SUMMARY Interleukin 9 (IL-9) is a pleiotropic cytokine that can regulate autoimmune responses by enhancing regulatory CD4+FoxP3+ T regulatory (Treg) cell survival and T helper 17 (Th17) cell proliferation. Here, we analyzed the costimulatory requirements for the induction of Th9 cells, and demonstrated that Notch pathway cooperated with TGF-β signaling to induce IL-9. Conditional ablation of Notch1 and Notch2 receptors inhibited the development of Th9 cells. Notch1 intracellular domain (NICD1) recruited Smad3, downstream of TGF-β cytokine signaling, and together with recombining binding protein (RBP)-Jκ bound the Il9 promoter and induced its transactivation. In experimental autoimmune encephalomyelitis (EAE), Jagged2 ligation regulated clinical disease in an IL-9-dependent fashion. Signaling through Jagged2 expanded Treg cells and suppressed EAE when administered before antigen immunization, but worsened EAE when administered concurrently with immunization by favoring Th17 cell expansion. We propose that Notch and Smad3 cooperate to induce IL-9 and participate in regulating the immune response.

The transcriptional repressor B-cell lymphoma 6 (BCL6) is required for the development of T helper (Th) follicular cells and it has been shown to suppress Th2 cell differentiation. We demonstrate that BCL6 is a key regulator of Th9 cell development. BCL6 expression is transiently downregulated in polarized Th9 cells and forced expression of BCL6 in Th9 cells impairs Th9 cell differentiation. In contrast, BCL6 knockdown up-regulated interleukin (IL)-9 production in Th9 cells. The function of BCL6 in Th9 cells is under the control of IL-2/Janus kinase 3 (JAK3)/signal transducer and activator of transcription 5 (STAT5) signaling pathway. Using chromatin immunoprecipitation (ChIP), we show that in Th9 cells, BCL6 and STAT5 bind to adjacent motifs in the Il9 promoter. Furthermore, we found that STAT5 binding was associated with the abundance of a permissive histone mark at the Il9 promoter, while under conditions where BCL6 binding was predominant a repressive histone mark was prevalent. The effects of STAT5 and BCL6 on IL-9 transcription were further demonstrated using an IL-9-luciferase reporter assay where BCL6 repressed STAT5-mediated Il9 transactivation. In experimental autoimmune encephalomyelitis (EAE), forced expression of BCL6 in myelin oligodendrocyte glycoprotein (MOG)35–55-specific Th9 cells resulted in decreased IL-9 production and induction of IFNγ causing an exacerbation of the clinical disease. Our findings demonstrate a novel role of BCL6 in the regulation of Th9 cell development and their encephalitogenicity.

While activated inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs) are potent T cell suppressors, non-activated IMCs and IDCs promote T cell activation and T helper (Th) 1/Th17 cell differentiation. In this study, we investigated how to reduce the pro-inflammatory properties of IMCs and IDCs, and convert them into immune regulatory DCs. We found that interleukin (IL)-4 and retinoic acid (RA) treatment of GM-CSF-differentiated IDCs synergistically induced the expression of aldehyde dehydrogenase family1 subfamily A2 (Aldh1a2), a rate-limiting enzyme for RA synthesis in DCs. IL-4+RA-treated IDCs upregulated CD103 expression and markedly reduced the production of pro-inflammatory cytokines upon activation. IL-4+RA-treated IDCs strongly induced CD4+Foxp3+ regulatory T cell (Treg) differentiation and suppressed Th1 and Th17 differentiation. Mechanistically, the transcription factors Stat6 and RARβ play important roles in Aldh1a2 induction. In addition, IL-4 and RA signaling pathways interact closely to enhance the regulatory function of treated DCs. Adoptive transfer of IL-4+RA-treated DCs significantly increased Treg frequency in vivo. Direct treatment with IL-4 and RA also markedly suppressed actively induced EAE. Our data demonstrate the synergistic effect of IL-4 and RA in inducing a regulatory phenotype in IDCs, providing a potential treatment strategy for autoimmune diseases.