Progressing to Alopecia Universalis Lymphocyte-Mediated Alopecia Areata T+ A Mouse Model of

14 Pages·2011·2.92 MB·English

RESEARCH ARTICLE | JANUARY 01 2012
A Mouse Model of Clonal CD8+ T Lymphocyte-Mediated Alopecia Areata
Progressing to Alopecia Universalis 
Rajshekhar Alli; ... et. al
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J Immunol (2012) 188 (1): 477–486. w
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https://doi.org/10.4049/jimmunol.1100657 loa
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The JournalofImmunology
+
A Mouse Model of Clonal CD8 T Lymphocyte-Mediated
Alopecia Areata Progressing to Alopecia Universalis
Rajshekhar Alli,* Phuong Nguyen,* Kelli Boyd,*,† John P. Sundberg,‡ and
Terrence L. Geiger*
Alopeciaareataisamongthemostprevalentautoimmunediseases,yetcomparedwithotherautoimmuneconditions,itisnotwell
studied.ThisinpartresultsfromlimitationsintheC3H/HeJmouseandDEBRratmodelsystemsmostcommonlyusedtostudythe
disease,whichdisplayalowfrequencyandlateonset.Wedescribeanovelhigh-incidencemodelforspontaneousalopeciaareata.
The 1MOG244 T cell expresses dual TCRA chains, one of which, when combined with the single TCRB present, promotes the
developmentofCD8+Tcellswithspecificityforhairfollicles.RetroviraltransgenicmiceexpressingthisTCRdevelopspontaneous
D
o
alopeciaareataatnearly100%incidence.Diseaseinitiallyfollowsareticularpattern,withregionallycyclicepisodesofhairloss w
n
andregrowth,andultimatelyprogressestoalopeciauniversalis.AlopeciadevelopmentisassociatedwithCD8+Tcellactivation, loa
d
e
migrationintotheintrafollicularregion,andhairfollicledestruction.ThediseasemaybeadoptivelytransferredwithTlympho- d fro
cytesandisclassIandnotclassIIMHC-dependent.PathologicTcellsprimarilyexpressIFNGandIL-17earlyindisease,with m
h
dramaticincreasesincytokineproductionandrecruitmentofIL-4andIL-10productionwithdiseaseprogression.Inhibitionof ttp
individualcytokinesdidnotsignificantlyalterdiseaseincidence,potentiallyindicatingredundancyincytokineresponses.These ://jou
rn
results therefore characterize a new high-incidence model for alopecia areata in C57BL/6J mice, the first to our knowledge to a
applyamonoclonalTCR,andindicatethatclassIMHC-restrictedCD8+Tlymphocytescanindependentlymediatethepathologic ls.aa
i.o
response. TheJournalof Immunology,2012,188: 477–486. rg
/jim
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u
A n
lopeciaareata(AA)presentsasatypicallypatchy,non- melanocytesprecededdamagetohairfolliclekeratinocytesledto ol/a
scarringhairloss,althoughitmayprogresstototalhair the suggestion that melanocytes are initial targets of autoreactive rticle
loss (alopecia universalis) (1). Lifetime incidence is lymphocytes(1,9).Theabsenceofwidespreadmelanocytedestruc- -p
d
greaterthan1%.Murinemodelsandhumanskintransplantedinto tion duringAA, however, suggests a morecomplex specificityre- f/1
8
8
NOD/SCIDmicesupportanautoimmune,Tcell-dependentcause lationship.Indeed,althoughAAisinrarecasesassociatedwith /1
/4
in which a breakdown of immune privilege is followed by de- vitiligo, it is also associated with other autoimmune diseases 77
/1
structionofhairfollicles(2,3).Indeed,acasereporthasdescribed andmostcommonlypresentsasanisolatedcondition(10,11). 34
8
the cure of a person with AA by allogeneic hematopoietic stem Furthermore,intheC3H/HeJmousemodelofdisease,whitehairs, 15
6
celltransplantation,supportinganimmunologicorigin(4).During created bylocalized freezebranding, werenot spared(12). /11
0
AA, class I and class II MHC are upregulated, and CD8+ and TheC3H/HeJmouseistheprimaryanimalmodelofspontaneous 06
5
CD4+lymphocyticinfiltratessurroundandpenetrateaffectedfol- AA(2).Femalemicegreaterthan6moofagehavealowfrequency 7.p
d
licles (5). CD8+ T cells predominatewithin the follicular epithe- (,1%) of a spontaneous AA-like disease, though this can reach f b
liumduringactivedisease(6–8).Ithasbeensuggested,although .20%by12moofage(13,14).OlderDEBRratssimilarlydevelop y gu
e
notproved,thatCD8+Tlymphocytesareprimarilyresponsible AA,althoughatahigherfrequency(15).Ineachcase,CD4+and st o
for the follicular damage, whereas CD4+ T cells provide help CD8+Tcellsarerequiredfordisease,althoughtheultimateeffec- n 2
1
fortheCD8+response(1). torsoffolliculardamagehavenotbeenconclusivelyidentified.Low Fe
b
The Ag(s) responsible for AA are unknown. Preservation of frequency and late disease onset in these systems are significant ru
a
amelanotic hairs in AA and a report that damage to hair bulb impedimentstostudiesofdiseasecauseandpathogenesis. ry 2
0
We describe a new model for AA in which clonal C57BL/6J- 23
derived CD8+ T lymphocytes independently mediate follicular
*Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN destruction. While evaluating the TCR expressed by a myelin-
38105;†DepartmentofPathology,VanderbiltUniversity,Nashville,TN37232;and
‡TheJacksonLaboratory,BarHarbor,ME04609 specific T cell, we identified a dual TCR, one of which guided
specificityagainstamyelinAg.Surprisingly,theseconddirected
ReceivedforpublicationMarch8,2011.AcceptedforpublicationOctober22,2011.
CD8+ Tcellsnot againstmyelin, butselectivelyagainst hair fol-
ThisworkwassupportedbyNationalInstitutesofHealthGrantsR01AI056153(to
T.L.G.)andR01AR056635(toJ.P.S.),theAmericanLebaneseSyrianAssociated licles. Retroviral transgenic (retrogenic) mice on a Rag1tm1Mom
Charities/St.JudeChildren’sResearchHospital(toR.A.,P.N.,K.B.,andT.L.G.),and (Rag12/2) background, in which T cells exclusively express this
theNorthAmericanHairResearchSociety(toK.B.). TCR,developalopeciaat∼6–7wk,withanearly100%incidence
AddresscorrespondenceandreprintrequeststoDr.TerrenceL.Geiger,Department by4mo.AlopeciamimicsthatseeninpatientswithreticularAA
ofPathology,St.JudeChildren’sResearchHospital,262St.JudePlace,D-4047,
Memphis,TN38105.E-mailaddress:[email protected] and ultimatelyprogresses toalopecia universalis.
