Efficient T cell–B cell collaboration guidesautoantibody epitope bias and onset of celiac diseaseRasmus Iversena,b,1, Bishnudeo Royb,2, Jorunn Stamnaesa,b, Lene S. Høydahla,b, Kathrin Hnidab, Ralf S. Neumanna,b,Ilma R. Korponay-Szabóc, Knut E. A. Lundina,d, and Ludvig M. Sollida,b,1

aKG Jebsen Coeliac Disease Research Centre, University of Oslo, NO-0372 Oslo, Norway; bDepartment of Immunology, Oslo University Hospital, NO-0372Oslo, Norway; cCeliac Disease Center, Heim Pál National Pediatric Institute, HU-1089 Budapest, Hungary; and dDepartment of Gastroenterology, OsloUniversity Hospital, NO-0372 Oslo, Norway

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved June 11, 2019 (received for review January 27, 2019)

B cells play important roles in autoimmune diseases throughautoantibody production, cytokine secretion, or antigen presenta-tion to T cells. In most cases, the contribution of B cells as antigen-presenting cells is not well understood. We have studied theautoantibody response against the enzyme transglutaminase 2(TG2) in celiac disease patients by generating recombinant anti-bodies from single gut plasma cells reactive with discrete antigendomains and by undertaking proteomic analysis of anti-TG2 serumantibodies. The majority of the cells recognized epitopes in the N-terminal domain of TG2. Antibodies recognizing C-terminal epitopesinterfered with TG2 cross-linking activity, and B cells specific for C-terminal epitopes were inefficient at taking up TG2-gluten com-plexes for presentation to gluten-specific T cells. The bias towardN-terminal epitopes hence reflects efficient T-B collaboration.Production of antibodies against N-terminal epitopes coincided withclinical onset of disease, suggesting that TG2-reactive B cells withcertain epitope specificities could be the main antigen-presentingcells for pathogenic, gluten-specific T cells. The link between B cellepitopes, antigen presentation, and disease onset provides insightinto the pathogenic mechanisms of a T cell-mediated autoimmunecondition.

B cells | autoantibodies | antigen presentation | celiac disease

The role of B cells in autoimmune diseases is not restricted toproduction of autoantibodies. Self-reactive B cells may also

be involved in secretion of cytokines or presentation of antigento T cells. Thus, it has been suggested that B cells can be themain antigen-presenting cells (APCs) for CD4+ T cells in auto-immune diseases (1–3). The function of B cells as dominantAPCs under some circumstances can be explained by uptake ofantigen via specific binding to the B cell receptor (BCR),allowing efficient capture and accumulation of antigen for pre-sentation (4). Recently, it was shown that plasma cells are thedominant cell type presenting gluten antigen in the gut laminapropria of celiac disease patients, suggesting that B-lineage cellsare involved in stimulating pathogenic, gluten-specific T cells (5).One of the hallmarks of celiac disease is a highly specific au-

toantibody response against the enzyme transglutaminase 2(TG2) (6). Production of TG2-specific IgA and IgG is believedto result from collaboration between TG2-specific B cells andgluten-specific CD4+ T cells, facilitated by BCR-mediated up-take of TG2-gluten complexes (7). Gluten peptides are goodsubstrates for TG2, which targets glutamine residues in certainsequence contexts through a calcium-dependent reaction andeither converts them to glutamic acid by hydrolysis (deamida-tion) or cross-links them to protein lysine residues throughisopeptide-bond formation (transamidation) (8, 9). Notably,gluten-reactive CD4+ T cells in celiac disease specifically rec-ognize peptides that have been deamidated by TG2 and arepresented on disease-associated HLA-DQ molecules (HLA-DQ2.5, HLA-DQ2.2, or HLA-DQ8) (10–12).Here, we show that TG2-specific plasma cells in celiac disease

primarily target epitopes in the N-terminal region of the antigenand that this epitope bias reflects presentation of deamidated

gluten peptides to T cells by B cells binding enzymatically activeTG2. Specific targeting of N-terminal TG2 epitopes was associatedwith clinical onset of disease, suggesting that efficient collaborationbetween TG2-specific B cells and gluten-specific T cells is a pre-requisite for disease development.

ResultsPlasma Cells Targeting Distinct Regions of TG2 Have Particular V-GeneSignatures. TG2 consists of four structural domains and canadopt at least two distinct conformations depending on thebinding of effector molecules (Fig. 1A). Both celiac-patient se-rum IgA and mAbs cloned from TG2-specific gut plasma cellsshow IGHV-dependent targeting of defined nonlinear epitopes,primarily located in the N-terminal domain (15, 16). To identifyplasma cells targeting other regions of TG2, we constructed achimeric TG2 variant with the N-terminal domain replaced withthat of TG3 (TG3/TG2; Fig. 1B and SI Appendix, Fig. S1 A–F).By costaining IgA plasma cells in gut biopsies of celiac patientswith WT TG2 and TG3/TG2, we could identify populations re-active with either N-terminal or non–N-terminal TG2 epitopes(Fig. 1C). As expected, the majority of TG2-binding plasma cells

Significance

B cells have important antibody-independent functions and arenow recognized as key players in autoimmune diseases tradi-tionally thought to be T cell-mediated. The role of B cells asantigen-presenting cells, however, is not well understood. Bystudying the autoantibody response against the enzyme trans-glutaminase 2 in celiac disease, we have shown that B cellstargeting particular epitopes are selectively activated and thatthis epitope bias reflects efficient presentation of gluten antigento T cells. Production of antibodies against the preferred epitopecoincided with clinical onset of disease, suggesting that B cellswith this specificity can be main antigen-presenting cells forpathogenic gluten-specific T cells. Our study thus provides in-sight into the mechanisms controlling initiation of a T cell-mediated autoimmune condition.

