# Downmodulation of Vaccine-Induced Immunity and Protection against the Intracellular BacteriumFrancisella tularensis by the Inhibitory Receptor FcγRIIB

Authors: Brian J. Franz; Ying Li; Constantine Bitsaktsis; Bibiana V. Iglesias; Giang Pham; Raju Sunagar; Sudeep Kumar; Edmund J. Gosselin
Journal: Journal of Immunology Research
 (2015)

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## Abstract

Fc gamma receptor IIB (FcγRIIB) is the only Fc gamma receptor (FcγR) which negatively regulates the immune response, when engaged by antigen- (Ag-) antibody (Ab) complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft), a Category A biothreat agent. We utilized inactivated Ft (iFt) as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO) or wildtype (WT) mice were challenged with Ft-live vaccine strain (LVS). While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed.


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## Body

## 1. Introduction

Fc gamma receptor IIB (FcγRIIB), which is expressed on B cells, as well as other antigen (Ag) presenting cells (APC), is the only Fc gamma receptor (FcγR) known to negatively regulate the immune response and play an important role in B-cell regulation and antibody (Ab) production [1–7]. Thus, Ag-specific Ab generated in response to infection, immunization, or the administration of FcγR-targeted vaccines, when complexed with Ag, has the potential to interact with FcγRIIB and significantly downmodulate immunity and protection against infectious agents. Importantly, FcγRIIB is also expressed on dendritic cells (DCs) and macrophages and can negatively regulate cellular as well as humoral immunity [1–3, 6, 8]. Consistent with the regulation of cellular immunity, it has been demonstrated that utilizing aMycobacterium tuberculosis (MTB) model in which naïve wildtype (WT) and FcγRIIB knockout (KO) mice received an aerosol challenge of MTB, FcγRIIB KO mice exhibited an enhanced cellular immune response compared to their WT counterparts, which included increased IFN-γ production [9]. In another study, followingStaphylococcus aureus (SA) infection, naïve FcγRIIB KO mice were better protected against a primary intravenous infection with SA [10]. However, in contrast to the above results using naïve mice, if FcγRIIB KO mice were first immunized with a pneumococcal vaccine and then challenged with high doses ofStreptococcus pneumoniae, mortality rates were increased above that of WT mice, correlating with increased proinflammatory cytokine production [11]. Yet, in another study in whichPlasmodium berghei parasitemia was induced postimmunization, parasitemia was not impacted following intradermal immunization of FcγRIIB KO versus WT mice with aPlasmodium berghei vaccine [12]. Thus, while enhanced immunity and/or protection against infection was observed in naïve FcγRIIB KO versus WT mice, the absence of FcγRIIB had either negative or no impact on immunity and/or protection following immunization.To further clarify the role of FcγRIIB in the generation of protective immunity against infection, we investigated the impact of FcγRIIB onF. tularensis (Ft) challenge before and after immunization with inactivatedFt (iFt) in WT versus FcγRIIB KO mice.Ft is a gram-negative intracellular pathogen that in designated a Category A biothreat agent due to its extreme virulence [13–15]. Based on the published studies cited above using naïve FcγRIIB KO mice [9–11], we hypothesized that naïve mice challenged withFt would be better protected in the absence of FcγRIIB. In contrast, based on the published studies above using immunized FcγRIIB KO mice [11, 12], we predicted a negative or very limited impact of the absence of FcγRIIB on survival followingFt-challenge of iFt-immunized FcγRIIB KO versus WT mice. In fact, the opposite was observed in the case of both naïve and iFt-immunized mice. Specifically, there was no difference in survival of naïve FcγRIIB KO versus WT mice challenged withFt. In contrast, in the case of iFt-immunized FcγRIIB KO versus WT mice, protection was significantly improved in FcγRIIB KO mice. The latter correlated with enhancedFt-specific IgA productionin vivo, enhanced recall responses in the form of increased IFN-γ production byex vivo splenocytes incubated with iFt, and reducedin vivo proinflammatory cytokine production 5 days after challenge. Thus, these studies demonstrate for the first time that the presence of FcγRIIB can significantly dampen immunity and protection against infection following immunization. These are also the first studies to suggest a role of FcγRIIB in the regulation of Ag-specific IgA production. Potential explanations for the above observations are discussed.


## 2. Materials and Methods

### 2.1. Reagents

RPMI medium was obtained from Cellgro Mediatech Inc. (Manassas, Va) into which was added 10% heat inactivated fetal bovine serum (FBS) (Hyclone Thermo Scientific, South Logan, Utah), 1% Pen/Strep (Hyclone Thermo Scientific, South Logan, Utah), 5 mL of 200 mM glutamine (100X) (Cellgro Mediatech Inc., Manassas, Va), 5 mL of 100 mM sodium pyruvate (100X) (Gibco Invitrogen, Grand Island, NY), 5 mL nonessential amino acids (AA) (100X) (Cellgro Mediatech Inc., Manassas, Va), and 250μL of 0.1 M 2-mercaptoethanol (2-ME) (Bio-rad, Hercules, CA). Media was then filter sterilized with a 0.22 μm filter and stored at 4°C.Ft-live vaccine strain (LVS) was utilized in these studies and was cultured in Mueller Hinton Broth (MHB) medium consisting of 490 mL of distilled, deionized cell culture grade water (Cellgro Mediatech Inc., Manassas, Va), 10.5 g MHB (Becton Dickinson, Sparks, Maryland), 0.069 g anhydrous calcium chloride (Acros Organics, NJ), 0.105 g hydrous magnesium chloride (hexahydrate) (MP Biomedicals, Solon, OH), 5 mL of glucose (Sigma-Aldrich, St. Louis, Missouri), 5 mL of ferric pyrophosphate (Sigma-Aldrich, St. Louis, Missouri), and 10 mL isovitalex (Becton Dickinson, Sparks, Maryland). MHB media was adjusted to a pH of 6.8, filtered (0.22 μm), and stored at 4°C for up to two weeks. Red blood cell (RBC) lysis buffer contained 4.13 g of ammonium chloride (Sigma-Aldrich, St. Louis, Missouri), 0.5 g of potassium bicarbonate (Sigma-Aldrich, St. Louis, Missouri), and 18.5 mg of EDTA (Sigma-Aldrich, St. Louis, Missouri) diluted in 500 mL of distilled water. The pH of the solution was adjusted to 7.2, after which the solution was filter-sterilized using a 0.22 μm filter and stored at 4°C.


### 2.2. Splenocyte Isolation

Spleens were isolated from mice and passed through a 70μm cell strainer (Fisherbrand, Houston, TX). The single cell suspension was collected in a sterile petri dish containing approximately 3 to 5 mL of media. The cell suspension was washed and RBCs were lysed using a RBC lysis buffer. The cell suspension was then passed through a second 70 μm cell strainer (Fisherbrand, Houston, TX) and collected in a 50 mL conical centrifuge tube containing approximately 10 mL of media. Cells were spun for 5 minutes at 1500 revolutions per minute (RPM) and resuspended in RBC lysis buffer. After 3 to 5 minutes (min), the reaction was quenched through the addition of 20 to 40 mL of media. Splenocytes were again spun for 5 min at 1500 RPM. Following resuspension, any residual tissue was removed via pipet or by again passing the splenocyte suspension through a 70 μm filter (Fisherbrand, Houston, TX). Splenocytes were subsequently washed twice with fresh media twice and enumerated using trypan blue.


### 2.3. Mice

C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). Breeding pairs of FcγRIIB KO mice (B6; 129S4-Fcgr
2
b
tm
1
Ttk/J) on a C57BL background were obtained from Jackson Laboratories and bred in the Animal Resources Facility at Albany Medical College. Mice of 6–15 weeks old were used in order to identify significant differences under conditions that include a relatively broad range of ages. All animals were housed and cared for according to guidelines approved by the Institutional Animal Care and Use Committee.


