Proliferative tracing with single-cell mass cytometry optimizes generation of stem cell memory-like T cells

Selective differentiation of naïve T lymphocytes into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye-dilution assay for tracking cell proliferative history to mass cytometry, and uncouple division, time and regulatory protein expression in single naïve human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins that are controlled predominantly by division state or time, and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naïve T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK/ITK inhibitor administered before activation, to direct differentiation toward a T(SCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation.