IL-12 and NK Cells Are Required for Antigen-Specific Adaptive Immunity Against Initiated by CD8+ T Cells in the yoelii Model This information is current as of September 28, 2021. Denise L. Doolan and Stephen L. Hoffman J Immunol 1999; 163:884-892; ; http://www.jimmunol.org/content/163/2/884 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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† and Stephen L. Hoffman*

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-␥ 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-␥ and Downloaded from 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. The Journal of Immu- nology, 1999, 163: 884–892.

alaria is the most important parasitic disease of hu- been fully elucidated. The study presented here was designed to http://www.jimmunol.org/ mans, ranking with acute respiratory infections, diar- address this issue. M rheal diseases, and tuberculosis as one of the major When sporozoites are experimentally irradiated, they are able to causes of mortality world wide. Recent estimates (1) attribute 950 invade hepatocytes but are unable to mature to the stage that in- new infections and 5 deaths every minute to malaria. fects erythrocytes (4, 5). The infected hepatocyte is considered the Sporozoites are the stage of the parasite’s life cycle infective to primary target of irradiated sporozoite-induced protection, immune the host. Sterile protective immunity against Plasmodium spp. responses against parasite-derived peptides expressed on the sur- sporozoite challenge can be induced by immunization with radia- face of the infected hepatocyte have been demonstrated, CD8ϩ T tion-attenuated sporozoites in multiple model systems and humans. cells have been implicated as the principal effector cells, and

