Arthritis Research & Therapy

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Increased frequency of circulating IL-21 producing Th-cells in patients with granulomatosis with polyangiitis
Arthritis Research & Therapy 2013, 15:R70 doi:10.1186/ar4247

Wayel H Abdulahad (w.abdulahad@umcg.nl) Nikola Lepse (n.lepse@umcg.nl) Coen A Stegeman (c.a.stegeman@umcg.nl) Minke G Huitema (m.g.huitema@umcg.nl) Berber Doornbos-van der Meer (b.doornbos-van.der.meer@umcg.nl) Henko H Tadema (h.tadema@umcg.nl) Abraham Rutgers (a.rutgers@umcg.nl) Pieter C Limburg (p.c.limburg@umcg.nl) Cees GM Kallenberg (c.g.m.kallenberg@umcg.nl) Peter Heeringa (p.heeringa@umcg.nl)

ISSN Article type Submission date Acceptance date Publication date Article URL

1478-6354 Research article 16 January 2013 18 June 2013 24 June 2013 http://arthritis-research.com/content/15/3/R70

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Increased frequency of circulating IL-21 producing Th-cells in patients with granulomatosis with polyangiitis (GPA
Wayel H Abdulahad1, Nikola Lepse2, Coen A Stegeman3, Minke G Huitema1, e!be! "oo!nbos#$an de! Mee!1, Henko %adema1, Ab!aham &utge!s1, 'iete! C Limbu!g(, Cees GM )allenbe!g1, and 'ete! Hee!inga2*
1

"epa!tment o+ &heumatology and Clini,al -mmunology, .ni$e!sity o+ G!oningen, .ni$e!sity Medi,al

Cente! G!oningen, Han/eplein 1, 0113 G2, G!oningen, %he Nethe!lands*

2

"epa!tment o+ 'athology and Medi,al iology, .ni$e!sity o+ G!oningen, .ni$e!sity Medi,al Cente!

G!oningen, Han/eplein 1, 0113 G2, G!oningen, %he Nethe!lands*

3

"epa!tment o+ Neph!ology, .ni$e!sity o+ G!oningen, .ni$e!sity Medi,al Cente! G!oningen,

Han/eplein 1, 0113 G2, G!oningen, %he Nethe!lands*

(

"epa!tment o+ Labo!ato!y Medi,ine, .ni$e!sity o+ G!oningen, .ni$e!sity Medi,al Cente! G!oningen,

Han/eplein 1, 0113 G2, G!oningen, %he Nethe!lands* Wayel H Abdulahad :

w.abdulahad@umcg.nl

Nikola Lepse : n.lepse@umcg.nl Coen A Stegeman : Minke G Huitema :

c.a.stegeman@umcg.nl m.g.huitema@umcg.nl b.doornbos-van.der.meer@umcg.nl

e!be! "oo!nbos#$an de! Mee! : Henko %adema : Ab!aham &utge!s : 'iete! C Limbu!g 3

h.tadema@umcg.nl a.rutgers@umcg.nl

p*,*limbu!g4um,g*nl

Cees GM )allenbe!g 3 ,*g*m*kallenbe!g4um,g*nl 'ete! Hee!inga 3 p*hee!inga4um,g*nl

Co!!esponding autho!3 Wayel H Abdulahad

A!stract
"!#ecti$es% %he p!esent study aimed to e5plo!e a possible !ole +o! -L#21 p!odu,ing %h#,ells in the immunopathogenesis o+ g!anulomatosis 6ith polyangiitis 7G'A8* &ethods% 'e!iphe!al blood +!om (2 G'A#patients in !emission and 20 age#mat,hed healthy ,ont!ols 7HCs8 6e!e stimulated in vitro, and the +!e9uen,ies o+ -L#21 p!odu,ing %h ,ells 6e!e dete!mined by +lo6 ,ytomet!y* Sin,e %h11#,ells p!odu,e a lo6 le$el o+ -L#21, -L#11 6as also in,luded in the analysis* Gi$en that -L#21 is a hallma!k ,ytokine +o! % +olli,ula! helpe! ,ells 7%:H8, 6e ne5t e$aluated the e5p!ession o+ thei! key t!ans,!iption +a,to! ,l#; by &%#'C& and +lo6 ,ytomet!y* %o in$estigate the e++e,t o+ -L#21 on autoantibody#p!odu,tion, ' MCs +!om G'A#patients 6e!e stimulated in vitro 6ith A::<-L#21 and total -gG and ANCA le$els 6e!e measu!ed in supe!natants* -n addition, the e5p!ession o+ -L#21#!e,epto! on #,ells 6as analy/ed* 'esults% 'e!,entages o+ -L#21 p!odu,ing %h#,ells 6e!e signi+i,antly ele$ated in G'A#patients ,ompa!ed to HCs, and 6e!e !est!i,ted to ANCA#positi$e patients* %he e5p!ession o+ ,l#; 6as

signi+i,antly highe! in ANCA#positi$e G'A#patients, as ,ompa!ed 6ith ANCA#negati$e patients and HCs* -L#21 enhan,ed the p!odu,tion o+ -gG and ANCA in vitro in stimulated ' MCs +!om G'A#patients* No di++e!en,e 6as +ound in the e5p!ession o+ the -L#21#!e,epto! on #,ells bet6een ANCA#negati$e, ANCA#positi$e patients, and HCs* (onclusion% %he in,!eased +!e9uen,y o+ ,i!,ulating -L#21 p!odu,ing %h#,ells in ANCA#positi$e G'A# patients and the stimulating ,apa,ity o+ -L#21 on ANCA#p!odu,tion suggest a !ole +o! these ,ells in the immunopathogenesis o+ G'A* lo,kade o+ -L#21 ,ould ,onstitute a ne6 the!apeuti, st!ategy +o! G'A*

)ey *ords% G!anulomatosis 6ith polyangiitis, %h#,ells, -L#21, ANCA*

Introduction
G!anulomatosis 6ith polyangiitis 7G'A8 is an autoimmune $as,ulitis o+ small# to medium#si/ed blood $essels, asso,iated 6ith the p!esen,e o+ ,i!,ulating anti#neut!ophil ,ytoplasmi, autoantibodies 7ANCA8 that a!e mainly di!e,ted against p!oteinase 3=1#3>* Histopathologi,ally, G'A is ,ha!a,te!i/ed by g!anulomatous in+lammation and pau,i#immune $as,ulitis, in,luding ne,!oti/ing ,!es,enti, glome!uloneph!itis* Although the p!odu,tion o+ ANCA is di!e,tly att!ibutable to auto!ea,ti$e #,ells, the!e is e5tensi$e e$iden,e that %#,ells play a ,!iti,al !ole in G'A as 6ell* %he -gG sub,lass dist!ibution o+ ANCA, 6ith a p!eponde!an,e o+ the -gG1 and -gG( sub,lasses, suggests a % ,ell?dependent immune !esponse=(>* -n+ilt!ating %#,ells in g!anulomatous lesions as 6ell as pe!sistent %#,ell a,ti$ation ha$e been obse!$ed in G'A#patients=@, ;>* -n addition, an abe!!ant %#,ell phenotype and impai!ed !egulato!y %#,ell +un,tion a!e also !epo!ted in G'A#patients in !emission=1#0>, suggesting that e$en du!ing !emission, the immune system is dys!egulated* Mo!eo$e!, %#helpe! ,ell pola!i/ation 6ith an in,!ease in %h11 ,ells has been demonst!ated=1A, 11>* %h11 ,ells and thei! ,ytokine -L#11 ha$e been sho6n to play a ,!iti,al !ole in many in+lammato!y diseases* esides -L#11, %h11 ,ells ,an p!odu,e -L#21, a ,ytokine that is la!gely !esponsible +o! di++e!entiation o+ 13>* -t is ,ell ,lass s6it,hing and antibody p!odu,tion, and 6hi,h indu,es

,ells to6a!ds plasma ,ells by syne!gi/ing 6ith #,ell a,ti$ating +a,to! 7 A::8=12, ,on,ei$able that -L#21 may ,ont!ibute to the p!odu,tion o+

the!e+o!e

pathogeni, autoantibodies in G'A* Multiple studies in animal models indi,ate a pi$otal !ole o+ -L#21 in the pathogenesis o+ autoimmune diseases* Studies in a!th!itis models ha$e sho6n that blo,kade o+ the -L#21 a,ti$ity !edu,es Boint in+lammation and dest!u,tion=1(>* Subse9uent in$estigations demonst!ated that blo,king o+ the -L# 21 path6ay !edu,ed le$els o+ anti#ds"NA autoantibodies and p!e$ented !enal disease in mouse models o+ SLC=1@>* -n addition, mi,e de+i,ient in -L#21#!e,epto! e5p!ession 6e!e +ound to be p!ote,ted to a la!ge e5tent against the de$elopment o+ in+lammato!y bo6el disease 7- "8 and type#-

diabetes=1;, 11>* -nte!estingly, !e,ent genome#6ide asso,iation studies ha$e p!o$ided ,on$in,ing e$iden,e that geneti, $a!iants in the !egion on ,h!omosome (921 that ha!bo! the -L#21 and -L#2 genes a!e asso,iated 6ith ,h!oni, in+lammato!y diso!de!s, in,luding SLC, - " and pso!iasis=1D#2A>* %hus, -L#21 seems to play an impo!tant !ole in autoimmune diseases in gene!al and ,ould ,onstitute a no$el ta!get +o! the!apy* -L#21 is p!odu,ed mainly by a,ti$ated C"(E %h#,ells* &e,ent studies ha$e demonst!ated that -L#21, besides its p!odu,tion by %h11 ,ells, is p!edominantly se,!eted by a distin,t %h ,ell lineage, te!med +olli,ula! helpe! %#,ells 7%:H8 that e5p!ess the t!ans,!iption +a,to! ,l#; and a!e ,onside!ed to be spe,iali/ed p!o$ide!s o+ ,ell help=21>* C5pansion o+ ,i!,ulating % ,ells !esembling %:H ,ells has been

