HLA variants DQ2 and DQ8 were the genetic factors most closely associated with CD. The isolation in duodenal biopsies of T cell clones recognizing gluten peptides in association with these HLA molecules further confirmed the pathogenic role of these genetic variants[20
]. Furthermore, anti-gluten CD4 T cells produced large amounts of interferon gamma, which seemed to account for the typical mucosal damage seen in CD-affected mucosa. However, even this knowledge failed to explain why a HLA DQ2/DQ8 patient can present CD, yet another, not. In this scenario, the identification of the single antigen targeted in the Endomysial staining reaction was expected to permit a better knowledge of CD pathogenesis, and a better understanding of the origins of CD[21
Thus, the search for the “endomysial antigen” represented an amazing adventure for most researchers involved in CD in the 1990s. In 1997, Dieterich and colleagues found that the endomysial antigen involved in the autoimmune response in CD was the enzyme tissue transglutaminase or Type 2 transglutaminase (tTG or TG2)[22
]. Indeed, tTG is present in the endomysial net, where it stabilizes the connective tissue by catalyzing the link between glutamine and lysine of different structural proteins. This activity is very important in tissue repair processes and an increased activity of the enzyme can be evidenced in damaged tissues, including the mucosa in CD. Furthermore, tTG plays another important role, in the packaging of debris after cell apoptosis, which allows for the correct removal of apoptotic bodies containing inflammatory response materials.
Ludwig Sollid was the first to publicly hypothesize a model linking gluten to tTG and to anti-tTG autoantibodies. Briefly, when large amounts of gluten enter the mucosa because of increased epithelial permeability (may be favored by other factors, such as infections), the anti-gluten response causes mucosal damage, causing the release and activation of tTG. Gluten itself, due to its high content in glutamine, can be a target of tTG and can be cross-linked with other proteins, including tTG. As a consequence, macromolecular complexes containing both gluten peptides and tTG can be recognized by AGA-producing B cells, as well as by AEA-producing B cells. According to the “Sollid hypothesis”, B cells recognizing these macro-complexes, regardless of their antibody specificity will present gluten peptides to gluten-specific T cells. As a consequence, a single antigen can drive an immune response to many targets, overcoming the tolerance of the immune system, in a process also known as “antigen spreading”.
Another finding connecting tTG and gluten relies on the capacity of the enzyme to deaminate gluten-derived peptides increasing their affinity to the DQ2 and DQ8 HLA, thus worsening the consequences of anti-gluten immunity[23,24
]. Recently, measurement of the immune response to deaminated gliadin peptides (DGP)[25
] has been utilized to increase the performances of the AGA assay[26
]. This model could partly explain the role of environment, with gastrointestinal infections, in precipitating the pathogenic mechanisms of CD with a vicious circle of tissue damage, activation of tTG, entry and deamination of gluten, anti-gluten response and the spreading of autoantibodies. Hyper-production of IL-15 is associated with these mucosal changes, which could affect the production of the immunoregulatory cytokine TGF-beta[27,28
]. Even if this model does not illuminate the specific relationship between CD and other autoimmune disorders, it describes a dysregulated mucosal immunity, which is likely to interfere with the normal mechanisms of immune tolerance.
Apart from contributing to pathogenic knowledge, the identification of the main CD autoantigen allowed for a further improvement of diagnostics for CD by using ELISA assays based on human recombinant tTG (htTG). Using htTG, population screening has been performed starting from finger puncture producing as little as a few drops of blood in children from primary schools[27
], and more recently rapid tests have been produced for the consumer market. Due to the reliability of htTG assays, CD diagnosis can now be confirmed with just one jejunal biopsy without any need for repeating bioptic examinations after the start of the diet. In some cases, it is even thought that a confirmation by means of jejunal biopsy may not be necessary. In fact, considering that a strong correlation has been demonstrated between high levels of tTG antibodies and a higher grade of mucosal damage (Marsh score)[29,30
], the ESPGHAN is currently evaluating the possibility of making the diagnosis without a confirmatory jejunal biopsy in patients who have symptoms that can be referred to CD, if IgA-tTG antibodies are > 10x the upper normal limit, AEA and HLA DQ2 and/or DQ8 are positive.