CD4+ T cells play a dual role in the immune system where they act as both “helper” and “effector” cells. In their helper role, CD4+ T cells promote both B cell, CD4+ T cell and CD8+ T cell responses through their production of cytokines (i.e. IL-21, IL-2) and their ability to engage antigen presenting cells via expression of co-stimulatory cell surface proteins (i.e. CD40L) [1]. In their effector role, these cells can differentiate into unique T helper (Th) cell subtypes that produce a unique set of effector cytokines that promote inflammation, pathogen clearance and, when these cells become dysregulated or mistargeted, can lead to inflammatory or autoimmune disease [1], [2]. As their ability to differentiate (e.g. transition from the naïve/quiescent state to a helper or cytokine-producing effector cell) is necessary for all of the above functions, understanding the upstream and downstream signals that drive Th cell differentiation is critical for understanding how these cells function and how we can manipulate these signals to bolster immunity or dampen inflammation and disease. As such, this review will focus on our understanding and integration of factors that drive Th cell differentiation.

The activation and differentiation of Th cells is largely thought to require three major signals. The activation signal (i.e. the “on switch” for T cells), comes in the form of T cell receptor (TCR) recognition of their cognate peptide in the context of major histocompatibility complex class II (MHCII) molecules on the surface of an antigen presenting cell (APC)(termed “Signal 1”). Downstream of signal 1 lies a tyrosine kinase cascade that leads to the activation of NFAT, associated AP-1 transcription factors and NF-κB that underpin the activation cascade [3]. Signal 2 comes in the form of co-stimulatory interactions between the Th cell and APC. Major cell surface protein players in this process include B7 family members on the APC (CD80/CD86) and the co-stimulatory receptor CD28 on newly activated Th cells. Similar to TCR engagement, this interaction induces a signaling cascade resulting in the optimal and sustained activation of NF-κB. This further promotes cell survival and, in combination with NFAT factors, enhances IL-2 production and further proliferation [4]. Finally, Th cells require “Signal 3” to differentiate into cytokine-producing effector cells that typically comes in the form of cytokines derived from APCs or cytokines that are found in the local inflammatory milieu. Importantly, distinct cytokines activate specific JAK/STAT signaling pathways that guide the differentiation of CD4⁺ T cells into diverse Th subsets, each characterized by unique effector cytokine profiles [5]. For example, IL-12 activates JAK2/STAT4 signaling proteins and leads to the downstream activation of a set of transcription factors that drives the differentiation of IFN-γ-producing Th1 cells [6], [7]. While the cytokines and downstream transcription factors that lead to the differentiation of multiple Th cell subtypes (i.e., Th1, Th2, Th17) are relatively well-characterized, those that lead to the differentiation of IL-9-producing Th9 cells remain less well understood. Here, we will review how signals 1–3 and their downstream signaling pathways influence Th9 differentiation and function.

Th9 cells produce high levels of IL-9 and recent literature highlights their unique roles in protective immunity and inflammatory/autoimmune disease. In regard to protective immunity, Th cell-derived IL-9 has been shown to contribute to protection from parasite infection [8] and from certain types of cancers, where adoptive Th9 cell therapy has been highly effective against specific tumor types [9]. In regard to their roles in disease, mistargeted or dysregulated Th9 cell responses have been implicated in allergic disease, inflammatory bowel disease and autoimmunity [10]. Given the critical roles of Th9 cells in both protective immunity and disease, understanding the signals that govern their differentiation is essential for developing targeted therapies, either to enhance their anti-tumor functions or suppress their activity in autoimmune conditions. This review examines how Signals 1–3 shape Th9 development, identifies current knowledge gaps, and outlines potential future directions for research.