Molecular Determinants of T Cell Epitope Recognition to the Common Timothy Grass Allergen

Abstract

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-γ, 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, IFN-γ, IL-10, and IL-17 production.

Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals

“…Studies conducted in mice have demonstrated the development of allergic airway hyperresponsiveness mediated by T cells in the absence of IgE (10,11). Furthermore, data obtained from human studies have demonstrated a lack of correlation between antigen-specific IgE levels and T-cell responses (12)(13)(14)(15)(16).…”

“…PBMCs were isolated by density gradient centrifugation from one unit of blood (∼450 mL) and cryopreserved as described (12). For in vitro expansions, PBMCs were thawed and cultured in RPMI 1640 (Omega Scientific) supplemented with 5% human AB serum (Cellgro) at a density of 2 × 10 6 cells per mL in 24-well plates (BD Biosciences) and stimulated with TG pollen extract (50 μg/mL; Greer).…”

“…The production of IL-5 from cultured PBMCs in response to antigen stimulation was measured by ELISPOT as described (12). Briefly, 1 × 10 5 cells per well were incubated with peptide, peptide pool, or TG extract (10, 5, and 50 μg/mL, respectively).…”

“…In our previous study of T-cell responses against Timothy grass (TG) allergens (12), no correlation was detected between IgE levels and T-cell responses in TG pollen-allergic individuals. Furthermore, we found that one-third of the patients studied had no Th2 cell response against any of the known IgE-reactive proteins, despite strong responses against whole TG extract.…”

“…In a previous study (12), we determined the T-cell epitopes in the 10 known major IgE-reactive allergens present in TG pollen. Using 15-mer peptides overlapping by 10 residues that spanned the entire protein sequence of each of these allergens, we identified 20 dominant epitopic regions accounting for the majority of T-cell responses.…”