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. 2022 Apr 6;17(4):e0266543. doi: 10.1371/journal.pone.0266543

Injection of prototypic celiac anti-transglutaminase 2 antibodies in mice does not cause enteropathy

Christian B Lindstad 1,2, M Fleur du Pré 1,3, Jorunn Stamnaes 1,2,3, Ludvig M Sollid 1,2,3,*
Editor: Lucienne Chatenoud4
PMCID: PMC8985999  PMID: 35385534

Abstract

Background

Celiac disease is an autoimmune enteropathy driven by dietary intake of gluten proteins. Typical histopathologic features are villous flattening, crypt hyperplasia and infiltration of inflammatory cells in the intestinal epithelium and lamina propria. The disease is hallmarked by the gluten-dependent production of autoantibodies targeting the enzyme transglutaminase 2 (TG2). While these antibodies are specific and sensitive diagnostic markers of the disease, a role in the development of the enteropathy has never been established.

Methods

We addressed this question by injecting murine antibodies harboring the variable domains of a prototypic celiac anti-TG2 immunoglobulin into TG2-sufficient and TG2-deficient mice evaluating for celiac enteropathy.

Results

We found no histopathologic abnormalities nor clinical signs of disease related to the injection of anti-TG2 IgG or IgA.

Conclusions

Our findings do not support a direct role for secreted anti-TG2 antibodies in the development of the celiac enteropathy.

Introduction

Celiac disease is an autoimmune enteropathy driven by ingestion of gluten proteins. The celiac lesion is characterized by villous flattening, crypt hyperplasia and infiltration of inflammatory cells. Extraintestinal manifestations include skin disease, anemia, osteopenia, neurological symptoms and obstetric complications. The enzyme transglutaminase 2 (TG2) has a crucial role in the disease, both by catalyzing deamidation of gluten peptides into immunogenic T cell epitopes [1], and by being the target of a disease-specific autoantibody response [2]. Anti-TG2 IgA and IgG are sensitive and specific markers of the disease [3,4]. These antibodies disappear rapidly from the blood upon initiation of a gluten free diet [5]. When absent in serum, the antibodies may still be found in the intestinal tissue [6,7].

Anti-TG2 antibodies have previously been suspected to contribute to enteropathy as well as extraintestinal manifestations of celiac disease [8]. In support of this, the antibodies have been reported to deposit extracellularly both in the intestine and at extraintestinal sites [9,10]. Serum titers correlate with the degree of enteropathy [11]. Yet, a group of individuals defined as “potential celiac disease” have anti-TG2 in serum and in the intestinal mucosa despite normal mucosal histology [12].

If anti-TG2 antibodies play a role in development of enteropathy, they would be an interesting therapeutic target. In vitro studies have reported numerous biological effects of anti-TG2 antibodies, including inhibiting differentiation, inducing proliferation or reducing attachment of intestinal epithelial cells, as reviewed [8]. The in vivo evidence supporting a role of secreted anti-TG2 antibodies in celiac enteropathy is, however, scarce and inconclusive. When generating an anti-TG2 response in vivo by immunizing with TG2 [13] or expression of mini-antibodies using virus vectors [14], no significant pathology was observed. Kalliokoski et al. injected celiac IgA-deficient serum, total IgG or recombinant monoclonal anti-TG2 mini-antibodies [15,16]. They observed minor histologic changes and in one study minor clinical effects. Limitations of these studies are the use of non-physiological antibodies (mini-bodies or polyclonal human serum antibodies) as well as immunocompromized mouse strains. To conclusively address whether anti-TG2 antibodies as found in the serum of celiac patients play a direct role in the development of enteropathy, we injected TG2-sufficient and TG2-deficient mice with murine IgG or IgA harboring the variable domains of the prototypic celiac anti-TG2 antibody 679-14-E06 (from here denoted 14E06) [17]. We observed no evidence of enteropathy nor clinical signs of disease. Thus, this study does not support a direct role for anti-TG2 antibodies in development of celiac enteropathy.

Materials and methods

Generation of murine 14E06 antibodies

Hybridomas producing monoclonal murine antibodies with 14E06 variable domains were generated from naive B cells of 14E06 immunoglobulin knock-in mice as described [18]. The 14E06 antibody has equal affinity (5 nM) to mouse and human TG2 [18]. Antibodies were purified from culture supernatants using HiTrap protein L columns (GE), buffer exchanged to PBS and sterile filtered before storage at -20°C until use.

Mice

C57Bl/6 mice were purchased from Janvier Labs. Tgm2-/- mice on C57Bl/6 background [19] were kindly provided by G. Melino and bred in-house. Mice were age and sex-matched between groups and included in the experiment at 6 or 8 weeks of age. Each experimental group was split evenly between cages. Injections were performed cage-by-cage. Mice were kept at the Department of Comparative Medicine, Oslo University Hospital, Rikshospitalet (Oslo, Norway) under specific pathogen-free conditions. They were inspected daily by attending staff during the experiments and weighed at least every other day. All animal experiments were pre-approved by the Norwegian Food Safety Authority (Mattilsynet).

Experimental procedures and collection of samples

IgA or a mix of IgG2b and IgG2c were diluted in sterile PBS. For each injection, 200 μL was injected in the tail vein. Blood samples were collected from the lateral saphenous vein on day 0, 10 and 20 (Fig 1). For four IgA-injected mice, the third sample was taken on day 16 or 17, and a fourth sample on day 20 was obtained by postmortem cardiac puncture. Blood was allowed to coagulate for 1–2 hours, centrifuged at 900 g for 14 min at 4°C and serum was stored at -20°C. At the end of the experiment, the small intestine was extracted and the proximal 2 cm discarded. Boluses of feces were gently flushed out with ice-cold PBS. Samples from corresponding gut segments were fixed in 10% neutral buffered formalin (Sigma) for 24 hours, dehydrated and embedded in paraffin. The automated Tissue-Tek Paraform Sectionable Cassette System (Sakura) was used with orientation gels to ensure proper orientation.

Fig 1. Overview of experimental setup.

Fig 1

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Anti-TG2 was administered by intravenous injection at the indicated time points. Either a mix of IgG2b and IgG2c (100 μg each) or IgA (400 μg) or PBS was given each time. Blood was collected at indicated time points prior to injections. At the end of the experiment, samples of the small intestine were fixed in formalin or embedded in OCT and frozen. The figure was created using elements from Servier medical arts (www.smarts.servier.com).

