Introduction
Antigen-specific T-cells play a central role in the adaptive immune
system, promoting specific acute immune responses and the formation of
immunological memory. Analyzing not only frequency, but also phenotype
and function of these rare cells, represents a critical step to
understand the mechanisms of adaptive immunity in general, but also to
determine the specific immune status of the individual patient or to
diagnose infectious or auto-immune diseases. The high diversity of the
T-cell receptor, which allows for recognition of billions of different
antigens, leads to an extremely low frequency of T-cells, specific for a
single peptide-MHC ligand. This holds true even for pathogen-specific
memory compartments in the absence of acute infections, for which
specific T-cell frequencies in peripheral blood are typically far below
1%, but all the more for the naive repertoire (< 0.0005%)1,2. Hence, the analysis of antigen-specific T-cells
demands methods which are highly specific, but in parallel allow
processing of large cell numbers to enable quantification of these rare
events. For the last decade, techniques such as antigen-specific
proliferation via 3H-thymidine incorporation3 were applied, where antigen-specific T-cells have
been analyzed via bulk stimulation of peripheral blood mononuclear cells
(PBMC) with the respective antigen. However, in such assays, it is not
feasible to detect the phenotypic and functional properties of the
original reactive cells and therefore it is almost impossible to
determine the actual frequency and phenotype of these cells in the
starting cell sample or to exclude bystander effects. ELISPOT4, as an additional technical approach, reveals
information on a single cell level, but lacks the high throughput
capabilities to detect and quantify rare cells to an extent, where for
example food antigen-specific T-cells in healthy individuals can be
studied. Several studies aimed to characterize the systemic T-cell
response to decipher the role of circulating T-cells and their
functional impact 5,6. Until now, the specific
analysis of gluten-specific T-cells via flow cytometry depended on
tetramer analysis 5, which is limited to the knowledge
and availability of the antigen epitope 7, but also
the frequency of target cells, therefore suffering from a high
variability in cytokine analysis 8.
Among autoimmune diseases, celiac disease (CeD) represents a model
disease, since gluten has been identified as the disease-causing antigen
and elimination of gluten results in regeneration of the duodenal mucosa
and consecutive wellbeing of the patient 9. However,
the diagnosis in CeD patients who are already on a gluten-free diet
(GFD) remains challenging. Under a gluten-free diet (GFD),
tissue-transglutaminase (tTG) antibodies normalize and the small
intestinal villus atrophy regenerates. Until now, a burdening
re-challenge of the patients to gluten is mandatory for a valid
diagnosis. Yet, translocation of nutritional (and pathogenic) antigens
due to intestinal barrier breaches is described not exclusively for CeD,
but also for inflammatory bowel diseases (IBD). Although there are first
studies connecting GFD to improvement of patient wellbeing10 and even microbiota composition11 in IBD, until now, circulating nutritional
antigen-specific T-cells have not been analyzed in these patients. Since
the small intestine is the primary contact surface for food antigens and
hence for the immunological response, we analyzed the specific
nutritional T-cell response in the peripheral blood of patients with
small intestinal Crohn´s disease (CD), celiac disease as well as
ulcerative colitis (UC), respectively. To exclude the influence of a
non-intestinal inflammation, rheumatoid arthritis patients (RA) were
included as control.
We here applied the novel antigen-reactive T-cell enrichment (ARTE)7 technology to determine the specific nutritional
effector T-cell response in the peripheral blood. The ARTE technique is
based on the stimulation of PBMC with a defined antigen and the
subsequent up-regulation of the activation marker
CD154+, which is exclusively expressed on
antigen-specific CD4+ T-cells 12.
This method permits the detection of the entire antigen-specific
CD4+ T-cell response just by adding the antigen of
choice directly to PBMC without the need of in-vitro expanding the
reacting cells. The subsequent enrichment of CD154+cells enables further in-depth phenotyping of this rare cell population.
Thus, the ARTE technique allows a direct ex vivo cytometric - and
hence functional - analyses of gluten-specific, but also even rarer
nutritional antigen-specific T-cells.