CD161+ TH2 plays a role in driving eosinophilic inflammation in AR
In order to identify other atopy and AR-related subsets within lymphoid and myeloid populations, a flow cytometry panel was specifically designed for the detection of allergy-related PBMC subsets. [13] In contrast to whole blood analysis, PBMC clusters demonstrated non-significant associations with atopy and AR (Supplementary Figure 2 ). Given that TH2 was earlier identified as a disease-driving subset by transcriptomic sequencing, we gated for chemoattractant receptor homologue on TH2 (CRTH2+) positive cells to further enrich for key PBMC subsets with roles in disease progression of AR. CRTH2 is expressed by a small fraction (approximately 1.6%) of the PBMC population (Figure 2A ) and is typically expressed on TH2-associated immune cells such as TH2 and ILC2. [19] In line with transcriptomic analysis, PTGDR2 (gene encoding CRTH2) was also differentially expressed between non-AR and AR individuals (Table 1 ).
UMAP analysis segregated the entire CRTH2+ population into five distinct clusters representing basophils, TH2, ILC2, TC2 and non-classical monocytes (Figure 2B ). PhenoGraph clustering subdivided these five main clusters into 28 distinct subpopulations (K1-K28;Supplementary Figure 3 ). While all CRTH2+ subsets are reportedly involved in allergy and allergic diseases, cluster ‘K9’ is highlighted here as the sole subset directly associated with atopy (Figure 2C ). Cluster ‘K9’ (CD161+ TH2) is a TH2A-like subset [13] located within the TH2 cluster (Figure 2D and Supplementary Figure 3 ). Next, we interrogated CD161+ TH2 for associations with AR. Similar to eosinophils, frequency of CD161+ TH2 is significantly associated with both atopy and AR (Supplementary Figure 1B and Figure 2E ). Furthermore, we observed a positive correlation between the frequencies of CD161+ TH2 and eosinophils (Figure 2F ). Likewise, frequency of CD161+ TH2 is also positively correlated to titres of total IgE (Figure 2G ). Taken together, CD161+ TH2 cells may represent a key cellular subset driving atopy and airway inflammation in AR.