mTOR Promotes Antiviral Humoral Immunity by Differentially Regulating CD4 Helper T Cell and B ...

17 Pages·2017·4.49 MB·English

PATHOGENESIS AND IMMUNITY
crossm
mTOR Promotes Antiviral Humoral
Immunity by Differentially Regulating
CD4 Helper T Cell and B Cell Responses
LilinYe,a,bJunghwaLee,bLifanXu,aAta-Ur-RasheedMohammed,bWeiyanLi,b
J.ScottHale,bWendyG.Tan,bTuoqiWu,b*CarlW.Davis,bRafiAhmed,b
KoichiArakib D
o
InstituteofImmunology,ThirdMilitaryMedicalUniversity,Chongqing,Chinaa;EmoryVaccineCenterand w
DepartmentofMicrobiologyandImmunology,EmoryUniversitySchoolofMedicine,Atlanta,Georgia,USAb n
lo
a
ABSTRACT mTOR has important roles in regulation of both innate and adaptive im- d
Received19August2016 Accepted5 e
munity, but whether and how mTOR modulates humoral immune responses have d
December2016
yet to be fully understood. To address this issue, we examined the effects of rapa- Acceptedmanuscriptpostedonline14 fro
mycin, a specific inhibitor of mTOR, on B cell and CD4 T cell responses during acute December2016 m
infection with lymphocytic choriomeningitis virus. Rapamycin treatment resulted in CitationYeL,LeeJ,XuL,MohammedA-U-R,Li h
suppression of virus-specific B cell responses by inhibiting proliferation of germinal W,HaleJS,TanWG,WuT,DavisCW,AhmedR, ttp
ArakiK.2017.mTORpromotesantiviral
:
center (GC) B cells. In contrast, the number of memory CD4 T cells was increased in humoralimmunitybydifferentiallyregulating //
rapamycin-treated mice. However, the drug treatment caused a striking bias of CD4 C91D:e40h1e6l5p3e-r1T6.chetlltpans:d//dBocie.ollrgre/s1p0o.1n1s2e8s/.JVirol jvi.a
T cell differentiation into Th1 cells and substantially impaired formation of follicular JVI.01653-16. s
m
helper T (Tfh) cells, which are essential for humoral immunity. Further experiments EditorTerenceS.Dermody,Universityof
.
in which mTOR signaling was modulated by RNA interference (RNAi) revealed that B PittsburghSchoolofMedicine o
r
Copyright©2017AmericanSocietyfor g
cells were the primary target cells of rapamycin for the impaired humoral immunity /
Microbiology.AllRightsReserved. o
and that reduced Tfh formation in rapamycin-treated mice was due to lower GC B AddresscorrespondencetoKoichiAraki, n

cell responses that are essential for Tfh generation. Additionally, we found that [email protected]. M
rapamycin had minimal effects on B cell responses activated by lipopolysaccha- *Presentaddress:TuoqiWu,NationalHuman a
y
ride (LPS), which stimulates B cells in an antigen-independent manner, suggest- GenomeResearchInstitute,NationalInstitutes 6
ofHealth,Bethesda,Maryland,USA. ,
ing that rapamycin specifically inhibits B cell responses induced by B cell recep- 2
0
tor stimulation with antigen. Together, these findings demonstrate that mTOR 1
signals play an essential role in antigen-specific humoral immune responses by 9

b
differentially regulating B cell and CD4 T cell responses during acute viral infec- y

