on the surface of Plasmodium spp. infected murine hepatocytes in association with class II (27, 28) or class I (18, 23) MHC molecules, respectively, and that CD4+ T cells can be effective against Plasmodium spp. as demonstrated by active immunization and adoptive transfer experiments (28, 29). Following P. yoelii infection, a lymphoid population composed primarily of CD4+ T cells predominates in the extravascular hepatic compartment, and the absolute numbers of CD4+ T cells have been reported to be ~5- and 2.5-fold greater than those of CD8+ T cells at 30 h and 40 h postinfection, respectively (30). Even in normal mouse liver, a CD4+/CD8+ ratio of 2.8 has been reported (31). Thus, despite the fact that murine hepatocytes can present P. yoelii-derived, class II restricted peptides, and can be eliminated by CD4+ T cells that recognize P. yoelii-derived peptides complexed with class II MHC molecules on the surface of infected hepatocytes, it appears that immunization of BALB/c mice with irradiated P. yoelii sporozoites does not adequately induce this type of protective immune response.

Parasite-specific CTL are not sufficient for protection against sporozoite challenge

Because CD8+ CTL have for many years been considered critical effectors of preerythrocytic stage protection, it was of interest to determine whether a parasite-specific CTL response could be induced in the nonprotected IFN-{gamma} gko mice. Data presented in Fig. 1Go showed that high levels of CTL activity were detected despite the absence of IFN-{gamma}. It has been proposed that IFN-{gamma} is required for the generation and maturation of CTL (32) and it is known that several essential steps of the Ag-processing pathway are regulated by IFN-{gamma} (33, 34). However, our data demonstrate that IFN-{gamma} is not required for the induction of Plasmodium parasite-specific CTL. Consistent with this, normal CTL responses in the absence of IFN-{gamma} have been reported elsewhere (7, 35, 36).



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  		FIGURE 1. Parasite-specific CTL are induced in IFN-{gamma} gko mice despite the absence of IFN-{gamma}. Spleen cells from immunized IFN-{gamma} gko mice (A) or wild-type mice (B) were assayed for peptide-specific CTL against the immunodominant CTL epitope on the PyCSP (residues 280–288, SYVPSAEQI) (18 ) using a conventional chromium release assay at different E:T ratios. Percent lysis was determined as: [(experimental release - medium control release)/(maximum release - medium control release)] x 100.

 
Because IFN-{gamma} gko mice were not protected against sporozoite challenge despite the presence of high levels of CTL, our data indicate that in BALB/c mice parasite-specific CTL are not sufficient for protection. That CTL are not required for protective immunity cannot be excluded until studies of perforin knockout and Fas ligand-deficient mice (not yet available on the BALB/c background) are conducted. However, preliminary studies by ourselves (our manuscript in preparation) and others (37) indicate that sporozoite-induced protective immunity in the C57BL/6 rodent model is independent of both perforin and Fas.

Nevertheless, it is probable that some of the CD8+ T cells activated by infection may be cytotoxic; the presence of CTL specific for every Plasmodium falciparum parasite protein known to be expressed in the infected hepatocyte has been demonstrated in both naturally exposed and experimentally immunized humans (reviewed in Refs. 3, 38). The nature of the interaction dictates a tripartite complex be formed between the TCR on the CD8+ T cell and the peptide presented in the context of the MHC class I molecule expressed on the surface of the infected hepatocyte. By definition, all CD8+ CTL must express the CD3+CD8+CD4- cell surface markers. However, not all CD8+ T cells must be cytotoxic. In the BALB/c-P. yoelii system, we have established that CD8+ T cells per se are required, but that the cytotoxic function of CD8+ T cells is not sufficient.

NO is required for sporozoite-induced protective immunity

In vitro, IFN-{gamma} induces P. berghei (39) or P. yoelii (40) infected murine hepatocytes, and P. falciparum infected human hepatocytes (40) to produce NO. In vivo, IFN-{gamma} contributes to NO synthase (NOS) production by hepatocytes as well as other cells following infection with P. yoelii or P. berghei (41, 42) or other parasites (41, 43). Inducible NOS (iNOS) is considered a major mediator of cytotoxicity against intracellular parasites (reviewed in Ref. 44). Previously, Seguin et al. (21) demonstrated that protection in mice immunized with irradiated P. berghei sporozoites was dependent upon the inducible, but not constitutive, NO pathway, and that induction of iNOS in the liver was dependent on CD8+ T cells and IFN-{gamma}. To establish whether NO is important for sporozoite-induced protection in the P. yoelii model, sporozoite-immune mice were treated before challenge with aminoguanidine, a specific substrate inhibitor of iNOS. Protection was completely eliminated (Table IIIGo). Therefore, IFN-{gamma} induction of the L-arginine-dependent NO pathway in vivo, and subsequent elimination of infected hepatocytes or hepatic schizonts within those cells, is a necessary component for CD8+ T cell dependent protection in sporozoite-immunized mice.


