High-Dimensional Single-Cell Profiling of Tumor-Infiltrating CD4⁺ Regulatory T Cells

CD4⁺ T regulatory cells (Tregs) are a specialized subset of T lymphocytes, which promote immune homeostasis and tumor immunosuppression by restricting effector T cell immune responses. The characterization of context-specific Treg phenotypic heterogeneity is pivotal to determine their potential contributions to diseases. In the recent years, high-dimensional single-cell technologies, such as single-cell RNA sequencing, mass cytometry, or polychromatic flow cytometry, have played a central role in elucidating the heterogeneity of the Treg compartment at the cellular and molecular levels. Here we describe an example of high-dimensional flow cytometry analysis capable of defining an effector Treg subpopulation that positively correlates with cancer progression. Moreover, we provide a workflow template of high-dimensional single-cell analysis that is readily applicable to any leukocyte subpopulation.