tion and that rapamycin treatment alters the interplay of immune cell subsets in- g
u
volved in antiviral humoral immunity. e
s
t
IMPORTANCE mTOR is a serine/threonine kinase involved in a variety of cellular ac-
tivities. Although its specific inhibitor, rapamycin, is currently used as an immuno-
suppressive drug in transplant patients, it has been reported that rapamycin can
also stimulate pathogen-specific cellular immunity in certain circumstances. How-
ever, whether and how mTOR regulates humoral immunity are not well understood.
Here we found that rapamycin treatment predominantly inhibited GC B cell re-
sponses during viral infection and that this led to biased helper CD4 T cell differenti-
ation as well as impaired antibody responses. These findings suggest that inhibition
of B cell responses by rapamycin may play an important role in regulation of
allograft-specific antibody responses to prevent organ rejection in transplant recipi-
ents. Our results also show that consideration of antibody responses is required in
caseswhererapamycinisusedtostimulatevaccine-inducedimmunity.
KEYWORDS Bcellresponses,CD4Tcells,Th1/Tfhresponse,humoralimmunity,
immunization,mTOR,rapamycin,viralimmunity,viralinfection
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Asanevolutionarilyconservedserine/threoninekinase,mTORregulatesseveralkey
cellular processes, including cell growth, proliferation, and metabolism, in re-
sponsetovariousenvironmentalcues(1–3).Rapamycin,adrugthatspecificallyinhibits
mTOR function, is currently used in transplant patients as an immunosuppressant to
prevent immune responses against allografts (4, 5). One potential mechanism under-
lyingthistreatmentisthatrapamycininhibitstheproliferationofalloreactiveTcells(6),
but recent findings suggest that rapamycin has a variety of effects on the immune
system and that there may be more complex mechanisms resulting in immunosup-
pression (7). A better understanding of the exact target cells and mechanisms of
rapamycin-mediated immune suppression will facilitate the development of more
effectiveandrationalregimenstoinhibitalloreactiveimmuneresponseswithminimal
suppressionofpathogen-specificimmunity.
Despite generally being considered an immunosuppressive drug, rapamycin also
exhibits immunostimulatory effects in certain circumstances. Several reports have D
shownthatrapamycinenhancesinflammatorycytokinesecretionandantigenpresen- o
w
tationinmacrophagesandmyeloiddendriticcells(8,9).Moreover,inhibitingmTORin n
CD8 T cells can promote memory T cell differentiation in response to infection and lo
a
vaccination (10, 11). Thus, modulating mTOR activity has now become an attractive d
e
strategytoenhancedendriticcell-orCD8Tcell-mediatedimmunityduringvaccination. d
However, considering that most successful vaccines confer protective immunity by f
r
o
inducingantibodyresponses(12),itiscriticaltounderstandwhetherandhowmTOR m
regulates the generation of antigen-specific antibodies. Recent studies examined the
h
role of mTOR in B cells and found that mTOR signals are required for production of tt
p
high-affinityantibodies(13–15).ThesestudiesfocusedonBcellresponses(13–15),but :
/
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the interplay between CD4 T cells and B cells remains unclear in cases where mTOR jv
signals are inhibited in both CD4 T cells and B cells in vivo, such as by rapamycin i.a
s
treatment.Thus,examiningtherelationshipbetweenCD4TcellandBcellresponsesis m
importantforacomprehensiveunderstandingoftheeffectsofrapamycinonhumoral .o
immunity,sinceantibodyresponsesarecoordinatedbybothCD4TcellsandBcells. rg
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Following acute viral infections, antigen-specific naive CD4 T cells predominantly o
differentiateintoTh1andfollicularhelperT(Tfh)cells(16–19).Th1cellsplayacritical n

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roleinmediatingcellularimmunity,whileTfhcellsaretheprincipalTcellsthatprovide
a
necessary help to B cells for the initiation and maintenance of germinal center (GC) y

reaction. Although the mTOR pathway has been shown to regulate differentiation of 6
,

naive CD4 T cells into effector CD4 T cells (20–23), how in vivo rapamycin treatment 2
0
influences effector and memory CD4 T cell differentiation has yet to be fully under- 1
9
stood. Similar to that in CD4 T cells, the function of mTOR in B cell responses also
b
remainstobedetermined. y

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Inthepresentstudy,weattemptedtoexaminehowrapamycininfluencesBcelland
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CD4Tcellresponsesinvivobyusingamousemodelofacuteinfectionwithlympho- e
s
cytic choriomeningitis virus (LCMV). Our results showed that rapamycin treatment t
inhibitedthegenerationoflong-termantibodyresponsesbyreducinggerminalcenter
B cell formation. We also found that Tfh responses were significantly inhibited in
rapamycin-treatedmice,althoughthedrugtreatmentenhancedoverallmemoryCD4
Tcelldevelopment.Tofurtherdissecttheeffectofrapamycin,weinvestigatedtherole
ofmTORintrinsicallyinCD4TcellsandBcellsinthisstudy.OurresultsshowthatmTOR
promotesantiviralhumoralimmunitybydifferentiallyregulatingCD4helperTcelland
Bcellresponses.
RESULTS
RapamycininhibitsBcellresponsesduringviralinfectionandvaccination.To
understand the role of mTOR in humoral immunity during acute viral infections,
rapamycin was administered to mice infected with LCMV strain Armstrong, which
causesasystemicacuteinfection,withvirusbeingclearedwithin8daysafterinfection.
SerumIgMandIgGantibodiesspecificforLCMVwereexaminedatdays8,15,and
60 postinfection (p.i.). We found comparable serum IgM titers between treated and
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FIG1Rapamycin(Rapa)inhibitsBcellresponsesduringacuteviralinfection.B6micewereinfectedwithLCMVArmstrong,andrapamycinwas b
administeredfromdays(cid:2)1to30postinfection.(A)AnalysisofLCMV-specificserumIgM(left)andIgG(right)levelsattheindicatedtimepoints y