!epo!ted in patients 6ith SLC and in patients 6ith !heumatoid a!th!itis=22#2(>* %o date, no study has in$estigated the !ole o+ -L#21 p!odu,ing %h#,ells in G'A* %he!e+o!e, this study aimed to assess the +!e9uen,y o+ -L#21 p!odu,ing %h#,ells, and to e$aluate 6hethe! %:H ,ells o! %h11 ,ells a!e the maBo! sou!,e o+ -L#21 in G'A#patients* :o! this pu!pose, 6e e5amined the e5p!ession o+ both -L#21 and -L#11 in ,i!,ulating C"(E %#,ells o+ patients 6ith G'A* %o imp!o$e ou! unde!standing o+ the !ole o+ -L#21 p!odu,ing %h ,ells in autoantibody p!odu,tion 6e assessed thei! +!e9uen,ies in ANCA#positi$e and ANCA#negati$e patients, and studied e++e,ts o+ -L#21 on immunoglobulin and ANCA p!odu,tion in vitro*

&ethods

+tudy population, :o!ty#t6o patients 6ith G'A and 20 age# and se5#mat,hed healthy ,ont!ols 71D males, 11 +emales, mean age @; =S" F13> yea!s, !ange 2;#12 yea!s, PG A*1;8 6e!e in,luded in this study* %he diagnosis o+ G'A 6as established a,,o!ding to the de+initions o+ the Chapel Hill Consensus Con+e!en,e and patients +ul+illed the ,lassi+i,ation ,!ite!ia o+ the Ame!i,an College o+ &heumatology 7AC&8=2@, 2;>* Hnly patients 6ithout ,lini,al signs and symptoms o+ a,ti$e $as,ulitis and ,onside!ed to be in ,omplete !emission, as indi,ated by a s,o!e o+ /e!o on the i!mingham Ias,ulitis A,ti$ity S,o!e 7 IAS8, 6e!e in,luded in the study=21>* Se!ostatus +o! ANCA 6as +ollo6ed +o! se$e!al months in all patients, and patients 6ith a stable se!ostatus +o! ANCA 7positi$e o! negati$e8 +o! at least 3 months 6e!e in,luded in this study* ased on these ,!ite!ia, 23 patients 6e!e positi$e +o! '&3#ANCA, 6he!eas 10 6e!e ANCA#negati$e* %6enty#se$en patients 6e!e ,onside!ed to ha$e a histo!y o+ generalized disease in,luding !enal in$ol$ement, and 1@ patients localized disease, in 6hi,h the disease had been ,on+ined to the uppe! and<o! lo6e! !espi!ato!y t!a,t* None o+ the patients and ,ont!ols e5pe!ien,ed an in+e,tion at the time o+ sampling* Cle$en G'A#patients 7D ANCA#positi$e, and 3 ANCA#negati$e8 6e!e t!eated 6ith maintenan,e immunosupp!essi$e the!apy at the time o+ blood sampling* :ou! o+ them 6e!e t!eated 6ith only a/athiop!ine 72@#1AA mg<day8, one patient 6ith my,ophenolate mo+etil 71@AA mg<day8, and ; patients !e,ei$ed p!ednisolone 7@#1A mg<day8 in ,ombination 6ith a/athiop!ine 712@ mg<day8* 'a!ti,ipants in !itu5imab t!ials 6e!e e5,luded +!om the p!esent study* %he main ,lini,al and labo!ato!y data o+ the patients a!e summa!i/ed in %able 1* All patients and healthy indi$iduals p!o$ided in+o!med ,onsent and the study 6as app!o$ed by the lo,al Medi,al Cthi,s Committee o+ the .ni$e!sity Medi,al Cent!e G!oningen, .ni$e!sity o+ G!oningen 7NL8*

&easurement of A-(A titres and specificity, Anti#neut!ophil ,ytoplasmi, antibody 7ANCA8 tite!s 6e!e measu!ed by indi!e,t immuno+luo!es,en,e 7--:8 on ethanol#+i5ed human g!anulo,ytes a,,o!ding to standa!d p!o,edu!es as p!e$iously des,!ibed=2D>* ANCA tite!s highe! than 13(A 6e!e ,onside!ed positi$e* ANCA antigeni, spe,i+i,ity 6as dete!mined using an in#house ,aptu!e en/yme#linked immunoso!bent assay 7CL-SA8 as des,!ibed be+o!e=20, 3A>* !ie+ly, a 0;#6ell plate 6as ,oated 6ith goat#anti#mouse -g +o! (D hou!s* A+te! 6ashing, plates 6e!e in,ubated 6ith mouse mono,lonal antibody against human '&3 +o! 2 hou!s* A+te! 6ashing, the plate 6as in,ubated o$e!night at ( JC 6ith an e5t!a,t o+ human a/u!ophili, g!anules 6hi,h 6e!e isolated +!om neut!ophils o+ healthy dono!s* :u!the!, se!ial dilutions o+ se!um 76ith a sta!ting dilution o+ 131AA8 6e!e in,ubated +o! 1 hou!* %he plate 6as 6ashed, and the ,aptu!ed antibodies 6e!e dete,ted 6ith pu!i+ied :7ab82 goat# anti#human -gG ,onBugated to alkaline phosphatase* '#nit!ophenyl#phosphate disodium 6as used as a subst!ate, and the opti,al density 6as measu!ed at (A@ nm*

Anti!odies used in flow cytometry, %he +ollo6ing ,onBugated antibodies 6e!e used in +lo6 ,ytomet!y3 Allophy,o,yanin 7A'C8#,onBugated anti#C"3 7,lone .CH%18, pe!idin ,hlo!ophyll p!otein 7'e!C'8#,onBugated anti#C"D 7,lone S)18, 'hy,oe!yth!in 7'C8#,onBugated anti#-L#21#!e,epto! 7,lone 11A128, and 'e!C'#,onBugated anti#C"10 7,lone (G18, all pu!,hased +!om e,ton K "i,kinson

7Amste!dam, %he Nethe!lands8L 'C#,onBugated anti#-L#21 7,lone e io3A3#N28, Ale5a :luo!M (DD 7A(DD8#,onBugated anti#-L#11 7,lone e io;("CC118, and A(DD#,onBugated anti#:o5'3 7,lone 'CH1A18, all pu!,hased +!om e ios,ien,e 7San "iego, CA, .SA8L 'C#,onBugated anti# CL; 7,lone -C@A(;'8 6as obtained +!om &K" Systems 7Minneapolis, MN8*

+ample preparation and in vitro stimulation, Lithium hepa!ini/ed $enous blood 6as obtained +!om patients and healthy dono!s* -mmediately a+te! sampling, (AAL blood 6as mi5ed 6ith (AAL &'M-1;(A 7Camb!e5 io S,ien,e, Ie!$ie!s, elgium8, supplemented 6ith @ANg<ml gentamy,in 7Gib,o,

S,otland, .)8, and ali9uoted in t6o @mL polyp!opylene tubes 7 " ios,ien,es8 7(AAL pe! tube8* "iluted blood samples 6e!e stimulated ( hou!s 6ith @Ang<mL pho!bol my!istate a,etate 7'MAL Sigma#Ald!i,h, Steinheim, Ge!many8 and 2nM ,al,ium ionopho!e 7Ca#-oL Sigma#Ald!i,h8* As a negati$e ,ont!ol, one sample 6as kept in medium only 6ithout stimulation* :o! inhibiting ,ytokine !elease +!om the ,ells, 1AOg<ml o+ b!e+eldin A 7Sigma#Ald!i,h8 6as added to ea,h sample*

Intracellular .A(+-staining for cyto/ines, A+te! stimulation, ,ells 6e!e 6ashed in 6ash bu++e! =' S, @P :etal o$ine Se!um 7: S8, A*1P sodium a/ide 7Me!k, Ge!many8> and stained 6ith 'e!C'#

,onBugated anti#C"D and A'C#,onBugated anti#C"3 +o! 1@ minutes at !oom tempe!atu!e* Cells 6e!e +i5ed 6ith 1AANL Reagent A 7Caltag<-n$it!ogen*, !eda, %he Nethe!lands8 +o! 1A minutes* A+te!

