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The Journal of Immunology, 2007, 178: 2646-2650.
Copyright © 2007 by The American Association of Immunologists, Inc.

CUTTING EDGE

Cutting Edge: IFN-{gamma}-Producing CD4 T Lymphocytes Mediate Spore-Induced Immunity to Capsulated Bacillus anthracis1

Ian Justin Glomski*,{dagger}, Jean-Philippe Corre*,{dagger}, Michèle Mock*,{dagger} and Pierre Louis Goossens2,*,{dagger}

* Institut Pasteur, Unité des Toxines et Pathogénie Bactérienne, Paris, France; and {dagger} Centre National de la Recherche Scientifique, Unité de Recherche Associée 2172, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Virulent strains of Bacillus anthracis produce immunomodulating toxins and an antiphagocytic capsule. The toxin component-protective Ag is a key target of the antianthrax immune response that induces production of toxin-neutralizing Abs. Coimmunization with spores enhances the antitoxin vaccine, and inactivated spores alone confer measurable protection. We aimed to identify the mechanisms of protection induced in inactivated-spore immunized mice that function independently of the toxin/antitoxin vaccine system. This goal was addressed with humoral and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-{gamma}, and Ab-deficient (µMT–/–) or IFN-{gamma}-insensitive (IFN-{gamma}R–/–) mice. We found that humoral immunity did not protect from nontoxinogenic capsulated bacteria, whereas a cellular immune response by IFN-{gamma}-producing CD4 T lymphocytes protected mice. These results are the first evidence of protective cellular immunity against capsulated B. anthracis and suggest that future antianthrax vaccines should strive to augment cellular adaptive immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bacillus anthracis is a sporulating bacterium that is the etiological agent of anthrax. The primary virulence factors of B. anthracis include lethal toxin (LT)3 and edema toxin, which share the same cell-binding subunit named protective Ag (PA), and a poly-{gamma}-D-glutamic acid capsule (1). The toxins function to alter host cell signaling, and thereby modulate the immune responses of the host (1), whereas the capsule inhibits phagocytosis and is nonimmunogenic (1). Interestingly, eliminating toxin function does not decrease the virulence of capsulated strains in mouse models of infection, despite the fact that the toxins can kill mice and have effects at the cellular level (1, 2, 3). This highlights the importance of bacterial growth and dissemination, as a consequence of capsulation, in the mouse.

PA is a key immunogen of antianthrax vaccines that induces the production of toxin-neutralizing Abs (4). However, various animal models suggest that PA-based vaccines do not prevent infection from all strains of B. anthracis (5), suggesting that vaccines that offer a broader range of Ags (from toxins, spores, bacilli, and/or capsule) may be a more successful approach in vaccine development. Indeed, previous studies have shown that spores (3) and capsule (6) can both enhance protective immunity. These observations suggest that, though vital, anti-PA Abs are not the only, or completely sufficient, means by which an immune host may impede the development of anthrax.

IFN-{gamma} is a cytokine that is secreted by NK cells and T lymphocytes that activates phagocytes to more effectively kill pathogens, modulate chemotaxis, and up-regulate Ag presentation to promote the establishment of a Th lymphocyte type-1 (Th1) immune response (7). There are no reports examining the role of IFN-{gamma} in resisting B. anthracis infection in vivo, yet cell culture systems have shown that IFN-{gamma}-activated macrophages both kill B. anthracis more effectively and survive the toxic effects of LT better than untreated macrophages (8, 9). If cell culture studies translate to the in vivo systems, one would expect IFN-{gamma} to play a significant role in mediating host defenses against anthrax.

The dormant form of B. anthracis is the structurally complex spore. The spore is remarkably resistant to a number of noxious insults and is the infectious form of B. anthracis (1). From the site of infection, B. anthracis is transported to the draining lymph nodes and eventually invade deeper tissues, ultimately causing septicemia and toxemia (10). As such, the spore is the first form of B. anthracis that will interact with its host, and thus, the form to which the host defenses react in an attempt to terminate a nascent infection.

