Fig 1.
House dust mite proteins are highly immunogenic independent of allergy.
A) Heatmap of IgG levels against 40 house dust mite (HDM) proteins plus negative controls (BSA+ rHSA) in allergic donors (n = 21) and non-allergic donors (n = 16). Log 10 transformed fluorescence intensity measured in triplicates in a micro array immunofluorescent assay. B) Log 10 transformed mean fluorescent intensity (MFI) IgG levels for the individual HDM proteins are shown for allergic donors in red and for non-allergic donors in blue. C) Heatmap of IgA levels against the HDM proteins in allergic and non-allergic donors. D) Mean IgA levels towards the individual proteins. E) Heatmap of IFNγ induction by the HDM proteins in allergic and non-allergic donors. IFNγ from donor PBMCs stimulated for 5 days with individual HDM proteins quantified in a Uplex assay. F) Mean IFNγ response to each HDM protein for allergic and non-allergic donors. G) Heatmap of IL-17 induction by the HDM proteins in allergic and non-allergic donors measured with in a Uplex assay. H) Mean IL-17 response to each HDM protein for allergic and non-allergic donors. Error bars show the standard deviation of the log transformed values. Statistical significance was evaluated with Kruskal Wallis test, and Dunn’s post hoc test with Bonferroni correction and p-values >0.05 are indicated by *. Grey tiles denote missing data.
Fig 2.
Overlapping and restricted specificity of IgE and IG4 in HDM allergy.
A) Heatmap of IgE levels against 40 house dust mite (HDM) proteins in allergic donors (n = 21) and non-allergic donors (n = 16, n = 14 for the BAT assay) measured in triplicates in an immunofluorescent micro array-based assay. B) Log 10 transformed mean fluorescent intensity (MFI) IgE levels for the individual HDM proteins are shown for allergic donors (red) and for non-allergic donors (blue). C) Heatmap of IgG4 levels against the HDM proteins in allergic and non-allergic donors. D) Mean IgG4 levels towards the individual proteins. Error bars denote the standard deviation of the log transformed values. Statistical significance was evaluated with Kruskal Wallis test, and Dunn’s post hoc test with Bonferroni correction and p-values >0.05 are indicated by *. E) Heatmap of Basophil activation test (BAT) measured as percent of basophil cells with positive surface levels of CD63 in flow cytometry after stimulation with HDM proteins. Missing data is grey. F) Mean Basophil activation for each HDM protein. G) Concordance between specific IgE and IgG4 to HDM proteins for allergic donors (red) and for non-allergic donors (blue). H) Concordance between IgE and basophil activation. Individual log normalized IgE measurements and BAT percentages are shown for allergic donors as red dots. The lower limit of quantification marked by dashed lines is defined as the BSA value plus 10 standard deviations.
Fig 3.
TH2 responses are associated with allergy and characterized by broad specificity beyond known allergens.
A) Heatmap of IL-5 induction of 40 house dust mite (HDM) proteins in PBMCs from allergic donors (n = 19), and non-allergic donors (n = 13). IL-5 secreted from donor PBMCs stimulated for 5 days with individual HDM proteins was quantified in a Uplex assay. B) Mean IL-5 response to HDM proteins for allergic (red) and non-allergic donors (blue). C) Heatmap of IL-9 induction by the HDM proteins in allergic and non-allergic donors measured with a Uplex assay. D) Mean IL-9 response to HDM proteins for allergic and non-allergic donors. E) Heatmap of IL-10 induction by the HDM proteins in allergic and non-allergic donors measured with a Uplex assay. F) Mean IL-10 response to each HDM protein for allergic and non-allergic donors. G) Heatmap of IL-13 induction by the HDM proteins in allergic and non-allergic donors measured with a Uplex assay. H) Mean IL-13 response to each HDM protein for allergic and non-allergic donors. Error bars show the standard deviation of the log transformed values. Statistical significance was evaluated with Kruskal Wallis test, and Dunn’s post hoc test with Bonferroni correction and p-values >0.05 are indicated by *. Grey tiles denote missing data.
Fig 4.
Limited correlations in strength and frequency between antibody and T-cell responses.
Correlation matrix of Kendall’s tau-b correlations for A) Allergic donors and known house dust mite (HDM) allergens. B) Allergic donors and novel HDM proteins. C) Non-allergic donors and known allergens. D) non-allergic donors and novel HDM proteins. The dendrograms show the hierarchical clustering of the correlations.
Fig 5.
Overlap in specificity between IgE and IgG4 maintained by sublingual AIT.
A) Heatmap of levels of specific IgE against the panel of house dust mite (HDM) proteins (n = 40) for 38 HDM allergic individuals at baseline and after 9–12 months of SQ HDM SLIT-Tablet immunotherapy (SLIT). B) Specific IgG4 levels against the 40 HDM proteins. Antibodies were detected with isotype specific fluorescently tagged detection antibodies, values were log 10 transformed.
Fig 6.
IgE and IgG4 relationship in clinical study samples.
A) Sum of specific IgE against single HDM proteins (micro array), and Der p extract-specific IgE (ImmunoCAP), per sample. Shape indicates treatment group. Trendline indicates linear model, 95% confidence interval shown in grey. Dashed lines indicate 44 times the mean signal to BSA corresponding to the sum of background level for the micro array, and 0.1 kU/L lower limit of quantification for ImmunoCAP measurements. B) Sum of specific IgG4 against single HDM proteins (micro array), and Der p extract-specific IgG4 (ImmunoCAP), per sample. C) Specific IgE and IgG4 levels measured towards Der p extract (ImmunoCAP) and the sum of HDM protein reactivity (micro array) of active and placebo treated individuals at baseline (blue) and at follow-up (red). Boxes show median, 25th and 75th percentile levels, whiskers show highest or lowest value within 1.5 times the inter quartile range. D) IgE and IgG4 levels for all individual proteins for active SQ HDM SLIT and placebo treated individuals at baseline and at follow-up. Dashed lines mark the lower limit of quantification defined as the mean value of measured antibody against BSA plus 10 standard deviations. E) Mean percentage of measurements above the limit of quantification for each quadrant in D), error bars show the standard error of the mean (SEM).