### 2.4. Generation of iFt-Immunogen

iFt was used as immunogen. Briefly,Ft-LVS organisms were grown in MHB medium (Becton Dickinson, Sparks, Maryland) at 37°C to a density of 0.5–1 × 109 CFU/mL. 1 × 1010 CFU/mL live bacteria were then incubated in 1 mL of sterile PBS (Cellgro, Manassas, Virginia) containing 2% paraformaldehyde (Sigma-Aldrich, St. Louis, Missouri) for 2 hours at room temperature. The iFt-organisms were then washed with sterile PBS three times. Inactivation was verified by culturing a 100 μL sample (~1 × 109 iFt-organisms) on chocolate agar plates (Becton, Sparks, Maryland) for 7 to 10 days. The iFt preparations were stored at −20°C in PBS.


### 2.5. Immunization and Challenge Studies

Mice were divided into groups consisting of 6–8 mice per group. Each mouse was anesthetized by intraperitoneal (i.p.) injection of 20% ketamine (Vedco, St. Joseph, Missouri) plus 5% xylazine (Lloyd, Shenandoah, Iowa) diluted in sterile cell culture grade water (Cellgro, Manassas, Virginia). Mice were subsequently administered i.n. either 20μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS. Unless otherwise indicated, mice were immunized on day 0 and boosted on day 21. Immunized mice were then challenged on day 35 i.n. with either 4 × LD50 or 16 × LD50 of liveFt-LVS and subsequently monitored for survival for a minimum of 21 days.


### 2.6. Bacterial Burden Quantitation

Mice were immunized and subsequently challenged with a sublethal dose (approximately 0.4 × LD50) of liveFt-LVS. Five days after challenge, mice were sacrificed and tissues were collected. Tissues were homogenized with a mechanical bead beater and serially diluted with sterile PBS. Diluted tissue samples were plated on chocolate agar and CFU colonies were enumerated approximately 72 hours later.


### 2.7. Antibody Quantitation

Total mouse IgM, IgG, and IgA were measured by ELISA. Kits were purchased (Immunology Consultants Laboratory, Portland, OR) and the manufacturers protocol was followed. All reagents were brought to room temperature. Samples and standards were serially diluted and 100μL/well were added to plates precoated with anti-IgM, anti-IgG, or anti-IgA Abs. Plates were incubated at room temperature for 60 minutes. Following the incubation, plates were aspirated and washed, and 100 μL of the appropriate enzyme-Ab conjugate was added to each well. After 30 minutes, the plates were again washed and substrate solution was added. The reaction was stopped after 10 minutes with stop solution and the plates were read at 450 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, California).Ft-specific Ab production was also measured by ELISA. ELISA plates (Corning, Corning, New York) were coated with 50 μL/well of liveFt-LVS (5 × 107 CFU/mL in carbonate buffer containing 2.15 g sodium bicarbonate and 2.62 g sodium carbonate (Sigma-Aldrich, St. Louis, Missouri) diluted in 500 mL of sterile cell culture grade water (Cellgro, Manassas, Virginia) at pH 9.6–9.8), overnight at 4°C. The plates were then washed three times with 200 μL/well of PBS containing 0.5% bovine serum albumin (BSA) (Baxter Healthcare, Deerfield, Illinois) and 0.002% sodium azide (Sigma-Aldrich, St. Louis, Missouri). Plates were then blocked at 4°C for 2 hours with 200 μL/well of PBS containing 5% BSA and 0.02% sodium azide. Plates were again washed and serial dilutions of sera or bronchoalveolar lavage fluids (BALF) were added to the plates (50 μL/well) and incubated for 2 hours at 4°C. After washing, alkaline phosphatase conjugated anti-mouse Ab specific for total mouse Ab (Sigma-Aldrich, St. Louis, Missouri), IgG (Sigma-Aldrich, St. Louis, Missouri), IgA (Sigma-Aldrich, St. Louis, Missouri), IgG1 (Southern Biotech, Birmingham, AL), IgG2c (Abcam, Cambridge, MA), or IgM (Sigma-Aldrich, St. Louis, Missouri) were added and incubated for 1 hour at 4°C. Plates were washed and then 100 μL/well of alkaline phosphatase substrate (Sigma, St. Louis, MO) was added. All samples were read at 405 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, California) following a 5-second (sec) shake.


### 2.8. Adoptive Transfer Studies

Mice were immunized with iFt i.n. as described above and on day 35 mice were anesthetized and blood was collected via percutaneous cardiac puncture. Clotted blood samples were then spun at 8000 RPM for 10 minutes and serum was harvested and pooled. Following heat (complement) inactivation at 55°C for 30 minutes, serum was spun at 4000 RPM for 10 minutes, aliquoted, and frozen at −20°C until use. For adoptive transfer experiments, mice were administered i.p. 250 μL of PBS (vehicle), serum from PBS immunized mice, or serum iFt-immunized mice. Mice were then anesthetized and challenged with the indicated dose ofFt-LVS 24 hours after adoptive transfer. Survival was monitored for 21 days.


### 2.9.Ex Vivo Splenocyte Activation (Recall Response) Assay

On day 35 after primary immunization, spleen cells were harvested and diluted in medium to a concentration of 5 × 106 spleen cells/mL. 1000 μL of cells was added to wells of a 24-well plate (Costar Corning, Corning, NY) plate, each well containing an equivalent number of iFt-organisms. Alternatively 100 μL of cells was added to wells of a U-bottom 96-well plate (Falcon BD, Franklin Lakes, NJ) that contained a concentration range between 2.5 and 20 iFt per splenocyte. Plates were incubated at 37°C for up to 7 days in a humidity chamber to prevent medium evaporation. Supernatants were collected at 1, 3, 5, and 7 days and frozen at −20°C until they were analyzed. Samples were analyzed for cytokines via cytometric bead array (CBA) multiplex assay (BD Biosciences-BD Pharmingen, Sparks, Maryland). Data was acquired on a FACSArray Instrument and analyzed using CBA software version 1.0.1 (BD Immunocytometry Systems, Sparks, Maryland).


### 2.10. Cytokine Quantitation in Lungs and BALF

WT and FcγRIIB KO mice were immunized i.n. and on day 35 mice were given a sublethal challenge i.n. with approximately 0.4 × LD50 ofFt-LVS. Tissues were collected five days after challenge. Tissues were homogenized and centrifuged, and supernatants were collected. Luminex assay was performed on tissue supernatants to determinein vivo cytokine levels and to assess inflammation. The Luminex assay is a multiplex system for quantitation and detection of multiple cytokines in a single sample.


### 2.11. Statistical Analysis

The method of statistical analysis for each figure is described in the respective figure legends.


## 2.1. Reagents

RPMI medium was obtained from Cellgro Mediatech Inc. (Manassas, Va) into which was added 10% heat inactivated fetal bovine serum (FBS) (Hyclone Thermo Scientific, South Logan, Utah), 1% Pen/Strep (Hyclone Thermo Scientific, South Logan, Utah), 5 mL of 200 mM glutamine (100X) (Cellgro Mediatech Inc., Manassas, Va), 5 mL of 100 mM sodium pyruvate (100X) (Gibco Invitrogen, Grand Island, NY), 5 mL nonessential amino acids (AA) (100X) (Cellgro Mediatech Inc., Manassas, Va), and 250μL of 0.1 M 2-mercaptoethanol (2-ME) (Bio-rad, Hercules, CA). Media was then filter sterilized with a 0.22 μm filter and stored at 4°C.Ft-live vaccine strain (LVS) was utilized in these studies and was cultured in Mueller Hinton Broth (MHB) medium consisting of 490 mL of distilled, deionized cell culture grade water (Cellgro Mediatech Inc., Manassas, Va), 10.5 g MHB (Becton Dickinson, Sparks, Maryland), 0.069 g anhydrous calcium chloride (Acros Organics, NJ), 0.105 g hydrous magnesium chloride (hexahydrate) (MP Biomedicals, Solon, OH), 5 mL of glucose (Sigma-Aldrich, St. Louis, Missouri), 5 mL of ferric pyrophosphate (Sigma-Aldrich, St. Louis, Missouri), and 10 mL isovitalex (Becton Dickinson, Sparks, Maryland). MHB media was adjusted to a pH of 6.8, filtered (0.22 μm), and stored at 4°C for up to two weeks. Red blood cell (RBC) lysis buffer contained 4.13 g of ammonium chloride (Sigma-Aldrich, St. Louis, Missouri), 0.5 g of potassium bicarbonate (Sigma-Aldrich, St. Louis, Missouri), and 18.5 mg of EDTA (Sigma-Aldrich, St. Louis, Missouri) diluted in 500 mL of distilled water. The pH of the solution was adjusted to 7.2, after which the solution was filter-sterilized using a 0.22 μm filter and stored at 4°C.


