This article isprotected rights by All copyright. reserved. 10.1002/art.40doi: lead todifferences version between this andthe ofPleasec Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination This article undergone has for and buthas accepted been not review publication fullpeer directorsInc. of Atreca, Competing interests and Funding *To whom correspondence be should addressed: † 4 3 2 1 Affiliations: PhD NithyaLingampall Authors: Articletype : Full Length WILLIAMDR. ROBINSON (Orcid ID : 0000 Fred Research Hutchinson Cancer WA. Seattle, Center, VA Health Palo Alto Care System, CA. PaloAlto, ofImmunologyDivision and University, Rheumatology, Stanford ImmunologyStanford Stanford Program, University, CA. Stanford, AcceptedAuthors contributed equally study. tothis Article differentially drive U01 AI101981 4 , William H.Robinson T cell :
This researchwas supported by NIH Daniel R
- dependent affinity maturation i 578 . , BS . L .
: u W.H.R. is aconsultant is to,ownsequityW.H.R. ontheboard of in,andserves
, 2
,3 PhD
, Glanville Jacob autoreactive B cell responses in rheumatoid arthritis ,
MD PhD 1 ,2 ,3 † , Andrew N 1 - 0002 , 2,3 *
,
- PhD 0841 . McDavid \
1 NIAMS AR063676 R01 - , Chia 7148) w.
and and innate immune pathways
- , Hsin Ju
PhD
4 † , ,
Sarah Kongpachith PhD Stanford, CA. Stanford,
2 , 3 , Raphael Gottardo , U19 AI11049103
ite this a ite this
, rticle as
PhD 1
, ,2
,3
, This article isprotected rights by All copyright. reserved. arise from molecular twodifferent mechanisms. programs O Conclusion transcriptional n regulatory analysisCoexpression stimulate dependent downregulation hypermutation cell targeting tetramer of controls. andACPA RF Results expressed antibodies asand testedfor monoclonal immunoglobulin single developedWe Method mechanisms. investigate andBACPA RF cells molecular whether through breakdistinct tolerance which tolerance inthese Bincompletely is broken remain cells un citrullinated protein antibodies (ACPA) and factorsmechanisms rheumatoid butthe by (RF), R Objective Abstract heumatoid arthritis (RA) Acceptedur findings Article stages - cell RNA
Characterization of patient , rapid
. ACPA B cells utilized Bcells . ACPA suggesting that suggesting that responses
suggest
s antigen B cellsB were memoryreactivation
relative to RFrelative B to cells of CD72 and of CD72 -
seq repertoires uencing - .
specific Bcells atbothantigen specific that Inc - revealed tetramers to isolate ACPA toisolate tetramers ontrast, ACPA andACPA RFcells B
these autoantibodies inRA associatedwithincreased inflammation is characterizedactivation byof the etworks. enriched
and transcriptional programs. on 2,349 B cellson 2,349 from upregulation
that ACPA RF BRF cells
more class
- derived m through in the , and thesebyaccompanied differences were the - of genes thatpromote genes of peripheral peripheral
and RF
expressed transcriptional programsthat expressed - multiple innate immune pathwaysmultiple switch onoclonal antibod
are imprinted with
-
autoantigen and RF six patientsRA six - - ed more isotypes and exhibited
inexperienced and affinity B cell b lood of RA patients relativelood ofRApatients tohealthy
- Prominent producing B cells and cells Bproducing enriched genes belonggenesenriched todistinct
reactivity. B cellsB ies
class derstood. Herewe derstood.
to
distinct distinct confirmed ACPA and ACPA confirmed RF
immunoglobulins analyze their
- that switch
produce anti transcriptional ing - . matured B matured
and performed
somatic
T cell - were - -
This article isprotected rights by All copyright. reserved. findings identified producing stages of to organizeB by cells subtype w By using recover seropositive Here mediateddistinct by regulation reasonedwe that by pro promotesynovial inflammation and jointdestruction in mouse models activated challenges develop which survival by BCR signaling and costimulatory signals understanding remains B cell cell cytokines,and provide increasedactivity disease scores inRA autoantibodies (ACPA) and factor rheumatoid (RF) Auto R heu e
Acceptedthe continual Article ducing developed - depleting , we developed ,
antibodies area ofRA hallmark matoid arthritis (RA) synovitis ischaracterized and by chronic activation requires coordination of can be
and differentiation following acute the suggest that in
a subset of B cells with assessed with markersB surfacea subsetof cells and sequence
B cells continuouslyundergo of Bof cells distinct with
diverse paired BCR paired
RA patientsRA p
resent inotherautoimmune diseases therapies a LPS
uniquelycontribute tosystemic andare withassociated inflammation
regeneration of ACPA plasmablasts computational
the development
activation states of or or at
mechanisms
B cell antigen double different stages differentiationof and may tolerance mediated by two different molecular mechanisms must be must tolerance molecular by twodifferent mediated mechanisms limited microbial T cellT
in RA in variable region
and used of autoreactiveof B cells that would otherwise be
differentiation. inflammation - - stranded DNA programs tetramer staining reagents to to tetramer staining reagents
of howB cell help, contribute topathology as efficacy evidenced by the of
and characterize B cell classifier . (3)
infections
and persistence
. single
antigen (1) and include antiand include
many cell that distinguish RF from ACPA that distinguish , and dysregulated signaling eventscan promotethe
and (2)
. - (6,7)
cell tolerance is
Recent data suggest in mice, ( We applWe 4,5) .
- the driven Further, . - RNA In contrast, . RF generation hasbeen recapitulated through
- (“ the primary, antibody and secondary, extrinsic and intrinsic factors transcriptional
of in patients and chronic BCellNet providing evidence
ied
RF a - activation seq (scRNA broken B cells,B whichproduce -
these citrullinated protein antibodiescitrullinated protein nd ACPA ACPA are
isolate RF and ”)
(10) in RA. new methods to
inflammation
throughoutdisease that used thescRNA arise through
that ACPA that ACPA
profiles of individual Bprofiles cells. ofindividual . may - Based on seq) seq)
B cell
highly (8,9)
involve that RF joint destruction joint to deleted tolerized or
. Indeed, ACPA B cell activation is regulated
simultaneous specific for RA d ACPA
these observations, states
and RF and the loss toleranceof immunoglobulins
B cells becomeB cells differential (1)
RA andRA
auto response , , as evidenced , and
B cells can also antibodies, - seq profile seq profile our our - . - ly
and s
.
from
. O
RF,
ur B , This article isprotected rights by All copyright. reserved. bowtie2 (v.2.1.0) fromreads. raw Reads thehuman sequencing were genome aligned to Supplementary Materials. TruSeq dynabeads (LifeTechnologies) th Barcoded cDNA libraries werepooled and amplified template transcription of mRNA was autoantigen cells were considered ACPA sorted preparation as prevalent antigens citrullinated protein described in Stanford Blood Center Age factor of Rheumatologyclassification criteria 1987 for RA rheumatoid with individuals arthritis Stanford University accordingInvestigational protocolsapproved Board by tohumanat subject the Review Patients RA. broken and toactivateACPA RFcells B toproduceauto
Accepteden Article
- sonicated matched h matched
and/ - using a using V2 Single Library preparation and next Autoreactive cell B isolation design Study -
switched oligos containing and cell molecular identifiers and m and kits or - of Supplementary Materials Supplementary cyclic
. (Illumina) -
FACSAriaII - described in described cell RNA 5’ fragments5’ of cDNA molecules ealthy donors whoealthy were HIV negativefor donors or from were TB the obtained interest and d and interest ethods
(14)
citrullinated peptide (Supplem .
A . Duplicategenerated reads. by wereremovedand PCR final expression
(n=5) and
ll samples were and collectedconsent afterobtaining informed - seq pipeline primed using the
sequenced using theHiSeq2000 flow (Becton cytometer Dickinson) Supple
and uncoupled by + . ouble
RF entary1) Table -
or RF - mentary Materials - negative otherfor tetramerizedantigen the generation sequencing (n=6) . . . Rheumatoid Custom R scripts R extractedCustom cell and barcodes molecule Fourteen
+ (11) , and (iii) did not receivedid not , andB (iii) ACPA
STRT who
were prepared proteinwerecitrullinated tetramer for
were . Peripheral blood was obtained from . Peripheral was blood obtained r - estriction digest oligo -
(i) (i) if they double stained using PCR1 citrullinated peptidescitrullinated (400µ purified using MyOneC1 streptavidin - met at least the 4of 7American College factor tetramers were prep
, (ii) (12)
. Live CD3 , and cDNAs were barcoded using antibodies were sero .
( 2x100 Template - FWD
. (Supplementary Table 2)
L ibrar at puritycell single >99% )
- . and PCR1 CD14 Detailed methods arein the positive for rheumatoid positive
- and cell switching r ies were - ( positive for the positive -
- hg38 CD19 promote synovitis promote in depleting therapy - REV primers
constructed ) M) of highly
+ . everse ared
(13) B cells were
as using using .
and
using . . B
This article isprotected rights by All copyright. reserved. methods arein theSupplementa overlapping groups categories partial residual, whichthe is log substantiallyaffect transcription in the population of RA patients. standardiz differentially expressed if genesthat showed consistent differential expression across patients foraccount subtype. Furtherexplanation of plamablast (ASC),double negative, memoryand including Differential expression testing (9) monoclonal antibodies. Autoantigenwere described microarrays as performed previously D Immuno(Jackson determinedgenerating F(ab’) by IgL2IgG1IgKhuman with antibodies (Lake chains light reactivitywas or RF Pharma). from each generatedantibodies was first clustersautoreactiveCDR3 from Supplementary V, pr as described in was (v.1.2.25) calculated usingRSEM iagnostics). The for positivity threshold above 3SD was ofnegative themean control ovided
Accepted. Article D, and J regions;D, and J
Detailed methods areDetailed inthe Supplementary methods Materials. monoclonalRecombinant De novo Both Both models
a the most likelycellthe most type ed effect size
weredefined subset CDR3 cluster with complete cluster V CDR3
Supplementary Materials Materials
assembly of assembly of - stratified R .
P esearch). CCP bindingesearch). was CCP determinedusing ELISA CCP3 (Inova
artial residuals werefit inMAST a nd identification of clusters CDR3 nd identification
greater than 2. by o
they .
model, which rganizing the DEGs and 33 genes
. ( immunoglobulin V
i) - Wetested differential expression through expression aftercorrecting for nui ry Materials. were 2 fragments
for wereused antibody testing antibody
marginally differentially expressed at 10% and FDR T (16) any cell with anany cell with hestandardized effectsize selects geneslikely most to
account (15)
, version thetwo models is
- allow
region sequence reconstitution were sequenceregion reconstitution . The . The
, to calculatefunctional scores for each cell.
naïve and probing plates coated with human IgGandcoatedwith human probing plates s ing
H forprobability the of cells belonging to the 0.933 usingtwo a
BCellNet and V
subtypes us to estimateus to a classification . A preliminary ofcandidate list sc L
RNA w
-
regions of
in ere
and subtype was classification - Supplemen interest into
Both models -
sance performed asperformed described in seq profile
. The top VH . a global model Genes wereconsidered - ; classific step procedure factors. a series linear of models tary Materials 16 .
also returned a
ation of germlineation of
G potentially - ene functionalene VL pairings ,
which does not expresse to select function that . (
Detailed ii) trained
to beto had a d
-
Fc as
This article isprotected rights by All copyright. reserved. Accepted(Supplementary Figure 1 quantification donors and one healthy donor thenWe performed scRNA humoral autoimmunity. (18,19) individuals, and double subsets RA patientssignificant is compared healthyto controls. 0.14% rarein RA autoantigen tetramerization enabl previously demonstrated to reduce streptavidin previously demonstrated to be targeted by RA ACPA isolateTo RF and Article cells for flow cytometry to exam RA patients of Elevation Results FDR< expressed iftheir the CDR effects. by subtype and removing matrices 5% of theof .
Coexpression analysis. varies . The
scRNA Detaile blood - consistent - negative(DN) memory) and CD27
presence RFof and ASCs ACPA inRAblood epitopes Pearson correlation matrices bound fluorophores (Fig were compared within the16 genesets sampled .
