fc gamma receptor iib ( fcriib ) , which is expressed on b cells , as well as other antigen ( ag ) presenting cells ( apc ) , is the only fc gamma receptor ( fcr ) known to negatively regulate the immune response and play an important role in b - cell regulation and antibody ( ab ) production [ 17 ] .
ag - specific ab generated in response to infection , immunization , or the administration of fcr - targeted vaccines , when complexed with ag , has the potential to interact with fcriib and significantly downmodulate immunity and protection against infectious agents .
importantly , fcriib is also expressed on dendritic cells ( dcs ) and macrophages and can negatively regulate cellular as well as humoral immunity [ 13 , 6 , 8 ] .
consistent with the regulation of cellular immunity , it has been demonstrated that utilizing a mycobacterium tuberculosis ( mtb ) model in which nave wildtype ( wt ) and fcriib knockout ( ko ) mice received an aerosol challenge of mtb , fcriib ko mice exhibited an enhanced cellular immune response compared to their wt counterparts , which included increased ifn- production . in another study , following staphylococcus aureus ( sa ) infection , nave fcriib ko mice were better protected against a primary intravenous infection with sa .
however , in contrast to the above results using nave mice , if fcriib ko mice were first immunized with a pneumococcal vaccine and then challenged with high doses of streptococcus pneumoniae , mortality rates were increased above that of wt mice , correlating with increased proinflammatory cytokine production . yet , in another study in which plasmodium berghei parasitemia was induced postimmunization , parasitemia was not impacted following intradermal immunization of fcriib ko versus wt mice with a plasmodium berghei vaccine . thus , while enhanced immunity and/or protection against infection was observed in nave fcriib ko versus wt mice , the absence of fcriib had either negative or no impact on immunity and/or protection following immunization . to further clarify the role of fcriib in the generation of protective immunity against infection , we investigated the impact of fcriib on f. tularensis ( ft ) challenge before and after immunization with inactivated ft ( ift ) in wt versus fcriib ko mice .
ft is a gram - negative intracellular pathogen that in designated a category a biothreat agent due to its extreme virulence [ 1315 ] .
based on the published studies cited above using nave fcriib ko mice [ 911 ] , we hypothesized that nave mice challenged with ft would be better protected in the absence of fcriib .
in contrast , based on the published studies above using immunized fcriib ko mice [ 11 , 12 ] , we predicted a negative or very limited impact of the absence of fcriib on survival following ft - challenge of ift - immunized fcriib ko versus wt mice . in fact , the opposite was observed in the case of both nave and ift - immunized mice . specifically , there was no difference in survival of nave fcriib ko versus wt mice challenged with ft .
in contrast , in the case of ift - immunized fcriib ko versus wt mice , protection was significantly improved in fcriib ko mice .
the latter correlated with enhanced ft - specific iga production in vivo , enhanced recall responses in the form of increased ifn- production by ex vivo splenocytes incubated with ift , and reduced in vivo proinflammatory cytokine production 5 days after challenge .
thus , these studies demonstrate for the first time that the presence of fcriib can significantly dampen immunity and protection against infection following immunization .
these are also the first studies to suggest a role of fcriib in the regulation of ag - specific iga production .
( 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 250  l of 0.1  m 2-mercaptoethanol ( 2-me ) ( bio - rad , hercules , ca ) .
media was then filter sterilized with a 0.22  m filter and stored at 4c .
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  m ) , and stored at 4c 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 .
the ph of the solution was adjusted to 7.2 , after which the solution was filter - sterilized using a 0.22  m filter and stored at 4c .
spleens were isolated from mice and passed through a 70  m 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  m 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 .
following resuspension , any residual tissue was removed via pipet or by again passing the splenocyte suspension through a 70  m filter ( fisherbrand , houston , tx ) .
breeding pairs of fcriib ko mice ( b6 ; 129s4-fcgr2b / j ) on a c57bl background were obtained from jackson laboratories and bred in the animal resources facility at albany medical college .
mice of 615 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 .
briefly , ft - lvs organisms were grown in mhb medium ( becton dickinson , sparks , maryland ) at 37c to a density of 0.51  10  cfu / ml . 1  10  cfu / ml live bacteria were then incubated in 1  ml of sterile pbs ( cellgro , manassas , virginia ) containing 2% paraformaldehyde ( sigma - aldrich , st .
inactivation was verified by culturing a 100  l sample ( ~1  10  ift - organisms ) on chocolate agar plates ( becton , sparks , maryland ) for 7 to 10 days .
