Optimized phospho-flow cytometry for quantitative analysis of early TCR-proximal signaling: Implications for T cell...

T cells are central mediators of anti-tumor immune responses, with their activation critically depending on finely tuned, and kinetically controlled signaling through the T cell receptor (TCR)-CD3 complex. Early TCR-proximal phosphorylation events shape downstream pathways that govern T-cell proliferation, differentiation, and effector functions, including responses to cancer immunotherapies, thus playing a pivotal role for effective tumor surveillance and eradication. Despite their importance, accurately capturing these early signaling events, at the single-cell level, remains technically challenging. Here, we present an optimized flow cytometry-based phospho-profiling protocol to assess early TCR signaling dynamics, using ERK phosphorylation as a sensitive readout. By overcoming the challenges of traditional approaches, this protocol provides an easy, yet powerful tool to evaluate T cell functionality both systemically and within the tumor immune microenvironment (TIME). Its application holds significant promise for advancing our understanding of T cell biology and for guiding the development and likely refinement of next-generation cancer immunotherapies.