Identification of immunodominant epitopes of alpha-gliadin in HLA-DQ8 transgenic mice following oral immunization.
Celiac disease, triggered by wheat gliadin and related prolamins from barley and rye, is characterized by a strong association with HLA-DQ2 and HLA-DQ8 genes. Gliadin is a mixture of many proteins that makes difficult the identification of major immunodominant epitopes. To address this issue, we expressed in Escherichia coli a recombinant alpha-gliadin (r-alpha-gliadin) showing the most conserved sequence among the fraction of alpha-gliadins. HLA-DQ8 mice, on a gluten-free diet, were intragastrically immunized with a chymotryptic digest of r-alpha-gliadin along with cholera toxin as adjuvant. Spleen and mesenteric lymph node T cell responses were analyzed for in vitro proliferative assay using a panel of synthetic peptides encompassing the entire sequence of r-alpha-gliadin. Two immunodominant epitopes corresponding to peptide p13 (aa 120-139) and p23 (aa 220-239) were identified. The response was restricted to DQ and mediated by CD4+ T cells. In vitro tissue transglutaminase deamidation of both peptides did not increase the response; furthermore, tissue transglutaminase catalyzed extensive deamidation in vitro along the entire r-alpha-gliadin molecule, but failed to elicit new immunogenic determinants. Surprisingly, the analysis of the cytokine profile showed that both deamidated and native peptides induced preferentially IFN-gamma secretion, despite the use of cholera toxin, a mucosal adjuvant that normally induces a Th2 response to bystander Ags. Taken together, these data suggest that, in this model of gluten hypersensitivity, deamidation is not a prerequisite for the initiation of gluten responses.
Senger
Stefania
S
Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche, Avellino, Italy.
Maurano
Francesco
F
Mazzeo
Maria F
MF
Gaita
Marcello
M
Fierro
Olga
O
David
Chella S
CS
Troncone
Riccardo
R
Auricchio
Salvatore
S
Siciliano
Rosa A
RA
Rossi
Mauro
M
D000595:Amino Acid Sequence; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002446:Celiac Disease / Q000276:immunology; D016207:Cytokines / Q000032:analysis / Q000378:metabolism; D004195:Disease Models, Animal; D005903:Gliadin / Q000276:immunology*; D006683:HLA-DQ Antigens / Q000235:genetics*; D006801:Humans; D007114:Immunization; D016056:Immunodominant Epitopes / Q000737:chemistry*; D051379:Mice; D008822:Mice, Transgenic; D010446:Peptide Fragments / Q000008:administration & dosage / Q000276:immunology; D010449:Peptide Mapping; D021182:Wheat Hypersensitivity / Q000276:immunology
D016207:Cytokines; D006683:HLA-DQ Antigens; C109756:HLA-DQ8 antigen; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D005903:Gliadin