6ashing, the pellet 6as !esuspended in 1AANL pe!meabili/ation Reagent B 7Caltag<-n$it!ogen8 and labeled 6ith A(DD#,onBugated anti#-L#11 and 'C#,onBugated anti#-L#21 +o! 2A minutes in the da!k* A+te! staining, the ,ells 6e!e 6ashed and immediately analy/ed on :ACS#Calibu! +lo6 ,ytomete! 7 e,ton K "i,kinson8* "ata 6e!e ,olle,ted +o! 2 5 1A@ ,ells, and plotted using the Win#List so+t6a!e pa,kage 7Ie!ity So+t6a!e House -n,, MC, .SA8* e,ause stimulation !edu,es su!+a,e e5p!ession o+ C"( on %#,ells, C"(E%#,ells 6e!e identi+ied indi!e,tly by gating on C"3#positi$e and C"D#negati$e lympho,ytes* Gated C"(E %#,ells 6e!e +u!the! displayed as dot plot +o! e$aluation o+ int!a,ellula! ,ytokine p!odu,tion* %he unstimulated ,ont!ol sample 6as used as a guide +o! setting the linea! gates to dis,!iminate positi$e and negati$e populations*

Intracellular staining for transcription factors, 'e!iphe!al blood mononu,lea! ,ells 7' MCs8 +!om G'A#patients and HCs 6e!e p!epa!ed +!om hepa!ini/ed $enous blood by density#g!adient ,ent!i+ugation on Lymphop!ep 7A5is#Shield 'oC AS, Hslo, No!6ay8* Cells !e,o$e!ed +!om the g!adient inte!+a,e 6e!e 6ashed t6i,e in ' S and stained +o!

CL; and :o5'3 a,,o!ding to the manu+a,tu!e!Qs inst!u,tions 7e ios,ien,e staining set +o! t!ans,!iption +a,to!s8* !ie+ly, ' MCs 6e!e adBusted to 1 5 1A; ,ells in 1AAL and in,ubated 6ith app!op!iate ,on,ent!ation o+ A'C#,onBugated anti#C"3 and 'e!C'#,onBugated anti#C"D +o! 3A minutes at ( oC in the da!k, +ollo6ed by +i5ation and pe!meabili/aion in :i5<'e!m bu++e! 7e ios,ien,e8 +o! (@ minutes* Cells 6e!e then 6ashed t6i,e 6ith 1R pe!meabili/ation bu++e! 7e ios,ien,e8, and stained 6ith 'C#,onBugated anti# CL; and A(DD#,onBugated anti#:o5'3* A+te! in,ubation +o! 3A minutes in the da!k, the ,ell suspension 6as 6ashed and immediately analy/ed on :ACS#Calibu! +lo6 ,ytomete! 7 e,ton K "i,kinson8* Lympho,ytes 6e!e gated by +o!6a!d and side s,atte! patte!ns, and plotted using the Win#List so+t6a!e pa,kage 7Ie!ity So+t6a!e House -n,, MC, .SA8* -sotype mat,hed ,ont!ol antibodies o+ i!!ele$ant spe,i+i,ity 6e!e obtained +!om e ios,ien,e and &K" systems*

Immunofluorescent surface staining for IL-21' on 0-cells, :!esh blood samples +!om G'A#patients and HCs 6e!e labeled 6ith 'C#,onBugated anti#-L21&, and 'e!C'#,onBugated anti#C"10 +o! 1@ minutes in the da!k* Cells 6e!e su,,essi$ely t!eated 6ith 2 ml diluted :ACS lysing solution 7 ", Amste!dam, %he Nethe!lands8 +o! 1A minutes and then 6ashed t6i,e in 6ash#bu++e! and immediately analy/ed by +lo6 ,ytomet!y*

'-A isolation and 'eal-Time 'T-P(', C!yth!o,ytes 6e!e lysed and leuko,ytes 6e!e +i5ed and 6ashed t6i,e in 1P SA* &NA 6as isolated +!om total leuko,ytes 6ith %&-/ol !eagent 7-n$it!ogen8 a,,o!ding to the manu+a,tu!e!Qs inst!u,tions* "NAse t!eatment 7Ambion, Huntingdon, Camb!idgeshi!e, .)8 6as pe!+o!med and subse9uently ,"NA 6as synthesi/ed using M#MLI !e$e!se t!ans,!iptase and oligo 7d%8 1( to 1D as p!ime!* :o! measu!ement o+ m&NA +o! CL; and gly,e!aldehyde#3#phosphate dehyd!ogenase 7GA'"H8, 1 Ol o+ ,"NA in t!ipli,ate 6as used +o! ampli+i,ation by the %a9man &%#'C& system 7A - '!ism 10AAH%

Se9uen,e "ete,tion System, Applied

iosystems, :oste! City, CA, .SA8 6ith spe,i+i, %a9man iosystems8*

p!ime!s<p!obes 7 ,L#; =Hs AA1@33;DSm1> and GA'"H =Hs 000000A@Sm1>, Applied

Ampli+i,ation 6as pe!+o!med using standa!d ,onditions and ,al,ulations o+ +old indu,tion 6e!e pe!+o!med* We no!mali/ed gene e5p!ession to GA'"H and e5p!essed $alues !elati$e to ,ont!ol using the C% method*

(ell stimulation and total IgG production, ' MCs !e,o$e!ed +!om the g!adient inte!+a,e 6e!e 6ashed t6i,e in ' S and adBusted to 1A; ,ells<mL in &'M- 1;(A 7Lon/a, S6it/e!land8 supplemented 6ith 1AP +etal ,al+ se!um 7Lon/a, S6it/e!land8 and @A Og<mL gentami,in 7G- CH, -n$it!ogen8* Cells 6e!e ,ultu!ed in the p!esen,e o+ 1AA ng<mL !h-L#21 7-mmuno%ools GmbH, Ge!many8 and<o! 1AA ng<mL !h A:: 7'ep!o%e,h, .SA8 +o! 12 days at 31 TC 6ith @P CH2* A+te! 12 days, ,ultu!e supe!natants 6e!e ,olle,ted and total -gG 6as measu!ed using an in#house CL-SA as des,!ibed p!e$iously=31>* !ie+ly, Costa! 0;#6ell CL-SA plates 6e!e ,oated 6ith 2 mg<mL goat anti#human#-g antibody 7Southe!n iote,h, i!mingham, AL, .SA8 in ,a!bonate bu++e! 7A*A1M, pH 0*;8* 'lates 6e!e 6ashed 6ith 6ashing bu++e! 7A*A2@M %!is#HCl, A*1@M NaCl, A*A@P %6een#2A8 and blo,ked +o! 1 h 6ith blo,king<in,ubation#bu++e! 7A*A@M %!is#HCl, A*3M NaCl, A*A@P %6een#2A, 1P SA8* Cell ,ultu!e supe!natants 6e!e diluted in in,ubation bu++e!* 'u!i+ied human -gG 6ith a kno6n ,on,ent!ation 6as used as a standa!d sample* %he bound -gG 6as dete,ted 6ith goat# anti#human#-gG antibody ,onBugated 6ith alkaline phosphatase 7Sigma, St Louis, MH, .SA8* '# nit!ophenyl#phosphate disodium 6as used as subst!ate and opti,al density 6as !ead at (A@ nm using an Cma5 mi,!oplate !eade! 7Mole,ula! "e$i,es, Sili,on Ialley, CA, .SA8*

&easurement of in vitro production of P'1-A-(A In vitro '&3#ANCA -gG p!odu,tion in ' MC ,ultu!e supe!natants 6as measu!ed by 'hadia -mmunoCA'M 2@A analy/e! 7%he!mo :ishe! S,ienti+i,8 using CLiA%M '&3, and the le$els o+ '&3#ANCA -gG p!odu,tion 6e!e e5p!essed in !esponse units 7&.8*

+tatistical analysis, "ata a!e p!esented as median $alues, unless stated othe!6ise* %he nonpa!amet!i, Mann#Whitney . test 6as used to ,ompa!e data +!om patients 6ith that o+ healthy ,ont!ols* %he Wil,o5on mat,hed pai!s test 6as used +o! int!aindi$idual ,ompa!ison* Co!!elations 6e!e assessed using Spea!manQs !ank ,o!!elation ,oe++i,ient* %6o#tailed P#$alues less than A*A@ 6e!e ,onside!ed statisti,ally signi+i,ant*

