The E3 Ubiquitin Ligase Cbl-b Limits Nascent Th9 Differentiation

Introduction: Th9 cells are critical mediators of allergy and anti-cancer immunity. The E3 ubiquitin ligase Cbl-b modulates T cell activation via regulation of the T cell receptor (TCR) activation threshold as well as by inducing TGF-β sensitivity, which is a critical differentiation factor for Th9 differentiation. Even though some evidence shows that Cbl-b impairs Th9 differentiation by targeting IL-4 dependent STAT6 activation, a complete suppression of Th9 differentiation in the absence of both STAT6 and Cbl-b is not achieved, implying the involvement of additional mechanisms. In this study, we evaluate the role of Cbl-b in early stages of TGF-β dependent Th9 differentiation.

Methods: Th9 cells were generated from WT and cblb-deficient naïve CD4⁺ T cells. After maximum 3 days in presence of IL-4, TGF-β and anti-IFN-γ antibodies, differentiation was determined by the quantification of cytokines, mainly IL-9, and that of the two required transcription factors for Th9 differentiation, namely IRF4 and PU.1. Microarray assay revealed gene candidates that were further validated by mRNA and protein expression analysis. The functional role of Cbl-b was tested in a Th9-mediated murine lung allergy model, in which mice were challenged by intratracheal injections of house dust mite (HDM) extracts.

Results:cblb-deficient naïve T cells more efficiently differentiate into Th9 cells after 3 days in culture, express in parallel PU.1 more intensively compared to WT Th9 cells, while retaining similar expression levels of IRF4, another important Th9 differentiation factor. Increased IL-9 level is not based on cblb -deficient T cell hyperproliferation, as we show an increased IL-9 production per cell by using combination of CFSE with intracellular IL-9 staining. Microarray analysis revealed that RUNX1, a known transcriptional modulator of PU.1, is more rapidly down-regulated in cblb-deficient Th9 cells compared to WT Th9 cells. Accordingly, knocking down RUNX1 by siRNA in naïve CD4⁺ T cells and subsequently differentiating them into Th9 cells, also induces higher IL-9 expression at the mRNA and protein levels in RUNX1-depleted Th9 cells compared to control scrambled siRNA-nucleofected Th9 cells. In the HDM murine allergy model, cblb-deficient mice have a higher lung inflammation as mirrored by increased eosinophils in the BAL and in the lungs, as well as by increased IgE production in the blood. These are also paralleled by an increased IL-9 expression level in the lungs of the allergic cblb -deficient mice.

Conclusions: Cbl-b critically limits Th9 differentiation and may thus be a potential target to modify Th9 cell generation in allergy or cancer. Future studies will validate the molecular link that exists between Cbl-b and the RUNX1-dependent IL-9 expression as well as the in vivo significance of increased Th9 cell differentiation in cblb-deficient animal models of lung inflammation and cancer.