naïve, memory, antibody and between and computational reconstitution theof paired BCR for each Bcell In study, this
d methodsin are theSupplementary Materials. and virtuallyundetectable in healthy -
ACPA average corr seq
ed thedetection of
B cells analysis analysis (10,17) with previous observations defectiveof central and peripheral
B cells, biotinylated human IgG
patient ) . The ine the frequency marginal coexpression - seq on seq on , respectively w . Analysis of the elation population of usi s e 487 genes interestof (Fig , autoreactiveB sample ng a modified STRT sorted
the false ure ure differed from the
1A 1B). Autoantigen labeling dual with fluorophores d B cells expressing
ACPA werecalcu (Fig
blood ) - . Tet of positive detection fluorophoreof
+ ure B cell
(classical memory)compartments
, RF, and tetramer ACPA andACPA RF - -
cells from the CD27
due subtype,to tetramerspecificity, patient, and secreting autoreactive cells B cells provide
1C B cells fromcells B
frequencies reveal that
). . were lated - were tested for coexpression
Seq method that enables
Gene pairs wereconsidered differentially controls T -
Fc his small enrichment of RF and cells ACPA in reveals that While thefrequency RFof and background as well as individually for each pair of genes BCRs with moderate affinity for
s , comprising
a baseline seropositive
B cell - negative(Tet
- RA 14 correlation coefficient
( naïve, coupled to two sets of s
citrullinated peptides patients
as well asas well
to to o RF n average 0.2 non compare RA patientsRA - and cells ACPA reactiveB cells, - single ) have -
and affinity matured B (Fig are
in in
after stratifying cells from RA to isolatethese
ure t presentin all
active ongoing tolerancein molecule the blood of the blood of heaverages of characteristics ACPA
1A) 5% was
using at an
B cell
and are and
,
RA
This article isprotected rights by All copyright. reserved. ahave significantimpact on further our analyses theseof c inherentto transcriptomeanalysis (Figure1D). TheBCellNet du andcells RFB despite We observed a general correlation between serological autoantibody levels and the recovery CIT of subsequent analysis continuously light this,of rather than using quantized assignments to the mostlikely subtype, utilizewe the discrepancies significan the flowsorting discrepanciesbetween the fractions flow and classifier fractions whilealso parsing subtype ofrho 0.56 correlation Applying suchSEC61G as and EIF5A, were previously described in cytometric gating. In total, 159 genes (<2.5%) servea probability scorefor each cell, improving upon the scheme classifier various BCellNet ( 4.8E4 molecules percell with substantial no differencesbetween experimental groups o Supplementary Figure 2 f the autoreactivepopulations.
Acceptedring FACS. Article B cell allows usto considera continuum of differentiation states
we developedwe BCellNet predicts - s 0.89),indicating the that classifierrecapitulates flow scRNA
between
-
We alsoWe observed an enrichment in the classification of memory cells through valued posterior probabilities subtypeof membershipstratifying as co subsets to RA pathology, we and
- peripheral to heterogeneous B cells seq
( classification error from Supplementary T subtypefractions assessedflow by cytometry and the classifier (Spearman’s minor . , tly tly altered B cell sub The remaining bias inclassification through th
termed ).
-
dependent gene effects are novel
variancesin thenumber cellsof sorted dueto stochastic selection bulk B B cell BCellNet After aligning and deduplicating reads, werecovered an average of - level studies, such as MS4A1
subsets ( Supplementary able .
P organize
4 redicting subtypewith this transcriptomicclassificati us - isolated type classification (seeSupplementary Methods). ) BCellNet .
ing scRNA classifier
quantized subset identifications available in flow d ( from RAsamples Supple
B by cells subtype s predictors Fig .
However, dowe notbelieve these - ure3 seq profiles
accounted for much of the variability ells (see Supplementa mentary Figure 4
).
and
, likely
cytometry
( Supplementary T
TNFRSF13B (20) . is method likelymost did not demonstrates To evaluatethe contribution of
due to using a
and assignsubset a - identified ). sampling variability in The B cell transcriptomic
(21) ry re arere
able Methods)
variates for positive , while others
populations
3 certain ) .
Some .
In
on on , This article isprotected rights by All copyright. reserved. and a recognize CCP3 and only peptide(CCP3), a citrullinated peptide mimic used to diagnoseRA the 30 monoclonal antibodies from IgG cells b (Fig examinewhether autoreactivitypresent is in antigen CDR3 clustersas recombinantmonoclonal antibodies identifyto We populations. convergence suggests that between RFand Tet in 1 cells/cluster; P< significantly larger than Tet whileif all multiplicity w determineTo if the size of the CDR3 clusters thereforeputatively derive from thesame progenitor clus (Fig clustered investigateTo how affin an maturation variable BCR analysis reveals CDR3 convergence and autoantigen targets e comparede the number cellsof per
Accepted Articletibodies expressed from reconstituted V - ure ure
ter ter membership among cells that then Fc, and none of of protein microarray coated with over 300 i nd to 3 patients s 2A ands 2A 2B, - region genes expressed by indi expressed 42 representative RFand RF, ACPA, and Tet N . Supplementary Figure between autoreactivepopulations
I
human cells werein a single cluster,the multiplicity would be f each cellfrom a tetramergroup clustered separately, then the multiplicity would be 1, 0 B
.002 under hypergeometric sampling) (multiplicity ) ( ) of of the Supplementary Table
- IgG
multiplicities - antibodies from ity maturation shapescell B responses Fc two , whereas ACPA B ACPA cells - -
multiplicitiesin 2 B cells sharing >80% Levenshtein similarities intheir CDR3 sequences
of of the 12 RF antibodies b difference 6 areobserved ) citrullinated antigen . This represented
only only
exhibit identical germlinegenes and CDR3 lengths tetramergroup vidual B cells undergo extensiveaffinity maturation relative to Tet Tet 0 7 one .17 cells/cluster; P ). H /V -
Eleven B cells can
ACPA of of the 30 antibodiesfrom of L
and sequences RA autoantigens . 4 patients4 (multiplicity differences1.3 and 0.3 Hence,the measurethe
functional ch BCRs b out of the 12 antibodies , enabling i - nd to IgGnd to
inexperienced and (Supplementary Table 6) in each i the stringentmost , nd CCP3 nd CCP3 (Fig
and significantly larger than RFmultiplicities -
sorted cells b from the prominentmost V
( Supplementary Figure elevated <0.
CDR3 clusterto calculate thecluster magnitude of
- integrated againstthe autoantigen 04) Fc , we identif aracterization of recombinant their .
. (Fig
. Next, w N
N ure No significant differences o . frequency CDR3of
ure ne of the Tet i autoantigen targets ACPAcell B nd to
2 ACPA C -
experienced 2C) threshold analysis of ). e recoverede y
UsingCCP3 the ELISA
convergent evolution, cloned from RF cyclic citrullinated antigen hits . - We findalso that sorted cells b
(22)
5
m citrullinated - ).
antibodies . that p ultiplicities
H affinity
- V B cell s paired , , L
and we pairings and
reserved and i -
nd to stages sorted -
and RF BCR are 13 of
This article isprotected rights by All copyright. reserved. C different modules developmental stage(s) score for represents residual expression attributableto antigen specificity, modules basedupon previouslypublished literature. signat DEGs Performing glob both gene on expression) and subset RF responses, weapplied a elucidate the transcriptional profiles underlying the BCR differencesbetween RF and ACPA Functional B cells tend switching, havehigher mutation rates, and showevidence increasedof CDR3 convergence,while RF mutation rates than Tet mutated from germlinethan Tet differ being class mutation rateadjusted for subtype. used a mixed Because CSR through class whetherduration the affinityof maturation differs between ACPA RF and ACPA B cells differ in class matured isolation of consistent with previous antibodies fromcloned ACPA B cells werecross for 25 of the 30 citrullinated a ) . / Accepted Article ACPA
ence betweenence Tet than with either method alone ures.
