DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4+ T-Cell Population Structure
Figure 1
The DNA methylation profile of allergen-challenged CD4+ T-cells separates SAR patients from healthy controls.
(A) Allergen-challenge assay. Peripheral blood mononuclear cells were isolated from healthy individuals and SAR patients and challenged in vitro with either allergen (pollen) or diluent (PBS). One-week post-challenge total CD4+ T-cells were isolated by MACS negative cell sorting. Genomic DNA and total RNA were isolated from the purified cells and cDNA or bisulfite-converted DNA was applied to gene expression or DNA methylation arrays, respectively. (B) Unsupervised hierarchical clustering of gene expression data of CD4+ T-cells isolated after allergen-challenge of PBMCs from SAR patients (N = 21) and healthy control subjects (N = 21) collected outside the pollen season (left panel).. Sample annotation is illustrated by colored boxes below the dendrogram. Principle components analysis of the same gene expression data fails to cluster data by disease status (right panel). (C) Unsupervised hierarchical clustering of quantitative genome-wide DNA methylation data of CD4+ T-cell DNA isolated after allergen-challenge of PBMCs from SAR patients (N = 12) and healthy control subjects (N = 12) collected outside the pollen season (left panel). Repeated bootstrap resampling of the data to calculate P-values for each cluster revealed that the two main clusters (H & P) were significantly supported by the data (P<0.05). Principle components analysis of the same DNA methylation data also revealed clear separation by disease state along the main principle component (right panel).