Histology and immunohistochemistry

Paraffin embedded samples were cut into 2.5 μm sections. In hematoxylin/eosin-stained sections, villus height (Vh), crypt depth (Cd) and villus height/crypt depth ratio (Vh/Cd ratio) were measured only for well oriented villus-crypt pairs. Spanning at least three gut pieces, the mean values of the five Vh/Cd pairs with the longest villi were reported. If five valid measurements could not be obtained from one gut segment, the segment was excluded from the analysis. Number of excluded data points for duodenum/ileum in each group: WT IgG: 4/1, Tgm2-/- IgG: 6/1, WT IgA: 3/1, WT PBS: 2/0, Tgm2-/- PBS 1/0. For intraepithelial lymphocyte (IEL) counts, sections were stained for CD3 and counterstained with hematoxylin. As primary antibody, rabbit monoclonal anti-CD3, (SP7, Abcam) was used at 1:100. Samples were pretreated with Dako Target Retrieval Solution Citrate pH 6 (Agilent Technologies). For detection, rabbit on Rodent HRP (Biocare Medical) was used followed by development with 3,3′-diaminobenzidine. CD3+ IELs were expressed per 100 epithelial cells in a hotspot villus, reporting the mean of three measurements from thee different gut pieces. Slides were scanned with Pannoramic Midi and analyzed with Case Viewer (both 3DHISTECH) blinded to the investigator. Evaluation criteria were defined a priori.

Immunofluorescence

Unfixed small intestine was embedded in optimal cutting temperature (OCT) and snap frozen in liquid nitrogen. Six μm sections were adhered to SuperFrost slides by thaw-mounting and air-dried. To demonstrate binding of hybridoma-derived mouse 14E06 antibodies to mouse and human TG2, 6 μm unfixed tissue sections from WT mouse small intestine, or Tgm2-/- mouse small intestine pre-incubated with recombinant human TG2 or recombinant mouse TG2 (7 μg/ml), were stained with 3 μg/ml 14E06 mouse IgG2c followed by detection with donkey anti mouse IgG-Cy3 (Jackson ImmunoResearch) (S1 Fig). To assess co-localization between injected IgG and endogenous TG2, unfixed small intestinal tissue sections were blocked in 1.25% IgG-free BSA (Jackson Immunoresearch) in PBS and stained with goat-anti-mouse-IgG (Jackson ImmunoResearch) and rabbit-anti-mouse-TG2 (custom made antibody from Pacific Immunology) followed by detection with donkey anti-goat Alexa Fluor 488 (Jackson ImmunoResearch) and donkey anti-rabbit Cy3 (Jackson ImmunoResearch). To quantify and assess tissue deposition of injected IgG, unfixed sections were stained with anti-mouse-IgG2b-biotin and anti-mouse-IgG2c-biotin (both SouthernBiotech) (both at 3 μg/ml, as a mix or separately) followed by Streptavidin-Cy3 (2.5 μg/mL) (GE Lifesciences). Slides were counterstained with 40,6-diamidino-2-phenylindole (DAPI) and mounted with ProLong Diamond Antifade Mountant (ThermoFisher). Slides were imaged on an inverted Nikon fluorescence microscope (Nikon Eclipse Ti-S; Nikon, Tokyo, Japan) and images were processed in Fiji (ImageJ) [20]. Subepithelial antibody deposits were quantified in the small intestine of IgG-injected WT (n = 4) and Tgm2-/- mice (n = 4) as well as PBS injected WT (n = 1) and Tgm2-/- mice (n = 1). Fluoresence intensity was quantified from 4–8 villi per image and 1–2 images were anlyzed per mouse. Fluorescence intensity was measured in FIJI from unprocessed images aquired with identical microscope settings. Subepithelial regions of interest were defined using the freehand tool (linewith 5 pixels for 20x images and 10 pixels for 10x images) and integrated density was measured. Integrated density from a region drawn within the epitelial cell layer of the same villus was subtracted as background.

ELISA to evaluate anti-TG2 titers in serum

ELISA plates (Nunc) were coated with 5 μg/mL recombinant human TG2 [21] in PBS at 4°C overnight. After washing and blocking, plates were incubated with dilutions of mouse serum (1.5 hours at room temperature) followed by biotinylated goat anti-mouse IgG2b, IgG2c or IgA (SouthernBiotech, 1.5 hours at room temperature), then alkaline phosphatase-conjugated streptavidin (SouthernBiotech, 0.5 hours at room temperature) before development with phosphatase-substrate (Sigma). Optical density was determined at 405 nm. Absolute concentrations were estimated by comparing with dilutions of antibody and interpolating from standard curves.

Statistical methods and data visualization

Statistical comparisons and data visualization were done using GraphPad Prism 9.3.1 (GraphPad Software). For comparisons, individual Mann-Whitney tests were used. P < 0.05 was considered statistically significant, and no correction for multiple testing was applied. The study was powered to detect differences of >1 for Vh/Cd ratio and >10 for IEL counts with α = 0.05 and β = 0.20.

Results

Generation of anti-TG2 antibodies and choice of isotypes

The patient-derived 14E06 is a prototypic celiac anti-TG2 antibody [17,22]. Murine antibodies harboring the 14E06 variable domains were generated using hybridoma technology [18]. To maximize the chances of revealing a potential inflammatory effect, we chose to inject the main experimental groups with a mix of 14E06 IgG2b and IgG2c (see discussion). Based on the fact that the clinical presentation of IgA deficient celiac patients is similar to that of IgA-sufficient patients [23–25], we regarded IgA-injected mice mainly as a control group.

Overview of experimental setup and confirmation of injected anti-TG2 antibodies in serum and intestinal tissue

Experimental setup is outlined in Fig 1. Data were pooled from two independent experiments. Antibodies were injected intravenously at day 0, 5, 10 and 15. The main groups consisted of wild-type (WT, n = 14) and Tgm2-/- mice (n = 12) that received a mix of 100 μg IgG2b and 100 μg IgG2c each time. Additional groups included WT mice that received 400 μg IgA (n = 8) or PBS (n = 6), or Tgm2-/- mice that received PBS (n = 2). A higher dose of IgA was chosen because of short serum half-life [26]. In IgG-injected mice, high serum levels of both isotypes were detected on day 10 and 20 (Fig 2A and 2B). There were no statistically significant differences in serum levels between IgG-injected WT and Tgm2-/- groups. Surprisingly, no TG2-specific IgA was detected in serum on day 10 or 20 (Fig 2C). To confirm presence of injected IgA, blood was collected shortly after the 3rd antibody injection in a few mice. Small amounts of TG2-specific IgA could be detected in serum of 2/2 mice on day 16, while traces were detected in 1/2 mice on day 17 (Fig 2C), indicating rapid clearance. There was no reactivity to TG2 in serum of PBS-injected mice (Fig 2A–2C). Immunofluorescence staining of unfixed small intestine from WT mice injected with IgG revealed supepithelial IgG deposits that co-localized with endogenous extracellular matrix (ECM)-bound TG2 (Fig 3A). Immunofluoresence staining for mouse IgG2b and IgG2c confirmed that injected IgG reached the intestinal tissue and that ECM deposits were formed in the tissue in an antigen-dependent manner (Fig 3B and 3C). Weak signal was also observed in the small intestine of IgG-injected Tgm2-/- mice but no subepithelial deposits were detected. No signal was detected in PBS-injected mice.

