Different antibody-associated autoimmune diseases have distinct patterns of T follicular cell dysregulation

1 Vol.:(0123456789) Scien i ic Repo s | (2022) 12:17638 | h ps://doi.o g/10.1038/s41598-022-21576-8 www.na u e.com/scien i ic epo s Di e en an ibody‑associa ed au oimmune diseases ha e dis inc pa e ns o T ollicula cell dys egula ion Filipa Ribei o1,2,4, Vasco C. Romão1,3,4, Sa a Rosa1, Ká ia Jesus1, Ana Água‑Doce1, So ia C. Ba ei a1,3, Pa ícia Ma ins1,3, Susana Lopes da Sil a1,3, Ema Nob e3, Ma ia João Bugalho3, Vál e R. Fonseca1,5, João Eu ico Fonseca1,3,5 & Luis G aca1,2,5* Au oan ibodies a e p oduced wi hin ge minal cen e s (GC), in a p ocess egula ed by in e ac ions be ween B, T ollicula helpe (T h), and T ollicula egula o y (T ) cells. The GC dys egula ion in human au oimmuni y has been in e ed om ci cula ing cells, albei wi h con lic ing esul s due o di e se expe imen al app oaches. We applied a consis en app oach o compa e ci cula ing T and T h subse s in pa ien s wi h di e en au oimmune diseases. We ec ui ed 97 pa icipan s, including 72 pa ien s wi h Hashimo o’s hy oidi is (HT, n = 18), heuma oid a h i is (RA, n = 16), o sys emic lupus e y hema osus (SLE, n = 32), and 31 ma ched heal hy dono s (HD). We ound ha he equency o ci cula ing T ollicula subse s di e ed ac oss diseases. Pa ien s wi h HT had an inc eased equency o blood T h cells (p = 0.0215) and a educed T /T h a io (p = 0.0338) when compa ed wi h HD. This was no obse ed in pa ien s wi h sys emic au oimmune heuma ic diseases (RA, SLE), who had a educ ion in bo h T h (p = 0.0494 and p = 0.0392, espec i ely) and T (p = 0.0003 and p = 0.0001, espec i ely) cells, esul ing in an unchanged T /T h a io. Ac i a ed PD‑1+ICOS+T h and CD4+PD‑1+CXCR5–Tph cells we e aised only in pa ien s wi h SLE (p = 0.0022 and p = 0.0054), wi hou associa ion wi h disease ac i i y. Ou da a sugges ha GC dys egula ion, assessed by T ollicula subse s, is no uni o m in human au oimmuni y. Speci ic pa e ns o dys egula ion may become po en ial bioma ke s o disease and pa ien s a i ica ion. The inding ha T ollicula helpe (T h) and T ollicula egula o y (T ) cells con ol GC eac ions p omp ed he s udy o ci cula ing T ollicula subse s in human au oimmuni y1–4. T cells de i e om Foxp3+ T eg cells5–7, ha e a TCR epe oi e di ec ed o sel -an igens and dis inc om he epe oi e o T h cells8, ha e a supp essi e unc ion9,10, and a e essen ial o p e en he gene a ion o au oimmuni y in mice11–13. I was, he e o e, emp ing o specula e ha a dys egula ed T /T h a io could be implica ed in an ibody-media ed au oimmuni y. We p e iously demons a ed ha pa ien s wi h p ima y Sjög en’s synd ome (pSS) had an inc ease in ci cula ing T cells compa ed o heal hy dono s (HD)4,14. Mo eo e , a subse o pSS pa ien s wi h mo e au oan ibodies and ec opic lymphoid s uc u es (ELS) wi hin mino sali a y glands had a signi ican ly highe T /T h a io14. We demons a ed ha he gene a ion o ci cula ing T cells is a no mal p ocess ollowing a humo al esponse, namely in luenza accina ion4, and can explain he high T /T h a io ollowing ch onic in lamma o y eac ions, as in pSS4,14. Al hough o he esea che s subsequen ly con i med ou esul s15, he e we e also con lic ing epo s16. Fu he mo e, epo s ha e also been inconsis en in o he au oimmune diseases, sugges ing an inc ease o dec ease o he T /T h a io14,17–19. I has been di icul o compa e hose s udies as he expe imen al app oaches a e no di ec ly compa able. Fo ins ance, we p e iously ound ha ci cula