AOP ID and Title:

Short Title: Gluten intake leading to celiac disease

Authors

Rodríguez-Fernández, Pablo

Koning, Frits

Gil González, Aina

Moreno Andújar, Javier

Noriega Fernández, Estefanía

Fernandez Dumont, Antonio

Status

Abstract

Celiac disease is an immune-mediated disorder triggered by the ingestion of gluten in genetically susceptible individuals carrying human leukocyte antigen (HLA)-DQ2 or HLA-DQ8 molecules. This Adverse Outcome Pathway (AOP) describes the sequence of molecular and cellular events leading to celiac disease, beginning with key molecular initiating events (MIEs) and culminating in intestinal damage and disease manifestation.

The pathway is initiated by the formation of HLA-DQ2/8-gluten complexes, the generation of gluten-reactive T cell receptors, and the production of gluten- and transglutaminase 2 (TG2)-reactive B cell receptors. These MIEs facilitate the co-localization of gluten-reactive adaptive T-cells with antigen-presenting cells (APCs), an essential step in the immune response. This interaction triggers the activation of the innate immune response and subsequently leads to the activation of gluten-reactive CD4+ T cells. The cascade continues with the activation of gluten- and TG2-reactive B cells, which further amplifies the immune response and contributes to the disruption of the intestinal barrier. The final adverse outcome (AO) is the development of celiac disease, characterized by chronic intestinal inflammation, villous atrophy, and malabsorption.

The relationships between key events (KEs) in this AOP are supported by moderate levels of evidence, reflecting a well-characterized yet complex immunopathological process. Understanding this AOP provides valuable insights for risk assessment, the development of targeted therapies, and the refinement of strategies for gluten-related disorder management.

Background

In 2017, the EFSA GMO Panel published a guidance document (EFSA, 2017) that, for the first time, outlined a specific risk assessment strategy to predict the capacity of innovative or novel proteins to trigger celiac disease. This strategy, characterized by an integrated, stepwise, case-by-case approach, was made possible due to the well-documented pathogenesis of celiac disease and the known proteins involved. Specifically, gluten peptides presented by the disease-predisposing Human Leukocyte Antigen (HLA) class II molecules, HLA-DQ2 or HLA-DQ8, activate pro-inflammatory T-cells in the inflamed intestines of patients.

Ongoing efforts to refine risk assessment methodologies in this area are driven by new findings that suggest proteins from sources other than cereals may pose a hazard to individuals with celiac disease (Peterson et al., 2019) . This AOP is created to integrate the scientific knowledge into a conceptual framework in the regulatory context.

The risk assessment strategy developed for evaluating the potential of innovative or novel proteins to induce celiac disease is regarded as a benchmark, serving as an inspiration for the broader food safety assessment of novel proteins in the food sector.

Summary of the AOP

Events

Molecular Initiating Events (MIE), Key Events (KE), Adverse Outcomes (AO)

Key Event Relationships

Overall Assessment of the AOP

KER1: Formation of HLA-DQ2/8-gluten complexes leads to Co-localization of gluten-reactive adaptive T-cells with APC

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: The formation of the HLA-DQ2/8-gluten complex is a fundamental event for initiating the immune response in genetically predisposed individuals. The co-localization of gluten-reactive adaptive T cells with antigen-presenting cells (APCs) depends on the recognition of these complexes by the immune system. This relationship is essential for activating the adaptive immune system, a critical step in the development of celiac disease.
  Supporting Evidence: The interaction between gluten-HLA complexes and T cells is well-documented, and co-localization with APCs is required for T-cell activation. (Sollid, 2002; van de Wal et al., 1999)

KER2: Generation of gluten-reactive and TG2-reactive B cell receptors leads to Co-localization of gluten-reactive adaptive T-cells with APC

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: The generation of gluten-reactive and TG2-reactive B cell receptors facilitates the production of antibodies that contribute to the autoimmune response in celiac disease. While the direct influence on T-cell co-localization is less clear, the B-cell receptor generation is part of the broader immune response, influencing the progression of celiac disease. The co-localization of T-cells with APCs is indirectly impacted by the production of antibodies and antigen presentation.
  Supporting Evidence: While there is strong evidence for the generation of gluten-reactive B cells, the direct relationship with T-cell co-localization has moderate support, but it is still considered relevant for the disease process. (Kagnoff, 2007; Koning et al., 2010)

KER3: Generation of gluten-reactive T cell receptors leads to Co-localization of gluten-reactive adaptive T-cells with APC

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: Gluten-reactive TCR generation is a critical early event in the immune response to gluten. Once these TCRs are generated, the T-cells are able to recognize gluten peptides presented by APCs, facilitating their co-localization. This step is essential for initiating the adaptive immune response, a key event in the pathogenesis of celiac disease.
  Supporting Evidence: There is strong evidence for the role of gluten-reactive TCRs in initiating immune responses, and their interaction with APCs is fundamental for the disease process. (Zhang et al., 2010)

KER4: Co-localization of gluten-reactive adaptive T-cells with APC leads to Activation of the innate immune response

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: The co-localization of gluten-reactive T-cells with APCs activates the adaptive immune system, which in turn triggers innate immune pathways. Activation of the innate immune response amplifies the overall immune reaction, driving inflammation and tissue damage seen in celiac disease. Without this co-localization, the full immune activation needed for disease progression would not occur.
  Supporting Evidence: Studies indicate that activation of adaptive T-cells by APCs is tightly linked to subsequent activation of innate immune pathways. (Lundin et al., 1993; Anderson et al., 2011)

KER5: Activation of the innate immune response leads to Activation of gluten-reactive CD4+ T cells

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: The innate immune response plays a pivotal role in amplifying the activation of gluten-reactive CD4+ T cells, which is essential for driving the adaptive immune response in celiac disease. This relationship is critical because it ensures that the immune system's inflammatory reaction is properly mediated and directed toward the intestines.
  Supporting Evidence: The innate immune system is known to activate CD4+ T cells in response to antigenic stimulation, further promoting the inflammatory cascade in celiac disease. (Vella et al., 2012)

KER6: Activation of gluten-reactive CD4+ T cells leads to Activation of gluten- and TG2-reactive B cells

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: The activation of gluten-reactive CD4+ T cells is necessary to help activate B cells that produce gluten- and TG2-specific antibodies. These antibodies are markers of disease and contribute to the autoimmune responses that drive the pathology of celiac disease. Without T-cell activation, B-cell activation cannot occur, and the autoimmune response would be incomplete.
  Supporting Evidence: The interaction between activated T cells and B cells is well-established in the context of autoimmune diseases like celiac disease, where T-helper cells provide necessary signals for B cell activation. (Kagnoff, 2007; Koning et al., 2010)

KER7: Activation of gluten- and TG2-reactive B cells leads to Disruption of the intestinal barrier

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: The activation of gluten- and TG2-reactive B cells results in the production of antibodies, such as anti-TG2, which play a significant role in tissue damage. This damage contributes to the disruption of the intestinal barrier, a hallmark of celiac disease. Without B-cell activation, the autoimmune-mediated intestinal damage would be less pronounced, and the disease would not progress in the same way.
  Supporting Evidence: The presence of anti-TG2 antibodies and their involvement in intestinal injury is well-documented in celiac disease. (Lundin et al., 1993; Green & Cellier, 2007)

KER8: Disruption of the intestinal barrier leads to Celiac Disease

• Adjacency: Adjacent
• Evidence: Moderate
• Essentiality: High
  Rationale: The disruption of the intestinal barrier is the key event that allows gluten peptides and other immune activators to enter the mucosa, triggering the immune response and leading to celiac disease. This barrier disruption is essential for disease progression, as it creates the conditions for subsequent inflammation, villous atrophy, and clinical symptoms.
  Supporting Evidence: The breakdown of the intestinal barrier is considered a critical step in the pathogenesis of celiac disease. Without this disruption, immune activation would be limited, and disease symptoms would not manifest. (Anderson et al., 2011)

Domain of Applicability

Life Stage Applicability Taxonomic Applicability Sex Applicability

The AOP applies specifically to humans, as celiac disease is inherently linked to the HLA-DQ2/8 genotype, which is unique to humans. The described mechanisms are particularly relevant to individuals with genetic susceptibility.