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  		Table III. Sporozoite-induced protective immunity of BALB/c mice is dependent on NO and IL-12 and partially dependent on NK cells1

 
IL-12 is required for P. yoelii sporozoite-induced protective immunity

Data presented above and by others establish an essential role for CD8+ T cell secreted IFN-{gamma} in protection against sporozoite challenge. However, according to the proposed scenario, essentially every infected hepatocyte would have to be contacted by an Ag-specific CD8+ T cell. This hypothesis is supported by data indicating that for CD8+ CTL clones against the PyCSP to be protective in adoptive transfer, they must directly contact the infected hepatocyte (45). However, it has been established that infection with as few as one or two sporozoites of P. yoelii 17X NL will result in patent infection of 50% of BALB/c mice (ID50). Protective immunity in our studies is defined as the complete absence of blood-stage parasitemia. The sporozoite challenge dose used here was 5000 infectious sporozoites. In other studies (data not presented), we have demonstrated protection against challenge with as many as 100,000 infectious sporozoites. Therefore, it would be predicted that at least 2500 hepatocytes would be infected following challenge, and sterile immunity would dictate that every one of these 2500 infected hepatocytes must be contacted by a T cell. We reasoned that this scenario was not the most efficient mechanism for explaining the potent protection elicited by the irradiated sporozoite vaccine. This led us to speculate on the role of other molecules produced by non-T cells in the protective immunity found after immunization with irradiated sporozoites.

Other studies have demonstrated that systemic administration of recombinant IL-12 completely, in the absence of parasite Ag, protects against sporozoite challenge with P. yoelii in BALB/c mice (46) and Plasmodium cynomolgi in rhesus monkeys (47). IL-12 is a pleiotropic cytokine secreted by a wide variety of cells including dendritic cells, macrophages, and monocytes, and is thought to provide a functional bridge between innate resistance and Ag-specific adaptive immunity (reviewed in Refs. 48, 49). Receptors for the IL-12 heterodimer are found on activated CD4+ T cells, activated CD8+ T cells and NK cells, and IL-12 are known to regulate and promote Th1 type immune responses and enhance IFN-{gamma} production by T cells and NK cells (48). Accordingly, we speculated that IL-12 may play a role in the CD8+ T cell mediated IFN-{gamma} dependent protection of BALB/c mice induced by immunization with irradiated sporozoites. Consistent with this, we demonstrated that in vivo depletion of IL-12 completely abrogated sporozoite-induced protective immunity (Table IIIGo). This is the first demonstration for a role of IL-12 in Ag-specific adaptive immunity against preerythrocytic Plasmodium spp.

The absolute requirement for IL-12 was further confirmed by studying sporozoite-immunized IL-12 gko mice. IL-12 is a heterodimeric cytokine composed of two disulfide-linked subunits, p40 and p35, both of which are required for biological activity. Neither IL-12 p35 gko nor p40 gko mice are able to produce biologically active IL-12 (p70), but p35 gko mice express p40 at levels indistinguishable from wild-type mice. Here, we studied both IL-12 p35 gko and p40 gko mice generated on the BALB/c background. In our system, sporozoite-immunized p40 gko mice were not protected against challenge (Table IVGo). However, in two separate experiments, there was some suggestion of protection in the p35 gko mice (Table IVGo). Similar results were noted with p35 gko and p40 gko mice generated on the C57BL/6 background (data not shown). Because both p35 and p40 subunits are required for biologically active IL-12, these data are initially confusing, particularly since it has been reported that high affinity binding of p40 homodimers to the IL-12 receptor blocks the activity of biologically active IL-12, antagonizing the immune response and inhibiting CD4+ T cell function in vitro and in vivo (50). However, it has been reported recently (51, 52) that p40 homodimers markedly enhance rather than decrease CD8+ Th1 development and IFN-{gamma} production by CD8+ T cells. The results here in the P. yoelii-BALB/c model confirm that IL-12 is a critical component of the CD8+ T cell and IFN-{gamma}-dependent protective immunity induced by immunization with irradiated sporozoites. In addition, data are consistent with the proposal that p40 homodimers enhance IFN-{gamma} production by CD8+ T cells.