Author contributions: R.I., B.R., and L.M.S. designed research; R.I., B.R., J.S., L.S.H., andK.H. performed research; I.R.K.-S. and K.E.A.L. contributed new reagents/analytic tools;R.I., B.R., J.S., L.S.H., K.H., and R.S.N. analyzed data; and R.I. and L.M.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: Sequence data have been deposited at the European Genome-phenomeArchive, https://www.ebi.ac.uk/ega/home (accession no. EGAS00001003658). Proteomics datahave been deposited to the ProteomeXchange Consortium, http://www.proteomexchange.org/ (data set identifier PXD013777).1To whom correspondence may be addressed. Email: rasmus.iversen@medisin.uio.no orl.m.sollid@medisin.uio.no.

2Present address: Symbiosis School of Biological Sciences, Symbiosis International(Deemed University), Lavale, Mulshi, 412115 Pune, India.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1901561116/-/DCSupplemental.

Published online July 8, 2019.

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were specific to epitopes in the N-terminal domain, althougharound 30% of the cells consistently recognized other parts of theenzyme. To further characterize these cells, we sorted single gutplasma cells reactive with either N-terminal or non–N-terminalepitopes from five celiac patients and analyzed the paired heavyand light chain repertoires of 803 single cells by next-generationsequencing (SI Appendix, Table S1). In agreement with previousobservations, the population targeting N-terminal epitopes usedcertain IGHV gene segments with a particular bias toward IGHV5-51 (15) (Fig. 1D). Plasma cells recognizing non–N-terminal epi-topes, on the other hand, showed preferential usage of IGHV3-43.While IGHV5-51 heavy chains most often paired with certainkappa light chains, IGHV3-43 showed pairing with many differentlight-chain V segments (Fig. 1E).Plasma cells using either IGHV5-51 or IGHV3-43 had fewer

mutations than other TG2-specific cells in both heavy and lightchains (Fig. 1F), indicating that B cells using the two major IGHV

segments characteristic of N-terminal and non–N-terminal epi-topes have undergone less affinity maturation than other TG2-specific cells. This difference could possibly reflect a competitiveadvantage of B cells targeting the dominant epitopes during B cellactivation.

Binding Characteristics of mAbs Targeting Non–N-Terminal TG2Epitopes. To further characterize antibodies targeting non–N-terminal TG2 epitopes, we expressed sequences obtained fromindividual TG3/TG2-reactive plasma cells as recombinant hu-man IgG1 mAbs (Fig. 2A). By testing these mAbs in a compet-itive binding assay and including previously characterized anti-TG2 mAbs (15) (SI Appendix, Fig. S2A), we could identifycommon epitopes outside the N-terminal domain (SI Appendix,Fig. S2B). Importantly, the two tested IGHV3-43 mAbs bothbound to an area close to the intersection between the core andN-terminal domains (SI Appendix, Fig. S2C), suggesting that

10.5 4.82

0.9183.8

TG3/TG2

WT

TG2

N-terminal TG2 epitopes

Non-N-terminal TG2 epitopes

“Open” conformation “Closed” conformation

N

CoreC1 C2

Active site

A

C D

IGKV3-20IGKV4-1

IGKV1-39

IGKV1-12

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IGKV1-33

IGKV1-5IGKV1-9IGKV1D-16

IGKV6-21

IGKV3-11

IGLV3-10

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IGKV1-27IGLV2-11

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IGLV3-21IGLV3-9

IGKV1-13

IGKV3-15

IGLV2-14

IGLV5-45IGLV6-57IGLV7-43

IGLV1-51

IGLV3-19

IGKV2-28

5289

IGHV5-51N-term. epitopes

IGHV3-43Non-N-term. epitopes

E F

Naa 1-140

Coreaa 141-465

C1aa 466-585

C2aa 586-687

ns ns****

***Light chain

N-terminal epitopesNon-N-terminal epitopes

ns ns**

*

Nfrom TG3

Corefrom TG2

C1from TG2

C2from TG2

WT TG2

TG3/TG2

B

Non-N-terminal epitopesN-terminal epitopes

Nucleotide binding site

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IGHV5-5

1 (n= 18

4)