Essentiality of the Key Events

MIE1: Formation of HLA-DQ2/8-gluten Complexes

Essentiality: High
Rationale: The presence of HLA-DQ2/8 is a critical requirement for the development of celiac disease. Without these alleles, individuals cannot form gluten-HLA complexes, and celiac disease does not occur. The formation of this complex is a fundamental step in initiating the immune response against gluten in genetically predisposed individuals. (Sollid, 2002; van de Wal et al., 1999)

MIE2: Generation of Gluten-Reactive T Cell Receptors

Essentiality: High
Rationale: The generation of gluten-reactive TCRs is essential for the immune system to recognize gluten peptides. This step triggers the adaptive immune response, and individuals who lack gluten-reactive TCRs are unable to develop the disease. Clinical data consistently shows the presence of these TCRs in celiac patients, which play a direct role in the disease process (Zhang et al., 2010).

MIE3: Generation of Gluten-Reactive and TG2-Reactive B Cell Receptors

Essentiality: High
Rationale: B cells with receptors for both gluten and transglutaminase 2 (TG2) play a role in the immune response of celiac disease. These B cells contribute to the production of antibodies such as anti-TG2, which are a hallmark of celiac disease. The formation of these receptors is crucial for the onset of the disease as they facilitate the autoimmune response (Kagnoff, 2007; Koning et al., 2010).

KE1: Co-localization of Gluten Reactive Adaptive T-cells with APCs

Essentiality: High
Rationale: Co-localization of gluten-reactive T cells with antigen-presenting cells (APCs) is essential for the activation of T cells and the subsequent immune response. This interaction is necessary for the initiation of the adaptive immune response, which drives the inflammatory processes seen in celiac disease. Without this step, the disease cannot progress (Vella et al., 2012).

KE2: Activation of the Innate Immune Response

Essentiality: High
Rationale: Activation of the innate immune response is crucial for amplifying the immune response in celiac disease. This step helps recruit additional immune cells to the site of inflammation and promotes further activation of adaptive immune cells. Disruption of this pathway can prevent the development of disease (Lundin et al., 1993).

KE3: Activation of Gluten-Reactive CD4+ T Cells

Essentiality: High
Rationale: The activation of gluten-reactive CD4+ T cells is central to celiac disease pathology. These T cells recognize gluten peptides and drive the autoimmune response, leading to intestinal inflammation and damage. This step is directly linked to the development of disease symptoms and is essential for disease progression (Lundin et al., 1993).

KE4: Activation of Gluten- and TG2-Reactive B Cells

Essentiality: High
Rationale: The activation of gluten- and TG2-reactive B cells leads to the production of antibodies such as anti-TG2 and anti-gluten antibodies. These antibodies contribute to the pathological immune response in celiac disease and are markers of disease activity (Kagnoff, 2007; Koning et al., 2010).

KE5: Disruption of the Intestinal Barrier

Essentiality: High
Rationale: The disruption of the intestinal barrier is a key event in celiac disease and contributes to the leakage of antigens, including gluten peptides, into the intestinal mucosa. This leads to further activation of immune cells and is a critical step in disease pathogenesis. Without this disruption, the immune response would not be sufficiently activated to trigger celiac disease (Anderson et al., 2011).

AO: Celiac Disease

Essentiality: High
Rationale: Celiac disease is the adverse outcome of the AOP. It is characterized by chronic inflammation of the small intestine, leading to villous atrophy, malabsorption, and various systemic manifestations. Without the preceding key events, the disease cannot occur. Therefore, celiac disease as an outcome is directly dependent on the successful progression of the earlier KEs (Green & Cellier, 2007).

Considerations for Potential Applications of the AOP (optional)

This AOP holds significant translational value, particularly for:

• Diagnostic development: Insights into antigen presentation and immune responses support biomarker identification (e.g., TG2 autoantibodies).
• Therapeutic strategies: Potential interventions targeting gluten processing, HLA binding, or immune modulation.
• Regulatory applications: Could support safety assessments of gluten-derived products or alternative treatments.

References

• Bebi C, Urbani D, Evangelisti M, Grossi V, Russo F, Del Rio A. (2024). Outsourcing preparatory work based on a systematic literature review for the development of adverse outcome pathways (AOPs) relevant for the capacity of proteins to trigger celiac disease. EFSA Supporting publication 2024:EN-8570. doi: 10.2903/sp.efsa.2024.EN-8570.
• EFSA Panel on Genetically Modified Organisms (GMO), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, et al. (2017). Guidance on allergenicity assessment of genetically modified plants. EFSA Journal, 15(6):4862. doi: 10.2903/j.efsa.2017.4862.
• Petersen J, Ciacchi L, Tran MT, Loh KL, Kooy-Winkelaar Y, Croft NP, Hardy MY, Chen Z, McCluskey J, Anderson RP, Purcell AW, Tye-Din JA, Koning F, Reid HH, Rossjohn J. (2020). T cell receptor cross-reactivity between gliadin and bacterial peptides in celiac disease. Nature Structural & Molecular Biology, 27: 49–61. doi: 10.1038/s41594-019-0354-z.

Appendix 1

List of MIEs in this AOP

Event: 2252: Human leukocyte antigen DQ2/8-gluten complexes, formation

Short Name: Formation of HLA-DQ2/8-gluten complexes

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Human individuals with celiac disease, particularly those expressing HLA-DQ2 (including HLA-DQ2.5 and HLA-DQ2.2) or HLA-DQ8 (Sollid, 1989; Lundin, 1993; Dieterich, 1997; Molberg, 1997; Dieterich, 1998; Molberg, 1998; Nilsen, 1998; Van de Wal, 1998; Van de Wal, 1998b; Van de Wal, 1999; Arentz-Hansen, 2000; Vader, 2002; Vader, 2002b; Vader, 2003; Vader, 2003b; Meresse, 2004; Meresse, 2006; Tollefsen, 2006; Fallang, 2009; Qiao, 2011; Broughton, 2012; Di Niro, 2012; Sollid, 2012; Petersen, 2014; Qiao, 2014; Steinsbo, 2014). Patients with a genetic predisposition to celiac disease, especially those with specific haplotypes like DR3/DQw2, are most affected (Sollid, 1989; Nilsen, 1998; Van de Wal, 1998; Sollid, 2012).

Key Event Description

Celiac disease is an intestinal disorder triggered by gluten ingestion. It exclusively occurs in individuals who are positive for HLA-DQ2, HLA-DQ8, or both (Sollid, 1989; Dieterich, 1997). In patients with celiac disease, CD4⁺ T cells specifically recognize complexes formed between HLA-DQ2/8 molecules and modified gluten peptides (Molberg, 1997; Meresse, 2004; Sollid, 2012). The formation of these HLA-DQ2/8-gluten complexes is a prerequisite for T-cell activation (Molberg, 1998; Arentz-Hansen, 2000; Vader, 2002).

Upon gluten ingestion, proteolytic fragments are generated through enzymatic cleavage in the upper gastrointestinal tract. These fragments are subsequently modified by tissue transglutaminase (TG2), which converts specific glutamine residues into glutamic acid (Dieterich, 1997; Molberg, 1998). This modification introduces negatively charged residues, which are crucial for high-affinity binding of the modified peptides to HLA-DQ2 or HLA-DQ8, as these molecules preferentially bind peptides with such negatively charged residues (Vader, 2003; Sollid, 2012). This process underlies the immune response observed in celiac disease.

How it is Measured or Detected

• Peptide binding assays: To measure the direct binding of gluten peptides to HLA-DQ molecules (Sollid, 1989; Arentz-Hansen, 2000; Sollid, 2012).

• Mass spectrometry: To analyze deamidated gluten peptides and their interaction with HLA-DQ (Van de Wal, 1998b; Vader, 2002; Fallang, 2009).

• HLA-binding assays: To assess the stability and affinity of peptide-HLA complexes (Molberg, 1998; Vader, 2002; Sollid, 2012).

• T cell proliferation assays: To measure T cell response to gluten peptides (Lundin, 1993; Meresse, 2004; Tollefsen, 2006).

