Birth, life, and death of the MAGE3 hypothesis of alopecia areata pathobiology

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. 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