## 2.2. Splenocyte Isolation

Spleens were isolated from mice and passed through a 70μm cell strainer (Fisherbrand, Houston, TX). The single cell suspension was collected in a sterile petri dish containing approximately 3 to 5 mL of media. The cell suspension was washed and RBCs were lysed using a RBC lysis buffer. The cell suspension was then passed through a second 70 μm cell strainer (Fisherbrand, Houston, TX) and collected in a 50 mL conical centrifuge tube containing approximately 10 mL of media. Cells were spun for 5 minutes at 1500 revolutions per minute (RPM) and resuspended in RBC lysis buffer. After 3 to 5 minutes (min), the reaction was quenched through the addition of 20 to 40 mL of media. Splenocytes were again spun for 5 min at 1500 RPM. Following resuspension, any residual tissue was removed via pipet or by again passing the splenocyte suspension through a 70 μm filter (Fisherbrand, Houston, TX). Splenocytes were subsequently washed twice with fresh media twice and enumerated using trypan blue.


## 2.3. Mice

C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME). Breeding pairs of FcγRIIB KO mice (B6; 129S4-Fcgr
2
b
tm
1
Ttk/J) on a C57BL background were obtained from Jackson Laboratories and bred in the Animal Resources Facility at Albany Medical College. Mice of 6–15 weeks old were used in order to identify significant differences under conditions that include a relatively broad range of ages. All animals were housed and cared for according to guidelines approved by the Institutional Animal Care and Use Committee.


## 2.4. Generation of iFt-Immunogen

iFt was used as immunogen. Briefly,Ft-LVS organisms were grown in MHB medium (Becton Dickinson, Sparks, Maryland) at 37°C to a density of 0.5–1 × 109 CFU/mL. 1 × 1010 CFU/mL live bacteria were then incubated in 1 mL of sterile PBS (Cellgro, Manassas, Virginia) containing 2% paraformaldehyde (Sigma-Aldrich, St. Louis, Missouri) for 2 hours at room temperature. The iFt-organisms were then washed with sterile PBS three times. Inactivation was verified by culturing a 100 μL sample (~1 × 109 iFt-organisms) on chocolate agar plates (Becton, Sparks, Maryland) for 7 to 10 days. The iFt preparations were stored at −20°C in PBS.


## 2.5. Immunization and Challenge Studies

Mice were divided into groups consisting of 6–8 mice per group. Each mouse was anesthetized by intraperitoneal (i.p.) injection of 20% ketamine (Vedco, St. Joseph, Missouri) plus 5% xylazine (Lloyd, Shenandoah, Iowa) diluted in sterile cell culture grade water (Cellgro, Manassas, Virginia). Mice were subsequently administered i.n. either 20μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS. Unless otherwise indicated, mice were immunized on day 0 and boosted on day 21. Immunized mice were then challenged on day 35 i.n. with either 4 × LD50 or 16 × LD50 of liveFt-LVS and subsequently monitored for survival for a minimum of 21 days.


## 2.6. Bacterial Burden Quantitation

Mice were immunized and subsequently challenged with a sublethal dose (approximately 0.4 × LD50) of liveFt-LVS. Five days after challenge, mice were sacrificed and tissues were collected. Tissues were homogenized with a mechanical bead beater and serially diluted with sterile PBS. Diluted tissue samples were plated on chocolate agar and CFU colonies were enumerated approximately 72 hours later.


## 2.7. Antibody Quantitation

Total mouse IgM, IgG, and IgA were measured by ELISA. Kits were purchased (Immunology Consultants Laboratory, Portland, OR) and the manufacturers protocol was followed. All reagents were brought to room temperature. Samples and standards were serially diluted and 100μL/well were added to plates precoated with anti-IgM, anti-IgG, or anti-IgA Abs. Plates were incubated at room temperature for 60 minutes. Following the incubation, plates were aspirated and washed, and 100 μL of the appropriate enzyme-Ab conjugate was added to each well. After 30 minutes, the plates were again washed and substrate solution was added. The reaction was stopped after 10 minutes with stop solution and the plates were read at 450 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, California).Ft-specific Ab production was also measured by ELISA. ELISA plates (Corning, Corning, New York) were coated with 50 μL/well of liveFt-LVS (5 × 107 CFU/mL in carbonate buffer containing 2.15 g sodium bicarbonate and 2.62 g sodium carbonate (Sigma-Aldrich, St. Louis, Missouri) diluted in 500 mL of sterile cell culture grade water (Cellgro, Manassas, Virginia) at pH 9.6–9.8), overnight at 4°C. The plates were then washed three times with 200 μL/well of PBS containing 0.5% bovine serum albumin (BSA) (Baxter Healthcare, Deerfield, Illinois) and 0.002% sodium azide (Sigma-Aldrich, St. Louis, Missouri). Plates were then blocked at 4°C for 2 hours with 200 μL/well of PBS containing 5% BSA and 0.02% sodium azide. Plates were again washed and serial dilutions of sera or bronchoalveolar lavage fluids (BALF) were added to the plates (50 μL/well) and incubated for 2 hours at 4°C. After washing, alkaline phosphatase conjugated anti-mouse Ab specific for total mouse Ab (Sigma-Aldrich, St. Louis, Missouri), IgG (Sigma-Aldrich, St. Louis, Missouri), IgA (Sigma-Aldrich, St. Louis, Missouri), IgG1 (Southern Biotech, Birmingham, AL), IgG2c (Abcam, Cambridge, MA), or IgM (Sigma-Aldrich, St. Louis, Missouri) were added and incubated for 1 hour at 4°C. Plates were washed and then 100 μL/well of alkaline phosphatase substrate (Sigma, St. Louis, MO) was added. All samples were read at 405 nm using a microplate reader (VersaMax, Molecular Devices, Sunnyvale, California) following a 5-second (sec) shake.


## 2.8. Adoptive Transfer Studies

Mice were immunized with iFt i.n. as described above and on day 35 mice were anesthetized and blood was collected via percutaneous cardiac puncture. Clotted blood samples were then spun at 8000 RPM for 10 minutes and serum was harvested and pooled. Following heat (complement) inactivation at 55°C for 30 minutes, serum was spun at 4000 RPM for 10 minutes, aliquoted, and frozen at −20°C until use. For adoptive transfer experiments, mice were administered i.p. 250 μL of PBS (vehicle), serum from PBS immunized mice, or serum iFt-immunized mice. Mice were then anesthetized and challenged with the indicated dose ofFt-LVS 24 hours after adoptive transfer. Survival was monitored for 21 days.


## 2.9.Ex Vivo Splenocyte Activation (Recall Response) Assay

On day 35 after primary immunization, spleen cells were harvested and diluted in medium to a concentration of 5 × 106 spleen cells/mL. 1000 μL of cells was added to wells of a 24-well plate (Costar Corning, Corning, NY) plate, each well containing an equivalent number of iFt-organisms. Alternatively 100 μL of cells was added to wells of a U-bottom 96-well plate (Falcon BD, Franklin Lakes, NJ) that contained a concentration range between 2.5 and 20 iFt per splenocyte. Plates were incubated at 37°C for up to 7 days in a humidity chamber to prevent medium evaporation. Supernatants were collected at 1, 3, 5, and 7 days and frozen at −20°C until they were analyzed. Samples were analyzed for cytokines via cytometric bead array (CBA) multiplex assay (BD Biosciences-BD Pharmingen, Sparks, Maryland). Data was acquired on a FACSArray Instrument and analyzed using CBA software version 1.0.1 (BD Immunocytometry Systems, Sparks, Maryland).


## 2.10. Cytokine Quantitation in Lungs and BALF

WT and FcγRIIB KO mice were immunized i.n. and on day 35 mice were given a sublethal challenge i.n. with approximately 0.4 × LD50 ofFt-LVS. Tissues were collected five days after challenge. Tissues were homogenized and centrifuged, and supernatants were collected. Luminex assay was performed on tissue supernatants to determinein vivo cytokine levels and to assess inflammation. The Luminex assay is a multiplex system for quantitation and detection of multiple cytokines in a single sample.