postinfection.(B)(Left)FrequenciesofPNAhighFAShighB220(cid:3)germinalcenter(GC)Bcellsareshowninflowcytometryplots.(Right)Numbersof g
GCBcellsinthespleenattheindicatedtimepoints.(C)GChistologyinspleensectionsfromLCMV-infectedmicetreatedwitheithervehicleor u
e
rapamycinfromdays(cid:2)1to9andsacrificedonday10postinfection.Bars,10(cid:2)m.(D)NumbersofLCMV-specificantibody-secretingcells(ASCs) s
inbonemarrowonday60postinfection(rapamycintreatmentwasperformedondays(cid:2)1to30postinfection),calculatedbyELISPOTassay. t
Individualrepresentativewellsforvehicle-andrapamycin-treatedmiceareshownabovethebargraph.(E)LCMV-specificmemoryBcellnumbers,
analyzedonday60postinfection(rapamycintreatmentwasperformedondays(cid:2)1to30postinfection).(F)LCMVNP-specificIgGinserumon
day60afterLCMVinfectionwasdetectedbyELISAformicetreatedornotwithrapamycin.Foraffinitymeasurements,NP-capturedIgGwas
washedwithPBSor8Mureabeforedetection.(G)Todeterminerelativeaffinity,theratiooftheODwithureatotheODwithoutureawas
calculated at a 1:200 serum dilution. Data show means and standard errors of the means (SEM) (n (cid:4) 4 mice for each time point) and are
representativeoftwoindependentexperimentsforpanelsAtoE.ForpanelsFandG,datafromtwoindependentexperimentswerecombined
(n(cid:4)8miceforvehiclegroupandn(cid:4)5miceforrapamycingroup).
untreatedmiceatday8postinfection(Fig.1A,leftpanel).Althoughrapamycin-treated
micehadslightlyhigherlevelsofvirus-specificIgMtitersonday15afterinfection,IgM
responsesinbothgroupsweretransientandwerebelowthedetectionlimitonday60
after infection (Fig. 1A, left panel). In sharp contrast, rapamycin treatment led to
reduced LCMV-specific IgG titers (Fig. 1A, right panel). The significant reduction in
LCMV-specific IgG in rapamycin-treated mice was already seen at an early stage of
infection (day 8) (Fig. 1A, right panel). Although IgG titers were increased at day 15
postinfectioncomparedtothoseonday8forrapamycin-treatedmice,theyweremuch
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FIG 2Rapamycin inhibits IgG responses in protein and Ad5 immunization models. B6 mice were tp
immunizedwitheitherNP-KLH(precipitatedinalum)(A)orAd5(B).Rapamycinwasinjectedintraperi- :/
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toneallydailyfor30days.Serumwasharvestedontheindicateddays,andtheantigen-specificIgGtiter jv
wasquantifiedbyELISA.GraphsshowmeansandSEM(n(cid:4)5mice/timepointforeachgroup).Data i.
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representativeoftwoindependentexperimentsarepresented. s
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lower than those of control animals (Fig. 1A, right panel), suggesting that rapamycin rg
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inhibitsordelaysBcellactivation/proliferationduringtheearlystageofBcellresponses o
after viral infection. Importantly, this reduction was maintained at the memory stage, n

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andLCMV-specificIgGtitersinrapamycin-treatedmicewere10-foldlowerthanthose
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in vehicle controls at day 60 postinfection (Fig. 1A, right panel). The lower IgG titers y

during the memory stage for the rapamycin treatment group suggest that the drug 6
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may negatively regulate GC reaction, because the generation of long-term LCMV- 2
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specific IgG responses is strictly GC B cell dependent (24). In agreement with this 1
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notion,rapamycin-treatedmiceexhibitedamuchlowerfrequencyofPNAhighFAShigh
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GCBcellsatbothdays8and15postinfection(Fig.1B,leftpanel),andthetotalnumber y