Theonlineversionofthisarticlecontainssupplementalmaterial.
Materials and Methods
Abbreviationsusedinthisarticle:AA,alopeciaareata;B6,C57BL/6J;CFP,cyan
fluorescentprotein;HPC,hematopoieticprogenitorcell;LN,lymphnode;m,mouse; Mice
MSCV,murinestemcellvirus.
C57BL/6J (B6), B6.129P2-B2mtm1Unc/J (b2m2/2), and B6.129S7-
Copyright(cid:1)2011byTheAmericanAssociationofImmunologists,Inc.0022-1767/11/$16.00 Rag1tm1Mom/J(Rag12/2)micewereobtainedfromTheJacksonLaboratory
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100657
478 RETROGENICMODELOFALOPECIA
andbredunderspecificpathogen-freeconditions,includingalldetectable Cell proliferation
Helicobacterspp.B6.129S-H2dlAb1-Ea(Ab2/2)micewereprovidedby
Dr.P.Doherty(St.JudeChildren’sHospital,Memphis,TN).Experiments Spleenorlymphnode(LN)cellswereisolatedfromretrogenicorB6mice,
were performed in accordance with institutional animal care and use andCD8+TcellswerepurifiedusingPEanti-CD8Ab(clone53-6.7)and
procedures. anti-PE–coatedmicrobeads(MACS;MiltenyiBiotec).Cellswerecultured
at53104perwellin96-wellplateswith33105irradiatedsyngeneic
Media,reagents,Abs,andflowcytometry splenicAPCsandtheindicatedconcentrationsofanti-CD3and1mg/ml
anti-CD28,pulsedwith1mCi[3H]thymidineafter72h,andthenharvested
CellswereculturedinEagle’s–Hank’sAminoAcidMedium(BioSource) forscintillationcounting.Sampleswereanalyzedintriplicate.
supplementedwith10%heat-inactivatedPremiumFCS(BioWhittaker),
penicillin G (100 U/ml), streptomycin (100 mg/ml), L-glutamine 292 Cytokineanalysis
mg/ml (Invitrogen Life Technologies), and 50 mM 2-mercaptoethanol
(Fisher Biotech). mAbs specific for mouse CD4 (clone L3T4), CD8 PrimaryCD8+Tcells(53104)wereisolatedandstimulatedasdescribed
(clone53-6.7),CD25(clone7D4),TCRB(cloneH57-597),CD69(clone earlier.Culturesupernatantswerecollectedat24or48handanalyzedfor
H1.2F3), CD45RB (clone 16A), CD44 (clone IM7), CD3 (clone 145- IL-2,IL-4,IL-10,IFNG,andIL-17byBio-Plexassay(Bio-Rad).
2c11), and CD28 (clone 37.51) were obtained from BD Biosciences.
Real-timePCR
mAbsagainstmouseIL-10R(clone1B1.3A),IFNG(cloneXMG1.2),
TNFA(cloneXT3.11),andpolyclonalratIgG(cat.no.BE0094)were Skinspecimensweretakenfrom1MOG244.1micewithalopeciaorcontrol
purchased from BioXCell (West Lebanon, NH). Anti-mouse IL-17 B6mice.Hairwasshavedpriortospecimenremoval.For1MOG244.1mice
(clone 50104) was purchased from R&D Systems (Minneapolis, MN). withoutalopeciauniversalis,specimensweretakenattheedgesofactive
FlowcytometrywasperformedonaFACSCalibur(BDBiosciences)and lesions. Skin samples were flash frozen in liquid nitrogen and then ho- D
flow cytometric sorting on a MoFlo high-speed cell sorter (DakoCyto- mogenizedinTRIzol(Invitrogen)usinga19-gaugeneedle.RNAwasex- ow
n
mation). tractedusingPhase Lock gel(Eppendorf) andthe RNAeasyMini Kit loa
d
(Qiagen) following the manufacturers’ protocols. First-strand cDNAwas e
TCRcloning synthesizedusingtheSuperscriptIIIRT-PCRkit(Invitrogen).IL-2,IL-10, d fro
The1MOG244.1TCRwasclonedfromthe1MOG244hybridomaasde- IL-17, IFNG, and TNFAwere quantified as described after 45 cycles of m h
scclornibeeddin(1to6)a.vTaCriRanAtoafntdheTmCRurBinceDstNemAcseelplavriartuesd(MbySaC2VA)-dsreiqvuenenMceSCwVas- PacCtiRva(t1e9d)w.AithsCaopnoAsitaivnedc1o0nUtro/ml,lRrNhIAL-w2afsoris4o8lahte.dCfyrtoomkinBe6Cstpdleantaocwyeteres ttp://jo
u
I-GFP retroviral vector that replaces GFP with cyan fluorescent protein comparedwithGAPDHasaninternalcontrol,andthiswasnormalizedto rn
a
(CChFilPd)re(n1’7s)R.eTsheearcOhTH-1osTpCitRal,wMasempprohvisi,dTedN)b.y Dr. D. Vignali (St. Jude CcotmdpaatraatfivroemCtsiamppurlotaancehou(2s2lyDDaCnta).lyzed activated splenocytes using the ls.aai.o
rg
Retroviraltransduction ofTCR Abtreatment /jim
m
Retrovirus was produced as described (16, 18). Briefly, 10 mg each of Abs(250mg/mouse)againstmouse(m)IL-10R,mIFNG,mTNFA,mIL-17, uno
retroviralreceptorandhelperDNAconstructswerecotransfectedinto293 and polyclonal rat IgG were administered i.p. to five to eight mice per l/a
Tcellsusingcalciumphosphateprecipitation,andthecellswereincubated groupbeginning7wkafterHPCtransplantationandpriortotheonsetof rticle
inDMEMwith10%FCSfor48h.Supernatantwasthencollectedtwice diseasesymptoms.Administrationwasbiweeklyfor5wk.Thetreatment -p
d
dailyandusedtoinfectGP+E86retroviralproducercellsinthepresence conditionswithmIFNGandmTNFAAbswerevalidatedbydemonstrating f/1
osofr8temdgf/omrltphoelypbreresennec.eTroafnsCdFuPcedanGdPc+eEll8-f6receelslsupweernreatflaonwt-ucsyetdomfoertrtircaanllsy- tshpaetctsievreumcyftorokminetreinatevditrmoicaefte(3r dprpootesitndoGsincgl)eacroaunlcde.fuFlloyrdmepILle-t1e0tRheAreb-, 88/1/47