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3 (n= 94

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Non-IG

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IGHV1-1

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.0

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ific

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s

Fig. 1. Isolation and Ig sequencing of single gut plasma cells targeting distinct TG2 domains. (A) Surface representations of TG2 in two distinct confor-mational states depending on the binding of effector molecules (black stick representation). When the active site accommodates a peptide substrate, theenzyme adopts an “open” conformation [Protein Data Bank (PDB) code 2Q3Z] (13), whereas noncovalent binding of GDP induces a “closed” conformation(PDB code 1KV3) (14). Colors indicate individual structural domains. (B) Schematic representation of the domain organization of WT TG2 and chimeric TG3/TG2 used for staining of plasma cells. (C) Representative flow cytometry plot (Left) and mean distribution of TG2-reactive gut plasma cells (PCs) betweenpopulations targeting N-terminal and non–N-terminal TG2 epitopes in seven celiac patients (Right). (D) Average IGHV usage among clonotypes tar-geting N-terminal or non–N-terminal TG2 epitopes in five celiac patients. (E) Light-chain V-segment usage among IGHV5-51 clonotypes targeting N-terminal TG2epitopes and IGHV3-43 clonotypes targeting non–N-terminal TG2 epitopes. (F) V-segment mutation levels in heavy and light chain sequences among singleplasma cells targeting N-terminal or non–N-terminal TG2 epitopes. Sequences were grouped according to IGHV usage. Horizontal lines indicate mean number ofmutations, and differences between groups were analyzed by one-way ANOVA with Holm–Sidak multiple comparisons correction. *P < 0.05; **P < 0.01; ***P <0.001; ****P < 0.0001.

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most plasma cells targeting non–N-terminal TG2 epitopes recog-nize a region in the core domain located close to the N-terminaldomain.Unlike mAbs recognizing N-terminal or core-domain epi-

topes, mAbs specific to C-terminal epitopes were highly sensitiveto the conformational state of TG2 when assessing binding in thepresence of effector molecules (Fig. 2B and SI Appendix, TableS2). This observation agrees with available structural data showingthat primarily the C-terminal domains are involved in the grossconformational changes induced by TG2 binding of effectormolecules (13, 17). Although the conformation of TG2 present inthe extracellular environment has not been determined, our datawould support a model in which the enzyme exists in a dynamicequilibrium between different states, allowing interaction with allTG2-specific B cells. Overall, antibodies binding N-terminal and

non–N-terminal TG2 epitopes showed similar binding affinities(Fig. 2C and SI Appendix, Table S2).As previously observed for mAbs specific to N-terminal epi-

topes (18), all mAbs targeting non–N-terminal epitopes boundtissue-immobilized TG2 (Fig. 2D), suggesting that this is theantigenic form of TG2 in celiac disease. TG2 is mainly a cytosolicprotein but can also be found in the extracellular matrix (ECM).This association is primarily mediated by an unknown ECMcomponent, which specifically binds the second C-terminal do-main of TG2 (C2) (19). Curiously, very few plasma cells in celiacdisease recognized the C2 domain (Fig. 2E), suggesting thatECM-mediated epitope blocking prevents activation of most C2-reactive B cells.TG2-specific B cells may take up TG2-gluten complexes in the

form of covalently cross-linked adducts, generated through the

1407-12-C06(IGHV1-3:IGKV1-39)

0.0 0.5 1.00

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1407-10-F06(IGHV1-69:IGLV2-14)

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1390-6-C08(IGHV3-15:IGKV3-11)

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1393-4-A12(IGHV3-43:IGLV2-23)

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1390-6-E09(IGHV4-39:IGLV2-14)

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1407-11-E04(IGHV3-11:IGKV1-39)

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TG3/TG2

TG2 1-465

WT TG2

OD

405

Core epitopes C-terminal epitopes

Antibody (μg/ml)

Fab 1407-12-C06

Fab 1407-10-F06Fab 1390-6-C08

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1407-12-C06

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3. IP 1407-12-C06

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250150100755037

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2 C20

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.6

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(M)

Fig. 2. Binding characteristics of mAbs targeting non–N-terminal TG2 domains. (A) Binding between recombinant mAbs and different TG2 variants assessedby ELISA. Domain specificity was determined based on reactivity with truncated TG2 lacking the two C-terminal domains (TG2 1–465; SI Appendix, Fig. S1 A–F).(B) Surface plasmon resonance sensorgrams showing change in response units (RU) caused by binding and dissociation of Fab fragments using differentconformational states of TG2. When using TG2 covalently linked to an active-site inhibitor (iTG2) in the presence of Ca2+, only antibodies recognizing core-domain epitopes bound detectably, whereas all but one antibody showed binding in the presence of GDP. This antibody could still bind TG2 in the absence ofeffector molecules. (C) Comparison of TG2 affinities between Fab fragments specific to N-terminal (n = 9) and non–N-terminal (n = 7) epitopes. Plotted valueswere obtained with the TG2 conformation that, in each case, gave the highest affinity. Column heights indicate mean affinity, and statistical difference wasevaluated using an unpaired t test. (D) Immunofluorescence detection of mAbs bound to human recombinant TG3/TG2, which was immobilized on mouseTG2 KO small intestinal tissue sections. (E) Representative flow cytometry plot (Left) and mean distribution of TG2-reactive plasma cells (PCs) betweenpopulations targeting C2 and non-C2 epitopes in three celiac patients (Right). The two populations were identified by costaining with full-length TG2 and atruncated TG2 variant consisting of the C2 domain only. (F) Western blots showing cross-link formation between TG2 and a biotinylated 33mer glutenpeptide resulting in high-molecular-weight bands. Cross-linked TG2-gluten complexes were immunoprecipitated (IP) with the indicated mAbs, and proteinbands were visualized using either anti-TG2 mAb or streptavidin (SA).

