Small-intestinal TG2-specific plasma cells at different stages of coeliac disease

RESEARCH ARTICLE Open Access Small-in es inal TG2-speci ic plasma cells a di e en s ages o coeliac disease Minna Hie ikko 1 , Ou i Koskinen 1 , Kalle Ku ppa 2,3 , Kaija Lau ila 1 , Päi i Saa alainen 4 , Teea Salmi 1,5 , Tui e Ilus 1,6 , Heini Huh ala 7 , Ka i Kaukinen 1,8 and Ka i Lind o s 1* Abs ac Backg ound: In coeliac disease, inges ion o glu en induces he p oduc ion o ansglu aminase 2 (TG2)- a ge ed au oan ibodies by TG2-speci ic plasma cells p esen a high equency in he small in es inal mucosa in un ea ed disease. Du ing ea men wi h a glu en- ee die (GFD), he numbe o hese cells dec eases conside ably. I has no been p e iously in es iga ed whe he he cells a e also p esen p io o de elopmen o illous a ophy, o in non- esponsi e pa ien s and hose wi h die a y lapses. We aimed o de ine he equency o small bowel mucosal TG2- speci ic plasma cells in coeliac disease pa ien s wi h a ying disease ac i i y, and o in es iga e whe he he equency co ela es wi h se um and small in es inal TG2- a ge ing an ibodies as well as mucosal mo phology and he numbe o in aepi helial lymphocy es. Resul s: Mucosal TG2-speci ic plasma cells we e ound in 79% o pa ien s p io o de elopmen o mucosal damage, in all pa ien s wi h illous a ophy, and in 63% o he pa ien s a e 1 yea on GFD. In hese disease s ages, TG2-speci ic plasma cells accoun ed o median o 2.3, 4.3, and 0.7% o all mucosal plasma cells, espec i ely. A e long- e m ea men , he cells we e p esen in 20% o he pa ien s in clinical emission (median 0%) and in 60% o he pa ien s wi h poo die a y adhe ence (median 5.8%). In pa ien s wi h non- esponsi e coeliac disease despi e s ic GFD, he cells we e ound in only one (9%) subjec ; he cells accoun ed o 2.4% o all plasma cells. A posi i e co ela ion be ween he pe cen age o TG2-speci ic plasma cells and se um TG2 an ibody le els ( S =0.69,P<0.001) and he in ensi y o mucosal TG2- a ge ing IgA deposi s ( S = 0.43, P < 0.001) was obse ed. Conclusions: Ou esul s show ha TG2-speci ic plasma cells a e al eady de ec able p io o illous a ophy, and ha gene ally hei equency inc eases du ing o e disease. By con as , on GFD, he pe cen age o hese cells dec eases. O e all, he p esence o TG2-speci ic plasma cells in he small bowel mucosa mi o s he p esence o glu en in he die , bu he equency is no always pa allel o he le el o se um o in es inal TG2 an ibodies. These indings inc ease he knowledge abou he de elopmen o he TG2 plasma cell esponses especially in he ea ly phases o coeliac disease. Keywo ds: Coeliac disease, Glu en, T ansglu aminase 2, Au oan ibody, Small in es ine Backg ound In coeliac disease, die a y glu en in whea , ye, and ba ley unc ions as a d i ing an igen o an abno mal immune esponse ha de elops in gene ically suscep ible indi iduals ca ying he human leukocy e an igen (HLA)-DQ2 o -DQ8 haplo ypes. The disease is cha ac- e ised by small-bowel mucosal damage which de elops g adually om no mal illous mo phology o in lamma- ion and inally o illous a ophy wi h c yp hype plasia diagnos ic o coeliac disease. The in es inal damage is o en coupled wi h nume ous gas oin es inal symp oms, al hough a ious ex ain es inal mani es a ions a e also p e alen . A speci ic cha ac e is ic o coeliac disease is he gene a ion o immunoglobulin class A (IgA) an ibodies owa ds he main au oan igen, ansglu ami- nase 2 (TG2) [1]. These au oan ibodies a e gene ally ound in he ci cula ion o coeliac disease pa ien s [2] and as deposi s in he small in es inal mucosa below he subepi helial basemen memb ane and a ound blood essels [3]. In e es ingly, in es inal TG2- a ge ed deposi s can be de ec ed e en p io o mani es mucosal damage and in he absence o se um an ibodies [4–6]. * Co espondence: [email p o ec ed] 1 Celiac Disease Resea ch Cen e , Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, P.O. Box 100, 33014 Tampe e, Finland Full lis o au ho in o ma ion is a ailable a he end o he a icle © The Au ho (s). 