Perforin- and Granzyme-Mediated Cytotoxic Effector Functions AreEssential for Protection against Francisella tularensis followingVaccination by the Defined F. tularensis subsp. novicida �fopCVaccine Strain

Shilpa Sanapala,a* Jieh-Juen Yu,a Ashlesh K. Murthy,b Weidang Li,a M. Neal Guentzel,a James P. Chambers,a Karl E. Klose,a andBernard P. Arulanandama

South Texas Center for Emerging Infectious Diseases and Center of Excellence in Infection Genomics, University of Texas at San Antonio, San Antonio, Texas, USA,a andDepartment of Pathology, Midwestern University, Downers Grove, Illinois, USAb

A licensed vaccine against Francisella tularensis is currently not available. Two Francisella tularensis subsp. novicida (hereinreferred to by its earlier name, Francisella novicida) attenuated strains, the �iglB and �fopC strains, have previously been evalu-ated as potential vaccine candidates against pneumonic tularemia in experimental animals. F. novicida �iglB, a Francisellapathogenicity island (FPI) mutant, is deficient in phagosomal escape and intracellular growth, whereas F. novicida �fopC, lack-ing the outer membrane lipoprotein FopC, which is required for evasion of gamma interferon (IFN-�)-mediated signaling, isable to escape and replicate in the cytosol. To dissect the difference in protective immune mechanisms conferred by these twovaccine strains, we examined the efficacy of the F. novicida �iglB and �fopC mutants against pulmonary live-vaccine-strain(LVS) challenge and found that both strains provided comparable protection in wild-type, major histocompatibility complexclass I (MHC I) knockout, and MHC II knockout mice. However, F. novicida �fopC-vaccinated but not F. novicida �iglB-vacci-nated perforin-deficient mice were more susceptible and exhibited greater bacterial burdens than similarly vaccinated wild-typemice. Moreover, perforin produced by natural killer (NK) cells and release of granzyme contributed to inhibition of LVS replica-tion within macrophages. This NK cell-mediated LVS inhibition was enhanced with anti-F. novicida �fopC immune serum, sug-gesting antibody-dependent cell-mediated cytotoxicity (ADCC) in F. novicida �fopC-mediated protection. Overall, this studyprovides additional immunological insight into the basis for protection conferred by live attenuated F. novicida strains withdifferent phenotypes and supports further investigation of this organism as a vaccine platform for tularemia.

Live attenuated vaccines have played a significant role in controlof bacterial and viral infections (3, 4, 31, 32, 34, 38, 44). The

immune response to a live attenuated vaccine closely resemblesthat produced by a natural infection, usually comprising potentcellular and humoral responses (14, 71). Owing to their relativecomplex genetic nature, attenuated bacterial vaccines are oftendifficult to develop despite the availability of molecular techniques(34, 49, 70). To this end, there is a continued interest in develop-ment of a live attenuated vaccine against pneumonic tularemia.Francisella tularensis, the etiological agent of tularemia, is a Gram-negative facultative intracellular bacterium (11, 69). Untreatedcases of pneumonic tularemia caused by the virulent human typeA strains (Francisella tularensis subsp. tularensis) may have mor-tality rates of 30 to 60% (66). A live vaccine strain (LVS), derivedfrom a type B strain that is less virulent in humans (Francisellatularensis subsp. holarctica), has been used to immunize millionsof individuals in the former Soviet Union (62). However, LVS hasnot been licensed for general use due to phenotypic inconsistency(12, 23) and a lack of understanding of the nature of attenuation.Recently, our laboratory evaluated vaccine strains derived fromFrancisella tularensis subsp. novicida (herein referred to by itsearlier name, Francisella novicida) (U112 strain) in experimen-tal rodent models and found that protection can be achievedagainst heterotypic pulmonary LVS and SCHU S4 (type A)bacterial challenge (9, 53). F. novicida is genetically related totype A F. tularensis [98.1% homology between sequences com-

mon to U112 and the type A strain SCHU S4 (55)] and isavirulent in humans.

Using F. tularensis as a model pathogen, this study aimed atgaining additional immunological insight into the basis for pro-tection conferred by live attenuated bacterial vaccines. Specifi-cally, two live attenuated F. novicida mutant strains, namely, F.novicida �fopC (46) and F. novicida �iglB (9), were directly com-pared in order to characterize the mechanistic details underlyingthe respective protective efficacy against pulmonary F. tularensisLVS challenge. F. novicida �iglB is a Francisella pathogenicity is-land (FPI) mutant, deficient in intramacrophage growth andphagosomal escape (7, 36, 47). In contrast, F. novicida �fopC has adeficiency in the outer membrane lipoprotein (FopC), which hasbeen reported by us (46) to mediate evasion of gamma interferon

Received 19 January 2012 Returned for modification 12 February 2012Accepted 29 March 2012

Published ahead of print 9 April 2012

Editor: A. Camilli

Address correspondence to Bernard P. Arulanandam, Bernard.arulanandam@utsa.edu.