This protection (reviewed in Refs. 2 and 3) is effective against IFN-␥, and NO have been implicated as critical effector molecules by guest on September 28, 2021 challenge with massive doses of infectious sporozoites, is not (reviewed in Ref. 3). This has led to the hypothesis that CD8ϩ T strain-specific, is not genetically restricted because it is efficacious cells induced by immunization recognize parasite-derived peptide- in outbred and inbred mouse strains differing in genetic back- MHC complexes on the surface of infected hepatocytes and are ground as well as MHC-diverse humans, and persists for at least 9 activated to lyse the infected hepatocyte or release IFN-␥ that up- mo in humans. An ideal vaccine against malaria would mimic the regulates NO production by the infected hepatocytes, leading to protective immunity induced by immunization with irradiated elimination of the infected hepatocyte (3, 6). In this paper we es- sporozoites. Such a vaccine would prevent the development of tablish that CD8ϩ T cells play a critical role in triggering a novel clinical symptoms and the transmission of malaria. However, the mechanism of adaptive immunity which is absolutely dependent effector mechanisms of sporozoite-induced protection have not yet not only on IFN-␥ and NO, but also on IL-12 and in part on NK cells. We further demonstrate that, in BALB/c mice, parasite-spe- cific CD8ϩ CTL are not sufficient, and that CD4ϩ T cells are not sufficient or required for irradiated sporozoite elicited protective *Malaria Program, Naval Medical Research Center, Bethesda, MD 20889; and †Pan American Health Organization, Regional Office of the World Health Organization, immunity. Washington, DC 20037 Received for publication January 5, 1999. Accepted for publication May 5, 1999. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance Mice with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Naval Medical Research and Development Com- Female 4- to 8-wk-old BALB/cByJ mice were obtained from The Jackson mand work unit STO F 6.1 61102AA0100BFX and STO F 6.2 62787A00101EFX. Laboratory (Bar Harbor, ME). Outbred CD-1 mice were obtained from The experiments reported herein were conducted according to the principles set forth Charles River Laboratories (Wilmington, MA). Female 6- to 10-wk-old in the “Guide for the Care and Use of Laboratory Animals”, Institute of Laboratory IFN-␥ gene knockout mice (IFN-␥ gko)3 mice with a targeted homozygous Animal Resources, National Research Council, National Academy of Sciences Press, ␥ 1996. This work was supported by the Naval Medical Research and Development disruption of the IFN- gene (7) were generated on the BALB/cByJ back- Command work units 61102AA0100BFX and 62787A00101EFX. The opinions and ground and purchased from The Jackson Laboratory. Female and male 4- assertions herein are the private ones of the authors and are not to be construed as to 12-wk-old BALB/cByJ IL-12 p40 and p35 gene knockout (IL-12 gko) 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. 2 Address correspondence and reprint requests to Dr. Denise L. Doolan, Malaria Pro- 3 Abbreviations used in this paper: gko, gene knockout; PyCSP, P. yoelii circum- gram, Naval Medical Research Center, 12300 Washington Avenue, Rockville, MD sporozoite protein; PyHEP17, P. yoelii 17-kDa hepatocyte erythrocyte protein; iNOS, 20852. E-mail address: [email protected] inducible NO synthase.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 885 mice were generously supplied by Dr. Jeanne Magram (Hoffmann-La- OH). The anti-IL-12 mAb C17.8 (rat IgG2a) (17) was kindly provided by Roche, Nutley, NJ). The generation of these mice has been described pre- Drs. M. Wysocka and G. Trinchieri (Wistar Institute, Philadelphia, PA). viously (8, 9). Age- and sex-matched controls were used in all experiments. All Igs were purified from ascites (Harlam Bioproducts for Science, Indi- Studies were approved by the Naval Medical Research Institute’s Animal anapolis, IN) by 50% ammonium sulfate precipitation and final Ab con- Use Committee. centrations determined by optical density. Anti-asialo GM1 antiserum (rab- bit) was purchased from Wako Bioproducts (Richmond, VA). Parasites In vivo depletions Plasmodium yoelii (17X NL nonlethal strain, clone 1.1) was maintained by alternating passage of the parasites in stephensi mosquitoes and In vivo depletion regimes were designed so as to ensure that the treatments CD-1 mice. For irradiated sporozoite immunizations, P. yoelii 17X NL- were effective and reproducible (data not presented). Immunized mice were infected mosquitoes taken 14 days after an infectious blood meal were treated as follows. subjected to 10,000 rads of gamma radiation from a 137Cs source, and Undepleted. On days Ϫ7, Ϫ6, Ϫ5, Ϫ4, Ϫ3, Ϫ2, and 0 (relative to chal- sporozoites were isolated using a modification of the technique described lenge with P. yoelii sporozoites on day 0), mice received a single i.p. dose by Ozaki et al. (10). Briefly, infected mosquitoes were anesthetized at of 1.0 mg purified rat Ig. ,Ϫ20°C, washed in 80% ethanol, M199 (Quality Biologicals, Gaithersburg, CD4؉ T cell depletion. On days Ϫ7, Ϫ6, Ϫ5, Ϫ4, Ϫ3, Ϫ2, 0, and ϩ2 MD) containing fungizone (50 ␮g/ml), M199 containing penicillin (100 mice received a single i.p. dose of 1.0 mg of the anti-CD4ϩ mAb GK1.5. U/ml), and streptomycin (100 ␮g/ml), and then placed on a glass slide. The CD8؉ T cell depletion. On days Ϫ5, Ϫ4, Ϫ3, Ϫ2, and 0, mice received thorax of each mosquito was cut immediately anterior to the wings, the a single i.p. dose of 0.5 mg of the anti-CD8ϩ mAb 2.43. proboscis was separated from the upper body, and both segments from IFN-␥ depletion. On days Ϫ3, Ϫ2, Ϫ1, and ϩ2, mice received a single groups of 50 mosquitoes were suspended in a volume of 50 ␮l M199 and i.p. dose of 1.0 mg of the anti-IFN-␥ mAb XMG-6. centrifuged through a sterile column of glass wool in a microcentrifuge IL-12 depletion. At 12 h before and 3 h after challenge, mice received a tube for 2 min at 10,000 rpm. Each column was rinsed with 50 ␮l M199. single i.p. dose of 1.0 mg of the anti-IL-12 mAb C17.8. Downloaded from Sporozoite pellets were harvested after each centrifugation, and combined. NO depletion. Twice daily, commencing 24 h before sporozoite challenge Irradiated sporozoites were counted using a hemocytometer and diluted to and for 72 h postchallenge, mice were administered 50 mg aminoguanidine a final concentration of 100,000 sporozoites per 0.2 ml volume in M199 (Sigma, St. Louis, MO)/kg body weight in 0.5 ml PBS via gastric lavage. containing penicillin (100 U/ml) and streptomycin (100 ␮g/ml) without NK cell depletion. On days Ϫ2, 0, ϩ2, and ϩ4, mice received a single i.v. serum. dose of 200 ␮l of anti-asialo GM1 antiserum diluted 1:8 in 0.5ϫ PBS (25 For challenge of irradiated sporozoite-immunized mice, sporozoites ␮l stock; ϳ675 ␮g purified Ab). were harvested from nonirradiated P. yoelii 17X NL-infected mosquitoes 14 days after an infectious blood meal using the modified Ozaki technique, FACS analysis http://www.jimmunol.org/ and diluted to a final concentration of 5000 infectious sporozoites per 0.2 ϩ ϩ ml volume in M199 containing 5% FCS. For challenge of DNA-immu- The efficiency of anti-CD4 and anti-CD8 Ab depletion in vivo was nized mice, sporozoites were obtained 14 days after an infectious blood determined by performing single-color fluorescent activated cell sorting meal by hand-dissection of P. yoelii 17X NL-infected mosquito glands in using the FACScan (FAX 4000 Royal, Becton Dickinson Immunocytom- M199 medium containing 5% FCS and diluted to a final concentration of etry Systems, San Jose, CA). Spleen cells and/or PBMCs from Ab-treated 50 infectious sporozoites per 0.2 ml volume in M199 containing 5% FCS. and untreated mice were examined either at the time of challenge or when parasites were first detected in the peripheral blood. Approximately 1 ϫ DNA plasmids 106 cells of the population to be analyzed were stained with either anti- CD8ϩ FITC or anti-CD4ϩ FITC (PharMingen, San Diego, CA) for1hat