g
ofPNAhighFAShighBcellsinrapamycin-treatedmicewasabout10-foldlowerthanthat
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in vehicle-treated mice at both time points (Fig. 1B, right panel). Likewise, the drug e
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treatmentblockedtheformationofnormalGCclusters(Fig.1C).Inkeepingwiththese t
data,GC-derivedLCMV-specificantibody-secretingcells(ASCs)inthebonemarrowand
memoryBcellsinthespleenwerefoundtobegreatlydecreasedinrapamycin-treated
micecomparedtovehicle-treatedmiceatday60postinfection(Fig.1DandE,respec-
tively). We also measured affinity maturation of antibody by using an enzyme-linked
immunosorbent assay (ELISA) combined with a urea wash after the antibody had
boundtotheantigenontheplate(Fig.1FandG).Wefoundthatrapamycinmodestly
impairedaffinitymaturationofLCMV-specificIgG(Fig.1FandG).
We next tested whether the inhibitory effects of rapamycin on B cell responses
could be generalized to other immunization systems. To examine this, mice were
immunizedwithNP-KLHinthepresenceorabsenceofrapamycin,andnucleoprotein
(NP)-specificIgGtitersweremeasuredatdays8,15,and30postimmunization.Signif-
icantly lower IgG titers were observed in rapamycin-treated mice than in vehicle-
treated mice at day 8 postimmunization (Fig. 2A). Similar to the results for LCMV
infection, rapamycin-treated mice showed increased IgG titers at days 15 and 30
postimmunization,butthetitersneverreachedthelevelsseenforvehicle-treatedmice
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FIG3RapamycininhibitsearlybutnotlateBcellresponses.B6micewereinfectedwithLCMVArmstrong,and lo
rapamycinwasinjectedattheindicatedtimes.(A)LCMV-specificIgGtitersinserumfortheindicatedrapamycin a
treatmentperiods.Thetiterwasmeasuredatday31postinfection(rapamycintreatmentdays(cid:2)1to14or16to de
30) or day 121 postinfection (rapamycin treatment days 90 to 120). (B) Numbers of LCMV-specific antibody- d
secretingcells(ASCs),analyzedatday31postinfection.(C)NumbersofmemoryBcells(MBCs),analyzedatday31 f
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postinfection.(D)NumbersofLCMV-specificASCs,analyzedatday121postinfection.GraphsshowmeansandSEM o
(n(cid:4)5mice/group).Datarepresentativeoftwoindependentexperimentsarepresented. m

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(Fig.2A).Wealsoexaminedtheeffectofrapamycinduringvaccinationwithanonrep- :
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licatingadenovirusserotype5vector(Ad5).Consistently,rapamycintreatmentresulted jv
in reduced Ad5-specific IgG titers throughout the observation period, and the drug- i.a
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treatedmiceshowedabout5-foldlowerIgGtitersthanthoseofthevehiclecontrolsat m
day 30 postvaccination (Fig. 2B). Collectively, these data indicate that rapamycin .
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treatmentsignificantlyinhibitsIgGBcellresponsesafterviralinfectionorvaccinations. rg
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RapamycininhibitsearlybutnotlateBcellresponses.Next,inordertoexamine
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whichstageoftheBcellresponseisinhibitedbyrapamycin,wetreatedLCMV-infected n

micewithrapamycinduringtheearlyphase(days(cid:2)1to14p.i.),thelatephase(days M
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16to30p.i.),orthememoryphase(days90to120p.i.)ofBcellresponses.Incontrast y

to the severe inhibition of LCMV-specific IgG responses with rapamycin administered 6
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fromdays(cid:2)1to14,rapamycintreatmentatthelatephaseormemoryphaseofBcell 2
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responses had minimal to no effect on antibody titers as well as the formation of 1
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memoryBcells/long-livedplasmacells(Fig.3AtoD).ThesedatasuggestthatmTORis
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dispensableduringthelateandmemoryphasesofBcellresponsesbutiscrucialduring y

