Figure Legends
Figure 1. Eosinophilic allergic rhinitis (AR) in SSIC cohort is associated with high levels of HDM-specific IgE Whole blood leukocytes from SSIC cohort were stained and analysed with flow cytometry.(A) Representative scatterplot showing lymphocytes (green), monocytes (red) and granulocytes (blue) subsets in whole blood (n=216) and these subsets are analysed using unsupervised phenograph clustering and umap dimensionality reduction. (B) Umap clusters were annotated based on their lineage markers and (C) associated with atopy phenotype. P-value of the respective cluster was presented as blue/red shading based on statistical analysis. Red denotes direct correlation while blue denotes negative correlation. Scatter plots comparing (D) percentage of eosinophils in total leukocytes and(E) total plasma immunoglobulin E (IgE) between non-AR and AR individuals. Spearman correlation was performed in Figure 1Cwhile pairwise two-tailed Mann-Whitney U test was performed inFigure 1D-H . A p-value of less than 0.05 is considered statistically significant. (**p<0.01, ***p<0.001 )
Figure 2. CD161+ CD4+ TH2 cells are associated with AR. PBMCs of SSIC individuals were stained and analysed using flow cytometry.(A) CRTH2+ subsets from PBMCs (n=216) were gated and used for analysis by phenograph clustering and umap dimensionality reduction.(B) Umap clusters were annotated based on lineage markers and associated with (C) atopy phenotype. Statistical analysis was performed using Wilcoxon rank sum test. Umap clusters were shaded red according to statistical significance. (D) Representative scatterplot showing CD4 and CD161 expression on cluster K9 (derived from umap cluster in (C)) overlaid on total CRTH2+ PBMCs. Number in the plot refers to the representative percentage of cluster K9 of all CRTH2+ PBMCs. (E) Scatter plot comparing the numbers of peripheral CD161+ TH2 cells between non-AR and AR individuals. Two-tailed Mann-Whitney U test was performed. Association of peripheral CD161+ TH2 numbers with (F) whole blood eosinophil numbers and(G) total plasma IgE. Non-atopic individuals were shaded in grey while atopic individuals were shaded in black. Statistical analysis was performed using Spearman correlation. P-value of less than 0.05 is considered statistically significant. (**p<0.01, ***p<0.001 )
Figure 3. CD45RBloCD161+ TH2 subset produces high level of IL-5 and are associated with AR. PBMCs of SSIC individuals were stained and analysed using flow cytometry. (A)Scatterplots of CD45RB log mean fluorescence intensity (MFI) on CD161+ TH2 comparing between non-AR and AR individuals. (B)Scatterplot comparing plasma IL-5 level between non-AR and AR in SSIC individuals. Two-tailed Mann-Whitney U test was performed.(C) Scatterplot showing association of plasma IL-5 levels against CD45RB MFI on CD161+ TH2. PBMCs were pre-stained with CD45RB and stimulated using PMA/ionomycin. Intracellular staining was performed to determine IL-5 production using flow cytometry. (D) Paired analysis of IL-5 MFI on CD161- or CD161+ TH2 cells from non-AR or AR individuals. Wilcoxon paired analysis was performed. (E)Representative scatterplots showing expression of IL-5 and CD45RB in CD161- TH2 (cTH2) and CD161+ TH2 subsets. Numbers in quadrant represents percentage of cells in each quadrant of the plot. (F)Scatterplot showing association of CD45RBloIL-5+ cTH2 or CD161+ TH2 in non-AR or AR individuals. Statistical analysis was performed using one-way ANOVA followed by Tukey post test. P-values less than 0.05 are considered statistically significant. (**p<0.01, ***p<0.001 )
Figure 4. CD45RBloCD161+ TH2 subset is associated with active AR manifestation. Whole blood leukocytes from the pediatric cohort were stained and analysed with flow cytometry. Scatterplots comparing the percentage of (A) eosinophils and(B) CD161+ TH2 between non-AR and AR individuals. Statistical analysis was performed using two-tailed Mann-Whitney U test. PBMCs were pre-stained with cell surface markers and stimulated using PMA/ionomycin. Intracellular staining was performed to determine cytokine production using CyTOF. (C) Scatterplot showing association of CD45RBloIL-5+ cTH2 or CD161+ TH2 in non-AR or AR individuals. (D) Representative scatterplot showing gating of CD161+ TH2 from total CD4+ T cells to be used for umap cluster analysis in (E) . CD161+ TH2 cells were gated, downsampled to 200 cells per sample and analysed using unsupervised phenograph clustering with 38 markers. (E) Umap dimensionality reduction was performed and cells were segregated into different clusters. Numbers in the plot represent cluster numbers. (F)Scatterplot comparing percentage of cluster 3 (derived from(E) ) between non-AR and AR individuals. Statistical analysis was performed using two-tailed Mann-Whitney U test. P-value of less than 0.05 is considered statistically significant. (*p<0.05 ) (G) Umap scatterplot showing the distribution of cluster 3 (in red) in concatenated non-AR (n=6) and AR (n=14) samples respectively.
Figure 5. AR-associated CD45RBloCD161+ TH2 subset secreting diverse range allergy-related cytokines (A) Heatmap showing marker and cytokine expression on the different clusters derived from Figure 4E . (B) Scatterplots showing expression of hematopoietic prostaglandin D synthase (HPGDS), CD45RB, IL-5, IL-4 and IL-13 on cluster 3 overlaid on total CD161+ TH2.