ing T cells ha e an imma u e pheno ype, gi en he de elopmen al bi u ca ion o T cells wi hin human lymphoid issue4,20. Howe e , in some s udies, T cells a e selec ed wi hin OPEN 1Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina da Uni e sidade de Lisboa, Lisbon Academic Medical Cen e, A enida P o esso Egas Moniz, 1649-028 Lisboa, Po ugal. 2Ins i u o Gulbenkian de Ciência, Oei as, Po ugal. 3Rheuma ology Depa men , Hospi al de San a Ma ia, Cen o Hospi ala Lisboa No e, Lisbon Academic Medical Cen e, Lisboa, Po ugal. 4These au ho s con ibu ed equally: Filipa Ribei o and Vasco C. Romão. 5 These au ho s join ly supe ised his wo k: Vál e R. Fonseca, João Eu ico Fonseca and Luis G aca. *email: [email p o ec ed] 2 Vol:.(1234567890) Scien i ic Repo s | (2022) 12:17638 | h ps://doi.o g/10.1038/s41598-022-21576-8 www.na u e.com/scien i ic epo s/ CD45RO+ o PD-1+ cells17,21, excluding a la ge p opo ion o his cell popula ion. CD25 may also be a poo ma ke o T cells, especially he mos ma u e wi hin he GC, al hough ci cula ing T cells a e CD25+20,22–25. Con e sely, T h cells a e o en s udied wi hou exclusion o Foxp3+ cells, o selec ing hem as PD-1+, hus including bo h T ollicula subse s (T h and T ) as T h cells and/o excluding imma u e PD-1– T h cells26,27. Gi en ou esul s showing ha pSS pa ien s wi h ELS had a high T /T h a io, we add essed he hypo hesis ha o he au oimmune diseases wi h a ge o gan in ol emen , au oan ibodies’ p oduc ion and ELS would ha e a simila p o ile. Fo ha pu pose, we s udied pa ien s wi h Hashimo o’s hy oidi is (HT), an o gan-speci ic au oimmune disease wi h ELS o ma ion in hy oid gland. In addi ion, we aimed o in es iga e whe he hese indings could be eplica ed in o he , mo e sys emic, au oimmune diseases. Thus, using a consis en app oach, we included pa ien s wi h wo di e en au oimmune heuma ic diseases: heuma oid a h i is (RA), a sys emic disease wi h a clea -cu a ge (i.e., a icula ) in ol emen ; and sys emic lupus e y hema osus (SLE), he p o o ypical example o an au oan ibody-d i en sys emic au oimmune heuma ic disease. Pa ien s and me hods Human samples. We ec ui ed pa ien s wi h HT (n = 18) ollowed in he Endoc inology Depa men o Hospi al de San a Ma ia, Cen o Hospi ala Uni e si á io Lisboa No e (CHULN), Lisbon Academic Medical Cen e, Lisbon, Po ugal. Fu he , we included pa ien s wi h RA (n = 16) and SLE (n = 32), who we e ollowed in he Rheuma ology Depa men o he same hospi al. Only adul , non-p egnan , pa ien s ul illing he clinical classi ica ion c i e ia o HT (hypo hy oidism wi h he p esence o TPO an ibodies), RA (Ame ican College o Rheuma ology (ACR)/Eu opean Alliance o Associa ions o Rheuma ology (EULAR) 2010), o SLE (ACR 1997) we e included. We excluded pa ien s unde immunosupp essi e ea men o he han me ho exa e, hyd oxychlo oquine, o ≤ 7.5mg pe day o p ednisolone o equi alen . Pa ien s diagnosed as ha ing an in ec ious disease o diabe es, o who had ecei ed any accine in he p e ious mon h we e also excluded. Disease ac i i y was e alua ed using he Disease Ac i i y Sco e-28 (DAS-28) o RA pa ien s, and Sys emic Lupus E y hema osus Disease Ac i i y Index (SLEDAI) o SLE pa ien s. Rele an au oan ibodies and se ological ma ke s o disease/ disease ac i i y we e also collec ed. F esh blood samples we e collec ed in EDTA-coa ed ubes and p ocessed on he same day o low cy ome y analysis. Finally, we collec ed esh blood samples om 31 sex- and age- ma ched heal hy olun ee s. E hical app o al. This s udy was app o ed by he