TGFß determines morphology and key cellular processes of activated CD4+ T cells

During an immune response, cells are simultaneously exposed to multiple cytokine signals that collectively determine their phenotype. Transforming growth factor ß (TGFß) is a pleiotropic cytokine acting as a key regulator of T-cell differentiation with activating and suppressive effects on their immune function. Here we identify and analyze the cellular responses of CD4+ T cells to TGFß across signaling contexts by analyzing the responses of T cells to multiple cytokine mixtures in the presence or absence of TGFß. We found that TGFß had a profound dominant effect independent of the presence of other cytokines, modulating the expression of more than 4,000 genes. In the presence of TGFß, cells exhibit lower expression of translation-related and apoptosis-related genes, accompanied by increased survival of activated T cells. Notably, cells cultured in the presence of TGFß were smaller in size while preserving their proliferative ability. Accordingly, we identified a dense network of transcription factors that were modulated by TGFß, suggesting a core gene set connecting TGFß signaling to the regulation of T-cell size. We found N-Myc to be at the center of this network, and we directly show that TGFß regulates its gene expression level, protein level, and nuclear localization. Our work provides a system to study cell size control and demonstrate the profound effect of TGFß in the modulation and regulation of T-cell properties, expanding its role beyond guiding their phenotype. Overall design: This work examined the effect of TGFb1 on the cellular state and characteristics of CD4+ T cells. To distinguish the effect of TGFb from that of other cytokines we isolated naïve CD4+ T cells from spleens of mice and cultured the cells under different cytokine mixtures in the presence or absence of TGFb1. We then examined the gene expression profile of the CD4+ T cells using bulk RNA-sequencing in cells cultured under 10 cytokine conditions. As a control, we added a condition of naïve cells. Each condition had 3 repeats.