10

'esults

Increased percentage of circulating IL-212IL-13- cells in A-(A-positi$e GPA-patients compared to A-(A-negati$e patients and healthy controls We initially dete!mined the +!e9uen,y o+ -L#21 p!odu,ing C"( %#,ells in the pe!iphe!al blood o+ G'A# patients 7nG (28 and HCs 7nG 208 a+te! in vitro stimulation* %he pe!,entage o+ ,i!,ulating -L#21E %h# ,ells 6as signi+i,antly highe! in G'A#patients as ,ompa!ed 6ith the ,ont!ol g!oup 7:igu!e 1 8* H+ note, %h11 ,ells may p!odu,e -L#21 in addition to thei! signatu!e ,ytokine -L#11* Sin,e %h11 ,ells a!e in,!eased in G'A#patients=1A, 11>, 6e ne5t e5tended ou! analysis to in$estigate 6hethe! in,!eased -L# 21E %h#,ells in G'A#patients !esulted +!om an in,!ease in %h11 ,ells* %o this end, -L#11 staining 6as in,luded in the analysis to dete!mine 6hat pe!,entage o+ the total -L#21E %h#,ells a!e %h11 ,ells* .sing this app!oa,h, 6e assessed the +!e9uen,y o+ -L#21E-L#11#, -L#21E-L#11E, and -L#21#-L#11E ,ells 6ithin the C"( %#,ells in G'A#patients and HCs* As sho6n in +igu!e 1 7C, " and C8, G'A#patients in !emission had a signi+i,antly highe! pe!,entages o+ ,i!,ulating -L#21E-L#11#, -L#21E-L#11E, and -L#21#-L# 11E ,ells as ,ompa!ed 6ith the ,ont!ol g!oup* Ho6e$e!, the maBo!ity o+ ,i!,ulating C"(E %#,ells that p!odu,ed -L#21 only, 6e!e distin,t +!om %h11 ,ells, that is negati$e +o! -L#11* %o assess the possible !ole o+ -L#21E-L#11# %h#,ells in ANCA p!odu,tion, 6e ,ompa!ed thei! pe!,entage bet6een patients that 6e!e ANCA#positi$e 7nG 23L --: tite! U13(A8 o! ANCA#negati$e 7n G 108 at the time o+ in,lusion* Signi+i,ant in,!eases in the +!e9uen,ies o+ -L#21E-L#11# %h#,ells 6e!e obse!$ed in ANCA#positi$e patients in ,ompa!ison 6ith HCs and ANCA#negati$e patients, 6he!eas no signi+i,ant di++e!en,e 6as +ound bet6een ANCA#negati$e patients and HCs 7:igu!e 1:8* -n ,ont!ast, the pe!,entages o+ -L#21E-L# 11E and -L#21#-L#11E %h#,ells in ANCA#positi$e G'A#patients did not di++e! +!om those in ANCA# negati$e G'A#patients 7:igu!e 1G and H8* %hese !esults suggest that pe!sisten,e o+ -L#21E-L#11# %h# ,ells du!ing !emission plays a !ole in the ongoing humo!al autoimmune !esponse in ANCA#positi$e G'A#patients*

11

%o !ule out the possibility that the in,!eased p!opo!tion o+ -L#21E-L#11# %h#,ells in G'A#patients 6as the !esult o+ ,u!!ent t!eatment, the ANCA#positi$e patient g!oup 6as di$ided into t!eated and unt!eated patients, and the pe!,entages o+ -L#21E-L#11# %h#,ells 6e!e ,ompa!ed* No signi+i,ant di++e!en,es 6e!e obse!$ed bet6een t!eated and unt!eated patients 7data not sho6n8* We also ,ompa!ed the pe!,entage o+ -L#21E-L#11# %h#,ells bet6een ,u!!ently unt!eated ANCA#positi$e patients 6ith a histo!y o+ gene!ali/ed and lo,ali/ed disease* No di++e!en,e 6as +ound bet6een these patient g!oups 7data not sho6n8*

Increased frequencies of IL-212IL-13- Th-cells correlate positi$ely with Th13 response, -t has been !epo!ted that -L#21 is a key +a,to! !egulating the di++e!entiation o+ naV$e C"(E %#,ells into %h11 ,ells=32, 33>* -n o!de! to analy/e this !elation, 6e ,o!!elated pe!,entages o+ -L#21E-L#11# %h#,ells 6ith pe!,entages o+ te!minally di++e!entiated %h11 ,ells 7-L#21#-L#11E8 in G'A#patients 7nG (28 and HCs 7nG 208* -nte!estingly, a signi+i,ant positi$e ,o!!elation 6as obse!$ed bet6een -L#21E-L#11# %h#,ells and -L#21#-L#11E %h#,ells in both G'A#patients and HCs 7!G A*@D, PW A*AAA1 and !G A*31, PG A*A(, !espe,ti$ely8 7:igu!e 2A and 8*

Increased frequencies of 0(L-42 (562 T-cells in peripheral !lood of A-(A-positi$e GPA-patients Sin,e -L#21 is not the only ma!ke! +o! %:H ,ells, 6e +u!the! ,ha!a,te!i/ed the identity o+ ,i!,ulating -L# 21 p!odu,ing ,ells by analy/ing CL; e5p!ession, 6hi,h is ,onside!ed a maste! !egulato! and spe,i+i, t!ans,!iption +a,to! +o! %:H ,ells=3(, 3@>* %o this end, the e5p!ession o+ m&NA CL#; 6e!e assessed in ,i!,ulating leuko,ytes +!om G'A#patients and HCs by !eal#time &%#'C&* &est!i,ted numbe!s o+ patients and ,ont!ols 6e!e in,luded in this analysis due to insu++i,ient ,ell numbe!s* 'atients 6ith

12

ANCA#positi$e G'A 7nG 1A8 had a signi+i,antly highe! e5p!ession o+ m&NA CL#; than ANCA#negati$e patients 7nG ;8 and HCs 7nG 118 7:igu!e 3 8* -n addition, int!a,ellula! :ACS#staining +o! CL; 6ithin ,i!,ulating C"(E % ,ells ,on+i!med the in,!eased CL#; e5p!ession in ANCA#positi$e G'A#patients 7:igu!e 3A and C8* We ha$e also analy/ed the M:- 7mean +luo!es,en,e intensity8 o+ CL#; e5p!ession in C"(E %#,ells +!om patients and HCs and +ound that the e5p!ession le$el o+ CL#; pe! %h#,ell in G'A# patients 6as simila! to that in HCs 7data not sho6n8* %hus, CL#; e5p!ession is in,!eased in G'A# patients due to in,!eased +!e9uen,ies o+ ,i!,ulating CL#;E C"(E %#,ells* Sin,e in !e,ent studies a ne6 population o+ :o5'3E !egulato!y %#,ells has been des,!ibed that sha!es +eatu!es 6ith %:H ,ell by e5p!essing the t!ans,!iption +a,to! ,l#;, 6e also e$aluated 6hethe! the in,!ease in CL#;E %#,ells in G'A#patients 6as a !esult o+ an in,!ease in :o5'3E CL#;E %#,ells=3;, 31>* %his analysis sho6ed that the in,!ease in CL#; e5p!ession in G'A patients 6as !est!i,ted to %:H ,ells and although a lo6 pe!,entage o+ :o5'3E CL#;E %#,ells 6as +ound 7W A*3P8, no di++e!en,es in these ,ell +!e9uen,ies 6e!e obse!$ed bet6een G'A#patients and HCs 7data not sho6n8*

Proportions of IL-21-receptor e7pressing 0-cells do not differ !etween GPA-patients and 8( Sin,e it is 6ell kno6n that -L#21 a,ts on #,ells to suppo!t thei! e5pansion and antibody ,ells

p!odu,tion=3D#(A>, 6e ,ondu,ted +u!the! analysis to ,ompa!e the e5p!ession o+ -L#21& on

+!om G'A#patients and HCs* No di++e!en,es 6e!e seen in the pe!,entages o+ -L#21&E #,ells neithe! bet6een ANCA#positi$e patients 7nG 138 and ANCA#negati$e patients 7nG 1(8 no! bet6een patients and HCs 7nG 108 7:igu!e (8*

13

IL-21 induces IgG and A-(A production !y 0-cells from GPA-patients %o e5plo!e the inte!play bet6een -L#21 p!odu,ing %h#,ells and #,ells in G'A#patients, 6e

in$estigated the e++e,t o+ -L#21 on -gG antibody#p!odu,tion by #,ells +!om G'A#patients* &est!i,ted numbe!s o+ patients and ,ont!ols 6e!e en!olled in this analysis due to insu++i,ient ,ell numbe!s* ' MCs +!om G'A#patients 6e!e ,ultu!ed in vitro in the p!esen,e o! absen,e o+ e5ogenous -L#21 +o! 12 days and total -gG 6as measu!ed in supe!natants by CL-SA* Sin,e -L#21 p!omotes #,ell

di++e!entiation by syne!gi/ing 6ith A::=12, 13>, 6e 9uestioned 6hethe! the e++e,t o+ -L#21 on -gG p!odu,tion ,ould be augmented by adding A:: to the ,ultu!e* H+ note, autologous % ,ells in ou! ,ultu!e system a,t as a natu!al p!o$ide! o+ C"(A ligation +o! #,ells, as this ligation is !e9ui!ed +o! ,ell a,ti$ation, isotype s6it,hing and memo!y de$elopment* As sho6n in +igu!e @, -L#21 signi+i,antly enhan,ed the p!odu,tion o+ -gG in vitro in stimulated ' MCs +!om both ANCA#positi$e 7nG 18 and ANCA#negati$e 7nG ;8 G'A#patients, 6hile stimulation 6ith A:: alone did not !esult in in,!eased -gG p!odu,tion* %he ,ombination o+ A:: and -L#21 tended to in,!ease -gG p!odu,tion mo!e than -L#21 alone* Ne5t, 6e assessed the e++e,t o+ -L#21 plus A:: on in vitro p!odu,tion o+ '&3#ANCA* As sho6n in +igu!e @ , spontaneous '&3#ANCA p!odu,tion 6as obse!$ed in ,ultu!ed ' MCs +!om ANCA# positi$e patients 7nG 1;8 in ,ompa!ison 6ith ,ells +!om ANCA#negati$e patients 7nG 128* -mpo!tantly, -L#21 indu,es a signi+i,ant enhan,ement in '&3#ANCA p!odu,tion in ' MCs isolated +!om ANCA# positi$e patients in ,ompa!ison 6ith ANCA#negati$e patients* So it is ,on,ei$able that auto!ea,ti$e # ,ells 6e!e en!i,hed in the pe!iphe!al blood o+ ANCA#positi$e patients*