2018 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed. Hie ikko e al. BMC Immunology (2018) 19:36 h ps://doi.o g/10.1186/s12865-018-0275-7 Upon emo al o glu en om he die , he only cu en ly a ailable ea men , he clinical symp oms and his o- pa hological changes in he small in es ine esol e, and bo h he ci cula ing and in es inal an ibodies disappea wi hin 1 yea in mos pa ien s [7]. Howe e , a subse o pa ien s ails o espond o he die a y ea men and he illous a ophy pe sis s despi e a s ic glu en- ee die (GFD). The mos common eason o pe sis en illous a ophy is ei he ad e en o inad e en glu en in ake o , in a e cases, e ac o y coeliac disease [8]. TG2- a ge ing an ibodies we e long hough o be gene a ed by in es inal plasma cells [9–11], bu ecen da a sugges s ha hey migh also be p oduced in lymphoid issues ou side he gu [12]. In he small in es- ine, he TG2-speci ic plasma cells a e p esen a high equency du ing he ac i e disease [10,11], and hey dec ease conside ably wi hin 6–12 mon hs a e com- mencemen o a s ic GFD [11]. Howe e , no da a exis ega ding he p esence o hese cells in he ea ly phases o coeliac disease when he mucosal mo phology is s ill no mal. In addi ion, hei exis ence in non- esponding coeliac disease pa ien s o hose wi h die a y lapses has no been p e iously in es iga ed. Wi h his in mind, we enume a ed he TG2-speci ic plasma cells in un ea ed and ea ed coeliac pa ien s wi h a ying deg ees o disease ac i i y, and in es iga ed whe he he numbe o hese cells co ela es wi h se um TG2 an ibody le els, he in ensi y o mucosal TG2- a ge ing IgA deposi s, and small in es inal mucosal mo phology and in lamma ion. Me hods Pa ien s and s udy design The s udy coho comp ised 46 coeliac disease pa ien s who unde wen uppe gas oin es inal endoscopy a he Depa men o Gas oen e ology and Alimen a y T ac Su ge y o Tampe e Uni e si y Hospi al (Table 1). Fi een o he pa ien s we e clinically suspec ed o ha ing coeliac disease based on gas oin es inal symp oms and posi i e coeliac disease-speci ic au oan ibodies (endomy- sial and/o TG2 an ibodies) despi e ha ing no mal small bowel mucosa ( illous heigh c yp dep h a io (Vh/ C D) ≥2). These pa ien s we e p ospec i ely ollowed up while hey con inued on a no mal glu en-con aining die Table 1 Demog aphic da a and he small-bowel mucosal and se ological indings o pa ien s pa icipa ing in he s udy P ospec i ely s udied coeliac pa ien s (n= 15) Long- e m ea ed coeliac pa ien s (n= 31) Disease con ols (n= 25) CD p io o a ophy n=14 O e CD n=15 1 yea GFD n=11 Pa ien s in emission n=15 Non- esponding CD n=11 Pa ien s wi h die a y lapses n=5 Glu en sensi i i y n=18 Dyspepsia n=7 Female; n (%) 10 (71) 10 (67) 7 (64) 10 (67) 7 (64) 5 (100) 16 (89) 1 (14) Age; median ( ange), yea s 55 (16–70) 55 (17–71) 57 (28–72) 59 (24–66) 49 (40–76) 51 (31–77) 49 (24–65) 47 (24–76) Du a ion o GFD; median ( ange), yea s 00 1(1–1) 8 (3–34) 7 (3–24) 10 (9–17) 0 0 HLA-DQ2 o -DQ8-posi i e; n (%) 14 (100) 15 (100) 11 (100) 15 (100) 11 (100) 4 a (100) 9 (50) 1 (14) EmA; median ( ange), i e 1:100 (0– 1:2000) 1:100 (0– 1:4000) 