* Present address: Center for Infectious Diseases and Vaccinology, BiodesignInstitute at Arizona State University, Tempe, Arizona, USA.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00036-12

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(IFN-�)-mediated signaling and by others (35, 56) to play a role iniron acquisition and to be an important virulence factor for type AF. tularensis. Moreover, F. novicida �fopC replicated similarly towild-type F. novicida U112 in primary macrophages that had notbeen stimulated with IFN-� (46), suggesting that the bacteriumlikely escaped from phagosomes and replicated within the cytosol.Given the difference in phagosomal escape between F. novicida�iglB and F. novicida �fopC, the host may likely utilize differentantigen-processing pathways and T-cell subsets to generate pro-tective immunity.

To this end, the major histocompatibility complex class I(MHC I) pathway samples intracellular antigens (such as proteinssecreted or degraded from cytosolic F. novicida �fopC bacteria) topresent to cytotoxic T lymphocytes (CD8� T cells) (24). On theother hand, the MHC II pathway interacts with endocytic exoge-nous antigens (such as antigens generated from F. novicida �iglBin the phagosomes) for presentation to helper T cells (CD4� Tcells) (24). Given that the initial priming mechanisms for the twoattenuated mutant vaccine strains may be different, we sought toinvestigate whether these strains utilized different host immunecomponents to induce protection against pulmonary F. tularensisLVS challenge with a panel of knockout mice, including MHC I,MHC II, CD4� T cells, CD8� T cells, and perforin, a potent cyto-toxic effector molecule produced primarily by CD8� T cells andnatural killer (NK) cells. In these studies, we found an importantprotective role for perforin following oral F. novicida �fopC butnot F. novicida �iglB vaccination against pulmonary F. tularensisLVS infection. The protection conferred by F. novicida �fopC vac-cination may be mediated by NK cells via the release of perforinand granzymes. This is the first report that definitively describesdissimilar host-protective mechanisms mediated by two live at-tenuatedF.novicidavaccinestrainswithmajordifferencesinphago-somal escape phenotypes.

MATERIALS AND METHODSBacteria. F. tularensis subsp. novicida strain U112 was provided by FrancisNano (University of Victoria, Victoria, Canada). F. tularensis subsp. hol-arctica LVS (lot 703-0303-016) was obtained from Rick Lyons (Universityof New Mexico). The iglB and fopC mutants of F. novicida U112 weregenerated as reported previously (36, 46). All strains were grown at 37°Cin tryptic soy broth (TSB) or on tryptic soy agar (TSA) (BD Biosciences,San Jose, CA), each supplemented with 0.025% sodium pyruvate, 0.025%sodium metabisulfite, 0.025% ferrous sulfate, and 0.1% L-cysteine. Ali-quots of bacteria were stored at �80°C in TSB containing all supplementsand glycerol (24%).

Mice. C57BL/6 mice (4 to 8 weeks) were purchased from the NationalCancer Institute (Frederick, MD). C57BL/6 MHC I �2-microglobulin�/�

mice (30), MHC II H2�/� mice (39), �MT (B-cell-deficient) mice (29),CD4�/�-T-cell mice (43), CD8�/�-T-cell mice (18), Fc�R�/� mice (68),and perforin�/� mice (26) were purchased from the Jackson Laboratory(Bar Harbor, ME). Mice were housed and bred at the University of Texasat San Antonio Animal Facility. Animal care and experimental procedureswere performed in compliance with Institutional Animal Care and UseCommittee (IACUC) guidelines. Experiments were performed with age-matched groups of animals.

Immunization and challenge. Mice (5 to 7 animals/group) were anes-thetized and orally vaccinated, using a 22-gauge, 25-mm-long, 1.25-mm-tip feeding needle (Fine Science Tools Inc., Foster City, CA) (22), witheither 103 CFU of live F. novicida iglB or fopC mutant cells in 100 �l ofphosphate-buffered saline (PBS) or PBS alone (mock vaccination). The50% lethal doses (LD50) of �iglB and �fopC strains administered intrana-sally were greater than 107 CFU and between 104 and 105 CFU, respec-

tively (9, 46). Vaccinated mice were allowed to rest for 4 weeks and thenchallenged intranasally (i.n.) with 4 to 5 LD50 of LVS (LD50 � �8,000CFU [data not shown]) in 25 �l PBS. Within individual experiments, thesame dose of LVS was used for each respective test variable. The actualvaccination and challenge doses administered were determined by dilu-tion plating on TSA with supplements. Animals were monitored daily formorbidity and mortality.

Determination of bacterial dissemination of LVS in F. novicida�fopC- and F. novicida �iglB-vaccinated mice. C57BL/6 mice wereorally vaccinated with 1,000 CFU of F. novicida �fopC or F. novicida �iglBin 100 �l of PBS on day 0 and challenged i.n. with 5 LD50 of LVS on day 28.Groups of mice (n � 3 per group) were euthanized on days 2 and 5postchallenge. Lungs, livers, and spleens were removed, and the bacterialcounts in the homogenized tissues were determined by dilution plating.