14

5iscussion
-n the p!esent study, 6e demonst!ate an in,!ease in the pe!,entage o+ ,i!,ulating -L#21 p!odu,ing %h#,ells in G'A#patients* We +ound that ele$ated +!e9uen,ies o+ -L#21 p!odu,ing %h#,ells 6e!e !est!i,ted to ANCA#positi$e G'A patients and that these ,ells 6e!e distin,t +!om %h11 ,ells* We also ,on+i!med that -L#21 ,an enhan,e the p!odu,tion o+ -gG and ANCA in vitro* H$e! the past +e6 yea!s, %h11 ,ells ha$e ,hallenged the ,lassi,al %h1<%h2 pa!adigm, and ha$e been impli,ated in a g!o6ing numbe! o+ autoimmune and in+lammato!y diseases=(1>* &e,ently, a distin,t %h#,ell subset te!med %:H and ,ha!a,te!i/ed by ele$ated e5p!ession le$els o+ multiple su!+a,e p!oteins and ,l#; as 6ell as enhan,ed -L#21 se,!etion, has been identi+ied as t!ue helpe! ,ells +o! antibody !esponses* We and othe!s ha$e p!e$iously demonst!ated that ,i!,ulating %h11 ,ells a!e signi+i,antly in,!eased in G'A#patients e$en du!ing 9uies,ent disease=1A, 11>* Ho6e$e!, data a!e la,king to suppo!t a !ole o+ -L#21#p!odu,ing %h#,ells in G'A* Sin,e %h11 ,ells also p!odu,e -L#21, 6e in$estigated 6hethe! %h11 ,ells in G'A a!e a sou!,e o+ -L#21* St!ikingly, the maBo!ity o+ ,i!,ulating C"( %#,ells that p!odu,ed -L#21 6e!e distin,t +!om %h11 ,ells, indi,ating that othe! %h#,ell subsets su,h as %:H ,ells a!e the sou!,e o+ this ,ytokine* -mpo!tantly, the e5pansion o+ %:H ,ells in G'A patients 6as ,on+i!med by in,!eased ,L; e5p!ession* %o the best o+ ou! kno6ledge, this is the +i!st !epo!t demonst!ating an in,!ease in the +!e9uen,y o+ ,i!,ulating -L#21 p!odu,ing %h#,ells in G'A suggesting that %:H ,ell#de!i$ed -L#21 may ,ont!ibute to disease pathogenesis $ia stimulation o+ 7auto8antibody p!odu,tion* %:H ,ells a!e ,onside!ed to be the maBo! sou!,e o+ -L#21 and seem to be an impo!tant subset +o! adapti$e immune !esponses, although the!e a!e ,on+li,ting !epo!ts on thei! mode o+ a,tion in vivo* -t has been demonst!ated that -L#21 p!odu,ing %h#,ells indu,e %h11 de$elopment and p!oli+e!ation=32, 33>, 6hi,h has been sho6n to p!omote ge!minal ,ente! 7GC8 +o!mation in a R"2 mouse model o+ autoimmunity=(2>* -n ag!eement 6ith these +indings, 6e demonst!ate a signi+i,ant positi$e !elationship bet6een -L#11E-L#21# %h#,ells and -L#11#-L#21E %h#,ells in pe!iphe!al blood o+ G'A#

15

patients* -t seems likely that in,!eased %h11 ,ells in G'A#patients a!e the !esult o+ an enhan,ed %:H !esponse, 6hi,h in tu!n may pa!ti,ipate in g!anuloma +o!mation and $as,ula! damage* %he !ole o+ -L# 21 in $as,ulitis 6as p!e$iously suggested by Chen and ,o6o!ke!s=(3>* -n thei! study, mi,e de+i,ient in inte!+e!on !egulato!y +a,to!#(, a p!otein that inhibits -L#11A p!odu,tion, !apidly de$eloped la!ge# $essel $as,ulitis and sho6ed in,!eased -L#21 synthesis in addition to in,!eased -L#11A p!odu,tion=(3>* Mo!eo$e!, a !ole o+ -L#21 in !e,!uitment o+ %h11 ,ells to in+lamed tissues has been !epo!ted by Ca!uso and ,o6o!ke!s=((> by sho6ing that -L#21 indu,es gut epithelial ,ells to se,!ete ma,!ophage in+lammato!y p!otein#3X 7M-'#3X8, a ,hemokine that mediates %h11 ,ell homing to the skin, Boints, and mu,osal tissues* Gi$en that endothelial ,ells a!e kno6n to p!odu,e M-'#3X, it is possible that -L# 21 in G'A#patients enhan,es the mig!ation and a,,umulation o+ %h11 ,ells into the $as,ula! 6all !esulting in in+lammation* esides, -L#21 6as sho6n to enhan,e g!an/yme e5p!ession=(@> and

in,!ease pe!+o!in#mediated ,ytoto5i,ity by human C"D %#,ells=(;> and N) ,ells=(1>* -t is the!e+o!e ,on,ei$able that -L#21 ,an ,ont!ibute to $essel inBu!y and disease p!og!ession in G'A#patients* %his is an a!ea 6o!th o+ +u!the! in$estigation* -n ,ont!ast to the p!o#in+lammato!y !ole o+ %:H ,ells, !e,ent studies ha$e identi+ied a distin,ti$e population o+ %:H ,ells that displays a !egulato!y +un,tion and supp!esses the di++e!entiation o+ GC #,ells in +olli,les in vivo* %his subset 6as te!med +olli,ula! !egulato!y % ,ells 7%:&8, 6hi,h e5p!ess the !egulato!y t!ans,!iption +a,to! FoxP3 in addition to thei! spe,i+i, lineage t!ans,!iption +a,to! CL#;=3;, 31>* As ,i!,ulating FoxP3E %#,ells a!e in,!eased in G'A#patients=1>, it is ,on,ei$able that the obse!$ed in,!ease in %:H ,ells in patients is due to an in,!ease in %:& ,ells that ,o# e5p!ess FoxP3 and CL;* We ha$e in$estigated this possibility but +ound that the in,!ease in

,i!,ulating %:H ,ells in G'A#patients ,annot be e5plained by in,!ease in %:& ,ells 7data not sho6n8* -n ou! study, in,!eased +!e9uen,ies o+ %:H ,ells 6e!e obse!$ed in patients 6ho 6e!e ANCA positi$e at the time o+ in,lusion* %his suggests the in$ol$ement o+ -L#21 in the p!o,ess o+ autoantibody p!odu,tion in G'A* %hese data a!e in line 6ith p!e$ious !epo!ts sho6ing that %:H ,ells

16

a,t di!e,tly on #,ells th!ough the -L#21<-L#21& path6ay, and that -L#21 is a potent indu,e! o+ ,lass# s6it,h !e,ombination and plasma ,ell di++e!entiation=30, (D, (0>* %he e5p!ession o+ -L#21& on #,ells +!om ANCA#positi$e and ANCA#negati$e G'A#patients 6as ,ompa!able, 6hi,h suggests that both patient populations ha$e the same ability to !espond to -L#21* Ho6e$e!, in vitro stimulation 6ith -L# 21 enhan,ed the p!odu,tion o+ ANCA in ,ell ,ultu!es +!om ANCA#positi$e patients only, although enhan,ed total -gG#p!odu,tion 6as obse!$ed in both patient g!oups* So it is ,on,ei$able that auto!ea,ti$e #,ells 6e!e en!i,hed in the pe!iphe!al blood o+ ANCA#positi$e patients* %his might be ,lini,ally !ele$ant as 6ell sin,e ANCA positi$e patients a!e at in,!eased !isk +o! disease !elapse=@A, @1>* -n this study, patients 6e!e e$aluated +o! the dist!ibution o+ %:H ,ells du!ing !emission* We ha$e p!e$iously sho6n that a,ti$ated %#,ells a!e p!esent at the time o+ ,lini,ally 9uies,ent disease=0, 1A>* :u!the!mo!e, du!ing a,ti$e disease e++e,to! %#,ells appea! to mig!ate to6a!ds in+lamed tissue=@2>* %he!e+o!e, in o!de! to study dysbalan,e o+ %#,ells in G'A#patients using pe!iphe!al blood samples, 6e ,hoose to sele,t patients 6ithout o! 6ith lo6 dosages o+ immunosupp!essi$e medi,ation and at the time o+ ,lini,ally 9uies,ent disease*

