Immunotherapeutic potential of the immunodominant T-cell epitope of lipocalin allergen Bos d 2 and its analogues

Abstract

Lipocalin allergens, which contain most of the important animal-derived respiratory sensitizers, induce T helper type 2 (Th2) deviation, but the reasons for this are not clear. To explore the prospects for peptide-based allergen immunotherapy and to elucidate the characteristics of the immunodominant epitope of Bos d 2, BALB/c mice were immunized with a peptide containing the epitope, peptides containing its analogues, peptides from the corresponding regions of other lipocalin proteins, and peptides with a homologous sequence. We observed that murine spleen cells recognized the immunodominant epitope of Bos d 2, p127-142, in almost the same way as human Bos d 2-specific T cells did. Enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) analyses showed that p127-142 and a corresponding peptide from horse Equ c 1 induced a Th2-deviated cellular response, whereas a homologous bacterial peptide from Spiroplasma citri induced a Th0-type response. Interestingly, the spleen cell response to the bacterial peptide and p127-142 was cross-reactive, that is, able to induce reciprocally the proliferation and cytokine production of primed spleen cells in vitro. More importantly, the peptides were able to skew the phenotype of T cells primed with the other peptide. Our results suggest that modified peptides can be useful in allergen immunotherapy.

Figure 1

Proliferative responses of spleen cells from BALB/c mice primed with p127–142 of Bos d 2 upon stimulation with its alanine-substituted analogues. The results are presented as mean counts per minute (c.p.m.) (± standard error of the mean) for four independent experiments. The peptide concentrations were 15 µm (solid bars) and 1·5 µm (open bars). The medium background has been subtracted (< 1400 c.p.m. in all tests).

Figure 2

Proliferative responses of spleen cells from BALB/c mice primed with p127–142 upon stimulation with the truncated analogues of the peptide. The results are expressed as mean percentages (± standard error of the mean) of the response to p127–142 for three independent experiments. The peptide concentration was 15 µm. The proliferative response to p127–142 was > 2100 counts per minute (c.p.m.) and the background was < 885 c.p.m. in all tests.

Figure 3

Proliferative responses of purified spleen T cells (1 × 10⁵ cells/well) from BALB/c mice with I-A^d or I-E^d-transfected fibroblasts as feeder cells [LA(d) and LE(d), respectively; 0·5 × 10⁵ cells/well] upon stimulation with p127–142. The results are presented as mean counts per minute (c.p.m.) ± standard error of the mean for three independent experiments.

Figure 4

Proliferative responses of spleen cells from BALB/c mice primed with p127–142 or peptides obtained through database searches upon in vitro stimulation. The peptide concentration was 50 µm. The results are expressed as mean stimulation index (± standard error of the mean) for three independent experiments. The background proliferation in wells without a stimulant was < 950 counts per minute in all experiments.

Figure 5

Proliferative responses and cytokine production of spleen cells from BALB/c mice primed with p127–142, SP7 or SP12 upon in vitro stimulation. The cytokine production analysed using the enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) is expressed as mean spot number/10⁶ spleen cells [± standard error of the mean (SEM)] and the proliferation as mean counts per minute (c.p.m.) (± SEM) for at least four independent experiments. In each experiment, three to five mice per peptide group were primed. The spot numbers in the unstimulated cultures remained < 4 spots/10⁶ spleen cells in all experiments (data not shown).

Figure 6

Proliferative responses of spleen cells from three mouse strains (BALB/c, C57BL/6 and CBA) primed with p127–142 in Freund's adjuvant (CFA) or treated with phosphate-buffered saline (PBS) upon stimulation with p127–142 (or SP10). Results are presented as mean stimulation index (± standard error of the mean) for two independent experiments. The background levels were < 2900 counts per minute (c.p.m.) for BALB/c mice, < 1000 c.p.m. for C57BL/6 mice and < 3300 c.p.m. for CBA mice in all experiments.