The DNA vaccines encoding the P. yoelii circumsporozoite protein 4°C. Unstained and FITC controls were included for each sample. Stained by guest on September 28, 2021 (PyCSP) and P. yoelii hepatocyte erythrocyte protein 17 kDa (PyHEP17) cells were washed three times, resuspended in paraformaldehyde (0.5%, genes have been described previously (6, 11, 12). Briefly, the full-length v/v) and stored at 4°C until analyzed. PyCSP or PyHEP17 genes were cloned into the VR1012 vector, with ex- CTL analysis pression of the encoded gene being driven by a CMV immediate/early gene promoter. Spleen cells from immunized mice, harvested after the third immunization, Immunizations and challenges were incubated at a concentration of 5 ϫ 106 cells in 2 ml of RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 2 For irradiated sporozoite immunizations, mice were immunized three times mM L-glutamine, 50 ␮M 2-ME, 50 U/ml penicillin, and 50 U/ml strepto- at 2-wk intervals i.v. in the tail vein with 100,000 irradiated sporozoites in mycin (cRPMI) in a 24-well plate in the presence of 2.5 ␮M of the 16-mer a total volume of 200 ␮l M199 without serum. For DNA immunizations, PyCSP synthetic peptide (residues 280–296) containing the previously de- mice were immunized three times at 3-wk intervals i.m. in each tibialis fined 9-mer CTL epitope (residues 280–288, SYVPSAEQI) (18). Rat T- anterior muscle with 50 ␮g of each plasmid DNA construct in a total stim (2.5% v/v) (Collaborative Biomedical Products, Bedford, MD) was volume of 50 ␮l saline, or unmodified VR1012 plasmid. Two weeks after added at 48 h as a source of IL-2. At 7 days, these effector cells were used the third immunization, irradiated sporozoite-immunized mice were chal- in a conventional 6-h chromium release assay. Target cells were MHC- lenged with 5000 infectious sporozoites, and DNA-immunized mice were matched (H-2d) P815 mastocytoma cells (ATCC TIB 64) pulsed overnight challenged with 50 infectious sporozoites by tail-vein injection. Different with the 9-mer CTL epitope or no peptide and labeled with 100 Ci 51Cr challenge doses were used for the different vaccines because the challenge (sodium chromate solution). Percent lysis was determined as: [(experimen- dose was selected so as to ensure that all naive control mice were infected tal release Ϫ medium control release)/(maximum release Ϫ medium con- but that vaccine-induced sterile protection was not completely over- trol)] ϫ 100. whelmed. Giemsa-stained thin blood films were examined on days 5–14 Cytokine analysis 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 ␹2 test (uncorrected) or Fish- Pooled sera from experimental mice were assayed for circulating murine er’s Exact test (two-tailed) (if the expected cell value was less than five) IFN-␥ using a commercially available kit (Intertest-␥; Genzyme, Cam- (Epi Info Version 6.04b, Centers for Disease Control, Atlanta, GA). bridge, MA), as described by the manufacturer. Concentrations were cal- culated by interpolation from standard curves based on recombinant cyto- Antibodies kine dilutions run in parallel on the same plate.

Purified control rat Ig was purchased from Rockland Company (Gilberts- Results and Discussion ϩ ville, PA). The anti-CD4ϩ mAb (mAb) GK1.5 (rat IgG2a) (13) was ob- CD8 T cells are required for P. yoelii sporozoite-induced tained from American Type Culture Collection (Manassas, VA) (TIB207). protective immunity The anti-CD4ϩ mAb YTA3.1.2 (rat IgG2b) (14) was provided by Dr. H. Waldmann (Sir William Dunn School of Pathology, Oxford, UK.) The anti-CD8ϩ mAb 2.43 (mouse IgG2a) (15) was obtained from ATCC Initially, we confirmed that immunization of BALB/c mice with (TIB210). The anti-IFN-␥ mAb XMG-6 (rat IgG1) (16) was provided by radiation-attenuated P. yoelii sporozoites conferred solid protec- Dr. F. Finkelman (University of Cincinnati Medical Center, Cincinnati, tive immunity against challenge with 5000 infectious P. yoelii 886 ROLE OF IL-12 AND NK CELLS IN PROTECTION AGAINST MALARIA

ϩ Table I. Sporozoite-induced protective immunity of BALB/c mice is CD4 T cell secreted IFN-␥ is not required for protection ϩ ϩ dependent on CD8 T cells and IFN-␥, but not CD4 , T cells against sporozoite challenge

