BACKGROUND: The SQ tree SLIT-tablet (containing birch extract) proved clinically significant effects during the pollen season for birch as well as alder/hazel. Immune outcomes of this treatment for allergens from multiple birch homologous trees needs further investigation. We hypothesize that birch pollen extract AIT modulate a highly cross-reactive immune response and that this may be the basis for the observed clinical cross-protection. METHODS: Blood samples were collected from 397 birch allergic patients during SQ tree SLIT-tablet or placebo treatment (1:1) for up to 40 weeks. Serum-IgE and IgG4 specific to birch, and birch homologous tree pollens from alder, hazel, hornbeam, beech, and chestnut were measured by ImmunoCAP. IgE-Blocking Factor (IgE-BF) for alder, birch, and hazel during treatment were measured by Advia Centaur and blocking effects for birch and all these birch homologous tree pollens were further investigated by basophil activation (BAT). Antibody readouts were investigated in patient subsets. T-cell responses (proliferation) to allergen extracts and peptide pools (group 1 allergens) were investigated in T-cell lines from 29 untreated birch pollen allergic individuals. RESULTS: Significant Pearson correlations between serum-IgE toward birch, alder, hazel, hornbeam, and beech were observed (r-values>0.86). T-cell reactivity was observed throughout the birch homologous group. Almost identical kinetics for changes in IgE towards birch, alder, and hazel were observed during treatment and similar species-specific changes were seen for serum-IgG4 . IgG4 reactivity toward birch and alder, hazel, hornbeam, and beech, correlated significantly at end-of-treatment (r-values>0.72). Treatment resulted in similar IgE-BF kinetics for alder, birch, and hazel and blocking of BAT for multiple trees in most actively treated patients investigated. CONCLUSIONS: Systematic analyses of T-cell and antibody cross-reactivities before and during birch pollen extract AIT provides the immunological basis for the observed clinical effect of SQ tree SLIT-tablet treatment of tree pollen allergy induced by multiple trees in the birch homologous group.