cells, ACPA and Tet -
differential regulation To examinethe switched than Tet RF and ACPA B cells to useunswitched isotypes and and mutation rates vary between subtypes,which themselves vary by specificity, we - - effects logisticregressio switch recombination (CSR) rates and accumulated somatic mutations the on BCR. implicating dysfunctional central and peripheral tolerancein RA , RFand T -
B cell were concentrated within thenaïve al and subset
- - populati
where
and RF hurdle model to identify et B cells reports -
cumulative cells - ntigen
B cells differential
. Notably, - (Fig B cells (Figures ons stratified comparisons
- for each subtype
B cells, while (9)
- in the primary and secondar - switch and somatic mutation frequencies ures stratified analyse
. - for all patients W . Both gene sets
sorted antibodies (Fig and 3.7 This confirms thatthe RF and ACPA tetramer reagents n to measure theunique effect antigenof on e f functional
3C 3C and D i RF and ACPA nd that autoimmune
have fewermutations. - fold
ACPA 3A and3A - reactive with non ).
454 ACPA impact ( greater SupplementaryFigure
Thus, at global (
s
B cell were The partialThe residual differentially expressed allowed usto identify
B) and memory compartments
B cells (Fig B
regulation occurs
. Wealso find that ACPA of theof DEGs, we organized DEGs into cells haveover 3.8 odds compared to RF
s relevant to identifying autoreactiveB cell ure ure non i.e.
are in
2 4 B cel y - D A
significantly regressing the out effect subsetof RF and ACPA B
). ) ( was used to -
citrullinated antigens,is as
and RF Bcells, as measured We doWe note thatseveral Supplementary ls undergo isotypemore class all subsets, inclu
for each gene,which 7 . ) The m , pinpointing the RF B cells - a greate fold greater odds of ha . genes(DEGs) calculatea module We .
ve ve
B cells but - cell ost ost significantly
1% next askednext
Tables 8 and 9 CSR and (Fig ding r amountof responses
are higher ures non 3%
find
between 4 enabled
less - B and affinity no . To - ). This article isprotected rights by All copyright. reserved. Accepted critical for promoting upregulation of in ACPA memory B cells (LRRFIP1), and B subsets genesof despitenot having higheraverage module scores relativeto Tet cell activation pathway Differences in switch and mutation rates upregulation relativeto (Fig DBN1, a SOX11 highlyp responses Article RF B cells express transcriptional programs associated with naïve programmed for class matur molecules (CD84, JAM2 and ITGB2) which prolong B:T interactions cell and in not RF cells the differentiation of restingcells, B compartment which and Tet population, module scores antibodyfor responseand ACPAprimary responses initiate class
ure responses
). stage ation all
5D - RF memory roliferative IgMresponsesand suppressesgerminal center transcription factor BCL6, and
naïve promoteantibody class . ) IFN signaling (IFI6, PSMB10), and ACPA . Within the
(Fig
(29,30)
of
and B cell
B B cell (26) - (33) ure regulated positiveregulators of SOX11 and BACH2 inRF mem , which is
sustained inmemory
. In the . In the memory compartment, . T 5D B s
populations costimulation drive ACPAgeneration and versatility RF to multiple innate . cells ranscriptional regulator BACH2 germinal center - I ) nnateimmune mediators providecostimulatory signals th switching through B cell
(27,28) (Fig a feature of gene,are . exhibit B cell
ure6B
activation module, the transcription factor SOX11, . Together, thesesignatures suggest that
activation module, wedetect (Figure - switched res alter ). In contrast,
elevated is
NF
responses unswitched - ed downregulated in switch programs and downregulate CD72 s ACPA B -
T cellT kB 5A and B) expression
DAP12 DAP12 signaling signaling (REL in memoryRF cells orycells B providesan explanation for ponses
help cells (Fig ACPA
and survival B cell RF and cells ACPA exhibit differentially expressed . Within these modules,
and facilitated
of
is
B cell memory (23
also signature
responses ure naïve –
and 25)
significantly upregulated in RF memory activation
(SIGLEC14) 5 IgM responses and rapid recall
C (34 , naïve A
and memory HSP90AA1)(Fig ). Additionally B are relativeto
cells show coordinated ) genes .
by CD72 downregulation in the (Fig highly elevated inthe
CPA are ure 5D , none of involved in ACPA
elevation of adhesion elevated relativeto
thereby MSH5, JAK3, and EXOC4, memoryACPA Tet ACPA , CD72, which inhibits )
ure6B B cell
(31,32) which promotes at canat - which .
B cells (Figure populations but In the
MyD88 signaling affinity
the responses are ), which), is . Theselective augment are enriched ir n
naïve lowclass aïveACPA -
the
B cells ACPA s 6A
B
RF -
This article isprotected rights by All copyright. reserved. suggesting different molecular mechanisms mediate theloss of tolerancein ACPA versus RFB cells. demonstratethat ACPA andcells RFB are imprinted with distinct transcriptional programs, defineRF of development therapeutic modalities that specifically target dysregul Accumulating clinical evidenceimplicates pathogenic a role for Bcells in RA Discussion response pathways and that facilitaterapid recall responses. in contrastto ACPA B cells, RF Bcells upr innate immune genesupregulated in RF memory cells help prime rapid recall responses. between theseinnate immune genes,SOX11, and markers ASC differentiation,of suggests that that thesegenes are all individual memory RFB cells i The foundWe evidence that RF coexpress cells genes belonging to multiple characterized by a robust germinal center response. secondary responses. that JAK3 linksgerminal center signals with class network in which receivesignals from follicular helper T cells, are whether D response toler of networks within ACPA and RF DEGs and to provide insights into the mechanisms underlyingloss the ACPAand RF B cells. Distinct antibody response and innate immune ) , revealingco a AcceptedB cell Article nnate immunegenes ancein ACPA as compared to RF Bcells
tolerancein RA using upstream
- and memory compartments,
and ACPA ACPA
will be critical sustained for remission.
- memory regulated transcriptional networkspecific to ACPA develop effectors -
specific immunoglobulin usage We analyzed single Thus, a
significantly
B cells PSMB10, LRRFIP1, IFI6, and SIGLEC14 areall coexpressed, implying that
of JAK3of signaling were coexpressed with JAK3. exhibit ntigen antigen
( Figure6D - experienced ACPAcells B exhibit a transcriptional program coexpressed with PRDM1 and coordinate -
tetramers ACPA B cells coexpress - egulate transcriptional programs involved ininnate immune cell gene coexpression to identify transcriptional regulatory )
(35,36)
(Supplementary Table 10)
upregulat
- transcriptional regulatory networks characterize to switch functions in both isolate
. and transcriptional programs.