• Tetramer staining: To detect gluten-specific T cells bound to HLA-DQ (Qiao, 2011; Broughton, 2012).

• Flow cytometry: To analyze cell surface markers for gluten peptide presentation (Meresse, 2004; Di Niro, 2012; Steinsbo, 2014).

• Immunohistochemistry & Immunofluorescence: To visualize tTG-gluten complexes (Dieterich, 1997; Di Niro, 2012).

• ELISA: To detect IgA anti-tTG antibodies as a marker for gluten interaction (Dieterich, 1998; Di Niro, 2012).

• Surface plasmon resonance (SPR): To measure TCR affinity for HLA-DQ-gluten complexes (Vader, 2002b; Broughton, 2012; Petersen, 2014).

• HPLC: To purify gluten peptides for further analysis (Van de Wal, 1998b; Van de Wal, 1999; Vader, 2002).

• Serological typing & ASO probes: To identify HLA-DQ alleles in patients (Sollid, 1989; Vader, 2003).

References

• Arentz-Hansen H, Körner R, Molberg Ø, Quarsten H, Vader W, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM, McAdam S. (2000). The intestinal T cell response to α-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med. 191:603-612.

• Broughton SE, Petersen J, Theodossis A, Scally SW, Loh KL, Thompson A, van Bergen J, Kooy-Winkelaar Y, Henderson KN, Beddoe T, Tye-Din JA, Mannering SI, Purcell AW, McCluskey J, Anderson RP, Koning F, Reid HH, Rossjohn J. (2012). Biased T cell receptor usage directed against human leukocyte antigen DQ8-restricted gliadin peptides is associated with celiac disease. Immunity. 37:611-621.

• Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, Huang M, Iversen R, du Pré MF, Qiao SW, Lundin KE, Wilson PC, Sollid LM. (2012). High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med. 18:441-445.

• Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. (1997). Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med. 3:797-801.

• Dieterich W, Laag E, Schöpper H, Volta U, Ferguson A, Gillett H, Riecken EO, Schuppan D. (1998). Autoantibodies to tissue transglutaminase as predictors of celiac disease. Gastroenterology. 115:1317-1321.

• Fallang LE, Bergseng E, Hotta K, Berg-Larsen A, Kim CY, Sollid LM. (2009). Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation. Nat Immunol. 10:1096-1101.

• Lundin KE, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, Thorsby E, Sollid LM. (1993). Gliadin-specific, HLA-DQ(alpha 10501,beta 10201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med. 178:187-196.

• Meresse B, Chen Z, Ciszewski C, Tretiakova M, Bhagat G, Krausz TN, Raulet DH, Lanier LL, Groh V, Spies T, Ebert EC, Green PH, Jabri B. (2004). Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in celiac disease. Immunity. 21:357-366.

• Meresse B, Curran SA, Ciszewski C, Orbelyan G, Setty M, Bhagat G, Lee L, Tretiakova M, Semrad C, Kistner E, Winchester RJ, Braud V, Lanier LL, Geraghty DE, Green PH, Guandalini S, Jabri B. (2006). Reprogramming of CTLs into natural killer-like cells in celiac disease. J Exp Med. 203:1343-1355.

• Molberg Ø, Kett K, Scott H, Thorsby E, Sollid LM, Lundin KE. (1997). Gliadin specific, HLA DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac disease patients, but not from controls. Scand J Immunol. 46:103-109.

• Molberg Ø, McAdam SN, Körner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KE, Sjöström H, Sollid LM. (1998). Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med. 4:713-717.

• Nilsen EM, Jahnsen FL, Lundin KE, Johansen FE, Fausa O, Sollid LM, Jahnsen J, Scott H, Brandtzaeg P. (1998). Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology. 115:551-563.

• Petersen J, Montserrat V, Mujico JR, Loh KL, Beringer DX, van Liempt M, Thompson A, Mearin ML, Schweizer J, Kooy-Winkelaar Y, van Bergen J, Drijfhout JW, Kan WT, La Gruta NL, Anderson RP, Reid HH, Koning F, Rossjohn J. (2014). T-cell receptor recognition of HLA-DQ2-gliadin complexes associated with celiac disease. Nat Struct Mol Biol. 21:480-488.

• Qiao SW, Christophersen A, Lundin KE, Sollid LM. (2014). Biased usage and preferred pairing of alpha- and beta-chains of TCRs specific for an immunodominant gluten epitope in coeliac disease. Int Immunol. 26:13-19.

• Qiao SW, Raki M, Gunnarsen KS, Loset GA, Lundin KE, Sandlie I, Sollid LM. (2011). Posttranslational modification of gluten shapes TCR usage in celiac disease. J Immunol. 187:3064-3071.

• Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E. (1989). Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med. 169:345-350.

• Sollid LM, Qiao SW, Anderson RP, Gianfrani C, Koning F. (2012). Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules. Immunogenetics. 64:455-460.

• Steinsbø Ø, Henry Dunand CJ, Huang M, Mesin L, Salgado-Ferrer M, Lundin KE, Jahnsen J, Wilson PC, Sollid LM. (2014). Restricted VH/VL usage and limited mutations in gluten-specific IgA of coeliac disease lesion plasma cells. Nat Commun. 5:4041.

• Tollefsen S, Arentz-Hansen H, Fleckenstein B, Molberg Ø, Raki M, Kwok WW, Jung G, Lundin KE, Sollid LM. (2006). HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease. J Clin Invest. 116:2226-2236.

• Vader W, de Ru A, van der Wal Y, Kooy Y, Benckhuijsen W, Mearin L, Drijfhout JW, van Veelen P, Koning F. (2002). Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med. 195:643-649.

• Vader W, Stepniak D, Bunnik EM, Kooy Y, de Haan W, Drijfhout JW, van Veelen PA, Koning F. (2003). Characterization of cereal toxicity for celiac disease patients based on protein homology in grains. Gastroenterology. 125:1105-1113.

• Vader W, Stepniak D, Kooy Y, Mearin ML, Thompson A, Spaenij L, Koning F. (2003). The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T-cell responses. Proc Natl Acad Sci U S A. 100:12390-12395.

• Vader W, Kooy Y, van Veelen P, de Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. (2002). The gluten response in children with recent onset celiac disease. A highly diverse response towards multiple gliadin and glutenin-derived peptides. Gastroenterology. 122:1729-1737.

• van de Wal Y, Kooy Y, van Veelen P, Pena S, Mearin L, Papadopoulos G, Koning F. (1998). Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J Immunol 161:1585-1588.

• van de Wal Y, Kooy Y, van Veelen P, Pena S, Mearin L, Papadopoulos G, Koning F. (1998). Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin. Proc Natl Acad Sci U S A. 95:10050-10054.

• van de Wal, Y., Kooy, Y.M.C., Veelen, van P., August, S.A., Drijfhout, J.W. and Koning, F. (1999). Glutenin is involved in the gluten-driven mucosal T cell response. Eur. J. Immunol. 29, 3133-3139.

Event: 2253: Gluten-reactive T cell receptors, generation

Short Name: Generation of gluten-reactive T cell receptors

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Individuals with celiac disease, especially those expressing HLA-DQ2 (such as HLA-DQ2.5) or HLA-DQ8 (Sollid et al., 1989; Lundin et al., 1993; Molberg et al., 1997; Vader et al., 2002). The generation of these T cell receptors is specific to patients with a genetic predisposition to celiac disease (Sollid et al., 1989; Molberg et al., 1997; Qiao et al., 2011; Di Niro et al., 2012). Celiac disease patients with HLA-DQ2.2 are still susceptible to generating gluten-reactive T cell receptors, but their risk of developing the disease is generally lower than for those carrying HLA-DQ2.5 (Sollid et al., 1989; Vader et al., 2003; Tollefsen et al., 2006; Qiao et al., 2014).