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transamidation activity of the enzyme. mAbs recognizing N-terminal and core-domain epitopes were found to recognize suchproducts (20) (Fig. 2F). Two of four mAbs reactive with C-terminal epitopes, however, were not able to pull down co-valent TG2-gluten complexes (Fig. 2F). Consequently, B cellstargeting some C-terminal TG2 epitopes would not be able totake up cross-linked TG2-gluten complexes and present peptidesto gluten-specific T cells.

Antibody Binding to C-Terminal TG2 Epitopes Interferes with EnzymaticActivity and Impairs Antigen Presentation to T Cells. TG2-specificmAbs targeting the main N-terminal epitopes do not inhibit thedeamidation and transamidation activities of the enzyme (18, 21).To test if the same is true for mAbs specific to non–N-terminalepitopes, we assessed the effect of mAb binding on TG2-mediateddeamidation of gluten peptide (Fig. 3 A and B) and formation ofTG2-gluten transamidation products (Fig. 3 C and D). Comparedwith mAbs targeting core epitopes close to the N-terminal domain,mAbs recognizing more C-terminal epitopes generally had a neg-ative effect on TG2 enzymatic activity. The observed effect wasstatistically significant for transamidation, but not for deamidation.Of note, TG2-catalyzed formation of covalent complexes with glu-ten through transamidation is believed to be important for theability of TG2-specific B cells to interact with gluten-specific T cells.To test if the different effects of mAb binding on TG2 enzy-

matic activity would influence T-B collaboration, we assessed theactivation of gluten-specific hybridoma T cells by transducedlymphoma B cells expressing HLA-DQ2.5 in combination with aBCR specific to either an N-terminal or a C-terminal TG2 epi-tope (Fig. 3 E and F). While both B cell types were able topresent peptide to T cells upon incubation with chemically cross-linked TG2-gluten complexes (Fig. 3E), the cells with N-terminalspecificity had a clear advantage when incubated with active TG2and gluten peptide (Fig. 3F). This difference was particularlypronounced when excess TG2 in solution was washed away be-fore the addition of gluten peptide, leaving only the fractionbound to the BCR. These results indicate that the modest neg-ative effects on TG2 activity displayed by mAbs targeting C-terminal epitopes translate into a dramatic reduction in theability of B cells targeting C-terminal epitopes to take up andpresent deamidated gluten peptides to T cells, compared with Bcells targeting N-terminal epitopes. Thus, it is likely that theoverrepresentation of plasma cells targeting certain N-terminalor core-domain epitopes reflects superior antigen presentationby B cells with these specificities.

Production of Hallmark Antibodies Coincides with Celiac DiseaseOnset. In several autoimmune conditions, including celiac dis-ease, autoantibodies can be detected before the occurrence ofsymptoms (22–24). Notably, patients with “potential” celiac disease(i.e., with detectable autoantibodies but no or mild intestinal in-flammation) have significantly lower levels of anti-TG2 antibodiesthan untreated patients with severe inflammation (Fig. 4A and SIAppendix, Fig. S3A). To test whether these, presumably early, an-tibodies in individuals likely to develop celiac disease reflect theantibody response in the fully established disease state, we purifiedthe TG2-specific IgA fraction from serum of patients with differentdegrees of intestinal inflammation and assessed IGHV-family us-age by mass spectrometry (Fig. 4B and SI Appendix, Fig. S3 B andC). Compared with non–TG2-specific antibodies, anti-TG2 anti-bodies from both potential celiac patients and patients withestablished, untreated disease showed an increase in IGHV5-familyantibodies and a decrease in IGHV families that are rarely used byTG2-specific plasma cells (18, 26). Curiously, IGHV5 was the onlyantibody family for which we observed a correlation between usageamong anti-TG2 antibodies and the degree of mucosal tissuechanges (SI Appendix, Fig. S3B). In particular, the IGHV5 patterncould be ascribed to anti-TG2 serum antibodies using the hallmarkIGHV5-51 gene segment (Fig. 4C). IGHV5-51 usage also reflectedthe total level of anti-TG2 serum IgA (Fig. 4D). Moreover, byassessing binding of patient serum IgA to different regions of TG2,

we observed a preference for N-terminal epitopes, which wasparticularly pronounced in some children with recently developedceliac disease compared with adults with established, untreateddisease (SI Appendix, Fig. S4 A–C). In addition, children with astrong IgA response to the N-terminal domain showed a corre-sponding N-terminal IgG response (SI Appendix, Fig. S4D). Theseresults suggest that the preferential targeting of certain N-terminalTG2 epitopes is most prominent shortly after disease onset andthat the IgA and IgG responses could be coordinated in the earlydisease phase. The wave of antibodies with N-terminal specificitiesproduced initially may effectively block N-terminal TG2 epitopes,thus allowing increased production of non–N-terminal TG2 epi-topes as the disease progresses.Taken together, these results suggest that excessive production