Fig 2. Anti-TG2 in serum.

Fig 2

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Serum was obtained on day 0, 10 and 20 and analyzed for anti-TG2 antibodies by ELISA. Absolute concentrations of IgG2b (a), IgG2c (b) and IgA (c) were estimated by interpolating from standard curves. For four IgA-injected mice, the third serum sample was taken either on day 16 (n = 2) or day 17 (n = 2) and a fourth sample was taken on day 20 by postmortem cardiac puncture. Arrows indicate time of antibody injections. Dots and bars represent mean +/- SD. Data represent all mice from the two independent experiments.

Fig 3. Injected anti-TG2 antibodies reach the intestinal tissue.

Fig 3

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(a) Immunofluorescence staining of frozen sections of small intestine obtained on day 20 show deposition of mouse IgG (cyan) in the basement membrane of IgG injected WT mice that co-localizes with ECM staining for endogenous TG2 (magenta). No IgG deposits were detected in PBS injected WT mice and no clear ECM deposits were observed in IgG-injected Tgm2-/- mice. Nuclei counterstained with DAPI are shown in blue. (b) Distribution of IgG2b and IgG2c in the small intestine of WT mice injected with IgG (top panels). Weak antibody signal is detected also in the intestine of Tgm2-/- mice while no signal is seen in PBS-injected mice, which indicates that antibody presence in tissue does not per se depend on presence of cognate antigen. (c) Quantification of subepithelial fluoresence signal intensity from staining for IgG2b and IgG2c together (top) or separately (bottom). Each dot represents mean fluorescence intensity calculated from one image as described in materials and methods. Bar graphs show the group mean fluorescence intensity with standard error of mean. Scale bars represent 100 μm.

Clinical parameters

No signs of disease or distress were observed through the study period. The weigth gain in the different experimental groups are depicted in Figs 4 and S2. Testing weight change on day 20 of the IgG-injected WT group to each control group revealed no statistically significant differences. No obvious diarrhea occurred in any cage, although this was not evaluated in a systematic fashion.

Fig 4. Injection of anti-TG2 antibodies does not impair weight gain.

Fig 4

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The graphs report weight as % change from baseline for WT mice and Tgm2 deficient mice receiving injections of IgG. Dots and bars represent mean +/- SD. Data represent all mice from the two independent experiments.

Tissue architechture and IEL counts

Samples of small intestine were obtained on day 20. Vh, Cd and Vh/Cd ratio were measured as demonstrated in Fig 5A. In duodenum, there was no statistically significant difference between the IgG-injected WT group and any control group (Fig 5B). In ileum, Vh and Vh/Cd ratio were slightly lower in IgG-injected Tgm2-/- mice compared to IgG-injected WT mice (p = 0.022 and 0.046, respectively (Fig 5C). These differences were not considered biologically relevant. Next, IELs were counted (Fig 6A). In duodenum, there were no statistically significant differences between the WT-IgG group and any of the control groups (Fig 6B). In ileum, the IEL count was significantly higher in the IgG-injected Tgm2-/- group compared to the IgG-injected WT group (p = 0.006, Fig 6B). However, the difference was not considered biologically relevant. Taken together, the histologic evaluation revealed no signs enteropathy in any group.

Fig 5. No difference in mucosal architecture between groups.

Fig 5

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(a) Examples of Vh and Cd measurements. Formalin-fixed paraffin-embedded samples stained with hematoxylin/eosin. Representative images show well-oriented pairs of villus and crypt from duodenum and ileum. Numbers indicate length of corresponding bars. (b, c) Vh, Cd and Vh/Cd ratio in duodenum (b) and ileum (c) of the different experimental groups. The WT IgG group was compared to each control group by individual Mann-Whitney tests. Data are pooled from two independent experiments. Bars represent mean +/- SD. *P ≤ 0.05. n.s.: Not significant.

Fig 6. IEL count in duodenum and ileum.

Fig 6

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(a) Examples of staining for anti-CD3. Formalin-fixed paraffin-embedded sections stained with anti-CD3 and counterstained with hematoxylin. Representative images show CD3+ cells in duodenum and ileum. (b) CD3+ IELs per 100 epithelial cells in well-oriented villi in duodenum and ileum. The WT IgG group was compared to each control group by individual Mann-Whitney tests. Data represent all mice from the two independent experiments. Bars represent mean +/- SD. **P ≤ 0.01. n.s.: Not significant.

Discussion

In this study we found no evidence for a direct role of secreted anti-TG2 in the pathogenesis of celiac enteropathy as evaluated by standard histologic criteria and clinical parameters. Our approach has several advantages compared to previous in vivo studies. Injecting murine immunoglobulins permits immunocompetent recipients and eliminates the need to introduce foreign proteins, virus vectors or adjuvant. Also, even though human IgGs bind mouse Fc-receptors with affinities comparable to mouse IgGs [27], the clinical outcome may still differ when species-incompatible istotypes are used.

Effector functions of IgG antibodies are mediated through their interaction with Fcγ-receptors on the cell surface and by interactions with the complement system. C57Bl/6 mice express IgG1, IgG2b, IgG2c and IgG3. Of note, these are not direct homologues to the IgG subclasses in humans. IgG2 subtypes are generally considered the most potent mediators of cellular cytotoxicity and complement activation. Although not formally characterized, IgG2c is believed to have comparable properties to IgG2a. As opposed to IgG1 and IgG3, IgG2a (and hence, probably IgG2c) and IgG2b bind to all stimulatory mouse FcγRs [27,28]. Moreover, mouse IgG1 does not activate complement, and has even been implicated with anti-inflammatory properties [29]. As such, the apporach of Di Niro et al. [14] using IgG1-based mini-antibodies may have been suboptimal.

If anti-TG2 antibodies were pathogenic, the duration of exposure necessary to develop enteropathy would be unknown. Minor effects were reported already on day eight in the studies by Kalliokoski et al. [15,16]. Enteropathy is usually detectable in celiac patients after two weeks of gluten challenge. At this time, serum anti-TG2 is still normal or mildly increased [30–32]. However, high local concentrations in the intestinal tissue could be present earlier. We believe our trial length of 20 days would be sufficient to detect a significant contribution by anti-TG2 to enteropathy.

Immunoglobulin serves biological roles as secreted and water-soluble antibodies operating in extracellular fluids, but also as the antigen receptor of B-cells being anchored in the cell membrane as a transmembrane protein. This study is only addressing the role of anti-TG2 immunoglobulins as secreted antibodies. An involment of anti-TG2 immunoglobulins in the pathogenesis of celiac disease as B-cell receptor is likely [33]. Further, our study is also only addressing the role of anti-TG2 immunoglobulins in relation to enteropathy. Anti-TG2 immunoglobulin may have effects elsewhere in the body, effects which could very well explain many of the extraintestinal manifestations of celiac disease [34].