## 2.11. Statistical Analysis

The method of statistical analysis for each figure is described in the respective figure legends.


## 3. Results

### 3.1. Naïve FcγRIIB KO Mice Exhibit Increased Levels of Total Serum IgG versus That of Naïve WT Mice

Given the key role Ab plays in initiating FcγRIIB-mediated downmodulation of the immune response, we sought to verify prior studies demonstrating increased levels of total serum IgG in FcγRIIB KO versus WT mice [16]. We also examined serum levels of total IgM and total IgA, as well asFt-specific IgM and IgG, since mice have propensity for low basal levels of naturalFt-specific Ab [17]. As shown in Table 1, we observed no difference in total IgM levels between WT and FcγRIIB KO mice. However, naïve FcγRIIB KO mice exhibited higher levels of total serum IgG than their WT counterparts. Total IgA also appeared somewhat elevated in FcγRIIB KO versus WT mice. In regard to the presence of naturalFt-specific IgM and IgG Ab, there were no significant differences between FcγRIIB KO versus WT mice, although the median titer forFt-specific IgG was higher in FcγRIIB KO mice (Table 1).Table 1
Relative differences in Ab levels between naïve WT and naïve FcγRIIB KO mice.
Experimental group
Response measured
WT mice (median response)
KO mice (median response)
Fold changec
Naïve before challenge
Total serum IgMa
451
441
1.0
Total serum IgGa
758
1134
1.5*
Total serum IgAa
14
20
1.4
Ft-specific serum IgMb
88
85
1.0
Ft-specific serum IgGb
33
92
2.8
a
μg/mL of Ab.
bAb titer.
cFold change was caculated by deviding KO mice (median response) by WT mice (median response).
*
P < 0.05.


### 3.2. There Is No Difference in Survival of Naive FcγRIIB KO versus Naïve WT Mice Challenged withFt-LVS

Based on published studies demonstrating a beneficial effect on the immunity and/or protection of naïve FcγRIIB KO versus WT mice following infectious disease challenge [10, 11], we expected that the absence of FcγRIIB would enhance survival of naïve FcγRIIB KO mice challenged withFt-LVS. In fact, we observed no significant difference in survival between naïve FcγRIIB KO and WT mice at all challenge doses tested (Figures 1(a)–1(d)). Thus, the elevated median response levels of naturalFt-specific serum IgG Ab in naïve FcγRIIB KO mice versus WT mice (Table 1) had no impact on survival (Figures 1(a)–1(d)).Figure 1
There is no difference in survival of naive FcγRIIB KO versus naïve WT mice challenged withFt-LVS. Naïve WT C57BL/6J and FcγRIIB KO mice were challenged i.n. with 0.5 × LD50 (a), 1 × LD50 (b), 2 × LD50 (c), or 4 × LD50 (d) ofFt-LVS and survival was monitored for 25 days after challenge. Panel (a) represents 8 mice/group while panels (b)–(d) represent between 14 and 25 mice/group. Statistical analysis was determined by a contingency table analysis and two-tailed Fisher’s exact test on survival at day 25.
(a)
(b)
(c)
(d)


### 3.3.Ft-Specific IgA, but Not IgG, Is Significantly Increased in FcγRIIB KO versus WT Mice following iFt-Immunization

A number of published studies have demonstrated that in the absence of FcγRIIB, Ag-specific IgG production is enhanced upon immune stimulation [18–20]. Thus, we examined the production ofFt-specific Ab, including IgG, IgA, and IgM, in the serum and BALF of FcγRIIB KO versus WT mice immunized with iFt. In some cases, the titers obtained were highly variable between individual mice, which likely reflects not only normal mouse-to-mouse variation but also differences in immune responsiveness due to differences in the age of the individual mice and its effects on B cell repertoires [21]. Despite the latter, we did observe a significant increase in the production of totalFt-specific serum Ab in FcγRIIB KO versus WT mice following iFt-immunizations (Figure 2(a)). While there were no significant differences in the levels ofFt-specific serum IgG, IgG2c, IgG1, or IgM in the serum of these mice (Figures 2(b), 2(c), 2(d), or 2(e), resp.),Ft-specific IgA levels in serum were significantly increased (Figure 2(f)). While there is not a dramatic increase in the median response in total anti-Ft Ab titer between PBS and iFt-immunized FcγRIIB KO mice (Figure 2(a)), there is a significant increase in the median levels of IgG2c in iFt-immunized FcγRIIB KO mice versus PBS-immunized FcγRIIB KO mice (P
<
0.01) (Figure 2(c)). This suggests that despite the former observation regarding total anti-Ft Ab, iFt-immunized FcγRIIB KO mice produce increased levels of potentially high affinity IgG2c anti-Ft Ab. Lastly, there were no significant differences in the levels ofFt-specific IgG in the BALF of these mice, while the levels ofFt-specific IgA in the BALF of iFt-immunized FcγRIIB KO mice were significantly higher than that of WT mice (Figure 2(h)). The presence ofFt-specific Abs in naïve (Table 1) or unimmunized mice has been previously observed in mouse [17] and humans [22, 23] and is likely due to the presence of B1 cells producing natural antibodies stimulated in response to normal flora or self-Ag [24, 25].Figure 2
Ft-specific IgA, but not IgG, is significantly increased in FcγRIIB KO versus WT mice following iFt-immunization. FcγRIIB KO and WT mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. Serum was then collected on day 35 and levels of totalFt-specific Ab (a),Ft-specific IgG (b),Ft-specific IgG2c (c),Ft-specific IgG1 (d),Ft-specific IgM (e), andFt-specific IgA (f) were determined via ELISA. Titers ofFt-specific IgG (g) andFt-specific IgA (h) in BALF were similarly determined. Each symbol represents a single mouse. The 50% maximum of the highest experimental OD was determined and all experimental OD values were transformed to their respective log values. A log (agonist) versus response-variable slope (four parameters) nonlinear regression (curve fit) was performed on the log values, and the unknowns were interpolated. The inverse log was taken on the interpolated unknowns to determine the final titers for each individual mouse sample (error bars represent median with interquartile range of data distribution). Statistics were performed by adding 0.1 to the determined titer value and taking the log of this new value to correct for heteroscedasticity. A one-way ANOVA along with a Tukey’s multiple comparison test was performed, and median and interquartile range is graphed. ∗
∗
∗
P
<
0.001; ∗
P
<
0.05.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


### 3.4. iFt-Immunized FcγRIIB KO Mice Exhibit Increased Protection against a LethalFt-LVS Challenge as Compared to Their WT Counterparts

Given the integral role Ag-specific IgG plays in FcγRIIB-mediated immune modulation and the absence of increased levels ofFt-specific IgG in iFt-immunized FcγRIIB KO versus WT mice (Figure 2), we sought to determine whether there would be any impact of the presence versus absence of FcγRIIB on protection of iFt-immunized mice. Despite the lack of a significant increasedFt-specific IgG in FcγRIIB KO versus WT mice and in contrast to studies using naïve mice (Figure 1), at a challenge dose of 4 × LD50, the survival of iFt-immunized FcγRIIB KO mice was significantly better than that of WT mice (100% versus 50% resp.) (Figure 3(a)). Similar to that observed in naïve mice (Figure 1), there was no significant difference in survival between FcγRIIB KO and WT mice immunized with PBS (Figure 3(a)). When the challenge dose was increased to 16 × LD50, the overall level of survival decreased. However, a slight increase in the survival of iFt-immunized FcγRIIB KO versus WT mice was still apparent (Figure 3(b)). The increased survival also correlated with a reduction in the median bacterial burden in iFt-immunized FcγRIIB KO (Figure 3(c)).Figure 3
iFt-immunized FcγRIIB KO mice exhibit increased protection against a lethalFt-LVS challenge as compared to their WT counterparts. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. On day 35 mice were challenged i.n. with approximately 4 × LD50 (a) or 16 × LD50 (b) ofFt-LVS. Survival was then monitored for 21 to 25 days. Panel (a) represents between 12 and 14 mice/group while panel (b) represents between 17 and 20 mice/group. Statistical analysis was determined by a contingency table analysis and two-tailed Fisher’s exact test on survival at day 21. ∗
∗
P
<
0.01. In addition, bacterial burden in the lungs of challenged mice was also determined. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. On day 35 mice were given a sublethal challenge i.n. with approximately 0.4 × LD50 ofFt-LVS. Five days after challenge lungs were collected, homogenized, and plated on chocolate agar to determine bacterial burden as described in Section 2. Each symbol represents a single mouse. Statistical analysis of the tissue bacterial burden was done via a nonparametric one-way ANOVA (Kruskal-Wallis test) coupled with a Dunn’s multiple comparison after test. While there appeared to be a substantial reduction in bacterial burden in the majority of iFt-immunized FcγRIIB KO versus WT mice, the difference was not significant based on this analysis.
(a)
(b)
(c)