0(0–1:50) 0 (0) 0 (0–1:5) 1:200 (0– 1:2000) 0 (0) 0 (0) TG2 abs; median ( ange), U/ml 9.4 (3.3- > 100) 11.9 (4.2- > 100) 3.9 (0–8.6) 0.5 (0–2.8) 1.3 (0–9.9) 56.9 (12.9- > 100) 1.4 (0–4.1) 0.6 (0–2.8) Mucosal TG2-IgA deposi s p esen ; n (%) 14 (100) 14 a (100) 7 b (88) 5 (33) 10 a (100) 3 c (100) 4 (22) 0 (100) Vh/C D; mean (95% CI), a io 2.9 (2.6–3.1) 1.4 (1.1–1.7) 3.4 (2.4–4.5) 3.4 (3.2– 3.6) 0.2 (0.0–0.4) 0.5 (−0.3–1.3) 3.5 (3.0– 4.1) 3.5 (3.0–4.1) CD3 + IELs; median ( ange), cells/mm 54 (12–79) 67 (38–116) 39 (23–80) 42 (25–77) 60 (30–109) 50 (38–69) 21 (7–59) 26 (16–40) αβ + IELs; median ( ange), cells/mm 30 (12–50) 43 (21–75) 22 (14–43) 31 (20–46) 44 (26–105) 38 (35–56) 16 (5–26) 22 (17–31) γδ + IELs; median ( ange), cells/mm 18.8 (0–38.5) 23.7 (14–58.7) 13.6 (1.4– 56.3) 14.0 (7.3– 27.8) 12.1 (0–37.8) 13.0 (4.4–20.5) 2.7 (0– 10.2) 1.6 (0.7– 16.1) abs an ibodies, CD coeliac disease, CI con idence in e al, EmA endomysial an ibodies, GFD glu en- ee die , GS glu en sensi i e; IgA immunoglobulin A; IELs in aepi helial lymphocy es; TG2-abs, ansglu aminase 2 an ibodies; Vh/C D illous heigh c yp dep h a io Re e ence alues se a 2.0 o Vh/C D, 37 cells/mm o CD3 + IELs, 25 cells/mm o αβ + IELs, and 4.3 cell/mm o γδ+ IELs (Jä inen e al., [15]) Cu -o alue o TG2 an ibodies ≥5 AU/ml a Da a missing om one pa ien b Da a missing om h ee pa ien s c Da a missing om wo pa ien s Hie ikko e al. BMC Immunology (2018) 19:36 Page 2 o 7 o 1 yea , du ing which illous a ophy de eloped (Vh/ C D < 2) in all pa ien s. The ea e , he pa ien s s a ed a GFD, and a e 1 yea on he die , hei mucosal mo ph- ology had eco e ed. Small bowel samples om 14 o he pa ien s a he ime o he no mal mucosal mo ph- ology, all 15 pa ien s a he ime o he illous a ophy, and 11 o he pa ien s a e 1 yea on a GFD we e a ail- able o he cu en s udy (Table 1). Fu he mo e, 15 coeliac disease pa ien s on a long- e m GFD wi hou symp oms and e incing ull his ological eco e y, 11 non- esponsi e coeliac disease pa ien s wi h pe sis en illous a ophy despi e a s ic GFD, and i e pa ien s wi h poo die a y adhe ence we e in es iga ed. Twen y pa ien s wi h sel - epo ed glu en sensi i i y expe iencing abdominal symp oms a e con- sump ion o glu en-con aining p oduc s [13] and se en pa ien s wi h dyspepsia se ed as he non-coeliac con- ols in he s udy. All con ols had been excluded o coeliac disease, as demons a ed by nega i e se ology and no mal small bowel mucosal mo phology. The demog aphic da a and small-bowel mucosal and se o- logical indings o all subjec s a e epo ed in Table 1. The s udy p o ocol was app o ed by he E hics Commi ee o he Pi kanmaa Hospi al Dis ic , Tampe e, Finland, and w i en in o med consen was ob ained om all pa icipa ing subjec s. Small-in es inal mucosal mo phology and in lamma ion Small-in es inal mucosal biopsies we e ob ained upon uppe gas oin es inal endoscopy. Fo mo phological s udies, one o malin- ixed biopsy sample was s ained wi h haema oxylin and eosin o de e mine he illous heigh -c yp dep h a ios (Vh/C D) acco ding o a p e i- ously published p ocedu e [14]. A a io o ≥2 was con- side ed no mal. One o he biopsies was subme ged in op imal cu ing empe a u e compound (OCT; Tissue-Tek, Saku a Fine ek Eu ope, Holland), ollowed by snap- eezing in liquid ni ogen. The ea e , 5-μm- hick sec ions we e cu . Acco ding o an es ab- lished p o ocol [15], he sec ions we e s ained o CD3 + , αβ + , and γδ + in aepi helial lymphocy e (IEL) subse s. The e e ence alues we e 37 cells/mm, 25 cells/mm, and 4.3 cells/mm o CD3 + IELs, γδ + IELs