Key Event Description

For T cell recognition of the HLA-DQ2/8-gluten complexes, T cell receptors specifically tuned to recognize these complexes must be generated (Molberg et al., 1997; Arentz-Hansen et al., 2000; Vader et al., 2002; Broughton et al., 2012; Qiao et al., 2014). This occurs through gene rearrangement during T cell development (Sollid et al., 1989; Molberg et al., 1997; Molberg et al., 1998; Vader et al., 2002). Notably, T cell receptors specific for the immunodominant gluten epitopes exhibit distinct characteristics, which are consistently shared among patients with celiac disease (Lundin et al., 1993; Dieterich et al., 1997; Molberg et al., 1997; Molberg et al., 1998; Vader et al., 2002; Vader et al., 2002b; Meresse et al., 2004; Tollefsen et al., 2006; Fallang et al., 2009; Qiao et al., 2011; Broughton et al., 2012; Petersen et al., 2014).

How it is Measured or Detected

• TCR sequencing: To identify the specific gene sequences of gluten-reactive T cell receptors (Sollid et al., 1989; Vader et al., 2002; Qiao et al., 2011; Qiao et al., 2014).

• T cell proliferation assays: To measure the activation and proliferation of gluten-reactive T cells in response to gluten peptides (Lundin et al., 1993; Molberg et al., 1998; Meresse et al., 2006; Fallang et al., 2009).

• Flow cytometry: To detect TCR expression and cell surface markers on gluten-reactive T cells (Meresse et al., 2004; Broughton et al., 2012).

• Tetramer staining: To identify gluten-reactive T cells by binding HLA-peptide complexes to T cells (Molberg et al., 1997; Tollefsen et al., 2006).

• Cytokine production assays: To measure cytokine release (e.g., IFN-γ) to assess T cell activation (Nilsen et al., 1998; Meresse et al., 2004).

• Mass spectrometry: To analyze deamidated gluten peptides and their interactions with TCRs (van de Wal et al., 1998; Vader et al., 2003).

• Chromium release assays: To measure the cytotoxicity of gluten-specific CD8+ T cells (Molberg et al., 1998; Tollefsen et al., 2006).

References

• Arentz-Hansen H, Körner R, Molberg Ø, Quarsten H, Vader W, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM, McAdam S. (2000). The intestinal T cell response to α-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med. 191:603-612.
• Broughton SE, Petersen J, Theodossis A, Scally SW, Loh KL, Thompson A, van Bergen J, Kooy-Winkelaar Y, Henderson KN, Beddoe T, Tye-Din JA, Mannering SI, Purcell AW, McCluskey J, Anderson RP, Koning F, Reid HH, Rossjohn J. (2012). Biased T cell receptor usage directed against human leukocyte antigen DQ8-restricted gliadin peptides is associated with celiac disease. Immunity. 37:611-621.
• Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, Huang M, Iversen R, du Pré MF, Qiao SW, Lundin KE, Wilson PC, Sollid LM. (2012). High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med. 18:441-445.
• Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. (1997). Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med. 3:797-801.
• Dieterich W, Laag E, Schöpper H, Volta U, Ferguson A, Gillett H, Riecken EO, Schuppan D. (1998). Autoantibodies to tissue transglutaminase as predictors of celiac disease. Gastroenterology. 115:1317-1321.
• Fallang LE, Bergseng E, Hotta K, Berg-Larsen A, Kim CY, Sollid LM. (2009). Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation. Nat Immunol. 10:1096-1101.
• Lundin KE, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, Thorsby E, Sollid LM. (1993). Gliadin-specific, HLA-DQ(alpha 10501,beta 10201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med. 178:187-196.
• Meresse B, Chen Z, Ciszewski C, Tretiakova M, Bhagat G, Krausz TN, Raulet DH, Lanier LL, Groh V, Spies T, Ebert EC, Green PH, Jabri B. (2004). Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in celiac disease. Immunity. 21:357-366.
• Meresse B, Curran SA, Ciszewski C, Orbelyan G, Setty M, Bhagat G, Lee L, Tretiakova M, Semrad C, Kistner E, Winchester RJ, Braud V, Lanier LL, Geraghty DE, Green PH, Guandalini S, Jabri B. (2006). Reprogramming of CTLs into natural killer-like cells in celiac disease. J Exp Med. 203:1343-1355.
• Molberg Ø, Kett K, Scott H, Thorsby E, Sollid LM, Lundin KE. (1997). Gliadin specific, HLA DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac disease patients, but not from controls. Scand J Immunol. 46:103-109.
• Molberg Ø, McAdam SN, Körner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KE, Sjöström H, Sollid LM. (1998). Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med. 4:713-717.
• Nilsen EM, Jahnsen FL, Lundin KE, Johansen FE, Fausa O, Sollid LM, Jahnsen J, Scott H, Brandtzaeg P. (1998). Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology. 115:551-563.
• Petersen J, Montserrat V, Mujico JR, Loh KL, Beringer DX, van Liempt M, Thompson A, Mearin ML, Schweizer J, Kooy-Winkelaar Y, van Bergen J, Drijfhout JW, Kan WT, La Gruta NL, Anderson RP, Reid HH, Koning F, Rossjohn J. (2014). T-cell receptor recognition of HLA-DQ2-gliadin complexes associated with celiac disease. Nat Struct Mol Biol. 21:480-488.
• Qiao SW, Christophersen A, Lundin KE, Sollid LM. (2014). Biased usage and preferred pairing of alpha- and beta-chains of TCRs specific for an immunodominant gluten epitope in coeliac disease. Int Immunol. 26:13-19.
• Qiao SW, Raki M, Gunnarsen KS, Loset GA, Lundin KE, Sandlie I, Sollid LM. (2011). Posttranslational modification of gluten shapes TCR usage in celiac disease. J Immunol. 187:3064-3071.
• Sollid LM, Markussen G, Ek J, Gjerde H, Vartdal F, Thorsby E. (1989). Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med. 169:345-350.
• Sollid LM, Qiao SW, Anderson RP, Gianfrani C, Koning F. (2012). Nomenclature and listing of celiac disease relevant gluten T-cell epitopes restricted by HLA-DQ molecules. Immunogenetics. 64:455-460.
• Steinsbø Ø, Henry Dunand CJ, Huang M, Mesin L, Salgado-Ferrer M, Lundin KE, Jahnsen J, Wilson PC, Sollid LM. (2014). Restricted VH/VL usage and limited mutations in gluten-specific IgA of coeliac disease lesion plasma cells. Nat Commun. 5:4041.
• Tollefsen S, Arentz-Hansen H, Fleckenstein B, Molberg Ø, Raki M, Kwok WW, Jung G, Lundin KE, Sollid LM. (2006). HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease. J Clin Invest. 116:2226-2236.
• Vader W, de Ru A, van der Wal Y, Kooy Y, Benckhuijsen W, Mearin L, Drijfhout JW, van Veelen P, Koning F. (2002). Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med. 195:643-649.
• Vader W, Stepniak D, Bunnik EM, Kooy Y, de Haan W, Drijfhout JW, van Veelen PA, Koning F. (2003). Characterization of cereal toxicity for celiac disease patients based on protein homology in grains. Gastroenterology. 125:1105-1113.
• Vader W, Stepniak D, Kooy Y, Mearin ML, Thompson A, Spaenij L, Koning F. (2003). The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T-cell responses. Proc Natl Acad Sci U S A. 100:12390-12395.
• Vader W, Kooy Y, van Veelen P, de Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. (2002). The gluten response in children with recent onset celiac disease. A highly diverse response towards multiple gliadin and glutenin-derived peptides. Gastroenterology. 122:1729-1737.
• van de Wal Y, Kooy Y, van Veelen P, Pena S, Mearin L, Papadopoulos G, Koning F. (1998). Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J Immunol 161:1585-1588.
• van de Wal Y, Kooy Y, van Veelen P, Pena S, Mearin L, Papadopoulos G, Koning F. (1998). Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin. Proc Natl Acad Sci U S A. 95:10050-10054.
• van de Wal, Y., Kooy, Y.M.C., Veelen, van P., August, S.A., Drijfhout, J.W. and Koning, F. (1999). Glutenin is involved in the gluten-driven mucosal T cell response. Eur. J. Immunol. 29, 3133-3139.