Lisbon Academic Medical Cen e E hics Commi ee ( e e ence no. 505/14). In o med consen was ob ained om all pa icipan s. All me hods we e pe o med in acco dance wi h ele an guidelines and egula ions. Cell isola ion and low cy ome y. Pe iphe al blood mononuclea cells (PBMCs) we e isola ed by Ficoll g adien medium (His opaque-1077; Sigma-Ald ich) using SepMa e ubes (S em-Cell Technologies). Cells we e s ained wi h an i-CD4 (OKT4, BioLegend), an i-CD45RO (UCHL1, BioLegend), an i-CD25 (BC96, eBioscience), an i-CD185/CXCR5 (J252D4, BioLegend), an i-CD279/PD-1 (EH12.2H7, BioLegend), an i-CD278/ICOS (C398.4A, BioLegend), an i-CCR6 (G034E3, BioLegend), an i-CXCR3 (G025H7, BioLegend) and an i-Foxp3 (PCH101, eBioscience). In acellula Foxp3 s aining was pe o med using he Foxp3 Fix/Pe m Ki (eBioscience) acco ding o he manu ac u e ’s ins uc ions. Samples we e acqui ed on a BD LSRFo essa cy ome e (BD Biosciences) and u he analyzed on FlowJo so wa e (T eeS a ). S a is ical analysis. The no mali y o da a was e alua ed using he Shapi o–Wilk no mali y es . K uskal– Wallis one-way analysis o a iance (ANOVA) es wi h Dunn’s compa ison pos - es was applied o pai wise mul iple compa isons, and wo-way ANOVA wi h Dunne ’s es was pe o med o mul iple compa isons. P alues less han 0.05 we e conside ed signi ican . S a is ical and g aphic analyzes we e pe o med using G aphPad P ism so wa e. Resul s The ci cula ing T /T h a io is he e ogeneous among dis inc au oimmune diseases. One o he majo challenges in compa ing s udies o ci cula ing T ollicula cells in au oimmune diseases is he inconsis ency o he app oaches applied o selec hese cells by low cy ome y, namely he dispa i y in he ma ke s used. To main ain a consis en s a egy o analyze T ollicula subse s by low cy ome y, allowing compa abili y among diseases, we in es iga ed he p o ile o ci cula ing T h cells (de ined as CD4+CXCR5+CD45RO+CD25–Foxp3–) and T cells (de ined as CD4+CXCR5+CD25+ Foxp3+) in dis inc an ibody-media ed au oimmune diseases (Fig.1A). We included pa ien s wi h HT, ep esen ing an o gan-speci ic au oimmune disease; RA, an au oimmune heuma ic disease wi h a ge in ol emen ; and SLE, he p o o ypical au oan ibody-d i en sys emic au oimmune disease (Table1). All pa ien s wi h HT had hypo hy oidism wi h he p esence o hy oid-speci ic au oan ibodies, and he majo i y we e ea ed wi h le o hy oxine. Pa ien s wi h RA and SLE we e in line wi h he na u e o an es ablished disease coho , wi h low disease ac i i y and an expec ed equency o au oan ibodies. Su p isingly, he equency o T h and T cells wi hin he CD4+ T cell popula ion a ied subs an ially among he di e en diseases (Fig.1A,B). Pa ien s wi h HT showed an inc eased equency o T h cells compa ed o HD (p = 0.0215). In con as , he equency o ci cula ing T h cells was dec eased in pa ien s wi h RA and SLE (p = 0.0494 and p = 0.0392, espec i ely). Fu he mo e, RA and SLE pa ien s also displayed educed equency o T cells (p = 0.0003 and p = 0.0001, espec i ely), which was no obse ed in HT pa ien s. Simila esul s we e obse ed wi h he absolu e cell numbe s (Supplemen a y Fig.S1). These indings con as wi h ou p e ious esul s in pa ien s wi h pSS, who had simila T h and highe T equencies han HD14. 