Here C coexpressed with the MSH5/ ated pathways in autoreactive oexpression between thesegenes suggests
ion of w disease e
investig MSH5, CD84, and JAK3
genes (Fig - CCND2 relevantcells B ate the
B cell . . ACPA ure In both the T
Both IL21R and CD40,
he coexpression (3,37,38)
ment.also We differential regulation responsepathways.
6D
prima ). Further, findwe
and Our findings CD84/
B cell , and (Fig ry and primary scRNA
Therefore ure JAK3
asked s 6C and - seq
to ,
This article isprotected rights by All copyright. reserved. mechanisms whether maturation ACPAof occurs ingerminal centers,f antibody class genesthat prolong B:Tcell interactions (CD84, JAM2, and ITGB2) and molecul ACPA potential cells transcriptional program rates, class Once activated, naïve received by ACPAcells B cells. that b a restrainto ACPA developmentThe ACPAof appears to stem fr th regulation overwhelming (40) reported and may polymorphisms in BCR signaling genes duelikely to healthyindividuals but not in R IgM (39) While
oth study by Makrygiannakis and colleagues found that citrullinat Accepted Articleis
+ , the near . Systemic inflammat occurs /IgD CD72, aninhibitory co
suggests dependence theon IL21R/JAK3 pathways ACPA for production and RA and non
+ This may ACPA and
self patients + this inoccurs
explanation for why tofacitinib, which targ
- RF cells inRApatientsindicates that
reactive
of ACPAof cells - in thebone marrow swi
. a
ly
-
combination genetic of and environmental factors. Defectiveregulation through switching (MSH5). apoptotic or receptor enable transduction of tching rates, and CDR convergence
exclusivedetection of (44) ACPA Tet - B cell RA inflamed tissues permit B -
. cells cell Further
B cells undergo extensive affinity maturation
convent activation s
begins
ory mediators
, as well precursors leakinto the periphery atlow a frequency reminiscent of those (43) thesurvival autoreactiveof immature B cells that are - receptor, emphasizing .
E ional germinal centerr
A patients at the at or or the xpressio , ,
While these transcriptional signatures suggest that affinity and from though this effect was not significantin s our - ; ; yet only RApatientspersistently develop ACPA editing
is
naïve , activating CD27 periphery.
as wellaltered as receptor editing havebeen previo downregulated innaïve ACPA B cells relative to RFand Tet can also promote the survival autoreactiveof cells B by n of the co . Thissuggestsa defect in
the importanceof - promo stage IgM
proces om in ACPA +
B cell
uture studies will benecessary to determine /IgD germinal center multiple , f
that are tion - uturestudies ses receptor CD21 ets results JAK3, inlower /RF + eactions
ACPA signals that override tolerogenicsignals
by
(41) precursors defectivetolerance autoreactive associated with expression of .
B While dataindicateour that T cell
, cells and the elevation CD27of
through ed protein levels s will be needed decipherto whether .
was help, we find an upregulation in T
are as evidenced by high mutation central tolerancein RA he elevation JAK3of in down T cell predominantly ectopic
- help.
regulat es thatpromote rem mechanisms
in healthyindividuals tudy T normally
are - provides a cell help ission rates In support this, of .
increased ed
(42) deleted
between
robust removed differential , or other .
that fail that is usly ACPA We
- in
in in
find
B -
B
This article isprotected rights by All copyright. reserved. tolerance. important to providefurther insights into studies comparinggene the expression of RFB cells between individuals with purpose engulfingof (45) antibody specificity for diagnosing RA investigation. Given the presence of RFin other inflammatorydiseases and its relatively lower etiologyThe behind RFproduction in multiple inflammatory contextsremains an area activeof mount rapid re correlates with markers of ASC, implying that RF memorygenerates a reservoir of cells that can in responseto diverse inflammatory stimuli. innate immune Our dataindicatethat hypermutation affinity mayintrinsically be antibodies and immunecomplexes. tenup IgG to molecules that expressIgM and extensive affinity maturation driving theACPA response,the RFresponse with In contrast ACPAto inflammation, and citrullinated protein availability combinatorially createan env formationACPA during relapse citrullinated epitope, drivesACPA the response proteins suggest I Accepted Article dentif . Thus, RF eleva broad ication of maturation, RFcells B can differentiate into memorycells B ASCs or
B activation cell and autoantibody production - mediated ”
to ACPA innate immune activation, rapid recall . call responses.
pathways (
s shared transcriptional programs for
that exposure
tion , theRF inflammation by facilitating th reactivewith antigen exhibit
RF s in itspenta
in RA may represent anexpanded pool of naturally occurringcells B
B cells e.g. B cell
- lower mutation rates. The (4,5) antibody complexes to
MyD88, IFN, DAP12)
(37) tospectrum a of mountrapid secondaryresponses with co
responseis characterizedtranscriptional by programs associated IgG , results our suggestRF that could mericform . The low mutation rates suggest that certain - Hence, Fc. W
whether RF hereas U wepropose that pregulation theseof signatures in RF memory , enables responses RF to aggregatesecreted , inagreement with a previous study that examined
citrullinated proteins
e clearancee antibodiesof and i responses T cellT ,
s ACPA enable antigen presentation to uggest in fact .
bias help causesACPA to undergoprolonged
B cells
ing toward , and resultsfrom dysregulated Bcell
differential
that RF B cells
that ASC differentiat
the IgM isotype,which can bind represent
target diversecitrullinated
ratherthan specific a B cell is characterized by - stimulation from multiple with minimal
and without RA will
areprimed to expand a response to resolve
imprinting, enhanced V(D)J mmunecomplexes ironment conducive ion
T cells.T Future .
Counter combinations somatic
B cells B cells for the to the be
This article isprotected rights by All copyright. reserved. and areaccessionaccessible number through materialsData and availabili Lu,Lingampalli, Glanville, Kongpachith,Gottardo, McDavid,Robinson, Ju and data interpretationAnalysis of Lu,Lingampalli, Kongpachith, Ju of data Acquisition Lu,Robinson McDavid,Gottardo, conception and Study design Author contributi A.Gomez,L. thankS.Elliott, We Z. Blum,Weng and andinput. for discussions insightful Acknowled as studies tolerance followed response inthe primary by robust innate activation immune pathwa pr O ur findingsreveal that ograms atdifferent stages of
Accepted Articlethe impacttherapiesRF oftargeted and B onACPA activation cell will
gments further ons define
ACPA andACPA RFcells B h ow
ty dev ACPA andB promot ACPA RF cells : y
Raw sequencing data haveinNCBI deposited been dbGaP
s while ACPA B cellsbys while ACPA are characterized of loss elopment, with RF Belopment, characterized by cells with of activation
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are imprinted with submission inprocess submission affinity
- e matured
tissue damagetissue in distinct distinct transcriptional .