ϩ No. Protected/ The absolute requirement for both CD8 T cells and IFN-␥ in the a b c Treatment No. Challenged % Protection p Value effector mechanism of sporozoite-induced protective immunity ϩ None 16/16 100 dictates that the IFN-␥ is secreted by CD8 T cells, and that the interaction of CD8ϩ T cells with the peptide-MHC complex on the — 94 16/15 ءControl Ab Anti-CD8ϩ mAb† 0/16 0 0.0000 ϩ ‡ surface of the infected hepatocyte (18, 23) is critical. Data here Anti-CD4 mAb 16/16 100 — establish that CD4ϩ cells are not adequate or required for the ef- Anti-IFN-␥ mAb§ 0/10 0 0.0000 fector arm of protective immunity, at least in BALB/c mice (Table a Mice were immunized with irradiated sporozoites and immune mice treated I). However, IFN-␥ can be produced by CD4ϩ and CD8ϩ T cells ,† ;control rat Ig ,ء :before challenge with 5000 infectious sporozoites, as indicated anti-CD8ϩ mAb 2.43; ‡, anti-CD4ϩ mAb GK1.5; §, anti-IFN-␥ mAb XMG-6. as well as by NK cells in response to bacterial or parasitic infection b All naive mice (n ϭ 16) developed patent blood-stage parasitemia. (24). Furthermore, it is known that class II as well as class I MHC c p values are calculated using X2 test (uncorrected) or Fisher’s Exact test (two- tailed) molecules are expressed on the surface of virally infected human hepatocytes (25, 26), that both CD4ϩ T cells and CD8ϩ T cells can recognize parasite-derived peptides presented on the surface of sporozoites, as assessed by the complete absence of blood-stage Plasmodium spp. infected murine hepatocytes in association with parasitemia (Table I). We further established that in vivo depletion class II (27, 28) or class I (18, 23) MHC molecules, respectively, ϩ ϩ of CD8 T cells completely eliminated protective immunity (Ta- and that CD4 T cells can be effective against Plasmodium spp. as Downloaded from ϩ ble I). Depletion of CD4 T cells had no effect. These data em- demonstrated by active immunization and adoptive transfer exper- ϩ ϩ phasize the critical role of CD8 T cells, but not CD4 T cells, in iments (28, 29). Following P. yoelii infection, a lymphoid popu- the effector arm of preerythrocytic stage protection in BALB/c lation composed primarily of CD4ϩ T cells predominates in the mice, as reported previously (19). extravascular hepatic compartment, and the absolute numbers of CD4ϩ T cells have been reported to be ϳ5- and 2.5-fold greater IFN-␥ is required for sporozoite-induced protective immunity ϩ

than those of CD8 T cells at 30 h and 40 h postinfection, respec- http://www.jimmunol.org/ tively (30). Even in normal mouse liver, a CD4ϩ/CD8ϩ ratio of In previous studies in the model, sporozoite- 2.8 has been reported (31). Thus, despite the fact that murine hepa- induced protective immunity of A/J (20) and BALB/c mice (21) tocytes can present P. yoelii-derived, class II restricted peptides, was abrogated by in vivo depletion of IFN-␥. However, one study ϩ and can be eliminated by CD4 T cells that recognize P. yoelii- (22) in the P. yoelii model reported that IFN-␥ receptor knockout derived peptides complexed with class II MHC molecules on the (IFN-␥RϪ/Ϫ) mice on a (C57BL/6 ϫ 129) background failed to surface of infected hepatocytes, it appears that immunization of develop protective immunity after a single immunization with ir- BALB/c mice with irradiated P. yoelii sporozoites does not ade- radiated sporozoites but were protected after multiple immuniza- ␥ quately induce this type of protective immune response.

tions. We elected to further study the role of IFN- in the P. yoelii by guest on September 28, 2021 BALB/c model. Accordingly, sporozoite-immune BALB/c mice Parasite-specific CTL are not sufficient for protection against were depleted in vivo of IFN-␥ using the anti-IFN-␥ mAb XMG-6. sporozoite challenge Results (Table I) showed that IFN-␥ was absolutely required for the protective immunity induced by immunization with irradiated Because CD8ϩ CTL have for many years been considered critical P. yoelii sporozoites. effectors of preerythrocytic stage protection, it was of interest to The critical role for IFN-␥ was confirmed using IFN-␥ gko on a determine whether a parasite-specific CTL response could be in- BALB/c background. These mice have a targeted disruption of the duced in the nonprotected IFN-␥ gko mice. Data presented in Fig. IFN-␥ gene and are therefore unable to mount an IFN-␥ response 1 showed that high levels of CTL activity were detected despite the (7). In these experiments, wild-type mice and IFN-␥ gko mice absence of IFN-␥. It has been proposed that IFN-␥ is required for were immunized with irradiated sporozoites and challenged with the generation and maturation of CTL (32) and it is known that infectious sporozoites in parallel. Before challenge, wild-type mice several essential steps of the Ag-processing pathway are regulated were treated with a mAb against IFN-␥ or a control Ab, or left by IFN-␥ (33, 34). However, our data demonstrate that IFN-␥ is untreated. Wild-type mice (untreated and control) were solidly not required for the induction of Plasmodium parasite-specific protected against challenge with 5000 sporozoites, whereas wild- CTL. Consistent with this, normal CTL responses in the absence of type mice depleted of IFN-␥ and IFN-␥ gko mice were not pro- IFN-␥ have been reported elsewhere (7, 35, 36). tected (Table II). Therefore, in BALB/c mice immunized with P. Because IFN-␥ gko mice were not protected against sporozoite yoelii sporozoites, as with BALB/c and other inbred strains im- challenge despite the presence of high levels of CTL, our data munized with P. berghei sporozoites, CD8ϩ T cells are critical indicate that in BALB/c mice parasite-specific CTL are not suffi- effector cells and IFN-␥ is a critical mediator of sporozoite-in- cient for protection. That CTL are not required for protective im- duced protective immunity. munity cannot be excluded until studies of perforin knockout and