Event: 2254: Gluten-reactive and transglutaminase 2 reactive B cell receptors, generation

Short Name: Generation of gluten-reactive and TG2-reactive B cell receptors

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Humans, with a female to male proportion of approximately 2 to 1

Key Event Description

The presence of TG2-specific antibodies is a hallmark of celiac disease and is commonly used in its diagnosis of celiac disease (Fleur du Pre et al., 2020). Additionally, antibodies targeting deamidated gluten peptides are frequently detected in patients with celiac disease. The persistent production of these deamidated gluten- and TG2-reactive antibodies contributes to chronic inflammation and tissue damage in the small intestine.

For the antibody-mediated recognition of deamidated gluten and TG2, B cell receptors must be generated during B cell development. Similar to T cell receptors, this process occurs through gene rearrangement. During this process, constant and variable gene segments are joined, encoding distinct light and heavy chains. Antibodies consist of two light and two heavy chains, with a structure that includes two antigen-binding sites formed by the variable regions and a single constant region. Notably, specific variable gene segments encoding TG2-specific antibodies are consistently shared among patients with celiac disease.

How it is Measured or Detected

Gene rearrangement itself can be detected through molecular biological techniques. In practice, however, it is much more common to detect antibodies specific for TG2 and deamidated gluten with enzyme-linked immunosorbent assay (ELISA) or rapid test kits.

References

• Arentz-Hansen H, Körner R, Molberg Ø, Quarsten H, Vader W, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM, McAdam S. (2000). The intestinal T cell response to α-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med. 191:603-612.

• Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, Huang M, Iversen R, du Pré MF, Qiao SW, Lundin KE, Wilson PC, Sollid LM. (2012). High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med. 18:441-445.

• du Pré MF, Blazevski J, Dewan AE, Stamnaes J, Kanduri C, Sandve GK, Johannesen MK, Lindstad CB, Hnida K, Fugger L, Melino G, Qiao SW, Sollid LM. (2020). B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2. J Exp Med. Feb 3;217(2):e20190860. doi: 10.1084/jem.20190860. 

• Fallang LE, Bergseng E, Hotta K, Berg-Larsen A, Kim CY, Sollid LM. (2009). Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation. Nat Immunol. 10:1096-1101.

• Lundin KE, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, Thorsby E, Sollid LM. (1993). Gliadin-specific, HLA-DQ(alpha 10501,beta 10201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med. 178:187-196.

• Vader W, Kooy Y, van Veelen P, de Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. (2002). The gluten response in children with recent onset celiac disease. A highly diverse response towards multiple gliadin and glutenin-derived peptides. Gastroenterology. 122:1729-1737.

List of Key Events in the AOP

Event: 2275: Gluten reactive adaptive T-cells with antigen presenting cells, co-localization

Short Name: Co-localization of gluten reactive adaptive T-cells with APC

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

• Taxonomic: Homo sapiens
• Life-stage: any
• Sex: Female to Male ration: approximately 2 to 1

Key Event Description

For the initiation of adaptive responses, the cellular components required need to be simultaneously present in lymphoid structures present in the target tissue/organ. In the case of the gastrointestinal system these lymphoid structures are the Peyer’s patches and the mesenteric lymph nodes. The relevant cellular components are professional antigen presenting cells like dendritic cells, antigen specific B cells and T cells expressing gluten-reactive T cell receptors. Both dendritic cells and antigen specific B cells can endocytose and degrade complex antigens, resulting in peptides of variable length. Upon intracellular binding of such peptide antigens to HLA-class II molecules, like HLA-DQ2.5 and HLA-DQ8, such HLA-peptide complexes will be transported to the cell surface of the antigen presenting cells, where they can be recognized by T cells expressing T cell receptors specific for the HLA-peptide complex. Thus, once all relevant cellular components are present in the lymphoid structures, the stage is set for the induction of a gluten-specific T cell response.

How it is Measured or Detected

Detection methods could include the measureing of T-cell activation by T cell stimulation assays (e.g. cytokine secretion assays, cytotoxicity assay), or could also include quantification of peptide-HLA-DQ complexes by ELISA (e.g. against HLA-peptide complex) or mass spectrometry (e.g. peptide mass fingerprinting).

References

Parham P. The Immune System, Fifth Edition. ISBN: 978-0-393-53334-7

Event: 2255: Innate immune response, activation

Short Name: Activation of the innate immune response

Key Event Component

AOPs Including This Key Event

Biological Context

Organ term

Domain of Applicability

Homo sapiens

Key Event Description

The groundwork for the development of celiac disease is established. However, an adaptive T and B cell response is only initiated after innate immune activation. While the exact nature of the agent causing innate immune activation remains unknown, it could involve exposure to viruses or bacteria, particularly when this occurs in the gastrointestinal tract, or exposure to environmental factors, such as gluten.

Certain infections may induce inflammation that promotes the activation of self-reactive B cells. Preexposure to IL-15, a cytokine upregulated in inflammatory and infectious conditions (Meresse et al., 2006; Fehniger et al., 2001), plays a key role in this process. IL-15 activates NK cells and intraepithelial lymphocytes (IELs) (CD8+ T cells), which become cytotoxic, damaging epithelial cells in the intestine. This epithelial damage allows gluten peptides to cross into the lamina propria, further perpetuating the immune response and inflammation.
Nilsen et al. (1998) hypothesized that a Th1-like profile, characterized by predominantly high levels of IFNγ, results from various types of intestinal immune responses. This was recently observed in studies following intestinal astrovirus infection (Molberg et al., 1998) and in cases of cow’s milk-sensitive enteropathy.
Additionally, infections can cause molecular mimicry, where pathogen-derived antigens resemble self-antigens. This can trigger an immune response that cross-reacts with the body’s own tissue, contributing to the development of autoimmune conditions such as celiac disease (Petersen et al., 2020).
 

References

• Fehniger, T.A., and M.A. Caligiuri. (2001). Interleukin 15: biology and relevance to human disease. Blood. 97:14–32.

• Meresse B, Curran SA, Ciszewski C, Orbelyan G, Setty M, Bhagat G, Lee L, Tretiakova M, Semrad C, Kistner E, Winchester RJ, Braud V, Lanier LL, Geraghty DE, Green PH, Guandalini S, Jabri B. (2006). Reprogramming of CTLs into natural killer-like cells in celiac disease. J Exp Med. 203:1343-1355.

• Molberg Ø, Nilsen EM, Sollid LM, Scott H, Brandtzaeg P, Thorsby E, Lundin KEA. (1998). CD41 T-cells with specific reactivity against astrovirus isolated from normal human small intestine. Gastroenterology 114:115–122.

• Nilsen EM, Jahnsen FL, Lundin KE, Johansen FE, Fausa O, Sollid LM, Jahnsen J, Scott H, Brandtzaeg P. (1998). Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology. 115:551-563.

• Petersen J, Ciacchi L, Tran MT, Loh KL, Kooy-Winkelaar Y, Croft NP, Hardy MY, Chen Z, McCluskey J, Anderson RP, Purcell AW, Tye-Din JA, Koning F, Reid HH, Rossjohn J. (2020). T cell receptor cross-reactivity between gliadin and bacterial peptides in celiac disease. Nat Struct Mol Biol. Jan;27(1):49-61. doi: 10.1038/s41594-019-0353-4. 

 

Event: 2260: Gluten-reactive CD4+ T cells, activation

Short Name: Activation of gluten-reactive CD4+ T cells

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Homo sapiens

Key Event Description

In the intestinal mucosa of celiac disease patients, gluten-specific CD4+ T cells recognize gliadin antigens, particularly those deamidated by the tissue transglutaminase enzyme (TG2) (Lundin et al., 1993; Arentz-Hansen et al., 2000). These antigens are presented by antigen-presenting cells (APCs) expressing HLA-DQ2 or HLA-DQ8 molecules (Arentz-Hansen et al., 2000; Broughton et al., 2012). Antigen presentation activates the gluten-specific CD4+ T cells, initiating a cascade of immune responses.

Upon activation, gluten-specific CD4+ T cells undergo rapid clonal expansion and differentiation into a pro-inflammatory population that secretes cytokines such as interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) (Nilsen et al., 1998). Additionally, B cells with B cell receptors (BCRs) specific for TG2-gliadin complexes can present these complexes to gluten-specific CD4+ T cells, further stimulating their activation (Di Niro et al., 2012).