of antibodies that target the main N-terminal epitope and use the

1407-12-C06679-14-D041393-4-A12679-14-A03

1407-10-F061390-6-C081390-6-E091407-11-E04693-2-B01679-14-F01693-10-E02

More N-terminal More C-terminal

75100

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** ** ns **

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Fig. 3. Effect of epitope binding on TG2 enzymatic activity and T-B col-laboration. (A) TG2-mediated deamidation of synthetic gluten peptide wasmeasured at different time points by mass spectrometry. (B) Effect of mAbstargeting different regions of TG2 on gluten peptide deamidation. TG2 ac-tivity is given relative to the deamidation measured in the presence of anon–TG2-specific mAb (693-2-F04). (C) TG2-mediated transamidationassessed by the formation of cross-links between TG2 and FITC-labeledgluten peptide followed by SDS/PAGE and detection of fluorescent gelbands (Left), the intensity of which could be quantified (Right). (D) Effect ofpreincubation with mAbs on TG2-mediated cross-link formation. TG2 activityis given relative to the total fluorescence intensity measured in presence ofmAb 693–2-F04 shown in C. Data shown in A–D are based on average resultsfrom two independent experiments. Column heights indicate means, anddifferences between groups were analyzed using an unpaired t test. **P <0.01. (E and F) Collaboration between gluten-specific hybridoma T cellsrecognizing a deamidated epitope and lymphoma B cells expressing HLA-DQ2.5 in combination with the indicated BCRs assessed by detection of se-creted IL-2. (E) The B cells were incubated with chemically cross-linkedcomplexes of TG2 and deamidated gluten peptide (33merE) before the ad-dition of T cells. (F) Combinations of active TG2 and a nondeamidated glutenpeptide (33merQ) were used for incubation with B cells. TG2 was eitherpresent in solution together with the peptide or prebound to the BCR beforeaddition of peptide. The figure shows representative data from one of twoindependent experiments. Error bars indicate SD based on culture triplicates.

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IGHV5-51 gene segment coincides with initiation of the de-structive immune response in celiac disease. As the inflammationis believed to be driven by gluten-reactive CD4+ T cells, our dataindicate that the activation of B cells specific to particular TG2epitopes goes hand in hand with activation of pathogenic T cells.

DiscussionFueled by the beneficial effect of anti-CD20 therapy, non–antibody-mediated contributions of B cells are receiving in-creasing attention in autoimmune diseases traditionally thought tobe T cell-mediated, such as multiple sclerosis (27). In keeping withthe clinical observations, knockout of MHC-II expression on Bcells resulted in amelioration of T cell-mediated symptoms inmouse models of multiple sclerosis (2) and systemic lupus eryth-ematosus (28), pointing to an important role of B cells as APCs inthese diseases. In lupus-prone mice, self-reactive B cells wereshown to proliferate and interact with T cells at extrafollicular sitesrather than in germinal centers (29). This type of B cell activationis associated with short-lived antibody responses, reflecting thatthe formation of long-lived plasma cells homing to the bonemarrow is germinal-center–dependent (30). A similar type of Bcell activation appears to take place in celiac disease, as anti-TG2antibodies disappear within a few months after patients com-mence a gluten-free diet (31). In addition, TG2-specific plasma

cells isolated from the gut lesion of celiac patients were found tocarry fewer Ig mutations than other plasma cell populations, in-dicating that they have undergone limited affinity maturation (18,26, 32). Strikingly, TG2-specific cells using the preferred IGHVsegments, IGHV3-43 and IGHV5-51, displayed even lower muta-tion levels than other TG2-specific plasma cells. This finding mayreflect that B cells using these gene segments target epitopes thatallow efficient uptake of TG2-gluten complexes and interactionwith gluten-specific T cells. Hence, such B cells may require fewermutations and lower affinity than other TG2-binding B cells beforedifferentiating into plasma cells because they have an advantage inthe competition for T cell help. Alternatively, antibodies usingIGHV3-43 or IGHV5-51 could have an inherent TG2 affinity,which favors their early production and obviates the need for ac-cumulated Ig mutations. However, both mAbs using IGHV5-51and mAbs using other IGHV segments retained an ability tobind TG2 when reverted to their presumed germline configuration,and IGHV5-51mAbs on average had lower avidity than other anti-TG2 mAbs, speaking against an inherent high affinity of antibodiesusing the preferred IGHV segments (18).Anti-TG2 antibodies are highly specific and sensitive markers

for celiac disease (33). However, a pathogenic role of the anti-bodies themselves has not been established (34). Rather, it isbelieved that destruction of the intestinal epithelium is mediatedby intraepithelial CD8+ T cells, which are under control of gluten-reactive CD4+ T cells (35). The CD4+ T cells recognize epitopesthat have been deamidated by TG2, but it is not known how theyare activated or where deamidation takes place. Here, we haveshown that B cells binding catalytically active TG2 can be excellentAPCs for T cells in the presence of nondeamidated gluten pep-tide. As both antigen uptake and deamidation rely on formation ofTG2-gluten enzyme-substrate complexes, the process directly linksgluten internalization by TG2-specific B cells to deamidation andantigen presentation. The efficiency of gluten presentation byTG2-specific B cells depended on the epitope recognized by theBCR and reflected the catalytic activity of BCR-bound TG2.Hence, the ability of B cells to interact with catalytically activeTG2 and take up TG2-gluten complexes explains the bias towardcertain N-terminal and core-domain epitopes in celiac disease.Low levels of autoantibodies can often be detected years be-