Table II. Irradiated sporozoite induced immunity in IFN-␥ gko micea

No. Protected/ Strain Treatment No. Challenged % Protection p Value

Wild-typeϩ/ϩ None 8/8 100 Wild-typeϩ/ϩ Control Ab 8/8 100 — Wild-typeϩ/ϩ Anti-IFN-␥ mAb 0/8 0 0.0002 IFN-␥Ϫ/Ϫ None 0/8 0 0.0002

a All naive wild-type mice (n ϭ 8) developed patent blood-stage parasitemia. The Journal of Immunology 887

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

No. Protected/ Treatmentb No. Challenged % Protection p Value

None 16/16 100 — 94 16/15 ءControl Ab Anti-iNOS† 0/10 0 0.0000 Anti-IL-12 mAb‡ 0/16 0 0.0000 Anti-NK cells§ 5/10 50 0.0038

a Data are from the same experiment presented in part in Table I. b Mice were immunized with irradiated sporozoites and immune mice treated ,† ;control rat Ig ,ء :before challenge with 5000 infectious sporozoites, as indicated aminoguanidine; ‡, anti-IL-12 mAb C17.8; §, anti-asialo GM1 antiserum.

FIGURE 1. Parasite-specific CTL are induced in IFN-␥ gko mice de- spite the absence of IFN-␥. Spleen cells from immunized IFN-␥ gko mice necessary component for CD8ϩ T cell dependent protection in (A) or wild-type mice (B) were assayed for peptide-specific CTL against sporozoite-immunized mice. the immunodominant CTL epitope on the PyCSP (residues 280–288, Downloaded from SYVPSAEQI) (18) using a conventional chromium release assay at different E:T ratios. Percent lysis was determined as: [(experimental re- IL-12 is required for P. yoelii sporozoite-induced protective lease Ϫ medium control release)/(maximum release Ϫ medium control immunity release)] ϫ 100. Data presented above and by others establish an essential role for CD8ϩ T cell secreted IFN-␥ in protection against sporozoite chal-

Fas ligand-deficient mice (not yet available on the BALB/c back- lenge. However, according to the proposed scenario, essentially http://www.jimmunol.org/ ground) are conducted. However, preliminary studies by ourselves every infected hepatocyte would have to be contacted by an Ag- specific CD8ϩ T cell. This hypothesis is supported by data indi- (our manuscript in preparation) and others (37) indicate that sporo- ϩ zoite-induced protective immunity in the C57BL/6 rodent model is cating that for CD8 CTL clones against the PyCSP to be protec- independent of both perforin and Fas. tive in adoptive transfer, they must directly contact the infected Nevertheless, it is probable that some of the CD8ϩ T cells ac- hepatocyte (45). However, it has been established that infection tivated by infection may be cytotoxic; the presence of CTL specific with as few as one or two sporozoites of P. yoelii 17X NL will for every Plasmodium falciparum parasite protein known to be result in patent infection of 50% of BALB/c mice (ID50). Protec- expressed in the infected hepatocyte has been demonstrated in both tive immunity in our studies is defined as the complete absence of by guest on September 28, 2021 naturally exposed and experimentally immunized humans (re- blood-stage parasitemia. The sporozoite challenge dose used here viewed in Refs. 3 and 38). The nature of the interaction dictates a was 5000 infectious sporozoites. In other studies (data not present- tripartite complex be formed between the TCR on the CD8ϩ T cell ed), we have demonstrated protection against challenge with as and the peptide presented in the context of the MHC class I mol- many as 100,000 infectious sporozoites. Therefore, it would be ecule expressed on the surface of the infected hepatocyte. By def- predicted that at least 2500 hepatocytes would be infected follow- inition, all CD8ϩ CTL must express the CD3ϩCD8ϩCD4Ϫ cell ing challenge, and sterile immunity would dictate that every one of surface markers. However, not all CD8ϩ T cells must be cytotoxic. these 2500 infected hepatocytes must be contacted by a T cell. We In the BALB/c-P. yoelii system, we have established that CD8ϩ T reasoned that this scenario was not the most efficient mechanism cells per se are required, but that the cytotoxic function of CD8ϩ for explaining the potent protection elicited by the irradiated T cells is not sufficient. sporozoite vaccine. This led us to speculate on the role of other molecules produced by non-T cells in the protective immunity NO is required for sporozoite-induced protective immunity found after immunization with irradiated sporozoites. Other studies have demonstrated that systemic administration of In vitro, IFN-␥ induces P. berghei (39) or P. yoelii (40) infected recombinant IL-12 completely, in the absence of parasite Ag, pro- murine hepatocytes, and P. falciparum infected human hepato- tects against sporozoite challenge with P. yoelii in BALB/c mice cytes (40) to produce NO. In vivo, IFN-␥ contributes to NO syn- (46) and Plasmodium cynomolgi in rhesus monkeys (47). IL-12 is thase (NOS) production by hepatocytes as well as other cells fol- a pleiotropic cytokine secreted by a wide variety of cells including lowing infection with P. yoelii or P. berghei (41, 42) or other dendritic cells, macrophages, and monocytes, and is thought to parasites (41, 43). Inducible NOS (iNOS) is considered a major provide a functional bridge between innate resistance and Ag-spe- mediator of cytotoxicity against intracellular parasites (reviewed in cific adaptive immunity (reviewed in Refs. 48 and 49). Receptors Ref. 44). Previously, Seguin et al. (21) demonstrated that protec- for the IL-12 heterodimer are found on activated CD4ϩ T cells, tion in mice immunized with irradiated P. berghei sporozoites was activated CD8ϩ T cells and NK cells, and IL-12 are known to dependent upon the inducible, but not constitutive, NO pathway, regulate and promote Th1 type immune responses and enhance and that induction of iNOS in the liver was dependent on CD8ϩ T IFN-␥ production by T cells and NK cells (48). Accordingly, we cells and IFN-␥. To establish whether NO is important for sporo- speculated that IL-12 may play a role in the CD8ϩ T cell mediated zoite-induced protection in the P. yoelii model, sporozoite-immune IFN-␥ dependent protection of BALB/c mice induced by immu- mice were treated before challenge with aminoguanidine, a spe- nization with irradiated sporozoites. Consistent with this, we dem- cific substrate inhibitor of iNOS. Protection was completely elim- onstrated that in vivo depletion of IL-12 completely abrogated inated (Table III). Therefore, IFN-␥ induction of the L-arginine- sporozoite-induced protective immunity (Table III). This is the dependent NO pathway in vivo, and subsequent elimination of first demonstration for a role of IL-12 in Ag-specific adaptive im- infected hepatocytes or hepatic schizonts within those cells, is a munity against preerythrocytic Plasmodium spp. 888 ROLE OF IL-12 AND NK CELLS IN PROTECTION AGAINST MALARIA