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 20  l of pbs ( vehicle ) or 2  10  ift - organisms in 20  l 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 live ft - lvs and subsequently monitored for survival for a minimum of 21 days .
mice were immunized and subsequently challenged with a sublethal dose ( approximately 0.4  ld50 ) of live ft - lvs .
diluted tissue samples were plated on chocolate agar and cfu colonies were enumerated approximately 72 hours later .
kits were purchased ( immunology consultants laboratory , portland , or ) and the manufacturers protocol was followed .
samples and standards were serially diluted and 100  l / well were added to plates precoated with anti - igm , anti - igg , or anti - iga abs .
following the incubation , plates were aspirated and washed , and 100  l of the appropriate enzyme - ab conjugate was added to each well .
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 ) .
elisa plates ( corning , corning , new york ) were coated with 50  l / well of live ft - lvs ( 5  10  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 9.69.8 ) , overnight at 4c .
the plates were then washed three times with 200  l / well of pbs containing 0.5% bovine serum albumin ( bsa ) ( baxter healthcare , deerfield , illinois ) and 0.002% sodium azide ( sigma - aldrich , st .
plates were then blocked at 4c for 2 hours with 200  l / 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  l / well ) and incubated for 2 hours at 4c .
after washing , alkaline phosphatase conjugated anti - mouse ab specific for total mouse ab ( sigma - aldrich , st .
louis , missouri ) , igg1 ( southern biotech , birmingham , al ) , igg2c ( abcam , cambridge , ma ) , or igm ( sigma - aldrich , st .
plates were washed and then 100  l / well of alkaline phosphatase substrate ( sigma , st .
all samples were read at 405  nm using a microplate reader ( versamax , molecular devices , sunnyvale , california ) following a 5-second ( sec ) shake .
clotted blood samples were then spun at 8000  rpm for 10 minutes and serum was harvested and pooled . following heat ( complement ) inactivation at 55c for 30 minutes , serum was spun at 4000  rpm for 10 minutes , aliquoted , and frozen at 20c until use . for adoptive transfer experiments ,
250  l of pbs ( vehicle ) , serum from pbs immunized mice , or serum ift - immunized mice .
mice were then anesthetized and challenged with the indicated dose of ft - lvs 24 hours after adoptive transfer .
, spleen cells were harvested and diluted in medium to a concentration of 5  10  spleen  cells / ml .
1000  l 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  l 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 37c 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 20c 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 ) .
wt and fcriib ko mice were immunized i.n . and on day 35 mice were given a sublethal challenge i.n . with approximately 0.4  ld50 of ft - lvs .
luminex assay was performed on tissue supernatants to determine in 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 .
the method of statistical analysis for each figure is described in the respective figure legends .
given the key role ab plays in initiating fcriib - mediated downmodulation of the immune response , we sought to verify prior studies demonstrating increased levels of total serum igg in fcriib ko versus wt mice .
we also examined serum levels of total igm and total iga , as well as ft - specific igm and igg , since mice have propensity for low basal levels of natural ft - specific ab .
as shown in table 1 , we observed no difference in total igm levels between wt and fcriib ko mice .
however , nave fcriib ko mice exhibited higher levels of total serum igg than their wt counterparts .
total iga also appeared somewhat elevated in fcriib ko versus wt mice . in regard to the presence of natural ft - specific igm and igg ab , there were no significant differences between fcriib ko versus wt mice , although the median titer for ft - specific igg was higher in fcriib ko mice ( table 1 ) .
based on published studies demonstrating a beneficial effect on the immunity and/or protection of nave fcriib ko versus wt mice following infectious disease challenge [ 10 , 11 ] , we expected that the absence of fcriib would enhance survival of nave fcriib ko mice challenged with ft - lvs .
in fact , we observed no significant difference in survival between nave fcriib ko and wt mice at all challenge doses tested ( figures 1(a)1(d ) ) .
thus , the elevated median response levels of natural ft - specific serum igg ab in nave fcriib ko mice versus wt mice ( table 1 ) had no impact on survival ( figures 1(a)1(d ) ) .
a number of published studies have demonstrated that in the absence of fcriib , ag - specific igg production is enhanced upon immune stimulation [ 1820 ] .
thus , we examined the production of ft - specific ab , including igg , iga , and igm , in the serum and balf of fcriib 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 . despite the latter ,
we did observe a significant increase in the production of total ft - specific serum ab in fcriib ko versus wt mice following ift - immunizations ( figure 2(a ) ) . while there were no significant differences in the levels of ft - 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 fcriib ko mice ( figure 2(a ) ) , there is a significant increase in the median levels of igg2c in ift - immunized fcriib ko mice versus pbs - immunized fcriib ko mice ( p < 0.01 ) ( figure 2(c ) ) .