duction. dosingwasvalidatedbydemonstratingthatserumacquiredfromsimilarly 7/1
treatedmicecouldspecificallystainmousemacrophageata1:50dilution. 34
8
Generationofretrogenicmice ThemIL-17Abwasdosedbasedonconditionspreviouslydescribed(20– 1
5
22). 6/1
Retrogenicmiceweregeneratedasdescribed(18).Briefly,bonemarrow 10
cells were harvested from the femurs of Rag12/2 mice 48 h after the 06
5
administration of 0.15 mg 5-fluorouracil per gram of body weight. The Results 7.p
pooledcellswereculturedincompleteEagle’s–Hank’sAminoAcidMe- Productionof1MOG244.1retrogenic mice df b
diumcontaining20%FCSsupplementedwithIL-3(20ng/ml),IL-6(50 y g
cnegl/lmslw),earendthsetnemcocceullltufarecdtofro(r5a0nnagd/mdilt)iofnoarl4488hhawti3th7˚1C20in05ra%dCirrOa2d.iaTthede W1MeOidGen2t4i4fieTdtcweollphryobdruidctoimveal.yOrenaerraTnCgRedAT,CwRheAncthraaninssduwciethdinwtihthe uest on
GitoPr+cEe8l6lsr(eHtroPvCirsa)lwperoreduhcaerrvceesltlesd.,TwheasthreandswduicthedPhBeSm,aatnoaployizeetidcbpyroflgeonw- the single rearranged TRBV13-2+ TRBJ2-7+ TCRB into T cells, 21 F
ccdyioptnoioemnr.teMtRryiacgfe1ow2r/eC2rFemPa,incaaenlydazteinadjeracatttei4od–8oinfwtotkwsoaufbtreelerctiHhpaiPellCnyttirmrarniacdsepialpateendrtab(ti4oo5nn0efromardaTr)rCorewR- cdTorConRcfeyArtre,eddgildyscpnoeopctir.fioTcteiCtiynRA(f1o6rc)h.tahTienhieamlslmeelcuiocnniezdxi,ncagluTsAiRognA,Vims8ynDeo-lt2in+rooTbluRigsAto,Jda3en7nd+- ebruary 20
2
engraftment. 3
dual TCR expression has been hypothesized to facilitate T cell
Alopeciaareata escapefromtolerance(23).ToanalyzefunctionallyTCRexpres-
singthesecondTCRAchain,wegeneratedretrogenicmiceselec-
Cohorts ofretrogenic micewerefollowed for 6mo oras indicated,and
tively expressing it. The TCR A and B chains, denoted as
clinicalsignsofalopeciaandbodyweightweremeasuredthreetimesper
week.Hairloss wasmonitoredandscoredas follows:0,,5%hairloss 1MOG244.1,wereintroducedintoaretroviralvectorseparatedby
from total body surface area; 1, 5–20%; 2, 20–40%;3, 40–60%;4, 60– theThoseaasignavirus2Asequence(Fig.1).The2Aallowsfor
80%;5,.80%.Foradoptivetransferdisease,CD8+Tcellswereisolated stoichiometric translation of both TCR chains from a single cis-
bnyummbaegrnoeftcicelblsewaderseetlreacntisofenrr(eMdAinCtrSa;veMnoiluteslnyyiinBtoioutnemc)a,nainpdulathteediRndagic1a2te/2d wtrohnich(2w4)e.rTehtehecnontrsatnruspctlawntaesdtrinantosdRuacge1d2i/n2tomBic6e.Rag12/2 HPCs,
mice,450rad-irradiatedB6mice,orunirradiatedB6mice.Alternatively,
103106totalsplenocytes(∼1.53106Tlymphocytes)weretransferred
into450rad-irradiatedB6b2m2/2orAb2/2mice. Developmentofalopeciain1MOG244.1mice
1MOG244.1 mice showed no gross abnormalities until ∼6–7 wk
Histology
after HPC transplant, when individuals began experiencing hair
TissueswereharvestedandfixedovernightinFekete’ssolution,processed loss.At2mo,greaterthan50%ofmicedevelopedalopecia,and
routinely,embeddedinparaffin,andsectionedat4mm.Gramstainsand by4movirtuallyallhad developedalopecia (Fig.2A).Hairloss
periodicacid–Schiffstainswereperformedtoruleoutbacterialandfungal
didnotresultfrombarbering,asasimilarincidenceofdiseasewas
infection, respectively. Immunohistochemical labelingwas performed on
a Lab Vision autostainer. Complete necropsies were performed on two observed in mice housed individually (data not shown). Neither
micefromeachgrouptoidentifyconcurrentlesions. was the alopecia a nonspecific consequence of the retrogenesis.
TheJournal ofImmunology 479
FIGURE 1. 1MOG244.1 retroviral vector. The
1MOG244.1 TCR A and B chain cDNAwere linked
withaT.asigna2Asequenceaspreviouslydescribed
(16).CDR3aminoacidandnucleotidesequencesare
shown.InvariantsequencefortheTRAV8D-2TRAJ37
TCRA and TRBV13-2 TRBJ2-7 TCRB is accessible
throughtheImMunoGeneTicsresource(http://imgt.
cines.fr).TheMSCVretroviralvectorincorporatesan
IRES-linkedCFP.
ThisphenotypehasnotbeenobservedwithotherclassI-orclass developedcompletehairlossordied,progressedtoalopeciauni-
II-restricted TCRs produced in our or other laboratories (16,25– versalis.Amedianofthreecyclesofhairlossandregrowth,asde-
27).Further,miceretrogenic fortheOVA-specific,class IMHC- finedbyachangeinoveralldiseasescore$1,wasseen(TableI).
restrictedOT-1TCR(28)producedsimultaneouslyandco-housed Therefore, 1MOG244.1 retrogenic mice develop a disease that
withacohortof1MOG244.1micedidnotdevelopalopecia(Fig. resembles reticular AAprogressing toalopecia universalis.