In a mouse model of celiac disease, B cells were found to be important in the pathogenesis by testing mice that were made devoid of B cells by genetic manipulation[35]. Yet in the same mouse model with B cells present, circulating anti-TG2 antibodies could not be detected [36]. These observations support the notion that circulating anti-TG2 antibodies are not implicated in generation of the celiac enteropathy.

Recent observations from the above mentioned animal model [36] and from a clinical trial with a TG2 inhibitor [37] support the notion that TG2 is engaged in the pathogenesis of celiac disease, and that the catalytic activity of the enzyme is involved. The antibodies that celiac disease patients make against TG2, as is the case for the 14E06 antibody, do not interfere with the catalytic activity of TG2 [17]. Observing effects of celiac patient antibodies that would implicate inhibition of enzyme activity would thus be unexpected.

Based on accumulated in vivo data, we believe that anti-TG2 immunoglobulins in the form of secreted antibodies do not play a major role in the development of enteropathy in celiac disease. Therefore, efforts to discover novel therapeutics are probably better directed elsewhere. Of note, anti-TG2 immunoglobulins may still play an important role in pathogenesis of celiac disease as the antigen receptor of B cells which present antigen to T cells [33]. Also, the contribution of anti-TG2 to extraintestinal manifestations of celiac disease has not been investigated in detail. This would be an interersting topic for future research.

Supporting information

S1 Fig. Confirmation of mouse TG2 reactivity of mAb 14E06.

Hybridoma-derived 14E06 (mouse IgG2c) binds to endogenous TG2 in the ECM of mouse small intestine (left panel). Mouse 14E06 (IgG2c) also binds to recombinant human or mouse TG2 immobilized in the ECM of Tgm2-/- mouse small intestine (middle and right panel). Nuclei were counterstained with DAPI. Scale bar represents 100μm.

(TIF)

Click here for additional data file. (1.5MB, tif)
S2 Fig. Weight change in control groups.

The graph reports weight as % change from baseline for the different groups as indicated. Dots and bars represent mean +/- SD. Data represent all mice of each group from the two independent experiments.

(TIF)

Click here for additional data file. (77.8KB, tif)
S1 Dataset

(XLSX)

Click here for additional data file. (56.3KB, xlsx)

