ORIGINAL RESEARCH
A B-Cell Gene Signa u e Co ela es Wi h he Ex en o
Glu en-Induced In es inal Inju y in Celiac Disease
Mi chell E. Ga be ,
1,2
Alok Saldanha,
3
Joel S. Pa ke ,
4,5
Wendell D. Jones,
6
Ka i Kaukinen,
7,8,9
Kaija Lau ila,
7
Ma ja-Leena Lähdeaho,
9,10
Pu esh Kha i,
11,12
Chai an Khosla,
2,13,14
Daniel C. Adelman,
1,15
and Ma kku Mäki
7,9,16
1
Al ine Pha maceu icals, Inc, San Ca los, Cali o nia;
2
Depa men o Chemis y,
11
Ins i u e o Immuni y, T ansplan a ion and
In ec ion,
12
Di ision o Biomedical In o ma ics, Depa men o Medicine,
13
Depa men o Chemical Enginee ing,
14
S an o d
ChEM-H, S an o d Uni e si y, S an o d, Cali o nia;
3
In e Sys ems Co po a ion, Camb idge, Massachuse s;
4
Linebe ge
Comp ehensi e Cance Cen e ,
5
Depa men o Gene ics, Uni e si y o No h Ca olina, Chapel Hill, No h Ca olina;
6
EA
Genomics, Di ision o Q
2
Solu ions, Mo is ille, No h Ca olina;
16
Tampe e Cen e o Child Heal h Resea ch,
7
Uni e si y o
Tampe e Facul y o Medicine and Li e Sciences, Tampe e, Finland;
10
Depa men o Pedia ics,
8
Depa men o In e nal
Medicine,
9
Tampe e Uni e si y Hospi al, Tampe e, Finland;
15
Di ision o Alle gy/Immunology, Depa men o Medicine,
Uni e si y o Cali o nia San F ancisco, San F ancisco, Cali o nia
SUMMARY
A glu en challenge in celiac pa ien s p o ides a unique
oppo uni y o s udy he immunology associa ed wi h he
ansi ion om ela i e heal h o au oimmuni y. This s udy
showed ha a B-cell popula ion in pe iphe al blood co e-
la ed in e sely wi h glu en-dependen small in es inal
lesions, implica ing a p o ec i e mechanism.
BACKGROUND & AIMS: Celiac disease (CeD) p o ides an
oppo uni y o s udy au oimmuni y and he ansi ion in im-
mune cells as die a y glu en induces small in es inal lesions.
METHODS: Se en y- h ee celiac disease pa ien s on a long-
e m, glu en- ee die inges ed a known amoun o glu en
daily o 6 weeks. A pe iphe al blood sample and in es inal
biopsy specimens we e aken be o e and 6 weeks a e ini i-
a ing he glu en challenge. Biopsy esul s we e epo ed on a
con inuous nume ic scale ha measu ed he illus-
heigh – o–c yp -dep h a io o quan i y glu en-induced
in es inal inju y. Pooled B and T cells we e isola ed om whole
blood, and RNA was analyzed by DNA mic oa ay looking o
changes in pe iphe al B- and T-cell gene exp ession ha
co ela ed wi h changes in illus heigh o c yp dep h, as
pa ien s main ained a ela i ely heal hy in es inal mucosa o
de e io a ed in he ace o a glu en challenge.
RESULTS: Glu en-dependen in es inal damage om baseline
o 6 weeks a ied widely ac oss all pa ien s, anging om no
change o ex ensi e damage. Genes di e en ially exp essed in B
cells co ela ed s ongly wi h he ex en o in es inal damage.
A ela i e inc ease in B-cell gene exp ession co ela ed wi h a
lack o sensi i i y o glu en whe eas hei ela i e dec ease
co ela ed wi h glu en-induced mucosal inju y. A co e B-cell
gene module, ep esen ing a subse o B-cell genes analyzed,
accoun ed o he co ela ion wi h in es inal inju y.
CONCLUSIONS: Genes comp ising he co e B-cell module
showed a ne inc ease in exp ession om baseline o 6 weeks
in pa ien s wi h li le o no in es inal damage, sugges ing ha
hese indi iduals may ha e moun ed a B-cell immune esponse
o main ain mucosal homeos asis and ci cum en
inflamma ion. DNA mic oa ay da a we e deposi ed a he GEO
eposi o y (accession numbe : GSE87629; a ailable: h ps://
www.ncbi.nlm.nih.go /geo/). (Cell Mol Gas oen e ol Hepa ol
2017;4:1–17; h p://dx.doi.o g/10.1016/j.jcmgh.2017.01.011)
Keywo ds: O al Tole ance; Mucosal Immuni y; Au oimmuni y;
Regula o y B Cell.
Human beings inges a wide a ie y o ood p o eins
in he die ha a e conside ed o eign wi h espec
o he immune sys em. Immune cells in he small in es ine
su ey he con en s in he in es inal en i onmen looking
o pa hogens. O al ole ance is a mechanism ha balances
he need o p omo e ole ance o o ally adminis e ed,
o eign, ye ha mless, ood p o eins wi h he need o p o ide
a hos de ense agains ha m ul pa hogens in he in es ine.
1
Al hough no well unde s ood, o al ole ance o a ood
p o ein is an ac i e immune esponse whose unc ion is o
supp ess inflamma o y immune esponses o he same ood
p o ein when p esen ed o he immune sys em o a second
ime. In celiac disease (CeD), a lack o o al ole ance
de elops o a amily o ce eal p o eins collec i ely e e ed
o as glu en,
2
esul ing in a pa hogenic and inflamma o y
immune esponse.
The small in es inal mucosa consis s o an epi helium
and i s unde lying s uc u es, which a e immedia ely adja-
cen o he in es inal lumen and in con ac wi h diges ed
ood. To inc ease su ace a ea o nu ien abso p ion, he
mucosa p ojec s finge -like ex ensions called illi in o he
lumen o he gu . A he base o he illi a e p oli e a i e
c yp s. In pa ien s wi h CeD, glu en inges ion esul s in
blun ing o he illi and hype ophy o elonga ion o he
c yp s. The a io o he heigh o he illi (Vh) o he dep h o
he c yp (Cd), exp essed as Vh:Cd, has been used o
quan i y he ex en o in es inal damage in CeD.
