Use of multiple peptides containing T cell epitopes is a feasible approach for peptide-based immunotherapy in Can f 1 allergy

Abstract

We have previously shown that the major dog allergen Can f 1 contains seven T cell epitope regions, none of which was preferentially recognized. To identify the immune characteristics of Can f 1 epitopes and to verify their suitability for peptide-based allergen immunotherapy, short-term T cell lines were generated with epitope-containing peptides from peripheral blood mononuclear cells of Can f 1 skinprick test-positive allergic and healthy control subjects. The lines were examined for their proliferative capacity and cytokine production upon stimulation with the allergen peptide, a homologous peptide from human tear lipocalin (TL) and Can f 1 and TL proteins. Can f 1 peptides induced proliferation of T cells and gave rise to T cell lines with comparable efficiencies. In particular, the T cell lines of allergic subjects induced with p33-48 and p107-122 favoured the production of interferon-gamma and interleukin-10, respectively. A greater number of Can f 1-specific T cell lines were generated from allergic than from healthy individuals. Two p107-122-induced Can f 1-specific T cell lines also reacted to a homologous peptide of human TL. Our results suggest that several T cell epitope-containing peptides should be used in combination for specific immunotherapy in Can f 1 allergy.

Figure 1

Proportion of positive results as percentages in 12 parallel peripheral blood mononuclear cell (PBMC) cultures stimulated with Can f 1 peptides. Specific proliferation of PBMCs of dog-allergic and non-allergic subjects was determined using the mean counts per minute (cpm) + 3 SD of 12 negative control cultures as a cut-off. Thereafter, a percentage of positive cultures per peptide was calculated for each subject (e.g. 1 positive culture in 12 = 8·3%). Results are presented as the mean of individual percentages ± SEM per peptide.

Figure 2

Proliferative responses of the peptide-specific T cell lines of Can f 1 skinprick test-positive allergic (a) and non-allergic control (b) subjects upon stimulation with p33–48 and p107–122. Peripheral blood mononuclear cells were isolated from heparinized blood samples and stimulated with p33–48 or with p107–122. After the first stimulation cycle, specificity of the cultures was determined using the thymidine incorporation test. Lines with stimulation indices ≥ 2 were re-stimulated for further study. The reactivity of these T cell lines to the peptide used in the induction was examined at 0–50 µm. Results are presented as mean counts per minute ± SEM.

Figure 3

Peptide concentrations needed for inducing half-maximal proliferative responses (EC₅₀) in the T cell lines of Can f 1 skinprick test-positive allergic and healthy control subjects. Can f 1 peptide-specific T cell lines were stimulated at 0–50 µm with the peptide used in the induction of the lines. Functional T cell receptor (TCR) avidity of the lines was studied by determining EC₅₀ values from four-parameter dose–response curves using non-linear curve fitting., A high EC₅₀ value refers to low functional TCR avidity (i.e. a greater Ag concentration is needed to obtain the half maximal response) and vice versa. Horizontal lines represent medians and arrowheads indicate the T cell lines responsive to Can f 1 protein.

Figure 4

Proliferative responses of two p107–122-induced T cell lines upon stimulation with the Can f 1 peptide p107–122 and the corresponding tear lipocalin (TL) peptide. Can f 1 peptide-specific T cell lines were stimulated with the peptide used in the induction of the lines and with the corresponding human TL peptide at 0–50 µm. The functional T cell receptor (TCR) avidity of the lines was studied as for the data in Fig. 3. A high EC₅₀ value refers to low functional TCR avidity and vice versa. The results are representative of two independent experiments and indicated as mean counts per minute ± SEM.