Molecular markers distinguishing T Cell subtypes with TSDR strand-bias methylation

ORIGINAL RESEARCH published: 05 No embe 2018 doi: 10.3389/ immu.2018.02540 F on ie s in Immunology | www. on ie sin.o g 1No embe 2018 | Volume 9 | A icle 2540 Edi ed by: Remy Bosselu , Na ional Cance Ins i u e (NCI), Uni ed S a es Re iewed by: Sid P. Ke ka , Boeh inge Ingelheim, Uni ed S a es Elizabe h Wohl e , Uni e si y a Bu alo, Uni ed S a es *Co espondence: João F. Lace da [email p o ec ed] †P esen Add ess: Eka e ina Minskaia, Ad anced The apeu ics, Sco ish Na ional Blood T ans usion Se ice, The Jack Copland Cen e, Edinbu gh, Uni ed Kingdom Ana I. S. Viei a, Flow Cy ome y Pla o m, Champalimaud Founda ion, Lisbon, Po ugal ‡These au ho s sha e senio au ho ship Special y sec ion: This a icle was submi ed o T Cell Biology, a sec ion o he jou nal F on ie s in Immunology Recei ed: 17 May 2018 Accep ed: 15 Oc obe 2018 Published: 05 No embe 2018 Ci a ion: Minskaia E, Sa ai a BC, Soa es MMV, Aze edo RI, Ribei o RM, Kuma SD, Viei a AIS and Lace da JF (2018) Molecula Ma ke s Dis inguishing T Cell Sub ypes Wi h TSDR S and-Bias Me hyla ion. F on . Immunol. 9:2540. doi: 10.3389/ immu.2018.02540 Molecula Ma ke s Dis inguishing T Cell Sub ypes Wi h TSDR S and-Bias Me hyla ion Eka e ina Minskaia1†‡, Ba ba a C. Sa ai a1, Ma ia M. V. Soa es1, Ri a I. Aze edo1, Ruy M. Ribei o2, Saumya D. Kuma 1, Ana I. S. Viei a1† and João F. Lace da1*‡ 1Faculdade de Medicina da Uni e sidade de Lisboa, Ins i u o de Medicina Molecula –João Lobo An unes, Lisbon, Po ugal, 2Depa men o de Bioma emá ica, Faculdade de Medicina da Uni e sidade de Lisboa, Lisbon, Po ugal Human egula o y CD4+CD25+FOXP3+T cells (T eg) play impo an oles in he main enance o sel - ole ance and immune homeos asis in a ious disease se ings and a e also in ol ed in he supp ession o e ec i e immune esponses. These cells a e he e ogeneous in pheno ype and unc ion, and he abili y o eliably dis inguish be ween a ious FOXP3-exp essing subpopula ions can a ec he de elopmen o success ul he apies. This s udy demons a es ha hypome hyla ed CpG si es, p esen in ou egions o he FOXP3 locus, CAMTA1 and FUT7 gene egions, can be used o dis inguish se e al subse s o T eg om con en ional CD4+T lymphocy es (Tcon) in dono s o bo h gende s. We desc ibe a p e iously un epo ed s and-bias hemime hyla ion pa e n in FOXP3 p omo e and TSDR in dono s o bo h gende s, wi h he coding s and being deme hyla ed wi hin p omo e and me hyla ed wi hin TSDR in all CD4+lymphocy e sub ypes, whe eas he empla e s and ollows he p e iously desc ibed pa e n o me hyla ion wi h bo h egions being mo e deme hyla ed in T eg sub ypes and mos ly me hyla ed in Tcon. This s and-speci ic app oach wi hin he TSDR may p o e o be ins umen al in co ec ly de ining T eg subse s in heal h and in disease. Keywo ds: FOXP3,CAMTA,FUT7, egula o y T lymphocy es, epigene ics,s and-bias me hyla ion INTRODUCTION Regula o y T cells (T eg) play c ucial oles in he main enance o sel - ole ance and immune homeos asis in diseases such as alle gy and au oimmune diso de s (AID). These cells a e also in ol ed in he supp ession o e ec i e immune esponses agains in ading pa hogens and au ologous cance ous cells (1,2). Human CD4+CD25+FOXP3+T cells a e he e ogeneous in pheno ype and unc ion and include es ing ( T eg) and ac i a ed (aT eg) T eg cells, as well as non-supp essi e T eg-like cells (3,4). Despi e his, o many yea s FOXP3 exp ession alone has been used as a speci ic T eg ma ke un il i was deemed insu icien o iden i ica ion o supp essi e T eg cells. The abili y o eliably dis inguish be ween a ious FOXP3-exp essing subpopula ions and o unde s and hei oles in immunological diseases, cance and in ec ions can os e he apies ha ei he aim o boos he supp essed immune esponses o o dampen he abno mally acu e immune esponses. Towa d his goal, Miya a e al. (5) used he combina ion o CD25 and CD45RA o isola ing h ee human T eg subse s: FOXP3lowCD45RA+CD25++ supp essi e in i o T eg, FOXP3hiCD45RA−CD25+++ e ec o (eT eg) cells and cy okine-sec e ing non-supp essi e Minskaia e al. Epigene ics T Cell Sub ypes FOXP3lowCD45RA−CD25++ T cells. La e , CD15s (sialyl Lewis x) was iden i ied as a bioma ke o mos supp essi e FOXP3high eT eg cells (6). A combina ion o CD15s and CD45RA was ins umen al in he isola ion o dis inc CD4+CD127lowCD25+FOXP3+T cell sub ypes: naï e CD45RA+CD15s−T eg, highly supp essi e CD45RA−CD15s+ eT eg and a non-supp essi e CD45RA−CD15s−subse . Toge he wi h his one ace yla ion and non-coding RNAs, DNA me hyla ion can ei he s ably o empo a ily al e gene exp ession depending on he immedia e physiological equi emen s o he o ganism. Se e al egula o y egions on FOXP3 locus a e e y impo an playe s in he T eg- speci ic epigenome: wo conse ed non-coding sequences (CNS 1 and 3) a e in ol ed in his one ace yla ion while h ee o he egions - ups eam enhance , p oximal p omo e and CNS 2 (known as FOXP3 TSDR) con ibu e o FOXP3 exp ession ia deme hyla ion and we e p oposed as addi ional molecula ma ke s ha can help dis inguish T eg om con en ional T lymphocy es (Tcon), as well as di e en T eg ma u a ion s ages (7–9). A he same ime, changes in T cell DNA me hyla ion pa e ns ha e been epo