We report two human Th2 subpopulations, both expressing IL-4 and IL-13, but differing in IL-5 expression: a minority IL-5+ Th2 (IL-4+, IL-5+, IL-13+) and the majority IL-5− Th2 (IL-4+, IL-5−, IL-13+) subpopulation. The generalizability of this finding is underscored by its ubiquity across the wide range of experimental systems and disease entities used in the study. IL-5 expression was restricted to a small minority of Th2 cells in both non-allergic controls and Th2 skewed eosinophilic subjects. These data are most consistent with a hierarchical model of Th2differentiation and gene expressioncharacterized by sequential transcriptional regulation in which IL5 gene activation represents the most highly differentiated stage. IL-5+ Th2 cells demonstrated greater chromatin accessibility at the IL5p and greater overall GATA3 expression, relative to IL-5− Th2 cells, suggesting that hierarchical differentiation is due to sequential epigenetic changes. Our findings demonstrate that IL-5+ Th2 cells are highly differentiated Th2 cells, whose differentiation requires multiple rounds of antigenic stimulus. These results suggest a possible mechanism whereby Th2 dominant disease states characterized by recurrent Ag exposure would thus favor the generation of IL-5+ Th2 cells, which may preferentially drive eosinophilic inflammation.
We have previously reported the disease association of IL-5+ Th2 cells with allergic EGID (15
). However, this current report provides more thorough characterization of these cells as being true subpopulations. The generalizability of these findings to all human Th2 responses is underscored by the presence of these subpopulations across a wide variety of experimental approaches, including multiple culture systems (6h ex vivo
, Th2 lines, in vitro
differentiated Th2 cells), Ags (polyclonal activators, allergens), assay systems (ICCS, cytokine secretion staining, PCR), and subject populations (allergic asthma, EGID, non-atopic controls) (). The hereditability and durability of these Th2 subpopulationsin vitro
, their different phenotypic and epigenetic signatures, and their dissynchronous appearance in Th2 differentiation cultures, all support the thesis that these are distinct entities. These data, together with established T cell biology (2
), suggest that these subpopulation differences are intrinsic to the cells, rather than due to heterogeneity of activation conditions resulting in different cytokines being expressed.
Notably, evidence for IL-5 expression restricted to a minority Th2 subpopulation can be gleaned from previous human (18
) and murine (33
) studies, further supporting the generalizability of these findings. Such Th2 heterogeneity is further supported by the finding that IFN-γ blockade enhances in vitro
Th2 differentiation, resulting in a greater fraction of IL-5+ Th2 cells (33
). One reason that these Th2 subpopulations have not been appreciated is that the 1-2 rounds of in vitro
Th2 differentiation used in most investigations are insufficient to generate large numbers of IL-5+ Th2 cells (36
). Conversely, the relative ease of detection of human IL-5+ Th2 cells ex vivo
() may reflect the chronicity of human Ag exposure that is more likely to yield highly differentiated Th2 cells.
These subpopulations could represent two dichotomous lineages (4
), polyfunctional Th2 cells (22
), or a continuum of hierarchical Th2 differentiation states with IL-5+ Th2 being the most highly differentiated Th2 cells. Multiple findings from this work support a hierarchical model for the expression and differentiation of IL-5+ Th2 cells. First, IL-5+ Th2 cells were almost exclusively IL-4+, IL-5+, IL-13+ triple positive cells within the larger Th2 population (-, Supplemental Fig. 2
). Second, this hierarchy is apparent in the greater per cell IL-4 and IL-13 expression as Th2 cells progressed from single positive to double positive to triple positive (; ). Third, both phenotyping and in vitro
differentiation studies indicate that IL-5+ Th2 cells are more highly differentiated ( and ). Lastly, IL-5+ Th2 cells demonstrate epigenetic changes and greater GATA3 expression, consistent with greater chromatin accessibility at the Th2 locus (). A dichotomous lineage model would generate clones that were either wholly IL-5+ or IL-5−, whereas sorted Th2 clones displayed a range of IL-5 expression, indicating a continuum of differentiation states(). The polyfunctional phenotype (37
) is conferred by greater expression of effector genes in any combination, whereas our findings clearly indicate a hierarchy of Th2 cytokine expression with IL-5 at the extreme. Intriguingly, IL-17A and IL-17F are coexpressed in a similar pattern to IL-5 and IL-4/13,(38
) suggesting that comparable hierarchical expression may occur within the Th17 lineage.
The findings in this current work also support a probabilistic model of cytokine expression, in which the chromatin configuration and transcription factor binding to a given allele confer a probability of that gene being expressed (39
). The finding that clones derived from the IL-4+, IL-5− sort exhibit a wide range of IL-5 expression () are most consistent with IL-5 being expressed in a probabilistic manner. In this model, a single cell whose IL5
loci have an intermediate probability of expression clonally expands into a population with intermediate IL-5 expression. In contrast, the high coexpression of IL-4 and IL-13, most dramatically exhibited in the Th2 cytokine secretion sorted cells ( and ), suggest probabilities of IL-4 and IL-13 gene expression approaching 100%.