Preparation of single-cell suspensions from spleens. Immunizedmice were sacrificed on day 14, and spleens were removed under asepticconditions, disrupted with a 3 ml syringe plunger, and passed through a0.76-�m cell strainer. Single-cell suspensions were prepared, and eryth-rocytes were lysed with ammonium chloride. Cells were washed, viabilitywas assessed by trypan blue exclusion, and cells were resuspended in Dul-becco’s modified Eagle medium (DMEM; Mediatech, Fairfax, VA) con-taining 10% (wt/vol) fetal bovine serum (FBS; HyClone, Logan, UT) sup-plemented with L-glutamine (D10). The T-lymphocytes and NK cells wereenriched from single-cell suspensions of splenocytes using an EasySepmouse T cell enrichment kit and an EasySep mouse NK cell enrichmentkit (Stemcell Technologies, Vancouver, Canada), respectively. For exper-iments involving antigen-presenting cells (APCs), splenocytes weretreated with mitomycin C (25 �g/107 cells) for 20 min at 37°C in 5% CO2,followed by 2 h of incubation to separate the adherent APCs from non-adherent lymphocytes.

Generation of bone marrow-derived macrophages (BMDM) and invitro infection. Four- to five-week-old mice were euthanized, and femurswere removed and flushed with D10 supplemented with penicillin (100U/ml) and streptomycin (100 �g/ml). Cells were collected by centrifuga-tion and resuspended in D10 containing 10% culture medium from theL929 hybridoma cell line (6), seeded into 75-cm2 tissue culture flasks, andincubated at 37°C with 5% CO2 to allow differentiation into BMDM.Adherent cells were harvested after 6 to 7 days and stained with phyco-erythrin (PE)-conjugated CD11b (BD Biosciences), and flow cytometrywas used to determine the purity of the cell population. We routinelyobtained 95% CD11b� cells from the preparations.

For bacterial infection, BMDM were seeded at 2 105/well in sterile96-well plates in D10 and incubated at 37°C in 5% CO2. After 2 h ofincubation, Francisella bacteria were diluted from frozen stocks, added toBMDM at a multiplicity of infection (MOI) of 10:1 or 100:1, and incu-bated for 2 h to allow bacterial uptake. After 3 washes with DMEM, theBMDM were then incubated for 1 h in D10 supplemented with 50 �g/mlgentamicin to kill extracellular bacteria. Cells were washed three times andlysed with 0.2% deoxycholate solution (3-h time point) or incubated inD10 for additional 21 h prior to lysis (24-h time point). Intracellularbacteria were enumerated by plating serial dilutions of lysates on TSAcontaining supplements.

For coculture experiments, T cells or NK cells enriched from spleno-cytes were added to LVS-infected (10 MOI) BMDM at a 1:1 ratio (200�l/well) following gentamicin treatment. Cultures were incubated at 37°Cin 5% CO2 for the remainder of the experiment. LVS replication withinBMDM was determined as described above. Supernatants also were col-lected for measurement of cytokines and/or granzyme at 3, 6, 12, 24,and/or 72 h based on the specific experimental conditions. IFN-�, inter-leukin 1� (IL-1�), and granzyme enzyme-linked immunosorbent assays(ELISAs) (BD Pharmingen, San Diego, CA) were used to determine cyto-kine levels in the supernatants according to manufacturer’s recommen-dations.

Statistical analysis. Data from all assays were evaluated using anANOVA one-way test, except that the Kaplan-Meier test was used for

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survival analysis. The data are presented as means � standard deviations.Each experiment was independently done at least twice.

RESULTSF. novicida �fopC but not F. novicida �iglB escaped fromphagosomes in primary macrophages. We previously character-ized two F. novicida (strain U112) mutants, F. novicida �iglB andF. novicida �fopC, which showed distinctive intramacrophagereplication phenotypes (9, 46). As shown in Fig. 1A, the �iglBstrain was attenuated in growth (less than a 1-log-unit increasefrom 3 h to 24 h after inoculation) in BMDM, whereas the �fopCstrain, like its wild-type parent, U112, displayed marked replica-tion (3- to 4-log-unit increase). Lack of intracellular replicationforFrancisella�iglBhasbeencorrelatedwithitsdeficiencyinphago-somal escape (7). To assess whether F. novicida �fopC escapedfrom phagosomes and replicated in the cytosol, as seen with theU112 parental strain (57, 58), we analyzed IL-1� secretion byBMDM following bacterial infection. Release of IL-1� in infectedmacrophages as a result of inflammasome activation followingescape of Francisella from the phagosome into the cytosol has beenreported (8, 20, 21, 41). We observed a comparable high level(3,000 pg/ml) of IL-1� released by BMDM upon live U112 andF. novicida �fopC infection but not with UV-inactivated bacteria

(data not shown), suggesting escape of live F. novicida �fopC cellsfrom phagosomes (Fig. 1B). In contrast, minimal amounts ofIL-1� secreted by F. novicida �iglB-infected BMDM confirmedthat F. novicida �iglB failed to disrupt phagosomes, similarly toother reported FPI-associated mutants (58, 59). Negligibleamounts of IL-1� were secreted by uninfected macrophages (Fig.1B) as well as those infected with UV-inactivated bacteria (datanot shown). Collectively, such evidence indicates differences inthe cytosolic presence and replication between the F. novicida�iglB and F. novicida �fopC strains, which might lead to somedistinct immune responses due to differences in antigen process-ing and presentation by the host cells.