ed in diseases such as alle gies, mul iple scle osis and heuma oid a h i is (10,11). Howe e , as FOXP3 gene is encoded on Xp11.23, mos s udies op ed o use male dono s in o de o a oid he a i ac s o he inac i a ion o X ch omosome (Xi). The e o e, p ecise egula ion o FOXP3 exp ession in emale dono s emains somewha o an enigma—ye emales comp ise he majo i y o pa ien s wi h AID and show a s onge esponse o in ec ions han males. CAMTA1, encoded on ch omosome 1q3.6 and no a ec ed by Xi, was able o di e en ia e CD25highCD45RA−CD4+T eg om CD25−CD45RA+CD4+Tcon (7) and, he e o e, has he po en ial o dis inguish T cell subse s in bo h male and emale dono s. In his s udy, we cha ac e ized he epigene ic p o ile o hema opoie ic s em cells (CD34+) and ou popula ions o CD4+CD25+T cells: CD45RA+CD15s−FOXP3low (naï e nT egs), CD45RA−15s−FOXP3low (non-supp essi e T eg-like cells), CD45RA−CD15s+FOXP3high (eT egs) and Tcons isola ed om pe iphe al blood o heal hy male and emale dono s. As α(1,3)- ucosyl ans e ase 7 (FUT7) media es syn hesis o CD15s exp essed in bo h eT egs and CD34+cells (12), FUT7 p omo e was expec ed o be deme hyla ed in hese cell popula ions o allow o p o ein exp ession. Toge he wi h CAMTA1 in onic egion 3, FUT7 p omo e was es ed o i s po en ial o ac as an addi ional and/o al e na i e o FOXP3 molecula ma ke . Th ee p e iously desc ibed egions on FOXP3 locus: ups eam enhance , p oximal p omo e and TSDR (T eg-speci ic deme hyla ed egion), we e also s udied oge he wi h he ou h egion, ha we now e m p eTSDR. As DNA me hyla ion was shown o a y among indi iduals and e en be ween wins (13,14), we a emp ed o cha ac e ize epigene ic changes in all six gene egions om he i e cell popula ions o each dono in o de o ob ain comp ehensi e in o ma ion speci ic o each indi idual. Using bisulphi e con e sion o genomic DNA (gDNA) ollowed by sequencing o indi idual clones was ins umen al in deciphe ing he me hyla ion s a us o indi idual CpG posi ions and he in ica e pa e ns con olling gene exp ession in CD34+cells and T lymphocy e subse s. MATERIALS AND METHODS Isola ion o Human PBMCs and Flow Cy ome y Pe iphe al blood samples we e ob ained om young heal hy male (M1-6) and emale (F1-5) olun ee s. None o he dono s had known au oimmune o gene ic condi ions. Pe iphe al blood mononuclea cells (PBMCs) we e p epa ed by Ficoll g adien cen i uga ion (15). CD34+cells (dono s M4-6 and F1-5) we e i s en iched using he EasySepTM Human CD34 Posi i e Selec ion ki (STEMCELL Technologies) ollowing he manu ac u e ’s ins uc ions. In o de o inc ease he pu i y o he magne ically isola ed CD34+ ac ion, he cells we e u he s ained wi h CD34 FITC (Mil enyi Bio ec) and so ed by luo escen ac i a ed cell so ing (FACS) on a BD FACSA iaIII. Tcon and T eg subpopula ions we e pu i ied om he nega i e ac ion ob ained om he EasySepTM CD34 selec ion p o ocol as ollows: cells we e incuba ed o 25 min a oom empe a u e in PBS (2% human se um) wi h p e- i a ed amoun s o he ollowing an ibodies: an i- hCD3 (-Pe CP, clone OKT3, eBioscience), an i-hCD4 (-APC, clone RPA-T4, eBioscience), an i-hCD45RA (-FITC, Mil enyl Bio ec), an i-hCD25 (-Pe-Cy7, BD Biosciences), an i-hCD127 (-APCe780, clone eBioRDR5, eBioscience), an i-hCD15s (- PE, BD Biosciences). Cells we e hen washed and so ed on a BD FACSA iaIII. Cells ob ained om he EasySep CD34 nega i e ac ion we e u he used o in acellula s aining o FOXP3. Following he su ace s aining using he same an ibody combina ion as desc ibed abo e o cell so ing, cells we e s ained wi h an i-hFOXP3 (eFluo 450, clone PCH101, eBioscience) using he FOXP3 S aining Bu e Se (e-Bioscience) acco ding o he manu ac u e ’s ins uc ions. Da a was acqui ed on he BD FACSA iaIIu. Fo analysis o CD34+cells, whole blood samples we e su ace s ained o 20 min a oom empe a u e wi h he same an ibodies as abo e excep o an i- hCD4 (Pe CPCy5.5, clone OKT4, eBioscience), an i-hCD45RA (APC, clone T6D11, Mil enyl Bio ec), an i-hCD3 (BV510, cloneUCHT1, BD Ho izon). In acellula s aining o FOXP3 (eFluo 450, clone PCH101, eBioscience) was pe o med using RBC lysis, ixa ion and pe meabiliza ion eagen s (eBioscience), acco ding o he manu ac u e ’s ins uc ions. Samples we e acqui ed on a BD LSR Fo essa low cy ome e (BD Biosciences). Da a we e analyzed using FlowJo R LLC. Isola ion o Genomic DNA gDNA om CD34+cells and ou popula ions o CD4+T cells we e isola ed by he Quick-gDNA MiniP ep ki (Zymo Resea ch) ollowing manu ac u e ’s ins uc ions. B ie ly, cells we e esuspended in 100 µl o PBS hen lysed in 400 µl o Genomic Lysis Bu e o 10 min. Sample was loaded in o p o ided Zymo-Spin column and cen i uged a 10,000 g o 1 min. The column was hen washed in wo s eps: (i) 200 µl o DNA P e-Wash Bu e and (ii) 500 µl o gDNA Wash Bu e . F on ie s in Immunology | www. on ie sin.o g 2No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes gDNA was elu ed om he column in 45 µl o DNA Elu ion Bu e . Bisulphi e T ea men and Me hyla ion Analysis De e mina ion o me hyla ion s a us o indi idual CpG si es ac oss en i e amplicons can be achie ed by BS ea men o gDNA ollowed by sequencing o indi idual clones, each ep esen ing one DNA molecule om one cell in a gi en cell popula ion. The ac ha BS ea men con e s all