The release of IFNγ upregulates the expression of HLA class II molecules on APCs, enhancing the efficiency of gluten peptide presentation (Meresse et al., 2006). This creates a feedback loop that amplifies antigen presentation and intensifies the T cell-mediated immune response to gluten.

How it is Measured or Detected

• Isolation from Intestinal Biopsies: Gluten-reactive T cells can be isolated from patients with celiac disease but not from healthy controls.
• T Cell Activation Measured by Phenotyping: Activated T cells, specifically CD25+ (expressing the IL-2 receptor α-chain), can be identified through phenotyping. When small intestinal biopsies from celiac disease patients on a gluten-free diet are challenged ex vivo with gluten, CD4+ T cells in the lamina propria become activated and express CD25 (Lundin et al., 1993).
• Proliferation Assays: Proliferation assays are performed using antigen-presenting cells (APCs), specific peptides or digested gliadin, and T cells labeled with a marker such as tritiated thymidine (³H). After incubation, plates are harvested, and the incorporation of ³H is measured to assess T cell proliferation (Arentz-Hansen et al., 2000, Broughton et al., 2012, Fallang et al., 2009).
• In Vitro T/B-Cell Cooperation Assay: T/B-cell cooperation is assessed by culturing A20 B-cells with TCR transfectants in the presence of various complexes and conditions. Murine IL-2 secretion, a marker of T cell activation, is measured by ELISA as the readout (Di Niro et al., 2012).
• Additional Applications of ELISA: ELISA is also used to quantify cytokines in biopsies, providing insights into immune responses (Nilsen et al., 1998).
• Quantification of Cytokine mRNA Expression: Competitive PCR is employed to compare cytokine production at the mRNA level. mRNA is reverse-transcribed, amplified by PCR, and visualized on agarose gels. Band intensities are analyzed to determine the ratios of cytokine expression (Nilsen et al., 1998).

References

• Arentz-Hansen H, Körner R, Molberg Ø, Quarsten H, Vader W, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM, McAdam S. (2000). The intestinal T cell response to α-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med. 191:603-612.

• Broughton SE, Petersen J, Theodossis A, Scally SW, Loh KL, Thompson A, van Bergen J, Kooy-Winkelaar Y, Henderson KN, Beddoe T, Tye-Din JA, Mannering SI, Purcell AW, McCluskey J, Anderson RP, Koning F, Reid HH, Rossjohn J. (2012). Biased T cell receptor usage directed against human leukocyte antigen DQ8-restricted gliadin peptides is associated with celiac disease. Immunity. 37:611-621.

• Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, Huang M, Iversen R, du Pré MF, Qiao SW, Lundin KE, Wilson PC, Sollid LM. (2012). High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med. 18:441-445.

• Fallang LE, Bergseng E, Hotta K, Berg-Larsen A, Kim CY, Sollid LM. (2009). Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation. Nat Immunol. 10:1096-1101.

• Lundin KE, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, Thorsby E, Sollid LM. (1993). Gliadin-specific, HLA-DQ(alpha 10501,beta 10201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med. 178:187-196.

• Meresse B, Curran SA, Ciszewski C, Orbelyan G, Setty M, Bhagat G, Lee L, Tretiakova M, Semrad C, Kistner E, Winchester RJ, Braud V, Lanier LL, Geraghty DE, Green PH, Guandalini S, Jabri B. Reprogramming of CTLs into natural killer-like cells in celiac disease. J Exp Med 2006;203:1343-1355. 

• Molberg Ø, Kett K, Scott H, Thorsby E, Sollid LM, Lundin KE. (1997). Gliadin specific, HLA DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac disease patients, but not from controls. Scand J Immunol. 46:103-109.

• Molberg Ø, McAdam SN, Körner R, Quarsten H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P, Lundin KE, Sjöström H, Sollid LM. (1998). Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med. 4:713-717.

• Nilsen EM, Jahnsen FL, Lundin KE, Johansen FE, Fausa O, Sollid LM, Jahnsen J, Scott H, Brandtzaeg P. (1998). Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology. 115:551-563.

Event: 2256: Gluten-reactive B cells and transglutaminase 2-reactive B cells, activation

Short Name: Activation of gluten- and TG2-reactive B cells

Key Event Component

AOPs Including This Key Event

Biological Context

Cell term

Organ term

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Activation of B cells specific to tissue transglutaminase 2 (TG2) are well documented in humans. Although Irish setter can develop partial lymphocyte infiltration in response to wheat diet, it is not a CD4 T cell mediated disease. Monkeys can produce anti gliadins IgA and IgG but the levels of these antibodies do not change with removal or reintroduction of dietary gluten. In mice there is a clear antibody response to TG, but not in a gluten dependent way (Marietta et al., 2011).

Key Event Description

B cells specific to tissue transglutaminase 2 (TG2) are activated in a CD4+ T-cell-dependent manner. Gluten-specific T cells, once activated, provide the necessary "help" to TG2-reactive B cells, facilitating their activation and differentiation into plasma cells that produce anti-TG2 antibodies in the lamina propria (Di Niro et al., 2012). TG2-specific B cells appear to undergo limited affinity maturation, even under chronic antigen exposure, suggesting that their activation relies on naive B cells and is sustained by ongoing gluten exposure (Di Niro et al., 2012; Steinsbo et al., 2014). The crosslinking of TG2 with B-cell receptors (BCRs) may lower the activation threshold for naive TG2-specific B cells, enhancing their activation and subsequent proliferation. This contributes to the high abundance of plasma cells secreting anti-TG2 antibodies, creating a feedback loop that further amplifies antigen presentation to T cells (Di Niro et al., 2012).

Mechanistic Insights

The selection of high-affinity B cells during affinity maturation depends on peptide presentation to T cells. Evidence from Di Niro et al. (2012) supports the necessity of T-cell help, as mutations reducing affinity were linked to a loss of function. B cells engineered to express anti-TG2 BCRs were shown to process and present TG2-gliadin complexes, activating gluten-specific T cells derived from celiac patients, confirming a T-cell-dependent model for antibody generation (Sollid et al., 1997; Sollid et al., 2002; Di Niro et al., 2012).

Further insights by Fleur du Pré et al. (2020) reveal that while most autoreactive B cells are typically removed or silenced through central and peripheral tolerance mechanisms, in celiac disease these controls fail. Anti-TG2 B cells survive and produce autoantibodies when T-cell help is available. These autoreactive B cells act as antigen-presenting cells (APCs), driving the anti-gluten T-cell response, creating an amplification loop central to disease pathogenesis.

Failure of Tolerance Mechanisms

In healthy individuals, autoreactive B cells are controlled through receptor editing, apoptosis, or anergy (Gay et al., 1993; Tiegs et al., 1993; Nemazee and Bürki, 1989; Goodnow et al., 1988). However, in celiac disease, TG2-reactive B cells escape these mechanisms. Some autoreactive B cells may remain "ignorant" in the absence of sufficient antigenic stimulation but become pathogenic when gluten-derived peptides and TG2 form complexes. This failure enables both TG2- and gluten-reactive B cells to survive and contribute to the disease state (Fleur du Pre et al 2020).

How it is Measured or Detected

B cell activation can be evaluated by measuring the generation of monoclonal antibodies. Single plasma cells can be isolated from intestinal biopsies and cultured or sorted with gluten peptide tetramers. The resulting monoclonal antibodies can be analyzed for their reactivity to gluten and TG2 antigens, by ELISA or AlphaLISA​ (Di Niro et al., 2012). Alternatively, fluorescently labeled peptides (e.g., biotinylated gliadin peptides) can be used in flow cytometry to sort and analyze gluten-specific IgA+ plasma cells, allowing for the detection and characterization of B cell activation in celiac lesions​ (Di Niro et al., 2012).

References

• Arentz-Hansen H, Körner R, Molberg Ø, Quarsten H, Vader W, Kooy YMC, Lundin KEA, Koning F, Roepstorff P, Sollid LM, McAdam S. (2000). The intestinal T cell response to α-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med. 191:603-612.