Antigen presentation by APC following F. novicida �fopCand F. novicida �iglB infection. Gene knockout (KO) mice withdeficient MHC I or MHC II were used to study whether differentpathways were used to present F. novicida �fopC and F. novicida�iglB antigens. APCs were generated from naïve wild-type (WT),MHC I KO, and MHC II KO mice by treating the total splenocytepopulation with mitomycin C, followed by removal of nonadher-ent cells. APCs (2 105/well) were then infected (MOI of 10) withlive or UV-inactivated F. novicida �fopC or F. novicida �iglB for 2h, treated with gentamicin for an additional 1 h, and then washedto remove all extracellular bacteria. These antigen-loaded APCswere incubated for 72 h with T-lymphocytes enriched fromspleens of WT mice which had been inoculated orally with 1,000CFU of F. novicida �fopC, F. novicida �iglB, or PBS. Followingincubation, IFN-� concentrations in the culture supernatantswere assayed, and these served as an indicator of T-cell activationby the APCs.

As shown in Fig. 2, T cells from PBS-treated (mock-vacci-nated) mice, regardless of whether they were cocultured withAPCs isolated from WT, MHC I KO, or MHC II KO mice andpreviously infected with F. novicida �fopC, F. novicida �iglB, orhen egg lysozyme (HEL, an unrelated antigen used as a control),had minimal IFN-� induction, as expected. In contrast, T cellsobtained from WT mice primed by oral vaccination with F. novi-

FIG 1 Phenotypic differences between F. novicida �iglB and F. novicida �fopCin bacterial replication and IL-1� production in BMDM. BMDM were in-fected (MOI, 10 or 100) with WT U112, F. novicida �fopC, or F. novicida �iglB.(A) Cell lysates were dilution plated at 3 and 24 h postinfection for bacterialenumeration. Differences in bacterial numbers were significant (*, P � 0.05;**, P � 0.01) for F. novicida �iglB compared to U112 or F. novicida �fopC at 24h at MOIs of 10 and 100. (B) Supernatants were collected at 6, 12, and 24 hpostinfection for determination of IL-1� levels by ELISA. Differences in IL-1�production at 12 and 24 h between F. novicida �iglB and either U112 or F.novicida �fopC were significant (***, P � 0.001). Results are representative ofthree independent experiments with triplicates in each group.

FIG 2 Presentation of F. novicida �iglB and F. novicida �fopC antigens byMHC I and II. T cells obtained from WT mice orally immunized with F.novicida �fopC, F. novicida �iglB, or PBS were incubated for 72 h with respec-tive WT, MHC I�/�, or MHC II�/� APCs that had been infected with bacteria(live or UV inactivated) and treated with mitomycin C. IFN-� production bythe T cells was measured by ELISA. APCs incubated with an unrelated antigen,hen egg lysozyme (HEL), also were included for comparison. Results are rep-resentative of two independent experiments with triplicates in each group.

Vaccine Immunity against Tularemia Requires Perforin

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cida �fopC or F. novicida �iglB, upon incubation with WT APCsinfected with respective bacterial mutant strains, exhibited similarelevated levels of IFN-� production, indicating that WT APCseffectively processed and presented both F. novicida �fopC and F.novicida �iglB antigens. Similarly, comparable IFN-� levels wereobserved in T cells primed with F. novicida �fopC and F. novicida�iglB, upon activation by respective mutant-infected APCs fromeither MHC I or MHC II KO mice, although levels for both mu-tants were lower in MHC II KO mice. Additionally, there was nosignificant difference in IFN-� production by primed T cells uponactivation by either live or UV-inactivated mutant-infected APCsfrom WT, MHC I�/�, or MHC II�/� mice. Collectively, these datasuggest that there may be cross antigen presentation of the twomutant strains by MHC I and II pathways.

Roles of CD4� and CD8� T cells in F. novicida �fopC and F.novicida �iglB oral vaccination-mediated protection againstintranasal LVS challenge. In the ex vivo system described above,we observed no difference in antigen processing and presentationby major MHC pathways following F. novicida �fopC and F. novi-cida �iglB uptake by APCs. Thus, we then determined whetherCD4� and CD8� T cells played comparable roles in F. novicida�fopC and F. novicida �iglB vaccination-mediated protection.Mice (WT, CD4�/�, and CD8�/�) were vaccinated orally with F.novicida �fopC, F. novicida �iglB, or PBS and challenged i.n. witha lethal dose (5 LD50) of LVS on day 28. Organs (lungs, livers, andspleens) were collected on days 2 and 5 postchallenge and homog-enized to determine bacterial burdens. In mock-vaccinated mice,although there was 1 log unit less LVS in the spleens of CD4�/�

