A subset of T cells, termed Th2 cells, is required for the generation of allergic immune responses. Th2 cells express three signature cytokines, IL-4, IL-5, and IL-13, each of which makes a specific contribution to allergic pathology. IL-4 drives Th2 differentiation and plays a dominant role in antibody class switching to IgE, IL-5 is the major eosinophil active cytokine, and IL-13 drives eosinophil-rich tissue inflammation through its upregulation of adhesion molecules and chemokines. We hypothesized heterogeneity within the Th2 lineage would yield Th2 subpopulations with different cytokine expression, effector functions and roles in disease pathogenesis. We used polychromatic flow cytometry for IL-4, IL-5 and IL-13 to demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5+ (IL-5+, IL-4+, IL-13+) and majority IL-5- (IL-5-, IL-4+, IL-13+) Th2 population. The generalizability of these findings to all human Th2 responses was demonstrated by the presence of these subpopulations across a wide variety of experimental approaches, using multiple culture systems, antigens, assay systems, and subject populations. Sorted IL-5+ and IL-5- Th2 cells retained their respective IL-5 bias after one week of culture, demonstrating that this differential IL-5 expression is a hereditable and durable property of these Th2 subpopulations. The relationship between these subpopulations and Th2 differentiation was examined by performing serial rounds of differentiation under Th2 polarizing conditions. In vitro differentiation initially yielded IL-5- Th2 cells, but required multiple rounds of differentiation to generate IL-5+ Th2 cells. There was a consistent and markedly delayed acquisition of the IL-5+ relative to the IL-5- Th2 phenotype. Additionally, IL-5+ Th2 cells expressed phenotypic markers (PD-1+, CD27-) consistent with their being highly differentiated T cells. This suggests the possibility that Th2 dominant diseases characterized by chronic Ag exposure may preferentially drive IL-5+ Th2 cell differentiation and eosinophilic inflammation. The durability and delayed acquisition of the IL-5+phenotype in vitro suggests that epigenetic changes underlie the differences in IL-5 expression. IL-5+ Th2 cells expressed greater GATA-3, the Th2 lineage specific transcription factor, suggesting epigenetic control of IL5 gene expression. This was confirmed by performing chromatin immunoprecipitation on purified IL-5+ and IL-5- Th2 subpopulations which demonstrated greater GATA-3 and H3K4me3 binding to the IL5 promoter in IL-5+ relative to IL-5- Th2 cells. Conversely, H3K27me3 binding to the IL5 promoter was greater in IL-5- Th2 cells. In sum, these findings demonstrate an open chromatin configuration in the IL5 promoter specific to IL-5+ Th2 cells, which is consistent with the greater IL-5 expression by this subpopulation. In summary, these findings demonstrate that Th2 lineage heterogeneity is a generalizable and durable feature of human immune responses. 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. This project has been completed and will be terminated at the end of the current fiscal year (September 30th, 2012).

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