Follicular helper T cells may play an important role in the severity of primary Sjögren's syndrome

  Follicula helpe T cells may play an impo an ole in he se e i y o p ima y Sj¨og en’s synd ome K isz ina Szabo, Gabo Papp, Sando Ba a h, Edi Gyimesi, An onia Szan o, Ma gi Zehe PII: S1521-6616(13)00063-6 DOI: doi: 10.1016/j.clim.2013.02.024 Re e ence: YCLIM 7124 To appea in: Clinical Immunology Recei ed da e: 13 No embe 2012 Accep ed da e: 26 Feb ua y 2013 Please ci e his a icle as: K isz ina Szabo, Gabo Papp, Sando Ba a h, Edi Gy- imesi, An onia Szan o, Ma gi Zehe , Follicula helpe T cells may play an impo an ole in he se e i y o p ima y Sj¨og en’s synd ome, Clinical Immunology (2013), doi: 10.1016/j.clim.2013.02.024 This is a PDF file o an unedi ed manusc ip ha has been accep ed o publica ion. As a se ice o ou cus ome s we a e p o iding his ea ly e sion o he manusc ip . The manusc ip will unde go copyedi ing, ypese ing, and e iew o he esul ing p oo be o e i is published in i s final o m. 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ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1 Follicula helpe T cells may play an impo an ole in he se e i y o p ima y Sjög en’s synd ome K isz ina Szabo*, Gabo Papp MD, PhD*, Sando Ba a h PhD, Edi Gyimesi PhD, An onia Szan o MD, PhD and Ma gi Zehe MD, PhD, DSc Di ision o Clinical Immunology, Ins i u e o Medicine, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, Deb ecen, Hunga y * The i s wo au ho s con ibu ed equally o his wo k E-mail add esses: K isz ina Szabo: k sz [email protected]; Gabo Papp: [email p o ec ed]; Sando Ba a h: [email p o ec ed]; Edi Gyimesi: egy[email p o ec ed]; An onia Szan o: [email p o ec ed]; Ma gi Zehe : [email p o ec ed] Co espondence and ep in eques : Ma gi Zehe MD, PhD, DSc Di ision o Clinical Immunology, Ins i u e o Medicine, Medical and Heal h Science Cen e , Uni e si y o Deb ecen Add ess: Mo icz Zs. s . 22. H-4032 Deb ecen, Hunga y Tel/Fax: +36-52-255-218 Email: [email p o ec ed] ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 2 Abs ac The aim o his s udy was o in es iga e he possible ole o ollicula helpe T (TFH) cells in he pa hogenesis o p ima y Sjög en’s synd ome (pSS) by analyzing immune-compe en cells and se ological ma ke s wi h special emphasis on clinical symp oms. We en olled 50 pSS pa ien s and 16 heal hy indi iduals in he s udy. Pa ien s had ele a ed a io o pe iphe al TFH cells, howe e , when di iding pa ien s in o wo g oups de ined by he p esence o ex aglandula mani es a ions (EGMs), only pa ien s wi h EGMs di e ed om con ols signi ican ly. Mo eo e , TFH cell pe cen ages co ela ed posi i ely wi h bo h ac i a ed T cell and T 1 cell alues. On he con a y, TFH cell pe cen ages showed nega i e co ela ion wi h bo h IgM and IgG memo y B cell p opo ions. Ele a ed TFH pe cen ages we e obse ed in he an i-SSA/SSB posi i e pa ien s, and also in pa ien s wi h highe IL-12, IL-21 le els and ocus sco e alues. Inc eased TFH cell p opo ions seem o ha e an impo an ole in disease de elopmen . ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 3 Keywo ds p ima y Sjög en’s synd ome (pSS); ollicula helpe T cells (TFH); memo y B cells; ac i a ed T cells; au oan ibodies Abb e a ions AID: ac i a ion-induced cy idine deaminase; APC: allophycocyanin; BAFF: B cell ac i a ing ac o ; Bcl-6: B cell lymphoma 6 p o ein; Blimp-1: B lymphocy e induced ma u a ion p o ein 1; CD: Clus e o Di e en ia ion; CCR7: C-C chemokine ecep o ype 7; C-X-C chemokine ligand 13: CXCL13; C-X-C chemokine ecep o 5: CXCR5; DCs: dend i ic cells; EGMs: ex