the early phase, when B cells are actively proliferating with antigen stimulation and g
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formGC. e
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Effect of rapamycin on expression of survival/apoptosis-related molecules in t
GC B cells. The reduction in GC B cell number by rapamycin treatment may be
attributedtolowersurvivalofthesecellsthanofcellsexposedtovehicletreatment.To
test this, we analyzed the expression of the survival/apoptosis-associated molecules
Mcl-1,Bcl-xl,Bim,andactivepan-caspaseonGCBcellsinspleensfromrapamycin-or
vehicle-treated mice on day 10 after infection. We observed comparable expression
levelsofMcl-1,Bcl-xl,andBimonGCBcellsfrombothgroupsandlowerlevelsofactive
pan-caspase for the rapamycin-treated group than for the vehicle-treated group (Fig.
4AtoD).Sinceactivepan-caspaseisamarkerofcellsundergoingapoptosis,ourdata
indicatethatapoptosisofGCBcellsisnottheprimaryreasonfortheimpairedBcell
responsesinducedbyrapamycintreatmentduringacuteviralinfection.
RapamycininhibitstheproliferationofgerminalcenterBcells.Oneofthekey
features of GC B cells is their ability to proliferate rapidly and extensively during the
early phase of the B cell response. To examine whether rapamycin inhibits GC B cell
proliferation, LCMV-infected mice that had been treated with rapamycin or vehicle
were given bromodeoxyuridine (BrdU) for 3 h before sacrifice on days 9 and 11
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FIG4 Effectofrapamycinonexpressionofsurvival/apoptosis-relatedmoleculesingerminalcenter(GC)Bcells. D
LCMVArmstrong-infectedB6miceweretreateddailywithrapamycinfromday(cid:2)1postinfection.Flowcytometry o
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analysisofthesurvival/apoptosis-relatedmoleculesBim(A),Bcl-xl(B),Mcl-1(C),andactivatedpan-caspase(D)on
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PNAhighFAShighB220(cid:3)germinalcenterBcellsfromvehicle-orrapamycin-treatedmicewasperformedonday10 lo
postinfection.Datarepresentativeoftwoindependentexperimentsarepresented(n(cid:4)4mice/group). a
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postinfection.TheamountofBrdUincorporatedintoPNAhighFashighBcellswas2-to f
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3-foldlowerinrapamycin-treatedmiceatbothtimepoints(Fig.5A),indicatingthatthe m
proliferation of GC B cells was inhibited by rapamycin. Moreover, we found that
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compared to control mice, rapamycin-treated mice had a smaller fraction of CD86low tt
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CXCR4highdark-zoneGCBcells(Fig.5B),whichpossessintrinsicallyhigherproliferation :
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potentialthantheirlight-zonecounterparts(25).Wealsoanalyzedtheexpressionofthe jv
transcription factor B cell lymphoma 6 (Bcl-6; increases the proliferation of PNAhigh i.a
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Fashigh B cells), and we found that rapamycin-treated mice had fewer Bcl-6(cid:3) PNAhigh m
Fashigh B cells than vehicle-treated mice (Fig. 5C). Finally, rapamycin treatment also .o
impaired ongoing GC B cell proliferation when administered from days 9 to 14 rg
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postinfection (Fig. 5D). Together, these data show that rapamycin inhibits the prolif-
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erationofGCBcells,andthisseemstobeaprimarycauseoftheimpairedformation n

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ofmemoryBcellsandlong-livedplasmacells.
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Rapamycin enhances the formation of virus-specific CD4 T cell memory. The y

results described above clearly establish that rapamycin treatment inhibits B cell 6
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responses. Because CD4 T cell help is required for optimal B cell responses, we next 2
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examined if rapamycin-treated mice would develop normal CD4 T cell responses. 1
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LCMV-specific CD4 T cell responses were monitored using the I-Ab GP tetramer.
66–77 b
Interestingly, the administration of rapamycin from day (cid:2)1 to day 30 postinfection y