and CD8�/� mice than in those of WT mice, we observed nosignificant difference in mortality among these three groups ofmice following a lethal LVS challenge (all mice succumbed to in-fection by day 10 [data not shown]), which may have been due tothe relatively high LVS burdens in the target organs that led touncontrolled inflammation and death (40, 73). In contrast, allmice (WT, CD4�/�, and CD8�/�) vaccinated with either F. novi-cida �fopC or F. novicida �iglB had significantly reduced LVSburdens on days 2 and 5 in the primary challenge site (lungs) andin secondary targeted organs (livers and spleens) compared tomock-vaccinated mice (Fig. 3). However, in CD8�/� mice, greaterbacterial burdens were evident in the lungs and livers of F. novicida�fopC-vaccinated mice than F. novicida �iglB-immunized mice,and the increased organ burdens correlated with reduced survivalrate (66.7% for F. novicida �fopC immunization versus 100% forF. novicida �iglB immunization [data not shown]), suggestingthat the CD8 component may be required for F. novicida �fopC-but not F. novicida �iglB-mediated protection against LVS chal-lenge.

Perforin is required for F. novicida �fopC-mediated protec-tion against LVS challenge. Given that CD8� T cells may controlLVS infection in F. novicida �fopC-vaccinated mice and perforinis a key effector of CD8� cell granule-mediated cytotoxicity (26,33), we sought to determine the role of perforin in inducing pro-tective immunity following oral F. novicida �fopC vaccination.WT and perforin KO (PRF�/�) mice were vaccinated with F. novi-cida �fopC, F. novicida �iglB, or PBS and challenged i.n. with 5LD50 of LVS on day 30. Bacterial burdens in the lungs, livers, andspleens were measured at days 2 and 5 after LVS challenge, andsurvival was monitored for 30 days. We observed consistently highLVS burdens in all organs of PBS control WT mice at day 2 post-challenge (Fig. 3 and 4), which might be due to the high challenge

inoculum (38,000 to 44,000 CFU) used as described in Materialsand Methods. Vaccination of WT mice with either F. novicida�fopC or F. novicida �iglB resulted in significant reductions ofLVS in all target organs compared to mock-vaccinated mice (Fig.4, top). In contrast, while PRF�/� mice retained their ability tocontrol LVS growth effectively when vaccinated with F. novicida�iglB, the deficiency in perforin abrogated the reduction of LVSburdens mediated by F. novicida �fopC immunization, resultingin organ burdens as high as those in the mock-vaccinated animals(Fig. 4, bottom). Although no significant increase of LVS burdensfrom day 2 to day 5 was observed in target organs of F. novicida

FIG 3 Role of CD4� and CD8� T cells in F. novicida �fopC- and F. novicida�iglB-mediated protection (bacterial burden) against intranasal LVS chal-lenge. Mice (3 per time point) were vaccinated orally with 1,000 CFU of F.novicida �fopC or F. novicida �iglB or mock treated with PBS on day 0 and i.n.challenged with a lethal dose of LVS (44,000 CFU, �5 LD50) on day 28. Lungs,livers, and spleens were collected on days 2 and 5 after LVS challenge to deter-mine the bacterial burdens. Significant differences in LVS burdens on day 2and day 5 in three tested organs from either F. novicida �fopC- or F. novicida�iglB-vaccinated mice, compared to those in mock-vaccinated mice, are indi-cated (*, P � 0.05; **, P � 0.01; ***P � 0.001). The difference in LVS burdensin the lungs and livers between F. novicida �fopC- and F. novicida �iglB-vaccinated CD8�/� mice was also significant (*, P � 0.05; ***, P � 0.001).Results are representative of two independent experiments.

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�fopC-vaccinated PRF�/� mice, high levels of bacterial burdencorrelated with high mortality (67%) of these perforin-deficientmice following lethal LVS challenge, compared to a 17% mortalityrate in the F. novicida �fopC-vaccinated WT mice (Fig. 5). Theseresults suggest that perforin plays an important role in F. novicida�fopC-mediated protective immunity against LVS infection.There was no difference in survival between F. novicida �iglB-immunized, LVS-challenged WT and PRF�/� mice, suggestingthat while perforin is essential for F. novicida �fopC-mediatedprotection, it has a minimal role in F. novicida �iglB-generatedprotective immunity.