3–8
In se e e
cases, illi sh ink comple ely wi h ex ensi e c yp elonga-
ion, esul ing in a fla mucosa and a Vh:Cd measu emen
app oaching ze o.
HLA-DQ is an impo an gene ic ac o ha p edisposes
indi iduals o CeD and ype 1 diabe es.
9,10
A o al o
5%–10% o indi iduals wi h ype 1 diabe es de elop CeD,
11
which is significan ly highe han he chance o de eloping
CeD in he o e all Caucasian popula ion, es ima ed o be
1%.
12
Mos CeD pa ien s (90%) exp ess HLA-DQ2.5,
whe eas he emainde exp ess HLA-DQ2.2 o HLA-DQ8.
13
Glu en pep ides ha a e deamida ed by he sel -p o ein
ansglu aminase 2 (TG2) bind s ongly o HLA-DQ and
he esul ing complex is p esen ed o HLA-DQ– es ic ed
CD4þT cells,
14
esul ing in a T-cell esponse o deamida ed
glu en.
15
In addi ion o he T-cell esponse, glu en-
dependen , disease-specific B cells appea ea ly du ing
disease pa hogenesis. They p ecede gu damage, o en a e
p edic i e o impending disease,
16–20
and p oduce an i-
bodies specific o deamida ed glu en and TG2.
21
I is un-
clea whe he glu en-dependen au o-an ibodies agains
TG2 con ibu e o he disease and he e is li le e idence
ha T cells wi h specifici y o sel -an igens d i e he
disease.
The B cell ecognizes a specific p o ein an igen, such as
glu en o TG2, h ough di ec in e ac ions wi h i s B-cell
ecep o con aining a memb ane-bound immunoglobulin.
22
The immunoglobulin de e mines an igen specifici y and is
unique o each B-cell clone. B-cell ecep o signaling con-
ibu es s ongly o B-cell p oli e a ion and di e en ia ion. B
cells can be an igen-p esen ing cells
23
and ha e he abili y
o exp ess HLA-DQ2 o HLA-DQ8. Because glu en pep ides
a e excellen subs a es o TG2 and he wo p o eins o m a
ansien co alen complex, he possibili y exis s ha a
deamida ed glu en- o TG2-specific B cell binds he complex
h ough he B-cell ecep o , in e nalizes, and hen p esen s
glu en on HLA-DQ2 o HLA-DQ8 a he B cell su ace o a
glu en-specific HLA-DQ2–o HLA-DQ8– es ic ed CD4þT
cell.
15,24
In his scena io, he esul ing B cell is a unc ional
an igen-p esen ing cell whose cell pheno ype, as de e -
mined by B-cell– ecep o signaling and di ec B- and T-cell
co-s imula o y in e ac ions, ul ima ely may be unde he
con ol o glu en.
An inflamma o y, glu en-induced immune esponse in
he gu may p opaga e sys emically in pe iphe al blood.
The plausible a ficking o B and T cells and an i-TG2 o
si es beyond he gu may help o explain se e al sys emic
clinical mani es a ions o CeD,
25
including de ma i is he -
pe i o mis, a glu en-dependen blis e ing skin condi ion.
CeD also may mani es as a bone disease,
26
in he cen al
ne ous sys em as a axia and b ain a ophy,
27
o as an
isola ed subclinical
28
o se e e li e disease.
29
E idence
indica es ha immunecellsmig a e oand om hegu in
pe iphe al blood. Fo example, disease-specific T cells
exp essing he gu -homing b7 in eg in mig a e ansien ly
o he pe iphe y upon glu en challenge in CeD pa ien s;
hese T cells a e inflamma o y in na u e.
30,31
The pe iph-
e al blood he e o e may be a good sou ce o ob ain bio-
ma ke s o he disease.
I is no clea how glu en in e ac s wi h mechanisms o
pe iphe al immune ole ance and whe he B o T cells a e
esponsible o dis up ing he ole ogenic en i onmen o
he small in es ine. The objec i e o his wo k was o
de e mine i pe iphe al blood B and T cells modi y
gene exp ession in esponse o a 6-week glu en challenge in
pa ien s wi h ea ed CeD, and o co ela e any changes in
pe iphe al B- o T-cell gene exp ession wi h he ex en o
glu en-induced his ological damage o he small in es ine.
Ma e ials and Me hods
All au ho s had access o he s udy da a and e iewed
and app o ed he final manusc ip .
Abb e ia ions used in his pape : Cd, c yp dep h; CeD, celiac disease;
cRNA, complemen a y RNA; TG2, ansglu aminase 2; TR, T-cell
ecep o ; Vh, illus heigh ; Vh:Cd, a io o illus heigh o c yp dep h.
Mos cu en a icle
©
2017 The Au ho s. Published by Else ie Inc. on behal o he AGA
Ins i u e. This is an open access a icle unde he CC BY-NC-ND
license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
2352-345X
h p://dx.doi.o g/10.1016/j.jcmgh.2017.01.011
2 Ga be e al Cellula and Molecula Gas oen e ology and Hepa ology Vol. 4, No. 1
Human Clinical S udy
In es inal biopsy specimens and whole blood samples
we e collec ed om a single clinical si e a he Uni e si y o
Tampe e, Tampe e, Finland, wi h pa ien consen (e hics
app o al ETL R09084M, ETL R10135M, and Eud a CTs
2009-012221-10 and 2010-023127-23). Fo each ime
poin , small-bowel biopsy specimens (4–7 specimens) we e
sampled om he descending duodenum, sec ioned, and
sco ed by he same e alua o using s anda dized mo pho-
me ic ools.
5,32
O he 73 pa ien s included in his analysis,
37% we e male and 63% we e emale. The age ange was
23–74 yea s wi h a median age o 59 yea s. Fo 6 weeks,
pa ien s inges ed 6 g/day (20 pa ien s), 3 g/day (26
pa ien s), o 1.5 g/day (27 pa ien s) whea glu en wi h a
meal. A he baseline ime poin , pa ien s (85%) we e
nega i e o an ibodies agains ansglu aminase 2. All
pa ien s comple ed he ull 6-week s udy.