non- me hyla ed cy osines (C) in o u acils (U) while me hyla ed Cs (mC) emain unchanged means ha he wo DNA s ands in BS DNA a e no longe complemen a y. Only one s and o BS DNA is ampli ied by each p ime se , wi h he e e se p ime binding he chosen a ge s and and he esul ing ampli ied s and se ing as a empla e o he o wa d p ime . In addi ion o s and-speci ici y, p ime s a e designed in such a way ha biased ampli ica ion o non-me hyla ed s ands is a oided and only BS DNA is ampli ied. To a oid any po en ial bias a ec ing he downs eam sequencing esul s, gDNA om he i e cell popula ions: CD34+, CD45RA+15s−, CD45RA−15s−, CD45RA−15s+, and Tcons, was isola ed and BS ea ed a he same ime ollowed by BS PCR and cloning o he six gene egions. Con e sion o gDNA om he i e cell popula ions was pe o med by EZ DNA Me hyla ion Ligh ning Ki (Zymo Resea ch) which esul s in o e 99.5% C o U con e sion o non-me hyla ed esidues while o e 99.5% o mC a e p o ec ed. B ie ly, 130 µl o Ligh ning Con e sion Reagen was added o 20 µl o gDNA and he samples we e incuba ed a 95◦C o 8 min ollowed by 54◦C o 60 min. DNA samples we e loaded in o Zymo-Spin IC Columns con aining 600 µl o M-Binding Bu e , mixed well and cen i uged a 10,000 g o 30 s (as all o he cen i uga ion s eps). Following he i s wash s ep wi h 100 µl o M-Wash Bu e , he samples we e incuba ed wi h 200 µl o M-Desulphona ion Bu e a oom empe a u e o 15 min. Columns we e washed wice wi h 200 µl o M-Wash Bu e . Con e ed BS- ea ed DNA was elu ed in 12 µl o M-Elu ion bu e and immedia ely used o PCR analyses. Remaining BS- ea ed DNA was s o ed a −20◦C o la e use. The six gene egions we e ampli ied using non- me hyla ion-, BS- ea ed DNA-, coding s and-speci ic p ime s (Supplemen a y Table 1). Thi y PCR eac ions o he six gene egions we e pe o med in pa allel con aining 1.5 µl o BS DNA and Phusion U Ho S a PCR mix (The mo Fishe Scien i ic) in a o al olume o 25 µl. A e ini ial dena u a ion a 98◦C o 30 s, ampli ica ion consis ed o 45 cycles a 98◦C o 10 s, 58-60◦C o 20 s, and 72◦C o 45 s. The PCR p oduc s ob ained we e gel pu i ied using NZYGelpu e ki (NZY ech) ollowing he manu ac u e ’s ins uc ions and cloned in o he pGEM-T Easy ec o (P omega) ia NcoI and NsiI es ic ion si es o ensu e di ec ional cloning. Plasmid DNAs om 22–24 clones we e isola ed using plasmid NZYMinip ep ki (NZY ech) and 20–22 posi i e clones (unless o he wise s a ed), con i med by es ic ion diges ion wi h he abo e-men ioned enzymes, we e sequenced using e e se SP6 p ime : 5′-GTGACACTATAGAATACTC-3′ (NZY sequencing and S ab ida). Sequences (AB1 iles con aining ch oma og ams) we e aligned o each gene egion’s e e ence sequence using SeqMan so wa e (DNA S a Lase gene 8). All non-me hyla ed cy osines we e iden i ied by he p esence o a T nucleo ide (n ) in BS-con e ed sequences while mCs we e iden i ied by he p esence o a C n . E iciency o bisulphi e con e sion was con i med by con e sion o non-CpG Cs o Ts. The pe cen age o me hyla ion in each CpG posi ion was de e mined by de ining he p opo ion o mCs in he o al o 20 (2 mCs ou o 20=10% me hyla ion). As he six gene egions we e ampli ied om he same BS DNA empla e, he me hyla ion di e ences e lec he a e age me hyla ion s a us o he cell popula ion. S a is ical Analysis To analyze he di e ences in me hyla ion be ween Tcon and he h ee T eg popula ions (CD45RA+CD15s−, CD45RA−CD15s−, and CD45RA−CD15s+) o he six genes s udied, we used a mixed-e ec s app oach, aking in o conside a ion he pai ed na u e o he da a. Fo each gene, he di e ence in me hyla ion alues be ween Tcon and each o he T eg popula ions was calcula ed and hen his di e ence was es ed o signi ican di e ences om ze o (indica ing a di e ence in me hyla ion be ween he wo popula ions being compa ed), wi h indi idual and CpG posi ion conside ed as andom e ec s. This analysis was done using he unc ion lme om he package nlme o R (h ps://c an. -p ojec .o g/). Since in each case we used he me hyla ion o Tcon o h ee es s, we co ec ed o mul iple compa isons and only conside ed as signi ican hose esul s wi h p<0.016 (equi alen o α=0.05/3). We used he same app oach o compa e he me hyla ion o he op and bo om s ands. Hie a chical Clus e ing Hie a chical Agglome a i e Clus e ing was pe o med using Euclidean dis ance ma ix and comple e linkage algo i hm. Dis ance ma ix was calcula ed on a e age me hyla ion o each CpG posi ion ac oss all dono s o each genomic elemen . Hea map package in R was used o gene a e hea maps (16). S udy App o al E hical app o al by he ins i u ion’s E hics Commi ee was ecei ed p io o he beginning o he s udy. W i en in o med consen was ecei ed om pa icipan s p io o inclusion in he s udy. RESULTS Isola ion o CD34+and CD4+T Cell Subpopula ions Cell popula ions we e isola ed om he pe iphe al blood o heal hy dono s by FACSo ing. The mean pu i y o CD34+ cells a e FACSo was >95%, as shown in a ep esen a i e do plo in Figu e 1A. T eg and Tcon cells we e iden i ied as CD3+CD4+CD25B igh CD127Low and CD3+CD4+CD25Low cells, espec i ely. T eg cells we e u he pu i ied in o he ollowing subpopula ions: CD45RA+CD15s−(naï e T eg), CD45RA−CD15s−(non-supp essi e T eg-like cells) and F on ie s in Immunology | www. on ie sin.o g 