Acknowledgments

We thank Liv Kleppa, Bjørg Simonsen, Marie Kongshaug Johannesen and Alisa Dewan for excellent technical assistance and support. We thank the staff at the Department of Comparative Medicine, Oslo University Hospital, Rikshospitalet, for animal husbandry and care, and staff at the Department of Pathology, Oslo University Hospital (Rikshospitalet and Radiumhospitalet), for preparation of histologic samples.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by grants from the Research Council of Norway (project 275053 to L.M.S.), the European Commission (project ERC-2010-Ad-268541 to L.M.S.), and the University of Oslo. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Molberg O, McAdam SN, Korner R, Quarsten H, Kristiansen C, Madsen L, et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med. 1998;4(6):713–7. doi: 10.1038/nm0698-713 [DOI] [PubMed] [Google Scholar]
  • 2.Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med. 1997;3(7):797–801. doi: 10.1038/nm0797-797 [DOI] [PubMed] [Google Scholar]
  • 3.Leffler DA, Schuppan D. Update on serologic testing in celiac disease. Am J Gastroenterol. 2010;105(12):2520–4. doi: 10.1038/ajg.2010.276 [DOI] [PubMed] [Google Scholar]
  • 4.Al-Toma A, Volta U, Auricchio R, Castillejo G, Sanders DS, Cellier C, et al. European Society for the Study of Coeliac Disease (ESsCD) guideline for coeliac disease and other gluten-related disorders. United European Gastroenterol J. 2019;7(5):583–613. doi: 10.1177/2050640619844125 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Sulkanen S, Halttunen T, Laurila K, Kolho KL, Korponay-Szabó IR, Sarnesto A, et al. Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease. Gastroenterology. 1998;115(6):1322–8. doi: 10.1016/s0016-5085(98)70008-3 [DOI] [PubMed] [Google Scholar]
  • 6.Koskinen O, Collin P, Korponay-Szabo I, Salmi T, Iltanen S, Haimila K, et al. Gluten-dependent small bowel mucosal transglutaminase 2-specific IgA deposits in overt and mild enteropathy coeliac disease. J Pediatr Gastroenterol Nutr. 2008;47(4):436–42. doi: 10.1097/MPG.0b013e31817b6dec [DOI] [PubMed] [Google Scholar]
  • 7.Salmi TT, Collin P, Korponay-Szabó IR, Laurila K, Partanen J, Huhtala H, et al. Endomysial antibody-negative coeliac disease: clinical characteristics and intestinal autoantibody deposits. Gut. 2006;55(12):1746–53. doi: 10.1136/gut.2005.071514 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Rauhavirta T, Hietikko M, Salmi T, Lindfors K. Transglutaminase 2 and transglutaminase 2 autoantibodies in celiac disease: a review. Clin Rev Allergy Immunol. 2019;57(1):23–38. doi: 10.1007/s12016-016-8557-4 [DOI] [PubMed] [Google Scholar]
  • 9.Korponay-Szabó IR, Halttunen T, Szalai Z, Laurila K, Király R, Kovács JB, et al. In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut. 2004;53(5):641–8. doi: 10.1136/gut.2003.024836 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Maglio M, Tosco A, Auricchio R, Paparo F, Colicchio B, Miele E, et al. Intestinal deposits of anti-tissue transglutaminase IgA in childhood celiac disease. Dig Liver Dis. 2011;43(8):604–8. doi: 10.1016/j.dld.2011.01.015 [DOI] [PubMed] [Google Scholar]
  • 11.Taavela J, Kurppa K, Collin P, Lähdeaho ML, Salmi T, Saavalainen P, et al. Degree of damage to the small bowel and serum antibody titers correlate with clinical presentation of patients with celiac disease. Clin Gastroenterol Hepatol. 2013;11(2):166–71.e1. doi: 10.1016/j.cgh.2012.09.030 [DOI] [PubMed] [Google Scholar]
  • 12.Tosco A, Aitoro R, Auricchio R, Ponticelli D, Miele E, Paparo F, et al. Intestinal anti-tissue transglutaminase antibodies in potential coeliac disease. Clin Exp Immunol. 2013;171(1):69–75. doi: 10.1111/j.1365-2249.2012.04673.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Freitag T, Schulze-Koops H, Niedobitek G, Melino G, Schuppan D. The role of the immune response against tissue transglutaminase in the pathogenesis of coeliac disease. Autoimmun Rev. 2004;3(2):13–20. doi: 10.1016/S1568-9972(03)00054-5 [DOI] [PubMed] [Google Scholar]
  • 14.Di Niro R, Sblattero D, Florian F, Stebel M, Zentilin L, Giacca M, et al. Anti-idiotypic response in mice expressing human autoantibodies. Mol Immunol. 2008;45(6):1782–91. doi: 10.1016/j.molimm.2007.09.025 [DOI] [PubMed] [Google Scholar]
  • 15.Kalliokoski S, Caja S, Frias R, Laurila K, Koskinen O, Niemelä O, et al. Injection of celiac disease patient sera or immunoglobulins to mice reproduces a condition mimicking early developing celiac disease. J Mol Med. 2015;93(1):51–62. doi: 10.1007/s00109-014-1204-8 [DOI] [PubMed] [Google Scholar]
  • 16.Kalliokoski S, Piqueras VO, Frías R, Sulic AM, Määttä JA, Kähkönen N, et al. Transglutaminase 2-specific coeliac disease autoantibodies induce morphological changes and signs of inflammation in the small-bowel mucosa of mice. Amino Acids. 2017;49(3):529–40. doi: 10.1007/s00726-016-2306-0 [DOI] [PubMed] [Google Scholar]
  • 17.Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, et al. High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med. 2012;18(3):441–5. doi: 10.1038/nm.2656 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.du Pré MF, Blazevski J, Dewan AE, Stamnaes J, Kanduri C, Sandve GK, et al. B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2. J Exp Med. 2020;217(2). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.De Laurenzi V, Melino G. Gene disruption of tissue transglutaminase. Mol Cell Biol. 2001;21(1):148–55. doi: 10.1128/MCB.21.1.148-155.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. doi: 10.1038/nmeth.2019 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Stamnaes J, Pinkas DM, Fleckenstein B, Khosla C, Sollid LM. Redox regulation of transglutaminase 2 activity. J Biol Chem. 2010;285(33):25402–9. doi: 10.1074/jbc.M109.097162 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Iversen R, Di Niro R, Stamnaes J, Lundin KE, Wilson PC, Sollid LM. Transglutaminase 2-specific autoantibodies in celiac disease target clustered, N-terminal epitopes not displayed on the surface of cells. J Immunol. 2013;190(12):5981–91. doi: 10.4049/jimmunol.1300183 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Cataldo F, Marino V, Ventura A, Bottaro G, Corazza GR. Prevalence and clinical features of selective immunoglobulin A deficiency in coeliac disease: an Italian multicentre study. Italian Society of Paediatric Gastroenterology and Hepatology (SIGEP) and "Club del Tenue" Working Groups on Coeliac Disease. Gut. 1998;42(3):362–5. doi: 10.1136/gut.42.3.362 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Collin P, Mäki M, Keyriläinen O, Hällström O, Reunala T, Pasternack A. Selective IgA deficiency and coeliac disease. Scand J Gastroenterol. 1992;27(5):367–71. doi: 10.3109/00365529209000089 [DOI] [PubMed] [Google Scholar]
  • 25.Heneghan MA, Stevens FM, Cryan EM, Warner RH, McCarthy CF. Celiac sprue and immunodeficiency states: a 25-year review. J Clin Gastroenterol. 1997;25(2):421–5. doi: 10.1097/00004836-199709000-00004 [DOI] [PubMed] [Google Scholar]
  • 26.Vieira P, Rajewsky K. The half-lives of serum immunoglobulins in adult mice. Eur J Immunol. 1988;18(2):313–6. doi: 10.1002/eji.1830180221 [DOI] [PubMed] [Google Scholar]
  • 27.Dekkers G, Bentlage AEH, Stegmann TC, Howie HL, Lissenberg-Thunnissen S, Zimring J, et al. Affinity of human IgG subclasses to mouse Fc gamma receptors. MAbs. 2017;9(5):767–73. doi: 10.1080/19420862.2017.1323159 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Bruhns P. Properties of mouse and human IgG receptors and their contribution to disease models. Blood. 2012;119(24):5640–9. doi: 10.1182/blood-2012-01-380121 [DOI] [PubMed] [Google Scholar]
  • 29.Lilienthal GM, Rahmöller J, Petry J, Bartsch YC, Leliavski A, Ehlers M. Potential of murine IgG1 and human IgG4 to inhibit the classical complement and Fcγ receptor activation pathways. Front Immunol. 2018;9:958. doi: 10.3389/fimmu.2018.00958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Leffler D, Schuppan D, Pallav K, Najarian R, Goldsmith JD, Hansen J, et al. Kinetics of the histological, serological and symptomatic responses to gluten challenge in adults with coeliac disease. Gut. 2013;62(7):996–1004. doi: 10.1136/gutjnl-2012-302196 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Leonard MM, Silvester JA, Leffler D, Fasano A, Kelly CP, Lewis SK, et al. Evaluating responses to gluten challenge: a randomized, double-blind, 2-dose gluten challenge trial. Gastroenterology. 2020. doi: 10.1053/j.gastro.2020.10.040 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sarna VK, Skodje GI, Reims HM, Risnes LF, Dahal-Koirala S, Sollid LM, et al. HLA-DQ:gluten tetramer test in blood gives better detection of coeliac patients than biopsy after 14-day gluten challenge. Gut. 2018;67(9):1606–13. doi: 10.1136/gutjnl-2017-314461 [DOI] [PubMed] [Google Scholar]
  • 33.Iversen R, Sollid LM. Autoimmunity provoked by foreign antigens. Science. 2020;368(6487):132–3. doi: 10.1126/science.aay3037 [DOI] [PubMed] [Google Scholar]
  • 34.Yu XB, Uhde M, Green PH, Alaedini A. Autoantibodies in the extraintestinal manifestations of celiac disease. Nutrients. 2018;10(8). doi: 10.3390/nu10081123 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Lejeune T, Meyer C, Abadie V. B Lymphocytes Contribute to Celiac Disease Pathogenesis. Gastroenterology. 2021;160(7):2608–10.e4. doi: 10.1053/j.gastro.2021.02.063 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Abadie V, Kim SM, Lejeune T, Palanski BA, Ernest JD, Tastet O, et al. IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease. Nature. 2020;578(7796):600–4. doi: 10.1038/s41586-020-2003-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Schuppan D, Mäki M, Lundin KEA, Isola J, Friesing-Sosnik T, Taavela J, et al. A randomized trial of a transglutaminase 2 inhibitor for celiac disease. N Engl J Med. 2021;385(1):35–45. doi: 10.1056/NEJMoa2032441 [DOI] [PubMed] [Google Scholar]

Decision Letter 0

Lucienne Chatenoud

24 Jan 2022

PONE-D-21-39201Injection of prototypic celiac anti-transglutaminase 2 antibodies in mice does not cause enteropathyPLOS ONE

Dear Dr. Lindstad,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit an extensively revised version of the manuscript that addresses all the points raised during the review process including the inclusion of additional experimental data (see issue #3 below)..

In particular three major issues have been raised by one of the reviewers.