3 Vol.:(0123456789) Scien i ic Repo s | (2022) 12:17638 | h ps://doi.o g/10.1038/s41598-022-21576-8 www.na u e.com/scien i ic epo s/ Con a y o wha we had hypo hesized, pa ien s wi h HT had a signi ican educ ion in T /T h a io (p = 0.0338) (Fig.1C), a he han an inc ease, as epo ed o pSS, an au oimmune disease which also has p edominan a ge -o gan (i.e., sali a y/lac imal gland) in ol emen 14. O no e, he low T /T h a io in HT pa ien s was mainly d i en by an inc ease in he equency o blood T h cells (Fig.1A–C), which we e p e iously shown o be unchanged in pa ien s wi h pSS14. No co ela ion was obse ed be ween he equency and he numbe o T h cells wi h he se um an ibody i e s o HT pa ien s (Supplemen a y Fig.S2). Fu he mo e, he educ ions in ci cula ing T h and T cells obse ed in RA and SLE esul ed in T /T h a ios simila o heal hy con ols. The esul s conce ning RA a e o e all in acco dance wi h wha we ha e p e iously demons a ed o pa ien s wi h es ablished RA in emission/low disease ac i i y28. No ably, he T /T h a io om RA and SLE pa ien s showed a signi ican in e indi idual a ia ion (Fig.1C). We could no ind any clinical pa ame e s, such as disease du a ion, disease ac i i y, au oan ibody s a us, o ea men ha could explain he in e indi idual a iabili y o he T /T h a io among hese pa ien s (Supplemen a y Figs.S3 and S4). Ne e heless, he T /T h a io in pa ien s wi h RA in emission (DAS28 ≤ 2.6) ends o be highe when compa ed o pa ien s who a e no in emission, as p e iously epo ed28. As an icipa ed, we did no ind signi ican di e ences ega ding he equency o CD4+ T cells (Fig.1D). O e all, hese esul s show ha di e en an ibody-media ed au oimmune diseases ha e dis inc pa e ns o ci cula ing T h and T cells in ol ed in he egula ion o GCs, sugges ing ha hese diseases may ha e dis inc o ms o GC dys egula ion. Ac i a ed PD‑1+ICOS+ T h cells a e inc eased in some, bu no all, au oimmune diseases. A e assessing he o e all T h and T popula ions, we in es iga ed he equency o T h subse s. Fi s , we quan i ied he equency o ac i a ed T h cells, de ined as PD-1+ICOS+ cells wi hin he T h ga e (Fig.2A). The PD-1+ICOS+ cells ep esen he popula ion o T h cells ha ansien ly inc eases in ci cula ion sho ly a e immune s imula ion, such as ha p o ided by in luenza accina ion29–31. We ound a end o a highe equency o PD-1+ICOS+ T h cells in pa ien s wi h o gan-speci ic and sys emic au oimmune disease, in compa ison o heal hy con ols (Fig.2A and Supplemen a y Fig.S1). This inc ease was, howe e , s a is ically signi ican only o pa ien s wi h SLE (p = 0.0022) (Fig.2A and Supplemen a y Fig.S1). No co ela ion be ween PD-1+ICOS+ T h cells o RA and SLE pa ien s wi h disease ac i i y was obse ed, possibly due o he low numbe o pa ien s wi h high disease ac i i y, as hese a e es ablished disease coho s (Supplemen a y Fig.S5). Mo eo e , no co ela ion o disease ac i i y wi h o al T h cells, bo h in numbe and pe cen age, was also obse ed (Supplemen a y Fig.S5). Figu e1. The ci cula ing T /T h a io is he e ogeneous among di e en au oimmune diseases. (A) Rep esen a i e plo s and (B) pooled da a o he equency o CD4+CXCR5+CD25–Foxp3– T h cells and CD4+CXCR5+CD25+Foxp3+ T cells in pe iphe al blood o pa ien s wi h Hashimo o’s hy oidi is (HT, n = 18), heuma oid a h i is (RA, n = 16), sys emic lupus e y hema osus (SLE, n = 32), and sex- and age-ma ched heal hy dono s (HD, n = 31). (C) Blood T /T h a io and (D) equency o CD4+ T cells in HT, RA, SLE, and sex- and age-ma ched HD. Each da a poin ep esen s one indi idual subjec ; ba s ep esen mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, K uskal–Wallis one-way analysis o a iance (ANOVA) es wi h Dunn’s compa ison pos - es . 4 Vol:.