### 3.5. Adoptive Transfer of Serum from iFt-Immunized FcγRIIB KO and WT Mice to Naïve FcγRIIB KO and WT Mice, Respectively, Does Not Replicate the Differences in Survival Observed in iFt-Immunized FcγRIIB KO versus WT Mice

We surmised that while there were no differences inFt-specific IgG levels in iFt-immunized FcγRIIB KO versus WT mice (Figure 2), the observed increases inFt-specific IgA in the serum and BALF of FcγRIIB KO versus WT mice might be responsible for the increased protection observed (Figure 3). To test this, we adoptively transferred serum from iFt-immunized FcγRIIB KO and WT mice into their naive counterparts, in an effort to recapitulate the difference in survival obtained in Figure 3. As demonstrated previously by others [26], adoptive transfer of serum to naïve mice did increase protection againstFt-LVS challenge. However, a significant increase in protection in naïve FcγRIIB KO mice receiving serum from iFt-immunized FcγRIIB KO mice versus that of WT mice was not observed (Table 2).Table 2
Summary of adoptive transfer survival studies.
Group
Donor
Immunogen
Recipient
Percent protection
Mouse number
Statistical comparison
Significance (P valuea)
A
WT
PBS
WT
6.67
n = 15
B
KO
PBS
KO
26.67
n = 15
A versus B
P = 0.3295
C
WT
iFt
WT
53.33
n = 15
D
KO
iFt
KO
62.5
n = 16
C versus D
P = 0.7224
aThe P values were determined by performing a contingency table analysis and two-tailed Fisher’s exact test on survival at day 21 after challenge.


### 3.6. Splenocytes from iFt-Immunized FcγRIIB KO versus WT Mice Exhibit Enhanced IFN-γ, IL-10, and TNF-α Production in Response to iFt AddedEx Vivo

Given that IFN-γ, a Th1 cytokine, has been shown to play an important role in protection againstFt-infection [13, 27–30] and that FcγRIIB has been shown to downmodulate cellular immune responses, including Th1 responses [9, 11], we examined the impact of FcγRIIB’s absence on the production of IFN-γ by splenocytes from iFt-immunized FcγRIIB KO versus WT mice restimulatedex vivo with iFt. Consistent with the increased protection observed in iFt-immunized FcγRIIB KO versus WT mice (Figure 3) and a critical role for IFN-γ in mediating protection againstFt-infection, within three days following restimulation with iFt, splenocytes from iFt-immunized FcγRIIB KO mice exhibited increased levels of IFN-γ compared to that of WT splenocytes (Figure 4(a)). This remained the case at a series of iFt/splenocyte ratios ranging between 2.5 and 20 : 1 (Figure 4(b)). Furthermore, in this same experiment, we also examined the production of IL-10 and IL-17, since IL-10 restrains IL-17-induced lung pathology following pulmonaryFt-LVS infection [31]. We demonstrate that iFt-immunized FcγRIIB KO mice produce increased levels of IL-10 and lower levels of IL-17A than iFt-immunized WT mice, consistent with the ability of IL-10 to limit IL-17 induced pathology in FcγRIIB KO mice (Figures 4(c) and 4(d)), leading to increased survival (Figure 3). Finally, we demonstrate that iFt-immunized FcγRIIB KO mice produce higher levels of TNF-α compared to iFt-immunized WT mice (Figure 4(e)), another cytokine that also plays an essential role in survival against a primaryFt-infection [32].Figure 4
Splenocytes from iFt-immunized FcγRIIB KO versus WT mice exhibit enhanced IFN-γ, IL-10, and TNF-α production in response to iFt addedex vivo. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. (a) Splenocytes were then harvested from individual mice on day 35 and stimulated at a ratio of one iFt per splenocyte. Supernatants were then harvested at the indicated days. (b) FcγRIIB KO and WT mice were immunized and splenocytes were harvested as previously described. Splenocytes were then stimulated at increasing iFt to splenocyte ratios. Supernatants were harvested five days later and analyzed for IFN-γ, (c) IL-10, (d) IL-17A, and (e) TNF-α via CBA assay. A two-way ANOVA and a Bonferroni posttest was performed on the CBA determined sample concentrations to determine statistical significance, and mean and standard deviation is graphed. ∗
∗
∗
P
<
0.001; ∗
∗
P
<
0.01; ∗
P
<
0.05.
(a)
(b)
(c)
(d)
(e)


### 3.7. Levels of Proinflammatory Cytokines in the Lungs of iFt-Immunized FcγRIIB KO versus WT Mice Were Reduced 5 Days after Challenge

We have previously observed that although immunization with iFt initially stimulates increased production of inflammatory cytokines, including IFN-γ, on days 1–3, an overall reduction in such cytokines occursin vivo between days 5 and 7 after challenge. Furthermore, the latter decrease correlates with increased protection [13, 33]. Therefore, we also analyzed inflammatory cytokine levels in the lungs of iFt-immunized FcγRIIB KO versus WT 5-day mice after challenge. Consistent with our prior observations, significantly lower levels of IFN-γ and MCP-1 in the lungs and TNF-α in the BALF of iFt-immunized FcγRIIB KO versus WT mice were observed (Figures 5(c), 5(e), and 5(h), resp.). There were also reductions in the median cytokine levels for IL-6 and TNF-α in the lungs of these same mice (Figures 5(a) and 5(g), resp.), as well as IL-6 (Figure 5(b)), IFN-γ (Figure 5(d)), and MCP-1 (Figure 5(f)) in BALF.Figure 5
Levels of proinflammatory cytokines in the lungs of iFt-immunized FcγRIIB KO versus WT mice were reduced 5 days after challenge. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. On day 35 mice were given a sublethal challenge i.n. with approximately 0.4 × LD50. Five days after challenge lung tissue was collected, homogenized, and centrifuged and supernatants were collected. Luminex assay was performed on tissue supernatants to determinein vivo cytokine levels. Each symbol represents a single mouse. A value of 0.1 was added to the average cytokine concentration to correct for heteroscedasticity. A one-way ANOVA along with a Tukey’s multiple comparison test was performed on the log of the average cytokine concentration of lung and BALF samples from individual mice to determine statistical significance, and median and interquartile range is graphed. ∗
P
<
0.05.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.1. Naïve FcγRIIB KO Mice Exhibit Increased Levels of Total Serum IgG versus That of Naïve WT Mice

Given the key role Ab plays in initiating FcγRIIB-mediated downmodulation of the immune response, we sought to verify prior studies demonstrating increased levels of total serum IgG in FcγRIIB KO versus WT mice [16]. We also examined serum levels of total IgM and total IgA, as well asFt-specific IgM and IgG, since mice have propensity for low basal levels of naturalFt-specific Ab [17]. As shown in Table 1, we observed no difference in total IgM levels between WT and FcγRIIB KO mice. However, naïve FcγRIIB KO mice exhibited higher levels of total serum IgG than their WT counterparts. Total IgA also appeared somewhat elevated in FcγRIIB KO versus WT mice. In regard to the presence of naturalFt-specific IgM and IgG Ab, there were no significant differences between FcγRIIB KO versus WT mice, although the median titer forFt-specific IgG was higher in FcγRIIB KO mice (Table 1).Table 1
Relative differences in Ab levels between naïve WT and naïve FcγRIIB KO mice.
Experimental group
Response measured
WT mice (median response)
KO mice (median response)
Fold changec
Naïve before challenge
Total serum IgMa
451
441
1.0
Total serum IgGa
758
1134
1.5*
Total serum IgAa
14
20
1.4
Ft-specific serum IgMb
88
85
1.0
Ft-specific serum IgGb
33
92
2.8
a
μg/mL of Ab.
bAb titer.
cFold change was caculated by deviding KO mice (median response) by WT mice (median response).
*
P < 0.05.