1- "There is no discussion on the cross-reactivity between human and mouse TG2. While figure 3a shows much stronger binding of the IgG antibodies to WT than to TG2-/- intestine, IgG binding in figure 3b seems very weak. How reproducible is the binding of anti-TG2 IgG to mouse TG2 and here to mouse intestinal tissues? Can it be quantified? What is the affinity of the anti-human TG2 antibody for mouse TG2? Why is there some weak IgG binding in TG2-/- mice (as indicated by the authors). Where is it localized? Does it colocalize with TG2? Is the localization of TG2 shown in figure 3b expected? The authors have previous reported that TG2 is largely present in epithelial cells. Here binding seems to be in the basement membrane. Can these discrepancies be discussed."

2- "Since TG2 is activated by tissue damage or inflammation, is it conceivable that the effect of the ant-TG2 antibody may be different in these pathological conditions (notably in the case of IgG, if complement becomes available locally). If so, should this be specifically addressed?"

3- "Only one anti-TG2 specificity was tested. The authors indicate that they use a prototypic anti-TG2 antibody. Yet they have produced a spectrum of antibodies and described their binding to different epitopes. It is conceivable that different antibodies may have different impact. The authors have shown that, in CeD most antibodies bind to the N-terminus and interfere minimally with TG2 activity. Yet is it sufficient to test only one specificity to draw such definitive conclusions. This question is also raised by the lack of indication on the reactivity of anti-human TG2 antibodies with mouse TG2. Of note it has been suggested that anti-TG2 antibodies may participate in extra-intestinal manifestations of CeD, notably in dermatitis herpetiformis as the skin lesions contain deposits of anti-TG antibodies but no T cells. The article showing a possible role of anti-TG2 antibody in dermatitis herpetiformis used human skin grafts in immunodeficient mice (Zone et al 2011 DOI 10.4049/jimmunol.1003273)."

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Reviewer #1: Yes

Reviewer #2: Partly

**********

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Reviewer #1: Yes

Reviewer #2: N/A

**********

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Reviewer #1: No

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

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Reviewer #1: The authors present very intersting and original data concerning the potential role of anti-transglutaminase 2 secreted antibodies (anti-TG2) in vivo, in an experimental murine model. Their findings are not in favour of a major role of anti-TG2 immunoglobulins in the form of secreted antibodies in the development of enteropathy in celiac disease. This manuscript is written in an intelligible fashion and in standard english. Please note two very little form orthography remarks : lignes 129 "follwed" and 278 "disdease".

Most important, I couldn't visualise Figure 4 and Figure S1, this is why I recommend Minor Revision.

Reviewer #2: In this article, Borgen et al further explore the role of the B cell response to TG2, the celiac disease autoantigen. This enzyme plays a key role in CD by deamidating gluten peptides, a post-translational modification indispensable for the activation of DQ2/8 restricted gluten specific CD4+ T cells that are instrumental in disease pathogenesis. Strikingly, active CeD is associated with a massive intestinal plasma cell response to TG2. The latter response has been extensively characterized by the authors. They have notably provided compelling evidence that the anti-TG2 antibody response can be assimilated to a hapten-carrier B cell response where TG2 becomes recognized when bound to gluten. Abadie et al have also recently shown in a CeD mouse model that B cell depletion prevents intestinal damage. Yet it is still unclear whether anti-TG2 antibodies have a pathogenic role in CeD and if so how. On the one hand, the authors have provided strong evidence that anti-TG2 B cells may participate in CeD pathogenesis by binding gluten peptides and thereby promoting their presentation to CD4+ T cells (after endocytosis and loading into HLA-DQ molecules). On the other hand, a limited number of ancient articles suggested that anti-TG2 antibodies may exert deleterious effects directly by interacting with TG2 in the intestinal tissues, a finding that contrasts with the fact that anti-TG2 antibodies can be detected in the intestine of patients with latent CeD who have no lesions.

Here the authors investigate whether anti-TG2 antibodies can induce intestinal damage in B6 mice following intravenous injection of one the numerous anti-TG2 monoclonal antibodies that they have generated. They use the same antibody in diverse murine versions (IgG2 b , IgG2c and IgA), and they analyze the intestine after repeating the injections for 21 days.

Based on the demonstration that the injected anti-TG2 IgG antibodies can be detected in the intestine of wild type B6 but only at a much lesser degree in TG2-/- mice and that the antibodies do not induce any obvious histological intestinal lesions in WT mice, the authors conclude that anti-TG2 antibody has no direct pathogenic role in intestinal lesions.

Although this reviewer could be easily convinced that anti-TG2 antibodies do not induce directly tissue damage in the intestine, additional experiments and comments seem useful to support such a very strong and definitive conclusion

1- There is no discussion on the cross-reactivity between human and mouse TG2. While figure 3a shows much stronger binding of the IgG antibodies to WT than to TG2-/- intestine, IgG binding in figure 3b seems very weak. How reproducible is the binding of anti-TG2 IgG to mouse TG2 and here to mouse intestinal tissues? Can it be quantified? What is the affinity of the anti-human TG2 antibody for mouse TG2? Why is there some weak IgG binding in TG2-/- mice (as indicated by the authors). Where is it localized? Does it colocalize with TG2? Is the localization of TG2 shown in figure 3b expected? The authors have previous reported that TG2 is largely present in epithelial cells. Here binding seems to be in the basement membrane. Can these discrepancies be discussed.

2- Since TG2 is activated by tissue damage or inflammation, is it conceivable that the effect of the ant-TG2 antibody may be different in these pathological conditions (notably in the case of IgG, if complement becomes available locally). If so, should this be specifically addressed?

3- Only one anti-TG2 specificity was tested. The authors indicate that they use a prototypic anti-TG2 antibody. Yet they have produced a spectrum of antibodies and described their binding to different epitopes. It is conceivable that different antibodies may have different impact. The authors have shown that, in CeD most antibodies bind to the N-terminus and interfere minimally with TG2 activity. Yet is it sufficient to test only one specificity to draw such definitive conclusions. This question is also raised by the lack of indication on the reactivity of anti-human TG2 antibodies with mouse TG2. Of note it has been suggested that anti-TG2 antibodies may participate in extra-intestinal manifestations of CeD, notably in dermatitis herpetiformis as the skin lesions contain deposits of anti-TG antibodies but no T cells. The article showing a possible role of anti-TG2 antibody in dermatitis herpetiformis used human skin grafts in immunodeficient mice (Zone et al 2011 DOI 10.4049/jimmunol.1003273).

4- Are IgG relevant to the induction of lesions in the intestine of celiac patients

As indicated by the authors, conclusion concerning the IgA version of the antibody is difficult as very little IgA is detectable in the serum. Based on the fact that IgA deficient patients can develop (are prone to) celiac disease, the authors conclude that it is not important to address the role of IgA. I nevertheless wonder whether this suggestion is fully valid as it is likely that patients with IgA deficiency have IgM against TG2 in their intestine, the role of which is not addressed here. Is it clear that CeD patients have prominent anti-TG2-IgG in the intestine? Since the authors only work during 3 weeks, would it be pertinent to use hybridomas as backpack to provide continuously IgA antibodies to the mice and circumvent their short half-life.

