CD45RBlo CD161+ TH2 promotes IL-5 production and disease progression
As cellular phenotype is crucial to functional characteristics, we further refined the phenotype of the CD161+ TH2 subset driving AR. Of particular interest is the activation status of this cellular subset. Interestingly, surface expression of marker CD45RB was significantly downregulated on CD161+ TH2 cells of AR individuals compared to non-AR individuals, indicative of memory and differentiation characteristics (Figure 3A ).
To assess the inflammatory profile of AR, we quantified IL-5, IL-13, IL-17A, IL-6 and TNFα plasma levels by ultra-sensitive SiMoA array. AR cases had significantly elevated plasma IL-5 levels as compared to non-AR cases, which confirmed Type 2 inflammation in AR (Figure 3B and Supplementary Figure 4 ). As the key survival factor of eosinophils, IL-5 plays a pivotal role in eosinophilic allergic diseases. [20-23] Given that TH2A and pe-TH2 populations were previously reported to be major producers of IL-5, [13, 24] we probed for IL-5 secretion in AR-relevant CD161+ TH2 subset. Interestingly, high circulatory plasma IL-5 levels were only observed in individuals with low CD45RB (CD45RBlo) expression on CD161+ TH2 cells (Figure 3C ), suggesting that CD45RBlo CD161+ TH2 could be responsible for IL-5 secretion. To clarify the cell subsets responsible for IL-5 secretion, PBMCs were stimulated with PMA and Ionomycin, and respective cytokine secretion was analysed using flow cytometry. As stimulation results in an upregulation of CD45RB, PBMCs were pre-stained with surface markers (including CD45RB) prior to activation so that steady state phenotype could be preserved. IL-5 expression was then quantified by intracellular staining following stimulation. While we note a small population of IL-5 secreting conventional CD161- TH2 (cTH2), IL-5 secretion was significantly elevated in CD161+ TH2 cells (Figure 3D and E ). Strikingly, IL-5 secretion was exclusively found within the CD45RBlo subset in both cTH2 and CD161+ TH2 (Figure 3E ). These findings confirm CD45RBloCD161+ TH2 as the main producers of IL-5.
Importantly, the proportion of IL-5 producing CD45RBloCD161+ TH2 cells was significantly elevated in AR as compared to non-AR (Figure 3F ). In contrast, there was no significant difference in the proportion of IL-5 producing CD45RBlo cTH2 between the non-AR and AR individuals. Taken together, these results suggest a role for IL-5 producing CD45RBlo CD161+ TH2 cells in the pathogenesis of AR.