1
1
Le e o he Edi o
P esence o MAGE-A3 speci ic T cells in alopecia a ea a - s udy o he
possibili y o AA an igens -
Taisuke I oa*, Ma a Be olinib, A suko Funakoshia, Na suho I oa, Ta suya Takayamac,
Tamas Bi od, Ral Pausb,e§ and Yoshiki Toku aa§
aDepa men o De ma ology, Hamama su Uni e si y School o Medicine, 1-20-1
Handayama, Higashi-ku, Hamama su 431-1192 Japan
bDepa men o De ma ology, Uni e si y o Lübeck, D-23538 Lübeck, Ge many
cDepa men o U ology, Hamama su Uni e si y School o Medicine, 1-20-1
Handayama, Higashi-ku, Hamama su 431-1192 Japan
dDE-MTA “Lendüle ” Cellula Physiology Resea ch G oup, Depa men o Physiology,
Uni e si y o Deb ecen, Medical and Heal h Science Cen e , Resea ch Cen e o
Molecula Medicine, Deb ecen, Nagye dei k . 98, H-4032 Hunga y
eIns i u e o In lamma ion and Repai , Uni e si y o Manches e , and he De ma ology
Cen e, Royal Sal o d Hospi al, Manches e , Uni ed Kingdom
§ con ibu ed equally
2
2
*Co esponding au ho a : Depa men o De ma ology, Hamama su Uni e si y School
o Medicine, 1-20-1 Handayama, Higashi-ku, Hamama su 431-1192 Japan
Tel.: +81 53 435 2303, ax.: +81 53 435 2368
E-mail add ess: [email p o ec ed]
Sho i le: MAGE3 and alopecia a ea a
Keywo ds: Alopecia a ea a; melanocy e-associa ed p o ein; MAGE; CTLs; immune
p i ilege
3
3
Dea Edi o ,
In his dis inguished jou nal, we ou inely ead disco e y s o ies ha ha e
been ul ima ely c owned by success. Ins ead, he e we would like o sha e ou ials and
ibula ions along a less elici ous, ye e y ins uc i e and educa ional esea ch jou ney
ha b ough us close o wha we hoped o be a clinically impo an ad ance in
unde s anding he pa hobiology o alopecia a ea a (AA), a issue-speci ic, T
cell-dependen au oimmune disease [1].
Mos cu en ly a ailable e idence sugges s ha , upon in e e on (IFN)--induced
collapse o he hai ollicle’s (HF’s) physiological immune p i ilege (IP), as ye
uniden i ied ollicula au oan igens a e exposed o p eexis ing au o eac i e CD8+ T cells
by ec opically exp essed majo his ocompa ibili y (MHC) class I molecules wi hin he
epi helium o anagen hai bulbs [1,2]. Pep ides de i ed om melanogenesis-associa ed
au oan igens exp essed only by melanin-p oducing anagen HFs a e pe suasi e
candida es as key au oan igens in AA [3]. The e o e, ocusing on well-in es iga ed
MHC class I- es ic ed melanocy e- ela ed an igens known o be ecognized by CD8+ T
cells is a sensible AA esea ch s a egy (supplemen a y ex S1).
We hus hypo hesized ha i should be possible o de ec cy o ox ic CD8+ T cells
(CTLs) di ec ed agains MHC class-I es ic ed au oan igens ( y osinase, MAGE-A2,
and MAGE-A3 (MBL)), using pen ame echnology [4] (Supplemen al ex S2). To es
his hypo hesis, pe iphe al blood mononuclea cells (PBMCs) we e ob ained om
4
4
Japanese heal hy con ols and AA pa ien s (Supplemen a y Table S1).