• Di Niro R, Mesin L, Zheng NY, Stamnaes J, Morrissey M, Lee JH, Huang M, Iversen R, du Pré MF, Qiao SW, Lundin KE, Wilson PC, Sollid LM. (2012). High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat Med. 18:441-445.

• du Pré MF, Blazevski J, Dewan AE, Stamnaes J, Kanduri C, Sandve GK, Johannesen MK, Lindstad CB, Hnida K, Fugger L, Melino G, Qiao SW, Sollid LM. (2020). B cell tolerance and antibody production to the celiac disease autoantigen transglutaminase 2. J Exp Med. Feb 3;217(2):e20190860. doi: 10.1084/jem.20190860. 

• Fallang LE, Bergseng E, Hotta K, Berg-Larsen A, Kim CY, Sollid LM. (2009). Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation. Nat Immunol. 10:1096-1101.

• Gay D, Saunders T, Camper S, and Weigert M. (1993). Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177:999–1008. 10.1084/jem.177.4.999

• Goodnow CC, Crosbie J, Adelstein S, Lavoie TB, Smith-Gill SJ, Brink RA, Pritchard-Briscoe H, Wotherspoon JS, Loblay RH, Raphael K. (1988). Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature. 334:676–682. 10.1038/334676a0

• Lundin KE, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O, Thorsby E, Sollid LM. (1993). Gliadin-specific, HLA-DQ(alpha 10501,beta 10201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med. 178:187-196.

• Marietta EV, David CS, Murray JA. (2011). Important lessons derived from animal models of celiac disease. Int Rev Immunol. Aug;30(4):197-206. doi: 10.3109/08830185.2011.598978.

• Nemazee DA, and Bürki K. (1989). Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature. 337:562–566. 10.1038/337562a0

• Sollid LM, Molberg O, McAdam S, Lundin KE. (1997). Autoantibodies in coeliac disease: tissue transglutaminase--guilt by association?. Gut 
  Dec;41(6):851-2. doi: 10.1136/gut.41.6.851.

• Sollid LM. (2002). Coeliac disease: dissecting a complex inflammatory disorder. Nat Rev Immunol. 2:647–655. 

• Steinsbø Ø, Henry Dunand CJ, Huang M, Mesin L, Salgado-Ferrer M, Lundin KE, Jahnsen J, Wilson PC, Sollid LM. (2014). Restricted VH/VL usage and limited mutations in gluten-specific IgA of coeliac disease lesion plasma cells. Nat Commun. 5:4041.

• Tiegs SL, Russell DM, and Nemazee D. (1993). Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177:1009–1020. 10.1084/jem.177.4.1009

• Vader W, Kooy Y, van Veelen P, de Ru A, Harris D, Benckhuijsen W, Pena S, Mearin L, Drijfhout JW, Koning F. (2002). The gluten response in children with recent onset celiac disease. A highly diverse response towards multiple gliadin and glutenin-derived peptides. Gastroenterology. 122:1729-1737.

• van de Wal Y, Kooy Y, van Veelen P, Pena S, Mearin L, Papadopoulos G, Koning F. (1998). Small intestinal T cells of celiac disease patients recognize a natural pepsin fragment of gliadin. Proc Natl Acad Sci U S A. 95:10050-10054.

Event: 1931: Intestinal barrier, disruption

Short Name: Disruption of the intestinal barrier

Key Event Component

AOPs Including This Key Event

Stressors

Biological Context

Organ term

Domain of Applicability

Sex Applicability

Key Event Description

A proper definition (and related ontology) of the intestinal barrier and permeability would benefit the understanding of this biological event central in many diseases. However, it is generally accepted that the intestinal barrier is a multilayer system encompassing :

- a chemical barrier able to detoxify bacterial endotoxins,

- a mucus layer providing a physical barrier against bacteria,

- an one-cell-thick epithelial layer which physical barrier function is ensured by epithelial cell integrity and by tight junction proteins (occludins, claudins and zonulins), adherence junctions and desmosomes ^2,4,5

- the cellular immune system present in the lamina propria underlying the epithelial cell layer

- the antibacterial proteins secreted by the specialized intestinal epithelial cells or the Paneth cells.

Together with the chemical barrier of the mucosal layer and the cellular immune system, the intestinal epithelial cell layer has actually two barrier functions:^1–3

1. It acts as a physical barrier against external factors (pathogens, toxins),
2. It acts as a selective barrier by regulating the absorption of essential dietary nutrients and  ions, meaning their transport from the lumen into the blood.

Intestinal permeability⁶ describes the movement of molecules across the intestinal barrier from the lumen to the blood (Figure 1), and as such, is the measurable feature of the intestinal barrier.

Figure 1. Created with Biorender.com

Molecules can cross the epithelium via paracellular or transcellular route. Transcellular permeability encompass passive diffusion from the apical to the basal side (from the lumen to the blood), vesicle-mediated transcytosis and uptake mediated by a membrane receptor. Paracellular permeability is regulated by the tight junctions between adjacent cells and by the integrity of the epithelium.

Alteration or disruption of one or more layers of the intestinal barrier leads to increased intestinal permeability, also called intestinal hyperpermeability or “leaky gut”, enhancing the transport of pathogens, toxins (such as lipopolysaccharides), undigested nutrients and the translocation of bacteria of the gut microbiota from the intestinal lumen into the systemic circulation³.

How it is Measured or Detected

The definition of intestinal permeability being relatively broad includes altered paracellular route, regulated by TJ proteins, transcellular routes involving membrane transporters and channels, and endocytic mechanisms. Paracellular intestinal permeability can be assessed in vivo via different molecules and via putatiive blood biomarkers and ex vivo in Ussing chambers combining electrophysiology and probes of different molecular sizes. The latter is still the gold standard technique for assessing the epithelial barrier function, whereas in vivo techniques are also broadly used despite limitations (doi: 10.3389/fnut.2021.717925).

In humans.

Virtually all in vivo methods to assess paracellular intestinal permeability rely on the urinary excretion of orally ingested probes. Several markers, including different sizes of PEG, ⁵¹CrEDTA, and especially sugars have been used, each with advantages and disadvantages (doi: 10.3389/fnut.2021.717925). Intestinal Permeability Assessment (IPA) directly measures the ability of two non-metabolized sugar molecules (lactulose and mannitol) to permeate the small intestinal barrier by paracellular passage (sign of perturbed TJ-lactulose) or by transcellular passage (giving information of the whole epithelial absorptive area-mannitol), respectively. The patient drinks a premeasured amount of those sugars and 6h after, the ratio of Lactulose/Mannitol levels is measured in the urine ¹¹.

Levels in plasma/serum or in feces of:

• Markers of epithelial cell damage, such as intestinal fatty acid binding protein (FABP)
• Markers of tight junction alterations, such as zonulin levels (doi: 10.1080/21688370.2016.1251384)
• Microbial translocation, such as peptidoglycans and lipopolysaccharides (LPS) and gut microbiota alteration.

In vitro systems¹²

Transepithelial electrical resistance (TEER) or the Lucifer Yellow (LY) leakage assay are techniques to measure barrier integrity and permeability of a cell layer¹³. Caco-2 cells are human epithelial colorectal adenocarcinoma cells with a structure and function similar to the differentiated small intestinal epithelial cells (e.g. exhibit microvilli). Caco-2 cells can be plated in wells as monolayers^14,11. Other cell lines can be used, such as intestinal epithelial cells (IEC) or primary epithelial cells from human intestinal biopsies¹². Co-culturing of enterocyte-like cells with immune cells in three-dimensional structure and within a microfluidic gut-on-chip has been shown to reflect better the physiology of the gut epithelium. Epi-Intestinal^TM is an example of 3D human primary cell-based organotypic small intestinal model which allows evaluation of TEER and LY leakage assay (doi: 10.1007/s11095-018-2362-0).

In vivo system

In mice, one way to study intestinal paracellular permeability is by measuring the ability of fluorescein isothiocyanate (FITC)-dextran to cross from the lumen into the blood. After gavaging mice with FITC-dextran, the concentrations are measured in collected serum samples (doi: 10.3791/57032).

References

1.            Chelakkot, C., Ghim, J. & Ryu, S. H. Mechanisms regulating intestinal barrier integrity and its pathological implications. Exp. Mol. Med. 50, (2018).