## 3.2. There Is No Difference in Survival of Naive FcγRIIB KO versus Naïve WT Mice Challenged withFt-LVS

Based on published studies demonstrating a beneficial effect on the immunity and/or protection of naïve FcγRIIB KO versus WT mice following infectious disease challenge [10, 11], we expected that the absence of FcγRIIB would enhance survival of naïve FcγRIIB KO mice challenged withFt-LVS. In fact, we observed no significant difference in survival between naïve FcγRIIB KO and WT mice at all challenge doses tested (Figures 1(a)–1(d)). Thus, the elevated median response levels of naturalFt-specific serum IgG Ab in naïve FcγRIIB KO mice versus WT mice (Table 1) had no impact on survival (Figures 1(a)–1(d)).Figure 1
There is no difference in survival of naive FcγRIIB KO versus naïve WT mice challenged withFt-LVS. Naïve WT C57BL/6J and FcγRIIB KO mice were challenged i.n. with 0.5 × LD50 (a), 1 × LD50 (b), 2 × LD50 (c), or 4 × LD50 (d) ofFt-LVS and survival was monitored for 25 days after challenge. Panel (a) represents 8 mice/group while panels (b)–(d) represent between 14 and 25 mice/group. Statistical analysis was determined by a contingency table analysis and two-tailed Fisher’s exact test on survival at day 25.
(a)
(b)
(c)
(d)


## 3.3.Ft-Specific IgA, but Not IgG, Is Significantly Increased in FcγRIIB KO versus WT Mice following iFt-Immunization

A number of published studies have demonstrated that in the absence of FcγRIIB, Ag-specific IgG production is enhanced upon immune stimulation [18–20]. Thus, we examined the production ofFt-specific Ab, including IgG, IgA, and IgM, in the serum and BALF of FcγRIIB KO versus WT mice immunized with iFt. In some cases, the titers obtained were highly variable between individual mice, which likely reflects not only normal mouse-to-mouse variation but also differences in immune responsiveness due to differences in the age of the individual mice and its effects on B cell repertoires [21]. Despite the latter, we did observe a significant increase in the production of totalFt-specific serum Ab in FcγRIIB KO versus WT mice following iFt-immunizations (Figure 2(a)). While there were no significant differences in the levels ofFt-specific serum IgG, IgG2c, IgG1, or IgM in the serum of these mice (Figures 2(b), 2(c), 2(d), or 2(e), resp.),Ft-specific IgA levels in serum were significantly increased (Figure 2(f)). While there is not a dramatic increase in the median response in total anti-Ft Ab titer between PBS and iFt-immunized FcγRIIB KO mice (Figure 2(a)), there is a significant increase in the median levels of IgG2c in iFt-immunized FcγRIIB KO mice versus PBS-immunized FcγRIIB KO mice (P
<
0.01) (Figure 2(c)). This suggests that despite the former observation regarding total anti-Ft Ab, iFt-immunized FcγRIIB KO mice produce increased levels of potentially high affinity IgG2c anti-Ft Ab. Lastly, there were no significant differences in the levels ofFt-specific IgG in the BALF of these mice, while the levels ofFt-specific IgA in the BALF of iFt-immunized FcγRIIB KO mice were significantly higher than that of WT mice (Figure 2(h)). The presence ofFt-specific Abs in naïve (Table 1) or unimmunized mice has been previously observed in mouse [17] and humans [22, 23] and is likely due to the presence of B1 cells producing natural antibodies stimulated in response to normal flora or self-Ag [24, 25].Figure 2
Ft-specific IgA, but not IgG, is significantly increased in FcγRIIB KO versus WT mice following iFt-immunization. FcγRIIB KO and WT mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. Serum was then collected on day 35 and levels of totalFt-specific Ab (a),Ft-specific IgG (b),Ft-specific IgG2c (c),Ft-specific IgG1 (d),Ft-specific IgM (e), andFt-specific IgA (f) were determined via ELISA. Titers ofFt-specific IgG (g) andFt-specific IgA (h) in BALF were similarly determined. Each symbol represents a single mouse. The 50% maximum of the highest experimental OD was determined and all experimental OD values were transformed to their respective log values. A log (agonist) versus response-variable slope (four parameters) nonlinear regression (curve fit) was performed on the log values, and the unknowns were interpolated. The inverse log was taken on the interpolated unknowns to determine the final titers for each individual mouse sample (error bars represent median with interquartile range of data distribution). Statistics were performed by adding 0.1 to the determined titer value and taking the log of this new value to correct for heteroscedasticity. A one-way ANOVA along with a Tukey’s multiple comparison test was performed, and median and interquartile range is graphed. ∗
∗
∗
P
<
0.001; ∗
P
<
0.05.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 3.4. iFt-Immunized FcγRIIB KO Mice Exhibit Increased Protection against a LethalFt-LVS Challenge as Compared to Their WT Counterparts

Given the integral role Ag-specific IgG plays in FcγRIIB-mediated immune modulation and the absence of increased levels ofFt-specific IgG in iFt-immunized FcγRIIB KO versus WT mice (Figure 2), we sought to determine whether there would be any impact of the presence versus absence of FcγRIIB on protection of iFt-immunized mice. Despite the lack of a significant increasedFt-specific IgG in FcγRIIB KO versus WT mice and in contrast to studies using naïve mice (Figure 1), at a challenge dose of 4 × LD50, the survival of iFt-immunized FcγRIIB KO mice was significantly better than that of WT mice (100% versus 50% resp.) (Figure 3(a)). Similar to that observed in naïve mice (Figure 1), there was no significant difference in survival between FcγRIIB KO and WT mice immunized with PBS (Figure 3(a)). When the challenge dose was increased to 16 × LD50, the overall level of survival decreased. However, a slight increase in the survival of iFt-immunized FcγRIIB KO versus WT mice was still apparent (Figure 3(b)). The increased survival also correlated with a reduction in the median bacterial burden in iFt-immunized FcγRIIB KO (Figure 3(c)).Figure 3
iFt-immunized FcγRIIB KO mice exhibit increased protection against a lethalFt-LVS challenge as compared to their WT counterparts. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. On day 35 mice were challenged i.n. with approximately 4 × LD50 (a) or 16 × LD50 (b) ofFt-LVS. Survival was then monitored for 21 to 25 days. Panel (a) represents between 12 and 14 mice/group while panel (b) represents between 17 and 20 mice/group. Statistical analysis was determined by a contingency table analysis and two-tailed Fisher’s exact test on survival at day 21. ∗
∗
P
<
0.01. In addition, bacterial burden in the lungs of challenged mice was also determined. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. On day 35 mice were given a sublethal challenge i.n. with approximately 0.4 × LD50 ofFt-LVS. Five days after challenge lungs were collected, homogenized, and plated on chocolate agar to determine bacterial burden as described in Section 2. Each symbol represents a single mouse. Statistical analysis of the tissue bacterial burden was done via a nonparametric one-way ANOVA (Kruskal-Wallis test) coupled with a Dunn’s multiple comparison after test. While there appeared to be a substantial reduction in bacterial burden in the majority of iFt-immunized FcγRIIB KO versus WT mice, the difference was not significant based on this analysis.
(a)
(b)
(c)


## 3.5. Adoptive Transfer of Serum from iFt-Immunized FcγRIIB KO and WT Mice to Naïve FcγRIIB KO and WT Mice, Respectively, Does Not Replicate the Differences in Survival Observed in iFt-Immunized FcγRIIB KO versus WT Mice

We surmised that while there were no differences inFt-specific IgG levels in iFt-immunized FcγRIIB KO versus WT mice (Figure 2), the observed increases inFt-specific IgA in the serum and BALF of FcγRIIB KO versus WT mice might be responsible for the increased protection observed (Figure 3). To test this, we adoptively transferred serum from iFt-immunized FcγRIIB KO and WT mice into their naive counterparts, in an effort to recapitulate the difference in survival obtained in Figure 3. As demonstrated previously by others [26], adoptive transfer of serum to naïve mice did increase protection againstFt-LVS challenge. However, a significant increase in protection in naïve FcγRIIB KO mice receiving serum from iFt-immunized FcγRIIB KO mice versus that of WT mice was not observed (Table 2).Table 2
Summary of adoptive transfer survival studies.
Group
Donor
Immunogen
Recipient
Percent protection
Mouse number
Statistical comparison
Significance (P valuea)
A
WT
PBS
WT
6.67
n = 15
B
KO
PBS
KO
26.67
n = 15
A versus B
P = 0.3295
C
WT
iFt
WT
53.33
n = 15
D
KO
iFt
KO
62.5
n = 16
C versus D
P = 0.7224
aThe P values were determined by performing a contingency table analysis and two-tailed Fisher’s exact test on survival at day 21 after challenge.