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substantiallyenhancedthenumberofvirus-specificCD4Tcellsatday60postinfection
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(Fig.6A).Weobservedsimilarnumbersofantigen-specificCD4Tcellsatthepeakofthe e
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responses(days8to15p.i.)betweenbothgroups,butrapamycintreatmentincreased t
thesurvivalofantigen-specificCD4Tcellsduringthecontractionphase(Fig.6A).This
reduced contraction is consistent with previously published data showing that rapa-
mycin enhanced memory CD8 T cell formation (10). To examine the functionality of
thesememoryCD4Tcellsinducedinthepresenceofrapamycin,westimulatedspleen
cells with the LCMV GP peptide. Mice treated with rapamycin had around 3-fold
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higherfrequenciesofIFN-(cid:3)(cid:3)TNF-(cid:4)(cid:3)andIFN-(cid:3)(cid:3)IL-2(cid:3)CD4Tcellsthanmicetreated
with the vehicle control (Fig. 6B). Thus, rapamycin was shown here to enhance the
formationoffunctionalantigen-specificmemoryCD4Tcells.
Rapamycin treatment alters the balance between Th1 and Tfh cell differenti-
ation. The observation that rapamycin markedly impaired antigen-specific B cell re-
sponsesdespitealargernumberofmemoryCD4Tcellspromptedustoinvestigateif
the drug treatment would modulate the differentiation program of antigen-specific
CD4Tcells.AcuteLCMVinfectionisknowntoinducetwodistinctCD4Tcellsubsets:
Th1andTfhcells(17).Therefore,acriticalquestiontoaddresswaswhetherthebalance
of the differentiation of antigen-specific Th1 and Tfh subsets would be altered in
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FIG5RapamycininhibitsgerminalcenterBcellproliferation.B6micewereinfectedwithLCMVArmstrong.(A)Rapamycinwasadministereddailyfromday g
(cid:2)1ofinfection,andBrdUwasgivenintraperitoneally3hpriortosacrifice.ThepercentagesofBrdU(cid:3)cellsinPNAhighFAShighgerminalcenter(GC)Bcellsat /
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theindicatedtimepointsareshownintheflowplots.(B)Flowcytometryanalysisofdark-zoneGCBcells(CXCR4highCD86low;gatedonGCBcells)atday8 n
postinfection (rapamycin treatment was performed on days (cid:2)1 to 7). The bar graph shows percentages of dark-zone B cells among total GC B cells. (C)
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FrequenciesofBcl-6(cid:3)cellswithinthePNAhighFAShighB220(cid:3)GCBcellpopulationonday8postinfection(rapamycintreatmentwasperformedondays(cid:2)1to
a
7)asanalyzedbyflowcytometryandsummarizedinabargraph.(D)ThefrequencyofPNAhighFAShighGCBcellsisshownforLCMV-infectedmicetreatedwith y
rapamycinorvehiclefromdays9to14postinfection(left;gatedonBcells).BrdUstainingisshownontherightforthesePNAhighFAShighGCBcells(3-hpulse 6
ofBrdU)at15dayspostinfection.GraphsshowmeansandSEM(n(cid:4)4or5mice/group).Datarepresentativeofthreeindependentexperimentsarepresented. ,

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rapamycin-treated LCMV-infected mice in which GC B cell responses were compro- y

mised. To extensively analyze Tfh responses, we took advantage of the LCMV I-Ab g
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GP -specific T cell receptor (TCR)-transgenic Smarta CD4 T cell system. Smarta e
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chimericmiceweremadebyadoptivelytransferringnaiveSmartaCD4TcellsintoB6 t
mice,andthesechimericmiceweresubsequentlyinfectedwithLCMV.Consistentwith
the results seen with endogenous virus-specific memory CD4 T cells, rapamycin in-
creasedthenumberofmemorySmartaCD4Tcells(datanotshown).Usingthissystem,
we first analyzed CXCR5 expression, which is one of the key features of Tfh cells,
directing the migration of primed Tfh cells to B cell follicles (26). We found that
rapamycinstronglyinhibitedCXCR5expressiononSmartacellsatday8postinfection
(Fig.7A),suggestingthatTfhcellgenerationwasimpairedinrapamycin-treatedmice.
TofurthercharacterizeTfhdifferentiationinthepresenceofrapamycin,theexpression
of several other canonical markers of Tfh cells was examined. ICOS and PD-1 are
well-known cell surface markers of Tfh cells, and we observed markedly decreased
numbersofICOShighCXCR5highandPD-1highCXCR5highantigen-specificCD4Tcellsin
rapamycin-treatedmice(Fig.7BandC).Suchdecreasednumbersofantigen-specificTfh
cells were also confirmed by lower expression of Bcl-6 (Fig. 7D), the transcriptional
factor governing Tfh lineage differentiation (27). Together, these data indicate that
rapamycintreatmentimpairsformationoftheTfhcellpopulation.
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FIG 6 Rapamycin enhances virus-specific CD4 T cell responses. B6 mice were infected with LCMV
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Armstrong, and rapamycin was administered from days (cid:2)1 to 30 postinfection. (A) Numbers of I-Ab n
GP66–77tetramer-positiveLCMV-specificCD4Tcellsinthespleen(n(cid:4)4micepergroup).(B)Cytokine lo
production by LCMV-specific CD4 T cells following stimulation with the GP peptide at 35 days a
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postinfection (gated on CD4 T cells). Data representative of three independent experiments are d
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presented. d

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GiventhatonlyTh1andTfhsubsetsareinducedinresponsetoLCMVinfection,the