2.            Groschwitz, K. R. & Hogan, S. P. Intestinal barrier function: Molecular regulation and disease pathogenesis. J. Allergy Clin. Immunol. 124, 3–20 (2009).

3.            Ghosh, S. S., Wang, J., Yannie, P. J. & Ghosh, S. Intestinal barrier dysfunction, LPS translocation, and disease development. J. Endocr. Soc. 4, 1–15 (2020).

4.            Sturgeon, C. & Fasano, A. Zonulin, a regulator of epithelial and endothelial barrier functions, and its involvement in chronic inflammatory diseases. Tissue Barriers 4, 1–19 (2016).

5.            Sturgeon, C., Lan, J. & Fasano, A. Zonulin transgenic mice show altered gut permeability and increased morbidity/mortality in the DSS colitis model. Ann N Y Acad Sci 1397, 130–142 (2017).

6.            Bischoff, S. C. et al. Intestinal permeability - a new target for disease prevention and therapy. BMC Gastroenterol. 14, 1–25 (2014).

7.            Qiu, W. et al. PUMA-mediated intestinal epithelial apoptosis contributes to ulcerative colitis in humans and mice. J. Clin. Invest. 121, 1722–1732 (2011).

8.            Hering, N. A., Fromm, M. & Schulzke, J. D. Determinants of colonic barrier function in inflammatory bowel disease and potential therapeutics. J. Physiol. 590, 1035–1044 (2012).

9.            Giron, L. B. et al. Plasma Markers of Disrupted Gut Permeability in Severe COVID-19 Patients. medRxiv 2020.11.13.20231209 (2021).

10.          Prasad, R. et al. Plasma microbiome in COVID-19 subjects: an indicator of gut barrier defects and dysbiosis Ram. BioRxiv (2021).

11.          Aguirre Valadez, J. M. et al. Intestinal permeability in a patient with liver cirrhosis. Ther. Clin. Risk Manag. 12, 1729–1748 (2016).

12.          Fedi, A. et al. In vitro models replicating the human intestinal epithelium for absorption and metabolism studies: A systematic review. J. Control. Release 335, 247–268 (2021).

13.          Lea, T. Epithelial Cell Models; General Introduction. in The Impact of Food Bioactives on Health: in vitro and ex vivo models (eds. Verhoeckx, K. et al.) 95–102 (Springer International Publishing, 2015). doi:10.1007/978-3-319-16104-4_9

14.          Li, B. R. et al. In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability. J. Vis. Exp. 1–6 (2018). doi:10.3791/57032

15.          Ayehunie, S. et al. Human Primary Cell-Based Organotypic Microtissues for Modeling Small Intestinal Drug Absorption Seyoum. Pharm. Res. 35, 72 (2019).

List of Adverse Outcomes in this AOP

Event: 2257: Celiac disease

Short Name: Celiac disease

Key Event Component

AOPs Including This Key Event

Biological Context

Domain of Applicability

Taxonomic Applicability Life Stage Applicability Sex Applicability

Homo sapiens. Although irish setters have shown increased intestinal permeability, partial villous atrophy and intraepithelial infiltration with lymphocytes in connection with gluten in the diet. However, this disease is paroxysmal gluten-sensitive dyskinesia (PGSD) and it is connected primarily with the nervous system, not the small intestine. There is no serological test for PGSD (Lowrie et al., 2018).

Key Event Description

The disease is characterized by an inappropriate immune response leading to changes in the gut (crypt hyperplasia and villous atrophy), stomachache, malabsorption (accompanied by impaired growth in young children), diarrhea, and tiredness. Interestingly, extraintestinal symptoms represent a substantial proportion of the clinical manifestations of the disease (dermatitis herpetiformis, arthritis, neurological symptoms, anemia…) (Dieterich et al., 1998; Lindfors et al., 2019).

How it is Measured or Detected

The basis for the diagnosis of celiac disease is a combination of serology testing and the determination of small intestinal mucosal morphology forms (e.g. endoscopy and biopsy). The most common serological tests various serological tests are EmAs (antibodies specific for TG2 in the endomysium, which is a form of perivascular connective tissue) and TG2-Ab assays (ELISA), reaching a sensitivity of 98.1% and a specificity of 94.7% in patients with biopsy-confirmed cases (Dieterich et al., 1997; Dieterich et al., 1998).

Importantly, some patients are IgA deficient and around 10% of patients are seronegative. Although for these cases the gold standard is the biopsy, other tests are:

• For patients IgA deficient, EMAs and TG2 assay with IgG, considering that IgG may be elevated due to other autoimmune diseases, and the predictive value is lower (Dieterich et al., 1998)
• HLA typing is useful as the disease is unlikely when individuals do not carry HLA-DQ2 or HLA-DQ8 (Lindfors et al., 2019).
• Detection of intestinal TG2-targeted celiac IgA isotype autoantibody deposits in intestinal mucosal tissue samples is helpful but requires frozen biopsy samples.
• Detection of T cells. A 3-day gluten challenge induces the mobilization of memory T cells reactive against gliadin, which can be detected by IFNγ enzyme-linked immunospot (ELISPOT) assay. Otherwise, T cells can be detected with HLA-DQ–gluten tetramers by flow cytometry.

Regulatory Significance of the AO

Celiac disease may be considered as a public health problem as it increases the overall mortality risk, reduces quality of life and yields extensive negative economic consequences. Although majority of patients experienced a good and long life after the diagnosis, a subgroup may develop complications such as T-cell lymphoma (Lindfors et al., 2019; Dieterich et al., 1997).

Although Swedish epidemiological study of coeliac disease in the mid-1980s88 suggests that coeliac disease may be prevented by the early introduction of small quantities of gluten into the diet of young children, two systematic reviews and meta- analyses have concluded that the timing of gluten introduction and the duration or maintenance of breastfeeding do not influence the development of coeliac disease. The use of primitive wheat varieties (kamut, einkorn and others) or the use of oats to reduce the clinical symptoms have not been shown in proper trials (Lindfors et al., 2019).

About 20–50% of patients with coeliac disease have persistent or recurrent symptoms despite a long- term gluten- free diet, usually due to other gastrointestinal disorders (irritable bowel syndrome, lactose intolerance…) or inadvertent gluten exposure. To avoid this last one, US FDA in 2013 or EU 828/2014 enforced regulations to labelling products defining “gluten free” as less than 20 mg/kg when measured by an approved system for testing, normally gluten-analysis R5 ELISA (Mendez), as less than 20ppm is considered safe in celiac disease patients. Although there are a good range of products available, these products are often inadequately labelled, less palatable, and more expensive, causing non-adherence to a strict gluten free diet. Managing the disease involves an active effort from the patient to regulate feelings, actions and reactions during any social activity that involves food (Lindfors et al., 2019).

References

• Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, Schuppan D. (1997). Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med. 3:797-801.
• Dieterich W, Laag E, Schöpper H, Volta U, Ferguson A, Gillett H, Riecken EO, Schuppan D. (1998). Autoantibodies to tissue transglutaminase as predictors of celiac disease. Gastroenterology. 115:1317-1321.
• Meresse B, Chen Z, Ciszewski C, Tretiakova M, Bhagat G, Krausz TN, Raulet DH, Lanier LL, Groh V, Spies T, Ebert EC, Green PH, Jabri B. (2004). Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in celiac disease. Immunity. 21:357-366.
• Lindfors K., Ciacci C., Kurppa K., Lundin K. E. A., Makharia G. K., Mearin M. L., Murray J. A., Verdu E. F., Kaukinen K. (2019). Coeliac disease. Nature Reviews Disease Primers, 5(1), Article 3. https://doi.org/10.1038/s41572-018-0054-z
• Lowrie M, Garden OA, Hadjivassiliou M, Sanders DS, Powell R, L Garosi L. (2018). Characterization of Paroxysmal Gluten‐Sensitive Dyskinesia in Border Terriers Using Serological Markers. J Vet Intern Med. Feb 9;32(2):775–781. doi: 10.1111/jvim.15038

Appendix 2

List of Key Event Relationships in the AOP