Perforin plays an important role in T-cell- and NK cell-me-diated inhibition of LVS intramacrophage replication. Cyto-toxic T lymphocytes and NK cells are the major cell types thatproduce perforin upon activation (42, 61). To dissect the potentialmechanism of perforin in control of LVS infection in F. novicida�fopC-vaccinated mice, we applied an in vitro killing coculturesystem to compare the inhibition of LVS intramacrophage repli-cation in perforin competent and deficient T cells and NK cells.Total T cells were enriched from spleens of either F. novicida�fopC- or mock-immunized WT or PRF�/� mice and coculturedfor 21 h with WT BMDM which had previously been infected withLVS (2-h infection and 1-h gentamicin treatment). The levels ofviable LVS within the BMDM were determined, and the produc-tion of granzymes, proteases delivered by perforin which triggerapoptosis of target cells (10), as well as IFN-�, a potent cytokineproduced by T cells that controls Francisella replication (2, 13, 46,48), were measured in the culture supernatant at the end of cocul-ture. LVS increased by 3 log units from 3 to 24 h in BMDM thatwere cultured alone (Fig. 6A). In contrast, there was a 25-fold(1.4-log-unit) reduction of LVS replication in BMDM cocultured

with F. novicida �fopC-primed WT T cells (a 1.6-log-unit increasefrom 3 h to 24 h) compared to WT PBS-treated T cells (3-log-unitincrease from 3 h to 24 h). BMDM cocultured with LVS WT F.novicida �fopC-primed PRF�/� T cells exhibited only a 3-fold(0.5-log-unit) inhibition of LVS replication, suggesting that per-forin played an important role in control of LVS intracellular rep-lication. Granzyme levels were markedly higher in WT, F. novicida�fopC-vaccinated T cells cocultured with LVS-infected BMDMthan the minimal granzymes produced by PRF�/� F. novicida�fopC-primed T cells (Fig. 6B). On the other hand, comparablehigh levels of IFN-� were produced by both WT and PRF�/� F.novicida �fopC-primed T cells (Fig. 6C), suggesting that perforin-mediated inhibition of LVS replication in BMDM cells may beIFN-� independent but granzyme dependent. Since CD8� cellsare the most predominate perforin-secreting T-cell type and PRF-mediated bacterial clearance was evident in our total T-cell pop-ulation, a role of CD8� cells in PRF-mediated bacterial clearancein F. novicida �fopC-vaccinated mice seems likely.

Similar results were observed in LVS-infected BMDM cocul-tured with NK cells enriched from spleens of naïve WT andPRF�/� mice. Replication of LVS within BMDM over a period of24 h was markedly decreased in the presence of WT NK cells butnot PRF�/� NK cells, suggesting that NK cell-mediated LVS kill-ing required perforin (Fig. 7A, left). This NK cell-mediated LVSkilling could be further enhanced by preincubation of NK cellswith F. novicida �fopC immune sera (Fig. 7A, right). In theseexperiments, NK cells were incubated with serum from mock-immunized or F. novicida �fopC-immunized mice (1:20 dilution)for 20 min before coculture with BMDM. As shown in Fig. 7A, WTNK cells incubated with sera from PBS-treated mice significantlyreduced LVS replication in BMDM compared to non-serum-

FIG 4 Perforin is required for control of LVS replication and dissemination inF. novicida �fopC-vaccinated mice. WT and PRF�/� mice were vaccinatedorally with 1,000 CFU of F. novicida �fopC or F. novicida �iglB or mock treatedwith PBS on day 0 and i.n. challenged with a lethal dose of LVS (38,000 CFU,�5 LD50) on day 28. Lungs, livers and spleens (3 mice per time point) werecollected on days 2 and 5 after LVS challenge to determine the bacterial bur-dens. In WT mice (top), the difference in LVS burdens in all organs betweenPBS and mutant (F. novicida �fopC or F. novicida �iglB)-vaccinated mice weresignificant (P � 0.001). In PRF�/� mice (bottom), LVS burdens were signifi-cantly (P � 0.001) reduced only when mice were vaccinated with F. novicida�iglB, not F. novicida �fopC, compared to PBS treatment. Results are repre-sentative of two independent experiments.

FIG 5 Perforin is required for F. novicida �fopC-mediated protection againstLVS lethal challenge. WT and PRF�/� mice were vaccinated orally with 1,000CFU of F. novicida �fopC or F. novicida �iglB or mock treated with PBS andchallenged i.n. with a lethal dose of LVS (35,000 CFU, �4 LD50) on day 28.Mice were monitored for 30 days for survival. There were significant (P �0.05) differences in survival between F. novicida �fopC- and F. novicida �iglB-vaccinated PRF�/� mice. Results are representative of two independent exper-iments.

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treated NK cells (5.7 � 1.5 104 versus 1.8 � 0.2 106 CFU/wellat 24 h). Incubation of WT NK cells with serum from F. novicida�fopC-immunized mice further reduced LVS replication withinBMDM (8.5 � 1.9 103 CFU/well at 24 h). Deletion of perforintotally abrogated NK cell-mediated LVS killing even in the pres-ence of F. novicida �fopC immune sera (Fig. 7A, right). Similar toT cells, NK cell perforin-mediated LVS killing was associated withgranzyme secretion and was independent of IFN-� production(Fig. 7B and C).

Collectively, our data suggest that perforin- and granzyme-mediated control of LVS replication in macrophages by T and NKcells is one of the mediators of the immune response against F.tularensis infection induced by F. novicida �fopC vaccination.