## 3.6. Splenocytes from iFt-Immunized FcγRIIB KO versus WT Mice Exhibit Enhanced IFN-γ, IL-10, and TNF-α Production in Response to iFt AddedEx Vivo

Given that IFN-γ, a Th1 cytokine, has been shown to play an important role in protection againstFt-infection [13, 27–30] and that FcγRIIB has been shown to downmodulate cellular immune responses, including Th1 responses [9, 11], we examined the impact of FcγRIIB’s absence on the production of IFN-γ by splenocytes from iFt-immunized FcγRIIB KO versus WT mice restimulatedex vivo with iFt. Consistent with the increased protection observed in iFt-immunized FcγRIIB KO versus WT mice (Figure 3) and a critical role for IFN-γ in mediating protection againstFt-infection, within three days following restimulation with iFt, splenocytes from iFt-immunized FcγRIIB KO mice exhibited increased levels of IFN-γ compared to that of WT splenocytes (Figure 4(a)). This remained the case at a series of iFt/splenocyte ratios ranging between 2.5 and 20 : 1 (Figure 4(b)). Furthermore, in this same experiment, we also examined the production of IL-10 and IL-17, since IL-10 restrains IL-17-induced lung pathology following pulmonaryFt-LVS infection [31]. We demonstrate that iFt-immunized FcγRIIB KO mice produce increased levels of IL-10 and lower levels of IL-17A than iFt-immunized WT mice, consistent with the ability of IL-10 to limit IL-17 induced pathology in FcγRIIB KO mice (Figures 4(c) and 4(d)), leading to increased survival (Figure 3). Finally, we demonstrate that iFt-immunized FcγRIIB KO mice produce higher levels of TNF-α compared to iFt-immunized WT mice (Figure 4(e)), another cytokine that also plays an essential role in survival against a primaryFt-infection [32].Figure 4
Splenocytes from iFt-immunized FcγRIIB KO versus WT mice exhibit enhanced IFN-γ, IL-10, and TNF-α production in response to iFt addedex vivo. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. (a) Splenocytes were then harvested from individual mice on day 35 and stimulated at a ratio of one iFt per splenocyte. Supernatants were then harvested at the indicated days. (b) FcγRIIB KO and WT mice were immunized and splenocytes were harvested as previously described. Splenocytes were then stimulated at increasing iFt to splenocyte ratios. Supernatants were harvested five days later and analyzed for IFN-γ, (c) IL-10, (d) IL-17A, and (e) TNF-α via CBA assay. A two-way ANOVA and a Bonferroni posttest was performed on the CBA determined sample concentrations to determine statistical significance, and mean and standard deviation is graphed. ∗
∗
∗
P
<
0.001; ∗
∗
P
<
0.01; ∗
P
<
0.05.
(a)
(b)
(c)
(d)
(e)


## 3.7. Levels of Proinflammatory Cytokines in the Lungs of iFt-Immunized FcγRIIB KO versus WT Mice Were Reduced 5 Days after Challenge

We have previously observed that although immunization with iFt initially stimulates increased production of inflammatory cytokines, including IFN-γ, on days 1–3, an overall reduction in such cytokines occursin vivo between days 5 and 7 after challenge. Furthermore, the latter decrease correlates with increased protection [13, 33]. Therefore, we also analyzed inflammatory cytokine levels in the lungs of iFt-immunized FcγRIIB KO versus WT 5-day mice after challenge. Consistent with our prior observations, significantly lower levels of IFN-γ and MCP-1 in the lungs and TNF-α in the BALF of iFt-immunized FcγRIIB KO versus WT mice were observed (Figures 5(c), 5(e), and 5(h), resp.). There were also reductions in the median cytokine levels for IL-6 and TNF-α in the lungs of these same mice (Figures 5(a) and 5(g), resp.), as well as IL-6 (Figure 5(b)), IFN-γ (Figure 5(d)), and MCP-1 (Figure 5(f)) in BALF.Figure 5
Levels of proinflammatory cytokines in the lungs of iFt-immunized FcγRIIB KO versus WT mice were reduced 5 days after challenge. WT C57BL/6J and FcγRIIB KO mice were immunized i.n. with 20 μL of PBS (vehicle) or 2 × 107 iFt-organisms in 20 μL of PBS on day 0 and boosted on day 21. On day 35 mice were given a sublethal challenge i.n. with approximately 0.4 × LD50. Five days after challenge lung tissue was collected, homogenized, and centrifuged and supernatants were collected. Luminex assay was performed on tissue supernatants to determinein vivo cytokine levels. Each symbol represents a single mouse. A value of 0.1 was added to the average cytokine concentration to correct for heteroscedasticity. A one-way ANOVA along with a Tukey’s multiple comparison test was performed on the log of the average cytokine concentration of lung and BALF samples from individual mice to determine statistical significance, and median and interquartile range is graphed. ∗
P
<
0.05.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)


## 4. Discussion

### 4.1. The Impact of FcγRIIB on Primary Infection

A very limited number of studies thus far have focused on the role of FcγRIIB in resolving primary infections [9–12]. Specifically, they suggest that, during primary infection, the absence of FcγRIIB results in an improved immune response to infection, which can be beneficial to the host [9–11]. In contrast, other studies suggest that, following immunization, the absence of FcγRIIB leads to an overproduction of cytokines and potential septic shock [11] or no differences in immunity and/or protection [11, 12]. However, our studies using theFt-infectious disease model demonstrate that the absence of FcγRIIB has little or no impact on the outcome of survival following primary infection, while its absence following vaccination and challenge increases the protective efficacy of the vaccine. A number of likely explanations exist for our observations. In regard to primary infection of naïve mice, our studies and those of others have demonstrated FcγRIIB deficient mice exhibit higher levels of total IgG [6, 16]. Our studies also demonstrate that there is no significant difference inFt-specific IgG or total IgA Abs in naïve FcγRIIB KO versus WT mice (Table 1). The latter, in addition to the fact that the formation ofFt-anti-Ft Ab complexes would be necessary to actively engage FcγRIIB and impact immunity and protection, may explain a failure to see differences in protection between naïve FcγRIIB KO and WT mice. Furthermore, the generation ofFt-specific IgG in response to infection would not be expected to occur until approximately day 7 after infection [26], the same point at which mice generally succumb to infection (Figure 1).