B- and T-Cell Isola ion
CD19þB cells and CD3þT cells we e pu ified oge he
om eshly p epa ed, enous whole blood (14 mL) using
whole-blood CD19 and CD3 magne ic mic obeads and he
whole-blood column ki as specified by he manu ac u e
(Mil enyi Bio ec, Be gisch Gladbach, Ge many). EDTA was
used as a blood an icoagulan . The final cell pelle con ain-
ing pu ified B and T cells was esuspended in TRIzol (Fishe
Scien ific) eagen (5 mL) o lyse he cells and p o ec he
RNA om deg ada ion. The dena u ed B- and T-cell lysa e
was s o ed a -70C o up o 3 mon hs be o e pu ifica ion o
o al RNA. Fo con enience, whole-blood samples may be
s o ed on ice o up o 1 hou be o e adding magne ic beads.
P epa a ion ime om he poin o adding magne ic beads o
eezing he B- and T-cell lysa e was less han 2 hou s.
To al RNA P epa a ion
Pooled B- and T-cell lysa es (5 mL) dena u ed in TRIzol
we e ex ac ed wi h chlo o o m (0.2 ol) and he c ude RNA
was p ecipi a ed om he aqueous phase wi h he addi ion
o isop opanol (0.7 ol). The RNA pelle was pu ified u he
using he RNeasy Plus Ki (Qiagen, Hilden Ge many)
acco ding o he manu ac u e ’s specifica ions. Pu ified o al
RNA was elu ed om he spin column wi h he addi ion o
2 aliquo s (35 mL) RNase- ee wa e . An Agilen BioAnalyze
(San a Cla a, CA) was used o de e mine o al RNA yield
(median, 2.2 mg), pu i y (median A260/A280, 2.1), and size
(median RNA in eg i y numbe , 9.5) o all 146 samples.
Pu ified o al RNA was s o ed a less han -60C.
Mic oa ays
RNA samples we e con e ed in o labeled a ge com-
plemen a y RNA (cRNA) using he Illumina To alP ep-96
RNA Amplifica ion Ki (Ambion). B iefly, 100 ng o o al
RNA was con e ed o double-s anded complemen a y
DNA using an oligo-d(T) p ime -adap o . This complemen-
a y DNA was pu ified using magne ic beads and used as a
empla e o in i o ansc ip ion using T7 RNA polyme ase
and bio in–u idine iphospha e. The esul ing bio inyla ed
cRNA was pu ified using magne ic beads and quan ified by
spec opho ome y. Fo hyb idiza ion, 750 ng o pu ified
bio inyla ed cRNA was added o Hyb idiza ion Cock ail
Bu e (Illumina), applied o a ays, and incuba ed a 58C
o 16 hou s. A e hyb idiza ion, a ays we e washed and
s ained using s anda d Illumina p ocedu es be o e scanning
on an Illumina iScan ins umen using he Di ec Hyb Ti
se ing. Scanned images we e p ocessed by he Gene
Exp ession module o GenomeS udio ( 1.6; Illumina) using
de aul pa ame e s wi hou no maliza ion.
A o al o 146 RNA samples we e analyzed by mic oa ay
ep esen ing 73 pa ien s wi h high-quali y mic oa ay da a
a baseline and 6-week ime poin s (146 a ays). The 146
a ays included 39 om HT-12 e sion 3 and 107 om
HT-12 e sion 4 (Illumina). The e sion 3 a ays we e dis-
con inued by he manu ac u e , equi ing he change o
e sion 4. Only p obes ha ing iden ical sequence con en
be ween e sions we e e ained. Po en ial a i ac s caused
by he 2 a ay e sions and ba ch we e emo ed by
assuming he ba ch medians o a gi en p obe we e equal
(co a ia es we e no associa ed wi h ba ch). This was
acili a ed by he ac ha bo h baseline and 6-week ime
poin s o 60 o 73 pa ien s (82%) we e analyzed on he
same ba ch. A e median shi ing each p obe ela i e o
ba ch medians, p obe quan ile no maliza ion was pe -
o med ac oss all samples. Backg ound hen was sub ac ed
wi h a mono onic ans o m o p obe alues below back-
g ound o he (0,1) in e al so ha all no malized p obes
had posi i e alues be o e log ans o ma ion. A e
no maliza ion, ba ch adjus men , and backg ound sub ac-
ion, he di e ence in exp ession be ween baseline and he
6-week ime poin s was calcula ed as log
2
(6-wk/baseline)
o each subjec esul ing in a ma ix o 20,624 ea u es and
73 samples.
Gene Lis s
Acco ding o Bindea e al,
33
genes highly en iched in T
cells we e seg ega ed in o immune subpopula ions: T cell,
T CD8, T gd, T helpe , T helpe 1, T helpe 2, T helpe 17, T
cen al memo y, T e ec o memo y, T ollicula helpe , and
T egula o y. These ca ego ies we e aken e ba im om
he publica ion as mul iple lis s ep esen ing T-cell sub-
popula ions and used sepa a ely o analyze he CeD da a se .
The T- egula o y ca ego y was cha ac e ized by one gene
(FOXP3), which was no p esen on he Illumina a ay and
he e o e d opped om he analysis. Acco ding o Newman
e al,
34
genes highly en iched in T cells also we e seg ega ed
in o immune subpopula ions: CD8, CD4 nai e, CD4 memo y
es ing, CD4 memo y ac i a ed, T ollicula helpe , T egu-
la o y, and T gd. In his case, genes ep esen ing T-cell
subpopula ions we e consolida ed in o one gene lis . Genes
highly en iched in B cells we e aken e ba im om he
publica ion as a single lis (Bindea e al
33
) o he B-cell
subpopula ions we e consolida ed in o a single lis (New-
man e al
34
). Bindea e al
33
also p o ided gene lis s ep e-
sen ing a di e se se o immune cell exp ession pheno ypes,
including mas , na u al kille , neu ophils, mac ophage, eo-
sinophils, dend i ic, and cy o oxic cells, as well as SW480
cance cells and no mal mucosa, which we e included as
July 2017 B Cell Gene Signa u e in Celiac Disease 3
con ols. The Uni e si y o No h Ca olina IgG gene lis was
used p e iously as a p edic o in b eas cance .