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RA, as well RA, Future
This article isprotected rights by All copyright. reserved. Supplementary Table AcceptedSupplementary Table9 Supplementary Table8 Supplementary Table7 Supplementary Table6 Supplementary Table5 Supplementary Table4 Supplementary Table3 Supplementary Table2 Supplementary Table Supplementary Figure Supplementary Figure ArticleSupplementary Figure Supplementary Figure Supplementary Figure Supplementary Figure Supplementary Figure of List from RApatients. genesfor each single cell library. workflow. Model same samples. frequencies (
Supplementary - based assembly.
C lassifier
1. 1. 10. Subsetted single 7 6 5. 4. 3. 2. Single1. . . Genesused for transcriptomic subset prediction. . Cell and molecule identifier oligonucleotidedesign. . Subset . Global . Monoclonal antibody expression an . Clustering of antigen . BCR reconstitution and IG isotyperecovery.
. . B cell RA patientand healthy don
Differential expression tests for gene modules. Analysis of multi Pair Comparison of the distribution of transcriptomic subset classifier A Distribution of molecule counts(UMIs), pre
Figures Tables and pplication theof transcriptomic F ed immunoglobulin variable
raction) flowto cytometry subset frequencies ( subset classifier
- ted ACPA and cell RNA
ACPA and RF
- seq library preparation and computationa
- RF B cell - specific CDR3 clusters. cell co - specific byB cells applied healthyto and RA B cell
expression testing.
differentially expressed transcripts. or
differentially expressed transcripts. demographic and clinical data - region reconstitution using hidden Markov
B cell d functional characterization.
paired HC and LC
subsetclassifier to CD19
- deduplicated reads(reads), and
B
cells.
F low
CDR3 F l de raction)from the .
-
multiplexing sequences +
B cells .
This article isprotected rights by All copyright. reserved. strand Islam12. S, Kjällquist U, MolinerA, Zajac FanP, J 2012;7:e35296. spreading inthepre Sokolove11. J,Bromberg R, DeaneKD, Lahey LJ, Derber LA, Chandra PE,et al. Autoantibody epitope patients with rheumatoid arthritis. Identification and characterisation citrof 10. Kerkman FabrePF, Voort E, EIH van der, ZaldumbideA, Rombouts Rispens Y, etT, al. 2014;66:2706 plasmablast antibody repertoires inrheumatoid arthritis. Tan 9. Y 2006;116:961 citrullinated proteins enhancetissue injuryin experimental autoimmune arthritis. Kuhn8. KA, Kulik TomookaL, B, Bra A required for anti 7. lipopolysaccharides. Izui6. S,Eisenberg RA, Dixon FJ. IgM rheumatoid factors in miceinjected with bacterial 1980;142:250 with deter Salonen5. EM, Vaheri A, Suni J,O. Wager Rheumatoid factor inacute viral infections: interference Rheumatol Int systemic lupus erythematosus:association with clinical and laboratory parameters. study SLE group. Witte4. HartungT, SachseK, C, Matthias T, Fricke M, Kalden JR, al. et Rheumat 2004;350:2572 Efficacy of B Edwards3. JCW,Szczepanski Szechinski L, ArthritisRheumatol Hoboken NJ potentiator of anti Sokolove2. J, Johnson DS, Lah 2219. McInnes1. IB, Schett G.ThePathogenesis Rheumatoidof Arthritis. References
2009;106:12061 AcceptedKono HaraldssonDH, MK, Lawson BR, Pollard KM, Koh YT, Du X, al. et Endosomal TLR signaling is Article
- specificsingle - C, Kongpachith S, Blum LK,Ju C mination IgM,of IgG, and IgA antibodies in an enzymeimmunoassay. -
cell – – –
2000;19:107 2715. 973. 255. –
2581. - - targeted therapy with rituximab inpatients with rheumatoi nucleic acid and rheumatoid factor autoantibodiesin lupus. – - citrullinated protein antibody mediated in
12066. -
J Immunol Baltim Md 1950 clinical - cell RNA 5′ end sequencing.
– phasepredicts progression rheumatoid to arthritis. 111. ey LJ, WagnerCA, Cheng Thiele D, GM, al.et Rheumatoid factor as a
2014;66:813
Ann Rheum Dis schler KJ, Arend WP, Robinson WH, al.Antibodieset against - H, Lahey LJ, Lu al. DR, et Barcode ullinated antigen J, Filipowicz – 821.
1979;122:2096 - B, Lönnerberg P, al.Highlyet multiplexed and Nat Protoc
2016;75:1170 - Sosnowska A, Emery CloseP, DR, etal. Arthritis Rheumatol Hoboken NJ - specificB cells in peripheral blood of
2012;7:813 flammation in rheumatoid arthritis.
– 2102. – 1176. N Engl J Med
– - 828. enabl d arthritis. Proc Natl Acad U Sci S
oid factors in ed sequencing of PloS One J Infect Dis 2011;365:2205 J Clin Invest N Engl J Med
–
This article isprotected rights by All copyright. reserved. 2005;35:2325 naive B differentiation cell by down 26. Yamazaki T, Nagumo H, Hayashi T, SuganeK, Agematsu K.CD72 Mediated Signaling. Jabara25. HH, Buckley RH, Roberts JL, LefrancG, LoiseletJ, Khalilet G, al. Role of JAK3in CD40 pathway. 24. O’SheaJJ, Gadina M 7198. in Msh5 the regulation Igof switch class recombination. Sekine23. H, Ferreira RC, Pan biological sequence Huang22. Y, NiuGao B, Y, Fu Li L, CD W. Front Immunol 21. Kaminski WeiDA, C, Qian Rosenberg Y, AF, San 2014;157:714 Detection Uncovers Progression and Regulatory Coordination inHumancell B Development. 2 checkpointsin rheumatoid arthritis. 19. Menard SamuelsL, J, Ng Y with rheumatoid arthritis. Samuels18. J,Ng Y with autoreactivity. al. Rheumatoid arthritis synovial tissue harbours dominantB 17. Doorenspleet ME,Klarenbeek PL, Hair MJH de,Schaik BDC van, Esveldt REE, Kam 24, 2017. sequencing data. Available2015. at: http://dspace.mit.edu/handle/1721.1/100458. Accessed April frameworkfor assessing transcr Finak16. G, McDavid A, Yajima M, Deng J,Gersuk V, ShalekAK, et al.MAST: a flexible statistical referencegenome. 15. B, Li Dewey CN. R 359. 14. Langmead B,Salzberg SL. Fastgapped Browserat UCSC. 13 0. Bendall0. SC, Davis KL, Amir ED, Tadmor MD, Simonds Chen EF, TJ, et al.Single
Accepted Article . Kent WJ, Sugnet CW,Furey TS, Roskin KM, Pringle TH,Zahler AM, et al.Human The Genome
Cell
– – 2002;109 Suppl:S121
725. 2334. 2012;3. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3467643/. Genome Res -
BMC Bioinformatics s. S, CoupillaudPaget C, D, Meffre E. Impaired early B ce Ann Rheum Dis Blood
SEM: accurate transcript quantification from RNA Bioinformatics , SchreiberRD. Cytokinesignaling in2002: newsurprises inJak/Stat the
J Exp Med 1998;92:2435 - - Hammarström Q, Graham RR, Ziemba B, Vries SSde,et al.Role for
S, Meffre E. Inflammation 2002;12:996 iptional changes and characterizing heterogeneity insingle -
- 2013:annrheumdis 131.