We sought to elucidate the mechanisms of the adaptive immune protection induced by immunization with formaldehyde-inactivated spores (FIS) against B. anthracis infection (3). To focus our study on the immune response that functioned independently of toxin neutralization, we chose to study resistance of mice to capsulated toxin-inactivated bacteria (11). We approached this goal with humoral transfer, splenocyte and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-{gamma}, and knockout mice that do not produce Abs (µMT–/–) (12) or sense IFN-{gamma} (IFN-{gamma}R–/–) (13). We found that, unlike the Ab-based protection against toxins, a CD4 T lymphocyte response that produces IFN-{gamma} was essential for protecting mice from infection. These results are the first evidence of protective cellular immunity against capsulated B. anthracis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial strains, reagents, software, and mice

The strains used in this study are B. anthracis vaccine strain RPLC2, a noncapsulated B. anthracis strain that produces wild-type PA and mutation-inactivated LT and edema toxin, and the challenge strain RPG1, a capsulated derivative of RPLC2 carrying pXO2 from B. anthracis strain American Type Culture Collection (ATCC) 4229 (11).

Spores were produced and purified using previously described methods (14). Purified RPLC2 spores were inactivated at 2 x 109 spores/ml in 4% paraformaldehyde (Sigma-Aldrich), incubated at 37°C with agitation for 4 h, washed, and stored in deionized water (3). Inactivated spores were quantified using a Malassez counting chamber.

RPMI 1640 medium plus GlutaMAX I with 50 µM 2-ME, 100 µg/ml penicillin/streptomycin (Invitrogen Life Technologies), and 10% heat-inactivated FBS (BioWest) (complete RPMI 1640) were used for cell culture.

Female B6CBAF1, C57BL/6J, and BALB/c mice were purchased from Charles River Laboratories, housed in specific pathogen-free conditions at the Pasteur Institute, and cared for in accordance with European animal welfare regulations. B6CBAF1 mice were used because they were first-generation heterozygotes of both C57BL/6 and CBA mice, thus allowing the B6CBAF1 to accept tissue transfers from either parental strain. C57BL/6 µMT–/– (B6.129S2-Igh-6tm1Cgn) (12) were a gift from H. Strick (Institut Pasteur, Unité des Cytokines et Développement Lymphoïde, Paris, France). B6.129S7-IfnrltmlAgt/J (IFN-{gamma}R–/–) (13) mice were purchased from The Jackson Laboratory.

Statistical analysis and graphing was performed with GraphPad Prism 4 software. Kaplan-Meier survival curves were analyzed using log rank analysis, and Th cell reactivation assays were analyzed with Student’s t test.

Immunoreagents

Anti-mouse IFN-{gamma} Abs and the recombinant protein standard used in the cytokine ELISA were described previously (15). ELISA to establish serum titer to spores were performed as previously described and represent the mean titer of serum from five mice (3). Hamster anti-mouse CD3e clone 145-2C11 and anti-CD28 clone 37.51, and rat FITC-conjugated anti-mouse CD4 clone H129.19 were purchased from BD Pharmingen.

Immunization and infection with B. anthracis spores

Mice were immunized s.c. at day 0 and 14 with a solution of 1 x 108 FIS and 0.3% aluminum hydroxide gel (Eurobio), or adjuvant only. All B6CBAF1 and C57BL/6 (LD50 5 x 105 and 3 x 105, respectively) mice were challenged s.c. on day 35 with 1 x 108 (200 LD50) RPG1 spores, with the exception of IFN-{gamma}R–/– mice (LD50 3 x 104), which were infected with 2 x 105 spores because of their greater sensitivity to infection.

