TRANSCRIPTIONAL CONTROL OF T HELPER CELL DIFFERENTIATION
Creators: name: Gonzalez, Daniel Alejandro Canaria
Publisher: name: Purdue University Graduate School
Year: 2023
Subjects: Cellular immunology
Immunology not elsewhere classified
Signal transduction
Biochemistry and cell biology not elsewhere classified

description: IL-9-producing CD4+ T helper (Th9) cells contribute to inflammatory responses during infection, anti-cancer responses and autoimmune disease. Thus, elucidating the signals that regulate their differentiation is critical for understanding the roles of Th9 cells in protective immunity and disease. Th9 cells differentiate in response to IL-4, TGF-β and IL-2, where IL-2 signaling through STAT5 is crucial for transactivating <em>Il9</em> locus. While the roles of IL-4 and TGF- β-mediated signaling are relatively well understood, how IL-2 signaling contributes to Th9 cell differentiation outside of directly inducing the <em>Il9</em> locus remains less clear. I found that human allergen-induced Th9 cells exhibited a strong signature of STAT5-mediated gene repression that was associated with inhibition of a Th17-like transcriptional signature. Likewise, blockade of IL-2/STAT5 signaling increased IL-17 and RORγt expression in murine Th9 cells <em>in vitro</em>. Interestingly, development of this Th17-like phenotype was independent of STAT3. While STAT3 was not required for IL-17 expression, it was required for their long-term persistence. These results suggest that IL-2/STAT5 signaling controls the balance between Th9 and Th17-like cell differentiation in vitro and during allergy. Additionally, I found that murine Th9 cells cultured in a low IL-2 environment had reduced IL-9 production and a diminished NF-kB-associated transcriptional signature, suggesting that IL-2 signaling is associated with NF-kB activation in Th9 cells. Interestingly, NF-kB activation via IL-1β stimulation enhanced Th9 differentiation under IL-2 limiting conditions and promoted their inflammatory potential in a mouse model of Lung inflammation. Mechanistically, we found that IL-2- limiting conditions enhanced IL-1β receptor expression and that IL-1β/NF-kB signaling increased the sensitivity to IL-2 and silenced the expression of the anti-Th9 transcription factor BCL6. Together, these findings indicate that IL-1β /NF-kB signaling can promote Th9 cell differentiation in IL-2-limiting conditions and that this pathway may be targeted to enhance Th9 differentiation and their inflammatory function. Collectively, these data revealed two novel roles for the IL-2/STAT5 axis in Th9 cells. The Thymocyte associated High Mobility Group (HMG) box, known as TOX has been previously described to have paramount functions in the development of all the lineages of CD4+ T cells during thymic selection, during CD8+ T cell exhaustion and in Tfh cell differentiation and function. However, the role of TOX in non-Tfh CD4+ T cells in the periphery has not been addressed. In these studies, I found that CD4+ T cells express TOX in the steady state in secondary lymphoid organs like spleen, lymph nodes, and Peyer’s patches. Specifically, TOX was expressed remarkably in Tfh, Th1, Treg cells, and other non-Tfh unidentified Th cells, as well as Th2 cells in the lungs. Transcriptomics analyses using bulk RNA-seq revealed that TOX minimally alters s gene expression, however it revealed for the first time, that TOX induced genes associated with cell migration i.e., <em>Xcl1</em> <em>Ccl3</em>, <em>Ccl4</em> and also the inhibitory cytokine <em>Il10</em>. The induction of IL-10 and CCL3 was validated at the protein levels, and mechanistic studies revealed that the induction of these molecules required the transcription factor BATF, indicating for the first time a mechanism of TOX-mediated functions. Together, these data shed light in novel roles of TOX in CD4+ T cell function and opens the door for future functional and mechanistic studies that may be relevant during health and disease.