DISCUSSION
There has been growing evidence of heterogeneity within the human TH2
subsets discovered over the years. [12,
13, 25]
Prussian and colleagues first reported high levels of IL-5+ producing
TH2 subset in patients with allergic eosinophilic gastroenteritis.
[12] This population was named
’peTH2’ and was defined by their expression of HPGDS and CD161.
[12] In parallel, the importance of
IL-5 producing TH2 subset was further confirmed in mouse models of
allergic airway inflammation and atopic dermatitis.
[26,
27] Wambre et al defined a
subpopulation of human allergen-specific TH2 subset which they coined as
TH2A. [13] TH2A was identified as a
memory subset that is over-represented in allergic individuals and
secretes IL-5, IL-4 and IL-13 in response to stimulation.
[13] As different cell markers were
used across different groups to identify the subsets, this raises the
question whether the identified TH2 populations pertinent for allergic
diseases across these studies were homogenous.
In this study, we used unsupervised clustering tools (UMAP and
PhenoGraph) on CRTH2+ PBMCs and identified a TH2A-like subset that
correlated directly to the markers of atopy. A common feature between
the different TH2 subsets reported in literature was the production of
IL-5. [13,
19, 27]
Similarly, we were also able to detect IL-5 in our identified CD161+ TH2
subset. Strikingly, IL-5 production was only limited to the
CD45RBlo T cell population. As
CD45RBlo T cells are typically terminally
differentiated memory cells, this suggests a need for repetitive antigen
stimulation before CD161+ TH2 subsets are capable of IL-5 secretion.
This is supported the studies by Upadhyaya et al. and Islam et al, who
found that the ability of TH2 cells to secrete IL-5 requires multiple
rounds of in vitro stimulation.
[25,
27] Similar requirements for repeated
allergen exposure is needed to polarize IL-5 producing TH2 cells.
Intriguingly, CD45RBloCD161+ TH2 population was found
to be directly correlated to AR in both cohorts with active and
self-reported AR. Not only does this show the persistence of the
CD45RBloCD161+ TH2 population, it also implies the
pertinence of this population in the pathogenesis of AR and possibly
other allergic diseases. Both SSIC and active AR cohorts described in
this study were collected in Singapore, whereby majority of the
individuals are sensitized against HDM.
[10] HDM is a perennial allergen in
tropical nations such as Singapore, thus T cells in atopic individuals
undergo constant stimulation. [11]
This could explain the strong association observed between CD45RB
expression on CD161+ TH2 cells and atopy markers despite the fact that
not all subjects demonstrated active AR symptoms during the collection
of SSIC cohort.
A high dimensional 40-marker panel was used to further characterize the
CD161+ TH2 population. As mentioned previously, pre-staining of cell
surface markers was performed prior to stimulation in order to preserve
the steady state cell identities. We identified a specific subset within
the CD161+ TH2 population that associated strongly with AR. Other than
exhibiting a terminally differentiated phenotype (defined by low
expression of CD27 and CD45RB with concomitant high expression of CD45RO
and ICOS) with intracellular expression of HPGDS, this subset was also
able to secrete multiple cytokines upon stimulation. Amongst several
others, this included allergy related cytokines such as IL-5, IL-4 and
IL-13. Taken together, our current study unifies the markers previously
reported for allergic-specific TH2 subsets and provides clarity for the
pathogenic TH2 subset previously reported in different allergic
diseases. The persistence of CD45RBlo CD161+ TH2 may
allow for public health surveillance of allergic individuals. Moreover,
these cells may also be leveraged as a biomarker for the effectiveness
of immunotherapy as well as a potential disease target in the treatment
of AR and allergic diseases.