Ini ially, his app oach yielded auspicious esul s: MAGE-A3- eac i e CD8+ T cells
we e ound o be signi ican ly inc eased in PBMCs in he acu e phase o AA wi h
mul i ocal lesions (AAM) and alopecia a ea a o alis (AAT) compa ed o heal hy
con ols, ch onic phase o AAM, o AAT/alopecia a ea a uni e salis (AU) (Figu e 1a
and S1a,b, Supplemen a y Tex S3) (p=0.025 by K uskal-Wallis ANOVA). Fu he mo e,
skin in il a ing T cells o an acu e phase AA lesion om one pa ien , which we e
isola ed as p e iously desc ibed [5], also showed an inc eased numbe o MAGE-A3
speci ic CTLs (Figu e 1b) as ha in pe iphe al blood nuclea cells (PBMCs) om he
same AA pa ien s (Figu e 1c) compa ed o he a e age equency o MAGE-A3+ T cells
in PBMCs om con ol subjec s (Figu e 1a). This pilo inding sugges ed an en ichmen
o MAGE-A3+ CTLs in lesional AA skin.
Nex , we p obed whe he such CTLs can p oduce IFN- a e s imula ion wi h
MAGE-A3 (supplemen a y ex S4). Indeed, IFN- p o ein exp ession was signi ican ly
inc eased in CD8+ T cells om acu e phase AA pa ien s co-cul u ed wi h MAGE-A3
compa ed o heal hy con ols (Figu e 1d and supplemen a y Figu e S1c,d). Mo eo e ,
he pe cen age o MAGE-A3 speci ic CTLs in PBMCs was also moni o ed in an acu e
phase AAT pa ien (n=1, pa ien 16) du ing he ea men wi h o al 20 mg/day
p ednisolone o 60 days. Be o e he ea men , he pa ien su e ed om AAT and
2.30% o CD8+ T cells eac ed wi h MAGE-A3 (Figu e 1e). Six y days a e he
5
5
ea men , ma ked hai eg ow h had occu ed while he pe cen age o
MAGE-A3- eac i e CTLs dec eased o 0.49% (Figu e 1 ).
A his poin , we wonde ed whe he , con a y o he con en ional wisdom ha
MAGE-A3 is only exp essed by cance cells [6,7], MAGE-A3 is also exp essed in
human HFs, a leas unde in lamma o y condi ions. This had been encou aged by he
epo ha es is, placen a, e al o a y and wounded skin may also exp ess MAGE
amily membe s, besides melanoma and o he cance s [8,9]. Finally, i s
immunohis ological analyses conduc ed in one Japanese AA pa ien (Supplemen a y
ex S5) sugges ed ha AA-a ec ed HFs exp essed MAGE-A3-like immuno eac i i y
wi h an i-human MAGEs (Y-18) (Figu e 2a).
A his poin we go qui e exci ed: No only seemed he adop ed sho cu -s a egy o
iden i y bo h, one hypo hesis-d i en, ca e ully selec ed pu a i e key au oan igen as well
as he au o eac i e CTLs ha ecognize i , o ha e wo ked. Bu his also appea ed o
gene a e u he expe imen al suppo o he IP collapse hypo hesis o AA pa hogenesis,
which s ipula es a key ole o MHC class-I p esen ed, melanocy e- ela ed au oan igens
ecognized by CTLs. Finally, his would ha e been he i s demons a ion ha
MAGE-A3 is exp essed also by adul human scalp HFs, a leas unde p oin lamma o y
condi ions. Gi en, howe e , ha he demons a ion o in a ollicula MAGE-A3 gene
and p o ein exp ession is a co ne s one suppo ing he scena io ske ched abo e, we
decided o un addi ional analyses and con ols.

6
6
Fi s , by qRT-PCR analysis o mRNA ex ac ed om ei he heal hy human scalp HFs o
lesional skin, using app op ia e p ime s and con ols (Supplemen a y ex S6), ailed o
e eal MAGE-A3 ansc ip s abo e he de ec ion h eshold o ou assay (Figu e 2b).
