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.