(onclusions
-n ,on,lusion, the data p!esented he!e demonst!ate a p!ominent in,!ease o+ ,i!,ulating %:H ,ells in ANCA#positi$e G'A#patients* %he key ,ytokine o+ these %:H ,ells, that is -L#21, ,ont!ibutes to the p!odu,tion o+ ANCA autoantibodies in vitro* %hese data suppo!t the notion that %:H ,ells a!e asso,iated 6ith the pathogeni, p!o,ess in G'A#patients and may ,onstitute a no$el ta!get +o! the!apeuti, inte!$ention*

17

A!!re$iations
ANCA3 anti#neut!ophil ,ytoplasmi, autoantibodiesL A(DD3 Ale5a :luo!M (DDL A'C3 Allophy,o,yaninL A::3 #,ell a,ti$ating +a,to!L IAS3 i!mingham Ias,ulitis A,ti$ity S,o!eL Ca#-3 ,al,ium ionopho!eL CL-SA3 en/yme#linked immunoso!bent assayL :CS3 +etal ,al+ se!umL :-%C3 +luo!es,ein

isothio,yanateL%:H3 +olli,ula! helpe! %#,ellsL G'A3 g!anulomatosis 6ith polyangiitisL HC3 healthy ,ont!olL -g3 immunoglobulinL --:3 indi!e,t immuno+luo!es,en,eL -L#213 inte!leukin#21L -L#21&3 inte!leukin#21 !e,epto!L ' 3 pe!iphe!al bloodL ' MC3 pe!iphe!al blood mononu,lea! ,ellsL ' S3 phosphate#bu++e!ed salineL 'C3 'hy,oe!yth!inL 'e!C'3 pe!idin ,hlo!ophyll p!oteinL 'MA3 pho!bol my!istate a,etateL '&33 p!oteinase 3*

(ompeting interests
%he autho!s de,la!e that they ha$e no ,ompeting inte!ests*

Authors9 contri!utions
All autho!s ,ont!ibuted to the design, a,9uisition o+ data, analysis and inte!p!etation o+ data* WHA ,ont!ibuted to ,on,ept and design, pe!+o!med the statisti,al analysis, and had +ull a,,ess to all o+ the data in the study and takes !esponsibility +o! the integ!ity o+ the data and the a,,u!a,y o+ the data analysis* WHA, NL, MGH, "$dM, and H% pe!+o!med the +lo6,ytomet!y, in vitro e5pe!iments, &%#'C& e5pe!iments, inte!p!etation o+ data, and d!a+ting o+ the manus,!ipt* CAS and A& ,ont!ibuted to ,on,ept and design, in,lusion o+ G'A#patients, analyses and inte!p!etation o+ ,lini,al data, and d!a+ting o+ the manus,!ipt* 'CL, 'H, and CGM) ,ont!ibuted to ,on,ept and design, inte!p!etation o+ data and !e$ising the manus,!ipt +o! impo!tant intelle,tual ,ontents* All autho!s !ead and app!o$ed the +inal manus,!ipt*

18

Ac/nowledgments
We a!e g!ate+ul to the patients and healthy dono!s +o! thei! ,o#ope!ation and pa!ti,ipation in this study* %he !esea!,h leading to these !esults has !e,ei$ed +unding +!om the Cu!opean .nion Se$enth :!ame6o!k '!og!amme 7:'1<2AA1#2A138 unde! g!ant ag!eement nJ 2;13D2, and +!om the G!oningen .ni$e!sity -nstitute +o! "!ug C5plo!ation 7G.-"C8*

19

'eference list

1*

:au,i AS, Haynes :, )at/ ', Wol++ SM3 *egener:s granulomatosis% prospecti$e clinical and therapeutic e7perience with ;< patients for 21 years* Ann Intern Med 10D3, =;31;# D@* $an de! Woude :Y, &asmussen N, Lobatto S, Wiik A, 'e!min H, $an Cs LA, $an de! GM, $an de! Hem G), %he %H3 Autoanti!odies against neutrophils and monocytes% tool for diagnosis and mar/er of disease acti$ity in *egener:s granulomatosis* Lancet 10D@, 13(2@#(20* Niles YL, M,Cluskey &%, Ahmad M:, A!naout MA3 *egener:s granulomatosis autoantigen is a no$el neutrophil serine proteinase* Blood 10D0, 3631DDD#1D03* !ou6e! C, %e!$ae!t YW, Ho!st G, Huitema MG, $an de! GM, Limbu!g 'C, )allenbe!g CG3 Predominance of IgG1 and IgG6 su!classes of anti-neutrophil cytoplasmic autoanti!odies (A-(A in patients with *egener:s granulomatosis and clinically related disorders* Clin Exp Immunol 1001, ;13310#3D;* )omo,si A, Lamp!e,ht ', Cse!nok C, Muelle! A, Holl#.l!i,h ), Seit/e! ., Moosig :, S,hnabel A, G!oss WL3 Peripheral !lood and granuloma (56(2 (52;(- T cells are a ma#or source of interferon-gamma and tumor necrosis factor-alpha in *egener:s granulomatosis* Am J Pathol 2AA2, 14>31111#112(* Lamp!e,ht ', Moosig :, Cse!nok C, Seit/e! ., S,hnabel A, Muelle! A, G!oss WL3 (52; negati$e T cells are enriched in granulomatous lesions of the respiratory tract in *egener:s granulomatosis* horax 2AA1, <431@1#1@1* Abdulahad WH, Stegeman CA, $an de! Geld ZM, "oo!nbos#$an de! M , Limbu!g 'C, )allenbe!g CG3 .unctional defect of circulating regulatory (562 T cells in patients with *egener:s granulomatosis in remission* Arthriti! Rheum 2AA1, <432ADA#2A01* Mo!gan M", "ay CY, 'ipe! )', )han N, Ha!pe! L, Moss 'A, Sa$age CH3 Patients with *egener:s granulomatosis demonstrate a relati$e deficiency and functional impairment of T-regulatory cells* Immunolog" 2A1A, 11>3;(#13* Abdulahad WH, $an de! Geld ZM, Stegeman CA, )allenbe!g CG3 Persistent e7pansion of (562 effector memory T cells in *egener:s granulomatosis* #idne" Int 2AA;, 3>303D# 0(1* Abdulahad WH, Stegeman CA, Limbu!g 'C, )allenbe!g CG3 +/ewed distri!ution of Th13 lymphocytes in patients with *egener:s granulomatosis in remission* Arthriti! Rheum 2AAD, <;3210;#22A@* Noguei!a C, Hamou! S, Sa6ant ", Hende!son S, Mans+ield N, Cha$ele )M, 'usey C", Salama A"3 +erum IL-13 and IL-21 le$els and autoantigen-specific Th13 cells are

2*

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ele$ated in patients with A-(A-associated $asculitis* $ephrol %ial ran!plant 2A1A, 2<322A0#2211* 12* Cttinge! &, Sims G', &obbins &, Withe!s ", :is,he! &%, G!amme! AC, )u,hen S, Lipsky 'C3 IL-21 and 0A..?0Ly+ synergi@e in stimulating plasma cell differentiation from a unique population of human splenic memory 0 cells* J Immunol 2AA1, 13;32D12#2DD2* )a!nell YL, Cttinge! &3 The Interplay of IL-21 and 0A.. in the .ormation and &aintenance of 8uman 0 (ell &emory* Front Immunol 2A12, 132* Zoung "A, Hegen M, Ma HL, Whitte!s MY, Albe!t LM, Lo6e L, Seni,es M, Wu 'W, Sibley , Leathu!by Z, !o6n %', Ni,ke!son#Nutte! C, )eith YC, Y!*, Collins M3 0loc/ade of the interleu/in-21?interleu/in-21 receptor pathway ameliorates disease in animal models of rheumatoid arthritis* Arthriti! Rheum 2AA1, <4311@2#11;3* He!be! ", !o6n %', Liang S, Zoung "A, Collins M, "unussi#Yoannopoulos )3 IL-21 has a pathogenic role in a lupus-prone mouse model and its !loc/ade with IL-21',.c reduces disease progression* J Immunol 2AA1, 13;33D22#3D3A* :ina ", Sa!!a M, :antini MC, &i//o A, Ca!uso &, Cap!ioli :, Stol+i C, Ca!dolini -, "otto!i M, oi!i$ant M, 'allone :, Ma,donald %%, Monteleone G3 'egulation of gut inflammation and th13 cell response !y interleu/in-21* &a!troenterolog" 2AAD, 11631A3D#1A(D* Spolski &, )ashyap M, &obinson C, Zu 2, Leona!d WY3 IL-21 signaling is critical for the de$elopment of type I dia!etes in the -"5 mouse* Proc $atl Acad 'ci ( ' A 2AAD, 1><31(A2D#1(A33* Sa6alha AH, )au+man )M, )elly YA, Adle! AY, Abe!le %, )ilpat!i,k Y, Wakeland C), Li [2, Wandst!at AC, )a!p "&, Yames YA, Me!!ill Y%, Lipsky ', Ha!ley Y 3 Genetic association of interleu/in-21 polymorphisms with systemic lupus erythematosus* Ann Rheum %i! 2AAD, 433(@D#(;1* :esten CA, Goyette ', S,ott &, Annese I, 2he!nako$a A, Lian Y, Le+eb$!e C, !ant S&, Cho YH, Sil$e!be!g MS, %aylo! )", de Yong "Y, Stokke!s 'C, M,go$e!n ", 'almie!i H, A,hka! Y', Ra$ie! &Y, "aly MY, "ue!! &H, WiBmenga C, Wee!sma &), &iou5 Y"3 Genetic $ariants in the region har!ouring IL2?IL21 associated with ulcerati$e colitis* &ut 2AA0, <;3100#DA(* Liu Z, Helms C, Liao W, 2aba LC, "uan S, Ga!dne! Y, Wise C, Mine! A, Malloy MY, 'ullinge! C&, )ane Y', Sa,,one S, Wo!thington Y, !u,e -, )6ok 'Z, Mente! A, )!uege! Y, a!ton A, Sa,,one NL, o6,o,k AM3 A genome-wide association study of psoriasis and psoriatic arthritis identifies new disease loci* PLo' &enet 2AAD, 63e1AAAA(1* C!a+t YC3 .ollicular helper T cells in immunity and systemic autoimmunity* $at Rev Rheumatol 2A12, ;3331#3(1*

