Naturally processed T cell–activating peptides of the major birch pollen allergen

Time kinetics of endolysosomal processing of Bet v 1. After 0.5 to 24 hours of incubation of Bet v 1 with endolysosomal proteases isolated from DCs, generated peptides were sequenced by mass spectrometry. Frequently recognized T-cell epitopes are framed in the amino acid sequence of Bet v 1, shown on top.

Optimizing the period for allergen pulsing of DCs. DCs from an individual with birch pollen allergy were incubated with Bet v 1 (5 μg/0.5 × 10⁶ cells) for 6, 11, 24, and 48 hours, respectively, and used to stimulate a Bet v 1–specific T-cell clone. T-cell proliferation (SI) is shown.

Naturally processed Bet v 1–derived peptides. Peptides eluted from purified HLA-DR:peptide complexes isolated from DCs of 4 patients with Bet v 1 allergy were sequenced by mass spectrometry. A, Amino acid sequence of Bet v 1. Frequently recognized T-cell epitopes are framed. B, HLA-DR–eluted peptides are shown in black; 12mer peptides inducing proliferative responses in Bet v 1–specific TCLs are shown in gray.

T cell–activating properties of naturally processed Bet v 1–derived peptides. Bet v 1–reactive TCCs were stimulated with 2.5 μmol/L of the epitope-containing 12mer peptide (black bars), an 18mer peptide representing the HLA-DR–eluted peptides (white bars), and Bet v 1 (gray bars). TCC218 was stimulated with FKYNYSVIEGGP and FKYNYSVIEGGPIGDTLE. TCC266, TCC334, TCC14VR, and TCC4R were stimulated with TLLRAVESYLLA and GETLLRAVESYLLAHSDA. A, Proliferative responses (SI) are shown. B, Cytokines (pg/mL) were measured by ELISA.

Structural determinants of the immunodominant T-cell epitope Bet v 1_142-153 (TLLRAVESYLLA). A, Zigzags in the aa sequence of Bet v 1 represent α-helices, arrows indicate β-sheets, and bars represent loops. Structure assignments were taken from Protein Data Bank file 1BV1. B, The loop (green) flanking the C-terminal α-helix (golden) containing Bet v 1_142-153 is shown in the 3-dimensional structure. C, Surface representation of the loop (green) flanking the C-terminal α-helix (golden) containing Bet v 1_142-153. Peptide C-N atoms in the loop region are yellow and blue. The arrow indicates the only solvent-accessible peptide bond. Molecular graphics in B and C were created with UCSF Chimera (PMID: 15264254).

Time kinetics of endolysosomal processing of Bet v 1. A, After 0.5 to 24 hours of incubation of Bet v 1 with endolysosomal proteases, 10 μL of the digestion reactions corresponding to 2.5 μg Bet v 1 were analyzed by SDS-PAGE and Coomassie staining. M, Marker (kd); B, Bet v 1 in digestion buffer without microsomal fractions. B, Grayscaled and inverted bands corresponding to intact Bet v 1 were quantified as integrated measures of intensity and size using the software Adobe Photoshop CS3. Protein degradation is shown as function of time.