(1234567890) Scien i ic Repo s | (2022) 12:17638 | h ps://doi.o g/10.1038/s41598-022-21576-8 www.na u e.com/scien i ic epo s/ I has also been epo ed ha ci cula ing T h cells can be subse ed in Th1-like, Th2-like, and Th17-like T h cells based on CCR6 and CXCR3 exp ession2. We did no ind signi ican di e ences ega ding he equency o T h1, T h2, and T h17 subse s in pa ien s wi h HT o SLE, compa ed o HD. Howe e , we obse ed a dec ease o T h17 cells in RA pa ien s (p = 0.0109), wi h a con e se inc ease o he T h2 cells (p = 0.0105) (Fig.2B). Ci cula ing CD4+PD‑1+CXCR5– Tph cells and CXCR5+CD25–Foxp3+ T ollicula cells a e inc eased in pa ien s wi h SLE. We nex assessed he equency o ci cula ing T pe iphe al helpe (Tph) cells, de ined as CD4+PD-1+CXCR5–, which accumula e in in lamed issues and also ha e he abili y o p o ide help o B cells32,33. We ound ha his popula ion o cells is especially inc eased among SLE pa ien s (p = 0.0054) (Fig.3A), as p e iously desc ibed by o he g oups34–36. Al hough he Tph equency among SLE pa ien s ollows a bimodal dis ibu ion, wi h a small subg oup o pa ien s displaying highe le els o ci cula ing Tph cells, we did no ind an associa ion wi h clinical pa ame e s, such as disease du a ion, disease ac i i y, au oan ibody s a us, o ea men (Supplemen a y Fig.S6). Finally, we assessed he equency o ci cula ing CD4+CXCR5+CD25–Foxp3+ cells. We called hese cells CD25– T cells, gi en he simila i ies wi h he pheno ype o bona ide T cells. We ound ha ci cula ing CD25– T cells we e inc eased only in pa ien s wi h SLE (p = 0.0096), wi h no di e ences obse ed o HT and RA (Fig.3B). Gi en he exclusi e associa ion o his cell popula ion wi h SLE, we pe o med a ROC analysis ha iden i ied he equency o CD25– T cells as a signi ican p edic o o SLE compa ed o HD (a ea unde he cu e (AUC): 0.744; p = 0.0009; Supplemen a y Fig.S7). Namely, alues o CD25– T cells equal o abo e 0.61% (o o al CD4+ T cells) ha e a sensi i i y o 62.5% and a speci ici y o 71% o he diagnosis o SLE, in compa ison wi h HD (Supplemen a y Fig.S7). Fu he , SLE pa ien s also had an inc ease in he o al popula ion o ci cula ing CD25– T eg cells (p = 0.0050) (Fig.3B). This inding sugges s ha he inc ease o he CD25– T cells does no explain he g ea e equency o o al CD25– T eg cells (independen ly o CXCR5 exp ession), which has been p e iously poin ed ou as a possible bioma ke o he s a i ica ion o SLE pa ien s ega ding enal in ol emen 37. Discussion In ecen yea s we wi nessed majo ad ances ega ding he unde s anding o he egula ion o humo al esponses leading o an ibody and au oan ibody p oduc ion. While T h cells p omo e GC o ma ion, a ini y ma u a ion, and p oduc ion o high-a ini y an ibodies, T cells egula e he magni ude o he GC esponse and p e en au oimmuni y in mice23,38,39. E en hough i is emp ing o assume ha au oimmuni y a ises when he T /T h balance is shi ed by he educ ion o T cells o inc eased T h cells, he e is e idence ha human au oimmuni y Table 1. Demog aphic and clinical cha ac e is ics o s udy pa icipan s. HD heal hy dono s, HT hashimo o’s hy oidi is, RA heuma oid a h i is, SLE sys emic lupus e y hema osus, DAS28 disease ac i i y sco e-28, SLEDAI sys emic lupus e y hema osus disease ac i i y index, An i-TG an i- hy oglobulin an ibodies, An i-TPO an i- hy oid pe oxidase an ibodies, An i-CCP an i-cyclic ci ullina ed pep ides, RF heuma oid ac o , ANA an inuclea an ibody. *The e we e no signi ican di e ences agains HD con ols. **An i-TG and an i-TPO i e s una ailable o 2 pa ien s. HD (n = 31) HT (n = 18) RA (n = 16) SLE (n = 32) Age, mean ± SD yea