35
All gene
lis s used o analyze he CeD da a se a e shown in
Supplemen a y Table 1.
In whole blood, he es ima e is ha T cells as ly
ou numbe B cells. In addi ion, he pu ified CD19þB-cell
popula ion used in his s udy should ha e li le o no ully
di e en ia ed CD19- plasma B cells, u he educing he
numbe o B cells. Gi en ha B and T cells we e pu ified
oge he , B- and T-cell genes we e selec ed om Newman
e al
34
based on he ollowing cha ac e is ic: highly en iched
in a leas one B- o T-cell subpopula ion ela i e o o he
leukocy es. In addi ion, o B-cell gene selec ion, he gene
had o show li le o no exp ession in T cells o exp ession
ha g ea ly exceeded ha in T cells because he con ibu-
ion o B cells o he o e all pool o o al RNA was ela i ely
small.
Genes ob ained om he A yme ix (San a Cla a, CA)
a ay pla o m (Bindea e al
33
and Newman e al
34
s udies) ha we e no exp essed in he CeD da a se o
no p esen on he Illumina a ay pla o m we e excluded
om analysis. The e we e compa ibili y issues wi h T-cell
ecep o (TR) gene segmen s be ween a ay pla o ms.
The T-cell gene lis ob ained om Newman e al
34
was
modified o e ain TR ansc ip s by dele ing A yme ix
TR p obes and subs i u ing all TR p obes on he Illumina
a ay i hese p obes we e exp essed in he CeD da a se .
I was common o genes ob ained om Bindea e al
33
and Newman e al
34
o co espond o mul iple Illumina
DNA mic oa ay p obes. All ele an p obes we e
included in he analysis.
Co ela ion Be ween Gene Lis s and DVh:Cd
App oxima ely one hi d o genes mapped o mo e han
one mic oa ay p obe. In hese cases, we ook wo di e en
app oaches. Fi s , he gene was consolida ed o a single
p obe by aking only he p obe wi h he g ea es SD o
exp ession ac oss he 73 pa ien s. The a ionale behind his
app oach is ha p obes ha pe o m poo ly show a nea ly
cons an , low le el o signal ac oss he da a se , and hus can
be sc eened ou by hei low a ia ion. This app oach was
e e ed o as genes. Second, he gene was analyzed using all
co esponding p obes, as opposed o he one wi h highes
a ia ion. This app oach was e e ed o as p obes. In bo h
cases, exp ession le els o all genes o p obes in a gi en lis
we e a e aged on log signal alues, and he a e age
exp ession p ofile ac oss 73 pa ien s was co ela ed o
DVh:Cd using Spea man ank co ela ion. Nea ly iden ical
esul s we e ob ained using he median a he han he
mean exp ession p ofiles o bo h genes and p obes (da a
no shown) o collapse a gi en gene lis . Fo de e mina ion
o he co e B-cell gene module, exp ession le els o single
p obes also we e co ela ed o DVh:Cd using Spea man ank
co ela ion.
S a is ical Analyses
Spea man co ela ions we e calcula ed in R using he
s anda d co . es ou ine. The significance o he co ela ion
was assessed using he S uden dis ibu ion by se ing
exac ¼FALSE. The GSA module in R was used o file
pa sing. The S uden es used o co ela ions wi h an i-
TG2 also was pe o med in R. Chi-squa ed analysis was
pe o med using Mic oso Excel (Redmond, WA).
Celiac Disease Se um An ibodies
Se um an ibodies di ec ed agains TG2-IgA we e
measu ed by enzyme-linked immunoso ben assay (Quan a
Li e h-TG-IGA; Ino a Diagnos ics, San Diego, CA).
32
The
posi i e h eshold was 20 in ensi y uni s.
Resul s
Glu en-Dependen In es inal Damage
The da a se consis ed o 73 CeD pa ien s ollowing a
s ic glu en- ee die o a leas one yea . Each pa ien
inges ed 6, 3, o 1.5 g whea glu en daily o 6 weeks. A
whole blood sample, which was used o pu i y B and T
cells, and in es inal biopsy specimens we e aken be o e
(baseline) and 6 weeks a e ini ia ing he glu en challenge.
The median Vh:Cd a baseline was 2.7 (see Table 1 o
pa ien da a). Ne change in in es inal biopsy om baseline
o 6 weeks, defined as DVh:Cd, showed wide a ia ion
ac oss all pa ien s om no change o sligh imp o emen o
ex ensi e mucosal damage (Figu e 1). The la ges DVh:Cd
(-2.9) was obse ed in 3 pa ien s who ansi ioned om a
ela i ely heal hy mucosa (Vh:Cd, 3.1) o a nea ly fla
mucosa (Vh:Cd, 0.2) in 6 weeks. Daily glu en dose o 2 o
hese pa ien s was 6 g ( oughly 2 slices o whea b ead).
Al hough he 6 g glu en dose in hese 2 pa ien s esul ed in
ex ensi e mucosal damage, in o he pa ien s i esul ed in
no damage (Figu e 1, blue ba s). Simila obse a ions we e
made o he o he 2 glu en doses, 3 g (yellow) and 1.5 g
(g ey). As a esul , glu en dose was dis ibu ed ac oss he
ull spec um o in es inal damage. Reg ession analysis o
DVh:Cd s glu en dose showed ha glu en dose explained
oughly 18% o he a ia ion in mucosal damage (adjus ed
R
2
, 0.18; P¼.0001). How an indi idual esponded o he
glu en challenge pa ly eflec ed he magni ude o he
glu en dose.
We we e in e es ed in wide a ia ions in glu en-induced
DVh:Cd ac oss he da a se , which we obse ed. The ques-
ion we in es iga ed in his s udy was whe he mucosal
damage co ela ed wi h an immune esponse, i espec i e
o he nominal amoun o glu en adminis e ed daily.
