RESUMO
BACKGROUND: Immunotherapy inhibits basophil histamine release, but the assay is cumbersome, and no one has studied the effects of immunotherapy withdrawal. OBJECTIVE: Intracellular fluorochrome-labeled diamine oxidase (DAO) was used as a novel functional readout of basophil histamine release after immunotherapy. Results were compared with conventional basophil surface expression of activation markers. METHODS: Subcutaneous immunotherapy (SCIT)-treated patients (n = 14), sublingual immunotherapy (SLIT)-treated patients (n = 12), participants who completed 3 years of treatment with grass pollen sublingual immunotherapy (the SLIT-TOL group; n = 6), patients with untreated seasonal allergic rhinitis (SAR; n = 24), and nonatopic control subjects (n = 12) were studied. Intracellularly labeled DAO(+) and surface expression of CD203c(bright), CD63(+), and CD107a(+) on chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes (CRTh2)-positive basophils were measured by means of flow cytometry. Serum IgG4 levels and serum inhibitory activity for IgE-allergen complex binding to B cells (IgE-FAB) and basophil histamine release were also determined. RESULTS: Proportions of allergen-stimulated DAO(+)CRTh2(+) basophils were higher in participants in the SCIT, SLIT, and SLIT-TOL groups (all P < .0001) compared with those in patients in the SAR group. Similarly, there were lower proportions of CRTh2(+) basophils expressing surface CD203c(bright) (all P < .001), CD63 (all P < .001), and CD107a (all P < .01). Rhinitis symptoms were lower in the SCIT, SLIT, and SLIT-TOL groups (P < .001) compared with those in the SAR group. Serum inhibitory activity for IgE-FAB and basophil histamine release were also significantly greater in all immunotherapy groups (P < .05) compared with the SAR group. CONCLUSION: These results support long-term clinical and immunologic tolerance during and after grass pollen immunotherapy. Intracellularly labeled DAO expression by basophils merits further investigation as a surrogate biomarker for monitoring efficacy and tolerance after immunotherapy.