Discussion
So far, the published data for ARTE have been focusing on bacteria- or fungi-specific antigens as well as house dust mites16. However, this methods allows also to study even rarer nutritional antigen-specific T-cells without in-vitro expanding of the reacting cells and without re-challenging the patients. Therefore, we apply this method for detection of the rare nutritional antigen-specific T-cells in peripheral blood, in order to analyze antigen reactivity for different clinical subgroups of CeD and IBD patients.
Recently, peripheral gluten-specific CD4+ T-cells were analyzed applying HLA-DQ2:gluten tetramers, thus identifying an increase of gluten-specific CD4+ T-cells in aCeD5,6. However, the here applied ARTE allows for a deeper analysis of the respective specific CD4+T-cells to distinguish different disease states of CeD. The diagnosis of CeD in patients who already follow a GFD is challenging, since tTG antibodies under GFD normalize and histological markers, as the small intestinal villous atrophy, regenerates. This clinical need is even growing, giving the increasingly popular gluten-free life style in the western world 17,18, or for first degree relatives, who frequently initiate a GFD when a household member is diagnosed with CeD. For the latter, the high risk of developing CeD has been proven in many studies 19,20 and surveillance for CeD is even recommended for first-degree relatives of a diagnosed patient where carriage of a risk gene has not been excluded 21,22. Work herein might be the first step towards identifying such cases, without a conventional burdening gluten re-challenge, since characteristic changes in cytokine expression in gliadin-specific CD4+ T-cells in the peripheral blood are present. For the rare subgroup of Refr patients, especially for type I, the specific immunological nature remains unclear. Diagnosis is still based on histopathology alone, while recent studies suggest a heterogeneous composition of different pathologies to be merged under this term. In this respect, the ARTE technique for gliadin-specific T-cells represents a unique research tool for future studies that has the potential to contribute to a subclassification of this disease group. Furthermore, our data reveal a specific immunological phenotype of gliadin-specific CD4+ T-cells from FDR regarding the hypersensitivity towards gluten, even if diagnosed as healthy due to their tTG status (without GFD). By demonstrating an active immune response against the pathogenic antigen, identification and even phenotyping of gluten-reactive T-cells from peripheral blood might represent an alternative, all the more in pediatric cases, where first time diagnosis is common, while invasive endoscopy is meant to be avoided. Thus, this novel approach could fulfill the clinical need for a noninvasive marker of CeD activity as clinical and research tool 23.
With regard to IBD, which shares the characteristics of barrier disruption 24 and subsequent intestinal inflammation in the lamina propria, we detected increased levels of gliadin-specific T-cells in the peripheral blood of active CD patients with concurrent small intestinal inflammation, paralleled by the highest frequency of antigen-specific T-cells expressing pro-inflammatory cytokines. This distinct occurrence suggests the small intestinal barrier disruption as major cause for the observed T-cell activation, since these cells express small intestinal homing markers. The homing to the ileum (α4β1) was described as essential pathway in CD 25. In line, only in these three patient groups of active small intestinal inflammation, the effector-memory T-cells outnumbered the naïve phenotype among gliadin-specific T-cells in the peripheral blood. Furthermore, peripheral T-cells from CD patients with small intestinal inflammation proved to be responsive to other major nutritional antigens26, while neither active UC nor CD or UC in remission showed any reaction. Again, only antigen-specific T-cells from active CeD, but not GFD patients demonstrated similar properties, corroborating on the one hand the leaky barrier of the affected small intestine as the site of food antigen translocation and subsequent T-cell activation. On the other hand, the significant effect of gliadin, but no other food antigen, in the GFD group further confirmed the singular antigen-driven nature of CeD. Nevertheless, based on surveys, it has been suggested, that long-term GFD improves gastrointestinal symptoms in active IBD patients 10. With the present study, we are able to convey cellular and functional data by demonstrating an enhanced gliadin-specific response of pro-inflammatory cytokines towards gliadin for CD patients
Our data strongly suggest, that small intestinal inflammation is key for the development of a nutritional antigen-specific T-cell response. Therefore, ARTE allows to distinguish CD with small intestinal inflammation from UC and CD in remission by a unique profile of circulating antigen-specific T-cells, raising the question, if a well-defined nutritional regimen (e.g. GFD) might have therapeutic potential in the setting of IBD. Hence, based on the analysis of the systemic immune response, an “anti-inflammatory” diet could be developed and monitored. In addition, this technique allows detailed analyses of gliadin-specific T-cells in such a high resolution that even healthy first-degree relatives can be discriminated and might thus provide a novel non-invasive diagnostic tool to identify symptom-free CeD patients on a gluten-free diet.