Genes Di e en ially Exp essed in B and T Cells
The expe imen al design was o isola e only CD19þB
cells and CD3þT cells om whole blood, which enabled a
simplified analysis o gene exp ession da a ocusing solely
on genes highly en iched in B and T cells. Wi h his design,
ou goal was o educe he noise associa ed wi h analyzing
global gene exp ession. Fo his pu pose, we used wo
published gene lis s de i ed om DNA mic oa ay analyses
o ac iona ed human leukocy es (Bindea e al
33
and
Newman e al
34
). The o e lap in he B-cell gene lis s
be ween Bindea e al
33
(23 genes) and Newman e al
34
4 Ga be e al Cellula and Molecula Gas oen e ology and Hepa ology Vol. 4, No. 1
Table 1.Pa ien Da a
Pa ien
ID
Vh:Cd
( ime,
0 days)
Vh:Cd
( ime,
42 days)
DVh:Cd
( ime 0 -
ime 42)
Glu en
challenge,
g/day
Age
ange, ySex
An i–TG2-IgA
( ime, 0 days)
An i–TG2-IgA
( ime, 42 days)
An i–TG2-IgA
old change
An i–TG2-IgA
posi i i y
Z 3.1 0.2 2.9 6 41–50 F 7 216 31 Pos
X 3.1 0.2 2.9 6 51–60 F 6 8 - Neg
AV 3.1 0.2 2.9 3 61–70 F 17 183 11 Pos
O 3.5 0.7 2.8 6 51–60 F 8 146 18 Pos
P 2.7 0.2 2.5 6 41–50 F 11 227 21 Pos
AS 3.2 0.7 2.5 3 51–60 M 11 8 - Neg
M 2.9 0.5 2.4 6 61–70 M 12 19 - Neg
Y 2.8 0.4 2.4 6 51–60 F 10 10 - Neg
G 2.6 0.3 2.3 6 31–40 M 6 240 40 Pos
W 2.6 0.3 2.3 6 41–50 F 6 53 9 Pos
AF 2.7 0.4 2.3 3 41–50 M 23 40 - Baseline pos
R 3 0.8 2.2 6 61–70 F 7 10 - Neg
AW 2.8 0.6 2.2 3 41–50 F 15 85 6 Pos
K 2.4 0.3 2.1 6 41–50 F 15 282 19 Pos
CM 2.8 0.7 2.1 1.5 41–50 F 10 72 7 Pos
AQ 2.4 0.4 2 3 61–70 F 10 22 2 Pos
AY 2.6 0.6 2 3 61–70 M 12 36 3 Pos
AC 2.3 0.3 2 3 61–70 M 7 11 - Neg
V 2.6 0.7 1.9 6 61–70 F 10 133 13 Pos
CX 2.6 0.7 1.9 1.5 61–70 F 12 34 3 Pos
AP 3 1.1 1.9 3 51–60 F 8 88 11 Pos
CS 2.5 0.6 1.9 1.5 61–70 F 9 8 - Neg
AH 2.7 0.9 1.8 3 41–50 F 10 114 11 Pos
H 2 0.4 1.6 6 51–60 F 109 365 - Baseline pos
AA 2.5 0.9 1.6 3 51–60 F 25 59 - Baseline pos
AG 2.5 0.9 1.6 3 51–60 M 11 23 2 Pos
BK 2.6 1 1.6 3 41–50 M 14 24 2 Pos
S 2.8 1.2 1.6 6 61–70 F 11 14 - Neg
AN 2.9 1.3 1.6 3 41–50 F 16 5 - Neg
AU 1.7 0.2 1.5 3 61–70 M 22 21 - Baseline pos
DK 3 1.5 1.5 1.5 61–70 F 9 8 - Neg
AL 2.7 1.3 1.4 3 51–60 M 11 52 5 Pos
AI 2.5 1.2 1.3 3 51–60 F 8 95 12 Pos
AK 2.9 1.6 1.3 3 61–70 F 15 7 - Neg
AE 2.4 1.1 1.3 3 71–80 M 8 67 8 Pos
T 2.9 1.7 1.2 6 61–70 M 7 15 - Neg
AD 2.3 1.1 1.2 3 51–60 F 50 41 - Baseline pos
DF 2.6 1.5 1.1 1.5 51–60 M 6 4 - Neg
L 1.4 0.3 1.1 6 61–70 M 23 209 - Baseline pos
AM 3.3 2.2 1.1 3 51–60 F 6 6 - Neg
CE 1.3 0.4 0.9 1.5 61–70 M 10 21 2 Pos
DC 2.9 2 0.9 1.5 51–60 F 7 5 - Neg
CA 3.6 2.8 0.8 1.5 31–40 F 8 7 - Neg
DZ 3.2 2.4 0.8 1.5 61–70 F 6 5 - Neg
CG 2 1.3 0.7 1.5 61–70 F 6 8 - Neg
DQ 2.9 2.2 0.7 1.5 61–70 F 4 3 - Neg
DY 3 2.3 0.7 1.5 61–70 F 7 5 - Neg
DB 2.9 2.2 0.7 1.5 41–50 M 9 6 - Neg
C 1.1 0.5 0.6 6 21–30 F 74 239 - Baseline pos
July 2017 B Cell Gene Signa u e in Celiac Disease 5

(38 genes) was 15 genes. The o e lap in he T-cell gene lis s
be ween he sum o all 11 T-cell subpopula ions om
Bindea e al
33
(185 genes) and he single T-cell lis om
Newman e al
34
(99 genes) was 33 genes. The wo s udies
ha e mo e ag eemen han one would expec by chance bu
less han ideal ag eemen in defining di e en ial B- and T-
cell gene exp ession. Lymphocy es a e di e se, he me hods
used o pu i y hese di e se cells a y, and di e en ial gene
exp ession is a ela i e measu e ha compa es exp ession
wi h o he leukocy es. Gi en hese complexi ies, i is bene-
ficial o use bo h s udies o define he mos ele an genes.