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"ol++ S, Abdulahad WH, West!a Y, "oo!nbos#$an de! M , Limbu!g 'C, )allenbe!g CG, iBl M3 Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus* Arthriti! Re! her 2A11, 113&1@1* Simpson N, Gatenby 'A, Wilson A, Malik S, :ul,he! "A, %angye SG, Manku H, Iyse %Y, &on,ado! G, Huttley GA, Goodno6 CC, Iinuesa CG, Cook MC3 A7pansion of circulating T cells resem!ling follicular helper T cells is a fi7ed phenotype that identifies a su!set of se$ere systemic lupus erythematosus* Arthriti! Rheum 2A1A, 42323(#2((* Ma Y, 2hu C, Ma , %ian Y, aidoo SC, Mao C, Wu W, Chen Y, %ong Y, Zang M, Yiao 2, Ru H, Lu L, Wang S3 Increased frequency of circulating follicular helper T cells in patients with rheumatoid arthritis* Clin %ev Immunol 2A12, 2>123D21(DA* Yennette YC, :alk &Y, And!assy ), a,on 'A, Chu!g Y, G!oss WL, Hagen CC, Ho++man GS, Hunde! GG, )allenbe!g CG, *3 -omenclature of systemic $asculitides, Proposal of an international consensus conference* Arthriti! Rheum 100(, 1331D1#102* Lea$itt &Z, :au,i AS, lo,h "A, Mi,hel A, Hunde! GG, A!end W', Calab!ese LH, :!ies Y:, Lie Y%, Light+oot &W, Y!*, *3 The American (ollege of 'heumatology 1==> criteria for the classification of *egener:s granulomatosis* Arthriti! Rheum 100A, 11311A1#11A1* Lu9mani &A, a,on 'A, Moots &Y, Yanssen A, 'all A, Cme!y ', Sa$age C, Adu "3 0irmingham Basculitis Acti$ity +core (0BA+ in systemic necroti@ing $asculitis* )JM 100(, ;33;11#;1D* %e!$ae!t YW, Mulde! L, Stegeman C, Clema Y, Huitema M, %he H, )allenbe!g C3 "ccurrence of autoanti!odies to human leucocyte elastase in *egener:s granulomatosis and other inflammatory disorders* Ann Rheum %i! 1003, <2311@#12A* %e!$ae!t YW, Golds,hmeding &, Clema Y", $an de! GM, Huitema MG, $an de! Hem G), %he %H, $on dem o!ne AC, )allenbe!g CG3 Autoanti!odies against myeloid lysosomal en@ymes in crescentic glomerulonephritis* #idne" Int 100A, 133100#DA;* Sa$ige Y, "ime,h W, :!it/le! M, Goeken Y, Hagen CC, Yennette YC, M,C$oy &, 'usey C, 'ollo,k W, %!e$isin M, Wiik A, Wong &3 Addendum to the International (onsensus +tatement on testing and reporting of antineutrophil cytoplasmic anti!odies, Cuality control guidelinesD commentsD and recommendations for testing in other autoimmune diseases* Am J Clin Pathol 2AA3, 12>3312#31D* %adema H, Abdulahad WH, Lepse N, Stegeman CA, )allenbe!g CG, Hee!inga '3 0acterial 5-A motifs trigger A-(A production in A-(A-associated $asculitis in remission* Rheumatolog" *+x,ord- 2A11, <>3;D0#;0;* )o!n %, ettelli C, Gao W, A6asthi A, Yage! A, St!om % , Hukka M, )u,h!oo I)3 IL-21 initiates an alternati$e pathway to induce proinflammatory T(8 13 cells* $ature 2AA1, 66;3(D(#(D1*

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Nu!ie$a &, Zang RH, Ma!tine/ G, 2hang Z, 'anopoulos A", Ma L, S,hluns ), %ian [, Wato6i,h SS, Yetten AM, "ong C3 Assential autocrine regulation !y IL-21 in the generation of inflammatory T cells* $ature 2AA1, 66;3(DA#(D3* Zu ", &ao S, %sai LM, Lee S), He Z, Sut,li++e CL, S!i$asta$a M, Linte!man M, 2heng L, Simpson N, Cllya!d Y-, 'a!ish -A, Ma CS, Li [Y, 'a!ish C&, Ma,kay C&, Iinuesa CG3 The transcriptional repressor 0cl-4 directs T follicular helper cell lineage commitment* Immunit" 2AA0, 113(@1#(;D* Nu!ie$a &-, Chung Z, Ma!tine/ GY, Zang RH, %anaka S, Matske$it,h %", Wang ZH, "ong C3 0cl4 mediates the de$elopment of T follicular helper cells* 'cience 2AA0, 12<31AA1# 1AA@* Chung Z, %anaka S, Chu :, Nu!ie$a &-, Ma!tine/ GY, &a6al S, Wang ZH, Lim H, &eynolds YM, 2hou RH, :an HM, Liu 2M, Neelapu SS, "ong C3 .ollicular regulatory T cells e7pressing .o7p1 and 0cl-4 suppress germinal center reactions* $at Med 2A11, 1330D3# 0DD* Linte!man MA, 'ie!son W, Lee S), )allies A, )a6amoto S, &ayne! %:, S!i$asta$a M, "i$eka! "', eaton L, Hogan YY, :aga!asan S, Liston A, Smith )G, Iinuesa CG3 .o7p12 follicular regulatory T cells control the germinal center response* $at Med 2A11, 13301@#0D2* 'ene Y, Gau,hat Y:, Le,a!t S, "!ouet C, Guglielmi ', oulay I, "el6ail A, :oste! ", Le,!on YC, Zssel H3 (utting edge% IL-21 is a switch factor for the production of IgG1 and IgG1 !y human 0 cells* J Immunol 2AA(, 1323@1@(#@1@1* Cttinge! &, Sims G', :ai!hu!st AM, &obbins &, da Sil$a ZS, Spolski &, Leona!d WY, Lipsky 'C3 IL-21 induces differentiation of human nai$e and memory 0 cells into anti!odysecreting plasma cells* J Immunol 2AA@, 13<31D;1#1D10* )u,hen S, &obbins &, Sims G', Sheng C, 'hillips %M, Lipsky 'C, Cttinge! &3 Assential role of IL-21 in 0 cell acti$ationD e7pansionD and plasma cell generation during (562 T cell-0 cell colla!oration* J Immunol 2AA1, 13=3@DD;#@D0;* Waite YC, Skokos "3 Th13 response and inflammatory autoimmune diseases* Int J In,lam 2A12, 2>123D10(;1* Hsu HC, Zang ', Wang Y, Wu [, Mye!s &, Chen Y, Zi Y, Guente!t %, %ousson A, Stanus AL, Le %I, Lo!en/ &G, Ru H, )olls Y), Ca!te! &H, Chaplin "", Williams &W, Mount/ Y"3 Interleu/in 13-producing T helper cells and interleu/in 13 orchestrate autoreacti$e germinal center de$elopment in autoimmune 0E52 mice* $at Immunol 2AAD, =31;;#11@* Chen [, Zang W, Gupta S, is6as ', Smith ', hagat G, 'e!nis A 3 I'.-6-!inding protein inhi!its interleu/in-13 and interleu/in-21 production !y controlling the acti$ity of I'.-6 transcription factor* Immunit" 2AAD, 2=3D00#011*