aglandula mani es a ions; ELISA: enzyme-linked immunoso ben assay; FITC: luo escein iso hiocyana e; FMO: Fluo escence Minus One; GC: ge minal cen e ; HLA: human leukocy e an igen; ICOS: inducible T cell co-s imula o ; IFN: in e e on; IL: in e leukin; NK: na u al kille ; PD-1: p og ammed cell dea h p o ein 1; PE: R-phycoe y h in; PE-Cy5: R-phycoe y h in-Cyanine dye 5; Pe CP-Cy5.5: Pe idinin-chlo ophyll p o ein- Cyanine dye 5.5; PI3K: Phospha idylinosi ol 3-kinase; pSS: p ima y Sjög en´s synd ome; RA: heuma oid a h i is; SAP: signaling lymphocy ic ac i a ion molecule (SLAM)- associa ed p o ein; SLE: sys emic lupus e y hema osus; STAT: signal ansduce and ac i a o o ansc ip ion p o ein; TFH: T ollicula helpe ; Tc: T cy o oxic; Th: T helpe ; T 1: Type 1 egula o y T; T eg: egula o y T ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 4 1. In oduc ion P ima y Sjög en´s synd ome (pSS) is a common sys emic au oimmune disease, cha ac e ized by lymphocy ic in il a ion and des uc ion o he exoc ine glands, p ima ily he lach ymal and sali a y glands. Besides he cha ac e is ic glandula symp oms, o he sys emic symp oms, deno ed as ex aglandula mani es a ions (EGMs) such as non-e osi e polya h i is, asculi is o myosi is may also occu du ing he disease cou se [1]. Humo al au oimmune esponses, B cell ac i a ion and au oan ibody p oduc ion a e key immune abno mali ies in pSS. Immunohis ological analysis o biopsies om mino sali a y glands usually demons a es he p esence o ec opic ge minal cen e s (GCs) in he disease. The numbe o GCs in sali a y glands co ela es wi h he se e i y o in lamma ion, and enhanced an i-Ro/SSA, an i-La/SSB au oan ibody-p oduc ion. Mo eo e , he o ma ion o ec opic GCs ca ies a highe isk o de eloping B cell lymphoma [2-4]. The selec ion o mu a ed high-a ini y GC B cells depends on he es imula ion wi h an igen a ayed on ollicula dend i ic cells and he p o ision o help by ollicula helpe T (TFH) cells [5]. TFH cells we e ini ially p oposed as a sepa a e lineage, based on hei ailu e o exp ess T helpe (Th) 1 /Th2/Th17 cy okines and lineage-speci ic ansc ip ion ac o s. La e in es iga ions showed ha TFH cells may exp ess cha ac e is ic cy okines o canonical helpe T e ec o subse s, including in e e on-gamma (IFN-γ), in e leukin-4 (IL-4) o IL-17 unde speci ic mic oen i onmen and acco dingly, TFH cells seem o be he e ogeneous and ha e a close ela ionship o Th1, Th2 o Th17 cells [6-9]. A ecen s udy demons a ed he abili y o TFH cells o o m memo y cells which di e en ia e upon ecall no only in o TFH bu con en ional helpe T cells as well [10]. Fu he mo e, wo elegan s udies showed ha mouse Th2 and na u al egula o y T cells (T eg) may ans o m in o TFH cells unde ce ain in i o condi ions [8, 11]. These obse a ions shed ligh on he de elopmen al plas ici y o TFH cells. The e o e, ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 5 di e ing om o he CD4+ T cell lineages, TFH cells a e mainly loca ed in seconda y lymphoid o gans and de ined by he exp ession o unique combina ion o cell su ace molecules [9]. The main ea u es o TFH cells a ise om he exp ession o he B-cell lymphoma 6 (Bcl-6) ansc ip ion ac o which egula es he exp ession o C-X-C chemokine ecep o 5 (CXCR5), CXCR4 and C-C chemokine ecep o ype 7 (CCR7) ha di ec s TFH cells in o he C-X-C chemokine ligand 13 (CXCL13) ich a eas o B cell ollicles. Mo eo e i induces he exp ession o inducible T cell co-s imula o (ICOS), p og ammed cell dea h p o ein 1 (PD-1), clus e o di e en ia ion (CD)40L and signaling lymphocy ic ac i a ion molecule (SLAM)- associa ed p o ein (SAP) which a e c i ical in T-B cell in e ac ion [9, 12-16]. In he ollicles, TFH cells p o ide su i al signals o GC B cells ia mul iple pa hways, including CD40L, IL- 4, IL-21, PD-1, and B cell ac i a ing ac o (BAFF), which compe e wi h Fas-FasL in e ac ions [14, 17, 18]. TFH cells a e equi ed o he o ma ion and main enance o GCs and o he gene a ion o mos memo y B cells and long-li ed plasma cells. The con ol o hese p ocesses hinges on TFH egula ion o mul iple B cell a e decisions, including cell dea h [14, 19]. IL-27 seems o be also impo an in TFH cell unc ion and no mal o pa hogenic GC esponses. In i o IL-27 ecep o is equi ed on CD4+ T cells o no mal TFH cell gene a ion, GC o ma ion and an ibody esponses [20]. Simila ly o pSS, ec opic lymphoid s uc u es ha e also been desc ibed in he a ge issues o o he au oimmune condi ions ha a e accompanied by B cell dis u bances and au oan ibody p oduc ion, such as sys emic lupus e y hema osus (SLE), heuma oid a h i is (RA) and au oimmune hy oidi is [21-23]. The be e unde s anding o he B cell dis u bances and de elopmen o ec opic GCs may p o ide new s a egies o B cell a ge ed he apies. In he p esen s udy, in o de o explo e he possible ole o TFH cells in he pa hogenesis o pSS, we analyzed a wide spec um o immune-compe en cells and se ological ma ke s wi h a special emphasis on he clinical symp oms. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 6 2. Ma e ial and me hods 2.1. Pa ien s Fi y pa ien s wi h pSS (2 male, 48 emale) we e en olled in he s udy, ec ui ed om he ou pa ien clinic o sys emic au oimmune diseases a he Di ision o Clinical Immunology, Ins i u e o Medicine, Medical and Heal h Science Cen e , Uni e si y o Deb ecen, whe e hey ecei ed egula ollow-up ea men . The diagnosis was based on he Eu opean- Ame ican consensus c i e ia. Six een age- and sex-ma ched heal hy indi iduals se ed as con ols [24]. Among pSS pa ien s, 25 su e ed om ex aglandula mani es a ions (EGMs), while 25 had only glandula symp oms. The dis ibu ion o EGMs o pSS pa ien s was as ollows: polya h i is n=19, Raynaud’s phenomenon n=11, lymphadenopa hia n=3, asculi is n=3, polyneu opa hia n=2 and myosi is n=1. No pa ien s, o con ols en olled in his s udy ook any immunosupp essi e o immunomodula ing medica ions, o had ongoing o p e ious in ec ions du ing he s udy. In o med w i en consen was ob ained om he subjec s, and he s udy has been app o ed by he E hics Commi ee o he Uni e si y o Deb ecen. All expe imen s ca ied ou we e in compliance wi h he Decla a ion o Helsinki. Da a on subjec s en olled in he s udy a e summa ized in Table 1. 2.2. De e mina ion o lymphocy e subpopula ions Fo pheno ypic analysis om hepa inized blood samples we used CD3- luo escein iso hiocyana e (FITC), CD4-FITC, CD8-R-phycoe y h in (PE), CD19-R-phycoe y h in- Cyanine dye 5 (PE-Cy5), and CD16+CD56-PE (BD Biosciences, San Diego, CA, USA and ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 7 Immuno ech, Beckmann Coul e Company, Ma seille, F ance) monoclonal an ibodies agains cell su ace ma ke s. The exp ession o T-lymphocy e ac i a ion ma ke s such as CD69-PE- Cy5 and human leukocy e an igen (HLA)-DR-PE we e also de e mined on CD3+ cells (BD Biosciences). The ollowing monoclonal an ibody combina ions we e used o pheno ypic cha ac e iza ion o nai e and memo y T cells: CD45RA-FITC/CD4-PE/CD62L-PE-Cy5 (Immuno ech) and CD45RA-FITC/CD8-PE/CD62L-PE-Cy5 (Se o ec, Ox o d, UK and Immuno ech). Fo iden i ica ion nai e and memo y B cells we used IgD-FITC/CD27- PE/CD19-PE-Cy5 (Beckman Coul e Inc, Fulle on, CA, USA and Immuno