We s a ed wi h comp ehensi e B- and T-cell gene lis s,
and ul ima ely se ou o efine hese lis s o ob ain a gene
signa u e ha co ela ed mo e specifically wi h
glu en-induced in es inal inju y in CeD.
The B- and T-cell gene lis s (see he Ma e ials and
Me hods sec ion and Supplemen a y Table 1) ob ained om
each publica ion we e used sepa a ely o analyze he CeD
da a se .
DVh:Cd Co ela es Wi h B-Cell Gene Exp ession
By using DNA mic oa ays, we measu ed genome-wide
gene exp ession in a pu ified pool o B and T cells aken
om each pa ien a baseline and a he 6-week ime poin s.
We calcula ed he ne change (baseline o 6 weeks) in B- and
T-cell gene exp ession o each o he published B- and
T-cell gene lis s and co ela ed his change wi h he ne
change in Vh:Cd.
Resul s showed ha he ne change in exp ession o
B-cell genes om Bindea e al
33
(co . -0.54; P¼8.7E-07)
and Newman e al
34
(co . -0.52; P¼1.9E-06) co ela ed
(co .) s ongly wi h DVh:Cd (Figu e 2Aand B). I was a
nega i e co ela ion, which means ha B-cell genes we e
exp essed ela i ely s ongly in pa ien s wi h li le o no
DVh:Cd and ela i ely poo ly in pa ien s wi h la ge DVh:Cd.
Two gene lis s om Bindea e al
33
ep esen ing T-cell sub-
popula ions showed weak posi i e co ela ion o DVh:Cd,
including Th1 (co . 0.34; P¼0.2.58E-03) and Th2 (co .
0.31; P¼7.21E-03). In his case, opposi e o B-cell genes,
Table 1.Con inued
Pa ien
ID
Vh:Cd
( ime,
0 days)
Vh:Cd
( ime,
42 days)
DVh:Cd
( ime 0 -
ime 42)
Glu en
challenge,
g/day
Age
ange, ySex
An i–TG2-IgA
( ime, 0 days)
An i–TG2-IgA
( ime, 42 days)
An i–TG2-IgA
old change
An i–TG2-IgA
posi i i y
CN 3.1 2.5 0.6 1.5 71–80 F 14 11 - Neg
AT 2.3 1.7 0.6 3 41–50 M 25 70 - Baseline pos
DG 2.9 2.5 0.4 1.5 61–70 F 3 3 - Neg
U 2.5 2.1 0.4 6 51–60 F 15 32 2 Pos
DH 3.3 3 0.3 1.5 61–70 F 6 4 - Neg
DW 2.6 2.3 0.3 1.5 61–70 M 8 6 - Neg
A 2.9 2.6 0.3 6 41–50 F 47 43 - Baseline pos
CB 3 2.7 0.3 1.5 31–40 F 17 32 2 Pos
CJ 3 2.8 0.2 1.5 61–70 F 8 7 - Neg
DR 2.9 2.7 0.2 1.5 61–70 F 8 6 - Neg
AO 2.9 2.8 0.1 3 51–60 M 14 16 - Neg
DJ 2.7 2.6 0.1 1.5 61–70 F 16 11 - Neg
Q 2.8 2.7 0.1 6 61–70 M 13 32 2 Pos
N 2.7 2.7 0 6 51–60 M 8 10 - Neg
DN 2.3 2.3 0 1.5 61–70 M 14 12 - Neg
DP 2.8 2.8 0 1.5 51–60 M 7 6 - Neg
DM 1.3 1.4 -0.1 1.5 41–50 F 13 12 - Neg
AJ 2.5 2.6 -0.1 3 51–60 F 13 16 - Neg
DX 2.9 3.1 -0.2 1.5 61–70 F 10 6 - Neg
CT 2.8 3 -0.2 1.5 71–80 M 15 23 2 Pos
AR 2.3 2.6 -0.3 3 51–60 M 13 22 2 Pos
DA 2.4 2.8 -0.4 1.5 51–60 F 36 22 - Baseline pos
AX 2.8 3.3 -0.5 3 51–60 M 20 19 - Baseline pos
AB 2.3 3.4 -1.1 3 41–50 M 9 9 - Neg
NOTE. Pa ame e s associa ed wi h he glu en challenge included he amoun o glu en inges ed daily o 42 days, he age
ange (median, 59 y; ange, 23–74 y), an ise a di ec ed agains TG2-IgA (an i-TG2-IgA) exp essed in in ensi y uni s o bo h
baseline and 42 days, and an i–TG2-IgA ha was posi i e (pos) o nega i e (neg) abo e h eshold (20 in ensi y uni s) a 42
days. Se e al pa ien s we e abo e h eshold o an i–TG2-IgA a baseline (baseline pos). An i–TG2-IgA old change was
exp essed in in ensi y uni s, which may o may no eflec linea i y pending assay alida ion.
F, emale; M, male.
6 Ga be e al Cellula and Molecula Gas oen e ology and Hepa ology Vol. 4, No. 1
T-cell genes we e exp essed ela i ely s ongly in pa ien s
wi h la ge DVh:Cd and ela i ely poo ly in pa ien s wi h
li le o no DVh:Cd. O he T-cell gene lis s defined by Bindea
e al
33
showed li le o no co ela ion, including T CD8þ,T
helpe , T, T cen al memo y, T e ec o memo y, T ollicula
helpe , T gd, and Th17. The T-cell gene lis om Newman
e al
34
also showed no co ela ion o DVh:Cd (co . 0.10;
P¼3.92E-01). No su p isingly, gene lis s co esponding o
o he leukocy es showed li le o no co ela ion because B
and T cells we e he only cells analyzed in his s udy. The
Uni e si y o No h Ca olina IgG gene signa u e,
35
which
con ains se e al known B-cell genes ha p edic a o able
ou comes in b eas cance pa ien s, showed a mode a e
nega i e co ela ion wi h DVh:Cd.