Cell and serum transfer

Two donor spleens for each recipient were dissociated in Dulbecco’s PBS (Invitrogen Life Technologies) in cell strainers with 70-µm pore diameters (BD Biosciences). RBC were lysed using hemolytic Gey’s solution (16). Single-cell suspensions of splenocytes were further purified in a 35% Percoll gradient (17). The splenocytes were purified using magnetic beads conjugated to anti-CD4 mAb (Miltenyi Biotec) and sorted with an AutoMACS machine. Cell purity was analyzed by flow cytometry (FACScan; BD Biosciences) on cells labeled with FITC anti-mouse CD4 mAb. Greater than 95% purity was consistently achieved. CD4-enriched and CD4-depleted fractions at 4 x 106 cells/ml were incubated overnight at 37°C in a 5% CO2 incubator (Heraeus Instruments), supplemented with 5 U/ml IL-2, to allow the elimination of magnetic beads. Two-thirds of the cells were transferred i.v. to the recipient mouse, and one-third was injected s.c. with the spore challenge.

A total of 600 µl of day-21 serum derived from FIS-immune mice was injected i.p. into naive mice 4 h before challenge.

In vivo depletion of CD4 T lymphocytes and IFN-{gamma}

A total of 250 µg of purified rat anti-mouse CD4 (clone GK1.5) (18) mAb was injected i.p. for three consecutive days to eliminate >98% of the circulating, splenic, and lymphatic CD4 T cells, as determined using flow cytometry. A total of 300 µg of purified anti-IFN-{gamma} (clone HB170; ATCC) (19) mAb was injected i.p. 6 h before infection and 12 h postinfection to eliminate IFN-{gamma}. Depletion of IFN-{gamma} was verified by testing the sensitization of mice to infection by Listeria monocytogenes (20). Total rat IgG (Jackson ImmunoResearch Laboratories) and IgG2b (Serotec) were used as controls.

Reactivation of CD4 T cells

Bone-marrow derived macrophages (BMDM) from B6CBAF1 and BALB/c mice were prepared as described previously (21). A total of 1 x 105 cells was added to a 96-well plate (TPP) and activated with 25 U/ml rIFN-{gamma} overnight. After washing, 1 x 105 CD4 T cells (prepared as described above) and 1 x 106 FIS were added. Anti-CD3 and anti-CD28 mAb were added as positive controls at 2 µg/ml and 10 µg/ml, respectively. Supernatants were collected after 3 days and stored at –20°C until ELISA quantification.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
FIS-induced protection is mediated by cellular immunity and not humoral immunity

To determine what immunological effectors conferred protection to FIS-immunized animals against B. anthracis infection, mice were immunized solely with FIS (3), and challenged with a capsulated nontoxinogenic B. anthracis (11). FIS vaccination significantly protected >80% of the mice (Fig. 1) and induced the production of anti-spore Abs, as previously shown (3). We thus questioned whether humoral immunity was the mediator of FIS-induced immunity. Transfer of FIS-immune serum did not protect naive mice from infection (Fig. 1A). Additionally, µMT–/– mice, which do not produce Abs (12), were protected by FIS immunization (Fig. 1B). Together, these data suggest that humoral immunity is not sufficient to protect FIS-immunized mice from infection by capsulated nontoxinogenic B. anthracis.


Figure 1
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  		FIGURE 1. Transfer of FIS-immune splenocytes protects naive mice from capsulated nontoxinogenic B. anthracis, whereas humoral immunity does not protect. A, FIS-immunized (FIS-immune), adjuvant-treated (control), or naive B6CBAF1 mice that received FIS-immune serum were challenged with 1 x 108 RPG1 spores (200 LD50s) s.c. ***, A significant difference in survival from naive controls (p = 0.0046; n = 14) from two independent experiments. B, FIS-immunized C57BL/6 µMT–/– (µMT FIS; n = 15), adjuvant-treated µMT (µMT control; n = 12), or FIS-immunized (B6 FIS, n = 10) or adjuvant-treated C57BL/6 control mice (B6 control; n = 8) were challenged as described in A. Each curve represents mice combined from two independent experiments. There was no significant difference between µMT FIS and B6 FIS. C, Splenocytes from FIS-immunized B6CBAF1 (transfer FIS immune cells) or adjuvant-treated (transfer control cells) mice were transferred to naive mice and challenged as described in A. Adjuvant only (control) and FIS-immune (FIS) controls were included. ***, A significant difference from adjuvant only controls (p = 0.0002); n = 18 from three independent experiments.

