Gastroenterology 2020;159:788–790
Evidence That Pathogenic Transglutaminase 2 in Celiac Disease
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Derives From Enterocytes
Rasmus Iversen,* Sunniva F. Amundsen,* Liv Kleppa, M. Fleur du Pré, Jorunn Stamnaes, and
Ludvig M. Sollid
K.G. Jebsen Coeliac Disease Research Centre, University of Oslo, and Department of Immunology, University of Oslo and Oslo
University Hospital, Oslo, Norway
T he enzyme transglutaminase 2 (TG2) plays a key
role in celiac disease (CeD) pathogenesis through
generation of immunogenic, deamidated gluten T-cell epi-
topes, and as a target of autoantibodies. The disorder is
caused by a maladaptive immune response to cereal gluten
proteins, and accumulating evidence suggest that in-
teractions between gluten-specific T cells and TG2-specific B
cells are important for driving the disease. A big remaining
question is where in the body TG2, which is a cytosolic
protein, encounters gluten and engages the immune system.
We here bring evidence that TG2 implicated in CeD is
derived from shed enterocytes.
A recent study of immunoglobulin knockin mice
expressing a celiac patient-derived anti-TG2 B-cell receptor
showed that there is no B-cell tolerance induction to TG2.1
This observation indicates that B cells are not exposed to
the enzyme during their development or while patrolling
the extracellular space of the body, raising the question
where the immune system meets TG2 in CeD. Knowing that
Peyer’s patches are major sites for induction of immune
responses to gut luminal antigens,2 we hypothesize that gut
luminal TG2 derived from shed enterocytes is the source of
pathogenic TG2 in CeD.
Methods
Enterocytes were isolated by incubating mouse small in-
testinal tissue in EDTA-containing buffer, resulting in 80% to
90% pure enterocytes (Supplementary Figure 1). The cells
were lysed by repeated freezing and thawing, and TG2 present
in the lysate was detected by Western blotting using a poly-
clonal rabbit anti-mouse TG2 antibody. Additional details as
well as TG2 activity and T-cell–B-cell collaboration assays are
described in the Supplementary Methods.
Results
To investigate TG2 expression in gut epithelium, we
generated lysates of mouse small intestinal enterocytes and
assessed the amount of TG2 present by Western blotting
(Figure 1A). The estimated amount of TG2 (0.15 pg per cell)
was in the same range as what was recently found by mass
spectrometry quantification of more than 5000 enterocyte
proteins in mice, placing TG2 among the 10% most abun-
dant proteins in small intestinal enterocytes3 (Figure 1B).
Because the role of TG2 in CeD is linked to its calcium-
dependent enzymatic activity, we next addressed whether
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TG2 from enterocytes could be catalytically active. After
incubation with a biotinylated gluten peptide in the pres-
ence of calcium, we assessed the incorporation of biotin into
proteins through TG2-mediated isopeptide-bond formation.
The peptide was covalently linked to a wide range of pro-
teins present in the lysate, confirming that active TG2 was
indeed present (Figure 1C). At least part of this TG2 was
available in a state that could be recognized by the immune
system as judged by immunoprecipitation with a mono-
clonal anti-TG2 antibody obtained from a patient with CeD
(Supplementary Figure 2A).
To test if TG2 from enterocyte lysate would be able to
facilitate interactions between TG2-specific B cells and
gluten-specific T cells, we incubated murine A20 B cells
expressing a TG2-specific or non–TG2-specific B-cell re-
ceptor with enterocyte lysate and non-deamidated gluten
peptide and assessed their ability to present the deamidated
peptide to gluten-specific hybridoma T cells. Under these
conditions, TG2-specific B cells efficiently activated T cells
specific to 2 different gluten epitopes (Figure 1D). Uptake
and presentation of gluten peptide by TG2-specific B cells
was dependent on presence of active TG2 (Supplementary
Figure 2B and 2C), indicating that antigen-specific B cells
can take up enzyme-substrate complexes formed between
enterocyte-derived TG2 and gluten followed by presentation
of deamidated gluten peptides to T cells.
Discussion
The continuous renewal of the gut epithelium is
accompanied by massive shedding of enterocytes. In
humans, the turnover is estimated to be 1011 cells per day
under normal physiological conditions and even greater
during inflammation.4 Many enterocytes are shed as live
cells with catalytically intact cytosolic enzymes.5,6 The
enterocytes presumably disintegrate in the lumen and
release their cytosolic content, which can be sampled by the
*Authors share co-first authorship.
Abbreviations used in this paper: CeD, Celiac Disease; TG2, Trans-
glutaminase 2.
Most current article
© 2020 by the AGA Institute. Published by Elsevier Inc. This is an open
access article under the CC BY license (http://creativecommons.org/
licenses/by/4.0/).
0016-5085
https://doi.org/10.1053/j.gastro.2020.04.018
August 2020 Luminal TG2 and Celiac Disease 789

