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.