 
Because humoral immunity did not protect mice, we hypothesized that cellular immunity functioned as the mediator of FIS-induced protection. To determine whether cellular immunity contributes to protection, splenocytes were isolated from FIS-immunized mice and transferred to naive mice, and challenged with B. anthracis. FIS-immunized splenocytes significantly protected naive mice from infection, whereas splenocytes from adjuvant-only treated mice did not (Fig. 1C).

FIS immunization induces the production of protective FIS-reactive MHC-restricted CD4 T lymphocytes

The spleen consists of a complex population of immune cells. Because the transfer of FIS-immune splenocytes protected naive mice, it was likely that a cellular effector of the adaptive immune response was responsible for the protective activity. Thus, to determine whether immune cells responded to FIS in a MHC-restricted manner, CD4 T lymphocytes were purified from FIS-immune mouse spleen and reactivated in cell culture with MHC-compatible or -incompatible BMDM and FIS. CD4 T lymphocytes were reactivated with MHC-compatible BMDM and FIS, and secreted significant quantities of IFN-{gamma}, whereas MHC-incompatible BMDM did not induce reactivation (Fig. 2A).


Figure 2
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  		FIGURE 2. CD4 T lymphocytes from FIS-immunized mice produce IFN-{gamma} in a MHC-restricted response to FIS and protect from infection. A, Purified FIS-immune or adjuvant-treated (control) CD4 T lymphocytes from B6CBAF1 mice were reactivated with FIS (multiplicity of infection = 10) and BMDM for 3 days in triplicate. Supernatants were tested by ELISA for IFN-{gamma} content expressed as mean ± SEM. BMDM from histocompatible B6CBAF1 or noncompatible BALB/c mice were used as APC. Anti-CD3 and anti-CD28 Abs were the positive control. Data are representative of three independent experiments. *, A significant difference from no-Ag controls (p = 0.012), and ***, p < 0.005. B, FIS-immune B6CBAF1 mice were treated in vivo with anti-CD4 Abs (FIS-Immune CD4 deoketed (depl)) or an irrelevant Ab control (FIS-Immune + control Ab), and then challenged as described in Fig. 1A. Anti-CD4 treatment completely eliminated CD4 T lymphocytes. Adjuvant (control) mice were treated with or without anti-CD4 Ab (control CD4 depl); n = 24 from three independent experiments. C, Splenocytes from FIS-immune B6CBAF1 mice were sorted into CD4+ and CD4– populations and transferred to naive mice and challenged as described in Fig. 1A. The *** in both B and C indicates a significant difference from the naive control (p < 0.0005); n = 22 from three independent experiments.

 
These results show that CD4 T lymphocytes produced IFN-{gamma} in a specific response to FIS, but were these cells playing a significant role in immune protection? This question was addressed using two different approaches: 1) in vivo CD4 T lymphocyte depletion with an anti-CD4 mAb (18), and 2) adoptive transfer of purified FIS-immune CD4 T lymphocytes. In vivo depletion of CD4 T lymphocytes significantly reduced the ability of FIS-immune mice to resist B. anthracis infection (Fig. 2B). Furthermore, transfer of purified FIS-immune CD4 T lymphocytes protected naive mice from B. anthracis challenge, whereas CD4-depleted splenocytes did not (Fig. 2C). These data strongly suggest that CD4 T lymphocytes are important mediators of the protective immune response induced by FIS immunization.