this suggests that despite the former observation regarding total anti - ft ab , ift - immunized fcriib ko mice produce increased levels of potentially high affinity igg2c anti - ft ab .
lastly , there were no significant differences in the levels of ft - specific igg in the balf of these mice , while the levels of ft - specific iga in the balf of ift - immunized fcriib ko mice were significantly higher than that of wt mice ( figure 2(h ) ) .
the presence of ft - specific abs in nave ( table 1 ) or unimmunized mice has been previously observed in mouse   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 ] . given the integral role ag - specific igg plays in fcriib - mediated immune modulation and the absence of increased levels of ft - specific igg in ift - immunized fcriib ko versus wt mice ( figure 2 ) , we sought to determine whether there would be any impact of the presence versus absence of fcriib on protection of ift - immunized mice .
despite the lack of a significant increased ft - specific igg in fcriib ko versus wt mice and in contrast to studies using nave mice ( figure 1 ) , at a challenge dose of 4  ld50 , the survival of ift - immunized fcriib ko mice was significantly better than that of wt mice ( 100% versus 50% resp . ) ( figure 3(a ) ) .
similar to that observed in nave mice ( figure 1 ) , there was no significant difference in survival between fcriib ko and wt mice immunized with pbs ( figure 3(a ) ) . when the challenge dose was increased to 16  ld50
however , a slight increase in the survival of ift - immunized fcriib 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 fcriib ko ( figure 3(c ) ) .
we surmised that while there were no differences in ft - specific igg levels in ift - immunized fcriib ko versus wt mice ( figure 2 ) , the observed increases in ft - specific iga in the serum and balf of fcriib ko versus wt mice might be responsible for the increased protection observed ( figure 3 ) . to test this , we adoptively transferred serum from ift - immunized fcriib 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 , adoptive transfer of serum to nave mice did increase protection against ft - lvs challenge .
however , a significant increase in protection in nave fcriib ko mice receiving serum from ift - immunized fcriib ko mice versus that of wt mice was not observed ( table 2 ) . given that ifn- , a th1 cytokine , has been shown to play an important role in protection against ft - infection [ 13 , 2730 ] and that fcriib has been shown to downmodulate cellular immune responses , including th1 responses [ 9 , 11 ] , we examined the impact of fcriib 's absence on the production of ifn- by splenocytes from ift - immunized fcriib ko versus wt mice restimulated ex vivo with ift .
consistent with the increased protection observed in ift - immunized fcriib ko versus wt mice ( figure 3 ) and a critical role for ifn- in mediating protection against ft - infection , within three days following restimulation with ift , splenocytes from ift - immunized fcriib ko mice exhibited increased levels of ifn- 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 pulmonary ft - lvs infection .
we demonstrate that ift - immunized fcriib 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 fcriib ko mice ( figures 4(c ) and 4(d ) ) , leading to increased survival ( figure 3 ) .
finally , we demonstrate that ift - immunized fcriib ko mice produce higher levels of tnf- compared to ift - immunized wt mice ( figure 4(e ) ) , another cytokine that also plays an essential role in survival against a primary ft - infection .
we have previously observed that although immunization with ift initially stimulates increased production of inflammatory cytokines , including ifn- , on days 13 , an overall reduction in such cytokines occurs in vivo between days 5 and 7 after challenge .
therefore , we also analyzed inflammatory cytokine levels in the lungs of ift - immunized fcriib ko versus wt 5-day mice after challenge .
consistent with our prior observations , significantly lower levels of ifn- and mcp-1 in the lungs and tnf- in the balf of ift - immunized fcriib 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- in the lungs of these same mice ( figures 5(a ) and 5(g ) , resp . ) , as well as il-6 ( figure 5(b ) ) , ifn- ( figure 5(d ) ) , and mcp-1 ( figure 5(f ) ) in balf .
a very limited number of studies thus far have focused on the role of fcriib in resolving primary infections [ 912 ] . specifically , they suggest that , during primary infection , the absence of fcriib results in an improved immune response to infection , which can be beneficial to the host [ 911 ] . in contrast , other studies suggest that , following immunization , the absence of fcriib leads to an overproduction of cytokines and potential septic shock   or no differences in immunity and/or protection [ 11 , 12 ] .
however , our studies using the ft - infectious disease model demonstrate that the absence of fcriib 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 nave mice , our studies and
those of others have demonstrated fcriib deficient mice exhibit higher levels of total igg [ 6 , 16 ] .
our studies also demonstrate that there is no significant difference in ft - specific igg or total iga abs in nave fcriib ko versus wt mice ( table 1 ) .