### 4.2. The Impact of FcγRIIB on Infection following Vaccination

In contrast to studies involving immunization againstS. pneumoniae and subsequent challenge [11], iFt-immunized mice are better protected when FcγRIIB is absent (Figure 3). This is also despite the lack of significant differences in the levels ofFt-specific IgG production in FcγRIIB KO versus WT mice following iFt-immunization (Figure 2). Importantly, however, both IgG and IgA can mediate protection againstFt-infection [13, 29, 30, 34–38]. Thus, the significant increase inFt-specific IgA production in iFt-immunized FcγRIIB KO versus WT mice (Figure 2) could explain the increased protection observed with iFt-immunized FcγRIIB KO versus WT mice. Never the less, a role forFt-specific IgA in mediating the increased protection observed (Figure 3) is not supported by adoptive transfer studies. Specifically, no significant difference in protection was observed in naïve recipient FcγRIIB KO versus WT mice following adoptive transfer of sera from iFt-immunized FcγRIIB KO or WT donors, respectively (Table 2). However, cellular immune responses can also play a crucial role in survival against a lethalFt-challenge [13, 27–30, 34–38]. In this regard, we do show splenocytes from iFt-immunized FcγRIIB KO mice incubated with iFt ex vivo produce substantially more IFN-γ, IL-10, and TNF-α than their WT counterparts, while producing less IL-17, which has been associated with increased pathology (Figure 4) [31]. Thus, this observation more likely explains the enhanced protection observed in iFt-immunized FcγRIIB KO mice. However, a role forFt-specific IgA cannot be totally excluded, in that our prior studies have demonstrated the requirement for both IgA and IFN-γ in protection of iFt-immunized mice followingFt-LVS challenge [13, 33]. Lastly, our studies, and those of others, have demonstrated that a reduction in the levels of proinflammatory cytokines 5–7 days after challenge, correlates with increased protection in theFt-infectious disease model [13, 29, 33, 37, 39]. Accordingly, we also observed a significant reduction in proinflammatory cytokines in the lungs of iFt-immunized FcγRIIB KO versus WT mice 5 days after challenge (Figure 5). Furthermore, this decrease in proinflammatory cytokines in lungs of iFt-immunized FcγRIIB KO versus WT mice at 5 days after challenge also correlated with a decrease in bacterial burden in the lungs of these animals, reflecting the ability of iFt-immunized FcγRIIB KO mice to better controlFt-infection than their WT counterparts (Figure 3).


### 4.3. Consideration of FcγRIIB’s Regulatory Role in the Development of Vaccines

We have recently demonstrated that targeting iFt to Fc receptor (FcR) in the form of monoclonal Ab- (mAb-) iFt complex administered i.n., provides superior protection to that of iFt [13]. However, a major concern regarding FcγR-targeted vaccines, when utilizing Fc to target vaccine Ags, is their ability to bind to FcγRIIB, as well as activating FcγRs, which could potentially dampen the response to vaccination and thereby limit efficacy. Thus, FcR-targeted vaccines that bypass FcγRIIB in favor of activating FcγR, such as one recently developed in our laboratory, which targets the activating FcγR (human FcγRI) [40], could significantly increase the efficacy of such vaccines. Alternatively, as we have demonstrated in this paper, FcγRIIB can also limit the stimulatory capacity of non-FcR-targeted vaccines. Thus, the use of FcγRIIB antagonists as vaccine adjuvants could significantly enhance the efficacy of vaccines in general.


## 4.1. The Impact of FcγRIIB on Primary Infection

A very limited number of studies thus far have focused on the role of FcγRIIB in resolving primary infections [9–12]. Specifically, they suggest that, during primary infection, the absence of FcγRIIB results in an improved immune response to infection, which can be beneficial to the host [9–11]. In contrast, other studies suggest that, following immunization, the absence of FcγRIIB leads to an overproduction of cytokines and potential septic shock [11] or no differences in immunity and/or protection [11, 12]. However, our studies using theFt-infectious disease model demonstrate that the absence of FcγRIIB has little or no impact on the outcome of survival following primary infection, while its absence following vaccination and challenge increases the protective efficacy of the vaccine. A number of likely explanations exist for our observations. In regard to primary infection of naïve mice, our studies and those of others have demonstrated FcγRIIB deficient mice exhibit higher levels of total IgG [6, 16]. Our studies also demonstrate that there is no significant difference inFt-specific IgG or total IgA Abs in naïve FcγRIIB KO versus WT mice (Table 1). The latter, in addition to the fact that the formation ofFt-anti-Ft Ab complexes would be necessary to actively engage FcγRIIB and impact immunity and protection, may explain a failure to see differences in protection between naïve FcγRIIB KO and WT mice. Furthermore, the generation ofFt-specific IgG in response to infection would not be expected to occur until approximately day 7 after infection [26], the same point at which mice generally succumb to infection (Figure 1).


## 4.2. The Impact of FcγRIIB on Infection following Vaccination

In contrast to studies involving immunization againstS. pneumoniae and subsequent challenge [11], iFt-immunized mice are better protected when FcγRIIB is absent (Figure 3). This is also despite the lack of significant differences in the levels ofFt-specific IgG production in FcγRIIB KO versus WT mice following iFt-immunization (Figure 2). Importantly, however, both IgG and IgA can mediate protection againstFt-infection [13, 29, 30, 34–38]. Thus, the significant increase inFt-specific IgA production in iFt-immunized FcγRIIB KO versus WT mice (Figure 2) could explain the increased protection observed with iFt-immunized FcγRIIB KO versus WT mice. Never the less, a role forFt-specific IgA in mediating the increased protection observed (Figure 3) is not supported by adoptive transfer studies. Specifically, no significant difference in protection was observed in naïve recipient FcγRIIB KO versus WT mice following adoptive transfer of sera from iFt-immunized FcγRIIB KO or WT donors, respectively (Table 2). However, cellular immune responses can also play a crucial role in survival against a lethalFt-challenge [13, 27–30, 34–38]. In this regard, we do show splenocytes from iFt-immunized FcγRIIB KO mice incubated with iFt ex vivo produce substantially more IFN-γ, IL-10, and TNF-α than their WT counterparts, while producing less IL-17, which has been associated with increased pathology (Figure 4) [31]. Thus, this observation more likely explains the enhanced protection observed in iFt-immunized FcγRIIB KO mice. However, a role forFt-specific IgA cannot be totally excluded, in that our prior studies have demonstrated the requirement for both IgA and IFN-γ in protection of iFt-immunized mice followingFt-LVS challenge [13, 33]. Lastly, our studies, and those of others, have demonstrated that a reduction in the levels of proinflammatory cytokines 5–7 days after challenge, correlates with increased protection in theFt-infectious disease model [13, 29, 33, 37, 39]. Accordingly, we also observed a significant reduction in proinflammatory cytokines in the lungs of iFt-immunized FcγRIIB KO versus WT mice 5 days after challenge (Figure 5). Furthermore, this decrease in proinflammatory cytokines in lungs of iFt-immunized FcγRIIB KO versus WT mice at 5 days after challenge also correlated with a decrease in bacterial burden in the lungs of these animals, reflecting the ability of iFt-immunized FcγRIIB KO mice to better controlFt-infection than their WT counterparts (Figure 3).


## 4.3. Consideration of FcγRIIB’s Regulatory Role in the Development of Vaccines

We have recently demonstrated that targeting iFt to Fc receptor (FcR) in the form of monoclonal Ab- (mAb-) iFt complex administered i.n., provides superior protection to that of iFt [13]. However, a major concern regarding FcγR-targeted vaccines, when utilizing Fc to target vaccine Ags, is their ability to bind to FcγRIIB, as well as activating FcγRs, which could potentially dampen the response to vaccination and thereby limit efficacy. Thus, FcR-targeted vaccines that bypass FcγRIIB in favor of activating FcγR, such as one recently developed in our laboratory, which targets the activating FcγR (human FcγRI) [40], could significantly increase the efficacy of such vaccines. Alternatively, as we have demonstrated in this paper, FcγRIIB can also limit the stimulatory capacity of non-FcR-targeted vaccines. Thus, the use of FcγRIIB antagonists as vaccine adjuvants could significantly enhance the efficacy of vaccines in general.


## 5. Conclusions

This study is the first to demonstrate that (1) the absence of FcγRIIB does not affect the susceptibility of mice to a primary infection with the intracellular Category A mucosal pathogenF. tularensis; (2) that in the absence of FcγRIIB, both humoral and cellular immunity are enhanced following immunization with the inactivated vaccine iFt; (3) that the level of Ag-specific IgA produced in response to vaccination can be impacted by the presence/absence of FcγRIIB; (4) that the enhanced immune responses observed (Ft-specific IgA and IFN-γ production) following iFt-immunization correlate with increased protection of FcγRIIB KO versus WT mice after lethal mucosal challenge withFt LVS. Thus, these studies further expand our knowledge regarding the role of FcγRIIB plays in the immune response to infection, while also providing further impetus for developing vaccines geared toward modulating the inhibitory activities of this receptor.



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*Source: 840842-2015-04-19.xml*