Inhibition of LVS replication in BMDM by NK cells is medi-ated via an antibody-dependent cellular cytotoxicity pathway.F. novicida �fopC-immune sera appeared to play an importantrole in enhancing the cytotoxic activity of NK cells in control ofLVS replication. NK cells express an activating Fc receptor(Fc�RIIIa) that mediates antibody-dependent cellular cytotoxic-ity (ADCC) and production of immune-modulatory cytokines inresponse to antibody-coated targets (67, 68). To confirm thatADCC was indeed employed by NK cells in the killing of F. tular-ensis, we compared LVS replication in BMDM cocultured withNK cells isolated from WT mice and from Fc�R�/� mice (68),

which are deficient in Fc�RI and Fc�RIII. We observed that LVSreplication increased the number of organisms by about 3 logunits in BMDM from 3 to 24 h, in contrast to almost no viable LVSincrease in BMDM cocultured with WT NK cells pretreated withF. novicida �fopC-immune serum. However, this marked LVS in-hibition was completely abrogated by coculture with Fc�R�/� NKcells (Fig. 8A). High levels of both granzyme (Fig. 8B) and IFN-�(Fig. 8C) secretion in coculture of BMDM with NK cells coincidedwith marked LVS inhibition by the WT NK cells. Additionally, asignificant reduction of granzyme (Fig. 8B) and IFN-� (Fig. 8C)production was found in infected BMDM cocultured withFc�R�/� NK cells compared to those cultured with WT NK cells,suggesting that both IFN-� and granzymes may be involved in NKcell Fc�R receptor-mediated LVS killing. Collectively, these re-sults further demonstrate that NK cells employ ADCC in controlof LVS infection.

FIG 6 Inhibition of LVS replication in BMDM by F. novicida �fopC-primed Tcells is perforin and granzyme dependent. LVS infected BMDM were cocul-tured with T cells enriched from spleens of PBS treated or F. novicida �fopCimmunized WT and PRF�/� mice. (A) Viable LVS within BMDM was enu-merated at 3 and 24 h after infection. Statistical significance between indicatedgroups (*, P � 0.05; **, P � 0.01). Granzyme (B) and IFN-� (C) levels in thesupernatants of cocultures were determined by ELISA. The difference in gran-zyme production between WT and PRF�/� T cells was significant (P � 0.001).Results are representative of two independent experiments.

FIG 7 Inhibition of LVS replication in BMDM by NK cells preincubated withanti-F. novicida �fopC serum is perforin and granzyme dependent. LVS-in-fected BMDM were cocultured with NK cells enriched from spleens of naïveWT or PRF�/� mice and either treated with serum from mice that had beenmock or F. novicida �fopC immunized or left untreated. (A) Viable LVS withinBMDM was enumerated at 3 and 24 h after infection. Replication of LVSwithin BMDM over a period of 24 h was markedly decreased (P � 0.05) incoculture with WT NK cells compared to coculture with PRF�/� NK cells andBMDM alone (left). LVS replication within BMDM was significantly reducedwhen cells were cocultured with immune serum-treated WT NK cells, com-pared to coculture with WT NK cells treated with serum from mock-immu-nized mice (**, P � 0.01) or immune serum-treated PRF�/� NK cells (***, P �0.001) (right). Granzyme (B) and IFN-� (C) levels in the supernatants ofBMDM alone or in BMDM-NK cell coculture were determined by ELISA. Thedifference in granzyme production between WT and PRF�/� NK cells wassignificant (P � 0.01). Results are representative of two independent experi-ments.

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DISCUSSION

In this study, we showed that the two live attenuated F. novicidamutant strains, F. novicida �fopC and F. novicida �iglB, utilizedifferent host immune components to induce protection againstpulmonary Francisella LVS challenge. Specifically, we demon-strated that perforin may play an essential role in F. novicida�fopC-mediated immunity but is dispensable for F. novicida�iglB-mediated protection against LVS infection. These analysesprovide additional insight into effector components that induceprotective immune responses against pulmonary tularemia.

Despite the significant differences in phagosomal escape andcytosolic bacterial replication, antigenic epitopes of F. novicida�fopC and F. novicida �iglB seem to be processed and presentedby APCs using both MHC I and MHC II pathways. In the case ofF. novicida �fopC, the bacterium escapes from phagosomes, rep-licates in the cytosol, and stimulates IL-1�, but it also is susceptibleto phagolysis upon activation of IFN-� (46). This may explain thepresentation of F. novicida �fopC epitopes by both MHC I and IIpathways. In contrast, small numbers of F. novicida �iglB may

escape and replicate in the cytosol, accounting for the minimalincrease in bacterial replication after 24 h within primary macro-phages. However, cross-presentation of exogenous (F. novicida�iglB) antigens by MHC I is also possible using cellular mecha-nisms, as reviewed by Ackerman and Cresswell (1). One suchmechanism is based on endoplasmic reticulum-mediated phago-cytosis. The MHC I peptide-loading complex is recruited tophagosomes, along with a Sec61 pore complex by which phagocy-tosed exogenous proteins are shuttled into the cytosol, and pro-teolysed by the proteasome, and the generated antigenic peptidesare then loaded onto the MHC I complex and presented to CD8�

T cells (19, 72). In support of cross-presentation, oral vaccinationof WT mice with F. novicida �fopC and F. novicida �iglB resultedin comparable serum anti-LVS IgG titers (S. Sanapala and B. P.Arulanandam, unpublished observations) and similar survivalrates following pulmonary LVS challenge. Additionally, compara-ble antigen-specific cell-mediated immune responses were in-duced following oral immunization with F. novicida �fopC and F.novicida �iglB, as seen by elevated IFN-� produced by F. novicida�fopC- or F. novicida �iglB-primed T cells upon cellular recallwith UV-inactivated LVS (Sanapala and Arulanandam, unpub-lished).

