Immune laboratory methods
Flow cytometry was used to analyze the CD4+ Th cell population in whole blood from the patients at randomization, and one and three years after completed ILIT. Peripheral blood mononuclear cells obtained from the patients at randomization and one year after ILIT were stimulatedin vitro with birch and timothy allergen. Levels of IL-5, IL-10, IL-13, IFN-γ, CCL17 and CXCL10 were quantified using Luminex. For detailed methods, experimental protocols, and statistical analyses, see the Methods section in this article’s supplementary information.
Statistics
Descriptive statistics for RQLQ, RTSS and MS are presented in medians and percentiles (p25 and p75), and in the graphs with medians and 95% confidence interval. Paired comparisons over time were calculated with Friedman’s test and adjusted with the Bonferroni correction for multiple comparisons. Descriptive statistics for IgE, IgG4, SPT and CAPT are presented in mean values and standard deviation (SD). Paired comparisons over time were calculated with repeated measures ANOVA with Bonferroni confidence interval adjustment. The answers to the 28 questions in RQLQ were explored with an item analysis, rendering a Cronbach’s Alpha at 0.933; thus the changes within the different domains of RQLQ were consistent. All analyses above were performed in SPSS version 25 (IBM Corp. Armonk, NY, USA).
All flow cytometry, cytokine and chemokine data were analyzed using GraphPad Prism, version 8.3.1 (GraphPad software, Inc., La Jolla, CA, USA), and non-parametric tests were used. Comparisons at the different time points within the treatment groups were calculated using paired Wilcoxon signed ranks test. Unpaired Mann Whitney U test was used to compare differences between the treatment groups at the different time points. The significance level was set at p < 0.05.