ech). We also in es iga ed CD4+CD25b igh T eg cells wi h he ollowing eagen s: CD4-FITC (Sigma Ald ich, S . Louis, MO, USA), CD25-PE-Cy5 (Immuno ech). Samples we e p ocessed acco ding o he Coul e Q-PREP p o ocol and sys em (Beckman Coul e Inc, Miami, FL, USA), as desc ibed p e iously [25, 26]. Measu emen s we e pe o med on a Coul e FC500 low cy ome e (Beckman Coul e Inc.). Iso ype-ma ched an ibodies we e used in all p ocedu es. The ollowing pe iphe al immune-compe en cell ypes we e in es iga ed: T cells (CD3+), T-helpe cells (CD4+), cy o oxic T (Tc) cells (CD8+), B cells (CD19+), ea ly- ac i a ed T lymphocy es (CD3+CD69+), la e-ac i a ed T lymphocy es (CD3+HLADR+), na u al kille (NK) cells (CD3-CD56+CD16+), NKT cells (CD3+CD56+ CD16+) and CD4+CD25b igh T eg cells (CD4+CD25b igh ). The B, T, T-helpe , ac i a ed T, NK, NKT and T eg cells we e quan i ied as hei pe cen age in he en i e lymphocy e popula ion. Nai e and memo y T cell subse s we e de e mined as hei pe cen ages in CD4+ o CD8+ cells, as he ollowings: nai e helpe T (CD4+CD45RA+CD62L+), cen al memo y helpe T (CD4+CD45RA–CD62L+), e ec o memo y helpe T (CD4+CD45RA–CD62L–), nai e cy o oxic T (CD8+CD45RA+CD62L+), cen al memo y cy o oxic T (CD8+CD45RA– CD62L+), e ec o memo y cy o oxic T (CD8+CD45RA–CD62L–) and e minally di e en ia ed e ec o memo y cy o oxic T cells (CD8+CD45RA+CD62L–). Nai e and ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 8 memo y B cell alues we e calcula ed as hei pe cen ages in CD19+ B cells as he ollowings: nai e B (IgD+CD27-), IgM memo y B (IgD+CD27+), IgG memo y B cells (IgD-CD27+). Fo he iden i ica ion o CD4+ T helpe cell subse s we used in acy oplasmic cy okine s aining me hod which has been desc ibed p e iously [25, 26]. The ollowing combina ions o monoclonal an ibodies we e used: IFN-γ-FITC/IL-4-PE/CD4-PE-Cy5 o CD8 PE-Cy5, IL-10- PE/CD4-PE-Cy5 o CD8 PE-Cy5 (all om BD Biosciences), IL-17-PE/IFN-γ-FITC/CD4-PE- Cy5 (R&D Sys ems, Minneapolis, MN, USA and BD Biosciences). Measu emen s we e pe o med and da a we e collec ed on a Coul e FC500 low cy ome e (Beckman Coul e Inc.). Iso ype-ma ched an ibodies we e used in all p ocedu es. The pheno ypes wi hin CD4+ cells we e de e mined as ollows: Th1 cells (CD4+ IFN-γ+ IL-4-); Th2 cells (CD4+ IFN-γ- IL- 4+); Th17 cells (CD4+ IFN-- IL17+) and Type 1 egula o y T cells (T 1) (CD4+ IL-10+). Cells we e quan i ied as hei pe cen age in he CD4+ lymphocy e popula ion. 2.3. Assessmen o TFH cells in he pe iphe al blood Fo he iden i ica ion o ci cula ing TFH cells we used CD4-allophycocyanin (APC), CXCR5- Alexa Fluo 488, ICOS-PE and PD-1-Pe idinin-chlo ophyll p o ein-Cyanine dye 5.5 (Pe CP- Cy5.5) (BD Biosciences) monoclonal an ibodies agains human cell su ace molecules. Fluo escence Minus One (FMO) con ols we e used in all p ocedu es. FMO con ols con ain e e y s ain in he panel excep he one we a e con olling, he e o e his me hod a e ideal o de e mining posi i e s. nega i e popula ion in he sample. The s ained cells we e assessed using he FACS Calibu low cy ome e (Bec on Dickinson, F anklin Lakes, NJ, USA) and da a analysis was pe o med using FlowJo So wa e (T ees a , Ashland, OR, USA). A leas 35 000 e en s pe sample we e analyzed wi hin he lymphocy e popula ion. TFH cells we e quan i ied as hei pe cen age in he CD4+ lymphocy e popula ion. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 15 pe cen ages o TFH cells 0.560 (0.130-1.690) % s. 0.905 (0.510-1.560) %, espec i ely, p = 0.0414) (Fig.3C). Whe eas, no signi ican di e ences was obse ed be ween he pa ien s wi h glandula symp oms in IL-21-nega i e g oup and he pa ien s wi h glandula symp oms in IL- 21-posi i e g oup (median pe cen ages o TFH cells 0.235 (0.030-0.600) % s. 0.310 (0.070- 0.390) %, espec i ely, p = 0.9666) (Fig.3C). We ound no signi ican di e ence in he se um le els o IL-27 be ween pSS pa ien s and heal hy indi iduals. 3.5. The in ensi y o ocus sco es in associa ion wi h TFH cells The his ological indings we e also examined in labial sali a y gland biopsies o 14 pSS pa ien s. Wi hin he g oup o pa ien s who su e ing only glandula symp oms he dis ibu ion o ocus sco es was as ollows: ocus sco e o 1 (n = 2) and ocus sco e o 2 (n = 4). In he g oup o pa ien s wi h EGMs he dis ibu ion o ocus sco es was as ollows: ocus sco e o 2 (n = 2), ocus sco e o 3 (n = 3) and ocus sco e o 4 (n = 3). We also examined he ela ionship be ween he pe cen ages o TFH cells and biopsy ocus sco es and we ound a signi ican posi i e co ela ion (R = 0.6984, espec i ely, p = 0.0055) (Fig. 4). ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 16 4. Discussion Cellula and humo al mechanisms behind he immune dis u bances cha ac e is ic o Sjög en’s synd ome a e s ill no known in de ails, bu e idence sugges s ha bo h T and B cells in il a ing he exoc ine glands play an impo an ole in he disease de elopmen . A complex in e play be ween lymphocy es, mac ophages, dend i ic cells (DCs) and bo h ac i a ed epi helial and endo helial cells leads o au oimmune issue damage and con ibu es o he disease p og ession [3, 27-29]. Sys emic ea u es o pSS, such as ci cula ing immune complexes, hype gammaglobulinemia, o gan-speci ic and non o gan-speci ic au oan ibodies, u he mo e, ec opic GCs in he a ec ed issues and enhanced isk o de eloping B cell lymphoma unde line he c ucial ole o B cells in he disease. The mos in ensi ely s udied T helpe cell ype in ecen yea s is he TFH cell, which has a majo ole in he p oli e a ion and di e en ia ion in o memo y o plasma cells o an igen-speci ic B cells, and in some cases e en con ibu es o he igge ing o hei apop osis. Recen s udies demons a ed ele a ed pe iphe al TFH cell pe cen ages in ce ain au oimmune condi ions, such as RA, SLE and au oimmune hy oidi is [30-32]. In ou p esen s udy, we assessed he equency o TFH cells in pSS pa ien s by de e mining CD4+CXCR5+ICOS+PD-1+ T cells. A e analysing he esul s o he whole pa ien popula ion, we ound an ele a ed a io o pe iphe al TFH cells, in e es ingly, when we di ided pa ien s in o wo g oups based on he p esence o absence o EGMs, only pa ien s wi h EGMs showed signi ican di e ences, while alues o pa ien s wi hou EGMs we e simila o heal hy con ols. O no e, a ecen s udy ocusing on SLE, has also in es iga ed a small g oup o pa ien s wi h Sjög en’s synd ome. Howe e , au ho s only iden i ied pe iphe al CD4+CXCR5+ICOShigh and CD4+PD-1high cells, no CD4+CXCR5+ICOS+PD-1+ TFH cells, mo eo e , labo a o y alues we e no e alua ed wi h he emphasis on clinical symp oms [30]. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 17 A ecen s udy demons a ed ha he p opo ions o CD4+ CXCR5+ CCR6+ T cells we e ele a ed in pSS pa ien s and co ela ed wi h he disease ac i i y. The wo kg oup’s esul s also showed ha hese cells exp essed he key ea u es o TFH cells, including PD-1, ICOS, CD40L, IL-21 and Bcl-6 [33]. Ou esul s also indica e ha p opo ions o TFH cells in pSS pa ien s show ele a ed alues. Mo eo e , ou s udy p o ed ha he a io o TFH cells is closely connec ed o he p esence o sys emic clinical symp oms in he pa ien s, consequen ly, TFH cells may play an impo an ole no only in he de elopmen o pSS, bu also