3No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes FIGURE 1 | Flow cy ome y analysis and pu i ica ion s a egy o T eg, Tcon, and CD34 cell popula ions. (A) Pu i y o CD34+cells a e FACSo ing. Rep esen a i e do plo showing he equency o CD34+cells wi hin a li e cell ga e, as de e mined by FSC and SSC analysis. (B) Rep esen a i e do plo s showing he ga ing s a egy o he isola ion o T eg and Tcon cells by FACSo . Fi s ly, FSC and SSC analysis was used o iden i y lymphocy es and o exclude double s. T eg cells we e isola ed as CD3+CD4+CD25B igh CD127Low and Tcon as CD3+CD4+CD25Low cells. T eg we e u he ga ed in o CD45RA+CD15s−(naï e T eg), CD45RA−CD15s− (non-supp essi e T eg-like cells), and CD45RA−CD15s+(eT eg). (C) Rep esen a i e do plo s showing he pu i y ob ained o each T cell popula ion a e FACSo . (D) FoxP3 exp ession le els wi hin CD45RA/CD15s T eg subpopula ions, as well as wi hin naï e Tcon cells. A ep esen a i e o e lay o Foxp3 exp ession wi hin hese T cell popula ions is shown. (E) Median Fluo escence In ensi y (MFI) o FoxP3 wi hin T eg subpopula ions o all indi iduals analyzed. As e isks deno e s a is ically signi ican di e ences be ween g oups (*p=0.01–0.05; ****p<0.0001). CD45RA−CD15s+(eT eg), as p e iously desc ibed (6). The mean pu i y o he isola ed popula ions was >95%. Rep esen a i e do plo s illus a ing he FACSo ga ing s a egy (Figu e 1B) and pu i y (Figu e 1C) o each subpopula ion a e shown. These pu i ied cell popula ions we e used o subsequen me hyla ion s udies. F on ie s in Immunology | www. on ie sin.o g 4No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes In addi ion, a PBMC sample was s ained wi h he same an ibody combina ion used o FACSo plus in acellula FoxP3. When we analyzed he exp ession le els o FoxP3 wi hin he T eg subse s de ined by CD45RA and CD15s exp ession, we con i med p e ious obse a ions (6) ha CD45RA+CD15s− cells exp ess he lowes , CD45RA−CD15s−cells in e media e and CD45RA−CD15s+cells he highes le els o FoxP3. This is illus a ed in a ep esen a i e o e lay o FoxP3 exp ession wi hin each o hese T eg subpopula ions, as well as wi hin naï e Tcon o compa ison (Figu e 1D). FoxP3 exp ession le els wi hin T eg subse s om all he dono s analyzed a e shown in Figu e 1E. Me hyla ion o CAMTA1 In onic Region In cells o he immune sys em, including T and B cells, Ca2+signals a e essen ial o di e se cellula unc ions including p oli e a ion, di e en ia ion, e ec o unc ion, gene ansc ip ion, and apop osis. The in acellula concen a ion o Ca2+is known o ac i a e se e al signaling p o eins and ansc ip ion ac o s (TFs) such as calcineu in and NFAT, CaMK and CREB, MEF2, and NFkB (17). Calcineu in–NFAT pa hway is in ol ed in T eg de elopmen and unc ion and is a ec ed by he absence o Ca2+signals (18). I is, he e o e, no coinciden al ha one o he egions in CAMTA 1, a Ca2+-dependen calmodulin- binding ansc ip ion ac o (19), was p oposed as an addi ional molecula ma ke dis inguishing T eg om Tcon (7). The me hyla ion s a us o 13 CpG si es wi hin he 470 bp CAMTA1 in onic egion 3 was assessed based on he op s and o BS DNA (Supplemen a y Tables 1,2). Two me hyla ion pa e ns we e obse ed. In he i s , ep esen ed by male dono M4 (Figu e 2B, le panel and Supplemen a y Table 3) and comp ising male dono s M1-4 and emale dono F2 (Figu e 2C), he i s eigh CpG si es (CpGs 1-8) we e mo e deme hyla ed in CD34+cells and he h ee subse s o T eg as compa ed o Tcon, as is e iden om he a e age me hyla ion pa e n (Figu e 2B, lowe panel).The a e age me hyla ion pe cen ages o CpGs 1-8 in hese dono s we e 37.8% (CD34+), 36.2% (CD45RA+CD15s−), 13.9% (CD45RA−CD15s−), and 35.8% (CD45RA−15s+) as compa ed o 74.1% in Tcon (Supplemen a y Table 3). In he second pa e n, ep esen ed by dono F4 (Figu e 2B, igh panel) and comp ising six dono s (M5, 6, and F1, 3-5), only wo CpG posi ions we e consis en ly mo e deme hyla ed in CD34+cells and T eg subse s as compa ed o Tcon. While in Tcon, CpGs 2 and 11 we e 70 and 74.2% me hyla ed, a e age me hyla ion le els o hese wo si es in he o he ou popula ions we e signi ican ly lowe : 25.8 and 33.3% (CD34+), 13.3 and 22.5% (CD45RA+CD15s−), 10.8 and 16.7% (CD45RA−CD15s−), and 7.5 and 11.7% (CD45RA−CD15s+), espec i ely (Figu e 2B, igh lowe panel and Supplemen a y Table 3). O e all, he en i e CAMTA1 egion in Tcon subse was hea ily me hyla ed (80%) as p esen ed by he a e age me hyla ion pa e n o ele en dono s (Figu e 2C, lowe panel and Supplemen a y Table 3), howe e , he main di e ences we e obse ed in CpGs 1- 11 (wi h he excep ion o CpG 9). A e age me hyla ion le els o mo e deme hyla ed CpGs 1-8 wi hin he i s 200 bp pa o CAMTA1 egion we e 56.7% (CD34+), 49% (CD45RA+CD15s−), 41% (CD45RA−CD15s−), and 48% (CD45RA−CD15s+) as compa ed o 78.4% in Tcon. CpG si es 2 and 11 we e mo e deme hyla ed in CD34+and T eg subse s: 24.4, 20.5, 10, and 15% in CpG 2 and 36.3, 44.5, 27.7, and 33.6% in CpG11 in CD34+, CD45RA+CD15s−, CD45RA−CD15s−, and CD45RA−CD15s+cells, espec i ely, as compa ed o 66.8 and 77.3% o hese CpG posi ions in Tcon (Supplemen a y Table 3). The di e ences in me hyla ion alues o all CpGs we e u he analyzed using he unc ion lme om he package nlme o R and he calcula ed di e ence was only conside ed as signi