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

Expt. Strain Treatment No. Protected/No. Challenged % Protection p Value

1a Wild-typeϩ/ϩ None 6/6 100 — IL-12 p40Ϫ/Ϫ None 0/8 0 0.0003 IL-12 p35Ϫ/Ϫ None 2/13 15.4 0.0010 2b Wild-typeϩ/ϩ Control Ab 4/10 40 — Wild-typeϩ/ϩ Anti-IL-12 mAb 0/10 0 0.043 IL-12 p40Ϫ/Ϫ None 0/10 0 0.043 IL-12 p35Ϫ/Ϫ Control Ab 2/12 16.7 0.229 IL-12 p35Ϫ/Ϫ Anti-IL-12 mAb 0/12 0 0.029

a In experiment 1, all naive wild-type mice (Expt. 1, n ϭ 16; Expt. 2, n ϭ 10), naive IL-12 p40Ϫ/Ϫ (Expt. 1, n ϭ 8) and naive IL-12 p35Ϫ/Ϫ (Expt. 1, n ϭ 5) developed patent blood-stage parasitemia. b In experiment 2, p values are calculated using Fisher’s Exact test (one-tailed).

The absolute requirement for IL-12 was further confirmed by gko and p40 gko mice generated on the C57BL/6 background (data studying sporozoite-immunized IL-12 gko mice. IL-12 is a het- not shown). Because both p35 and p40 subunits are required for erodimeric cytokine composed of two disulfide-linked subunits, biologically active IL-12, these data are initially confusing, par- p40 and p35, both of which are required for biological activity. ticularly since it has been reported that high affinity binding of p40 Downloaded from Neither IL-12 p35 gko nor p40 gko mice are able to produce bi- homodimers to the IL-12 receptor blocks the activity of biologi- ologically active IL-12 (p70), but p35 gko mice express p40 at cally active IL-12, antagonizing the immune response and inhib- levels indistinguishable from wild-type mice. Here, we studied iting CD4ϩ T cell function in vitro and in vivo (50). However, it both IL-12 p35 gko and p40 gko mice generated on the BALB/c has been reported recently (51, 52) that p40 homodimers markedly background. In our system, sporozoite-immunized p40 gko mice enhance rather than decrease CD8ϩ Th1 development and IFN-␥ ϩ were not protected against challenge (Table IV). However, in two production by CD8 T cells. The results here in the P. yoelii- http://www.jimmunol.org/ separate experiments, there was some suggestion of protection in BALB/c model confirm that IL-12 is a critical component of the the p35 gko mice (Table IV). Similar results were noted with p35 CD8ϩ T cell and IFN-␥-dependent protective immunity induced by guest on September 28, 2021