Nea ly iden ical esul s we e ob ained i espec i e o
whe he mul iple mic oa ay p obes co esponding o one
gene we e educed o a single p obe (Figu e 2Aand B)o
whe he all p obes we e analyzed (Figu e 2Cand D) (see
he Ma e ials and Me hods sec ion).
In addi ion o DVh:Cd, we co ela ed gene exp ession o
baseline and end-o -s udy (week 6) Vh:Cd o all 73 pa-
ien s. Resul s showed ha he ne change in exp ession o
B-cell genes om Bindea e al
33
(co . 0.49; P¼.1.16E-05)
and Newman e al
34
(co . 0.46; P¼4.71E-05) co ela ed
wi h end-o -s udy Vh:Cd (Figu e 2Eand F) and was only
sligh ly weake han ha obse ed o DVh:Cd. The co e-
la ion was posi i e, which sugges s ha a educ ion in B-cell
gene exp ession o e he 6-week glu en challenge (end-o -
s udy minus baseline) was associa ed wi h a smalle end-o -
s udy Vh:Cd. Th1 (co . -0.31; P¼.0084) and Th2 (co .
-0.30; P¼.011) gene exp ession was co ela ed nega i ely
wi h he end-o -s udy Vh:Cd, albei modes ly, which is
consis en wi h esul s ob ained om he DVh:Cd analysis.
The e was no co ela ion o gene exp ession wi h baseline
Vh:Cd (Figu e 2Gand H).
Se um An i-TG2 Co ela es Wi h DVh:Cd and he
Co e B-Cell Gene Module
Six y- wo o 73 pa ien s (85%) we e nega i e a
baseline o se um an ibodies di ec ed agains TG2
(an i–TG2-IgA) (Table 1, h eshold o posi i i y was 20
in ensi y uni s). Fo hose 62 pa ien s who we e nega i e
a baseline, 42% se ocon e ed o a posi i e alue o e
he cou se o a 6-week glu en challenge. By using a S u-
den es (unpai ed, 2-sided) o compa e means, and
excluding baseline-posi i e pa ien s, we de e mined
ha an i–TG2-IgA posi i i y co ela ed wi h DVh:Cd
(P¼.0029) and he co e B-cell gene module (P¼.0022).
These esul s showed ha an i-TG2 co ela ed wi h B-cell
gene exp ession as well as i did wi h DVh:Cd. A posi i e
an i-TG2 was associa ed wi h educed B-cell gene
exp ession and inc eased in es inal damage o e he
cou se o 6 weeks.
A Subse o B-Cell Genes D i es Co ela ion
o DVh:Cd
In he co ela ion analyses, p obes we e a e aged o
gene a e a mean exp ession p ofileac oss heen i egene
lis , which hen was co ela ed o DVh:Cd; indi idual genes
o p obes we e no analyzed sepa a ely. We asked whe he
all o a ac ion o he genes in he wo published B-cell
gene lis s om Bindea e al
33
and Newman e al
34
co e-
la ed wi h DVh:Cd. To define a co e se o B-cell genes ha
d i es he co ela ion, all p obes in he wo B-cell gene
lis s we e consolida ed in o a single lis and hen he
p obes we e anked sepa a ely based on hei abili y o
co ela e wi h DVh:Cd.The ewe e63uniquep obesbe-
ween he wo B-cell gene lis s, ep esen ing 48 unique
genes. O hese, 28 p obes ep esen ing 24 unique genes
significan ly co ela ed wi h DVh:Cd (P<.01). We defined
hese 28 p obes as a co e B-cell gene module ep esen ing
a subse o known B-cell genes (see Table 2 o genes). The
gene SPIB, p esen in bo h he Bindea e al
33
and Newman
e al
34
B-cell gene lis s, showed he s onges co ela ion o
DVh:Cd (co . -0.56; P¼2.5E-07) (Supplemen a y
Table 2).
Exp ession le els o all 28 p obes in he co e B-cell gene
module we e a e aged ac oss he da a se , and he mean
exp ession p ofile was co ela ed o DVh:Cd. The co e B-cell
gene module co ela ed s ongly wi h DVh:Cd (co . -0.59;
Figu e 1. Glu en-dependen in es inal mucosal inju y as a clinical end poin in a human clinical s udy. Vh:Cd, a his ologic
measu e o mucosal heal h, was de e mined a baseline and a he 6-week ime poin . DVh:Cd, defined as baseline minus he
6-week Vh:Cd, ep esen s in es inal damage (posi i e numbe ) o healing (nega i e numbe ) o e he 6-week ime ame. The
ba g aph shows he numbe o pa ien s (y-axis) wi h a gi en DVh:Cd (x-axis) o a o al o 73 pa ien s. The numbe o pa ien s
o a gi en DVh:Cd was subdi ided u he o indica e he amoun o glu en inges ed daily pe pa ien , which was 6 (blue, 20
pa ien s), 3 (yellow, 26 pa ien s), and 1.5 (g ey, 27 pa ien s) g ams o glu en daily o 6 weeks.
July 2017 B Cell Gene Signa u e in Celiac Disease 7
P¼3.3E-08). As expec ed, all 28 single p obes co ela ed
s ongly wi h he mean exp ession p ofile (Supplemen a y
Table 2). In con as , o he emaining 35 B-cell p obes
(24 unique genes) ha co ela ed poo ly as single p obes
(P>.01), in he agg ega e, he mean exp ession p ofile
ac oss all 35 p obes also co ela ed poo ly (co . -0.24; P¼
3.8E-02). We e e o he published B-cell genes ha
co ela ed poo ly wi h DVh:Cd as he non-co ela ing B-cell
gene lis (see Table 2 o genes). Co ela ions and P alues
compa ing he ela i e pe o mance o ele an gene lis s
a e summa ized in Table 3.