and αβ + IELs, espec i ely [15]. Se ological measu emen s and HLA geno yping Se um endomysial an ibodies (EmA) in IgA class we e de e mined by an indi ec immuno luo escence me hod exploi ing human umbilical co d as subs a e. A dilu ion o 1:≥5 was conside ed posi i e [16]. Se um IgA-class TG2 an ibodies we e measu ed by a comme cially a ail- able enzyme-linked immunoso ben assay (Celikey®, Phadia, F eibu g, Ge many) in samples dilu ed 1:100. A i e o ≥5 AU/ml was se as he cu -o o posi i i y. SSP DQB1 low- esolu ion ki (Ole up SSP AB, Sal sjö- baden, Sweden), DELFIA Celiac Disease Hyb idiza ion Assay (Pe kinElme Li e and Analy ical Sciences, Wallac Oy, Tu ku, Finland) o HLA- agging single-nucleo ide pep ides [17] we e used o HLA geno yping. Small-in es inal TG2-speci ic IgA deposi s Fo he de e mina ion o mucosal TG2- a ge ing IgA deposi s, ozen sec ions we e s ained wi h mouse monoclonal an i-TG2 an ibody (CUB7402; NeoMa ke s, F emon , Cali o nia, USA), ollowed by de ec ion wi h luo escein iso hiocyana e (FITC) -labelled abbi an i-human IgA an ibody (Dako A/S, Glos up, Denma k) [3]. Based on hei in ensi y along he base- men memb ane in he illous-c yp a ea, he deposi s we e g aded blinded as a nega i e, o a weak, mode a e, o s ong posi i e, as desc ibed p e iously [6]. Small-in es inal TG2-speci ic plasma cells An ea lie desc ibed echnique was used o de ec muco- sal TG2-speci ic plasma cells [10]. Ini ially, 5-μm- hick ozen sec ions we e ai -d ied o 20 min a oom empe a u e (RT). A e washing in PBS, he sec ions we e incuba ed wi h bio inyla ed human ecombinan TG2 (2 μg/ml; T002, Zedi a) o 45 min a RT. Bio inyl- a ion was pe o med using EZ-Link® Sul o-NHS-LC-Bio in (The mo Scien i ic, Wal ham, MA, USA) acco ding o he ins uc ions p o ided by he manu ac u e . The ea e , he sec ions we e incuba ed wi h hodamine-labelled s ep a idin (1:1000; KPL, Gai he sbu g, MD, USA) o 30 min a RT. Plasma cells we e iden i ied using a mouse monoclonal CD138 an i- body (1:25; B-A38, Bio-Rad), ollowed by goa an i-mouse IgG Alexa Fluo 488 (1:2000; A-11001, The mo-Fishe Scien i ic). S ainings we e analysed a 20x and 40x magni ica ion (Olympus BX60F5, Olympus Op ical Co. LTD, Japan) on wo consecu i e small in es- inal biopsy sec ions and he pe cen age o TG2-speci ic cells ou o all lamina p op ia plasma cells in he en i e sec ion was de e mined. S a is ical analyses Da a a e exp essed as he numbe o subjec s (n) and pe cen ages, o as medians and anges. S a is ical analyses we e pe o med using he Wilcoxon es o Mann–Whi ney es as app op ia e. Co ela ion was e alua ed using Spea man’s co ela ion. S a is ical es ing was pe o med using s a is ical analysis so wa e (IBM SPSS S a is ics, SPSS Inc., Chicago, IL, USA). A P- alue < 0.05 was conside ed s a is ically signi ican . Resul s O he 15 p ospec i ely s udied coeliac disease pa ien s, TG2-speci ic plasma cells we e al eady p esen in 11 ou Hie ikko e al. BMC Immunology (2018) 19:36 Page 3 o 7 o he 14 a ailable small bowel samples (79%) om he pa ien s be o e he de elopmen o illous a ophy. The median pe cen age o he cells was 2.3% ( ange 0– 12.7%) o all lamina p op ia plasma cells (Fig. 1a and b). A e con inuing on a glu en-con aining die o 1 yea and de eloping o e small bowel mucosal damage, all i een pa ien s had in es inal TG2-speci ic plasma cells, and he median pe cen age o he cells was 4.3% ( ange 1.8–8.8%) (P= 0.055 when compa ed o pa ien s wi h ea ly-s age coeliac disease). By con as , a e 1 yea on a GFD, he cells we e ound in 7 ou o 11 (64%) pa ien s wi h