s* 48.6 ± 17.4 49.0 ± 14.0 53.9 ± 15.7 52.8 ± 16.6 Female, no. (%)* 26 (81) 17 (94) 14 (88) 26 (81) Disease du a ion, mean ± SD yea s – 7.8 ± 12.7 12.1 ± 10.1 12.3 ± 8.4 DAS28, mean ± SD – – 2.9 ± 1.4 – SLEDAI, mean ± SD – – – 1.8 ± 1.8 An i-TG posi i e (> 40 U/mL), no. (%)** 12 (75) An i-TPO posi i e (> 60 U/mL, no. (%)** – 14 (88) – – An i-CCP posi i e, no. (%) – – 13 (81) – RF posi i e, no. (%) – – 15 (94) – ANA ≥ 1/160, no. (%) – – – 26 (81) An i-SSA posi i e, no. (%) – – – 6 (19) An i-SSB posi i e, no. (%) – – – 2 (6) An i-Sm posi i e, no. (%) – – – 5 (16) An i-RNP posi i e, no. (%) – – – 9 (28) An i-dsDNA posi i e, no. (%) – – – 14 (44) Me ho exa e, no. (%) – – 17 (100) 4 (13) P ednisolone, no. (%) – – 12 (75) 25 (78) Hyd oxychlo oquine, no. (%) – – 5 (31) 29 (91) Sul asalazine, no. (%) – – 4 (25) – Aza hiop ine, no. (%) – – – 6 (19) Le o hy oxin, no. (%) – 17 (94) – – 5 Vol.:(0123456789) Scien i ic Repo s | (2022) 12:17638 | h ps://doi.o g/10.1038/s41598-022-21576-8 www.na u e.com/scien i ic epo s/ Figu e2. Ac i a ed PD-1+ICOS+ T h cells a e expanded in SLE pa ien s. (A) Rep esen a i e plo s (le ) and pooled da a ( igh ) o he equency o ac i a ed PD-1+ICOS+ T h cells in he pe iphe al blood o HT (n = 18), RA (n = 16), and SLE (n = 32) pa ien s, and sex- and age-ma ched HD (n = 18). Each da a poin ep esen s an indi idual subjec ; ba s ep esen mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, K uskal–Wallis one-way analysis o a iance (ANOVA) es wi h Dunn’s compa ison pos - es (B) Rep esen a i e plo s (le ) and pooled da a ( igh ) o he dis ibu ion o Th1-like (CXCR3+CCR6–), Th2-like (CXCR3–CCR6–), and Th17-like (CXCR3– CCR6+) T h cell subse s in HT (n = 18), RA (n = 16), and SLE (n = 32) pa ien s, and sex- and age-ma ched HD (n = 19). *p < 0.05, **p < 0.01, ***p < 0.001, wo-way ANOVA wi h Dunne ’s mul iple compa isons es was pe o med. Figu e3. SLE pa ien s show an inc eased equency o ci cula ing Tph and CXCR5+CD25–Foxp3+ T ollicula cells. Rep esen a i e plo s (le ) and pooled da a ( igh ) o he equency o (A) Tph cells and (B) CXCR5+CD25– Foxp3+ T cells in he pe iphe al blood o HT (n = 18), RA (n = 16), and SLE (n = 32) pa ien s, and sex- and age- ma ched HD (A: n = 16; B: n = 31). Each da a poin ep esen s an indi idual subjec ; ba s ep esen mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, K uskal–Wallis one-way analysis o a iance (ANOVA) es wi h Dunn’s compa ison pos - es . 6 Vol:.(1234567890) Scien i ic Repo s | (2022) 12:17638 | h ps://doi.o g/10.1038/s41598-022-21576-8 www.na u e.com/scien i ic epo s/ can coexis wi h a blood T /T h a io shi ed in he opposi e di ec ion14. Se e al explana ions may accoun o his appa en ly coun e -in ui i e inding. One possibili y is ha he equency o T ollicula cells in blood may no di ec ly e lec hei incidence in he seconda y lymphoid o gans o ELS. S udies showing T ollicula cells in he a ec ed o gans in human au oimmuni y a e s ill a e, meaning ha his co ela ion be ween blood and issue has no been ye ully es ablished40,41. Ano he possibili y s ems om he obse a ion ha ch onic s imula ion by au oan igens, and he subsequen humo al ac i i y, can lead o pe sis en p oduc ion o ci cula ing T cells (as i occu s ollowing in luenza accina ion)4. Howe e , he e a e se e al epo s wi h inconsis en esul s, ei he o di e en diseases o ocused on he same disease, as di e en me hods a e used o de ine T and T h subse s. The same designa ion (T h o T ) has been used wi h cell popula ions wi h dispa a e pheno