Fig. 2SCIT provides therapeutic benefit. A Timeline for the long-term treatment group. BPE was injected subcutaneously on days 1, 8, and 15. On days 24–26, the mice were challenged with 0.1% birch pollen aerosol. After 1 day, AHR to methacholine was assessed. Mice were subcutaneously injected with 0.3 mg BPE adsorbed to 0.1 mg alum on days 32, 39, 46, 53, 60, 67, 74, and 81. On days 88–90, the mice were re-challenged for three consecutive days with 0.1% birch pollen extract aerosol, 30 min per day, as before. After 1 day, AHR to methacholine was assessed. After 24 h, the mice were euthanized for evaluation using the immunological parameters. The PBS treatment group: BPE was injected subcutaneously on days 1, 8, and 15. On days 24–26, the mice were challenged with 0.1% birch pollen aerosol. After 1 day, AHR to methacholine was assessed. Mice were subcutaneously injected with PBS adsorbed to 0.1 mg alum on days 32, 39, 46, 53, 60, 67, 74, and 81. On days 88–90, the mice were re-challenged for three consecutive days with 0.1% birch pollen extract aerosol, 30 min per day. After 1 day, AHR to methacholine was assessed. After 24 h, the mice were euthanized for evaluation using the immunological parameters. B Timeline for the short-term treatment group. After allergen sensitization, mice were subcutaneously injected with 0.3 mg birch pollen extract (BPE) without being adsorbed to alum on days 32, 34, 36, 38, 40, 42, 44, and 46. Then, the mice were challenged as described above. AHR to methacholine was evaluated on day 56. The next day, the mice were euthanized and evaluated. C Airway hyper-reactivity to methacholine is shown. The PBS treatment group and the long-term treatment group; the short-term treatment group and the long-term treatment group. D Cytokines in BALF are shown. E Birch pollen specific immunoglobulins (sIgE, sIgG1, sIgG2a) are measured by using ELISA. F Lung tissue eosinophilia and mucus production are shown. Lung specimens were stained with hematoxylin and eosin (H&E) and AB-PAS staining. Data are shown as means ± SD, ** represents the short-term treatment group and the long-term treatment group vs. the PBS treatment group, P < 0.01; * represents the short-term treatment group and the long-term treatment group vs. the PBS treatment group, P < 0.05.