Th2 differentiation cultures initially yielded predominantly IL-5− Th2 cells, but required multiple additional rounds of differentiation to generate IL-5+ Th2 cells ().This suggests thepossibilitythat Th2 dominant diseases characterized by chronic Ag exposure may preferentially drive IL-5+ Th2 cell differentiation and eosinophilic inflammation. This mechanism is further supported by our previous finding of IL-5+ and IL-5− dominant food Ag specific Th2 responses in EGID and peanut anaphylaxis, respectively (15
). Because EGID patients generally do not have anaphylaxis to foods, they may ingest greater amounts of allergenic foods, presumably driving the differentiation of IL-5+ Th2 cells. In contrast, peanut allergic patients avoid peanuts, likely leading to less Ag exposure, resulting in their having less differentiated IL-5− Th2 cells. Recent sporadic cases of EGID in peanut and milk anaphylactic patients undergoing oral immunotherapy may possibly reflect this enhanced differentiation of IL-5+ Th2 cells upon repeated food Ag exposure (41
). Similarly, the association of asthma with perennial allergen sensitization (e.g. house dust mites, cockroach) may reflect the generation of IL-5+ Th2 cells upon recurrent allergen inhalation (45
Although these results do not directly address Th2 plasticity, our finding that ≥90% of IL-4+ sorted cells retain the Th2 phenotype after 1-3 weeks in culture provides evidence of both the durability of the Th2 phenotype and the relative rarity of IL-4+, IFN-γ+ Th0-like cells in vivo
. Less differentiated or “early” Th2 cells are more plastic to Th1 differentiation than highly differentiated Th2 cells (5
). If IL-5+ Th2 cells are more highly differentiated, then one would expect them to be less plastic than IL-5− Th2 cells to cross-differentiation towards Th1, Th17 or Th9.
The molecular mechanisms underlying the hierarchical expression of IL-5 by Th2 cells are of particular interest. Chromatin accessibility at the IL5p is differentially regulated relative to either IL4 or IL13 (). These data are most consistent with a model of Th2differentiationcharacterized by an orderedsequence of transcriptional regulation in which increased chromatin accessibilityat theIL5p
is the last step.Our findings of greater overall GATA-3 expression in IL-5+ Th2 cells suggest that this difference may be due to IL5 gene expression requiring greater GATA-3 concentrations than that required for Th2 differentiation. Notably, IL-13, which is also highly GATA-3 dependent, was expressed at greater levels in IL-5+ vs. IL-5− Th2 cells (, , ). Previous reports demonstrating a GATA-3 gene dose effect in GATA3 heterozygotes indicate that a 2-fold difference in GATA-3 concentration is sufficient to affect Th2 differentiation and cytokine expression (46
). Similarly, a recent report demonstrating epigenetic changes at the GATA3 promoter in more highly differentiated Th2 cells further supports a critical role for GATA-3 (48
). Alternatively, hierarchical IL5 expression could be due to a limiting transcription factor other than GATA-3 that is uniquely required for IL5 gene activation, or to the IL5p itself being more resistant to chromatin remodeling. The clinical relevance of these epigenetic changes is further underscored by these studies having been performed in house dust mite specific T cell lines from allergic asthmatic subjects ().
This epigenetic model of IL5
expression in the human system is further supported by previous reports in murine Th2 cells demonstrating a closed configuration at the Il5p
relative to the Il4p
, as evidenced by greater DNA methylation (49
) and less H3K4me3 binding (50
) at the Il5p
. These prior studies used 1-2 round in vitro
differentiated, presumably “early” IL-5− Th2 cells, and thus would be expected to similarly demonstrate a relatively closed chromatin configuration at the Il5p
Luster and colleagues recently reported a subpopulation of CCL8 responsive CCR8+ Th2 cells with enhanced IL-5 expression (51
), which may represent the same IL-5+ Th2 cells characterized in this current work. In the previous report, the generation of CCL8 responsive murine Th2 cells required multiple rounds of in vitro
differentiation, analogous to our findings in . Notably, we found IL-5 expression was almost entirely limited to IL-4+, IL-5+, IL-13+ triple positive Th2 cells; whereas in the previous work, CCL8 responsive Th2 cells were largely characterized as IL-4−, IL-5+. This discrepancy may alternatively reflect differences in Th2 differentiation methods, human vs. mouse Th2 gene regulation, or Th2 ICCS methods.
This work has not specifically addressed the respective function of these Th2 subpopulations or their contribution to eosinophilic inflammation. Presumably, through their greater expression of IL-5, IL-5+ Th2 cells may have a pro-eosinophilic inflammatory function (13
). The disease association of IL-5+ Th2 cells with EGID provides additional evidence in that direction (15
). Notably, IL-5+ Th2 cells expressed greater per cell quantities of IL-4 and IL-13 than did IL-5− Th2 cells (, and ), and as such may have greater overall Th2 function. Notably, this study was limited to the three classic Th2 cytokines. It is possible that IL-5+ Th2 cells differentially express other genes in addition to IL-5, for example CCR8 (51
), that confer additional functional properties to this subpopulation.
In summary, these findings demonstrate a fundamental and generalizable heterogeneity in the Th2 lineage in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. These findings establish that IL-5+ and IL-5− Th2 cells respectively represent more and less highly differentiated Th2 cell subpopulations, with each having distinct phenotypic and epigenetic features. A potential consequence of this heterogeneity is that specific Th2 subpopulations may differentially contribute to Th2 driven immunopathology, and as such may represent distinct therapeutic targets.