a ailable samples, and he median pe cen age o he cells signi ican ly dec eased o 0.7% ( ange 0–2.9%, P= 0.003) when compa ed o he o e disease. In long- e m GFD- ea ed pa ien s in clinical emis- sion esponding well o die a y ea men , only a ew emaining TG2-speci ic plasma cells (median 0.0%, ange 0–1.1%) we e de ec ed in 3 ou o 15 (20%) o he pa ien s (Fig. 1a and b). In non- esponding coeliac disease pa ien s on a s ic GFD, he cells we e mos ly absen , being p esen in only one pa ien (9%) who had 2.4% o TG2-speci ic cells ou o all lamina p op ia plasma cells. O he coeliac disease pa ien s wi h die a y lapses, h ee ou o i e (60%) had TG2-speci ic plasma cells, he median being 5.8% ( ange 0–7.0%). No TG2-speci ic plasma cells we e ound in any o he non-coeliac con ol pa ien s wi h ei he glu en sensi i i y o dyspepsia. A posi i e co ela ion be ween he pe cen age o TG2-speci ic plasma cells and se um TG2 an ibody le els ( S = 0.690, P< 0.001, Fig. 2a) as well as EmA ( S = 0.712, P < 0.001) was obse ed when da a om all coeliac disease pa ien g oups we e included in he ana- lysis. Simila ly, he pe cen age o he TG2-speci ic plasma cells co ela ed wi h he in ensi y o he small in es inal IgA deposi s in all coeliac disease pa ien s ( S = 0.430, P< 0.001) (Fig. 2b). Howe e , he pe cen age o TG2-speci ic plasma cells did no co ela e wi h se um Fig. 1 a. The pe cen age o small-bowel mucosal ansglu aminase 2 (TG2) -speci ic plasma cells ou o all lamina p op ia plasma cells in he di e en pa ien g oups. b. Immuno luo escence s aining o ansglu aminase 2 (TG2) an ibodies and plasma cells in small-bowel mucosal sec ions. Rep esen a i e pic u e o a coeliac disease pa ien p io o illous a ophy showing posi i e s aining o TG2-speci ic plasma cells (a ows). Recombinan TG2 ( ed), plasma cell ma ke CD138 (g een), and hei colocalisa ion (yellow) a 20x magni ica ion. Scale ba = 100 μm. Abb e ia ions: CD, coeliac disease; GFD, glu en- ee die Hie ikko e al. BMC Immunology (2018) 19:36 Page 4 o 7 o mucosal TG2 an ibodies in any o he indi idual coeliac disease pa ien g oups (Addi ional ile 1Table S1). Conside ing all coeliac disease pa ien s, he e was no co ela ion be ween he pe cen age o he plasma cells and Vh/C D. O he IELs, he e was a modes co - ela ion be ween he pe cen age o TG2-speci ic plasma cells and γδ + IELs. De ailed in o ma ion abou he co e- la ions is p esen ed in Addi ional ile 1Table S1. Discussion In he cu en s udy, we ha e disco e ed ha TG2-speci ic plasma cells a e al eady p esen in mos pa ien s p io o de ec able mucosal damage. Mo eo e , we showed ha in he majo i y o cases he pe cen age o hese cells inc eases upon con inuous glu en in ake and he subsequen de elopmen o illous a ophy. On a s ic GFD, he pe cen age o hese cells declines. A e long- e m ea men , he cells a e mos ly absen bo h in pa ien s in clinical emission and in non- esponding pa ien s wi h pe sis en illous a ophy. By con as , TG2-speci ic plasma cells can be de ec ed in pa ien s wi h die a y lapses. In con ols, e en hose wi h glu en- ela ed symp oms, no cells we e de ec ed. Ou esul s om un ea ed and ea ed coeliac disease pa ien s a e in line wi h p e ious s udies [10,11], show- ing ha he amoun o TG2-speci ic plasma cells is ele- a ed in he small in es inal mucosa a he ime o diagnosis and his amoun dec eases on a GFD. I has ea lie been shown ha he pe cen age o hese cells ou o all lamina p op ia plasma cells accoun s o up o 24% in o e disease, being on a e age 10% [10,11]. Howe e , in he cu en s udy, he co esponding pe - cen ages