ican o hose esul s wi h p <0.016. This analysis demons a ed signi ican di e ences be ween all T eg sub ypes compa ed o Tcon (p=0.008 o CD45RA+CD15s−,p=0.0003 o CD45RA−CD15s−, and p=0.0004 o CD45RA−CD15s+). Me hyla ion o FUT7 P omo e The induc ion o CD15s on human hema opoie ic cells and lymphocy es accompanies ansc ip ional ac i a ion o FUT7 which is in ol ed in he las s ep o sialyl Lewis X syn hesis (12,20). FUT7 is highly exp essed in eT eg compa ed o o he FOXP3+o FOXP3−subpopula ions (5). T ansc ip ional egula ion o FUT7 plays an impo an ole in lineage-speci ic exp ession o CD15s among lymphocy e subpopula ions. Fo example, he gene a ion o E-selec in ligands on T cells unde going nai e- o-memo y ansi ion was shown o equi e FUT7 ac i i y (21). Human FUT7 p omo e was demons a ed o ha e binding si es o se e al TFs, six o which: T-be , GATA-3, Sp1, CBP/P300, HDAC-3, and HDAC-5 may o m ansc ip ional complex in human lymphoid cells (12,20). As FUT7 gene is encoded in he e e se s and o ch omosome 9, FUT7 p omo e egion was ampli ied wi h he e e se s and- speci ic p ime s (Supplemen a y Table 1). Due o he leng h o his egion and he ac ha longe amplicons canno be success ully p oduced as a esul o BS ea men , me hyla ion s a us o 19 indi idual CpGs was assessed wi hin wo amplicons, 500bp and 454bp in leng h, oge he comp ising FUT7 p omo e (Supplemen a y Table 2). Me hyla ion pa e n wi hin FUT7 p omo e egion was o e all simila among dono s M1-6 and F1-3 and is ep esen ed by dono F3 (Figu e 3B). Simila o dono F3, CpGs 1-7 o o he dono s (wi h he excep ion o CpG 5) did no show a ia ions in me hyla ion in all i e cell popula ions as demons a ed by he a e age me hyla ion p o ile o all dono s (Figu e 3C, lowe panel). CpG posi ions 8-19 we e mos ly deme hyla ed in CD34+cells (wi h he excep ion o CpG 13) wi h a e age me hyla ion le els o he wel e si es being 37.9% (CD34+), 54.1% (CD45RA−CD15s−), and 48.8% (CD45RA−CD15s+) as compa ed o CD45RA+CD15s− (69.6%) and Tcon (75%). CpGs 10–12, 14, and 15 we e mo e deme hyla ed in CD34+(38.2%), CD45RA−CD15s− (54.5%), and CD45RA−CD15s+(45%) popula ions while CD45RA+CD15s−and Tcon cells displayed highe me hyla ion le els (72.8 and 77.2%, espec i ely) (Figu e 3C, lowe panel, and Supplemen a y Table 3). O e all, nT eg (CD45RA+CD15s−) and Tcon subse s displayed a simila me hyla ed pa e n, while CD45RA−CD15s−, eT eg (CD45RA−CD15s+) and CD34+ cells displayed a simila deme hyla ed pa e n, which was F on ie s in Immunology | www. on ie sin.o g 5No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes FIGURE 2 | Me hyla ion pa e n o CAMTA1 in onic egion. CAMTA 1 in onic egion is mo e deme hyla ed in T eg subse s as compa ed o CD34+cells and Tcon wi hin he 200 bp egion con aining CpGs 1-8. CpGs 2 and 11 a e s ikingly mo e deme hyla ed in bo h T eg and CD34+cells. (A) Schema ic p esen a ion o CAMTA1 encoded on ch omosome 1q3.6. (B) Two me hyla ion pa e ns o CAMTA1 in i e cell popula ions: CD34+(1), CD45RA+CD15s−(2), CD45RA−CD15s− (3), CD45RA−CD15s+(4), and Tcon (5) o ep esen a i e dono s M4 (pa e n 1) and F4 (pa e n 2). A e age me hyla ion pe cen ages o dono s ollowing he wo pa e ns p esen ed in panels below. CpGs 1-13 a e numbe ed ela i e o he 5′-3′di ec ion o he coding ( op) s and. Each ho izon al line ep esen s DNA om one cell wi h unmodi ied C in ligh blue and mC in da k blue. Me hyla ion pe cen age o each CpG si e was calcula ed based on he numbe o mCs in a o al o 20 and summa ized in panels below. (C) Me hyla ion pa e ns o CAMTA1 in onic egion o indi idual dono s wi h he a e age me hyla ion pa e n o all dono s p esen ed in he panel below. See also Supplemen a y Figu es 1,3. cha ac e ized by hypome hyla ion o he i e in e nal CpG si es: 10–12, 14, and 15 wi hin he 225 bp egion o FUT7 p omo e . In compa ison o Tcon, he me hyla ion alues o all CpGs wi hin FUT7 egion we e signi ican ly di e en o CD45RA−CD15s+(p=0.0019) and CD45RA−CD15s−T eg (p=0.0098), bu no o CD45RA+CD15s−T eg (p=0.8889). FOXP3 Enhance FOXP3 gene exp ession is dependen on he ac i i y o se e al egula o y DNA elemen s and ou e o s we e concen a ed on he ou FOXP3 gene egions: enhance , p omo e , p eTSDR and TSDR, all o which we e ampli ied wi h he e e se s and-speci ic p ime s. Me hyla ion da a ob ained om emale dono s F1-5 is delibe a ely p esen ed in i s aw o ma , wi hou accoun ing o he possible Xi. The FOXP3 enhance is one o he h ee egula o y elemen s in he FOXP3 locus ha was shown o be con olled ia CpG me hyla ion (9,22). Loca ed abou 6 kb ups eam o he ansc ip ion s a si e (TSS), his 800 bp egion is ac ually pa o a p o ein phospha ase 1 egula o y subuni locus. Me hyla ion pa e n o he human FOXP3 enhance was shown o be di e en om ha o i s mu ine homolog (22), wi h bo h T eg and Tcon popula ions o heal hy dono s displaying simila p o iles. In pa ien s wi h a h i is, howe e , T eg subse s displayed highe deme hyla ion le els han in heal hy dono s (9), hus accoun ing o g ea e di e ences in me hyla ion be ween T eg and Tcon cells in his disease se ing. Highly CpG ich FOXP3 enhance con ains o e 50 CpG posi ions. Me hyla ion s a us o he i s 42 CpG si es was assessed in six dono s (M1-4 and F1, 2). Because o limi ed blood sample ma e