2A). The hair loss additionally did not accompany other clinical
SpontaneousTcellactivationin1MOG244.1mice D
manifestations ofillness. Theweight of 1MOG244.1 and control ow
OT-1micedidnotsignificantlydifferovera.120-dobservation WenextexaminedTlymphocyteengraftmentin1MOG244.1mice. nlo
a
period (Fig. 2B), and no abnormalities were detected other than T cells were predominantly CD8+, with few CD4+ or coreceptor ded
thoseaffectingtheskinin1MOG244.1micebynecropsyandhis- negative cells (Fig. 4A). Good splenic engraftment was seen in fro
m
tTmoColoRugseitrcastnarsnagianely.nDesiipssre(oadvsaoetkaiessnsoaptloonsphteaocnwieaoniu)n.stTihnehtenhroaetrfmiotradelol,yetshAenAo1-trMreesOqisuGtiar2ne4ta4Bn.61y mc1r8ei.ca5ese6pdriio1nr.6atgowe-tkmh,eastocconhrseeedt3(o1–f85.)d7ias6eloaps1ee.6,ciawcnkdmpTicoesctetwrlalintnhsupmmlaonbrte,ersscawodrveearen0c–ien1d-; http://journ
interventioninthe1MOG244.1 micefor itsinitiation. disease (Fig. 4B; 3.8 6 1.5 3 106 cells, score 0–1; 6.9 6 2.2 3 als.a
1MOG244.1retrogenichairlosswaspatchy,mosttypicallywith 106 cells, score 3–5). An increase in T cell blasts, determined as ai.o
acleardelineationofalopecicandnormalareas.Alopeciawas the percent of cells with increased forward and sidescatter mea- rg/jim
notlinearlyprogressive;measurementsoftheextentofhairloss surements, was also seen in 1MOG244.1 mice with more ad- m
u
idrneitsiiecnaudslieavriidnpuaamtlteomrsnticoeafndiAmemAalosinns(tFhruiagtme.da3nAas,)w.daFixfufierntrgheenartnmhdoawriera,pnsaiintmcghielpasartettexorpnethroie-f vwscaionthrceesdi0md–ui1sl;etaa5ns0ee.o1cuosm6lyppa9rr.eo7dd%uw,ciestdhcoOtrheTo-s13e–mw5i;ictheSu(nFpoipgol.er4meCaer,nl1yta6dl.5iFse6iags.5e.19aA%n)d,. nol/article-p
d
enced hair loss and regrowth independently (Fig. 3B); simulta- 1MOG244.1 AAwas associated with manifest adenopathy and f/1
8
neous loss and regrowth in different regions was often apparent. splenomegalycomparedwithcontrolOT-1orC57BL/6mice(Fig. 8/1
/4
This suggests that local skin factors, rather than a more general- 77
/1
ized state of immune responsiveness, was responsible for the re- 3
4
8
gionalperiodicity.Allmiceinacohortof20,followeduntilthey 15
6
/1
1
0
0
6
5
7
.p
d
f b
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0
2
3
FIGURE2. Spontaneousalopeciain1MOG244.1mice.Rag12/2mice
weretransplantedwithRag12/2HPCstransducedwithretrovirusencod-
ingthe 1MOG244.1orcontrolOT-1TCR. Micewere observedlongitu- FIGURE 3. Disease patterns in individual 1MOG244.1 mice. A, Plots
dinally.A,Kaplan–Meieranalysisofdisease-freesurvivalfor1MOG244.1 demonstratediseasescoresoffourrepresentativeindividual1MOG244.1
(n=63)andOT-1(n=10)TCRretrogenicmice.The1MOG244.1dataare mice.Scoreswerecalculatedbasedontotalbodyalopeciaasdescribedin
pooledfromseveralindependentlypreparedcohortsofmice.Ascore$1 MaterialsandMethodsanddemonstratethewaxingandwaningofdisease
(.5% surface hair loss) was considered a positive event. B, Average with ultimate progression to alopecia universalis. B, Regional hair loss
weight of groups of simultaneously produced 1MOG244.1 (n = 20) and fromfourrepresentativemiceisplotted.Thepresenceofalopeciawithin
OT-1(n=10)TCRretrogenicmiceisplotted.Nosignificantdifferenceis theneck,leftorrightflanks,andbackatanindividualevaluationpointare
seen. indicatedbythe♦,4,d,andNsymbols,respectively.
480 RETROGENICMODELOFALOPECIA
TableI. DevelopmentofAAin1MOG244.1mice
TimetoScore$1(d) TimetoScore=5(d) NumberofRelapses(n)
Maximal
RetrogenicTCR Mice(n) Mean Median Range Mean Median Range Mean Median Range Score
1MOG244.1 20 58616 53 38–118 13964 140 132–143 2.761.1 3 0–5 560
OT-1 10 NA NA NA NA NA NA NA NA NA 060
Theindicatednumbersof1MOG244.1orcontrolOT-1TCRretrogenicmiceweremonitoredforclinicalsignsofalopecia.Timetoinitialdisease,toalopeciauniversalis,and
thenumberofdiseaserelapses,indicatedbyachangeindiseasescore$1,arelisted.AAwasnotobservedintheOT-1mice.
NA,notapplicable.
4D), and moderately diminished TCR expression was frequently T cells in 1MOG244.1 mice (disease scores of 1–2) was alsoin-
apparentonTcellsfromdiseasedanimals(datanotshown).Cumu- creasedcomparedwiththatinage-matchedOT-1retrogenics(Sup-
latively,theseresultsindicatespontaneousactivationofTcells plementalFig. 1B,1C,p , 0.01 foreach). Notably, inindividual
in 1MOG244.1micewith thedevelopment ofAA. mice,TcellactivationmeasuredbyCD69expressionwaselevated
TheassociationofTcellactivationwithdiseasedevelopmentwas inskin-drainingLNcomparedwiththatinpairedspleens,implying
confirmedby immunophenotyping.Adramaticincreaseofsplenic thatTcellprimingismorepronouncedattheselocations(Fig.6, D
TcellsexpressingtheCD25andCD69activationmarkerswasseen p,0.05).Therefore,the1MOG244.1TCRguidesTcellstoward ow
n
lo
in concert with the progression of alopecia (Fig. 5). Disease was theCD8lineage.TheseTcellsarespontaneouslyandspecifically a
d
e
likewise associated with markedly increased proportions of cells activatedinretrogenicmiceanddifferentiateintomemorycellsin d fro
withamemory/activatedphenotypeasdefinedbydiminishedlev- association withthedevelopment of alopecia. m
h
els of CD45Rb and CD62L and increased CD44 compared with To determine whether the AA was associated with elevated ttp
C57BL/6Tcells.Thepercentageofmemory,CD69+,andCD25+ expression of skin-homing markers, we analyzed expression of ://jo
u
CD162andCCR10.However,CD162expressionwasnotaltered rna
relativetothatobservedonB6splenocytes,andCCR10wassimilar ls.a
a
or modestly diminished (data not shown). This suggests that the i.org
spontaneous activation of 1MOG244.1 T cells is not associated /jim
m
withtheacquisitionofaphenotypebiasingthecellstowardskin- u
n
o
specifichoming. l/a
Histopathologyof1MOG244.1mice rticle-p
d
Skin sections from mice affected by the cyclical alopecia had f/18
8
classicalfeaturesofAA.Characteristicsofcicatricialalopeciathat /1/4
7
cannormallybefoundinB6micewerealsoseen(29),althoughthe 7/1
3
microscopic lesions of AA predominated. Lymphocyte infiltrates 4
8
1
were observed in and around anagen and late-catagen stage hair 56
/1
follicles, extending from the bulge downward to just above the 10
0
hair bulb (Fig. 7A and not shown). Analysis of diseased skin, 65
7
however, failed to correlate disease presence or magnitude with .pd
the hair cycle phase of residual follicles (data not shown). Intra- f by g
follicular mononuclear cells expressing CD3 were readily detec- ue
ted, demonstrating that retrogenic T cells had infiltrated the fol- st o
n
licles(Fig.7B).Inthedermis,justadjacenttothehairfollicles, 21
F
lymphocyticinfiltratesweremixedwithmacrophagesandafew eb
ru
neutrophils(Fig.7anddatanotshown).Small,fragmented,con- a
densed, and membrane-bound nuclear material consistent with ry 20
2
apoptosiswasacommonfindingintheaffectedfollicularepithe- 3
lium.Apoptosiswasconfirmedinthesecellsbypositivelabeling
withacleavedcaspase3Ab(datanotshown).Specialstainsfor
bacteriaandfungiwereconsistentlynegativeinallsectionsof
skinexamined,indicatingthattheresponsedidnotresultfromsec-
ondaryinfections(datanotshown).