FIGURE 2. A, Circulating IFN-␥ 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-␥ by ELISA and compared with levels in naive wild-type mice that developed patent blood stage parasitemia. B, Circulating IFN-␥ 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-␥. The Journal of Immunology 889

Table V. DNA-induced protective immunity of BALB/c mice is dependent on IFN-␥, NO, IL-12, and NK cellsa

No. Protected/No. Challenged

Treatmentb PyCSP PyCSP ϩ PyHEP17 Total % Protection p Value

— 28 72/20 46/13 26/7 ءControl Ab Anti-IFN-␥ mAb† 0/10 0/29 0/39 0 0.0003 Anti-iNOS‡ 0/10 0/37 0/37 0 0.0004 Anti-IL-12 mAb§ 0/16 0/16 0/30 0 0.0020 Anti-NK cells¶ — 0/30 0/30 0 0.0020

a Data are the accumulated results of four experiments. All naive mice (n ϭ 26) and all mice immunized with control plasmid DNA (n ϭ 46) developed patent blood-stage parasitemia. b Mice were immunized with 100 ␮g/mouse plasmid DNA (PyCSP, or PyCSP ϩ PyHEP17) and immune mice treated before challenge with 50 control rat Ig; †, anti-IFN-␥ mAb XMG-6; ‡, aminoguanidine; §, anti-IL-12 mAb C17.8; ¶, anti-asialo GM1 ,ء :infectious sporozoites, as indicated antiserum. by immunization with irradiated sporozoites. In addition, data are sented here demonstrate that NK cells also play a crucial role in the consistent with the proposal that p40 homodimers enhance IFN-␥ Ag-specific adaptive immune response to Plasmodium spp. infec- production by CD8ϩ T cells. tion. Accordingly, we invoke a unique role for NK cells in Ag- It has been demonstrated previously that IL-12 p35 gko and p40 specific adaptive immunity initiated by parasite-specific CD8ϩ T Downloaded from gko mice can produce IFN-␥ and generate normal Th1 immune cells, which is distinct from the role of NK cells in innate responses, albeit at reduced levels compared with wild-type mice immunity. (8). Consistent with this, circulating IFN-␥ was detected following sporozoite challenge of immunized IL-12 p35 gko and p40 gko IL-12 and NK cells are required for DNA vaccine-induced mice (Fig. 2A). The magnitude of IFN-␥ was enhanced relative to protective immunity that of nonimmunized infectivity controls that developed patent http://www.jimmunol.org/ ϩ parasitemia, but reduced compared with that of immune wild-type The involvement of CD8 T cells, IFN-␥, IL-12, and NK cells in mice (Fig. 2A). These data suggest that IL-12 is required for op- protective immunity against P. yoelii sporozoites was further ver- timal production of IFN-␥, in our system. Furthermore, because ified by investigating the mechanism of protection induced by im- IL-12 gko mice were not protected despite the presence of IFN-␥, munization with plasmid DNA. Previously, we have reported (6) data indicate that the IFN-␥ secreted by activated CD8ϩ T cells is that the protection in B10.BR mice induced by immunization with not sufficient for protection and that IL-12 is required for the in- plasmid DNA encoding either PyHEP17 or a mixture of PyHEP17 ϩ duction of protective levels of IFN-␥. Accordingly, we propose ϩ PyCSP was absolutely dependent on CD8 T cells, IFN-␥ and that IFN-␥ precedes and initiates production of IL-12 and that this NO. In that strain, immunization with PyCSP DNA alone does not by guest on September 28, 2021 IL-12 in turn induces IFN-␥ in a positive feedback loop that rep- confer protection (6). Here, we extended those studies to demon- resents an important amplifying mechanism. strate the same dependence in the BALB/c strain. To determine Our data establish a critical role for IL-12 in the Ag-specific whether IL-12 and NK cells were also involved, BALB/c mice adaptive immune response to malaria. Other data from our labo- were immunized with either PyCSP DNA, or PyCSP ϩ PyHEP17 ratory (46, 47) are consistent with a role for IL-12 in the innate DNA, and depleted of IL-12 or NK cells. Results (Table V) show immune response to sporozoite challenge. It is likely that IL-12 that the protective immunity induced by immunization with plas- may be important in both innate and adaptive immunity, and that mid DNA was absolutely dependent on both IL-12 and NK cells, the innate response may influence the development of Ag-specific as demonstrated with irradiated sporozoite immunity. Treatment of immunity, because any IL-12 produced as a result of the innate DNA-immunized mice with anti-IL-12 Ab was found to com- immune response to sporozoite challenge would augment the pletely eliminate circulating IFN-␥ (Fig. 2B). In other studies, with IFN-␥ produced by both CD8ϩ T cells and NK cells. The precise other immunogens, in vivo depletion of IL-12 similarly resulted in dissection of this is beyond the scope of our report. a decrease or abrogation in circulating IFN-␥ (data not shown). Therefore, these data implicate IL-12 as critical for the optimal NK cells are required for sporozoite-induced protective induction of IFN-␥ following immunization with either irradiated immunity sporozoites or plasmid DNA.