IFN-{gamma} plays a vital role in resistance against capsulated B. anthracis

IFN-{gamma}, secreted primarily by T cells and NK cells, activates cellular defenses against infection. As shown above, CD4 T lymphocytes derived from FIS-immunized mice both secreted IFN-{gamma} in vitro and protected naive mice from capsulated nontoxinogenic B. anthracis. We thus questioned whether IFN-{gamma} was a mediator of FIS-induced protection from B. anthracis infection; two approaches were used: 1) adoptive transfer of FIS-immune CD4 T lymphocytes to IFN-{gamma}R knockout mice (13), which cannot sense IFN-{gamma}, and 2) in vivo elimination of secreted IFN-{gamma} (19). Interestingly, naive IFN-{gamma}R knockout mice were ~10-fold more sensitive to infection with the capsulated nontoxinogenic B. anthracis than their wild-type counterparts, with a LD50 of 3 x 104 spores vs 3 x 105, respectively, suggesting a role for IFN-{gamma} in innate immunity. Transfer of C57BL/6 FIS-immune splenocytes to naive syngeneic IFN-{gamma}R knockout mice did not protect them from infection, whereas transfer to naive C57BL/6 was protective (Fig. 3A). Furthermore, in vivo depletion of IFN-{gamma} eliminated most of the protection induced by FIS-immunization (Fig. 3B). Taken together, these data indicate that IFN-{gamma} is a vital component of the protective adaptive immune response as well as the innate response against infection with capsulated B. anthracis.


Figure 3
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  		FIGURE 3. IFN-{gamma} is required for resistance to B. anthracis infection. A, Splenocytes from FIS-immune C57BL/6 mice were transferred to IFN-{gamma}R–/– B6 mice (IFN-{gamma}R + FIS-Immune cells) or congenic control mice (B6 + FIS-Immune cells). B6 mice were challenged with spores as described previously, whereas IFN-{gamma}R–/– mice were challenged with 2 x 105 spores because they were innately more susceptible to infection (see Materials and Methods). *, A significant difference from naive B6 mice (p = 0.043); n = 16 from two independent experiments. B, FIS-immunized B6CBAF1 mice were treated with anti-IFN-{gamma} Abs to eliminate secreted IFN-{gamma} in vivo (FIS Immune + anti-IFN-{gamma}) or an isotype control Ab (FIS Immune + control Ab), and challenged as described in Fig. 1. Mice treated with adjuvant were used as controls (control). *, A significant difference between FIS-immune mice and anti-IFN-{gamma}-treated mice (p = 0.015); n = 16 from two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The human antianthrax vaccine is focused on inducing humoral immunity against the toxins secreted by B. anthracis (1). This antitoxin response has proven necessary for protection in numerous animal models of infection (1). However, the efficacy of antitoxin vaccines are enhanced when spores are added (3). Our aim was to determine what immune effectors were protecting FIS-immunized mice. In this study, we report that, unlike antitoxin immunity, anti-FIS humoral immunity was not sufficient to protect mice from challenge with a capsulated nontoxinogenic B. anthracis. Instead, CD4 T lymphocytes mediated this protection. These CD4 T lymphocytes reacted to FIS in a MHC-restricted manner with the production of IFN-{gamma}. Additionally, we found that IFN-{gamma} was required for both innate and FIS-based immunity to capsulated nontoxinogenic B. anthracis.

IFN-{gamma} has often been associated with resistance to both viral and intracellular bacterial pathogens, yet many generalized mechanisms of antimicrobial function are up-regulated by IFN-{gamma} as well (7). IFN-{gamma} increases production of reactive nitrogen and oxygen species and complement, and up-regulates Fc and complement receptors (7). Although generally considered to be an extracellular pathogen, recent reports have revealed a transient intracellular phase of B. anthracis infection, where some of the nascent spores escape from the macrophage phagosome into the cytosol before subsequently killing the host cell (22). Therefore, it is reasonable to hypothesize that the IFN-{gamma} secreted by FIS-reactive CD4 T lymphocytes may function to augment the activation of phagocytes to both increase spore killing (8), phagocytosis, and/or decrease bacterial escape from the phagosome, as was observed for L. monocytogenes (21). Although some studies have found that macrophages can produce IFN-{gamma} in vitro, this point remains controversial (23), and IFN-{gamma} production by macrophages was not observed in response to B. anthracis spores in this or previous studies (15).