ypic cha ac e is ics. Fo ins ance, T h cells a e o en conside ed as CD4+CXCR5+ cells, wi hou exclusion o Foxp3+ T cells26,27. These inconsis encies make i di icul o compa e he di e en s udies, hus no allowing o in e whe he he e is a uni o m T /T h dys egula ion ha explains he pa hogenesis o humo al imbalance in human au oimmuni y. We s udied a g oup o o gan-speci ic and sys emic au oimmune diseases, using a consis en analysis app oach, o in es iga e whe he common cha ac e is ics ega ding he equency o T and T h subse s could be es ablished. We ound ha he e is a ma ked he e ogenei y be ween di e en au oan ibody-media ed au oimmune diseases ega ding he equency o ci cula ing T and T h subse s. I emains unclea whe he he obse ed changes in T ollicula cells a e he cause o he consequence o he humo al immune dys egula ion unde lying hese diseases. As a esul , i is possible ha some a e ac ual au oan ibody-media ed condi ions, whe eas o he s a e diseases ha ha e de ec able au oan ibodies, bu a e no necessa ily media ed by hem, hus con ibu ing o he he e ogenei y we obse ed. This could be o e come by accessing he dys egula ion o he ge minal cen e esponse di ec ly in he a ec ed issues, bo h in he a ge o gan and in d aining lymphoid issue whe e au oan ibody p oduc ion occu s. Un o una ely, in he p esen s udy we compa ed di e en diseases wi h dis inc a ge issues. The e o e, a issue-based app oach would isk inc easing he he e ogenei y o he analysis, u he complica ing he in e p e a ion o ou and p e ious indings. Ou indings suppo he hypo hesis ha T ollicula cell dys egula ion di e s ac oss au oimmune diseases, bo h o gan-speci ic and sys emic. Howe e , we should unde line ha his s udy was no designed o iden i y blood bioma ke s o speci ic au oimmune diseases, based on T h/T cell pa e ns. Such a s udy would ha e limi ed applicabili y as HT, RA and SLE a e e y di e en diseases om a clinical poin o iew and, he e o e, he use o a bioma ke would likely no be clinically use ul. Howe e , he e may be some po en ial o hese ini ial da a o be u he explo ed wi hin each disease, aiming a pa ien s a i ica ion o e en pe sonalized ea men . Indeed, he di e se pa e ns obse ed wi hin a pa icula disease, aise he hypo hesis ha di e en subg oups o pa ien s ha e dis inc pa e ns o humo al dys egula ion, which possibly also a y o e ime. I is well es ablished ha some clinical ea u es, such as posi i i y o speci ic au oan ibodies o he p esence o ELS can occu in only a subg oup o pa ien s wi h he same disease, such as in RA o pSS14,42–44. This ype o he e ogenei y, linked o dis inc immunopa hogenesis, may p o ide an impo an explana ion o di e en esponses obse ed o he same ea men egimens. Thus, s a egies o u he s a i y pa ien s acco ding o he unde lying pa e n o immune dys egula ion, using T /T h as a p oxy, may con ibu e o de elop ailo ed indi idualized ea men s. In ac , we and o he s ha e p e iously shown ha he equency o ci cula ing PD-1+ICOS+ T h cells could be used o iden i y pSS pa ien s wi h g ea e disease ac i i y14,15, whe eas he T /T h a io could iden i y hose pa ien s wi h ELS wi hin sali a y glands14. This suppo s ou hypo hesis ha , in speci ic diseases, subpopula ions o T and T h cells may allow pa ien s a i ica ion. In addi ion, o he componen s o he in lamma o y esponse, namely se um cy okine p o iles, can po en ially con ibu e o a be e cha ac e iza ion o unique ea u es o dis inc au oimmune diseases and subg oups o pa ien s wi h he same disease, p o iding addi ional ools