ial, low gDNA quan i y om a e T eg subse s and mos ly di icul ies in cloning, de e mina ion o me hyla ion s a us wi hin his egion was no possible o o he dono s. Despi e he imp essi e numbe o CpG si es wi hin he 497 bp amplicon, he F on ie s in Immunology | www. on ie sin.o g 6No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes FIGURE 3 | Me hyla ion pa e n o FUT7 p omo e . CpG si es 10–12, 14, and 15 wi hin FUT7 p omo e display highe deme hyla ion le els in CD34+and e ec o T eg as compa ed o naï e T eg and Tcon, indica ing ha CpGs 10-15 can be used as an addi ional molecula ma ke o dis inguish hese cell popula ions. (A) Schema ic p esen a ion o FUT7 p omo e encoded in he e e se s and o ch omosome 9. (B) Me hyla ion pa e n o FUT7 p omo e in i e cell popula ions: CD34+(1), CD45RA+CD15s−(2), CD45RA−CD15s−(3), CD45RA−CD15s+(4), and Tcon (5) o ep esen a i e dono F3. CpGs 1-19 a e numbe ed ela i e o he 5′-3′di ec ion o he coding ( e e se) s and. Each ho izon al line ep esen s DNA om one cell wi h unmodi ied C in ligh blue and mC in da k blue. Me hyla ion pe cen age o each CpG si e was calcula ed based on he numbe o mCs in a o al o 20 and summa ized in he panel below. (C) Me hyla ion pa e ns o FUT7 p omo e o indi idual dono s wi h he a e age me hyla ion pa e n o all dono s p esen ed in he panel below. See also Supplemen a y Figu es 1,3. enhance egion demons a ed an in ica e me hyla ion pa e n ep esen ed by dono M4 (Figu e 4B). A e age me hyla ion le els o he mos deme hyla ed CpG si es 4, 9, 11, and 20 o dono M4 we e 11.2% (CD34+), 35% (CD45RA+CD15s−), 28.7% (CD45RA−CD15s−), 32.5% (CD45RA−CD15s+), and 81.2% (Tcon) (Figu e 4B and Supplemen a y Table 3). Simila o his dono , CpG posi ions 1–3, 5–8, and 14–19 we e mos ly me hyla ed in all cell popula ions o all dono s (Figu e 4C). Howe e , CpG posi ions 4, 9, 11, 20, and 24–28 demons a ed highe deg ee o deme hyla ion in CD34+cells and T eg sub ypes compa ed o Tcon as p esen ed by he a e age me hyla ion pa e n o his egion (Figu e 4C, lowe panel). A e age me hyla ion le els o CpGs 4, 9, 11, and 20 in all dono s we e 27.6% (CD34+), 36.7% (CD45RA+CD15s−), 35.5% (CD45RA−CD15s−), 28.5% (CD45RA−CD15s+), and 72.5% (Tcon) (Figu e 4C, lowe panel and Supplemen a y Table 3). S a is ical analysis u he con i med signi ican di e ences in me hyla ion alues o all CpGs wi hin FOXP3 enhance o CD45RA+CD15s−(p<0.00005), CD45RA−CD15s−T eg (p=0.0001), and CD45RA−CD15s+(p=0.0002), compa ed o Tcon. FOXP3 p eTSDR No much is known abou his egion excep ha pa o i was mo e deme hyla ed in CD25highCD45RA−CD4+T eg cells compa ed o CD25−CD45RA+CD4+Tcon (Amp6/7) (7). Conside ing he possibili y o i s hypome hyla ion in T eg sub ypes, i was also chosen o i s p oximi y o TSDR. Compa ed o Amp6/7, con aining 7 CpG si es, p eTSDR egion was ex ended o con ain 10 CpG si es wi hin 700 bp leng h (Supplemen a y Tables 1,2) and se ed as an addi ional in e nal con ol o TSDR me hyla ion bounda ies as i is loca ed immedia ely ups eam (hence he denomina ion we p opose, p eTSDR). Dono s M2, 4, 5, and F4, 5 ollowed he pa e n p esen ed o dono M6 (Figu e 5B, le panel) whe eby CpGs 2-4 we e hea ily me hyla ed in all i e cell popula ions while a ious deg ees o deme hyla ion we e obse ed in CpGs 5, 8, and 9 (Figu e 5C). As p esen ed by he a e age me hyla ion p o ile F on ie s in Immunology | www. on ie sin.o g 7No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes FIGURE 4 | Me hyla ion pa e n o FOXP3 enhance . CpGs 4, 9, 11, 20, and 24–28 wi hin FOXP3 enhance a e mo e deme hyla ed in CD34+cells and T eg sub ypes as compa ed o Tcon. (A) Schema ic p esen a ion o he FOXP3 enhance encoded in he e e se s and o Xp11.23. (B) Me hyla ion pa e n o he FOXP3 enhance in i e cell popula ions: CD34+(1), CD45RA+CD15s−(2), CD45RA−CD15s−(3), CD45RA−CD15s+(4), and Tcon (5), o ep esen a i e dono M4. CpGs 1-42 a e numbe ed ela i e o he 5′-3′di ec ion o he coding ( e e se) s and. Each ho izon al line ep esen s DNA om one cell wi h unmodi ied C in ligh blue and mC in da k blue. Me hyla ion pe cen age o each CpG si e was calcula ed based on he numbe o mCs in a o al o 20 and summa ized in panels below. (C) Me hyla ion pa e ns o he FOXP3 enhance o indi idual dono s wi h he a e age me hyla ion pa e n o all dono s p esen ed in he panel below. See also Supplemen a y Figu es 1,4. F on ie s in Immunology | www. on ie sin.o g 8No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes o all dono s (Figu e 5C, lowe panel), hese 3 si es we e mo e deme hyla ed in CD34+cells (19.6%) and wo T eg sub ypes: CD45RA-CD15s- (25.9%) and CD45RA−CD15s+(28.2%) while Tcon (49.6%) and CD45RA+CD15s−subse s (43.3%) displayed highe me hyla ion le els (Supplemen a y Table 3). In dono M6, he a e age me hyla ion le els o hese h ee CpG si es we e 10% (CD34+), 16.7% (CD45RA+CD15s−), 1.7% (CD45RA−CD15s−), 43.3% (CD45RA−CD15s+), and 76.7% (Tcon). In e es ingly, mo e CpG si es h oughou he en i e amplicon we e deme hyla ed in CD45RA+CD15s−and CD45RA−CD15s−popula ions o emale dono F3 (Figu e 5B, igh panel), wi h a e age me hyla ion le els o CpGs 1-10 being 33% o CD45RA+CD15s−and 47.5% o CD45RA−15s−as compa ed o 91.5% o Tcon. This egion-wide deme hyla ion was also p esen in