STAT5 Represses a STAT3-Independent Th17-like Program during Th9 Cell Differentiation

Abstract

IL-9–producing Th cells, termed Th9 cells, contribute to immunity against parasites and cancers but have detrimental roles in allergic disease and colitis. Th9 cells differentiate in response to IL-4 and TGF-β, but these signals are insufficient to drive Th9 differentiation in the absence of IL-2. IL-2–induced STAT5 activation is required for chromatin accessibility within Il9 enhancer and promoter regions and directly transactivates the Il9 locus. STAT5 also suppresses gene expression during Th9 cell development, but these roles are less well defined. In this study, we demonstrate that human allergy-associated Th9 cells exhibited a signature of STAT5-mediated gene repression that is associated with the silencing of a Th17-like transcriptional signature. In murine Th9 cell differentiation, blockade of IL-2/STAT5 signaling induced the expression of IL-17 and the Th17-associated transcription factor Rorγt. However, IL-2–deprived Th9 cells did not exhibit a significant Th17- or STAT3-associated transcriptional signature. Consistent with these observations, differentiation of IL-17–producing cells under these conditions was STAT3-independent but did require Rorγt and BATF. Furthermore, ectopic expression of Rorγt and BATF partially rescued IL-17 production in STAT3-deficient Th17 cells, highlighting the importance of these factors in this process. Although STAT3 was not required for the differentiation of IL-17–producing cells under IL-2–deprived Th9 conditions, their prolonged survival was STAT3-dependent, potentially explaining why STAT3-independent IL-17 production is not commonly observed in vivo. Together, our data suggest that IL-2/STAT5 signaling plays an important role in controlling the balance of a Th9 versus a Th17-like differentiation program in vitro and in allergic disease.

IL-1β promotes IL-9-producing Th cell differentiation in IL-2-limiting conditions through the inhibition of BCL6

“…IL-2/STAT5 signaling also contributes to Th9 cell differentiation through transcriptional repression where STAT5 represses expression of BCL6 that competes for STAT5 binding at the Il9 locus and limits Th9 differentiation ( 14 , 15 ). Further, we demonstrated that STAT5 suppresses a Th17-like phenotype in differentiating Th9 cells ( 16 ). Despite the importance of IL-2 in the differentiation process, we and others have noted that IL-9-producing Th cells often develop and act to induce protective or auto-immunity in IL-2-limiting environments.…”

“…The cells were cultured at 37°C for 4 days in plates coated with anti-CD3-(2μg/mL, 2C11, Bio X cell), with soluble aCD28 (4μg/mL, 37.51, Bio X cell), and the Th9-polarizing conditions: anti-IFNγ(10μg/mL), IL-4(20ng/mL), TGFβ(4ng/mL), anti-IL-10R(10μg/mL). IL-2 was neutralized using anti-IL-2 and anti-CD25 antibodies as previously published ( 16 ). In some conditions, we supplemented the cultures with NF-kB activators IL-1β (20ng/mL), IL-33 (20ng/mL), PAM3CSK4 (1μg/mL), DTA1 (5μg/mL) and OX40L (5μg/mL), and we used the NF-kB inhibitors QNZ (2nM, Cayman Chemical) and BAY 11-7082 (0.5μM, Cayman Chemical) with the respective dilution of DMSO as vehicle control.…”

“…In some conditions, we supplemented the cultures with NF-kB activators IL-1β (20ng/mL), IL-33 (20ng/mL), PAM3CSK4 (1μg/mL), DTA1 (5μg/mL) and OX40L (5μg/mL), and we used the NF-kB inhibitors QNZ (2nM, Cayman Chemical) and BAY 11-7082 (0.5μM, Cayman Chemical) with the respective dilution of DMSO as vehicle control. After 4 days in culture, we assessed cell number and cell viability in each well using trypan blue cell counting on a hemocytometer and the cytokine and TF expression through flow cytometry as previously described ( 16 ).…”

Perspectives of herbs and their natural compounds, and herb formulas on treating diverse diseases through regulating complicated JAK/STAT signaling

“…IL-2/STAT5 signaling contributed to monitoring the ratio of Th9/Th17-like in vitro and allergic disease. To be specific, STAT5 restricted a Th17-like program in the process of Th9 cell differentiation ( Canaria et al, 2021 ). As shown above, STAT5 regulates the development of inflammation and tumor-related diseases ( Surbek et al, 2021 ).…”

STAT proteins: a kaleidoscope of canonical and non-canonical functions in immunity and cancer

“…For example, IL-4-induced pSTAT6 mediates the repression of various genes involved in the alternative macrophage polarization, such as NLRP3 and IL1B , thereby inhibiting inflammasome stimulation and pyroptosis [ 71 ]. Similarly, IL-7-induced pSTAT5 binds to intronic tetrameric STAT sites in B cells to repress the transcription of the IGK gene to suppress immunoglobin gene rearrangement [ 70 ], while IL-2-induced pSTAT5 suppresses a Th17-like program during Th9 cell differentiation [ 83 ].…”