o a mo e e ined pa ien s a i ica ion. Ou s udy has se e al limi a ions. Pa ien s wi h dis inc diseases could no be ully ma ched o demog aphic o clinical cha ac e is ics, such as ea men o disease du a ion. Thus, i is possible ha speci ic d ugs o disease s age (i.e., ea ly/la e) may a ec T and T h subse s di e en ly. Ne e heless, we we e unable o ind an associa ion be ween he obse ed he e ogenei y in T and T h subse s and speci ic ea men s o o he clinical pa ame e s, such as disease du a ion o se ological ma ke s. In addi ion, he numbe o pa ien s included in each g oup may be ela i ely small, conside ing he desc ibed disease he e ogenei y, especially in RA and SLE. Analyzing a la ge numbe o pa ien s wi h such condi ions may o e come his issue, especially wi h ega d o pa ien s a i ica ion, hus allowing o mo e obus conclusions. Also, he he e ogenei y ound in T and T h subse s in pa ien s unde di e en ea men s (Supplemen a y Fig.S8) ein o ces he need o la ge s udies. I is also possible ha he pa e n o dys egula ion in issues wi h humo al esponses d i ing au oan ibody p oduc ion may di e om he con en o T ollicula subse s in ci cula ion. Ne e heless, he pe iphe al blood compa men sha es common ea u es ac oss diseases, namely he p esence o ci cula ing au oan ibodies and dys egula ed humo al esponses. In addi ion, i is easie o access and mo e ho oughly s udied, wi h equen claims o ci cula ing T h/T dys egula ion as a uni e sal hallma k au oan ibody p oduc ion—an assump ion we ha e shown canno be made. In conclusion, humo al dys egula ion in au oimmuni y canno be asc ibed o a common mechanism ela ed o T ollicula cells. Howe e , he he e ogenei y o T and T h subse s ound ac oss di e en au oimmune diseases, and wi hin speci ic pa ien subg oups, o e s an oppo uni y o explo e po en ial bioma ke s o he di e en diseases and o u he s udies on pa ien s a i ica ion. This may po en ially con ibu e o de eloping ailo ed he apeu ic s a egies and imp o ing pa ien ou comes. Da a a ailabili y The da a unde lying his a icle will be sha ed on easonable eques o he co esponding au ho L.G. 7 Vol.:(0123456789) Scien i ic Repo s | (2022) 12:17638 | h ps://doi.o g/10.1038/s41598-022-21576-8 www.na u e.com/scien i ic epo s/ Recei ed: 21 July 2022; Accep ed: 29 Sep embe 2022 Re e ences 1. Sage, P. T., Al a ez, D., Godec, J., Von And ian, U. H. & Sha pe, A. H. Ci cula ing T ollicula egula o y and helpe cells ha e memo y-like p ope ies. J. Clin. In es . 124, 5191–5204 (2014). 2. Schmi , N., Ben ebibel, S. 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Hi ing he b akes on ec opic lymphoid s uc u e o ma ion. Na . Re . Rheuma ol. 11, 621–621 (2015). 44. Bomba die i, M. & Pi zalis, C. Ec opic lymphoid neogenesis and lymphoid chemokines in Sjog en’s Synd ome: a he in e play be ween ch onic in lamma ion, au oimmuni y and lymphomagenesis. Cu . Pha m. Bio echnol. 13, 1989–1996 (2012). Acknowledgemen s We would like o hank he Biobank o Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, Lisbon Academic Medical Cen e, o hei help in sample collec ion. We also hank o he Flow Cy ome y Facili y o Ins i u o de Medicina Molecula João Lobo An unes, Faculdade de Medicina, Uni e sidade de Lisboa, Lisbon Academic Medical Cen e, o hei se ices and assis ance. We hank o he Endoc inology and Rheuma ology Depa men s o Hospi al de San a Ma ia, Cen o Hospi ala Uni e si á io Lisboa No e, Lisbon Academic Medical Cen e, o hei collabo a ions. 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