5- Recent work shows that inhibiting TG2 has positive effects both in an animal model quoted by the authors and in humans in a recent trial (Schuppan et al 2021 10.1056/NEJMoa2032441). Is this observation pertinent to discuss the lack of deleterious role of anti-TG2 antibodies?

6- The structure of the report seems strange to this reviewer as figure legends are directly inserted in the results section and are used directly to describe the results. Is it an acceptable format in Plosone? If not, the narrative in the result section needs to be thoroughly revised and implemented.

7- The authors use parametric tests (t-tests) for statistics. Although it is a minor point as comparison between treated animals and controls does not show any difference, it is somewhat surprising to this reviewer who would have chosen non parametric tests to compare normal versus disease conditions in the absence of demonstration that data follow a normal distribution.

**********

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Reviewer #1: No

Reviewer #2: Yes: Nadine Cerf-Bensussan

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PLoS One. 2022 Apr 6;17(4):e0266543. doi: 10.1371/journal.pone.0266543.r002

Author response to Decision Letter 0

28 Feb 2022

PONE-D-21-39201 Response to reviewer comments

Response to Editors comments

The three points raised by the Editor are addressed in the response to Reviewer #2, please see below.

Response to the reviewers’ comments

Reviewer #1:

The authors present very intersting and original data concerning the potential role of anti-transglutaminase 2 secreted antibodies (anti-TG2) in vivo, in an experimental murine model. Their findings are not in favour of a major role of anti-TG2 immunoglobulins in the form of secreted antibodies in the development of enteropathy in celiac disease. This manuscript is written in an intelligible fashion and in standard english. Please note two very little form orthography remarks : lignes 129 "follwed" and 278 "disdease". Most important, I couldn't visualise Figure 4 and Figure S1, this is why I recommend Minor Revision.

Author response:

We thank reviewer for pointing of these two typos. They are now corrected. We have ensured that Figure 4 and Figure S1 (now S2) can be fully visualized in the revised proof version.

Reviewer #2:

In this article, Borgen et al further explore the role of the B cell response to TG2, the celiac disease autoantigen. This enzyme plays a key role in CD by deamidating gluten peptides, a post-translational modification indispensable for the activation of DQ2/8 restricted gluten specific CD4+ T cells that are instrumental in disease pathogenesis. Strikingly, active CeD is associated with a massive intestinal plasma cell response to TG2. The latter response has been extensively characterized by the authors. They have notably provided compelling evidence that the anti-TG2 antibody response can be assimilated to a hapten-carrier B cell response where TG2 becomes recognized when bound to gluten. Abadie et al have also recently shown in a CeD mouse model that B cell depletion prevents intestinal damage. Yet it is still unclear whether anti-TG2 antibodies have a pathogenic role in CeD and if so how. On the one hand, the authors have provided strong evidence that anti-TG2 B cells may participate in CeD pathogenesis by binding gluten peptides and thereby promoting their presentation to CD4+ T cells (after endocytosis and loading into HLA-DQ molecules). On the other hand, a limited number of ancient articles suggested that anti-TG2 antibodies may exert deleterious effects directly by interacting with TG2 in the intestinal tissues, a finding that contrasts with the fact that anti-TG2 antibodies can be detected in the intestine of patients with latent CeD who have no lesions.

Here the authors investigate whether anti-TG2 antibodies can induce intestinal damage in B6 mice following intravenous injection of one the numerous anti-TG2 monoclonal antibodies that they have generated. They use the same antibody in diverse murine versions (IgG2 b , IgG2c and IgA), and they analyze the intestine after repeating the injections for 21 days.

Based on the demonstration that the injected anti-TG2 IgG antibodies can be detected in the intestine of wild type B6 but only at a much lesser degree in TG2-/- mice and that the antibodies do not induce any obvious histological intestinal lesions in WT mice, the authors conclude that anti-TG2 antibody has no direct pathogenic role in intestinal lesions.

Although this reviewer could be easily convinced that anti-TG2 antibodies do not induce directly tissue damage in the intestine, additional experiments and comments seem useful to support such a very strong and definitive conclusion

1- There is no discussion on the cross-reactivity between human and mouse TG2. While figure 3a shows much stronger binding of the IgG antibodies to WT than to TG2-/- intestine, IgG binding in figure 3b seems very weak. How reproducible is the binding of anti-TG2 IgG to mouse TG2 and here to mouse intestinal tissues? Can it be quantified? What is the affinity of the anti-human TG2 antibody for mouse TG2? Why is there some weak IgG binding in TG2-/- mice (as indicated by the authors). Where is it localized? Does it colocalize with TG2? Is the localization of TG2 shown in figure 3b expected? The authors have previous reported that TG2 is largely present in epithelial cells. Here binding seems to be in the basement membrane. Can these discrepancies be discussed.

Author response:

We thank the reviewer for bringing up these important points. We have previously demonstrated that the anti-TG2 antibody 679-14-E06 binds with high and equal affinity to human and mouse TG2 (KD about 5nM as determined by surface plasmon resonance; STable1 in du Pre et al. J Exp Med 2020; PMID31727780). This fact is now stated in the text (line 71 in the revised manuscript with track changes). We have also shown that 14-E06 expressed as human IgG1 recognizes endogenous mouse TG2 in unfixed frozen tissue-sections of intestine (Cardoso et al, FEBS 2015, PMID: 25808416). To demonstrate that hybridoma-derived 14-E06 as used in this study recognizes mouse TG2, we have included a supplementary figure that shows binding of 14-E06 mouse IgG2c to extracellular matrix bound TG2 in mouse small intestine (new S1 Figure). Thus, we are certain that the antibodies we have injected can bind to mouse TG2 with high affinity.

IgG deposits were reproducibly observed in the sub-epithelial extracellular matrix of wild-type (WT) mice injected with IgG. This signal co-localized with endogenous extracellular matrix bound TG2, as shown in our revised Figure 3a. IgG deposits were seen in all tissue sections analyzed from WT mice injected with IgG from which we had fresh frozen intestine (n = 4) (revised Figure 3b and c). Some variation in signal intensity within tissue sections likely reflect regional differences in IgG tissue concentration, whereas variation in overall signal staining intensity depends on the secondary antibody used for staining (revised Figure 3c). Presence of some IgG signal in Tgm2-/- mice injected with IgG suggests that antibody presence in the tissue is independent of cognate antigen presence. However, no extracellular IgG deposition was observed in absence of TG2. This has been clarified in line 196-197 and in the legend of Figure 3. From quantification of the sub-epithelial fluorescence signal (as per reviewer’s suggestions) we see a marked difference between IgG-injected WT and Tgm2-/- mice (revised Materials and Methods, and revised Figure 3c). As alluded to by the reviewer, the true in vivo localization of TG2 remains unknown as there is a clear discrepancy between the lack of B cell tolerance to TG2 (du Pre et al. J Exp Med 2020; PMID31727780) and the abundant extracellular TG2 staining that is observed in frozen tissue sections. We are currently working to resolve this discrepancy, and we do not consider this to be a scope of this paper.