we e lowe . Ou pa ien s had been ec ui ed o he s udy while s ill ha ing no mal mucosal mo phology, and hey de eloped o e illous a ophy wi hin a one-yea ollow-up on a glu en-con aining die . Thus, i is concei able ha he pa ien s had had la mucosal lesion o a easonably sho e ime han in he p e ious a b Fig. 2 Co ela ion be ween he pe cen age o small in es inal ansglu aminase 2 (TG2) -speci ic plasma cells and se um IgA class TG2 an ibody le els (a) and he in ensi y o small in es inal TG2- a ge ing immunoglobulin a (IgA) deposi s (b) in all coeliac disease pa ien s. G ading o IgA deposi s as ollows: 0 = nega i e, 1 = weak, 2 = mode a e, 3 = s ong Hie ikko e al. BMC Immunology (2018) 19:36 Page 5 o 7 s udies, which migh explain he lowe pe cen ages o TG2-speci ic plasma cells in ou s udy. TG2-speci ic plasma cells co ela ed posi i ely wi h se um TG2 an ibody le els when he da a o all coeliac disease pa ien s we e analysed oge he . This co ela ion mos likely mi o s he esponsi eness o he plasma cells o glu en exposu e which is no su p ising in he ligh ha TG2 an ibodies ha e been sugges ed o a ise by a hap en-ca ie -like mechanism in ol ing TG2-ca alysed gene a ion o glu en-TG2 complexes [18,19]. On he o he hand, co ela ions be ween he pe cen ages o he plasma cells and se um an ibody le els we e no de ec ed when di e en coeliac disease g oups we e analysed sepa - a ely; his is in ag eemen wi h p e ious indings in un ea ed coeliac disease pa ien s [11]. I has been p o- posed ha he lack o co ela ion could be explained by he p oduc ion o he an ibodies also ou side he gu [11]. This concep has ecen ly been u he suppo ed by he inding ha coeliac pa ien se um and in es inal TG2 an i- bodies a e clonally ela ed bu ha e di e en molecula composi ions, poin ing o di e en si es o o igin [12]. Such ex ain es inal p oduc ion o TG2 an ibodies could also explain why some pa ien s in ou s udy had se um TG2 an ibodies in he absence o in es inal TG2-speci ic plasma cells. Al hough small in es inal TG2-speci ic plasma cells a e no likely o be he majo sou ce o se um TG2 an i- bodies [12], i would be logical o assume ha he TG2 an ibodies bound o hei an igen in he small in es inal mucosa and p edic ing o hcoming mucosal damage a e p oduced locally by lamina p op ia TG2-speci ic plasma cells. In his s udy, we obse ed a co ela ion be ween he pe cen age o he plasma cells and he in ensi y o mucosal TG2-speci ic IgA deposi s, which suppo s his hypo hesis. In e es ingly, howe e , TG2-speci ic plasma cells we e mos ly absen in non- esponding coeliac pa ien s, e en hough hey all p esen ed wi h s ong TG2- a ge ing IgA deposi s in he small in es inal mucosa despi e a s ic GFD. I has been p oposed ha he long pe sis ence o small in es inal IgA deposi s in non- esponsi e pa ien s on a s ic die may be explained, o ins ance, by he high a idi y binding o he IgA an ibodies o small in es inal TG2 [6,7]. This could explain he p esence o IgA deposi s in he absence o TG2 an ibody-sec e ing plasma cells in ou non- esponsi e pa ien s. Howe e , i does no p o ide an explana ion o he p esence o IgA deposi s in he absence o plasma cells in he small subse o pa ien s p io o de elopmen o illous a ophy. Simila ly, i does no explain he p esence o weak IgA deposi s wi hou TG2-an ibody sec e ing plasma cells in a ew glu en-sensi i e con ol pa ien s. Whe he ex ain es inal p oduc ion o TG2 an ibodies occu ing o ins ance in he bone ma ow, spleen and lymph nodes con ibu es o he appea ance o mucosal IgA deposi s emains o be add essed in u u e s