fore the onset of symptomatic autoimmunity (22, 23). A possibleexplanation for this phenomenon is that a limited extent of T-Bcollaboration is not harmful until a certain threshold is reached.Thus, development of clinical disease may rely on expansion ofrare antigen-specific B cell clones, which give rise to potentiallyharmful autoantibodies and serve as efficient APCs for patho-genic T cells. We have shown that production of IGHV5-51 au-toantibodies targeting the preferred N-terminal TG2 epitopecoincides with clinical onset of celiac disease. Although wecannot rule out that formation of such antibodies is merely aconsequence of disease progression, our data suggest that B cellswith this specificity could be directly involved in stimulatinggluten-specific T cells. Thus, it is likely that the close connectionbetween production of anti-TG2 antibodies and development ofceliac disease reflects a role for B cells targeting permissive TG2epitopes as the main APCs for gluten-specific T cells. A similarrole of B cells is possible in other autoimmune diseases char-acterized by T cell-mediated tissue destruction.

Materials and MethodsPatients. Duodenal biopsies and/or peripheral blood samples were collectedfrom adult celiac patients who had given their informed consent. The di-agnosis of celiac disease was given according to the guidelines of the BritishSociety of Gastroenterology (36). Ethical approval of the experimental pro-cedures was obtained from the Regional Ethics Committee of South-EasternNorway (project 2017/2720). Serum samples from children with recentlydeveloped celiac disease were included from the Hungarian participants inthe PreventCD project (http://www.preventceliacdisease.com), a prospectivemulticenter study following at-risk infants from celiac families. Informedconsent was given by the parents, and ethical approval was provided by theEthics Committee of Heim Pál Children’s Hospital, Budapest, Hungary.

A B

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ns ns ns** ** ** *

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Fig. 4. TG2-specific serum antibodies in potential and established, un-treated celiac disease. (A) Anti-TG2 serum IgA and IgG levels determined asarea-under-the-curve (AUC) values by ELISA in patients with established,untreated celiac disease (n = 12) having severe intestinal inflammation(Marsh score 3A-C) and potential celiac patients (n = 12) characterized by thepresence of antibodies but no (Marsh 0) or mild (Marsh 1) inflammation (25).Plotted values were calculated from titration binding curves (SI Appendix,Fig. S3A). Column heights indicate mean antibody levels, and differencesbetween groups were analyzed by one-way ANOVA with Holm–Sidak mul-tiple comparisons correction. *P < 0.05; **P < 0.01; ****P < 0.0001. (B)Fraction of antibodies assigned to different IGHV families determined bymass spectrometry analysis of purified TG2-specific serum IgA or the flow-through (FT) fraction. Calculated frequencies are based on the summed in-tensities of all identified IGHV segments. Sera were obtained from potential(n = 6) or untreated (n = 6) celiac patients. Differences between groups wereanalyzed using a paired t test. (C) Fraction of antibodies using IGHV5-51determined by mass spectrometry analysis of the purified anti-TG2 IgA orTG2 FT fractions of patients with potential or untreated celiac diseasegrouped according to the degree of intestinal inflammation. Statistical dif-ference was evaluated by one-way ANOVA. (D) Correlation between thefraction of IGHV5-51 in purified anti-TG2 IgA and the level of anti-TG2 serumIgA determined as the EC50 value for binding of purified total serum IgA toTG2 in ELISA. Data points were fitted to a four-parameter dose–responsecurve, and the P value was calculated by Pearson correlation analysis.

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Flow Cytometry and Single-Cell Sorting. Biopsy specimens were processed intosingle-cell suspensions as previously described (32) and stained with a combina-tion of tetramerized, recombinant human TG2 (SI Appendix, Materials andMethods) and the following antibodies: goat anti-human IgA-FITC (SouthernBiotech), anti-human CD3-Brilliant Violet 570 (clone UCHT1, BioLegend), anti-human CD14-Brilliant Violet 570 (clone M5E2, BioLegend), anti-human CD27-Brilliant Violet 421 (clone O323, BioLegend). TG2 tetramers were prepared byincubating biotinylated WT TG2 and TG2 variants with Strep-Tactin-allophycocyanin (IBA) and streptavidin-PE (Thermo), respectively, using a 1:1molar ratio between TG2 and biotin-binding sites. Flow cytometry was per-formed using a FacsAriaII instrument (BD), and data were analyzed with FlowJov10. Single gut plasma cells (large, CD3−CD14−IgA+CD27+ lymphocytes) reactivewith WT TG2 only (N-terminal epitopes) or with both WT TG2 and TG3/TG2(non–N-terminal epitopes) were sorted and preserved as previouslydescribed (32).

Next-Generation Sequencing. Single-cell RT-PCR and sequencing librarypreparation were done as previously described (32). Paired-end sequencingof 300 bp was performed using Illumina MiSeq at the Norwegian Sequenc-ing Centre, Oslo, Norway (https://www.sequencing.uio.no/). Processing ofraw sequences and further data analysis, including clonal assignment, weredone using an in-house–developed pipeline as previously described (32).