2- Since TG2 is activated by tissue damage or inflammation, is it conceivable that the effect of the ant-TG2 antibody may be different in these pathological conditions (notably in the case of IgG, if complement becomes available locally). If so, should this be specifically addressed?

Author response:

We agree that an effect of complement activation cannot be excluded, and that both complement factors, TG2 and anti-TG2 IgG may be present in highly inflamed tissue such as in untreated celiac disease (PMID: 1537512). We have in our study addressed whether presence of anti-TG2 antibodies may play a role in development of enteropathy as has previously been suggested (PMID: 25209899, PMID: 23824706, PMID: 27503559). We find no evidence to support this notion.

3- Only one anti-TG2 specificity was tested. The authors indicate that they use a prototypic anti-TG2 antibody. Yet they have produced a spectrum of antibodies and described their binding to different epitopes. It is conceivable that different antibodies may have different impact. The authors have shown that, in CeD most antibodies bind to the N-terminus and interfere minimally with TG2 activity. Yet is it sufficient to test only one specificity to draw such definitive conclusions. This question is also raised by the lack of indication on the reactivity of anti-human TG2 antibodies with mouse TG2. Of note it has been suggested that anti-TG2 antibodies may participate in extra-intestinal manifestations of CeD, notably in dermatitis herpetiformis as the skin lesions contain deposits of anti-TG antibodies but no T cells. The article showing a possible role of anti-TG2 antibody in dermatitis herpetiformis used human skin grafts in immunodeficient mice (Zone et al 2011 DOI 10.4049/jimmunol.1003273).

Author response:

We have conclusively demonstrated that the antibody (14-E06) we have injected strongly binds to mouse TG2 (see response to #1). While the anti-TG2 response in celiac disease indeed can target multiple epitopes on TG2, 14-E06 represents the group of antibodies most frequently shared across patients (ref 17, PMID: 22366952). If anti-TG2 antibodies should play a role in development of intestinal remodeling, such an effect must be mediated by public antibodies shared across patients. Therefore, we consider mAb 14-E06 to be well suited to address this question. We do however agree with the reviewer that antibodies targeting other epitopes may have different effects on TG2. As we also state in our manuscript (line 300), we do not exclude a role for TG2 antibodies in the development of extra intestinal manifestations, but this warrants further investigation.

4- Are IgG relevant to the induction of lesions in the intestine of celiac patients

As indicated by the authors, conclusion concerning the IgA version of the antibody is difficult as very little IgA is detectable in the serum. Based on the fact that IgA deficient patients can develop (are prone to) celiac disease, the authors conclude that it is not important to address the role of IgA. I nevertheless wonder whether this suggestion is fully valid as it is likely that patients with IgA deficiency have IgM against TG2 in their intestine, the role of which is not addressed here. Is it clear that CeD patients have prominent anti-TG2-IgG in the intestine? Since the authors only work during 3 weeks, would it be pertinent to use hybridomas as backpack to provide continuously IgA antibodies to the mice and circumvent their short half-life.

Author response:

As long as we see IgG making tissue deposits in frozen sections of mice intestine, the specific antibody is indeed present and should be able to exert effects. The suggestion to introduce IgA hybridomas in vivo to secure continuous supply is interesting but we foresee issues to control equal production in different animals. Further we are unsure whether this approach would indeed allow us to prolong the time of exposure since visible tumors are present already 9-13 days after injection (PMID 9042427) and humane endpoints (i.e. maximum allowable tumor size) are expected to be reached shortly thereafter. In addition to these concerns, the timeline to obtain ethical approvals and finalize such studies would be long making it impossible to complete these studies within the scope of this paper.

5- Recent work shows that inhibiting TG2 has positive effects both in an animal model quoted by the authors and in humans in a recent trial (Schuppan et al 2021 10.1056/NEJMoa2032441). Is this observation pertinent to discuss the lack of deleterious role of anti-TG2 antibodies?

Author response:

We thank the reviewer for this suggestion. We agree with the reviewer that the recent observations in an animal model of celiac disease and a clinical trial of man strongly support the notion that TG2 is implicated in the pathogenesis of celiac disease. We have introduced a small paragraph on this point in the Discussion (line 309).

6- The structure of the report seems strange to this reviewer as figure legends are directly inserted in the results section and are used directly to describe the results. Is it an acceptable format in Plosone? If not, the narrative in the result section needs to be thoroughly revised and implemented.

Author response:

We confirm that this indeed is the desired format of PLoS ONE.

7- The authors use parametric tests (t-tests) for statistics. Although it is a minor point as comparison between treated animals and controls does not show any difference, it is somewhat surprising to this reviewer who would have chosen non parametric tests to compare normal versus disease conditions in the absence of demonstration that data follow a normal distribution.

Author response:

We agree that a non-parametric test could be appropriate in this setting, and we thank the reviewer for pointing this out. In the revised version of the manuscript, the non-parametric Mann-Whitney test has been used for all the comparisons reported. Importantly, our conclusions remain unchanged with this test.

Attachment

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Decision Letter 1

Lucienne Chatenoud

23 Mar 2022

Injection of prototypic celiac anti-transglutaminase 2 antibodies in mice does not cause enteropathy

PONE-D-21-39201R1

Dear Dr. Lindstad,

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Acceptance letter

Lucienne Chatenoud

28 Mar 2022

PONE-D-21-39201R1

Injection of prototypic celiac anti-transglutaminase 2 antibodies in mice does not cause enteropathy

Dear Dr. Lindstad:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Lucienne Chatenoud

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Confirmation of mouse TG2 reactivity of mAb 14E06.

    Hybridoma-derived 14E06 (mouse IgG2c) binds to endogenous TG2 in the ECM of mouse small intestine (left panel). Mouse 14E06 (IgG2c) also binds to recombinant human or mouse TG2 immobilized in the ECM of Tgm2-/- mouse small intestine (middle and right panel). Nuclei were counterstained with DAPI. Scale bar represents 100μm.

    (TIF)

    Click here for additional data file. (1.5MB, tif)
    S2 Fig. Weight change in control groups.

    The graph reports weight as % change from baseline for the different groups as indicated. Dots and bars represent mean +/- SD. Data represent all mice of each group from the two independent experiments.

    (TIF)

    Click here for additional data file. (77.8KB, tif)
    S1 Dataset

    (XLSX)

    Click here for additional data file. (56.3KB, xlsx)
    Attachment

    Submitted filename: Response To Reviewers.docx

    Click here for additional data file. (24.6KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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