CD34+cells o dono s F1 and F2, wi h a e age me hyla ion pe cen ages o CpGs 1-10 being 9 and 11.5%, espec i ely. Compa ed o Tcon, s a is ically signi ican di e ences in me hyla ion alues o all CpGs wi hin FOXP3 p eTSDR we e obse ed o CD45RA−CD15s+(p=0.0087) and CD45RA−CD15s−(p=0.0004) T eg, bu no o CD45RA+CD15s−T eg (p=0.2103). FOXP3 P omo e and TSDR Two egula o y egions in he FOXP3 locus, p omo e and TSDR, ha e been p e iously shown o be deme hyla ed in hymically de i ed nT eg and mos ly me hyla ed in induced T eg (iT eg) and Tcon cells, bo h in mice and humans (7,23–25). Se e al TFs, such as NFAT, AP-1, Foxo1 and 3 bind o he FOXP3 p omo e . Some o hese plus o he TFs, such as CREB, NF-kB, Runx1, STAT5, Ga a3, E s1, and FOXP3 i sel in e ac wi h TSDR, wi h CREB and NFAT, o example, in a deme hyla ion-dependen manne (23,24,26). Me hyla ion analysis o 10 CpG si es wi hin 451 bp egion o FOXP3 p omo e and 15 CpG si es wi hin 700 bp egion co e ing TSDR (Supplemen a y Tables 1,2) om he ou T lymphocy e subse s o dono M1 (Figu es 6B,E, le panels) was in ag eemen wi h p e iously published da a demons a ing almos comple e deme hyla ion o hese egions in T eg sub ypes and a high deg ee o me hyla ion in Tcon cells (Supplemen a y Table 3). Wi h he excep ion o CD45RA+CD15s−subse , which was hea ily me hyla ed wi hin TSDR and could accoun o indi idual di e ences, dono M2 displayed simila me hyla ion pa e n in bo h egions (Figu es 6C,F). A e age me hyla ion le els o CpGs 1-10 wi hin he p omo e egion o dono s M1 and M2 we e 9% (CD45RA+CD15s−), 1.7% (CD45RA−CD15s−), 7.5% (CD45RA−CD15s+) and 58% (Tcon) (Figu e 6B, le lowe panel), while o CpGs 1-15 wi hin TSDR egion hey we e 48.7% (CD45RA+CD15s−), 0.7% (CD45RA−CD15s−), 11.5% (CD45RA−CD15s+), and 85.7% (Tcon) (Figu e 6E, le lowe panel and Supplemen a y Table 3). Howe e , p omo e egion was deme hyla ed (Figu e 6C) and TSDR was me hyla ed (Figu e 6F) in dono s M3-6 and F1-5, wi hou signi ican di e ences be ween cell subse s and gende s. Wi hin he p omo e egion, he a e age me hyla ion le els o CpGs 1- 10 o hese dono s we e below 1% o CD34+cells and T eg sub ypes and sligh ly highe (6.6%) o Tcon (Figu e 6C, lowe panel and Supplemen a y Table 3). Wi hin TSDR, he a e age me hyla ion le els o CpGs 1-15 we e close o 100% in all cell popula ions (Figu e 6F and Supplemen a y Table 3). Sequences om he 700 bp TSDR egion had an equally high le el o non- CpG con e sion, indica ing ha mCs wi hin CpG si es we e in insic o he analyzed egion and we e no he consequence o incomple e con e sion. As expec ed, no signi ican di e ences in me hyla ion wi hin FOXP3 p omo e and TSDR we e obse ed o any o he T eg sub ypes in ela ion o Tcon (p=0.0401 and p=0.1465 o CD45RA+CD15s−,p=0.0438 and p=0.1184 o CD45RA−CD15s−, and p=0.0408 and p=0.1551 o CD45RA−CD15s+T eg, espec i ely o FOXP3 p omo e and TSDR). Hie a chical Clus e ing We applied an unbiased clus e ing me hod o examine he dis ance o cell sub ypes om each o he based on a e age CpG me hyla ion o each genomic elemen (Supplemen a y Figu e 1). Hie a chical clus e ing shows ha me hyla ion pa e ns o p omo e , enhance and TSDR egion o FOXP3 and CAMTA1 in onic egion o Tcon cells a e a hes om all o he T eg sub ypes, while o FOXP3 p eTSDR and FUT7 p omo e me hyla ion pa e ns show clus e ing o Tcon cells wi h CD45RA+CD15s−T eg sub ype. Clus e ing on CpG posi ions o each genomic elemen u he highligh s he posi ions wi h s iking me hyla ion di e ences ac oss he cell sub ypes (Supplemen a y Figu e 1). These CpG posi ions wi h hei a e age me hyla ion obse ed ac oss he ou cell popula ions a e summa ized in Figu e 7. S and-Speci ic Me hyla ion Biases Wi hin FOXP3 TSDR and P omo e P e iously obse ed me hyla ion s a us o FOXP3 p omo e and TSDR o dono s M3-6 and F1-5 was puzzling o se e al easons. Fi s , no changes we e in oduced in e ms o he wo k low (human ac o , eagen s, p o ocols). Second, all six gene egions we e ampli ied om he same ba ch o BS DNA om each cell popula ion. A he same ime, he six gene egions om he i e cell popula ions we e ampli ied om di e en ba ches o DNA (one om each cell popula ion). Thi d, p eTSDR egion was in en ionally in oduced as an addi ional in e nal con ol o bo h TSDR bounda ies and e iciency o BS PCR: he p ime s we e designed in such a way as o p oduce 700 bp amplicons (Supplemen a y Table 1). Fou h, me hyla ion pa e ns o FOXP3 p omo e and TSDR we e simila in hund eds o sequences analyzed (abou 110 sequences pe gene egion om each dono , o e 1,000 sequences pe gene egion in o al). These easons ins illed g ea con idence in ou da a and led us o conside he possibili y ha he obse ed me hyla ion pa e ns we e in insic p ope ies o he wo egions o dono s M3-6 and F1-5. To a oid any bias ela ed o BS ea men and ampli ica ion, a new ba ch o BS- ea ed DNA om dono s M2, F1, and F2 was used o de ec me hyla ion le els wi hin TSDR ampli ied ei he wi h wo op s and-speci ic o bo h op and e e se s and-speci ic p ime se s (Supplemen a y Figu e 2). F on ie s in Immunology | www. on ie sin.o g 9No embe 2018 | Volume 9 | A icle 2540 Minskaia e al. Epigene ics T Cell Sub ypes emale naï e and ac i a ed lymphocy es do no main ain Xi wi h he same ideli y as o he soma ic