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Figure 1. Enterocytes contain catalytically intact TG2, which can facilitate T-cell–B-cell interactions. (A) Representative
Western blot showing detection of TG2 in small intestinal enterocytes. Based on signal intensities, the amount of TG2 was
quantified in 2 different lysate preparations. Column heights represent mean amount of TG2 based on quantification of 2 or 3
different lysate dilutions with error bars indicating standard deviation. (B) Ranking of the expression levels (molecules per cell)
of more than 3600 proteins in mouse jejunum enterocytes based on quantitative mass spectrometry data reported by Arike
et al.3 The overlaid box plot indicates the 10th and 90th percentiles of the protein expression levels. (C) Western blot showing
detection of biotin in enterocyte lysate obtained from TG2 sufficient (Tgm2þ/þ) or TG2 knockout (Tgm2/) mice after incu-
bation with or without a biotinylated 34-mer gluten peptide and calcium to allow TG2-mediated isopeptide-bond formation.
The bands at w70 kDa and w120 kDa present in all lanes represent endogenously biotinylated proteins. (D) T-cell–B-cell
collaboration assay showing release of interleukin (IL)-2 from activated hybridoma T cells specific to the indicated gluten
epitopes after co-culture with A20 lymphoma B cells expressing HLA-DQ2.5 in combination with a TG2-specific or non–TG2-
specific B-cell receptor. Before addition of T cells, the B cells were pulsed with different concentrations of enterocyte lysate in
the presence of 2 mM CaCl2 and 25 mM gluten peptide containing the relevant non-deamidated T-cell epitope. Symbols
represent mean IL-2 concentration with indication of standard deviation based on culture triplicates.

immune system in the same way as microbial and food
antigens. Notably, soluble luminal molecules have direct
access to adaptive immune cells through the conduit system
of Peyer’s patches.7 Here, we have shown that mouse small
intestinal enterocytes contain a significant amount of TG2,
which can be catalytically active in the extracellular envi-
ronment. Moreover, TG2 expression has been demonstrated
in human enterocytes by single-cell transcriptome analysis.8
Hence, after a gluten-containing meal, both TG2 and gluten
peptides will be available to B cells in Peyer’s patches. B
cells with a TG2-specific receptor will therefore be able to
bind catalytically active TG2, which can react with gluten
peptides and form enzyme-substrate complexes. Thus,
following receptor-mediated endocytosis, TG2-specific B
cells can present deamidated gluten peptides on surface
MHC-II molecules. We believe that this mechanism allows
TG2-specific B cells to serve as important antigen-
presenting cells in CeD, leading to activation of disease-

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 790 Iversen et al Gastroenterology Vol. 159, No. 2

driving gluten-specific T cells and formation of
TG2-specific autoantibodies. Hence, luminal TG2 released
from shed enterocytes could form the basis for the adaptive
immune response in CeD.
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Received February 17, 2020. Accepted April 8, 2020.
Correspondence
Address correspondence to Ludvig M. Sollid, MD, PhD, Department of
Immunology, Oslo University Hospital–Rikshospitalet, 0372 Oslo, Norway.
e-mail: l.m.sollid@medisin.uio.no.

Acknowledgments
Supplementary Material We thank Chaitan Khosla for providing polyclonal rabbit anti-mouse TG2
antibody and Gerry Melino for providing TG2 knockout mice.
Note: To access the supplementary material accompanying
this article, visit the online version of Gastroenterology at CRediT Authorship Contributions
www.gastrojournal.org, and at https://doi.org/10.1053/j. Rasmus Iversen, PhD (Conceptualization: Supporting; Data curation: Lead;
Formal analysis: Equal; Investigation: Supporting; Methodology: Equal;
gastro.2020.04.018. Writing – original draft: Equal). Sunniva F. Amundsen, MSc (Data curation:
Equal; Formal analysis: Equal; Investigation: Lead; Methodology: Equal;
Writing – review & editing: Supporting). Liv Kleppa, PhD (Data curation:
References Supporting; Formal analysis: Supporting; Investigation: Supporting;
Methodology: Supporting; Writing – review & editing: Supporting). M. Fleur
1. du Pre MF, et al. J Exp Med 2020;217:e20190860. du Pré, PhD (Data curation: Supporting; Investigation: Supporting;
2. Mowat AM. Nat Rev Immunol 2003;3:331–341. Methodology: Supporting). Jorunn Stamnaes, PhD (Conceptualization:
Supporting; Data curation: Supporting; Investigation: Supporting;
3. Arike L, et al. Cell Rep 2020;30:1077–1087.e3. Methodology: Supporting; Supervision: Supporting). Ludvig M. Sollid, MD,
4. Williams JM, et al. Vet Pathol 2015;52:445–455. PhD (Conceptualization: Lead; Project administration: Lead; Supervision:
Lead; Writing – original draft: Equal).
5. Blander JM. FEBS J 2016;283:2720–2730.
6. Glaeser H, Drescher S, et al. Clin Pharmacol Ther 2002; Conflicts of interest
71:131–140. The authors disclose the following: Ludvig M. Sollid has privately or via his
employer been consultant during the two last years for Amagma
7. Chang JE, et al. Nat Immunol 2019;20:1506–1516. Therapeutics, ImmusanT Therapeutics, Intrexon Actobiotics, Bioniz
8. Wang Y, Song W, et al. J Exp Med 2020; Therapeutics, UCB Biopharma, Chugai Pharmaceutical, Merck and GSK.

217:e20191130. Funding
This work was supported by the Research Council of Norway (project 255053),
the University of Oslo World-leading research program on human immunology
(WL-IMMUNOLOGY) and the European Research Council (project ERC-2010-
Author names in bold designate shared co-first authorship. Ad-268541).

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