IL-2- and IL-4-dependent signaling play separate and sequential roles in the differentiation of GATA3-hi TH2 cells in vivo

Naive CD4+ T cells differentiation into T-helper type 2 (TH2) cells requires Interleukin 2 (IL-2) and IL-4-dependent signaling as well as presentation of allergens by dendritic cells (DCs). The role of IL-2 and IL-4 in TH2 differentiation has been mostly studied in vitro, however, these models do not account for the heterogeneity of TH responses and bypass the DC-derived signals that are necessary in vivo. We used cytokine-blocking antibodies and IL-4RaKO/IL-4RaWT mixed bone marrow chimeras to show that IL-4 signaling was not required for initial upregulation of GATA3 by CD4+ T cells after intradermal immunization, but was necessary for later TH2 cell expansion and further GATA3 upregulation. Single-cell transcriptomics and computational analyses confirmed that IL-4 signaling was not necessary for TH2 identity but promoted TH2 proliferation and expression of a pathogenic signature. Early IL-2 blockade prevented GATA3 upregulation without affecting TH2 proliferation or the differentiation of TH1 and TFH subsets after immunization. Adoptive transfer experiments showed that reduced competition for IL-4 in vivo drove extensive T cell proliferation and preferential expansion of the GATA3-hi population. Overall, our results suggest temporally and functionally distinct roles of IL-2 and IL-4 during TH2 differentiation: IL-2 is necessary for GATA3 upregulation, while IL-4 drives subsequent TH2 proliferation and licensing to effector activity. Therefore, IL-2 and IL-4 act in a co-ordinated manner to respectively promote TH2 differentiation, expansion, and effector commitment in the LN.