The progression through multiple hair cycles from patchy alo-
pecia to alopecia universalis was correlated to the microscopic
findings. In early hair cycles in haired skin adjacent to alopecic
areas, swarms of lymphocytes were present in and around intact
FIGURE4. Tcellengraftmentin1MOG244.1mice.A,Flowcytometric hair follicles as described earlier. As the disease and follicular
analysisofsplenocytesdemonstratesapredominantCD8+Tcellengraft-
destruction progressed overtime, the density of hair follicles de-
mentin1MOG244.1retrogenicmicecomparedwithB6controls.BandC,
clined,andtheabsenceofintactfolliclesbecamemoreprominent.
Totalnumbersofsplenic1MOG244.1CD8+Tcells(B)andpercentof
Inmicewithalopeciauniversalis,thealopecicareasweredevoidof
thesecellsthatare blasted(C),asdeterminedbyincreasedflowcyto-
follicleswithonlyremnantclustersofhairbulbsinthedeepdermis
metricallydeterminedforwardandsidescatter,areplotted.D,Grossphoto-
graphofLNandspleensfromarepresentative1MOG244.1mouse(score= and fewremaining lymphocytesinthedermaltissue.
3), age-matched OT-1 mouse, and C57BL/6 mouse indicates adenop- Withlesionprogression,superficialfibrousconnectivetissueor-
athyandsplenomegalyindiseased1MOG244.1animals. ganizedparalleltotheepidermisatthedermal–epidermaljunc-
TheJournal ofImmunology 481
FIGURE5. Increased1MOG244.1Tcellacti-
vationwithprogressivealopecia.Flowcytometry
plots frommicewiththeindicated diseasescore
orfromcontrolB6mice.Expressionoftheacti-
vation markers CD25 and CD69 on gated CD8+
Tcellsdemonstratesanincreasingpercentageof
activated T cells with disease progression. Ex-
pressionofCD62L,CD44,andCD45Rbindicates
D
progressiveincreasesinthepercentageofmemory o
w
Tlymphocytes.Representativedataareshown. nlo
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l/a
tion,asequelaoftheB6alopeciaanddermatitiscomplicatingthis Lymphopenia may promote T cell survival and homeostatic rticle
model. In areas interpreted as containing long-standing injury, expansion.Toexaminewhetherthelymphopeniain theRag12/2 -pd
therewerestretches of dermaltissue inwhich hair follicles were recipientsplayedaroleindiseasetransfer,isolatedCD8+Tcells f/188
absent. Remnant follicular structures consisted of mild linear fi- were transferred into either unirradiated (1 3 106 or 10 3 106 /1/4
7
brosis,dermalpapilla,andsmallclustersofmelanocytes.Insum- doses) or control sublethally irradiated (1 3 106 dose) C57BL/6 7/1
3
mary,histopathologywasconsistentwithsevereAAtogetherwith recipients. Whereas the irradiated recipients showed a disease 48
1
the sequelae of concurrent cicatricial alopecia and dermatitis incidencesimilartothatobservedaftertransferintotheRag12/2 56
/1
previouslydescribed inB6mice (29). mice,theunirradiated recipientsdidnotachieveaminimalscore 10
0
6
of 1. However, by 20–40 d after transfer, these mice manifested 5
ClassIMHCdependence ofTcellresponse substantial hair shedding with routine manipulation, something 7.pd
To establish better the independent role of T cells in alopecia de- not observed in control mice. The incidence and time course of f by g
velopment, we determined whether they could transfer disease to this shedding was T-cell dose dependent (Supplemental Fig. 2). ue
unaffectedmice.SplenicCD8+Tcellswereisolatedfrommicewith Similartoprimarydiseasein1MOG244.1mice,sheddingwasre- st on
1in.5te3rm1ed0i6aTtecheallisrwloesrse(tdriasnesafseerrsecdoir.ev.3i)n,toanodthdeorwseisseoufn7m.5an3ip1u0la5teodr pgiloentealr.eTchipeireenfotsr.e,HCoDw8e+veTr,cdelilsseacsaenptreannestfrearnAceAainndtosleyvmeprihtyoreis- 21 Feb
Rdiasge1a2se/2wmasicdeo(sFeigd.e8pAen).dAenlltm(liocweddeovseel,o6p3ed6A6Ad,;ahndigthimdeosteo,i4n3iti6al maCrkoendsliydeartitnegnutahtaetd.the T cells in 1MOG244.1 mice were pre- ruary 20
2
2 d; p = 0.001). All mice also eventually progressed to alopecia dominantly CD8+, we hypothesized that the alopecia would be 3
universalis, and time to this was likewise dose dependent (low class I MHC dependent. To test this, we transferred ∼1.5 3 106
dose,1336 17d;highdose,1086 2 d;p =0.03). 1MOG244.1TcellsintosublethallyirradiatedclassIMHC-defi-
cientb2m2/2orclassIIMHC-deficientIAb2/2mice.Whereas
7 of 8 IAb2/2 mice developed alopecia, 0 of 8 b2m2/2 recip-
ients did (Fig. 8B). Therefore, an exclusively class I MHC-
restricted response is adequate for alopecia development after
transfer; class II MHC is not required. Notably, in humans and
C3H/HeJ mice, the T cell infiltrate colocalizes with aberrant up-
regulation of class I MHC (30, 31), consistent with these obser-
vations.
Promiscuouscytokineproductionby1MOG244.1Tcells
Tobetterdelineatetheeffectorpotentialofthespontaneouslyac-
FIGURE6. CD69expressiononskin-drainingLNandsplenicTcells.
Pairedsplenicandskin-drainingLN(axial,cervical,andinguinal)CD8+ tivated 1MOG244.1 T cells, we analyzed their proliferative re-
Tcellsfrom1MOG244.1micewithvarieddiseasescores(range1–5)were sponsetostimulationwithCD3-andCD28-specificAbs.Similar
assessed for expression of the early T cell activation marker CD69. In- or only mildly diminished proliferative responses compared with
creasedexpressionwasobservedintheLNbypairedttest(p,0.05). thoseofcontrolB6CD8+Tcellswereseenregardlessofdisease
482 RETROGENICMODELOFALOPECIA
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FIGURE 8. Adoptive transfer of AA by 1MOG244.1 T cells. A, The ed
indicatednumbersofCD8+1MOG244.1Tcellsfrommicewithalopecia fro
were adoptively transferred i.v. into unmanipulated Rag12/2 mice and m h
raencailpyiseinstsofmdoisneiatosere-fdrefeorsutrhveivdaelviesloshpomwenn.tnof=a5loppeercigar.ouAp.KDaaptlaana–reMreeiper- ttp://jou
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irrersaednitaatteidve(4o5f0trwado)ebx2pmer2im/2enantsd.IBA,bD2/i2semasiec-efraefetesrutrrvainvsaflerooffssepmleinleotchyatlelys als.aa
including∼1.53106Tcellsfrom1MOG244.1micewithalopecia.Data i.org
arepooledfromtwoexperiments(n=8pergroup). /jim
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score 2 disease. The enhanced and promiscuous cytokine pro- l/a
duction of the 1MOG244.1 mice was not a consequence of ret- rticle
rogenesis.DirectcomparisonofTcellsfromscore31MOG244.1 -pd
and simultaneously produced OT-1 mice demonstrated markedly f/18
8
FIGURE 7. Clinical appearance and histopathology of 1MOG244.1 increased production of all cytokines by the 1MOG244.1 T /1/47
mice.A,Skinsections(boxes)wereisolatedfrommicewiththeindicated cells (Supplemental Fig. 3). Therefore, the cytokine profile of 7/1
3
alopeciascoresandaclinicallyunaffectedcontrolB6mousewithfollic- 1MOG244.1 T cells varies both quantitatively and qualitatively 48
1
ulardystrophyunrelatedtoalopeciaareata.H&E-stainedparaffinsections with disease state, a finding consistent with studies showing dy- 56
ofrepresentativefieldsatlowmagnification(320)andhighmagnification namicchangesinexpressionofimmuneresponsegenesbyarray /110
(360; enlargement of boxed area) are shown. B, Immunohistochemistry profilingofC3H/HeJmiceduringthecourseofskingraft-induced 065
7
withT-specificCD3Ab(brown)ofskinsectionsfrommicewiththein- AA (32). Early disease in 1MOG244.1 mice is primarily associ- .pd
d1iMcaOteGd24d4is.e1assekinscsoercetsi.onRsepisressheonwtant.ive data from a total of 24 analyzed aritpehderwailthTIcFeNllsG,wainthdplerossgerresasmivoeuinntcsreoafseIsLi-n17ILp-r1o7duanctdioTnc2bycyptoe-- f by gue
kineswith advancing disease. st o
n
score and inthe presence or absence of exogenousIL-2(Fig. 9). To assess for cytokine production in target tissue, RNA was 21
F
Therefore, disease progression in the 1MOG244.1 mice is not isolatedfromskinspecimensof1MOG244.1micewithalopeciaor eb
ru
associated with the development of overt anergy or proliferative from control B6 mice. TNFA, IFNG, IL-17, and IL-10 were an- a
defects amongTlymphocytes. alyzed by quantitative PCR and normalized to levels present in ry 20
2
Incontrasttotheproliferativeresponses,1MOG244.1cytokine ConA-stimulatedB6splenocytes(Fig.10B).Productionofsingle 3
production differed substantially from that of control CD8+ B6 cytokinesvariedsubstantiallybetweenindividualspecimens,with
T cells. IL-2 and IFNG generation by 1MOG244.1 T cells from often severallog differencesinquantities.
mice without disease was modestly elevated compared with that WiththeexceptionofpositivetestingforIFNGin1of4B6skin
from control cells (Fig. 10A). Neither 1MOG244.1 nor control samples, none of the cytokines were detectable in the control
cells produced detectable quantities of IL-4 or IL-10 (data not specimens.SignificantquantitiesofTNFA,IL-17,andIL-10were
shown).However,moderatequantitiesofIL-17wereproducedby observed in mice with disease scores of 1–3. IL-17 and IL-10
1MOG244.1Tcells,acytokinenotdetectedfromtheB6Tcells. production remained elevated in mice with alopecia universalis
Therefore,evenpriortoclinicaldiseasedevelopment,differentT (score=5),althoughTNFAproductionwasdiminished,with2of
effectorresponses are detected in1MOG244.1 and controlmice. 4miceshowingnodetectablecytokine.Thisisconsistentwiththe
In mice with intermediate disease (score = 2), dramatically presence of active inflammation and a regulatory response in the
increasedcytokineproductionwasobserved.An∼3-foldincrease skinof micewithactive disease.
in IL-2 production was accompanied by an ∼7-fold increase in To determine the role of individual cytokines, a single large
IL-17and.20-foldincreaseinIFNGproduction.Further,lowbut cohort of 1MOG244.1 retrogenic mice was segregated into sub-
detectable levels of IL-4 and IL-10 were seen. Progression to groupsthatweretreatedwithneutralizingAbsagainstTNFA(n=
severedisease(score=5)wasassociatedwithadditionaldramatic 7),IL-17(n=5),orIFNG(n=7),blockingIL-10RAb(n=7),or
enhancements in IL-17 (.10-fold), IL-4 (.100-fold), and IL-10 an Ab control (n = 8). Treatment began at 7 wk, at which time
(.10-fold) production compared with T cells from mice with alopeciawasfirstdetectedinthepopulation,andcontinuedfor5
TheJournal ofImmunology 483
D
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FIGURE 9. Proliferative response of 1MOG244.1 T cells. CD8+ T mu
n
lymphocyteswerepurifiedbymagneticbeadselectionfrom1MOG244.1 ol/a
mcoincceenotrractoionntrsolofBa6ntmi-iCceD3anadndide1ntmicga/lmnluamntbie-CrsDs2t8imiunlattheedpwrietshenvcaerieodr rticle-p
absenceof10U/mlrhIL-2.Proliferationwasmeasuredby[3H]thymidine df/1
8
incorporation at 72 h. Data shown are representative from separate ex- 8
/1
perimentsassessingmicewithdiseasescoresof0(A),3(B),or5(C). /4
7
7
/1
3
4
8
1
wk.Theindexdiseasedmiceusedtoidentifyfirstsymptomsinthe 5
6
population were excluded from treatments and further analyses. /11
0
0
Mice were monitored for disease incidence and severity in a 65
7
blinded manner, and mice from the different treatment regimens .p
d
were co-housed in individual pens to minimize environmental f by g
effects.Kaplan–Meierandarea-under-the-curveanalysesfailedto u
e
find significant differences between any of the groups (data not st o
n
shown). This suggests that either these cytokines primarily exert 21
F
theireffectspriortoalopeciadevelopmentorthattheydonotplay e
b
strong independent roles in alopecia progression and that redun- rua
dantpathways are available. ry 2
0
2
3
Discussion
FIGURE 10. Cytokine production by 1MOG244.1 T cells and in
AAisachronic,highlyprevalentdisfiguringcondition,whichan
1MOG244.1 skin. A, CD8+ T lymphocytes from 1MOG244.1 or control
abundance of data now indicates is primarily mediated by auto-
B6micewerepurifiedandstimulatedasinFig.9.At48h,cell-freecy-
reactiveTlymphocytes.Dissectingdiseasepathogenesishasbeen
tokinewas collected and concentrationsof the indicated cytokines mea-
hamperedbythelimitationsintheanimalmodelsystemsavailable.
