primary prevention of coeliac disease - Associazione Italiana ...

A DOMINANT EPITOPE IN GLUTEN PEPTIDES ?
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interferon gamma (IFNg) ELISpot assays using peripheral blood mononuclear cells
(PBMC). IFNgELISpot assays define the frequency of cells secreting IFNg, and are
capable of detecting peptide-specific T cell frequencies as low as 5-10 per million
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PBMC. Blood was collected from HLA-DQ2 healthy non-coeliac and coeliac subjects
after gluten free diet for at least 4 weeks, and then in the 12 days after commencing
gluten challenge (200 g white bread daily for ½ day [n=1], 3 days [n=10], or 10 days
[n=1]). Short-term gluten challenge was generally well tolerated by coeliac subjects,
11/12 subjects were able to consume 200g gluten bread for 3 or more days, and 8/12 had
only mild symptoms. One subject had abdominal cramps and vomited within 3 hours of
the first 2 slices of bread. All symptoms resolved within 1 to 3 days after ceasing gluten
challenge.
Prior to gluten challenge, no A-gliadin peptide elicited responses in the IFNg
ELISpot. However, in 11/12 coeliac subjects and 0/4 healthy control subjects there was
induction of IFNgELISpot responses on day 4-8 for one pool of overlapping peptides
only when treated with tTG. In all cases, IFNginduction was attributed to 2 peptides
overlapping by 10 aminoacids (A-gliadin 56-75). Aminoacid sequencing demonstrated
that only one glutamine residue in A-gliadin 56-75 was susceptible to tTG-mediated
deamidation (Q65). Truncations of A-gliadin 56-75 treated with tTG indicated that
residues 64-68 (PQLPY) were critical for bioactivity, and that the 17mer
QLQPFPQPQLPYPQPQS (57-73) was the optimal peptide. Bioactivity of
QLQPFPQPELPYPQPQS was identical to tTG-treated QLQPFPQPQLPYPQPQS,
demonstrating that bioactivity of this peptide was dependent upon a single deamidated
glutamine residue (QE65). Immunomagnetic bead depletion of PBMC prior to addition
of peptide showed that this peptide specific immune response was due to CD4 T cells
E
expressing the b,
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but not the aintegrin protein, indicating that A-gliadin 57-73 QE65
specific T cells express the abintegrin
4 7
associated with homing to the intestinal lamina
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propria . Pre-incubation of PBMC from HLA-DQ2 homozygous subjects with
antibody specific for HLA-DQ (but not HLA-DR or HLA-DP) blocks A-gliadin 57-73
QE65 responses, indicating HLA-DQ2 restriction.
In one subject who consumed only 2 slices of bread, IFNg-secreting T cells specific
for A-gliadin 57-73 QE65 were induced on day 6 and persisted until day 12. In another
subject who consumed 4 slices of bread daily for 10 days, A-gliadin 57-73 QE65-
specific T cells were also present on day 6-8. IFNgsecretion was only induced by A-
gliadin 57-73 QE65 in subjects who consumed bread for 3 days. However, PBMC
collected on day 11 from the subject who consumed bread for 10 days secreted IFNgin
response to 6 out of 10 pools of tTG-treated A-gliadin peptides (one pool included A-
gliadin 57-73). Hence, T cell epitope spreading occurs as early as 10 days after
commencing antigen challenge.
These studies indicated:
1. In A-gliadin, there is a hierarchy of T cell epitopes with only one dominant T cell
epitope.
2. T cells specific for the dominant epitope are present in peripheral blood only