Nex , when wo di e en , MAGE-A3-speci ic p ima y an ibodies (6C1 and 57B) we e
sys ema ically employed by immunohis ochemis y and an app op ia e MAGE-A3+
no mal human issue ( es is) [9,10] was used as posi i e con ol, along wi h igo ously
nega i e con ols (Supplemen a y ex S7), he p e iously de ec ed “MAGE-A3-like”
immuno eac i i y o AA HFs u ned ou o be nega i e (Figu e 2c- , Supplemen a y
Figu e S2d- , S2i-l) (Supplemen a y Tex S8). Finally, also a inal, semiquan i a i e
RT-PCR using di e en MAGE-A3 p ime s (Supplemen a y ex S6) ailed o show
MAGE-A3 mRNA in heal hy o AA skin (Figu e 2g). The e o e, unde physiological o
pa hological condi ions, human anagen scalp HFs do no exp ess MAGE-A3 on he
mRNA o p o ein le el, and he MAGE-A3- eac i e CTLs ound in AA pa ien s a e
likely o ha e p eexis ed be o e disease de elopmen .
In pu suing i , we p o ide he i s e idence o CD8+ CTLs ha do eac wi h
melanocy e-associa ed p o eins (he e: MAGE-A3) in HLA-A2402+ AA pa ien s and
show ha CD8+ T cells om AA pa ien s do exp ess he po en IP collapse induce ,
IFN- [2], upon s imula ion wi h MAGE-3A.
The appealing hypo hesis ha , a e HF-IP collapse, ec opic MAGE-A3 exp ession o
7
7
au o eac i e CD8+ T cells igge s a CTL-a ack on he HF, hus inducing he AA
pheno ype, may seem obsole e now. Ye , ha mo e MAGE-A3- eac i e CTLs a e
indeed p esen in acu e AAT and AAM pa ien s han in heal hy con ols o ch onic AA
pa ien s begs he ques ion whe he hese T cells a e in ol ed in AA pa hobiology, and
may hus be a wo hwhile he apeu ic a ge o u u e AA managemen , a e all.
8
8
Con lic s o in e es : None decla ed.
Acknowledgemen s
This s udy was suppo ed in pa by g an om DFG (GRK 1727/1) o RP and a DFG
PhD ellowship o MB (GRK 1727/1). We would like o hank P o . Giulio C. Spagnoli
(Onkologische Chi u gie, Ins i u e o Su gical Resea ch and Hospi al Managemen ,
Uni e si y Hospi al Basel, 4031 Basel, Swi ze land) o he gene ous gi o
an i-MAGE-3 monoclonal an ibody 57B, and M s. An je Win e -Keil o excellen
echnical assis ance.
9
9
Re e ences
1. Gilha A, E zioni A, Paus R. Alopecia a ea a. N Engl J Med 2012;366:1515-25.
2. I o T, I o N, Saa ho M, S ampachiacchie e B, Be e mann A, Bul one-Paus S, e
al. Collapse and es o a ion o MHC class-I-dependen immune p i ilege:
exploi ing he human hai ollicle as a model. Am J pa hol 2004;164:623-34.
3. Gilha A, Landau M, Assy B, Shalagino R, Se a imo ich S, Kalish RS.
Melanocy e-associa ed T cell epi opes can unc ion as au oan igens o ans e o
alopecia a ea a o human scalp explan s on P kdc(scid) mice. J In es De ma ol
2001;117:1357-62.
4. Yagi H, Hashizume H, Ho ibe T e al. Induc ion o he apeu ically ele an
cy o oxic T lymphocy es in humans by pe cu aneous pep ide immuniza ion.
Cance Res 2006;66:10136-44.
5. I o T, Hashizume H, Shimauchi T, Funakoshi A, I o N, Fukamizu H, e al.
CXCL10 p oduced om hai ollicles induces Th1 and Tc1 cell in il a ion in he
acu e phase o alopecia a ea a ollowed by sus ained Tc1 accumula ion in he
ch onic phase. J De ma ol Sci 2013;69:140-7.
6. Russo V, Pilla L, Lunghi F, C occhiolo R, G eco R, Cice i F, e al. Clinical and
immunologic esponses in melanoma pa ien s accina ed wi h
MAGE-A3-gene ically modi ied lymphocy es. In J Cance 2013; 132:2557-66.
7. Moelle I, Spagnoli GC, Finke J, Veelken H, Houe L. Up ake ou es o
umo -an igen MAGE-A3 by dend i ic cells de e mine p iming o naï e T-cell
sub ypes. Cance Immunol Immuno he 2012;61:2079-90.