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.