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negativeroleofrapamycininTfhdifferentiationsuggestedthatitmayinsteadpromote t
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Th1 differentiation. To test this hypothesis, we compared Th1 responses between p
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rapamycin-treated and untreated mice. We first measured the frequencies of gamma /jv
interferon (IFN-(cid:3))-producing cells in antigen-specific CD4 T cells after ex vivo stimula- i.
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tionwithpeptide.Inthepresenceofrapamycin,around75%ofantigen-specificCD4T s
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FIG 7 Rapamycin treatment modulates helper CD4 T cell differentiation. LCMV-specific transgenic CD4 T cells (Smarta cells; Thy1.1(cid:3)) were
adoptivelytransferredtoB6mice(Thy1.2(cid:3)),followedbyinfectionwithLCMVArmstrong.Thesemiceweretreatedwithrapamycinfromday(cid:2)1
today7.Smartacellsinthespleenwereanalyzedatday8postinfection.(A)CXCR5expressiononSmartacells.MFI,meanfluorescenceintensity.
(B)FlowcytometryanalysisofICOShighCXCR5(cid:3)andPD-1highCXCR5(cid:3)Tfhcells(gatedonSmartacells).(C)NumbersofTfhcells(ICOShighCXCR5(cid:3)),
summarizedfromthedatainpanelB.(D)PercentagesofBcl-6highCXCR5(cid:3)TfhcellsamongtotalSmartacells.(E)ProductionofIFN-(cid:3)bySmarta
cellsafterstimulationwiththeLCMVGP peptide.(F)FrequenciesofTh1cells(CXCR5(cid:2)granzymeB(cid:3))inSmartacells.Forallexperiments,n(cid:4)
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4to6mice/group.GraphsshowmeansandSEM.Dataarerepresentativeof3independentexperiments.
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mTORPromotesAntiviralHumoralImmuneResponse JournalofVirology
cellswerefoundtosecreteIFN-(cid:3),whereasonlyabout50%oftheircounterpartsfrom
vehicle-treated mice secreted IFN-(cid:3) (Fig. 7E). Next, the expression of another Th1
marker,granzymeB(GZB),wasalsoexaminedonantigen-specificCD4Tcellsobtained
fromLCMV-infectedmiceinthepresenceorabsenceofrapamycintreatment.Likewise,
thepercentageofGZB(cid:3)CXCR5lowcellswassubstantiallyincreasedbyrapamycin(Fig.
7F). Together, these data indicate that rapamycin skews virus-specific effector CD4 T
celldifferentiationfromTfhtoTh1subsets.
CD4Tcell-intrinsicinhibitionofmTORsignalsimpairsTh1formation.Ourdata
clearly show that inhibiting mTOR with rapamycin reduces both Tfh and B cell re-
sponses.SinceTfhandGCBcellsreciprocallyregulateeachother(28),itisimportant
tounderstandwhetherrapamycinintrinsicallyinhibitsBcellorCD4Tcellresponses.To
examinethis,weinhibitedmTORsignalingexclusivelyinantigen-specificCD4Tcellsby
usingaretroviralgeneknockdownsystem(Fig.8A).Retrovirus-transducedornontrans-
duced Smarta cells were adoptively transferred into B6 mice, followed by LCMV D
o
infection(Fig.8A).ThissystemallowedustodirectlycompareTh1andTfhdifferenti- w
ationpatternsoftransduced(GFP(cid:3))andnontransduced(GFP(cid:2))Smartacellswithinthe n
lo
sameanimal(Fig.8B).Incontrasttotheeffectofrapamycinadministration,wefound a
that mTOR knockdown in antigen-specific CD4 T cells resulted in inhibition of Th1 d
e
differentiation, as demonstrated by lower frequencies of the CXCR5low GZBhigh popu- d

f
lation(Fig.8C).Furthermore,mTORknockdownalsoinhibitedtheexpressionoftheTh1 r
o
lineage-associated transcription factor T-bet (Fig. 8D). Conversely, mTOR knockdown m

increasedthefrequenciesofTfhcells,characterizedasCXCR5highPD-1highcells(Fig.8E). h
t
t
Notably, all these results were faithfully recapitulated by the knockdown of raptor (a p
:
necessarycomponentofmTORC1),withadecreasedCXCR5lowGZBhighTh1subsetas //
jv
wellasreducedT-betandincreasedCXCR5(cid:3)PD-1highTfhpopulations(Fig.8FtoH). i.
a
Since we transferred Smarta transgenic CD4 T cells immediately after retrovirus s
m
transduction (before expressing green fluorescent protein [GFP]) in the above-
.
o
describedexperiments,howmanyretrovirus-transducedSmartacellswereadoptively
r
g
transferred into the recipient mice was unknown. This prevented us from examining /