in he disease p og ession. Rega ding o he T cell subse s, ou wo kg oup demons a ed p e iously ha p opo ions o ea ly ac i a ed T and T 1 cells a e ele a ed in he pe iphe al blood o pSS pa ien s [25, 26]. In ou p esen s udy, we ound posi i e co ela ions be ween he pe cen ages o TFH cells and ea ly- and la e-ac i a ed T cells, which indica e ha in pa allel wi h he ac i a ion o immune sys em, a s onge TFH cell expansion can be obse ed. TFH cells also showed a posi i e co ela ion wi h T 1 cell pe cen ages. Ele a ion in T 1 cell p opo ions could be a pa o an inc eased coun e - egula o y eac ion, p esumably compensa ing he de ailed, disp opo ional immune esponses. B cell hype eac i i y, de elopmen o au oan ibodies, and dis u bance in dis ibu ion o B cell sub ypes on he pe iphe y a e cha ac e is ics in pSS. Le els o ci cula ing IgM and IgG memo y B cells a e dec eased in pe iphe al blood; on he con a y hei p opo ions a e ele a ed in he in lamed issues, especially in sali a y glands [2, 34-37]. Ou esul s suppo pa ially he a o emen ioned obse a ions, since we ound a nega i e co ela ion be ween he p opo ions o TFH cells and IgM and IgG memo y B cells in he pe iphe al blood. We we e also in e es ed in he associa ions be ween he pe cen ages o TFH cells and he le els o IgG and au oan ibodies. We ound posi i e co ela ion be ween TFH cell alues and IgG le els, which may be he esul o he consequen ial B cell ac i a ion. Addi ionally, we obse ed ele a ed TFH pe cen ages in he an i-Ro/SSA and an i-La/SSB posi i e pa ien s, compa ed o ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 18 au oan ibody nega i e pa ien s and heal hy con ols. Mo eo e , he TFH p opo ions showed posi i e co ela ion wi h an i-Ro/SSA and an i-La/SSB i e s. Taken oge he , hese obse a ions a e in line wi h he concep ha he ele a ed TFH p o ile plays an impo an ole in au oan ibody p oduc ion. Recen s udies sugges ha di e en ia ion o TFH cells can be suppo ed by an in eg a ed model, which consis s o ac i a ion o cells, upholding o he ac i a ed condi ion and an en i ely pola ised s a e. Supposing ha myeloid DCs a e he only cell ypes capable o s imula ing nai e T cells, we can in e ha myeloid DCs ound in seconda y lymphoid issue, p oducing IL-12 cy okines play a cen al ole in inc easing amoun s o TFH cells in ce ain au oimmune diseases. A o me s udy showed ha ac i a ed myeloid DCs can induce he di e en ia ion o CD4+ nai e T cells in o TFH cells h ough he signal ansduce and ac i a o o ansc ip ion p o ein 4 (STAT4) pa hway ia IL-12 cy okine sec e ion. Fu he mo e, i has been demons a ed ha IL-12 is capable o inducing IL-21, CXCR5, ICOS and Bcl-6 exp ession o human CD4+ nai e T cells in i o, hus p omo ing B cell an ibody p oduc ion, al hough he capaci y o IL-12 o induce IL-21 cy okine exp ession is mos ly STAT3 dependen [38-41]. The e o e we s udied le els o soluble IL-12 cy okine in se a o pSS pa ien s. No e e y pa ien showed measu able le els o IL-12, hus we a anged hem in wo g oups based on se um IL-12 le els: IL-12 posi i e and nega i e g oups. Acco ding o ou esul s, IL-12 posi i e pa ien s had signi ican ly highe pe cen ages o TFH cells. Ano he impo an signal molecule o TFH cells is IL-21, which can con ibu e o ex ended su i al o TFH cells in an au oc ine manne , ia he ac i a ion o phospha idylinosi ol 3-kinase (PI3K). K oenke e al. ecen ly epo ed ha c-Ma , and no Bcl-6, induces he p oduc ion o IL-21, ne e heless, bo h ansc ip ion ac o s wo k oge he o igge he de elopmen o TFH cha ac e is ics [13]. Mo eo e , co-s imula ion con olled by IL-21 is