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  		Table IV. Irradiated sporozoite induced immunity in IL-12 gko mice

 
It has been demonstrated previously that IL-12 p35 gko and p40 gko mice can produce IFN-{gamma} and generate normal Th1 immune responses, albeit at reduced levels compared with wild-type mice (8). Consistent with this, circulating IFN-{gamma} was detected following sporozoite challenge of immunized IL-12 p35 gko and p40 gko mice (Fig. 2GoA). The magnitude of IFN-{gamma} was enhanced relative to that of nonimmunized infectivity controls that developed patent parasitemia, but reduced compared with that of immune wild-type mice (Fig. 2GoA). These data suggest that IL-12 is required for optimal production of IFN-{gamma}, in our system. Furthermore, because IL-12 gko mice were not protected despite the presence of IFN-{gamma}, data indicate that the IFN-{gamma} secreted by activated CD8+ T cells is not sufficient for protection and that IL-12 is required for the induction of protective levels of IFN-{gamma}. Accordingly, we propose that IFN-{gamma} precedes and initiates production of IL-12 and that this IL-12 in turn induces IFN-{gamma} in a positive feedback loop that represents an important amplifying mechanism.



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  		FIGURE 2. A, Circulating IFN-{gamma} is detected in sporozoite-immunized wild-type and IL-12 gko mice. Pooled sera from groups of immune wild-type mice, IL-12 p35 gko mice, and IL-12 p40 gko mice collected 3–4 days postchallenge were assayed for levels of circulating IFN-{gamma} by ELISA and compared with levels in naive wild-type mice that developed patent blood stage parasitemia. B, Circulating IFN-{gamma} in DNA-immunized wild-type mice is dependent on IL-12. Wild-type mice were immunized with PyCSP DNA (PyCSP DNA) or vector control DNA (VR1012) and then depleted of IL-12 in vivo by administration of an anti-IL-12 mAb (anti-IL-12) or not depleted (control Ab). Naive mice were not immunized and developed patent blood stage parasitemia. Pooled sera from each group collected 3–4 days postchallenge were assayed for levels of circulating IFN-{gamma}.

 
Our data establish a critical role for IL-12 in the Ag-specific adaptive immune response to malaria. Other data from our laboratory (46, 47) are consistent with a role for IL-12 in the innate immune response to sporozoite challenge. It is likely that IL-12 may be important in both innate and adaptive immunity, and that the innate response may influence the development of Ag-specific immunity, because any IL-12 produced as a result of the innate immune response to sporozoite challenge would augment the IFN-{gamma} produced by both CD8+ T cells and NK cells. The precise dissection of this is beyond the scope of our report.

NK cells are required for sporozoite-induced protective immunity

It is well established that IL-12 acts on both T cells and NK cells (48, 49), and that NK cells are a major producer of IFN-{gamma} (53). In particular, in parasitic and bacterial models, many reports demonstrate that IL-12 is essential for T cell and NK cell production of IFN-{gamma} and protective immunity (54, 55, 56, 57, 58, 59, 60). To determine whether NK cells were involved in the protective immunity induced by immunization with Plasmodium spp. parasites, we treated sporozoite-immune mice with anti-asialo GM1 antiserum before and during sporozoite challenge. This treatment was able to partially abrogate the protective immunity (Table VGo), providing the first demonstration of a critical role for NK cells in protective immunity against preerythrocytic stage malaria.


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  		Table V. DNA-induced protective immunity of BALB/c mice is dependent on IFN-{gamma}, NO, IL-12, and NK cells1

 
NK cells have been established as a crucial first line of defense against pathogens because they can exert their activity without prior sensitization by Ag (reviewed in Refs. 53, 60). Data presented here demonstrate that NK cells also play a crucial role in the Ag-specific adaptive immune response to Plasmodium spp. infection. Accordingly, we invoke a unique role for NK cells in Ag-specific adaptive immunity initiated by parasite-specific CD8+ T cells, which is distinct from the role of NK cells in innate immunity.

IL-12 and NK cells are required for DNA vaccine-induced protective immunity

The involvement of CD8+ T cells, IFN-{gamma}, IL-12, and NK cells in protective immunity against P. yoelii sporozoites was further verified by investigating the mechanism of protection induced by immunization with plasmid DNA. Previously, we have reported (6) that the protection in B10.BR mice induced by immunization with plasmid DNA encoding either PyHEP17 or a mixture of PyHEP17 + PyCSP was absolutely dependent on CD8+ T cells, IFN-{gamma} and NO. In that strain, immunization with PyCSP DNA alone does not confer protection (6). Here, we extended those studies to demonstrate the same dependence in the BALB/c strain. To determine whether IL-12 and NK cells were also involved, BALB/c mice were immunized with either PyCSP DNA, or PyCSP + PyHEP17 DNA, and depleted of IL-12 or NK cells. Results (Table VGo) show that the protective immunity induced by immunization with plasmid DNA was absolutely dependent on both IL-12 and NK cells, as demonstrated with irradiated sporozoite immunity. Treatment of DNA-immunized mice with anti-IL-12 Ab was found to completely eliminate circulating IFN-{gamma} (Fig. 2GoB). In other studies, with other immunogens, in vivo depletion of IL-12 similarly resulted in a decrease or abrogation in circulating IFN-{gamma} (data not shown). Therefore, these data implicate IL-12 as critical for the optimal induction of IFN-{gamma} following immunization with either irradiated sporozoites or plasmid DNA.