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Ca!uso &, :ina ", 'eluso -, Stol+i C, :antini MC, Gioia I, Cap!ioli :, "el Ie,,hio G, 'aolu/i HA, Ma,donald %%, 'allone :, Monteleone G3 A functional role for interleu/in-21 in promoting the synthesis of the T-cell chemoattractantD &IP-1alphaD !y gut epithelial cells* &a!troenterolog" 2AA1, 11231;;#11@* Liu Z, Zang , Ma Y, Wang H, Huang :, 2hang Y, Chen H, Wu C3 Interleu/in-21 induces the differentiation of human Tc22 cells $ia phosphorylation of signal transducers and acti$ators of transcription* Immunolog" 2A11, 1123@(A#@(D* Cbe!t CC3 Interleu/in 21 up-regulates perforin-mediated cytoto7ic acti$ity of human intra-epithelial lymphocytes* Immunolog" 2AA0, 12332A;#21@* Liu 2, Zang L, Cui Z, Wang R, Guo C, Huang 2, )an [, Liu 2, Liu Z3 Il-21 enhances -) cell acti$ation and cytolytic acti$ity and induces Th13 cell differentiation in inflammatory !owel disease* In,lamm Bo.el %i! 2AA0, 1<31133#11((* H/aki ), Spolski &, Cttinge! &, )im H', Wang G, [i C:, H6u ', Sha++e! "Y, Akilesh S, &oopenian "C, Mo!se HC, ---, Lipsky 'C, Leona!d WY3 'egulation of 0 cell differentiation and plasma cell generation !y IL-21D a no$el inducer of 0limp-1 and 0cl-4* J Immunol 2AA(, 1313@3;1#@311* Linte!man MA, eaton L, Zu ", &amis,al &&, S!i$asta$a M, Hogan YY, Ie!ma N), Smyth MY, &igby &Y, Iinuesa CG3 IL-21 acts directly on 0 cells to regulate 0cl-4 e7pression and germinal center responses* J Exp Med 2A1A, 2>333@3#3;3* Slot MC, %e!$ae!t YW, oomsma MM, Stegeman CA3 Positi$e classic antineutrophil cytoplasmic anti!ody ((-A-(A titer at switch to a@athioprine therapy associated with relapse in proteinase 1-related $asculitis* Arthriti! Rheum 2AA(, <132;0#213* 'ie!!ot#"eseilligny "C, 'ou,hot Y, 'agnou5 C, Coste Y, Guille$in L3 Predictors at diagnosis of a first *egener:s granulomatosis relapse after o!taining complete remission* Rheumatolog" *+x,ord- 2A1A, 6=321D1#210A* Abdulahad WH, )allenbe!g CG, Limbu!g 'C, Stegeman CA3 Frinary (562 effector memory T cells reflect renal disease acti$ity in antineutrophil cytoplasmic anti!odyassociated $asculitis* Arthriti! Rheum 2AA0, 4>32D3A#2D3D*

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Ta!le
Ta!le 1% (linical and la!oratory characteristics of GPA-patients at the time of !lood sampling,

No* o+ patients

(2

No* male<no* +emale

2;<1;

Age, mean FS" 7!ange8 yea!s

@0 F1( 72D#D18

No* 6ith lo,ali/ed<gene!ali/ed G'A

1@<21

No* positi$e<negati$e +o! '&3#ANCA\

23<10

No* &e,ei$ing<not !e,ei$ing t!eatment\\

11<31

No* o+ !elapses, median 7!ange8

A 7A#@8

"isease du!ation, median 7!ange8 months

112 72A#33(8

G'A G G!anulomatosis 6ith polyangiitisL '&3 G '!oteinase 3L ANCA G antineut!ophil ,ytoplasmi, antibody*

\ 'atients 6e!e ,onside!ed to be ANCA#positi$e 6hen ANCA#tit!es by --: 6e!e

g!eate! than 13(A

\\ :ou! patients 6e!e t!eated 6ith only a/athiop!ine, one 6ith my,ophenolate mo+etil, and
si5 patients !e,ei$ed p!ednisolone in ,ombination 6ith a/athiop!ine*

25

.igure legends

.igure 1% &ultiparameter flow cytometric detection of IL-21 and IL-13 in circulating (562 T-cells in GPA-patients and 8(s, Whole blood +!om G'A#patients and HCs 6as stimulated 6ith 'MA<Ca#-onopho!e and analy/ed +o! int!a,ellula! -L#21 and -L#11 ,ytokine e5p!ession* &ep!esentati$e :ACS plots o+ -L#21 $e!sus -L#11 e5p!ession in stimulated C"(E %#,ells +!om a G'A#patient 7!ight plot8 and an age# and se5#mat,hed HC 7le+t plot8 (A * Ialue in ea,h gate !ep!esents the pe!,entages o+ ,ytokine p!odu,ing ,ells* 'e!,entages o+ total -L#21 p!odu,ing %h#,ells in pe!iphe!al blood o+ G'A#patients 7nG (28 and HCs 7nG 208 (0 * 'e!,entages o+ ,i!,ulating -L#21E-L#11#, -L#21E-L#11E, and -L#21#-L#11E ,ells 6ithin the C"(E %#,ells in all G'A#patients and HCs 7(-A , o! in ANCA#positi$e 7nG 238 and ANCA#negati$e 7nG 108 G'A#patients (.8 * Ho!i/ontal lines !ep!esent the median pe!,entage* P#$alues 6e!e ,al,ulated using the nonpa!amet!i, Mann#Whitney .#test* ] G P W A*A@L ]]] G P W A*AAA@*

.igure 2% (orrelation !etween the percentages of IL-212IL-13- cells and IL-21-IL-132 cells within the (562 T-cells in peripheral !lood of 8(s (A D and GPA-patients (0 , Spea!man !ank ,o!!elation ,oe++i,ients 7!8 and P $alues a!e gi$en*

26

.igure 1% A7pression of transcription factors 0(L4 and .o7P1, (A &ep!esentati$e :ACS plots o+ CL; $e!sus :o5'3 e5p!ession in ,i!,ulating C"(E %#,ells +!om an ANCA#negati$e# 7!ight plot8 and an ANCA#positi$e# 7middle plot8 G'A#patient, and an age# and se5# mat,hed HC 7le+t plot8* Ialues in ea,h gate !ep!esent the pe!,entages o+ positi$e ,ells* (0 &elati$e m&NA e5p!ession o+ CL; in leuko,ytes +!om ANCA#positi$e# 7nG 1A8 and ANCA#negati$e# 7nG ;8 G'A# patients, and age# and se5#mat,hed HCs 7nG 118 6as analy/ed by !eal#time &%#'C& no!mali/ed to the housekeeping gene GA'"H* (( 'e!,entage o+ CL;E:o5'3# ,ells in ,i!,ulating C"(E %#,ells 6as dete!mined in ANCA#positi$e# 7nG 118 and ANCA#negati$e# 7nG 1A8 G'A#patients, and age# and se5#mat,hed HCs 7nG 1A8* a!s !ep!esent the mean $alues FS"* P#$alues 6e!e ,al,ulated using the nonpa!amet!i, Mann#Whitney .#test* ] G P W A*A@L ]] G P W A*AA@*

.igure 6% (omparison of IL-21' e7pressing 0-cells from GPA-patients and 8(s, (A &ep!esentati$e :ACS plots o+ -L#21& e5p!ession on C"10E #,ells +!om an ANCA#negati$e# 7!ight plot8 and an ANCA#positi$e# 7middle plot8 G'A#patient, and an age# and se5#mat,hed HC 7le+t plot8* Ialues in ea,h gate !ep!esent the pe!,entages o+ -L#21&E #,ells* (0 %he pe!,entage o+ -L#21&E #,ells 6as dete!mined in pe!iphe!al blood +!om ANCA#positi$e# 7nG 138 and ANCA#negati$e# 7nG 1(8 G'A# patients, and age# and se5#mat,hed HCs 7nG 108* a!s !ep!esent the mean $alues FS"* P#$alues 6e!e ,al,ulated using the nonpa!amet!i, Mann#Whitney .#test*

27

.igure <% IL-21 induces in vitro IgG and P'1-A-(A production !y 0-cells from GPA-patients, 7A8 ' MCs +!om ANCA#positi$e# 7nG 18 and ANCA#negati$e# 7nG ;8 G'A#patients 6e!e ,ultu!ed in the p!esen,e o+ !h-L#21 and<o! !h A::* Cultu!e supe!natants 6e!e ,olle,ted a+te! 12 days to measu!e total -gG by CL-SA* 708 %o assess the e++e,t o+ -L#21 on in vitro ANCA#p!odu,tion, ' MCs +!om ANCA# positi$e# 7nG 1;8 and ANCA#negati$e# 7nG 128 G'A#patients 6e!e stimulated in the p!esen,e o+ -L# 21< A::* A+te! 12 days, '&3#ANCA le$els 6e!e dete!mined by 'hadia -mmunoCA'M 2@A analy/e!* P# $alues 6e!e ,al,ulated using the Wil,o5on mat,hed pai!s test* ] G P W A*A@

28

Figure 2