necessa y o high le el exp ession o CXCR5, which is equi ed o mig a ion in o GCs [42, 43]. Ano he impo an ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 19 ole o IL-21 cy okine is o p omo e di e en ia ion o B cells in o plasma cells h ough he STAT3 pa hway wi h induca ing he B lymphocy e induced ma u a ion p o ein 1 (Blimp-1) ansc ip ion ac o [14]. Se um le els o IL-21 cy okine we e ound o be ele a ed in au oimmune hy oidi is and RA pa ien s [31, 32]. In ou s udy, no e e y pa ien had measu able le els o IL-21, hus we di ided hem in o wo g oups based on IL-21 le el in he se um: IL-21 posi i e and nega i e g oups. We ound ha pa ien s wi h highe TFH cell pe cen ages had ele a ed IL-21 le els, mo eo e , hese cy okine concen a ions co ela ed wi h he p esence o EGMs, hus suppo ing he heo y, ha IL-21 has an impo an ole in immune p ocesses egula ed by TFH cells. Addi ionally, ou s udy e ealed a close ela ionship be ween pe iphe al TFH cell pe cen ages and he ocus sco es o labial sali a y gland biopsies. O no e, pa ien s wi h EGMs had highe ocus sco es compa ed o pa ien s wi h only sicca symp oms. This in e es ing obse a ion sugges s ha he ele a ed equency o ci cula ing TFH cells may be associa ed wi h he se e i y o glandula in ol emen . ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 20 5. Conclusions Pe cen ages o pe iphe al TFH cells a e inc eased in pSS pa ien s su e ing om a mo e p onounced cou se o disease. Mo eo e , ou esul s e ealed clea co ela ions be ween ele a ed TFH cell pe cen ages, ce ain B cell sub ype p opo ions and au oan ibody le els. Taken oge he , ou obse a ions aise he possibili y ha al e a ion in TFH cell p opo ions may play an impo an ole in he disease de elopmen . The e o e, modula ion o TFH cells could be a po en ially powe ul elemen o he no el he apeu ic selec ion in pSS, by blocking TFH di e en ia ion and hei in e ac ion wi h B cells using selec i e agen s. We belie e ha u he in es iga ion o he complex unc ion o TFH cells will open new a enues o unde s and he de ailed B cell ope a ion and au oimmune p ocesses in pSS. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 21 Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. Au ho s’ con ibu ions KSz and GP pa icipa ed in he s udy design, pe o med labo a o y expe imen s, collec ed, s a is ically analyzed and in e p e ed he da a, and d a ed he manusc ip . SB and EGy pa icipa ed in he labo a o y expe imen s. ASz pa icipa ed in he in e p e a ion o clinical da a. MZ designed he s udy, in e p e ed he da a and ga e inal app o al o he e sion o be published. All au ho s ead and app o ed he inal manusc ip . Acknowledgemen s This wo k was suppo ed by G an No. K101470 om he Hunga ian Na ional Scien i ic Resea ch Fund (OTKA) and he TÁMOP-4.2.2.A-11/1/KONV-2012-0023 p ojec . The p ojec is co- inanced by he Eu opean Union and he Eu opean Social Fund. 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ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 31 Figu e 1 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 32 Figu e 2 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 33 Figu e 3 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 34 Figu e 4 ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 35 Highligh s  Inc eased pe cen ages o TFH cells a e associa ed wi h he se e i y o pSS.  TFH cell pe cen ages co ela es wi h he enhanced coun e - egula o y ac i i y.  TFH cell pe cen ages co ela es wi h ce ain B cell subse s and au oan ibody le els.  IL-12 and IL-21 ha e an impo an ole in immune p ocesses egula ed by TFH cells.