A novel mechanism of adaptive immunity against malaria

We have previously proposed (3, 6) that in DNA-immunized mice CD8+ T cells activated by interacting with the peptide-MHC complex on the surface of the infected hepatocyte secrete IFN-{gamma} that induces the infected hepatocyte to produce NO, eliminating the infected hepatocyte or intracellular parasite. In this paper, we provide the first demonstration of a role for NK cells and for IL-12 in adaptive immunity against Plasmodium spp. sporozoites. We establish that protection induced in BALB/c mice by immunization with irradiated sporozoites or plasmid DNA is mediated by NK cells as well as by CD8+ T cells, and is absolutely dependent on IFN-{gamma}, IL-12 and NO. To date there is no evidence for Ab-mediated immune mechanisms during the hepatic stage of the Plasmodium spp. parasite’s life cycle, although it has been speculated that Abs may kill the intrahepatic parasite, either directly, with complement, or via Ab-dependent cellular cytotoxicity (ADCC). However, we acknowledge the possibility that ADCC may play a role in preerythrocytic stage immunity, and that our data do not exclude such a role. Nevertheless, we believe that this has no bearing on the significance of our findings establishing a role for IL-12 and NK cells in protective immunity against sporozoite challenge.

Accordingly, in this report, we advance a novel feedback loop of adaptive immunity (Fig. 3Go). We propose that parasite-specific CD8+ T cells provide the critical initial trigger for the protective effector mechanism, via recognition of specific peptide-MHC complexes on the surface of the infected hepatocyte. Further, we propose that induction of IFN-{gamma} is a direct consequence of the CD8+ T cell activation, that IFN-{gamma} production precedes and initiates production of IL-12, and that the IL-12 in turn induces IFN-{gamma} production by NK cells (and perhaps other cells) in a positive feedback loop that represents an important amplifying mechanism. The IFN-{gamma}, via signal transducers associated with transcription, then activates NOS and induces the L-arginine-dependent NO pathway, subsequently eliminating the infected hepatocytes or the hepatic schizonts within those cells. In BALB/c mice, CD4+ T cells are not required or sufficient for the initial triggering of the effector mechanism, nor for the feedback induction of IFN-{gamma} (Fig. 3Go).



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  		FIGURE 3. Schematic of proposed mechanism of protective immunity induced by immunization of BALB/c mice with irradiated sporozoites or plasmid DNA.

 
In other parasitic infections, in contrast, it has been established that the induction of IFN-{gamma} synthesis by NK cells following infection is indirect, involving a secondary effect of IL-12. In the Toxoplasma gondii system, for example, enhanced IL-12 synthesis precedes IFN-{gamma} production, and the induction of IL-12 and other innate immune responses does not require IFN-{gamma} (61).

In this paper, we provide, to the best of our knowledge, the first demonstration of a feedback mechanism induced by parasite challenge of actively immunized animals. Furthermore, our proposal that NK cells constitute part of an Ag-specific adaptive immune response initiated by CD8+ T cells and dependent on IL-12 represents a novel mechanism of protective immunity against intracellular, bacterial and viral pathogens.


   Acknowledgments
 
We thank Mr. Arnel Belmonte and Mr. Romeo Wallace for providing the P. yoelii sporozoites, Mr. Salvador Doria and Ms. Tonette Bangura for excellent technical assistance, Dr. Jeanne Magram (Hoffmann-LaRoche) for providing the IL-12 gko mice, Drs. Maria Wysocka and Giorgio Trinchieri (Wistar Institute) for providing the anti-IL-12 mAb, Drs. Alan Sher and John Sacci for advice and critical discussion, and Dr. Eileen Villasante for artistic assistance.


   Footnotes
 
1 This work was supported by the Naval Medical Research and Development Command work unit STO F 6.1 61102AA0100BFX and STO F 6.2 62787A00101EFX. The experiments reported herein were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals", Institute of Laboratory Animal Resources, National Research Council, National Academy of Sciences Press, 1996. This work was supported by the Naval Medical Research and Development Command work units 61102AA0100BFX and 62787A00101EFX. The opinions and assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Navy or the naval service at large. This work was performed in part while D.L.D. held a National Research Council Associateship. Back

2 Address correspondence and reprint requests to Dr. Denise L. Doolan, Malaria Program, Naval Medical Research Center, 12300 Washington Avenue, Rockville, MD 20852. E-mail address: Back

3 Abbreviations used in this paper: gko, gene knockout; PyCSP, P. yoelii circumsporozoite protein; PyHEP17, P. yoelii 17-kDa hepatocyte erythrocyte protein; iNOS, inducible NO synthase. Back

Received for publication January 5, 1999. Accepted for publication May 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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