regulating X ArthritisRheum
2010;26:680 2005;201:1659
2011;12:323. -
– HIT Suite: a web serverfor clustering and comparing - 2440 read alignment with Bowtie 2. – 1006. .
- box binding protein1. –
z I. Advances inHuman B Cell Phenotypic Profiling. 682. –
2011;63:1237
1667. -
2012 - Proc NatlProc Acad U Sci A S independent defective Bearly cell tolerance
- - 202861. cell and plasma
– - 1245. mediated suppression humanof
- Seq data with or without a Eur JEur Immunol NatMethods
ll tolerancein patients - cell clones associated -
Cell Trajectory 2007;104:7193 pen AHC van, et
2012;9:357 - cell RNA Cell -
–
– This article isprotected rights by All copyright. reserved. Acceptedrituximab treatment inpatientswith rheumatoid arthritis. pharmacokinetics,development of human anti 43. Thurlings RM, Teng O, K,Vos Gerlag DM, Aarden L, StapelSO, et al.Clinical response, IgA antibodies to citrullinated antigens. rituximab inpatients rheumatoid with arthritis: Multiplexbead arrayreveals the kinetics IgG andof Ca 42. 2010;185:294 negative regulation surfaceof B cell receptor levels andcell activation. B 36. Ouchida R, Kurosaki T, Wang J like cell andcell B in rheumatoid arthritis. 35. Liu R, Wu Q, Su D, Che N, ChenH, Geng L, al.Aet regulatory effect of IL http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857169/. Accessed April 22, 2017. Function. Gerondakis34. S,SiebenlistU. Roles of the NF 2002;416:603 Chromatin 33. Leadbetter EA, Rifkin IR, Hohlbaum AM, BeaudetteShlomchik BC, MJ, Marshak reactions. human IgM memorycells B inearly inflammatory responses and secondarygerminal center Seifert32. M, Przekopowitz Taudien M, S,Lollies A,RongeV, Dree Articlecell differentiation. the transcription factor Bach2 contributes to predisposition IgG1of memory B cells toward plasma 31. Kometani K, Nakagawa R, Shinnakasu R, Kaji T, Ryb Biol Adapter Protein and IsCoupled to Signal Transduction from LymphocyteAntigen Receptors. 30. Larbolett SOX11 in Mantle Cell Lymphoma. Profiling and Chromatin Immunoprecipitation Identify SETMARDBN1, and HIG2 as Direct Targets of 29. Wang X, Björklund S,Wasik AM, Grandien A, Andersson P, Kimby E, et al. GeneExpression 4143. and Ly108 maintain germinal center Bcell tolerance. 28. Wong EB, Soni C,Chan AY, Domeier PP,Sh triggering proliferation of through CD11c/CD18. CD11c/CD18 integrin humanon lymphocytes. B Relation between attachment to fibrinogen and 27. Postigo AA, Corbí AL, Sánchez
1999;19:1539
mbridge G, Leandro MJ, Lahey LJ, Fairhead RobinsonT, WH, SokoloveJ. B cell depletion with Cold Spring Harb PerspectBiol Proc Natl Acad Sci - IgG complexes activate B cellsdual by engagement IgM of and Toll e O,e WollscheidSchweikert B, J,Nielsen PJ, Wienands J. SH3P7Cytoskeleton Isa – – 607. 301. – 1546.
Immunity
2015;112:E546
2013;39:136 - - PLOS ONEPLOS Madrid F,Landázuri Y. Arole for lysosomal J Autoimmun
2010;2. Available at: Art – 147.
– 2010;5:e14085. hritisRes Ther E555. wetank null, Abraham T, etal. B cell - κB Pathway in Lymphocyte Development and - chimaeric antibodies, and synovial tissueresponseto
J Exp Med
J Immunol Baltim Md 1950 2016;70: ouchkin A, Moriyama S, et al. Repression of MO de. MO Regulated expression and function of - associated protein transmembrane in 5 the
A 1991;174:1313
2012;14:R255.
nn Rheum Dis
22 – s B, et al.Functional capacities of 30.
J Immunol Baltim Md 1950
– 2010;69:409
- 1322. 21 on follicular T helper - likereceptors.
2015;194:4130
- - intrinsic CD84 Rot
hstein A. – 412.
Mol Cell Nature – -
This article isprotected rights by All copyright. reserved. Accepted by rheumatoid factor B cells. Roosnek45. E, Lanzavecchia A. Efficie seropositive [abstract]. 2016. Available http://acrabstracts.org/abstract/tofacitinibat: ArticleTreatment Outcomes inSeropositiveVersus Seronegative Patientsin Bird44. HallP, S,Nash ConnellP, CA, Kwok K, Witcombe D, Thirunavukkarasu K. Tofacitinib: Med lymphocyte responses Akatsu43. C, ShinagawaK, Numoto N, LiuZ, UcarAK, Aslam M, al.CD72et negatively regulatesB is an inflammation 42. Makrygiannakis D, Klint af, E Lundb to the Late Pre Pathways Responsible for Pre Nagaoka41. H, Takahashi Y, Hayashi R, N (PTPN22)is associated with rheumatoid arthritis. missensesingle Begovich40. AB, Carlton VEH, Honigberg SchrodiLA, Autoantibody Production by Early HumanCell B Precursors. 39. Wardemann H,Yurasov S,Schaefer A,Young JW, Meffre Nussenzweig E, MC. Predominant
2016:jem.20160560. - versus - - B Cell Stage. nucleotide polymorphism ina gene encodingprotein a tyrosinephosphatase - dependent process. - seronegative to thelupus
J Med Exp J Exp Med - B Cell Survival, Which Is Essential for the Developmental Progression - patients - related endogenous toll nt and selective presentation antigenof
erg IE, Löfberg R, Ulfgren A 2000;192:171
Ann Rheum Dis 1991;173:487 akamura T, Ishii Matsuda K, J, al. et Ras MediatesEffector - in - a - phase Am J HumGenet – SJ, Chokkalingam AP, AlexanderHC, et al.A - – 182. 3
489. - 2006;65:1219 ra -
population/. Accessed June 9, 2017.