Serum Antibody Purification and Mass Spectrometry. IGHV usage among se-rum IgA antibodies was assessed by liquid chromatography–tandem massspectrometry analysis of purified antibodies essentially as previously de-scribed (16). Total IgA was purified from 1 mL of serum followed by isolationof the TG2-binding fraction by incubation with 35 μg biotinylated TG2immobilized on 200 μL streptavidin-agarose (Sigma). After extensive washing,antibodies were eluted with 20 mM HCl, and TG2 reactivity was confirmed by

ELISA. The antibodies were reduced and alkylated before digestion withtrypsin (Promega). The generated peptides were analyzed based on duplicateruns using a Q Exactive mass spectrometer (Thermo). IGHV peptides wereidentified by searching against a database containing the amino acid se-quences of all human V-gene segments obtained from the International Im-MunoGeneTics Information System (37). The frequency of antibodies usingindividual IGHV segments was calculated from intensity-based absolutequantification values, obtained with MaxQuant software (38).

Data Availability. Sequence data have been deposited at the European Genome-phenome Archive, which is hosted by the European Bioinformatics Institute,under accession number EGAS00001003658. Proteomics data on purified serumantibodies have been deposited to the ProteomXchange Consortium via thePRIDE (39) partner repository with the data set identifier PXD013777.

Biochemical Analyses. Assays for antibody binding, TG2 activity, and T-Bcollaboration are described in the SI Appendix, Materials and Methods.

ACKNOWLEDGMENTS. We thank Gerry Melino for providing TG2 KO mice;Bjørg Simonsen, Marie K. Johannesen, and Saykat Das for technical assis-tance; and Maria Stensland (Proteomics Core Facility, Oslo University Hospi-tal) for conducting mass spectrometry analyses. Flow cytometry and cell-sorting experiments were performed at the Flow Cytometry Core Facility,Oslo University Hospital. This work was supported by grants from the Re-search Council of Norway through its Centre of Excellence funding scheme(project number 179573/V40 to L.M.S.), the South-Eastern Norway RegionalHealth Authority (projects 2014045, 2016113, and 2018067 to L.M.S.),Stiftelsen KG Jebsen (SKGJ-MED-017 to L.M.S.), and the Hungarian NationalResearch Fund (NKFI 120392 to I.R.K.-S.).

1. O. Chan, M. J. Shlomchik, A new role for B cells in systemic autoimmunity: B cellspromote spontaneous T cell activation in MRL-lpr/lpr mice. J. Immunol. 160, 51–59(1998).

2. N. Molnarfi et al., MHC class II-dependent B cell APC function is required for inductionof CNS autoimmunity independent of myelin-specific antibodies. J. Exp. Med. 210,2921–2937 (2013).

3. I. Jelcic et al., Memory B cells activate brain-homing, autoreactive CD4+ T cells inmultiple sclerosis. Cell 175, 85–100.e23 (2018).

4. S. Hong et al., B cells are the dominant antigen-presenting cells that activate naiveCD4+ T cells upon immunization with a virus-derived nanoparticle antigen. Immunity49, 695–708.e4 (2018).

5. L. S. Hoydahl et al., Plasma cells are the most abundant gluten peptide MHC-expressing cells in inflamed intestinal tissues from patients with celiac disease.Gastroenterology 156, 1428–1439.e10 (2019).

6. W. Dieterich et al., Identification of tissue transglutaminase as the autoantigen ofceliac disease. Nat. Med. 3, 797–801 (1997).

7. L. M. Sollid, O. Molberg, S. McAdam, K. E. Lundin, Autoantibodies in coeliac disease:Tissue transglutaminase: Guilt by association? Gut 41, 851–852 (1997).

8. L. W. Vader et al., Specificity of tissue transglutaminase explains cereal toxicity inceliac disease. J. Exp. Med. 195, 643–649 (2002).

9. B. Fleckenstein et al., Gliadin T cell epitope selection by tissue transglutaminase inceliac disease. Role of enzyme specificity and pH influence on the transamidationversus deamidation process. J. Biol. Chem. 277, 34109–34116 (2002).

10. O. Molberg et al., Tissue transglutaminase selectively modifies gliadin peptides thatare recognized by gut-derived T cells in celiac disease. Nat. Med. 4, 713–717 (1998).

11. Y. van de Wal et al., Selective deamidation by tissue transglutaminase strongly en-hances gliadin-specific T cell reactivity. J. Immunol. 161, 1585–1588 (1998).

12. L. M. Sollid, S. W. Qiao, R. P. Anderson, C. Gianfrani, F. Koning, Nomenclature andlisting of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ mole-cules. Immunogenetics 64, 455–460 (2012).

13. D. M. Pinkas, P. Strop, A. T. Brunger, C. Khosla, Transglutaminase 2 undergoes a largeconformational change upon activation. PLoS Biol. 5, e327 (2007).

14. S. Liu, R. A. Cerione, J. Clardy, Structural basis for the guanine nucleotide-bindingactivity of tissue transglutaminase and its regulation of transamidation activity. Proc.Natl. Acad. Sci. U.S.A. 99, 2743–2747 (2002).