Poin plo s showed he sca e associa ed wi h he ela-
ionship be ween DVh:Cd and he ne change in gene
exp ession o he single-p obe SPIB (Figu e 3A) and he
mean exp ession o all 28 p obes in he co e B-cell gene
module (Figu e 3B). Se en y- h ee pa ien s we e sca e ed
Figu e 2. Spea man ank
co ela ion o gene sig-
na u es wi h he ex en o
glu en-induced in es inal
inju y. Gene lis s ob ained
om h ee publica ions
co esponded o B- and T-
cell popula ions, o he leu-
kocy es, cance cells, and
no mal mucosa (y-axis). All
gene lis s we e ob ained
om Bindea e al,
33
excep
o B- and T-cell lis s om
Newman e al
34
(as indi-
ca ed) and he Uni e si y o
No h Ca olina (UNC) IgG
clus e om Fan e al.
35
(A)
The mean exp ession p o-
file o a gi en gene lis was
co ela ed wi h DVh:Cd
and he co ela ion was
epo ed on a scale o 0 o
1(x-axis). Mul iple mic o-
a ay p obes co espond-
ing o a single gene we e
consolida ed o a single
p obe by aking only he
p obe wi h he g ea es SD
o exp ession ac oss he
73 pa ien s. (B) Signifi-
cance o each co ela ion
in panel Awas exp essed
as a P alue. Gene signa-
u es also we e co ela ed
wi h (C)DVh:Cd, (E) end-
o -s udy Vh:Cd, and (G)
baseline Vh:Cd using mean
exp ession p ofiles and all
p obes ep esen ing a
gi en gene. Significance
o each co ela ion in
panels C,E, and Gwas
exp essed as a P alue in
panels D,F, and
H, espec i ely. aDC, ac i-
a ed dend i ic cell; DC,
dend i ic cell; iDC, imma-
u e dend i ic cell; NK,
na u al kille ; Tcm, T
cen al memo y; Tem,
T e ec o memo y; TFH,
T ollicula helpe ; Tgd, T
gamma del a.
8 Ga be e al Cellula and Molecula Gas oen e ology and Hepa ology Vol. 4, No. 1
ai ly uni o mly on bo h sides o he eg ession line o e he
en i e spec um o in es inal damage.
The Ex ended Co e B-Cell Gene Module
Genes ep esen ing he co e B-cell gene module and he
non-co ela ing B-cell gene lis we e ob ained exclusi ely
om he Bindea e al
33
and Newman e al
34
cu a ed gene
lis s. We asked whe he he co e B-cell gene module could
be used o disco e addi ional disease- ele an genes ha
we e no included in he Bindea e al
33
and Newman e al
34
published gene lis s. To his end, we ook he Spea man
co ela ion be ween he mean exp ession p ofile o he co e
B-cell gene module and all 20,624 p obes in he CeD da a
Figu e 2. (con inued).
July 2017 B Cell Gene Signa u e in Celiac Disease 9
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Recei ed Oc obe 24, 2016. Accep ed Janua y 20, 2017.
Co espondence
Add ess co espondence o: Mi chell E. Ga be , PhD. e-mail:
[email p o ec ed]om.
Acknowledgmen s
This wo k benefi ed om da a assembled by he Immgen conso ium.
Conflic s o in e es
These au ho s disclose he ollowing: Ma kku Mäki is a scien ific ad iso o
ImmusanT, Inc, and Celimmune LLC; Daniel Adelman is a scien ific ad iso
o ImmunogenX, Inc. The emaining au ho s disclose no conflic s.
Mi chell E. Ga be concep ualized he s udy; Mi chell E. Ga be , Alok Saldanha,
Joel S. Pa ke , and Ma kku Mäki we e esponsible o he me hodology; Alok
Saldanha and Joel S. Pa ke we e esponsible o he so wa e; Alok
Saldanha alida ed he s udy; Mi chell E. Ga be , Alok Saldanha, Joel S.
Pa ke , and Wendell D. Jones pe o med he o mal analysis; Mi chell E.
Ga be , Kaija Lau ila, and Ka i Kaukinen pe o med he in es iga ion;
Mi chell E. Ga be , Alok Saldanha, Joel S. Pa ke , Ma ja-Leena Lähdeaho,
Daniel C. Adelman, and Ma kku Mäki p o ided esou ces; Mi chell E. Ga be ,
Alok Saldanha, Joel S. Pa ke , Wendell D. Jones, and Kaija Lau ila cu a ed
da a; Mi chell E. Ga be w o e he o iginal d a ; Mi chell E. Ga be , Wendell
D. Jones, Pu esh Kha i, Chai an Khosla, Daniel C. Adelman, and Ma kku
Mäki e iewed he w i ing and edi ed he pape ; Mi chell E. Ga be , Alok
Saldanha, Joel S. Pa ke , and Wendell D. Jones we e esponsible o
isualiza ion; Mi chell E. Ga be , Chai an Khosla, Daniel C. Adelman, and
Ma kku Mäki supe ised he s udy; Mi chell E. Ga be , Daniel C. Adelman,
and Ma kku Mäki we e esponsible o p ojec adminis a ion; and Mi chell E.
Ga be , Alok Saldanha, Joel S. Pa ke , Chai an Khosla, Daniel C. Adelman
and Ma kku Mäki acqui ed unding. Ca ego ies we e defined by CRediT.
53
Funding
This s udy was unded by Al ine Pha maceu icals, Inc, and g an s om he
Ame ican Reco e y and Rein es men Ac ( o Al ine Pha maceu icals), om
he Compe i i e S a e Resea ch Financing o he Tampe e Uni e si y
Hospi al (M.M. and K.K.), om he Academy o Finland Resea ch Council o
Heal h (K.K.), and in pa om he Na ional Ins i u es o Heal h (R01
DK063158 o C.K.). M.E.G. was suppo ed pa ime du ing he w i ing o his
manusc ip by he Na ional Ins i u es o Heal h (R01 DK063158 o C.K.). All
au ho s cu en ly ha e no financial o nonfinancial compe ing in e es s in
Al ine Pha maceu icals, which had a ole in he clinical s udy design and
collec ion o biopsy and blood samples, bu no ole in da a collec ion,
analysis, o in e p e a ion associa ed wi h he biopsies, blood samples, and
genomic da a used in his s udy. Al ine Pha maceu icals had no ole in
w i ing his epo , o he decision o submi he epo o publica ion.
July 2017 B Cell Gene Signa u e in Celiac Disease 17