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.