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-gamma, 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-gamma, IL-10, and IL-17 production.

FIGURE 1

Representative pattern of CD4⁺ T cell cytokine secretion in response to Timothy grass-derived peptide pools. Peptides derived from Phl p 1, 2, 3, 4, 5, 6, 7, 11, 12, and 13 were synthesized as 15-mers overlapping by 10 residues and pooled in groups of ~20 peptides/pool. The distribution of CD4⁺ T cell secretion of IL-5 in response to these peptide pools from allergic donor U00032 is shown. Positive pools were defined as inducing a cytokine-specific response of ≥100 SFCs/10⁶ PBMCs, p ≤ 0.05, and SI ≥ 2.0.

FIGURE 2

Cytokine and Ig response patterns in donor cohorts. The patterns of IL-5, IFN-γ, IL-10, and IL-17 responses in the allergic, nonallergic, and SIT donor cohorts are shown in A and B. The majority of the responses, normalized for the size of the respective cohort, was observed in allergic donors and primarily IL-5 (A). Lesser overall responses were seen in the SIT and nonallergic donor cohorts, but as with the allergics, IL-5 represented the majority of the cytokine response. B shows the percent of the total SFC response in each cohort as a function of each cytokine. Median IgE and IgG responses in the different cohorts are shown in C.

FIGURE 3

Differing distribution of HLA class II binding affinities for microbial and allergen-derived T cell epitopes. The capacity of Phl p derived epitopes to bind (IC₅₀ nM) their respective HLA class II-restricting molecule is shown against the cumulative percent of epitopes with higher or lower affinities for their respective restricting molecule. Also shown is a similar analysis for a set of 46 HLA class II-restricted epitopes derived from various microbial sources.

FIGURE 4

Predictions of promiscuously binding peptides cover a large fraction of the response. The capacity of each Phl p peptide to bind our panel of HLA class II molecules was predicted using a panel of algorithms as described in the text. For each peptide, the number of HLA predicted to be bound in the top 20th percentile or better was recorded. Peptides were then ranked on the basis of the number of molecules predicted to be bound. Using the number of predicted bound alleles as selection criteria, the percent of total SFCs that would have been accounted for was determined. Selecting the top fifth of peptides that bind ≥10 alleles (vertical dashed line) would have accounted for 50% of the T cell response (SFCs).

FIGURE 5

Identified T cell responses are focused on a subset of Phl p allergens. The percent fraction of total Phl p response (SFC) obtained in the cohort of allergic donors specific for each Phl p Ag is shown. The majority of the Phl p response is directed toward Phl p 5. Responses to Phl p 1 and 3 also comprise a large fraction of the overall response.

FIGURE 6

Comparison of cytokine and IgE responses in allergic donors. The relationship between total CD4⁺ T cell cytokine response (IL-5, IFN-γ, IL-17, and IL-10) to overlapping peptides derived from Phl p 1, 2, 3, 4, and 5 are compared with the IgE serum Ab titers against the corresponding rPhl p protein.

FIGURE 7

Cytokine response patterns vary against different Phl p allergens. The total response (SFC) specific for each cytokine and Phl p Ag was tabulated. The Phl p 1 and 11 Ags were associated with exclusive IL-5 production, whereas Phl p 4 and 5 were associated with production of all four cytokines tested. The Phl p 2, 3, and 13 Ags were associated with production of both IL-5 and IFN-γ.