whether the number of GFP(cid:3) Tfh cells was increased by mTOR or raptor knockdown on
compared to the control level in this experimental setting. To address this issue, we M
sorted GFP(cid:3) retrovirus-transduced Smarta cells (raptor-specific short hairpin RNA a
y
[raptor-shRNA]orvectorcontrol)andthentransferredthesamenumberofthesecells 6
,
into recipient mice, followed by LCMV infection. The total number of raptor-shRNA-
2
transducedSmartacellswasmuchlowerthanthatofvector-transducedSmartacellson 0
1
day 8 after infection (Fig. 8J). In particular, the reduction in cell numbers was much 9

b
more severe for Th1 cells (Fig. 8K and L). Taken together, consistent with previous
y
observations(21,23),thedatashowthatselectiveattenuationofthemTORC1signaling g
u
pathwayinantigen-specificCD4TcellsinhibitsthegenerationofTh1cellsafteracute e
s
viralinfection.Inaddition,ourresultsalsosuggestthattheincreasedTh1cellformation t
observedinrapamycin-treatedmicewasnotduetoreducedmTORactivityinantigen-
specificCD4Tcells.
mTOR acts intrinsically in B cells to regulate germinal center responses. The
above-describedRNAinterference(RNAi)experimentsindicatethatTfhcellsarenotthe
primary target cells of rapamycin for the impaired humoral immunity observed in
drug-treated mice after viral infection or immunization. Therefore, we next asked if
rapamycin has any intrinsic effects on B cell responses. To address this issue, we
generatedrapamycin-insensitiveBcellsbyknockingdownFKBP12,whichisanessen-
tialbindingpartnerforrapamycintoinhibitmTORC1signals(2).Wetransducedbone
marrow (BM) hematopoietic stem cells (HSCs) with an FKBP12 shRNA retrovirus and
subsequently transferred the transduced HSCs into lethally irradiated recipient mice
(Fig.9A).Afterreconstitution,thesebonemarrowchimericmiceallowedustoanalyze
the intrinsic effect of rapamycin on B cells (Fig. 9A). In this experimental setting, we
observed that approximately 10% to 30% of total donor B cells were transduced, as
they were GFP positive (Fig. 9B). In addition, we observed normal matured B cell
February2017 Volume91 Issue4 e01653-16 jvi.asm.org 9
Yeetal. JournalofVirology
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FIG8InhibitingmTORsignalsintrinsicallyinantigen-specificCD4TcellsimpairsTh1cellformation.(A)
ExperimentaldesignformTORorraptorknockdowninLCMV-specifictransgenicCD4Tcells(Smarta
cells).Retrovirus-transducedSmartacellsweretransferredintoB6mice,followedbyLCMVArmstrong
infection.ThedifferentiationofSmartacellsintoTh1andTfhcellsinthespleenwasanalyzedatday10
postinfection.(B)Retrovirus-transduced(GFP(cid:3))andnontransduced(GFP(cid:2))Smartacellswerecompared
inthesameanimal.(CtoE)InmTORknockdownexperiments,thefrequencyofCXCR5(cid:2)granzymeB(cid:3)
Th1cells(C),theMFIofT-bet(D),andthefrequencyofPD-1highCXCR5(cid:3)Tfhcells(E)wereanalyzed.(F
toH)Inraptorknockdownexperiments,similaranalysesweredone.Fortheseexperiments,n(cid:4)4to6
animalspergroup.Datarepresentativeof3independentexperimentsarepresented.(I)Experimental
designfortheexperimentsperformedforpanelsJtoL.Retrovirus-transducedSmartacellswerecultured
for3days.GFP(cid:3)SmartacellsweresortedandthentransferredtoB6mice,followedbyLCMVArmstrong
infection.TotalnumbersofGFP(cid:3)Smartacells(J),Th1cells(K),andTfhcells(L)werecalculated.Data
representativeoftwoindependentexperimentsarepresented(n(cid:4)4mice/group).
populations(Fig.9C).TovalidatetheeffectofFKBP12knockdownonBcellsinresponse
to rapamycin, we compared mTOR activities by measuring phosphorylated S6 in
FKBP12 shRNA-transduced or non-shRNA-transduced B cells upon anti-B cell receptor
(anti-BCR) stimulation in the absence or presence of rapamycin. FKBP12 shRNA-
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