cells and a e p edisposed o become pa ially eac i a ed and o o e exp ess immuni y- ela ed genes (50,52). The e o e, i is possible ha silencing a he FOXP3 locus is incomple e and leaky Xi may ake place. In addi ion, eigh een X-linked miRNAs we e shown o be o e exp essed in CD4+T cells o women wi h lupus, i e o hose—in expe imen ally deme hyla ed CD4+T cells sugges ing ha deme hyla ion con ibu ed o he escape o Xi. Se e al o he emale biased miRNAs po en ially egula e FOXP3 exp ession wi h some miRNA binding si es loca ed in 3′UTR o FOXP3 ansc ip (53). To conclude, he indings in his s udy sugges ha hypome hyla ed CpG si es, p esen in ou egions o he FOXP3 locus, CAMTA1 and FUT7 gene egions, can po en ially be used o dis inguish subse s o CD4+T lymphocy es in bo h sexes. Wi h he excep ion o FUT7 p omo e , hese CpG si es also de ine CD34+cells as ha ing a “naï e T eg-like epigenome” ha do no , howe e , exp ess FOXP3 p o ein. We also desc ibe p e iously un epo ed s and-bias hemime hyla ion pa e n wi hin he human FOXP3 p omo e and TSDR in some dono s o bo h gende s. The coding s and is deme hyla ed wi hin he p omo e and me hyla ed wi hin he TSDR in all o he CD4+cell sub ypes, whils he empla e s and ollows he “con en ional” me hyla ion pa e n. These da a p o ide new insigh s in o he epigene ic con ol o CD4+T lymphocy es. FOXP3 exp ession and TSDR deme hyla ion a e classically used o con i ma ion o T eg lineage commi men and di e en ia ion s a us, and o p ognos ic pu poses in a ious disease se ings. While he e ec o he desc ibed hemime hyla ion pa e n on T eg unc ion will become he subjec o a new s udy, ou indings sugges ha he s and-speci ic app oach can be ins umen al in disclosing po en ial di e ences be ween subse s o T eg and Tcon. The s and-bias me hyla ion indings challenge cu en simpli ied in e p e a ions o TSDR me hyla ion as a T eg ma ke since op s and-based TSDR-deme hyla ed cells may be e e se-s and TSDR-me hyla ed. In ligh o ou esul s, we p opose ha i is essen ial, o (i) ob ain he TSDR coding e e se-s and me hyla ion da a i s , (ii) u he cla i y he p ecise TSDR me hyla ion pa e n o bo h s ands, and /o (iii) use CAMTA1 o FUT7 le els o me hyla ion as addi ional molecula ma ke s o clea ly dis inguish subse s o T eg om Tcon. Finally, ou indings will di ec ly impac bo h on T eg esea ch and on he clinical applica ion o T eg- ela ed he apies and p ognos ics in he ields o au oimmuni y, alle gy and cance . ETHICS STATEMENT This s udy was ca ied ou in acco dance wi h he ecommenda ions o he Lisbon No h Cen al Hospi als (CHLN) and Lisbon Academic Medical Cen e (CAML). The p o ocol was app o ed by he E hics Commi ee o Hospi al San a Ma ia, Lisbon, Po ugal ( e . 459/13). All subjec s ga e w i en in o med consen in acco dance wi h he Decla a ion o Helsinki. AUTHOR CONTRIBUTIONS EM co-designed he p ojec , designed and pe o med expe imen s, analyzed da a, and w o e he manusc ip . BS p o ided echnical assis ance. MS and RA p o ided assis ance wi h low cy ome y analysis. RR pe o med he s a is ical analysis. SK pe o med hie a chical analysis. AV p o ided echnical assis ance in cell so ing. JL designed he p ojec , discussed p og ess and c i ically ead he manusc ip . FUNDING This esea ch was unded by: Fundação pa a a Ciência e Tecnologia, Po ugal unde he Ha a d Medical School– Po ugal P og am p ojec Induc ion o Immune Tole ance in Human Allogeneic Hema opoie ic S em Cell T ansplan a ion (HMSP-ICT/0001/2011), Gabine e de Apoio à In es igação Cien í ica, Tecnológica e Ino ação, GAPIC (Faculdade de Medicina ULisboa, GAPIC-04-2016 P ojec 20160011), LISBOA- 01-0145-FEDER-007391 (p ojec co- unded by FEDER h ough POR Lisboa 2020—P og ama Ope acional Regional de Lisboa, PORTUGAL 2020 and Fundação pa a a Ciência e a Tecnologia) and ENLIGHT-TEN p ojec (Eu opean Union’s Ho izon 2020 esea ch and inno a ion p og amme unde he Ma ie Sklodowska-Cu ie g an ag eemen 675395). ACKNOWLEDGMENTS We a e g a e ul o P o esso Ma in D. Ryan o Uni e si y o S . And ews o c i ically eading he manusc ip and Sa a Buca o echnical assis ance. SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h ps://www. on ie sin.o g/a icles/10.3389/ immu. 2018.02540/ ull#supplemen a y-ma e ial REFERENCES 1. Miya a M, Amou a Z, Pa izo C, Badoual C, Do gham K, T ad S, e al. Global na u al egula o y T cell deple ion in ac i e sys emic lupus e y hema osus. J Immunol. (2005) 175:8392–400. doi: 10.4049/jimmunol.175.12.8392 2. Sakaguchi S, Yamaguchi T, Nomu a T, Ono M. Regula o y T cells and immune ole ance. Cell (2008) 133:775–87. doi: 10.1016/j.cell.2008.05.009 3. Allan SE, C ome SQ, C ellin NK, Passe ini L, S eine TS, Bacche a R, e al. Ac i a ion-induced FOXP3 in human T e ec o cells does no supp ess p oli e a ion o cy okine p oduc ion. In Immunol. (2007) 19:345– 54. doi: 10.1093/in imm/dxm014 4. 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(2013) 3:1–7. doi: 10.4172/2161-1149.1000114 Con lic o In e es S a emen : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . Copy igh © 2018 Minskaia, Sa ai a, Soa es, Aze edo, Ribei o, Kuma , Viei a and Lace da. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Immunology | www. on ie sin.o g 18 No embe 2018 | Volume 9 | A icle 2540