It is well established that IL-12 acts on both T cells and NK cells A novel mechanism of adaptive immunity against malaria (48, 49), and that NK cells are a major producer of IFN-␥ (53). In particular, in parasitic and bacterial models, many reports demon- We have previously proposed (3, 6) that in DNA-immunized mice strate that IL-12 is essential for T cell and NK cell production of CD8ϩ T cells activated by interacting with the peptide-MHC com- IFN-␥ and protective immunity (54–60). To determine whether plex on the surface of the infected hepatocyte secrete IFN-␥ that NK cells were involved in the protective immunity induced by induces the infected hepatocyte to produce NO, eliminating the immunization with Plasmodium spp. parasites, we treated sporo- infected hepatocyte or intracellular parasite. In this paper, we pro- zoite-immune mice with anti-asialo GM1 antiserum before and vide the first demonstration of a role for NK cells and for IL-12 in during sporozoite challenge. This treatment was able to partially adaptive immunity against Plasmodium spp. sporozoites. We es- abrogate the protective immunity (Table V), providing the first tablish that protection induced in BALB/c mice by immunization demonstration of a critical role for NK cells in protective immunity with irradiated sporozoites or plasmid DNA is mediated by NK against preerythrocytic stage malaria. cells as well as by CD8ϩ T cells, and is absolutely dependent on NK cells have been established as a crucial first line of defense IFN-␥, IL-12 and NO. To date there is no evidence for Ab-medi- against pathogens because they can exert their activity without ated immune mechanisms during the hepatic stage of the Plasmo- prior sensitization by Ag (reviewed in Refs. 53 and 60). Data pre- dium spp. parasite’s life cycle, although it has been speculated that 890 ROLE OF IL-12 AND NK CELLS IN PROTECTION AGAINST MALARIA Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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

Abs may kill the intrahepatic parasite, either directly, with com- Toxoplasma gondii system, for example, enhanced IL-12 synthesis plement, or via Ab-dependent cellular cytotoxicity (ADCC). How- precedes IFN-␥ production, and the induction of IL-12 and other ever, we acknowledge the possibility that ADCC may play a role innate immune responses does not require IFN-␥ (61). in preerythrocytic stage immunity, and that our data do not exclude In this paper, we provide, to the best of our knowledge, the first such a role. Nevertheless, we believe that this has no bearing on demonstration of a feedback mechanism induced by parasite chal- the significance of our findings establishing a role for IL-12 and lenge of actively immunized animals. Furthermore, our proposal NK cells in protective immunity against sporozoite challenge. that NK cells constitute part of an Ag-specific adaptive immune Accordingly, in this report, we advance a novel feedback loop of response initiated by CD8ϩ T cells and dependent on IL-12 rep- adaptive immunity (Fig. 3). We propose that parasite-specific resents a novel mechanism of protective immunity against intra- CD8ϩ T cells provide the critical initial trigger for the protective cellular, bacterial and viral pathogens. effector mechanism, via recognition of specific peptide-MHC com- plexes on the surface of the infected hepatocyte. Further, we pro- Acknowledgments pose that induction of IFN-␥ is a direct consequence of the CD8ϩ ␥ T cell activation, that IFN- production precedes and initiates pro- We thank Mr. Arnel Belmonte and Mr. Romeo Wallace for providing the duction of IL-12, and that the IL-12 in turn induces IFN-␥ pro- P. yoelii sporozoites, Mr. Salvador Doria and Ms. Tonette Bangura for duction by NK cells (and perhaps other cells) in a positive feed- excellent technical assistance, Dr. Jeanne Magram (Hoffmann-LaRoche) back loop that represents an important amplifying mechanism. The for providing the IL-12 gko mice, Drs. Maria Wysocka and Giorgio IFN-␥, via signal transducers associated with transcription, then Trinchieri (Wistar Institute) for providing the anti-IL-12 mAb, Drs. Alan activates NOS and induces the L-arginine-dependent NO pathway, Sher and John Sacci for advice and critical discussion, and Dr. Eileen subsequently eliminating the infected hepatocytes or the hepatic Villasante for artistic assistance. schizonts within those cells. In BALB/c mice, CD4ϩ T cells are not required or sufficient for the initial triggering of the effector References mechanism, nor for the feedback induction of IFN-␥ (Fig. 3). In other parasitic infections, in contrast, it has been established 1. World Health Organization. 1996. Malaria Fact Sheet No. 94. World Health ␥ Organization, Geneva, Switzerland. that the induction of IFN- synthesis by NK cells following in- 2. Nussenzweig, V., and R. S. Nussenzweig. 1989. Rationale for the development of fection is indirect, involving a secondary effect of IL-12. In the an engineered sporozoite malaria vaccine. Adv. Immunol. 45:283. The Journal of Immunology 891

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