the latter , in addition to the fact that the formation of ft - anti - ft ab complexes would be necessary to actively engage fcriib and impact immunity and protection , may explain a failure to see differences in protection between nave fcriib ko and wt mice . furthermore , the generation of ft - specific igg in response to infection would not be expected to occur until approximately day 7 after infection , the same point at which mice generally succumb to infection ( figure 1 ) .
in contrast to studies involving immunization against s. pneumoniae and subsequent challenge , ift - immunized mice are better protected when fcriib is absent ( figure 3 ) .
this is also despite the lack of significant differences in the levels of ft - specific igg production in fcriib ko versus wt mice following ift - immunization ( figure 2 ) .
importantly , however , both igg and iga can mediate protection against ft - infection [ 13 , 29 , 30 , 3438 ] .
thus , the significant increase in ft - specific iga production in ift - immunized fcriib ko versus wt mice ( figure 2 ) could explain the increased protection observed with ift - immunized fcriib ko versus wt mice .
never the less , a role for ft - 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 nave recipient fcriib ko versus wt mice following adoptive transfer of sera from ift - immunized fcriib ko or wt donors , respectively ( table 2 ) .
however , cellular immune responses can also play a crucial role in survival against a lethal ft - challenge [ 13 , 2730 , 3438 ] . in this regard , we do show splenocytes from ift - immunized fcriib ko mice incubated with ift ex vivo produce substantially more ifn- , il-10 , and tnf- than their wt counterparts , while producing less il-17 , which has been associated with increased pathology ( figure 4 ) .
thus , this observation more likely explains the enhanced protection observed in ift - immunized fcriib ko mice .
however , a role for ft - specific iga can not be totally excluded , in that our prior studies have demonstrated the requirement for both iga and ifn- in protection of ift - immunized mice following ft - lvs challenge [ 13 , 33 ] .
lastly , our studies , and those of others , have demonstrated that a reduction in the levels of proinflammatory cytokines 57 days after challenge , correlates with increased protection in the ft - infectious disease model [ 13 , 29 , 33 , 37 , 39 ] .
accordingly , we also observed a significant reduction in proinflammatory cytokines in the lungs of ift - immunized fcriib ko versus wt mice 5 days after challenge ( figure 5 ) .
furthermore , this decrease in proinflammatory cytokines in lungs of ift - immunized fcriib 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 fcriib ko mice to better control ft - infection than their wt counterparts ( figure 3 ) .
we have recently demonstrated that targeting ift to fc receptor ( fcr ) in the form of monoclonal ab- ( mab- ) ift complex administered i.n .
however , a major concern regarding fcr - targeted vaccines , when utilizing fc to target vaccine ags , is their ability to bind to fcriib , as well as activating fcrs , which could potentially dampen the response to vaccination and thereby limit efficacy .
thus , fcr - targeted vaccines that bypass fcriib in favor of activating fcr , such as one recently developed in our laboratory , which targets the activating fcr ( human fcri ) , could significantly increase the efficacy of such vaccines . alternatively , as we have demonstrated in this paper , fcriib can also limit the stimulatory capacity of non - fcr - targeted vaccines .
thus , the use of fcriib antagonists as vaccine adjuvants could significantly enhance the efficacy of vaccines in general .
this study is the first to demonstrate that ( 1 ) the absence of fcriib does not affect the susceptibility of mice to a primary infection with the intracellular category a mucosal pathogen f. tularensis ; ( 2 ) that in the absence of fcriib , 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 fcriib ; ( 4 ) that the enhanced immune responses observed ( ft - specific iga and ifn- production ) following ift - immunization correlate with increased protection of fcriib ko versus wt mice after lethal mucosal challenge with ft lvs . thus , these studies further expand our knowledge regarding the role of fcriib plays in the immune response to infection , while also providing further impetus for developing vaccines geared toward modulating the inhibitory activities of this receptor .
 fc gamma receptor iib ( fcriib ) is the only fc gamma receptor ( fcr ) 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 fcriib on immune protection against francisella tularensis ( ft ) , a category a biothreat agent . 
 we utilized inactivated ft ( ift ) as an immunogen . 
 nave and ift - immunized fcriib knockout ( ko ) or wildtype ( wt ) mice were challenged with ft - live vaccine strain ( lvs ) . 
 while no significant difference in survival between nave fcriib ko versus wt mice was observed , ift - immunized fcriib ko mice were significantly better protected than ift - immunized wt mice . 
 ft - specific iga in serum and bronchial alveolar lavage , as well as ifn- , il-10 , and tnf- production by splenocytes harvested from ift - immunized fcriib ko , were also significantly elevated . 
 in addition , ift - immunized fcriib 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 fcriib 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 . 