udies. Conclusions We conclude ha he TG2-speci ic plasma cells a e al eady p esen in he ea ly phases o coeliac disease when he mucosal mo phology is s ill no mal, hei pe cen age inc eases upon he de elopmen o illous a ophy and dec eases on a GFD. O e all, he equency o TG2 an ibody-sec e ing plasma cells in he di e en phases o coeliac disease e lec s he p esence o glu en in he die , bu he equency o hese cells is no always pa allel wi h se um TG2 an ibody le els o he in ensi y o small in es inal TG2- a ge ing IgA deposi s. Ou indings widen he unde s anding o small-bowel mucosal TG2-speci ic plasma cells in coeliac disease and hus p o ide u he insigh in o he gene a ion o TG2 an ibody esponses. Addi ional ile Addi ional ile 1: Table S1. Co ela ions be ween he pe cen age o TG2-speci ic plasma cells and o he s udy pa ame e s. (DOCX 15 kb) Abb e ia ions EmA: Endomysial an ibody; GFD: Glu en- ee die ; HLA: Human leukocy e an igen; IEL: In aepi helial lymphocy e; IgA: Immunoglobulin A; RT: Room empe a u e; TG2: T ansglu aminase 2; Vh/C D: Villous heigh -c yp dep h a io Acknowledgemen s No applicable. Funding This s udy was suppo ed by he Academy o Finland, he Finnish Medical Founda ion, he Resea ch Fund o he Finnish Coeliac Socie y, he Sig id Juselius Founda ion, he Founda ion o Pedia ic Resea ch, and he Compe i i e S a e Resea ch Financing o he Expe A ea o Tampe e Uni e si y Hospi al. A ailabili y o da a and ma e ials The da ase s gene a ed and/o analysed du ing he cu en s udy a e no publicly a ailable due o es ic ions de ined by he E hics Commi ee. Au ho s’con ibu ions Concei ed and designed he s udy: KL, KaKa. Concei ed, designed, and pe o med he expe imen s: MH, OK, KL, PS. Analysed he da a: MH. Pe o med he s a is ical analysis: MH, HH. Pa icipa ed in pa ien ec ui men and ma e ial sampling: OK, KuKa, TS, TI, KaKa. All au ho s ha e ead, e ised, and app o ed he inal manusc ip . E hics app o al and consen o pa icipa e The s udy p o ocol was app o ed by he E hics Commi ee o he Pi kanmaa Hospi al Dis ic , Tampe e, Finland, and w i en in o med consen was ob ained om all pa icipa ing subjec s. Consen o publica ion No applicable. Compe ing in e es s All au ho s ha e ead he jou nal’s policy on he disclosu e o po en ial con lic s o in e es and ha e none o decla e. Hie ikko e al. BMC Immunology (2018) 19:36 Page 6 o 7 Publishe ’sNo e Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a ilia ions. Au ho de ails 1 Celiac Disease Resea ch Cen e , Facul y o Medicine and Li e Sciences, Uni e si y o Tampe e, P.O. Box 100, 33014 Tampe e, Finland. 2 Tampe e Cen e o Child Heal h Resea ch, Uni e si y o Tampe e, Tampe e, Finland. 3 Depa men o Paedia ics, Tampe e Uni e si y Hospi al, Tampe e, Finland. 4 Depa men o Medical and Clinical Gene ics and he Resea ch P og ams Uni , Immunobiology, Uni e si y o Helsinki, Helsinki, Finland. 5 Depa men o De ma ology, Tampe e Uni e si y Hospi al, Tampe e, Finland. 6 Depa men o Gas oen e ology and Alimen a y T ac Su ge y, Tampe e Uni e si y Hospi al, Tampe e, Finland. 7 Facul y o Social Sciences, Uni e si y o Tampe e, Tampe e, Finland. 8 Depa men o In e nal Medicine, Tampe e Uni e si y Hospi al, Tampe e, Finland. Recei ed: 31 Augus 2018 Accep ed: 27 No embe 2018 Re e ences 1. Die e ich W, Ehnis T, Baue M, Donne P, Vol a U, Riecken E, e al. Iden i ica ion o issue ansglu aminase as he au oan igen o celiac disease. Na Med. 1997;3:797–801. 2. Sulkanen S, Hal unen T, Lau ila K, Kolho K, Ko ponay-Szabó IR, Sa nes o A, e al. 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