sured. B, RT-PCR was performed on skin specimens from 1MOG244.1
Both the C3H/HeJ mouse and DEBR rat model systems are micewithalopecia(scores1–3or5)orB6controls.Cytokinelevelswere
characterized by low-incidence spontaneous disease first devel- compared with simultaneous quantitative PCR performed on B6 spleno-
opinglateinlife.Althoughadoptivetransferoflymphocytesorskin cytesstimulatedwithConAfor48h,andrelativequantitywascalculated
grafting can transfer disease, understanding the pathogenesis of usingthecomparativeCtapproach.Circlesindicatedatafromindividual
incipientspontaneousalopeciarequiresanalysisofdenovodisease mice,andbarsindicategroupaverages.*p,0.05(byKruskal–Wallistest
development. We characterize a new high-incidence retrogenic usingDunn’smultiplecomparisontest).
model for AA. It is to our knowledge the first described TCR
transgenicAAmodelsystemandthefirstintheB6mousestrain. elucidated. McElwee and colleagues (33) found that s.c. injected
In both humans and rodents, AA is associated with the infil- CD8+ T cells from alopecic mice promoted local disease at the
trationofbothCD4+andCD8+Tcellsinthehairfollicle,though injectionsiteathighincidence,whereasCD4+Tcellscouldpro-
CD8+ cellsmore specifically localize totheintrafollicular region voke amoregeneralizedalopeciaalthoughatlowerincidence.
(6–8). The relative roles of these T cell subsets are not fully Thisstudydifferedfromoursinmousestrain(C3HversusB6)and
484 RETROGENICMODELOFALOPECIA
routeofcelladministration(s.c.versusi.v.),potentiallyexplaining transcriptomestudyat5-wkintervals(J.P.Sundberg,unpublished
differencesinoutcome.Thelocalizedreactioninthatstudymay data). IL-17 is prominently induced in the 1MOG 244.1 T cells
haveresultedfromimmuneexhaustioninthetransferredcellpop- during alopecia development, and though not well studied in the
ulation or a failure to migrate from the skin, effects obviated by contextofAA,itssignificanceinotherinflammatoryautoimmune
ourtransfersintolymphopenicmice,whichwillpromotecellsur- conditionsiswellrecognized.IL-10iswidelyconsideredtoplay
vivalandhomeostaticexpansion,andi.v.transfer,whichwillpro- aregulatoryroleinAAandwasfoundtobeupregulatedinC3H/
motedisseminationofthetransferredcells.Incontrast,instudies HeJ mice that were resistant to AA through grafting of alopecic
of human scalp explants, cooperation between CD4+ and CD8+ skin(40).However,C3HAAlesionalskingraftsplacedonIL102/2
T cells was required for reproducible hair loss (34). Likewise, mice paradoxically also showed decreased transfer of disease
depletion of CD4+ or CD8+ T cells with mAb proved transiently comparedwithwild-typecontrols(51).Therefore,thesecytokines
therapeuticinDEBRrats(35,36).Withdevelopmentofalopecia, have complex roles in AA, with seemingly mixed pro- and anti-
MHC class I and II is upregulated on follicular epithelium, po- inflammatory activities.
tentially supporting bothCD4+ and CD8+Tcellresponses (3). mAb inhibition of TNFA, IFNG, IL-17, and IL-10 in
OneroleforCD4+Tcellsistoprovidehelpfortheformationof 1MOG244.1micedidnotleadtosignificantdifferencesindisease
pathologicCD8+Tcells(37).Akeyfindingofouranalysisisthat incidenceorseveritycomparedwithcontrols,suggestingthatthey
class I MHC-restricted T cells are capable of independently me- donotplayessentialrolesintheCD8+TcellAAresponse.This
diatingalopeciadevelopmentandprogression.FewCD4+Tcells interpretation must be tempered, however, by the fact that mAb D
o
are present in 1MOG244.1 retrogenic mice (Fig. 4A). Further, inhibitionwasperformedforaperiodofjust5wkbeginningatthe w
n
purified adoptively transferred CD8+ T cells mediated disease timeofanticipatedclinicaldiseasedevelopment,anditispossible loa
d
ainfteclratsrsanIsMfeHrCin-tdoefiRcaiegn1t2b/22mm2i/c2erbeuctipfiaeinltesd. Itnocinodnutrcaest,alcolapsesciIaI tphoastsicbrlietitchaaltfcuyntocktiionnealarcetdiounnsdaoncccyuarmeoanrlgiecryttohkainnetshiisn.thItisismoadlseol ed from
h
MHC was not required for disease development after adoptive permits normal disease development despite blockade of indi- ttp
transfer. This implies that class I MHC-restricted 1MOG244.1 vidual cytokines. The generation of 1MOG244.1 retrogenics ://jo
u
CD8+ T cells are capable of independently producing effector in Rag2/2 mice bred for select cytokine deficiencies will be im- rna
responsesnecessaryforAAdevelopmentandfullprogression.An portant toevaluate further theroleofindividual cytokines. ls.a
a
alternativepossibilityisthattheCD4+Tcellsprovidedhelptothe Importantly,noAghasbeenidentifiedthatisrecognizedbyAA- i.org
CD8+ cells prior to transfer. We consider this improbable, as it associated T cells. The 1MOG244.1 T cell clone independently /jim
m
would seem unlikely that the very small number of these cells mediatesAAandmaythereforeserveasatoolforAgscreening. u
n
o
present would be effectively stimulated by a class I-restricted The loss of immune privilege in the hair follicle in association l/a
autoantigen in the absence of a CD8 coreceptor. More likely, with AA suggests that follicle-associated Ags stimulate autoim- rticle
the relatively high numbers of monoclonal 1MOG244.1 CD8+ munity. Melanocytes have also been hypothesized to be a target, -pd
T cells bypasses requirements for CD4+ T cell help, as has been anddecreasednumbersoffollicle-associatedmelanocytesareob- f/18
8
