Molecular Determinants of T Cell Epitope

We investigated the molecular determinants of allergen-derived T cell epitopes in humans utilizing the Phleum pratense (Timothy grass) allergens (Phl p). PBMCs from allergic individuals were tested in ELISPOT assays with overlapping peptides spanning known Phl p allergens. A total of 43 distinct antigenic regions were recognized, illustrating the large breadth of grass-specific T cell epitopes. Th2 cytokines (as represented by IL-5) were predominant, whereas IFN- g , IL-10, and IL-17 were detected less frequently. Responses from specific immunotherapy treatment individuals were weaker and less consistent, yet similar in epitope specificity and cytokine pattern to allergic donors, whereas nonallergic individuals were essentially nonreactive. Despite the large breadth of recognition, nine dominant antigenic regions were defined, each recognized by multiple donors, accounting for 51% of the total response. Multiple HLA molecules and loci restricted the dominant regions, and the immunodominant epitopes could be predicted using bioinformatic algorithms specific for 23 common HLA-DR, DP, and DQ molecules. Immunodominance was also apparent at the Phl p Ag level. It was found that 52, 19, and 14% of the total response was directed to Phl p 5, 1, and 3, respectively. Interestingly, little or no correlation between Phl p-specific IgE levels and T cell responses was found. Thus, certain intrinsic features of the allergen protein might influence immunogenicity at the level of T cell reactivity. Consistent with this notion, different Phl p Ags were associated with distinct patterns of IL-5, of protease inhibitors. a 2–4-d incubation, percent of MHC-bound radioactivity was determined by capturing MHC/peptide complexes on LB3.1 (DR), L243 (DR), SPV-L3 Ab-coated Optiplates Meriden, TopCount microscintillation against HLA-DR (LB3.1), DP (B7/21), or DQ (SVPL3) 30 min prior to peptide addition. Cytokine production in response to positive peptides was then measured in ELISPOTassays as described above. The pan MHC class I Ab (W6/32) was used as a control. To determine the specific HLA allele restriction, donor-derived T cells were expanded for 10 d using a single epitope peptide and were then sub-sequently incubated with peptide-pulsed EBV cell lines and/or fibroblasts expressing known HLA molecules also expressed in the donor from whom T cells were derived. Cytokine-specific ELISPOTassays were performed as described above to determine cytokine production and allele restriction determined by analyzing a matrix of negative and positive cytokine responses with the HLA-expressing EBV lines and fibroblasts used.