Differentiation of human T follicular helper cells in vitro requires co-operation between STAT3 and SMAD signaling cytokines that is restrained by IL-2

T follicular helper cells (TFH) are a CD4 T cell subset required for effective humoral immunity; promoting antibody responses to vaccines and natural infections. The mechanisms by which human TFH differentiate are not fully understood, and given the anatomical localization to the secondary lymphoid tissues, and asynchronous nature of in vivo TFH populations, studying TFH differentiation in vitro represents an attractive alternative, through which the mechanisms which govern TFH differentiation can be explored. Here we demonstrate reliable differentiation of in vitro TFH from naive human CD4 T cells in a STAT3 dependent fashion. Cooperation between STAT3 and SMAD signaling cytokines led to expression of the lineage defining transcriptional regulator BCL6, multiple downstream proteins required for TFH function and localization (including PD1 and CXCR5), and potent IL-21 production capacity by these in vitro TFH. Critically we found that autocrine production of IL-2 acted as an intrinsic brake on TFH differentiation in vitro; promoting proliferation but limiting the acquisition of a TFH phenotype resembling that found in germinal centers in vivo; neutralization of IL-2 resulting in the emergence of this phenotype. Overall, we show that human TFH expansion, differentiation and function relies on careful regulation of pro- and anti-TFH cytokine signaling.