observed elsewhere (38). Indeed, alopecia was only weakly in- servedincasesofAA(52).Likewise,stimulationofTcellswith /1/4
7
ducedaftertransferintowild-typerecipients,contrastingwiththe melanocyte-derived peptides enhanced AA in an experimental 7/1
more severe disease after transfers into Rag12/2 or sublethally model,andimmunotherapyofamousemodelofmelanomaledto 34
8
1
irradiatedmice.Diseaseinthesewild-typerecipientswasregional the development of alopecia (9, 53). However, AA is not intrin- 56
and associated with infiltrates of CD3+ cells. Potentially, the ho- sically associated with melanocyte loss, and though graying was /110
0
meostaticstimulusofthelymphodepletemicroenvironmenthelps seeninhairsinthe1MOG244.1model(Fig.7),thiswastypically 65
7
sustain the1MOG244.1 Tcellsandcompensates fortheabsence observed only after several rounds of hair loss and regrowth and .pd
omfoCnDos4p+ecTificcelClhDe4lp+.TThceeldlerveeslpoopnmseesn,tsoifmmilaordetolstyhsatteminstahsissesstsuindgy nfoorteucnoifnosrimdelryisteemno,rpearptriocublaabrlleytihnaetatrhlye d1iMseOasGe2s4t4at.1es.TWceeltlhsearree- f by gue
withCD8+cells,willassistinfullyevaluatingtheroleofThcells. specific for Ags that are not melanocyte associated, and that by- st o
n
Notably, unlike CD8+ T cells, both regulatory and effector func- standerlossoffollicularmelanocyteswithprogressivecyclesof 21
tions havebeen ascribedtoCD4+Tcellsin AA(39,40). alopecia leads to amelanosis. Keratins have also been implicated Feb
ru
Theeffectorpathwaysusedbythe1MOG244.1Tcellsremain asimmunologictargetsinAA(54)andmayserveasgoodcan- a
tobedefined.SeveralcytokineshavebeenimplicatedinAApatho- didates forscreeningwith the1MOG244.1 TCR. ry 20
2
genesis.TNFAaffectsskininmultipleways,andpolymorphisms Althoughalopeciauniversalisultimatelydevelopedinallmice, 3
inithave beenassociated with AA (41).Notably,despitebeing diseasewascyclicalandhighlyregional.Alopecicregionswerenot
generallyproinflammatory,blockadeofTNFAisnotonlyineffec- defined by dermatomes or other obvious borders and were often
tiveinAAbutalsohasbeenassociatedincasereportswithAA irregularinshapeandwithdistinctborders.Histologicstudieswere
development (42–45). Biologically, TNFA has been shown to unable to associate the follicular loss with specific hair follicle
block the IFN-mediated MHC upregulation in hair follicles that phases.Further,cyclicaldiseasewasregional,andatasingletime
promotes AA (3,46). A pathologicrole forIFNG is more estab- pointdistinctpatchesshowedhairlossorregrowth.Patchybaldness
lished in model systems, and the effector T cell response in AA is characteristic of the most common forms of alopecia areata in
appearsprimarilyTh1/Tc1(32,47).Ifng2/2C3H/HeJmicewere people, too, and the simultaneous growth and loss of hair is ap-
found to be resistant to AA (48). IFNG treatment was also asso- parent in the reticular variant of the disease. This seemingly ar-
ciated with enhanced disease in one study (49) but not a second bitraryregionalismmayindicatethatdiseaseisunassociatedwith
(50). It was therefore curious that relatively small amounts of eitherglobalcyclesofTcellfunctionalpotentialorwithhaircycle
IFNG were observed in skin sections in 1MOG244.1 mice (Fig. changes.Alternatively,studiesofhaircycleinmice,includingB6
10B). However, this may reflect the limited number of T cells mice,havedemonstratedcomplexhaircycledomainsinindividual
present within the skin mass, which may serve as the primary animals (55). Indeed, some mutant mice possess a cyclical alo-
source of the cytokine. Indeed, similar findings were present in peciawherehairshaftsdissociatefromthefolliclesduringspeci-
a cross-sectional transcriptome study done at 2-wk intervals of fichaircyclestages,andpatchinessisobservedinthesecircum-
theC3H/HeJskingraftmodel(32)andconfirmedinamorerecent stances.
TheJournal ofImmunology 485
Insummary,wedescribeanovelmousemodelforAAonthe 22. Iwanami,K.,I.Matsumoto,Y.Tanaka-Watanabe,A.Inoue,M.Mihara,Y.Ohsugi,
B6 background with similarities to the reticular variant though M. Mamura, D. Goto, S. Ito, A. Tsutsumi, et al. 2008. Crucial role of the
interleukin-6/interleukin-17 cytokine axis in the induction of arthritis by
progressing to alopecia universalis. Our findings indicate that glucose-6-phosphateisomerase.ArthritisRheum.58:754–763.
monoclonalCD8+Tcellscanindependentlymediatespontaneous 23. Ji,Q.,A.Perchellet,andJ.M.Goverman.2010.Viralinfectiontriggerscentral
nervoussystemautoimmunityviaactivationofCD8+Tcellsexpressingdual
AA in a class I MHC-dependent manner and that progressive
TCRs.Nat.Immunol.11:628–634.
disease is associated with systemic T cell expansion and degen- 24. Donnelly,M.L.,L.E.Hughes,G.Luke,H.Mendoza,E.tenDam,D.Gani,and
erate cytokine production. The 1MOG244.1 T cells further pro- M.D.Ryan.2001.The‘cleavage’activitiesoffoot-and-mouthdiseasevirus2A
site-directedmutantsandnaturallyoccurring‘2A-like’sequences.J.Gen.Virol.
vide an opportunity to better define Ags responsible for AA and
82:1027–1041.
disease pathophysiology. 25. Udyavar, A., R. Alli, P. Nguyen, L. Baker, and T. L. Geiger. 2009. Subtle
affinity-enhancingmutationsinamyelinoligodendrocyteglycoprotein-specific
TCRalterspecificityandgeneratenewself-reactivity.J.Immunol.182:4439–
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Disclosures
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