15. R. Iversen et al., Transglutaminase 2-specific autoantibodies in celiac disease targetclustered, N-terminal epitopes not displayed on the surface of cells. J. Immunol. 190,5981–5991 (2013).

16. R. Iversen et al., Strong clonal relatedness between serum and gut IgA despite dif-ferent plasma cell origins. Cell Rep. 20, 2357–2367 (2017).

17. R. Iversen, S. Mysling, K. Hnida, T. J. Jørgensen, L. M. Sollid, Activity-regulatingstructural changes and autoantibody epitopes in transglutaminase 2 assessed by hy-drogen/deuterium exchange. Proc. Natl. Acad. Sci. U.S.A. 111, 17146–17151 (2014).

18. R. Di Niro et al., High abundance of plasma cells secreting transglutaminase 2-specificIgA autoantibodies with limited somatic hypermutation in celiac disease intestinallesions. Nat. Med. 18, 441–445 (2012).

19. J. Stamnaes, I. Cardoso, R. Iversen, L. M. Sollid, Transglutaminase 2 strongly binds toan extracellular matrix component other than fibronectin via its second C-terminalbeta-barrel domain. FEBS J. 283, 3994–4010 (2016).

20. J. Stamnaes, R. Iversen, M. F. du Pré, X. Chen, L. M. Sollid, Enhanced B-cell receptorrecognition of the autoantigen transglutaminase 2 by efficient catalytic self-multi-merization. PLoS One 10, e0134922 (2015).

21. K. Hnida et al., Epitope-dependent functional effects of celiac disease autoantibodieson transglutaminase 2. J. Biol. Chem. 291, 25542–25552 (2016).

22. M. R. Arbuckle et al., Development of autoantibodies before the clinical onset ofsystemic lupus erythematosus. N. Engl. J. Med. 349, 1526–1533 (2003).

23. S. Rantapää-Dahlqvist et al., Antibodies against cyclic citrullinated peptide and IgArheumatoid factor predict the development of rheumatoid arthritis. Arthritis Rheum.48, 2741–2749 (2003).

24. T. T. Salmi et al., Immunoglobulin A autoantibodies against transglutaminase 2 in thesmall intestinal mucosa predict forthcoming coeliac disease. Aliment. Pharmacol.Ther. 24, 541–552 (2006).

25. G. Oberhuber, G. Granditsch, H. Vogelsang, The histopathology of coeliac disease:Time for a standardized report scheme for pathologists. Eur. J. Gastroenterol.Hepatol. 11, 1185–1194 (1999).

26. O. Snir et al., Analysis of celiac disease autoreactive gut plasma cells and their cor-responding memory compartment in peripheral blood using high-throughput se-quencing. J. Immunol. 194, 5703–5712 (2015).

27. R. Li, K. R. Patterson, A. Bar-Or, Reassessing B cell contributions in multiple sclerosis.Nat. Immunol. 19, 696–707 (2018).

28. J. R. Giles, M. Kashgarian, P. A. Koni, M. J. Shlomchik, B cell-specific MHC class II de-letion reveals multiple nonredundant roles for B cell antigen presentation in murinelupus. J. Immunol. 195, 2571–2579 (2015).

29. J. William, C. Euler, S. Christensen, M. J. Shlomchik, Evolution of autoantibody re-sponses via somatic hypermutation outside of germinal centers. Science 297, 2066–2070 (2002).

30. M. J. Shlomchik, F. Weisel, Germinal center selection and the development of memoryB and plasma cells. Immunol. Rev. 247, 52–63 (2012).

31. S. Sulkanen et al., Tissue transglutaminase autoantibody enzyme-linked immuno-sorbent assay in detecting celiac disease. Gastroenterology 115, 1322–1328 (1998).

32. B. Roy et al., High-throughput single-cell analysis of B cell receptor usage amongautoantigen-specific plasma cells in celiac disease. J. Immunol. 199, 782–791 (2017).

33. D. A. Leffler, D. Schuppan, Update on serologic testing in celiac disease. Am. J. Gas-troenterol. 105, 2520–2524 (2010).

34. K. Lindfors, M. Mäki, K. Kaukinen, Transglutaminase 2-targeted autoantibodies inceliac disease: Pathogenetic players in addition to diagnostic tools? Autoimmun. Rev.9, 744–749 (2010).

35. B. Jabri, L. M. Sollid, T cells in celiac disease. J. Immunol. 198, 3005–3014 (2017).36. J. F. Ludvigsson et al.; BSG Coeliac Disease Guidelines Development Group; British

Society of Gastroenterology, Diagnosis and management of adult coeliac disease:Guidelines from the British Society of Gastroenterology. Gut 63, 1210–1228 (2014).

37. M. P. Lefranc et al., IMGT, the international ImMunoGeneTics database. Nucleic AcidsRes. 27, 209–212 (1999).

38. J. Cox, M. Mann, MaxQuant enables high peptide identification rates, individualizedp.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Bio-technol. 26, 1367–1372 (2008).

39. Y. Perez-Riverol et al., The PRIDE database and related tools and resources in 2019:Improving support for quantification data. Nucleic Acids Res. 47, D442–D450 (2019).

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