Cytotoxic T lymphocytes and NK cells may employ severalmechanisms to clear intracellular bacteria, including cytokine re-lease (15), induction of apoptosis of the host cell (27), and anti-microbial activity (64). In this regard, IFN-� is essential for con-trol of F. tularensis infection, and NK cells are importantproducers of IFN-� during primary LVS infection in the lungs andliver (5, 37); however, the role of cytolytic activity of NK cells incontrol of Francisella infection is largely unknown. We show herethat perforin-deficient NK cells are impaired in effective inhibi-tion of LVS replication in macrophages. Perforin was originallythought to cause cell lysis by forming pores on the target cell mem-brane (51), but later studies favor the theory that perforin func-tions by enabling granzymes to escape from the endosomes intothe cytosol of the target cell (45, 50), where the granzymes triggercaspase activation and lead to cell apoptosis. In addition to medi-ating the cell death pathway, the internal peptide of granzyme B(HPAYNPK) displays bactericidal action in vitro, suggesting thepossibility of antibacterial activities (60). In this study, we ob-served minimal production of granzymes by PRF�/� T cells andNK cells upon coincubation with LVS-infected BMDM along withthe loss of the ability to control LVS replication, further suggestinga granzyme-dependent mechanism in perforin-mediated protec-tion. This was supported by the observation of greater bacterialdissemination and reduced survival rates in F. novicida �fopC-vaccinated PRF�/� mice than was observed following F. novicida�iglB immunization or in F. novicida �fopC-vaccinated WT mice.Thus, CD8� T cells and NK cells may play a greater role in F.novicida �fopC-mediated protection against LVS challenge thanin protection mediated by F. novicida �iglB, and this likely occursthrough cytolysis-induced mechanisms. CD8� T cell perforin-mediated cytotoxicity also has been reported to play a role in con-trol of other intracellular bacteria, such as Listeria monocytogenesand Mycobacterium tuberculosis (25, 63).

Of note, optimal control of LVS replication in BMDM by NKcells via perforin release was achieved in the presence of anti-F.novicida �fopC sera and was dependent on Fc�R receptor-medi-ated signaling. The importance of antibody in F. novicida �fopC-mediated protection is further supported by the increased suscep-

FIG 8 NK cell-mediated ADCC is Fc�R dependent. LVS-infected BMDMwere cocultured with WT and Fc�R�/� NK cells treated with serum frommock-immunized or F. novicida �fopC-immunized mice. (A) Viable LVSwithin BMDM was enumerated at 3 and 24 h after infection. LVS replicationwithin BMDM was significantly reduced when BMDM were cocultured withimmune serum-treated WT NK cells, compared to coculture with mock se-rum-treated WT NK cells (**, P � 0.01) or immune serum-treated Fc�R �/�

NK cells (***, P � 0.001). Granzyme (B) and IFN-� (C) levels in the superna-tants of BMDM alone or BMDM-NK cell cocultures were determined byELISA. The difference in granzyme and IFN-� production between WT andPRF�/� NK cells was significant (***, P � 0.001; *, P � 0.05). Results arerepresentative of two independent experiments.

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tibility to pulmonary LVS challenge in F. novicida �fopC-vaccinated B-cell-deficient mice (50% survival rate [Sanapala andArulanandam, unpublished]) compared to wild-type mice (100%survival). Antibody-mediated protection against F. tularensis alsohas been reported by us (48, 52, 54) and others (16, 17, 28, 65),showing that adoptive transfer of anti-F. novicida mutant, anti-LVS, or anti-Francisella LPS antibodies conferred partial pro-tection against LVS and type B Francisella challenge. Usinggenetically deficient mice, Kirimanjeswara et al. (28) furtherdemonstrated that Fc�R-mediated opsonophagocytosis playsan important role in antibody-mediated protection against i.n.LVS lethal challenge. Thus, specific antibodies generated by F.novicida �fopC immunization may contribute to the control ofLVS infection both by facilitating LVS uptake via Fc�R-medi-ated opsonophagocytosis into APCs and by LVS killing viaFc�R-dependent ADCC by NK cells.

Taken together, results of our study suggest that perforin- andgranzyme-mediated cytotoxic effector functions are essential forprotective immunity against F. tularensis following F. novicida�fopC vaccination.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health grantPO1 AI057986 and the Army Research Office of the Department ofDefense under contract no. W911NF-11-1-0136 to B.P.A., as well as1RO3AI088342 to A.K.M.

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