CD45RBlo CD161+TH2 is the central pathogenic cell type in active disease
As allergic manifestation in the aforementioned SSIC cohort were based on self-reported historical symptoms and is susceptible to recall bias, we therefore validated our findings in a second paediatric cohort with clinically diagnosed active AR manifestations (Supplementary Table 2B ). As expected, there are significantly higher levels of eosinophils and CD161+ TH2 observed in AR cases, thus confirming eosinophilic inflammation in active AR (Figure 4A, B ). In order to obtain a wide comprehensive phenotypic outlook of the disease-driving CD161+ TH2 subset, an extended 40plex mass cytometry allergy panel was designed to include antibodies specific for inflammatory cytokines and activation markers such as IL-2, IL-3, IL-4, IL-5, IL-9, IL-13, IL-17A, IL-22, IFN-γ, TNF-α and HPGDS [12] (Supplementary Table 1 ). As the stimulation required to induce cytokine production triggers major shifts in the expression of surface markers, most of the lineage and surface markers (including CD45RB) were stained prior to stimulation, which enabled depiction of the actualex vivo cellular phenotype.
Mass cytometry of PBMCs with the expanded 40-marker panel revealed all key populations as defined previously with the narrower FACS panel but at much higher granularity. Consistent with observations in the SSIC cohort (Figure 3F ), a significantly higher proportion of IL-5 producing CD45RBlo CD161+ TH2 was found in individuals with active AR compared to non-AR controls (Figure 4C ). This trend was not observed in CD161- cTH2.
To confirm our earlier observations in the SSIC cohort, we gated for CRTH2+ CD161+ cells and performed supervised phonograph clustering. A total of 9 sub-populations were segregated from the CD161+ TH2 subset (Figure 4D and E ). Of all sub-populations, Cluster 3 was found to be significantly increased in individuals with active AR (Figure 4F, G and Supplementary Table 5 ).
In line with expectations, ‘Cluster 3’ is indeed an IL-5 secreting CD45RBlo population (Figure 5A ). Moreover, this cluster appeared to be a highly differentiated population of mature CD161+ TH2 cells with an activated phenotype characterized by low expression of CD27, CD45RB, KLRG1 and high expression of CD45RO, HPGDS, ICOS, and CD38. The cytokine profile suggests that in addition to IL-5 production, this population also secretes a multitude of cytokines that includes IL-2, IL-3, IL-4, IL-9 and IL-13. Taking a deeper look, we further confirmed that cytokine secretion is highly correlative (Figure 5B ). Thus, the severity of eosinophilic airway allergies such as AR seems to be driven by an activated terminally differentiated CD161+ TH2 subset that is able to concomitantly secrete a complex set of inflammatory cytokines.