- Science likereceptor ligand7 Sm/RNP.
-
K, Klareskog L,al. et Citrullination 2004;75:330
2003;301:1374
– a Phase RA3 Population 1222. - treatment
- antibody complexes – 337. – - 1377.
outcomes
J Exp
- in - This article isprotected rights by All copyright. reserved. 95%confidence intervals. subset differences and patient variability using a generalized linear mixed model. Error b Class memory rates. Fig citrullinated microarray hitsfor positivereactivity represented is with ahorizontal gray line. respectively. The dotted green linedenotes a positive control monoclonal antibody. T expressed from RF, using absorbance val results(n=3) against from which recombinant antibodies were expressed are specificity cells with H.CDR3 and L.CDR3 sequences sharing >80%homology. Each cell, colored by antigen from which recombinant antibodies were expressed. Row labels denoteV Tet Heatmap showing mostfrequently utilized V Fig HD5 recovered from quality yields during library preparation and were excluded from analysis. No ACPA 2,349 single cells from seven sample. between RApatients anddonors. healthy A minimum 15,000of B cells wereanalyzed for each producing analyses of B cells isolation, single cell clas Fig F igure ure Acceptedure Articleure + sification singleof cells. , and Tet .
-
switch rates for antigen (A) s and Legends
2. 1. 3.
* =* , double negative memory,
Antigen RF and ACPA B cells exhibit distinctimmunoglobulin Interrogation of autoreactive Proportion of ,
RF is a node. P peptides - <0.05
or ACPA populations. Column labels denote antigen subject - experienced . (B) expressed
- ACPA (D RNA
H
RF ues G - . and
V ) Representative flowcytometry gating ray edgesconnect cells belonging th to (C)
L B cel and c antigen R04 and
, and by plotted - pairs arrangedV by , and Tet seq, and simultaneous immunoglobulin repertoireand transcriptomic
* = available corresponding Comparison of RF and
(A) l
ycl individual - subtype distribution P monoclonal antibodies. Binding is intensity shown for both experienced RF and
<0.05; **= - Schematicof the experimental workflowfor the tetramer ic experienced ( H - - D citrullinated peptide
B cells are represented using red, black,and bluesolid lines,
5 and5 R04, and no RF and antibody ACPA s B cell .
B cell T heACPA P ACPA
<0.01; n.s. not= significant B differ cells inclass H
H subsets
- genefamily. Red boxes identify prominent V i.e.
V subtype L
, RF, and Tet
pairs eachfor patient’sCIT ACPA - unswitched (IgM/D)and class
secreting using the non +
and RF
through (B)
-
B cell as determined by BCellNet 3 c
itrullinated antigens
highlighted - + Greedy, agglomerative clustering of single B for all specificpopulations arrangedpatient. by
cells wererecovered from subject R48 + )
BCellNet B cells from subject R01 produced poor B cell (D approa subtype frequencies inperipheral blood -
tetramersorting and transcriptomic samee cluster. gene B cells inR ) recombinant
Top - switch and somatic hypermutation s for each antigen specificity.
usage
ch in blue. citrullinated autoantigen
classifier
for theidentification of B cells (C) A
and
patients,adjusting for Somaticmutation rates - Tetramer (C) . antibodies, measured
Prominent clusters CDR3 convergence. forrecovered the -
switched (IgG/A/E) RepresentativeELISA . Antibodies +
c ells were hreshold for +
(CIT ars indicate - based H - - Tet V L
pairs + (B) ) , RF
and
(A)
-
This article isprotected rights by All copyright. reserved. ACPA Network displaying the coexpression between between Correlations with FDR theof edges indicate the Z relative1 to RF/ module genes, stratified by subset. Genes enriched in between in the networks from ACPA Fig differentialfor expression between differential expression between residual expression in the class Fig horizontal dotted lines denote FDR Modules consist of the averagepartial resid functional modules between genes expressed genes for all (A Fig bars indicate 95%confidence intervals. adjusting subset for differences and patient P for <0.01 ) ure ure ure Accepted Article antigen Subset - /RF memory switching and
innate immune module (47 genes). antibody response module (15 genes).
are known to beimmune
6. 5. 4. (D) Innate immune mediators enriched in RFB cells and comprise ACPA Aberrant B ACPA - - - stratified heatmap showing the Z Somatic mutation ratesfor the cell activation and innate immune experienced
B cells and RF ACPA B cell primary and secondary autoreactive
B in the - -
enriched genes. - cell activation and expression greater than or equal to Tet and RF Bcells
adjusted P B cells 2,349
responses. ACPA
B - score of the Pearson correlation c - cell activation module (25 genes).
ACPA in B cells from patients. , RF, and Tet - i ACPA related nnate immunegenes - values > 0.05 are omitted.
versus versus Tet q . ACPA exhibit - (A value cutoffs.
(A and RF
) . *** =
) Subset (B
Subset and RF * = immunoglobulins expressed by -
and - distinct uals from genesthat share cellular functions. Gray B cells by subtype. Error bars indicate mean SEM.± **= - to
scaled partial residual expression of the differentially
* =* - P P B cells
genesinvo (B - B <0.05 <0.001;not n.s. = significant
patient variability usinglinear a mixed model. Error - C stratified plot of averagepartial residual expression - - )
P cell activation and innateimmune stratified plot of averagepartial residual expression ) B cells
and RF versus Tet <0.05 Volcano plotsshowing differentially expressed
Of the 454 differentially transcriptional
in (B . ACPA
(C and(C D) antibody responsegenes. )
B cell
in ( Z lved in B - scores testing for differential expression ) B
/RF havedifferential expression Z Subset -
( cell activation genes.
oefficient for pairs of genes. C gene
) * =
Network displayingcoexpression the naïve Coexpression networks selectedof -
P - population. programs thatpromote antibody expression stratified plot averageof partial <0.05 -
(C
ACPA )
distinctcoexpression for all
(C ACPA
/RF ) expressed genes, 121
Z - signaturesin B cell Thecolor an
scorestesting for B cell , RF,and Tet (D
genesinvolved in )
subtypes.
Z responses. - scores testing
d weight RA -
B cells - score > .
( D )
This article isprotected rights by All copyright. reserved. Accepted Article
This article isprotected rights by All copyright. reserved. Accepted Article
This article isprotected rights by All copyright. reserved. Accepted Article
This article isprotected rights by All copyright. reserved. Accepted Article
This article isprotected rights by All copyright. reserved. Accepted Article