Inborn errors of immunity reveal molecular requirements for generation and maintenance of human CD4⁺ IL-9–expressing cells

Background: CD4⁺ T cells play essential roles in adaptive immunity. Distinct CD4⁺ T-cell subsets—T_H1, T_H2, T_H17, T_H22, T follicular helper, and regulatory T cells—have been identified, and their contributions to host defense and immune regulation are increasingly well defined. IL-9–producing T_H9 cells were first described in 2008 and appear to play both protective and pathogenic roles in human immunity. However, key requirements for generating human T_H9 cells remain incompletely defined. Objective: We sought to define signaling pathways that regulate IL-9 production by human CD4⁺ T cells. Methods: Human naive and memory CD4⁺ T cells were cultured under different conditions, and the molecular mechanisms regulating IL-9 induction were determined by assessing the ability of CD4⁺ T cells from a broad range of patients (n = 92) with pathogenic variants in key immune genes (n = 21) to differentiate into IL-9⁺ cells. Results: We identified 2 culture conditions that yielded IL-9–expressing cells from naive CD4⁺ T cells and amplified IL-9 production by in vivo–generated memory CD4⁺ T cells: TGF-β plus IL-4 (ie, T_H9 polarizing condition), and the combination of IL-21, IL-23, IL-6, IL-1β, and TGF-β (ie, T_H17 polarizing condition). Combining these conditions had a synergistic effect in generating IL-9⁺CD4⁺ T cells. IL-9 induction required STAT3-activating cytokines as well as intact signaling via the T-cell receptor and STAT5. Importantly, IL-9 induction was restrained by IFN-γ/STAT1 and IL-10. Conclusions: Our findings revealed critical molecules involved in inducing/restraining IL-9 production by human CD4⁺ T cells, thereby identifying pathways that could be targeted to modulate IL-9 in health and disease.