Cote et al. (24) reported that anti-PA Abs can increase the phagocytosis and destruction of spores. This observation and the data reported in this study, when taken together, may explain why mice immunized with both PA and FIS are more protected than those immunized with PA alone. We propose that IFN-{gamma}-activated macrophages eliminate spores more efficiently, by phagocytosing Ab-coated spores with their up-regulated Fc receptors, than they would in the absence of FIS treatment. In this manner, the host immune defenses are more likely to terminate infection before the bacteria are capable of producing the immunomodulatory toxins or protective capsule. However, this protective IFN-{gamma} pathway might only be functional when LT is absent or neutralized by anti-toxin Abs, because LT inhibition of CD4 T lymphocyte function has been reported (25, 26, 27).

In a previous publication, we demonstrated that naive splenic macrophages stimulated with B. anthracis spores produce IL-12 that induces IFN-{gamma} secretion by NK cells (15). Production of IL-12 and IFN-{gamma} by the innate immune system promotes the development of a Th1 adaptive immune response (28), which is dominated by cellular immunity, including IFN-{gamma} production by T lymphocytes, as we have observed in this study. Indeed, data derived from live-spore vaccinated humans suggests that the highly effective live-spore vaccine induces a cell-mediated immune response (29). We propose that the FIS vaccine induces the production of both IL-12 and IFN-{gamma} by the innate immune system, which subsequently leads to the establishment of a spore-specific Th1 immune response that effectively counters B. anthracis infection. The protective spore-associated Ags recognized by CD4 T lymphocytes remain to be determined, yet because the spore is a highly complex structure, it is likely that no single Ag will be solely responsible for the protective response. Although a majority of antianthrax vaccine studies have focused on anti-toxin Ab responses, our data suggest that further research into Th1 cellular immunity against B. anthracis may provide greater insight as to how to integrate more effectors of the immune system into a broader and more robust protection against B. anthracis infection.


   Acknowledgments
 
We thank Helene Strick at Institut Pasteur for providing the µMT–/– mice and Genevieve Milon at Institut Pasteur for providing the anti-IFN-{gamma} ascites.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by a Judith P. Sulzberger Post-Doctoral Fellowship from Pasteur Foundation of New York (to I.J.G.). Back

2 Address correspondence and reprint requests to Dr. Pierre Louis Goossens, Unité des Toxines et Pathogénie Bactérienne, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail address: pierre.goossens{at}pasteur.fr Back

3 Abbreviations used in this paper: LT, lethal toxin; PA, protective Ag; Th1, Th lymphocyte type-1; FIS, formaldehyde-inactivated spore; BMDM, bone marrow-derived macrophage. Back

Received for publication November 8, 2006. Accepted for publication December 22, 2006.


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 Discussion
 Disclosures
 References
 

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E. T. Harvill, M. Osorio, C. L. Loving, G. M. Lee, V. K. Kelly, and T. J. Merkel
Anamnestic Protective Immunity to Bacillus anthracis Is Antibody Mediated but Independent of Complement and Fc Receptors
Infect. Immun., May 1, 2008; 76(5): 2177 - 2182.
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C. K. Cote, J. Bozue, K. L. Moody, T. L. DiMezzo, C. E. Chapman, and S. L. Welkos
Analysis of a novel spore antigen in Bacillus anthracis that contributes to spore opsonization
Microbiology, February 1, 2008; 154(2): 619 - 632.
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I. J. Glomski, J.-P. Corre, M. Mock, and P. L. Goossens
Noncapsulated Toxinogenic Bacillus anthracis Presents a Specific Growth and Dissemination Pattern in Naive and Protective Antigen-Immune Mice
Infect. Immun., October 1, 2007; 75(10): 4754 - 4761.
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